Arindam K Dey

Adrien Nougarede

Flora Clement

Carole Fournier

Evelyne Jouvin-Marche

Marie Escudé

Dorothée Jary

Fabrice P Navarro

Patrice N Marche
email: patrice.marche@inserm.fr

Flora Clément

Ling Peng

Tuning the immunostimulation properties of cationic lipid nanocarriers for nucleic acid delivery

Keywords: Nanostructured lipid carrier, Antigen presenting cells, nucleic acid delivery, toxicity, surface charge 9.3.2 Immunotoxicity of Amphiphilic dendrimers Amphiphilic dendrimers, Immunotoxicity, Immunometabolism

Non-viral systems, such as lipid nanoparticles, have emerged as reliable methods to enable nucleic acid intracellular delivery. Lipid nanoparticles are currently in use for drug delivery. The use of cationic lipids in the formulation of lipid nanoparticles enable to form complexes with nucleic acid cargo and facilitate their uptake by target cells. However, due to their small size and highly charged nature, these nanocarriers systems can interact in vivo with antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages. As this might prove to be a safety concern for the development of therapies based on lipid nanocarriers, we sought to understand how they could affect APCs physiology. In the present study, we investigated the cellular and metabolic response of primary macrophages or DCs exposed to the neutral or cationic variant of the same lipid nanoparticle formulation. We demonstrated that macrophages were the most affected cells and that the cationic nanocarrier has a more substantial impact on their physiology, dependent on the positive surface charge. Our study provides a first model explaining the impact of charged lipid biomaterials on immune cells, and demonstrate that we can prevent the main adverse effects observed by finely tuning the load of nucleic acid cargo. Finally, we bring rational to calibrate the nucleic acid load of cationic lipid nanocarriers depending on whether immunostimulation is desirable with the intended therapeutic application, for instance, gene delivery or mRNA vaccines.

Acknowledgement

I would like to take this opportunity to extend my heartfelt gratitude towards the following people whose contributions have made this endeavor possible.

Firstly, I would like to express my sincere gratitude to the members of the jury Dr. Bernard Verrier, Dr.

Emilie Roger, Prof. Valérie Forest, Prof. Walid Rachidi, and Dr. Xavier Le Guevel, for taking the time and effort to evaluate this work.

I am thankful to my supervisor Dr. Patrice N Marche, for giving me the freedom to organize my research work and explore the subject. We had started working together five years back when I came to France as an Erasmus Master's student. His wisdom, enthusiasm, and support over the last few years has helped me understand and focus on the project and the subject. His stories about different discoveries in science, especially immunology, influenced me to build a stronger interest in the subject. Outside science, we spent many hours discussing French history, current politics, and different wines. We also traveled together many countries to attend different project meetings and gathered lifetime memories during those trips.

In the end, I will say, thank you for believing in me and help me to grow both personally and professionally. I will always be indebted to you. I am profoundly grateful to every member of Institute for Advanced Biosciences (IAB), Grenoble, who have helped me in making beautiful memories. Especially, I would like to thank present and former members of Team Marche (Alexis Gonon, Alexis LEROY, Benoît Manfroi, Bertrand Huard, Christian Villiers, Christine Charrat, Carole Fournier, Flora Clement, Herve Lerat, Laurie Baert, Lydie Carreres, Mashal Ahmed, Marion Ressejac, Mariam Mroweh, Mylene Pezet, Keerthi Kurma and Zuzana Macek Jílková), whose company I have enjoyed, and whose laugh and discussions I have digested. I would like to thank various platforms and facilities at IAB (Mylene Pezet, Alexei Grichine, and Jacques Mazzega: Microscopy platform) (Animal facility: Anthony Lucas) for great support. This Ph.D. work would not have been possible without financial support. I would like to thank the European Union's Horizon 2020 project "NEWDEAL" to support my Ph. D. project. I would like to acknowledge EURONANOMED European Research project "NANOGLIO" and institutional grants from Institut National I would like to express my utmost appreciation to Adrien Nougarede, Dorothée Jary, Fabrice P. Navarro from CEA-Leti, Grenoble, for providing lipid nanoparticles and Jiaxuan Chen, Ling Peng from Aix-Marseille Université, Marseille for providing the dendrimers. My deepest gratitude to Team NEWDEAL and Team NANOGLIO for productive discussion to conduct my experiments.

I would like to express my special thanks of gratitude to my school teacher Somnath Kundu. Your teaching style influenced me to opt for a career in biology.

I am delighted to have met so many amazing people during my time in Grenoble. Thank you, Pushpendra, Shashank, and Ritesh, to accompany me on our "TGVMax journey" to explore every corner of France and make wonderful memories which I will cherish for the rest of my life. A big part of my personal and professional growth belongs to the influence brought in my life by you all. I would like to mention how much you all mean to me by presenting a summary of the trip to my memory lane.

Finally, I am blessed to have my family and friends for always being there and supporting me no matter what. Warmest thanks to Keerthi for being a supportive colleague, a caring sister, and a fantastic roommate. My journey of life would remain incomplete if I have not met you in Grenoble. I am overwhelmed by the presence of my closest friends, Shailesh, Sayantan, Sumanta, Nikita, Aparajita, and Shreyasi, for standing beside me throughout my Ph.D. journey. Special Thanks to kaberi for her support and encouragement during the challenges of graduate school. Even being miles apart, you helped me to vent out the stress; you people are amazing. Thank you, Shailesh, for being an amazing travel buddy in several euro trips, which was a major energy source to withstand Ph.D. stress. I would like to express my most heartfelt thanks to my parents, Sujit Kumar Dey and late Rikta Dey, for their unconditional love and mental support throughout my life.

Résumé

Les caractéristiques uniques des nanomatériaux (NM) sont précieuses dans de nombreuses applications industrielles et biomédicales. Toutefois, les MR pourraient également donner lieu à une toLes phagocytes du xicité imprévue qui pourrait nuire au système biologique en particulier au système immunitaire. Les phagocytes du système immunitaire ont la capacité d'ingérer des matériaux étrangers ; ils sont donc appropriés pour l'étude de l'immunotoxicité car ils sont impliqués dans les réponses immunitaires non spécifiques et spécifiques.

Dans cette étude, nous avons étudié l'impact de différents nano matériaux (NM), telles que des nanoparticules d'or (AuNPs), des nanoparticules lipidiques neutres (NLCs) ou cationiques (CLCs), et des dendrimères amphiphiliques (ADs) sur les macrophages primaires (BMDMs) et des cellules dendritiques (BMDCs) par deux approches. Dans la première, nous avons recherché les effets directs de l'exposition. Dans la seconde, nous avons étudié les effets indirects sur l'activation du LPS ou de l'IL-4 des cellules exposées aux NMs.

Nos résultats concluent que tous les NMs testés n'ont pas modifié la capacité de phagocytose. Une analyse plus approfondie montre que tous les NMs ne montrent aucun, ou peu, effet direct sur l'activation des BMDC. Pour le BMDMs, les AuNPs, les CLCs et les ADs ont considérablement diminué l'activation cellulaire tandis que les NLCs n'ont d'effet. L'étude de la production de cytokine basée sur la production d'IL-6 et de TNF-, de la production de NO et du métabolisme cellulaire basée sur l'étude de la glycolyse et du métabolisme mitochondrial n'a également pas ou peu d'effet direct sur les BMDCs et les BMDMs. Cependant, nous avons remarqué une augmentation significative de la production de MCP-1 par les CLCs et les cellules exposées aux ADs, tandis que les AuNPs et les NLCs n'ont pas d'effet sur la production de MCP-1. De plus, nous avons noté que les CLCs et les NLCs à forte concentration pourraient légèrement augmenter la production de ROS, tandis que les AuNPs et les ADs ne modulaient pas la production de ROS par les BMDM et les BMDC.

L'étude des effets indirects de NMs a montré que quand les cellules exposées aux NMs sont stimulées par du LPS, nous avons observé une modulation de la réponse des BMDCs au niveau de l'activation cellulaire, la sécrétion de cytokine et de chimiokine, NO, et la production de ROS. Toutes les NMs n'affectent pas, ou peu, le métabolisme des BMDCs. D'autre part, nous avons enregistré une altération significative chez les BMDMs exposés aux NMs dans leur réponse à l'activation par le LPS au niveau de l'activation cellulaire, la sécrétion de cytokine et de chimiokine, la production de NO et le métabolisme cellulaire. Cependant, la production de ROS reste inchangée dans le cas des BMDMs exposés aux NMs.

L'étude in vitro de la présentation d'antigène, a indiqué que l'exposition à différents NMs modifie les cytokines produites par les cellules T, reflétant l'altération de la polarisation des lymphocytes T CD4 +. Dans l'ensemble, ces résultats démontrent que les NMs ne modifient pas directement l'activité phagocytique, la production de cytokine et de chimiokine, la production de NO et de ROS, le métabolisme cellulaire des cellules phagocytaires, mais lorsqu'elles sont confrontées à un signal d'activation comme le LPS ou l'IL-4, les cellules exposées aux NMs réagissent différemment. 2

List of Figures

The components of the innate and adaptive immune system.

3

The polarization of macrophages. 12 Six major metabolic pathways.

13 Glycolysis is the predominant source of energy in M1 macrophages.

14

In LPS activated M1-like macrophages, TCA cycle is broken in two places -after citrate and after succinate.

15

The role of nanoscience and nanotechnology in science and engineering.

List of tables

General Introduction

In the field of nanotechnology, nanoparticles (NPs) are defined as a small object which behaves as a unit with respect to its delivery and other properties. NPs (or Nanopowder or nanocluster or nanocrystal) are microscopic particles with three dimensions less than 100 nm [START_REF] Khan | Nanoparticles: Properties, applications and toxicities[END_REF]. It is observed that they have quite distinct properties because of their surface structure and small volume-to-surface area ratio, which can impact the solubility, shape, surface reactivity, and aggregation. The smaller the diameter of a spherical particle, the more the surface-to-volume ratio increases, leading to increased chemical reactivity.

This greatly affects the interactions with biological structures and living species. Engineered nanomaterials, particularly NPs, have been rapidly developed for the production of a wide variety of objects for a large range of applications, including industrial products, food, agriculture, and health.

Since long time extensive libraries of NPs, composed of an assortment of different sizes, shapes, and materials, and with various chemical and surface properties, have been used in different biomedical applications (McNamara & Tofail, 2017;[START_REF] Mudshinge | Nanoparticles: Emerging carriers for drug delivery[END_REF] As the field of nanotechnology is under constant and rapid growth and new additions continue to supplement these libraries. Owing to the fast-paced growth of nanomaterials, Nanotoxicology has emerged to evaluate safety during in the industrial processes and for the use of products. A functional understanding of the acute and chronic toxicological effects of engineered nanomaterials is not well understood and may represent a significant limitation to the nanomaterial development and application.

The immune system acts as a guardian of the body integrity either directly against exogenous intruders, which can be microbes during infections or indirectly against host cell modifications resulting from external stresses (UV, heat, chemicals, tissue destruction). Along with this task, the immune system ensures tolerance to the host components preventing auto-destruction. Defects in these missions are associated with a wide range of diseases: chronic inflammations, cancers, autoimmunity, and many more.

The immune system includes an innate and an adaptive contribution. The innate immune system is considered as the first line of defense against a foreign pathogen (Chaplin, 2010a). It includes several kinds of phagocytic cells whose main role is to engulf and digest cellular debris, pathogens, particles taken up from their environment. Neutrophils are the most abundant white blood cell in humans and mice, and they are the first immune cells recruited at the site of inflammation in response to chemotactic cues such as CXCL8 (interleukin-8) produced by stressed tissue cells and tissue-resident immune cells like macrophages. Macrophages are a subset of such phagocytes of the innate immune system. They contribute to a rapid and non-specific defense, efficient against most pathogens [START_REF] Klopfleisch | Macrophage reaction against biomaterials in the mouse model -Phenotypes, functions and markers[END_REF].

Dendritic cells (DCs) are defined as professional antigen-presenting cells (APCs). These cells act at the interface between innate and adaptive immune systems. After phagocytosis, the main function of these cells is to process and present antigens, associated with class II Major Histocompatibility Complex (MHC-II), to naïve T lymphocytes (LT), a process that may initiate the adaptive immune response. Upon activation by a foreign pathogen, DCs also secrete a wide variety of cytokines, which are implicated in the activation of several lymphocytes, including B, T, and NK cells [START_REF] Blanco | Dendritic cells and cytokines in human inflammatory and autoimmune diseases[END_REF]. The integrity of innate and adaptive immune cell population is necessary to ensure proper responses to an infection; these cells are thus likely the most relevant experimental models for system for toxicology studies.

Immune cells display a wide range of diversity in response to the microenvironment and adopt different phenotypes. Metabolic profile of these cells plays a very important role in their functions. While lipopolysaccharides (LPS) induced, classically activated cells are pro-inflammatory in nature and display enhanced glycolytic metabolism and take part in direct host-defense against pathogens, such as phagocytosis and secretion of pro-inflammatory cytokines and microbicidal molecules. On the other hand, IL-4 induces alternatively activated cells are anti-inflammatory in nature and show high mitochondrial oxidative phosphorylation (OXPHOS) and play an important role in the regulation of the immune response to parasites as well as allergic reactions [START_REF] Ley | M1 Means Kill; M2 Means Heal[END_REF][START_REF] O'neill | A guide to immunometabolism for immunologists[END_REF]. Growing evidence support the metabolic status of immune cells determines their functions, and perturbations of distinct metabolic networks are implicated in immune cell-associated diseases. In short, alteration of the metabolic profile of a particular cell type could lead to different disease conditions but can also be used to treat a particular kind of disease.

The presented works were designed to provide an accurate evaluation of the impact of some NPs on the immune system. We have selected macrophages and DCs as targets because of the roles in the regulations of the immune responses and the inflammation. The studied NMs are issued from collaborative works in the frame of National or European projects, including gold nanoparticles (AuNPs), neutral lipid nanoparticles (NLCs), cationic lipid nanoparticles (CLCs), and Amphiphilic dendrimers (ADs). We analyzed the toxicity, the functions, and the metabolic pathways of the exposed cells. Effects on macrophage and dendritic cell metabolism could lead to modifications in their respective functions that may propose cell reprogramming using NPs in the frame of novel therapeutic approaches for the treatment of inflammatory diseases. For this reason, we analyzed whether exposure to subtoxic doses of these nanoparticles leads to the alteration of the functions of the cells. We evaluated the effect of NPs on (1) Macrophages and DCs metabolism, (2) Macrophages and DCs activation, (3) cytokine production, (3) Redox profile, (4) the phagocytic capacity of macrophages, and ( 5) T cell activation by DCs (Figure 1).

Figure 1: Immunotoxicity assessment (Created with BioRender.com) BMDCs and BMDMs were cultured from mouse BM for 11 and 7 days, respectively. After harvesting, the cells were seeded either in 12, 24, or 96 wells plates from Falcon® or Seahorse XFe96 cell culture with AuNPs at 10 and or 50 µg/mL final concentrations. After 24 h of cell culture, cells were washed and stimulated with LPS or IL-4 for 24 h, and several downstream experiments were conducted according to the protocol.

In this manuscript, in the first part, I describe 1) The immune system and some of its components, 2) The importance of the metabolism governing different immune cell functions, and 3) A summary of different nanoparticles used in the study, including their usage for the treatment and the diagnosis of different diseases as well as their known impact on the immune system. In the second part, I present the results in the format of journal publications. Finally, a common discussion is presented at the end of the manuscript.

5. Immune System: Art of War

Introduction

Study of strategic warfare has shown that a detailed understanding of the enemy and one's own temperament is key to victory -by preparation, strategy, and carefully guided execution. Interestingly, all living organisms are connected with each other through a chain of interactions. Although many of them are harmless, some are not. Therefore, every living organism has developed a mechanism for identifying and neutralize threats from other species, commonly known as the "immune system." Likewise, in constant battle between metazoans and pathogenic invaders, evolution developed a strong "immune system" based on discrimination between self and infectious/pathogenic non-self [START_REF] Buchmann | Evolution of Innate Immunity: Clues from Invertebrates via Fish to Mammals[END_REF].

To fight the intruders of the immune system developed a highly effective arsenal of weapons through integrated systems broadly categorized as 'innate' or 'adaptive' immunity. Innate immunity, a primitive immunological mechanism for fighting against an intruding pathogen, recognizes the pathogenic non-self through a fixed number of germ-line-encoded receptors, which recognize structural components of microorganisms and viruses (Chaplin, 2010b). This achieves the "immediate immune response," which is triggered within minutes or hours of an attack. Adaptive immunity has been acquired during evolution by vertebrates and cartilaginous fishes, allowing specific immune responses towards molecular targets called antigens. The specificity relies on the clonal expansion of antigen-specific effector cells (B or T cells) selected for the expression of an antigen receptor (immunoglobulin and T cell receptor (TCR), respectively), which is generated by an original mechanism of gene rearrangements [START_REF] Smith | A Comparison of the Innate and Adaptive Immune Systems in Cartilaginous Fish, Ray-Finned Fish, and Lobe-Finned Fish[END_REF].

This enables the host to mount a specific immune response towards a large panel of targets. Furthermore, the immune system keeps memory of the specific immune allowing quick response in the case of secondary exposure to the antigens. [START_REF] Dranoff | Cytokines in cancer pathogenesis and cancer therapy[END_REF]))

Innate Immune response

The innate immune system is the first line of defense against the intruders. The system responds in the same way to all germs and non-self-agents. That is why the innate system is also known as a "non-specific immune system." Pathogens often enter through epithelial barriers such as the skin, gut, or respiratory tracks. The innate immune system could stop the intruders either by providing physical protection to the host by the skin and mucous membrane or by its specialized cellular system (defense system) and proteins.

Based on the type of defense barrier, the innate immune system can be classified into two categories (1) anatomic (epithelium and mucous membrane) and physiologic (temperature, low pH, and chemical mediators) barrier (2) endocytic and phagocytic barriers.

Table 1 summarizes the non-specific host-defense mechanisms for each of these barriers (Marshall et al, 2018).

Anatomic and Physiologic barriers

All the outer and inner surfaces of the organism act as a mechanical barrier and physically block the pathogens to invade. Skin is the single most crucial mechanical barrier of our body. The outer surface of the skin, known as the epidermis, prevents the pathogens from penetrating into the body. Other than skin, another crucial mechanical barrier of the innate system is the mucous membrane. These membranes line the gastrointestinal, urinary, respiratory, and reproductive tracts and prevent intruders' entry by a slimy mucous layer.

While the anatomical barrier physically blocks pathogens from entering the body. On the other hand, physiological barriers protect the body by maintaining body temperature, pH, and various soluble secretory products against a foreign pathogen.

Phagocytic and endocytic barriers

If a pathogen passes through physical barriers, the innate immune system activates specialized immune cells and proteins to take care of the intruder. For healthy survivability, multicellular organisms must identify dead or alive cells and the entry of microorganisms. To do that, they have developed several surveillances, defense / and repair mechanisms.

Defensive barriers

In 1989, Charles Janeway Jr. suggested that the presence of a set of receptors expressed by innate immune cells is responsible for identifying the conserved products of microbial origin [START_REF] Janeway | Approaching the asymptote? Evolution and revolution in immunology[END_REF]. After 25 years of extensive studies, intensive debates, and a Nobel Prize on this topic, it is unquestionable that Janeway's brilliant idea has changed the entire understanding of the immune system. Later on, his idea has led to the identification of so-called Pattern Recognition Receptors (PRRs), proteins responsible for recognizing molecules that are associated with pathogens (such as Pathogen-Associated Molecular Patterns-PAMPs). In mammals, PRRs can be grouped into at least five major categories: toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), C-type lectins (CLRs), nucleotide oligomerization (nod)-like receptors (NLRs), and non-melanoma 2 (AIM2)-like receptors (ALRs) [START_REF] Jang | An Overview of Pathogen Recognition Receptors for Innate Immunity in Dental Pulp[END_REF].

Another fantastic idea came from Polly Matzinger (Matzinger, 1994), who suggested that the immune system is less concerned with the origin of antigens (self vs. non-self) than with their interaction with our body (tissue damage vs. tissue homeostasis). In her "Danger Theory," Matzinger proposed that endogenous molecules are released or activated during tissue stress or damage and trigger or propagate the inflammatory response, which signals antigen-presenting cells to initiate the adaptive immune response. Today, the molecules described in her danger theory is known as DAMPs (Damage-Associated Molecular Patterns).

At least three broad-spectrum strategies are used by the innate immune system to identify the dead or alive cells and the entry of foreign intruders ( Table 2) [START_REF] Jang | An Overview of Pathogen Recognition Receptors for Innate Immunity in Dental Pulp[END_REF][START_REF] Turvey | Innate immunity[END_REF].

1. The innate immune system depends on a series of germline-encoded receptors to recognize 'microbial non-self.' These include components of microbial membranes, cell walls, proteins, and DNA and are termed pathogen-associated molecular patterns (PAMPs).

2. The innate immune system detects immunological 'danger' in the form of DAMPs. DAMPs represent typical metabolic by-products produced in the process of infection and inflammation [START_REF] Bianchi | DAMPs, PAMPs and alarmins: all we need to know about danger[END_REF].

3. Innate immune receptors detect "missing self" -molecules expressed by healthy cells but not expressed by infected cells or microorganisms. This inhibitory system is dependent on NK cells. [START_REF] Jang | An Overview of Pathogen Recognition Receptors for Innate Immunity in Dental Pulp[END_REF][START_REF] Turvey | Innate immunity[END_REF] Intruders can be recognized and stopped by specialized cells. The most important cells that take part in recognizing and stopping foreign intruders are known as phagocytes. Phagocytes are special kinds of scavenger cells that use phagocytosis to engulf bacteria, foreign particles, and dying cells to protect the body. They engulf pathogens and internalize them in a phagosome, which acidifies and fuses with lysosomes to destroy the contents. This mechanism is used by the immune system to remove potentially pathogenic material. The three most important phagocytic cells are neutrophils, monocytes/macrophages, and dendritic cells in the immune system.

Neutrophils

Neutrophils are the most abundant group of granulocytes in the blood and essential for acute inflammation. Notably, they are the first immune cells to arrive at the site of infection. Neutrophils are short-lived cells, and they usually undergo self-destruction followed by phagocytosis and the use of their granules. That is why they are the main component of pus seen with infection. Neutrophils produce significant amounts of antimicrobial peptides, enzymes, and reactive oxygen species to kill bacteria.

Additionally, they also produce enzymes involving tissue remodeling and repair after injury. Substantial quantity of neutrophil accumulation at the site of bacterial infection and tissue injury ensure the destruction and degradation of microbes and other antigenic materials. Therefore, it is clear that neutrophil plays an essential role in the clearance of microbial pathogens and repair of tissue injury.

Neutrophils are also crucial for initiating inflammation and recruiting other inflammatory cells by producing a large quantity of cytokines, Tumor Necrosis Factor (TNF) and interleukin-12 (IL-12), as well as specific chemokines (Chaplin, 2010b). This indicates an immunoregulatory role of neutrophils. Compared to neutrophils, eosinophils and basophils have lower phagocytic capacity and act mainly by releasing enzymes, toxic compounds, and pro-inflammatory, to eliminate the pathogens.

The other group of granulocytes are eosinophils and basophils. Eosinophils have prominent cytoplasmic granules that contain toxic molecules and enzymes that are especially active against helminths and other parasites. IL-5 stimulates the development of bone marrow-derived eosinophils and supports their survival in peripheral tissues, making them productive in most allergic responses (Chaplin, 2010b).

Basophils and mast cells are morphologically similar, but they represent distinct lineages. Based on the cell surface receptors for IgE (FcεRI), they are the main initiators of immediate hypersensitivity responses and host response to helminthic parasites, releasing histamine and other preformed mediators from their granules and generating large quantities of lipid mediators that induce tissue inflammation, edema, and smooth muscle contraction. Interestingly, Mast cells and basophils can release IL-4, indicating that they can play important roles in allergic immune responses [START_REF] Van Panhuys | Basophils Are the Major Producers of IL-4 during Primary Helminth Infection[END_REF].

Monocytes and macrophages

Monocytes are phagocytes found in the bloodstream. They circulate throughout the body, exit the circulation, and enter into infected or inflamed tissue. In the tissue, they differentiate into macrophages, which form the major resident population of phagocytes in healthy tissues. Both monocytes and macrophages are highly phagocytic for microbes and particles that have been marked for clearance by binding Ig and/or complement. More precisely, complement C3b and the IgG Fc fragment allow better uptake of opsonized particles. They arrive at the site of infection immediately after neutrophil mobilization, and in case of chronic inflammation and infection, they remain at the site for an extended period.

Based on the activating signal that initiates the differentiation of immature precursor cells to active macrophages, macrophages can adopt one of several phenotypes [START_REF] Benoit | Macrophage polarization in bacterial infections[END_REF]. For example, classically activated M1 macrophages produce large amounts of pro-inflammatory cytokines such as IL-1, IFN-γ, IL-6, IL-12, TNF to fight against infection or tissue stress by activating several antimicrobial mechanisms. M1 macrophages also produce reactive oxygen species (ROS) and nitric oxide (NO) to kill microbial pathogens. These ROS and NO are highly toxic to microorganisms but also nearby cells.

Therefore, the synthesis of these compounds must be highly regulated [START_REF] Nathan | Nonresolving inflammation[END_REF].

Alternatively, activated M2 macrophages carry out this regulation. Alternatively activated macrophages are induced by IL-4, IL-10, or IL-13, especially in the presence of glucocorticoid hormones, and express anti-inflammatory functions through their production of IL-10, the IL-1 receptor antagonist, and transforming growth factor β (TGF-β) [START_REF] Gordon | Alternative activation of macrophages[END_REF]. TGF-β1 stimulates the differentiation of fibroblasts into myofibroblasts, increases the synthesis of collagen by these myofibroblasts, and induces the Tissue Inhibitor of Metalloproteinase (TIMP, which inhibits the degradation of the extracellular matrix by metalloproteinase). Finally, M2 produces Platelet-Derived Growth Factor (PDGF), promoting the proliferation of myofibroblasts [START_REF] Wynn | Cellular and molecular mechanisms of fibrosis[END_REF]. It is important to consider that the macrophage acts in cooperation with many other cells of the immune system [START_REF] Nish | Host defense pathways: role of redundancy and compensation in infectious disease phenotypes[END_REF]. Thus, primary activation by a PAMPs is to induce the synthesis of interleukin as well as expression of distinct adhesion molecules such as LFA-3 (CD58), lymphocyte function-associated antigen (LFA)-1 (CD11a), intercellular adhesion molecule (ICAM)-1 (CD54), ICAM-2 (CD102) and costimulatory molecules B7.1 (CD80), B7.2 (CD86), or CD40 (Fröhlich, 2015b) (Figure 2). Altogether, the activation signal initiates the processing of cytosolic proteins to be presented by MHC I or MHC II to activate T cells.

PAMPs dependent, macrophage-mediated activation of T cells initiate CD4 + Helper 1 (Th1) response.

These T cells secrete IFN-γ, which stimulates the antibacterial activity of M1 macrophages, making a positive feedback loop. In contrast, IL-4 production during a response mediated by CD4 + Helper 2 (Th2) cells induces a macrophage polarization towards the M2 phenotype and local proliferation of macrophages, allowing a self-renewal of macrophages without recruiting the bone marrow monocytes [START_REF] Jenkins | Local macrophage proliferation, rather than recruitment from the blood, is a signature of TH2 inflammation[END_REF]. Similarly, the production IL-4 by granulocytes (mainly by polymorphonuclear eosinophil) seems to be necessary for M2 polarization in adipose tissues (Figure 3) [START_REF] Wu | Eosinophils sustain adipose alternatively activated macrophages associated with glucose homeostasis[END_REF]. [START_REF] Nakagawa | Role of microglial m1/m2 polarization in relapse and remission of psychiatric disorders and diseases[END_REF]) 5.2.2.3 Dendritic cells (DCs) DCs are heterogeneous populations of phagocytic cells of hematopoietic origin, except the follicular DCs, which are of mesenchymal origin. They are important innate immune cells that build a bridge between the innate and adaptive immune systems. Immature DCs circulate throughout the bloodstream and tissue and continuously sample the pathogens via micropinocytosis. The existence of numerous cytoplasmic projections from their surface gives them a wide surface area to volume ratio that allows close contact with multiple cells. These processes look similar to the dendrites of neurons, which gave dendritic cells their name.

Although DCs are well known phagocytic cells, DCs mainly specialize in the capture and presentation of antigens to T lymphocytes, to activate the adaptive response. DCs are the only cells capable of initiating the response of naive T lymphocytes. That is why they are called "professional" antigen-presenting cells.

To initiate a T cell response, DCs are equipped with major histocompatibility complex (MHC) and different co-stimulatory molecules necessary for the activation of naive T lymphocytes. They also secrete a large panel of chemokines involved in the attraction of other effector cells of the immune system at different phases of the inflammatory response [START_REF] Piqueras | Upon viral exposure, myeloid and plasmacytoid dendritic cells produce 3 waves of distinct chemokines to recruit immune effectors[END_REF] In mice, there are many precursors, namely CMPs, MDPs, and CDPs. CDPs divide into pre-cDCs and pre-pDCs. Pre-cDCs give rise to pre-cDC1 and pre-cDC2, which later differentiate into cDC1 and cDC2, respectively. Pre-pDCs develop into pDCs. Phenotypes of each cell and cytokines involved in the development process include ell myeloid progenitors (CMPs), macrophages and dendritic cells progenitors (MDPs), common myeloid progenitors (CDPs), granulocytes, macrophages and DCs progenitors (GMDPs), preconventional dendritic cells (pre-cDCs), pre-plasmacytoid dendritic cells (pre-pDCs), pre-conventional dendritic cells 1 (pre-cDC1), pre-conventional dendritic cells 2 (pre-cDC2), conventional dendritic cells 1 (cDC1), conventional dendritic cells 2 (cDC2), plasmacytoid dendritic cells (pDCs). (Adapted from [START_REF] Castell-Rodríguez | Dendritic Cells: Location, Function, and Clinical Implications[END_REF])

Conventional myeloid dendritic cells (mDCs):

To date, the development of DCs in bone marrow is well characterized in mice than in humans. Myeloid dendritic cells (mDCs) originating from CDPs are a group of professional antigen-presenting cells, responsible for identifying, processing, and presenting antigens to T cells. Morphologically, mDCs bear long dendrites. They are found in peripheral tissues, secondary lymphoid organs (spleen, lymph nodes, Peyer's patches, thymus), and blood and have a strong migratory capacity. They express TLRs 1 to 8 and TLR10 but not TLR9 [START_REF] Jarrossay | Specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells[END_REF]. These mDCs are also known as conventional DCs (cDCs). They are classified by their discreet roles and transcriptional identities and comprise two major subsets known as type 1 cDCs (cDC1s) and type 2 cDCs (cDC2s). The difference between cDC1 and cDC2 subsets is supported by their distinct genetic signature, which is essential for defining the specific functions of the cDC subsets [START_REF] Guilliams | Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny[END_REF].

The pro-inflammatory function of mDCs involves the production of inflammatory molecules and to prime the pro-inflammatory subsets of effector T cells. On the other hand, tolerogenic mDCs, counter inflammation by disrupting the activity of pro-inflammatory T cells and macrophages as well as by inducing immunosuppressive regulatory T cells. Differentiation of mDCs to acquire proinflammatory or regulatory phenotype depends on the surrounding microenvironment (Figure 5). [START_REF] Chistiakov | Myeloid dendritic cells: Development, functions, and role in atherosclerotic inflammation[END_REF].

More specifically, cDC1s are mainly involved in regulating cytotoxic T lymphocytes (CTLs) as they present exogenous, cell-associated antigens to CD8 + T cells. On the other hand, the cDC2 subset takes part in immune responses involving extracellular pathogens, parasites, and allergens by presenting the soluble antigens to CD4+ T cells [START_REF] Vu Manh | Investigating Evolutionary Conservation of Dendritic Cell Subset Identity and Functions[END_REF].

Although mouse and human mDCs share many evolutionarily conserved molecular pathways, they show some distinct differences in terms of the expression of their surface markers. Murine counterparts of human cDCs are shown in [START_REF] Robbins | Novel insights into the relationships between dendritic cell subsets in human and mouse revealed by genome-wide expression profiling[END_REF]).

Plasmacytoid dendritic cells (pDCs):

pDCs have an eccentric nucleus and prominent endoplasmic reticulum and Golgi (resembling a plasma cell)

for the production of type I interferon. In the inactive state, these cells look like plasma cells, round with a large cytoplasm; hence they are called Plasmacytoid DCs. pDCs are the majority subtype of blood DCs (0.4% of leukocytes) and are found in secondary lymphoid organs but not in peripheral tissues, unlike mDCs. Unlike mDCs, CD11c, CD33, CD11b, or CD13 markers are not expressed by pDCs [START_REF] Dzionek | BDCA-2, BDCA-3, and BDCA-4: three markers for distinct subsets of dendritic cells in human peripheral blood[END_REF][START_REF] Dzionek | BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen capture and is a potent inhibitor of interferon alpha/beta induction[END_REF]MacDonald et al, 2002). These cells have a very strong migratory capacity and are involved in the recognition and presentation of viruses. During stimulation by viruses [START_REF] Cella | Plasmacytoid dendritic cells activated by influenza virus and CD40L drive a potent TH1 polarization[END_REF][START_REF] Kadowaki | Natural interferon alpha/beta-producing cells link innate and adaptive immunity[END_REF] or by interleukin-3 (IL-3) and CD40L [START_REF] Grouard | Role of dendritic and follicular dendritic cells in HIV infection and pathogenesis[END_REF], these pDCs change in morphology and acquire dendrites similar to mDCs. They are characterized by the expression of α chain of IL-3 receptor (CD123), of the type C lectin BDCA-2 (CD303), and neuropilin-1 (CD304 or BDCA-4). However, they do not express the markers typical of mDCs such as CD13 or CD33. Similarly, CD11c and MHC-II are less present on the surface of pDCs than mDCs. The pDCs also express TLRs 1, 6, and 10, and more specifically, TLR7, and 9 [START_REF] Jarrossay | Specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells[END_REF][START_REF] Kadowaki | Natural interferon alpha/beta-producing cells link innate and adaptive immunity[END_REF]. These cells are also capable of secreting large quantities of type I interferon (IFN-α, IFN-β, and IFN-λ) in response to viruses and TLR7 and 9 stimuli.

Although Mouse and human mDCs share many evolutionarily conserved molecular pathways, but they differ in surface marker expression. Murine counterparts of human cDCs are shown in [START_REF] Robbins | Novel insights into the relationships between dendritic cell subsets in human and mouse revealed by genome-wide expression profiling[END_REF]).

Monocyte-derived inflammatory DCs:

During inflammation, monocytes can develop into a particular subtype called inflammatory DCs.

Inflammatory DCs expresses both CD1a, CD1c, and the mannose CD206 receptor. These cells produce the cytokines IL-1β, TNF-α, IL-6, and IL-23 and primarily activate the Th17 responses. In mice, inflammatory DCs were initially demonstrated in leishmaniasis and associated with other infections and inflammatory settings (Segura & Amigorena, 2013). In humans, inflammatory myeloid cells are reported in several disease conditions, including eczema, psoriasis, skin sensitization, allergic rhinitis, coeliac diseases, inflammatory bowel disease, and peritonitis [START_REF] Segura | Human inflammatory dendritic cells induce Th17 cell differentiation[END_REF]. This subpopulation of DCs is still needed to be explored.

Adaptive Immune response

The activation of the adaptive immune system (AIS) is dependent on the actions of the innate immune system and is crucial when innate immunity is not able to destroy the foreign intruder. AIS is always fascinating to immunologists because it offers a specific yet incredibly diverse system that can fight foreign intruders and has a 'memory' -the key to vaccination -which enables a rapid response to previously encountered pathogens.

AIS in mammals, which is centered on lymphocytes bearing antigen receptors, are generated by somatic recombination and appeared approximately 500 million years ago in jawed fish (Flajnik & Kasahara, 2010b). This dynamic network of defense system consists of several molecules, mechanisms, and tissues that are not present in jawless vertebrates.

Genetic information for the immunoglobulin polypeptide chain is found in the genome of the germ-line cell in several gene segments distributed along the chromosome. During bone marrow-derived lymphocytes development, these gene fragments are recombined, leading to the formation of a functional gene [START_REF] Tonegawa | Somatic generation of antibody diversity[END_REF].

The accuracy of adaptive immunity is accomplished by the proliferation of lymphocyte clones that bear immunoglobulin receptors for antigens of the pathogen and that are selected accordingly from an enormous pre-existing repertoire of cells with diverse receptors. Discovery of antibody answered the major questions relating to the generation of diversity lymphocyte clones in the 1970s with the detection of somatic hypermutation and variable-diversity-joining rearrangement (VDJ rearrangement) of antibody (or immunoglobulin (Ig) or B cell receptor (BCR)) genes (Maki et al, 1980).

T cell receptors (TCRs) were reported in the 1980s [START_REF] Hedrick | Sequence relationships between putative T-cell receptor polypeptides and immunoglobulins[END_REF] , and there was universal agreement that they shared a common ancestor with BCR genes. The three components (V, D, and J) of the variable regions of the TCRs and the BCRs are each encoded in several genomic copies. However, the recombination of V(D)J segment brings them together in a single exon and, in the process, introduces numerous small insertions and deletions at the junctions, generating the vast combinatorial diversity necessary to manage the enormous diversity of antigens. Recombination Activating Genes 1 and 2 (Rag1 and Rag2) encode the key enzyme required to develop the highly diversified antigen receptor repertoire of adaptive immunity [START_REF] Schatz | Pillars Article: The V(D)J Recombination Activating Gene, RAG-1. <em>Cell</em&gt[END_REF].

The advent of the recombination-activating gene (RAG) transposon and two rounds of full genome replication are the primary reason for the appearance of AIS to support the mammalian defense system (Flajnik & Kasahara, 2010b).

The key features of AIS are:

1. The recognition of specific "non-self" antigens and distinguishing them from "self" antigens.

2. The generation of pathogen-specific immunologic response to eliminate specific pathogens or pathogen-infected cells.

3. The development of an immunological memory that can quickly eradicate a specific pathogen upon reinfection.

The adaptive immune system is made up of (Figure 6):

1. T lymphocytes

B lymphocytes

T lymphocytes

T lymphocytes (also called T cells) are derived from hematopoietic stem cells in the bone marrow and then migrate to the thymus through the bloodstream, where they mature. As these cells mature in the thymus, that is why they are known as T cells. These cells express a set of unique antigen-binding receptors on their membrane, identified as the T-cell receptor (TCR). Each T-cell expresses a single form of TCR capable of rapid proliferation and differentiation if it receives the appropriate signals. For activation, T cells need the intervention of APCs (usually dendritic cells but also macrophages, B cells, fibroblasts, and epithelial cells) to recognize a particular antigen.

Three main functions performed by T cells are:

1. T cells use chemical messengers to trigger other immune cells to initiate the adaptive immune system (T helper cells).

T cells recognize and kill tumor cells and virus-infected cells (cytotoxic T cells).

3. Once the primary response is over, certain T cells become memory T cells. They can "memorize" which antigens were recognized and so that they could readily activate the adapted immune system for subsequent new exposure by the same antigens.

T Cell subsets

While all T cells follow the fundamental aspects of thymic development and the activation mechanisms, there is a remarkable heterogeneity of effector functions that are triggered in response to stimulation.

T cells can be broadly classified on the basis of CD4 or CD8 expression on their surface. In the thymus, most Naturally occurring mutations in the FOXP3 gene may give rise to a severe multisystem inflammatory disorder causing the production of self-reactive lymphocytes that cause a rare but severe disease IPEX (Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-Linked syndrome) in humans and scurfy in mice [START_REF] Zhou | FOXP3 and its partners: structural and biochemical insights into the regulation of FOXP3 activity[END_REF]. Treg plays a crucial role in suppressing activation, proliferation, and cytokine production of CD4+ T cells and CD8+ T cells, B cells, and dendritic cells by producing TGF-beta, IL-10, and adenosine [START_REF] Sakaguchi | Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease[END_REF].

Unlike CD4+ Helper T cells, CD8+ (cytotoxic) T cells are also generated in the thymus and express the T-cell receptor. However, instead of CD4 molecule, cytotoxic T cells express a dimeric co-receptor, CD8, usually composed of one CD8α and one CD8β chain, which serves as a co-receptor for MHC class I molecule found on all nucleated cells. The CD8 heterodimer binds to a conserved region (the α3 region) of MHC-I when an antigen is presented to the T cells.

CTLs mainly opt for three different mechanisms to kill target host cells.

1. Production of cytokines, primarily TNF-α and IFN-γ, which have anti-tumor and anti-viral microbial effects.

2. Production and release of cytotoxic granulates. These granules are also found in NK cells, contain mostly two families of proteins, perforin and granzymes. Perforin forms a pore within the target cell membrane, similar to the complementary membrane attack complex. This pore allows entry of granzymes into the contaminated or malignant cell. Granzymes are serine proteases that cleave the proteins inside the cell, shut down the production of viral proteins, and ultimately lead to the apoptosis of the target cell.

3. Destruction of infected cells is via Fas/FasL interactions. Activated CD8+ T cells express FasL on the cell surface, which binds to its receptor, Fas (CD95), on the surface of the target cell. This binding causes the Fas molecules to trimerise on the surface of the target cell, triggering the apoptosis of the target cell.

Gamma delta (γδ) T cells are recognized as 'unconventional' T cells expressing a heterodimeric TCR composed of γ and δ chains, and most of them are double negative (expressing neither CD4 nor CD8), with some variably CD4+ or CD8+. They are mostly found in the peripheral blood, and in human, they represent less than 5% of lymphocytes.

In contrast to αβ T cells, the majority of γδ T cells are activated in an MHC-independent manner. The antigens recognized by most γδ T cells are still unknown.

Some γδ T cells also recognize markers of cellular stress, resulting from infection or tumorigenesis. A small subpopulation of Natural Killer T (NKT) cells represents another subset of γδ T cells, which recognize nonpeptide antigens presented by nonclassical MHC molecules of the CD1 family. Activated NKT cells are capable of rapid production of cytokines, including IL-4, and play a key role in suppressing allergic pathogenesis.

B lymphocytes

Prenatal B cell development occurs in the fetal liver later, B lymphocytes (B cells) differentiate and mature from the hematopoietic stem cells of bone marrow to establish the adaptive humoral immunity. In the bone marrow, their antigen receptors (surface Ig) are arranged to from genetic building blocks involving RAG1/RAG2 similar to that used for the production of functional TCR in the case of T lymphocytes [START_REF] Thomas | Dynamic regulation of antigen receptor gene assembly[END_REF]. During the B cell development, the amino-terminal portion of each heavy chain is created by somatic recombination encoding a variable (VH), diversity (DH), and joining (JH) region (Chaplin, 2010a).

The amino-terminal portion of the light chain is created by the union of genes encoding variable and constant light chain components. The VDJ junctions formed by this recombination constitute the third hypervariable region that plays an important role in the site of antigen binding. These hypervariable sequences are combined into the Ig protein to form the antigen-binding domain of the molecule. As a result, each Ig has two identical antigen-binding sites. Carboxyl terminal portions of heavy and light chains are constant in each antibody subclass. Heavy chain constant regions combine to form the Fc domain of the molecule responsible for most of the Ig molecule's effector functions, including binding to the Fc receptors and activating the complement system (Chaplin, 2010a).

B cells are at the center of the adaptive humoral immune system and are responsible for producing antigen-specific immunoglobulin (Ig) under the direction of signals received from T cells and other cells, such as dendritic cells. Activated B cells multiply and transform themselves into plasma cells. These plasma cells quickly produce substantial amounts of antibodies and release them into the blood (Figure 8).

Because only the B cells that match the attacking germs are activated, only the specific required antibodies are produced.

Some activated B cells transform into memory cells and become part of the "memory" of the adaptive immune system. Dendritic cells play the most critical role and build a bridge between innate and adaptive immune systems.

DCs are heterogeneous populations of cells found in an immature state at sites of interaction with the environment, such as the skin and mucosa. They continually sample the environment by macropinocytosis.

Interaction with a foreign particle trigger the DCs maturation. This results in the relocation of the DCs to the draining lymph node site. Here, the class II-peptide complex's stability is enhanced to facilitate the peptide loading at the site of inflammation.

Expression of Costimulatory molecule is also induced by maturation, making dendritic cells the best antigen-presenting cells (APCs) capable of activating a naive T cell. Depending on the nature of the immune response DCs activate different subset of T cells. DCs-mediated T cell activation results in the differentiation of T cells into Th1 and Th2 phenotypes. Th1 cells secret IFN-and promote cell-mediated immunity and inflammation. Th2 cells generate IL-4 and enable B cells to switch isotypes during a humoral response (see fig. 3). Three major key players of the innate and adaptive immune communications are BCR, TCR, and MHC/HLA molecules.

Major histocompatibility complex:

Antigen receptors of lymphocytes recognize a family of cell-surface molecules on APCs that are collectively known as major histocompatibility complex (MHC). MHC molecules are transmembrane glycoproteins and are subdivided into two classes according to their function (Chaplin, 2010a).

MHC-I present antigenic peptides of intracellular origin (Marshall et al., 2018). These peptides can originate from intracellular pathogens (viruses or intracellular bacteria). MHC-I can also present four altered selfproteins, mutated or misfolded proteins generated during tumorigenesis. Antigenic peptides loaded on MHC-I are presented to CD8+ CTLs to destroy tumorigenic cells or virus-infected cells (Figure 9). MHC-I molecules are expressed in all somatic cells. Typically, at the start of the adaptive response to a virus, an infected DCs migrates from the site of infection to a secondary lymphoid organ (ganglion or spleen) [START_REF] Alvarez | Mechanisms and consequences of dendritic cell migration[END_REF]. There, the antigenic peptide loaded on MHC-I is presented to the naive CD8+ T lymphocyte. This step activates the specific CD8+ LT of this antigen, which leads to clonal expansion and differentiation into mature CTLs. From then on, the CTL is responsible for the elimination of all the cells presenting the same antigen on their MHC-I.

Unlike MHC-I molecules, the expression of MHC-II is restricted to APCs (DCs, macrophages) and B lymphocytes. MHC class II presents antigenic peptides of extracellular origin coming from bacteria, viruses, or extracellular parasites (Chaplin, 2010a). These antigenic peptides are generated during the phagocytosis of a pathogen or are captured in the microenvironment by endocytosis. During the resolution of the phagolysosome, the APCs can recover these peptides and load them on MHC-II. They present the peptide to naive or mature CD4 + T cells (Figure 9), which differentiate into T helper cells, initiating a wide range of pathogen-specific responses.

Of note, in human, MHC is termed as human leukocyte antigen [HLA] and MHC-I in human can be subdivided into HLA-A/B/C, and MHC-II can be divided into HLA-DP/DQ/DR.

T cell receptors (TCRs)

T-cell receptors (TCRs) are protein complexes located on T cells' surface that is responsible for the recognition of antigen fragments as peptides bound to MHC molecules.

The antigen-specific α and β chains of the TCR associate with invariant accessory chains serve to transduce signals when the TCR binds to antigen-MHC complexes. αβ TCRs recognize both the antigen (in the form of a peptide) and the MHC class I or II molecule to which it is bound. This is known as "major histocompatibility complex restriction" (MHC restriction) [START_REF] Zinkernagel | The discovery of MHC restriction[END_REF]. These α and β chains make up the CD3 complex, consisting of the transmembrane CD3γ, CD3δ, and CD3ε chains plus a largely intracytoplasmic homodimer of two CD3ζ chains (Figure 10). A translocon organization of both the α and β TCR genes is found in all animals (Flajnik & Kasahara, 2010a). The presence of a D segment in the β TCR locus and the absence of D segments and the presence of a large number of J segments in the α TCR locus are also evolutionarily conserved, at least from mammals to bony fish (Flajnik & Kasahara, 2010a).

The many J segments in the α locus allow extensive receptor editing in developing T cells, resulting in more significant opportunities for modification of the antigen receptor, followed by potential selection of self-MHC-restricted TCRs in the thymus.

γδ TCRs are quite different from αβ TCRs. The basic gene organization is conserved in gnathostomes, with only J segments in the γ gene, usually two D segments in the δ gene, and close linkage of the δ and α TCR genes. However, the receptor can be adapted in several ways. It was shown in mice that γδ T cells that arose early in ontogeny bear invariant receptors and home to sites, such as the skin, where they form the first line of defense and self-renew for the life of the organism.

Approximately 5-10% of T cells in the peripheral blood, lymph nodes, and spleen are CD4-CD8-. Some of these cells use αβ TCR, and others use γδ TCR. [START_REF] Franco | Basic Pharmacological and Structural Evidence for Class A G-Protein-Coupled Receptor Heteromerization[END_REF]).

B cell receptors (BCRs)

Binding of B-cell antigen receptor (BCRs) with antigen triggers a humoral immune response. B cells identify and attack pathogens with the aid of proteins called immunoglobulins (Ig). Five immunoglobulin isotypes (IgA, IgD, IgE, IgG, and IgM) can be either secreted (sIgs) or membrane-bound (mIgs) on the cell surface [START_REF] Friess | Structural Model of the mIgM B-Cell Receptor Transmembrane Domain From Self-Association Molecular Dynamics Simulations. Frontiers in Immunology 9: 2947 ➢ Fröhlich E (2015a) Value of phagocyte function screening for immunotoxicity of nanoparticles in vivo[END_REF]. Membrane-bound immunoglobulins (mIgA, mIgD, mIgE, mIgG, and mIgM) are components of the so-called B-cell receptor (BCR). Just like the TCR / CD3 complex, the BCR is also a complex of oligomers [START_REF] Li | The Coordination Between B Cell Receptor Signaling and the Actin Cytoskeleton During B Cell Activation[END_REF].

The mIg subunits bind the antigen, initiating the aggregation of the receptor, while α/β subunits initiate intracellular signaling [START_REF] Friess | Structural Model of the mIgM B-Cell Receptor Transmembrane Domain From Self-Association Molecular Dynamics Simulations. Frontiers in Immunology 9: 2947 ➢ Fröhlich E (2015a) Value of phagocyte function screening for immunotoxicity of nanoparticles in vivo[END_REF]. Initiation of BCR signaling promptly activates the Src family kinases Lyn, Blk, and Fyn as well as the Syk and Btk tyrosine kinases. This facilitates the formation of a 'signalosome' composed of the BCR, the tyrosine kinases, adaptor proteins such as CD19 and BLNK, and signaling enzymes such as PLCγ2, PI3K, and Vav (Figure 11) [START_REF] Dal Porto | B cell antigen receptor signaling 101[END_REF]. Signals emanating from the signalosome activate multiple signaling cascades that involve several kinases, GTPases, and transcription factors resulting in changes in cell metabolism, gene expression, and cytoskeletal organization. Abnormal BCR signaling is associated with the progression of lymphoma [START_REF] Niemann | B-cell receptor signaling as a driver of lymphoma development and evolution[END_REF] and immunological diseases, including autoimmune disorders [START_REF] Rawlings | Altered B cell signalling in autoimmunity[END_REF].

Immunometabolism

Immunometabolism relates to the creation of a correlated interplay between the metabolic and immunological functions. The knowledge of immunometabolism ensures the accuracy of a particular immune cell function, depending on the microenvironment the cell is facing.

One of the biggest obstacles we faced in introducing a link between metabolism with immunology is that most immunologists saw these metabolic pathways as scientifically interesting but unlikely to integrate these pathways on their own growing research interests into the complexity of the immune response.

Immunology by itself as a discipline has improved tremendously in the last 40 years. Notable developments include identifying whole new immune receptor systems (most notably pattern recognition receptors (PRRs), the identification of several cytokines and immune cell types, and a better understanding of different immune cell function.

But from the last 10 years, immunologists reinvent the metabolism by discovering the importance of intracellular metabolic pathways in immune cells that regulate their functions. Immunometabolism governs the adequate response of a particular immune cell towards particular pathogens. From the knowledge about metabolism, we know that six metabolic pathways play a key role in generating key products to promote cell survival or growth. These pathways are Tricarboxylic Acid Cycle (TCA cycle), the glycolytic pathway, pentose phosphate pathway (PPP), fatty acid oxidation (FAO), fatty acid synthesis and amino acid pathways (Figure 12). Each of them has a unique purpose in the cell and is regulated by cellular signaling pathways to link their activity to cellular needs. Immune cells are also tightly governed by these six key metabolic pathways, and each metabolic pathway is essential for a particular action of an immune cell type, depending on the surrounding microenvironment.

Although immunometabolism plays a role in all immune cell functions in this thesis, we only address the link of immunometabolism with DCs and macrophages. As a crucial component of the innate immune system, the DCs manage the aspect of immunity through their ability to look into the tissues and gather antigens [START_REF] Lawless | Glucose represses dendritic cell-induced T cell responses[END_REF]. The DCs also work detecting danger signals from tissues and the microbes within the bodies. In this instance, the provision of adequate handling for the immune responses in the body helps to bring in key management of antigens to preserve the integrity of the body (van der Windt et al, 2012). DCs have different functions to regulate the homeostatic processes within tissues and establish a system of immunity. However, handling the environment and forging reactions lead to the creation of a differentiation, which induces activation to ensure better functionality in the systems.

Appropriate functionality deals with the engagement of cellular environments and the creation of typical needs for all segments to define the course of an immune response [START_REF] Thwe | Cell-Intrinsic Glycogen Metabolism Supports Early Glycolytic Reprogramming Required for Dendritic Cell Immune Responses[END_REF]. The aspect of immunometabolism, therefore, encompasses diverse aspects to increase the functionality of the DCs.

Just like DCs, macrophages play an important role as a part of the innate immune system and response against infectious organisms, with a dual role of eliminating infectious agents by phagocytosis while mediating defensive and inflammatory pathways. However, on the other side, macrophages are also involved in the late response to infections by contributing to tissue debris clearance and "wound" repair.

Stimulation with TLR ligands, such as LPS and interferon-γ (IFNγ) macrophages polarize into pro-inflammatory M1 phenotype and secret several pro-inflammatory signaling factors, including cytokines and reactive nitrogen and oxygen species. Additionally, M1 macrophages show bactericidal and antitumor activity. On the other side, the Th2 cytokines, IL-4 and IL-13, polarize macrophages toward an antiinflammatory M2 phenotype. Briefly, the energy metabolism of M1 macrophages is mainly dependent on glycolysis, PPP, fatty acid synthesis, and a truncated TCA cycle, leading to the accumulation of succinate and citrate, whereas the metabolic profile of M2 macrophages is characterized by oxidative phosphorylation (OXPHOS), Fatty acid oxidation (FAO), reduced glycolysis and PPP(Mills & O 'Neill, 2016).

Notably, the immunometabolism process can lead to any of the appropriate metabolic pathways, which allow the immune cells to respond properly towards a particular immune response. The process of immunometabolism, therefore, affects the functionality of macrophages and DCs within the body.

Glycolysis in immunity

Glycolysis in macrophages and DCs requires the provision of oxygen or alternative procedures to ensure the achievement of the desired breakdowns. The use of the glycolytic procedure ensures the attaining of value for the key segments in immunometabolism and assurance of the value for each segment within the body [START_REF] Shapiro | Macrophages, meta-inflammation, and immuno-metabolism[END_REF]. Mitochondria within the macrophages and DCs are instructional in the provision of a platform to engage in signaling and metabolic activations (Domínguez-Amorocho et al, 2019). The activation of these cells depends on the ability to work with mitochondrial metabolism, which initiates the ability to break down glucose within the cells (Figure 13).

Glycolytic was initially correlated as an active immune signature in various cancers and highly glycolytic tumors. A metabolic switch from OXPHOS to glycolysis in the highly metabolically active cancer cells leads to a significantly increased amount of lactate production. This phenomenon is well described in the beginning of the last century by Otto Warburg, called the 'Warburg effect' [START_REF] Warburg | THE METABOLISM OF TUMORS IN THE BODY[END_REF]. Rate of glycolysis is often elevated during immune cell activation: activated T cells show increased rates of glycolysis [START_REF] Donnelly | Glucose, glycolysis and lymphocyte responses[END_REF][START_REF] Wang | The Transcription Factor Myc Controls Metabolic Reprogramming upon T Lymphocyte Activation[END_REF], and classically activated pro-inflammatory M1 macrophages show increased glucose metabolism resulting in increased lactate production (Jha et al, 2015a).

This switch is assumed to be important for cells; otherwise, glycolysis is less efficient in generating adenosine triphosphate (ATP). OXPHOS is much more effective than glycolysis because while glycolysis generates 2 molecules of ATP from 1 molecule of glucose, OXPHOS generates 36 ATP molecules from a single molecule of glucose. Conversely, it is also known that glycolysis could be rapidly activated by activation of the enzymes involved in this pathway. By contrast, initiating OXPHOS involves mitochondrial biogenesis, which is relatively more complex and probably slower process than glycolysis. That is why cells need to make ATP urgently switch to glycolysis.

Besides, glycolysis also rapidly generates biosynthetic intermediates to support rapid cell development.

Activating signals such as growth factors strongly enhanced the rate of glucose uptake and glycolysis, which supplies ATP, supports the TCA cycle and generates intermediates for PPP, glycosylation reactions, and synthesis of essential biomass constituents, including serine, glycine, alanine, and acetyl-CoA for lipid synthesis.

Enhanced glycolysis occurs in LPS activated macrophages and DCs [START_REF] Rodríguez-Prados | Substrate fate in activated macrophages: a comparison between innate, classic, and alternative activation[END_REF][START_REF] Villanueva | The action of hormones on the rat uterus and mammary gland gamma-glutamyltranspeptidase activity[END_REF], in NK cells [START_REF] Donnelly | mTORC1dependent metabolic reprogramming is a prerequisite for NK cell effector function[END_REF], in activated effector T cells [START_REF] Anderson | The effect of various saccharin forms on gastrointestinal tract, urine and bladder of male rats[END_REF], and in activated B cells [START_REF] Doughty | Antigen receptor-mediated changes in glucose metabolism in B lymphocytes: role of phosphatidylinositol 3-kinase signaling in the glycolytic control of growth[END_REF]. Therefore, enhanced glycolysis can be considered a crucial metabolic change in most immune cells undergoing rapid activation, for example, in response to stimulation of PRRs, cytokine receptors, or antigen receptors. More precisely, for macrophages, this involves phagocytosis and inflammatory cytokine production; for DCs, this includes antigen presentation (Everts et al, 2014b).

A closer look on the molecular insight associated with the signaling pathway that triggers glycolysis during immune cell activation revels LPS induces activation of hypoxia-inducible factor 1α (HIF1α), a key transcription factor that is responsible for the induction of several enzymes involved in glycolysis [START_REF] Tannahill | Succinate is an inflammatory signal that induces IL-1β through HIF-1α[END_REF]. Probably glycolysis also involves nuclear factor-κB (NF-κB) activation, as the ubiquitous isoform of phosphofructokinase-2 (uPFK2), which is involved in the regulation of glycolysis, is still activated by LPS in HIF1α-deficient cells [START_REF] Rodríguez-Prados | Substrate fate in activated macrophages: a comparison between innate, classic, and alternative activation[END_REF]. LPS can also rapidly initiate glycolysis in DCs by activating TANK-binding kinase 1 (TBK1) and/or inhibitor of NF-κB kinase ɛ (IKKɛ) and hexokinase 2, in a HIF1α-independent manner [START_REF] Huynh | Control of PI(3) kinase in Treg cells maintains homeostasis and lineage stability[END_REF].

Pyruvate kinase isoenzyme M2 (PKM2) also plays an important role in the enhancement of glycolysis in LPS-activated macrophages. This form of PKM is regulated to slow glycolytic flux and allows the diversion of glycolytic intermediates into biosynthetic pathways. PKM2 also plays a role in promoting the expression of HIF1α-dependent genes (Luo et al, 2011;[START_REF] Palsson-Mcdermott | Pyruvate kinase M2 regulates Hif-1α activity and IL-1β induction and is a critical determinant of the warburg effect in LPS-activated macrophages[END_REF], which encode the aforementioned glycolytic enzymes and inflammatory factors, such as IL-1β.

It has been observed that a small molecule that forces the tetrameric state of PKM2 (in which it cannot enter into the nucleus to promote the expression of HIF1α-dependent genes) leads to the reprogramming of macrophages to polarize towards M2-like in their gene-expression profiles [START_REF] Palsson-Mcdermott | Pyruvate kinase M2 regulates Hif-1α activity and IL-1β induction and is a critical determinant of the warburg effect in LPS-activated macrophages[END_REF]. This clearly indicates that inhibition of HIF1α alters the phenotype of the macrophage from a proinflammatory M1 phenotype to a pro-reparatory (or alternatively activated) M2 phenotype.

In macrophages, another important glycolytic enzyme is hexokinase 1, which acts as NLRP3 (NOD-, LRRand pyrin domain-containing 3) regulator (Moon et al, 2015). NLRP3 is an essential caspase 1 regulator that generates mature IL-1β and active IL-18 and induces a sort of cell death known as pyroptosis.

As mentioned above, in addition to its function in ATP synthesis, glycolysis is also involved in generating several biosynthetic intermediates. For example, the generation of glucose-6-phosphate, which enters into PPP.

The TCA cycle in immunity

The TCA cycle and oxidative phosphorylation is extensively studied in immune cells function. It has been observed that TCA cycle in recognizably different in subtypes of macrophage (Figure 14). For example, In M2 macrophages, TCA cycle is intact and coupled to oxidative phosphorylation. This enables M2 macrophages to generate Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) intermediates, which is necessary for the glycosylation of M2-associated receptors, such as the mannose receptor. Interestingly, in M1 macrophages TCA cycle is broken in two places, (1) after citrate (owing to a decrease in expression of isocitrate lyase) and (2) after succinate (Jha et al, 2015b;[START_REF] Tannahill | Succinate is an inflammatory signal that induces IL-1β through HIF-1α[END_REF].

Accumulated citrate in M1 macrophages is exported from the mitochondria via the citrate transporter to produce fatty acids, which are used for membrane biogenesis. This broken TCA cycle is also observed in activated DCs and appears to be important for their function, as these cells require substantial membrane production to support antigen presentation (Everts et al., 2014b). Additionally, it has been shown that the differentiation of monocytes into DCs in vitro and the development of DCs in lymphoid organs and peripheral tissues in vivo depend on fatty acid synthesis [START_REF] Rehman | Role of fatty-acid synthesis in dendritic cell generation and function[END_REF].

Excess citrate is used to produce nitric oxide and prostaglandins, which are the main effector molecules produced by macrophages [START_REF] Infantino | The mitochondrial citrate carrier: a new player in inflammation[END_REF]. A third metabolite generated from citrate is itaconic acid, which shows a direct bactericidal role against Salmonella enterica subspecies enterica serovar Typhimurium and Mycobacterium tuberculosis (Michelucci et al, 2013). This clearly demonstrates how a metabolic rewiring event can lead to the production of several metabolites with direct antimicrobial properties.

Succinate that accumulates in M1 macrophages as a result of the broken TCA cycle plays a key role in different proinflammatory cytokine production [START_REF] Tannahill | Succinate is an inflammatory signal that induces IL-1β through HIF-1α[END_REF]. The molecular mechanism underlying the inhibition of prolyl hydroxylases by succinate lead to the stabilization of HIF1α and the sustained production of IL-1β21. Altogether, these studies show that the TCA cycle alterations that occur in M1 macrophages lead to different mitochondrial metabolite accumulation that can promote their immune function. These events are closely linked with nitric oxide production, which is responsible for disrupting the electron transport chain [START_REF] Clementi | Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione[END_REF]. as materials with a length of 1-100 nm in at least one dimension; however, according to the EU Commission, NMs are usually described as "a manufactured or natural material that possesses unbound, aggregated or agglomerated particles where external dimensions are between 1-100 nm size range".

Although, until today, a single internationally accepted definition for NMs does not exist.

Because of the nanoscale size, NMs show quite distinct properties because of their small volume-tosurface area ratio and surface structure, which affects the surface reactivity, solubility, shape, and aggregation. The smaller the diameter of a spherical particle, the more the surface-to-volume ratio increases, leading to an increase of chemical reactivity. This significantly impacts on the interactions with biological structures and living species. Engineered nanomaterials, particularly nanoparticles (NPs), have been rapidly developed to produce a wide variety of objects for different applications, including industrial products, food, agriculture, and health (Figure 15). [START_REF] Kolahalam | Review on nanomaterials: Synthesis and applications[END_REF]).

Classification

NMs are commonly classified into different groups according to their morphology, size, and chemical properties. Based on physical and chemical characteristics, NMs can be classified into three groups.

Inorganic NMs

These NMs include metal and metal oxide NPs, Ceramics NMs, and Semiconductor NMs.

Metal-based NMs

Metal NMs are manufactured from metal precursors. These NMs can be synthesized using chemical, electrochemical or photochemical methods. Unique optical properties of Metal NMs is due to their wellknown surface plasmon resonance (SPR) properties. Due to their advanced optical properties, metal NMs are used in many disciplines. Most commonly used metals for NMs synthesis are cadmium (Cd), aluminum (Al), copper (Cu), cobalt (Co), iron (Fe), gold (Au), silver (Ag), zinc (Zn), and lead (Pb).

Metal oxide-based NMs

Metal oxide-based NMs are synthesized to alter the properties of their respective metal-based nanoparticles. For example, iron nanoparticles (Fe) instantly oxidize to iron oxide (Fe2O3) in the presence of oxygen at room temperature to increases its reactivity compared to iron nanoparticles. Most commonly synthesized Metal oxides, NMs are Cerium oxide (CeO2), Magnetite (Fe3O4), Titanium oxide (TiO2),

Aluminium oxide (Al2O3), Zinc oxide (ZnO), Silicon dioxide (SiO2) Iron oxide (Fe2O3). These NMs have shown exceptional property when compared to their metal counterparts.

Ceramics NMs

Ceramics NMs are inorganic non-metallic solids, which are synthesized by subsequent heat and cooling process. They can be found in amorphous, polycrystalline, solid, porous, or hollow forms (Sigmund et al., 2006). Therefore, due to their use in catalysis, photocatalysis, photodegradation of dyes, and imaging applications, these NMs draw special attention from the research community (Thomas et al., 2015).

Semiconductor NMs

Semiconductor materials have shown properties between metals and non-metals, making them useful in different applicative fields (Ali et al., 2017, Khan et al., 2017a). Semiconductor NPs possess wide bandgaps, which allow them to alter their properties with bandgap tuning. Therefore, they are very important materials in electronic devices, photocatalysis, and photo optics and (Sun, 2000).

Carbon-based NMs

Such NMs typically contain carbon and can be found in different morphologies such as hollow tubes, ellipsoids, or spheres. This group of carbon-based NMs mainly comprises of fullerenes (C60), carbon nanotubes (CNTs), carbon nanofibers, carbon black, graphene (Gr), carbon onions, Carbon dots, Nanodiamond and many more (Figure 16). Laser ablation, arc discharge, and chemical vapor deposition (CVD) are the commonly used production methods for these carbon-based NMs (except carbon black).

Organic NMs

These NMs are primarily made from organic matter, excluding carbon-based or inorganic-based NMs. The concept of non-covalent (weak) interactions for self-assembly and molecular design helps transform organic NMs into desired structures such as Dendrimers, micelles, liposomes, and ferritin, and other NMs.

These NMs are non-toxic, biodegradable, and some NMs such as micelles and liposomes also have a hollow core (Figure 17), which makes them sensitive to thermal and electromagnetic radiation such as heat and light. These distinctive qualities make them an ideal choice for drug delivery. Organic nanoparticles are most extensively used in the biomedical field, such as the drug delivery system, mainly because of targeted drug delivery. However, the cellular components are much smaller and are in the realm of sub-micron size.

Proteins, the key player of different biological functions, are about 5 nm, equivalent to the size of the smallest fabricated nanoparticles. Interestingly, nanotechnology offers manipulation of the regime of biological systems, which is correlated with both living systems and artificial devices.

The conceptual framework for nano-bio applications can be organized based on two main themes:

1. Nanotechnology allows new ways to understand and detect biological systems both in vitro and in vivo.

2. Nanotechnology enables new ways to treat diseased cells and cure patients.

For example, nanoscale devices can detect minute alterations in single-cell as well as molecular level. This exquisite sensitivity can be used to interpret single-cell heterogeneity at extremely high throughput, revealing distinguishable hierarchies and cell populations. On the other side, Nanomaterials can be engineered to deliver therapeutics precisely to specific locations while overcoming or avoiding biological obstacles, thus altering the inherent pharmacokinetics and biodistribution of the cargo.

Nanotechnology can be used for various biological applications such as Drug and gene delivery [START_REF] Dong | A Dual Targeting Dendrimer-Mediated siRNA Delivery System for Effective Gene Silencing in Cancer Therapy[END_REF][START_REF] Pantarotto | Immunization with Peptide-Functionalized Carbon Nanotubes Enhances Virus-Specific Neutralizing Antibody Responses[END_REF], Tissue engineering (de la [START_REF] De La Isla | Nanohybrid scratch resistant coatings for teeth and bone viscoelasticity manifested in tribology[END_REF], Probing of DNA structure (Mahtab et al, 1995), Fluorescent biological labels [START_REF] Chan | Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic Detection. Science 281: 2016 ➢ Chaplin DD (2010a) Overview of the immune response. The Journal of allergy and clinical immunology 125: S3-S23 ➢ Chaplin DD (2010b[END_REF], MRI contrast enhancement [START_REF] Weissleder | Ultrasmall superparamagnetic iron oxide: characterization of a new class of contrast agents for MR imaging[END_REF], Phagokinetic studies [START_REF] Parak | Cell Motility and Metastatic Potential Studies Based on Quantum Dot Imaging of Phagokinetic Tracks[END_REF], Hyperthermia [START_REF] Shinkai | Intracellular hyperthermia for cancer using magnetite cationic liposomes[END_REF], Purification and isolation of biological molecules and cells (Molday & Mackenzie, 1982) and many more.

Several NMs are used for the mentioned functions. Out of several NMs three of the NMs and their biomedical use is described below as a part of this Ph.D. thesis.

Gold nanoparticles (AuNPs)

Since long time, gold is used to treat various diseases like rheumatoid arthritis in recent days. Gold-based nanoparticles (AuNPs) are used in several biomedical applications. AuNPs with controlled properties are used in genomics and biosensors, immunoassays and clinical chemistry, photothermolysis of cancer cells and tumors, targeted delivery of drugs and antigens, and optical bioimaging of cells and tissues with stateof-the-art nanophotonic detection systems. These oscillations are known as Plasmon surface resonance.

The dominant characteristics of AuNPs include shape-related optoelectronic properties, a large surfaceto-volume ratio, excellent biocompatibility, and low toxicity, making them exemplary tools in biomedical applications (Figure 18). [START_REF] Wilson | The use of gold nanoparticles in diagnostics and detection[END_REF].

Photothermal therapy

Photothermal cell damage is a promising mechanism in tumor therapy [START_REF] Kim | Multifunctional Photonic Nanomaterials for Diagnostic, Therapeutic, and Theranostic Applications[END_REF] and the treatment of infectious diseases with minimal invasiveness. Because of the high absorption in the visible or near-IR region, AuNPs receive light and generate heat. The heat causes the death of malignant tumors (Manjunath et al, 2017;[START_REF] Narang | Electrochemical impediometric detection of anti-HIV drug taking gold nanorods as a sensing interface[END_REF]. In addition, AuNPs -antibody conjugates may be used for diagnostic and plasma photothermal therapy (PPTT), known as theranostic. Like PDT, the binding properties of AuNPs play a vital role in the cellular internalization process.

X-ray radiation therapy

AuNPs are also used to improve X-ray radiation therapy. A challenge in radiation therapy, in general, is that high-dose X-rays damage healthy cells. AuNPs, during X-ray irradiation, can act as dose amplifiers and / or generate radicals which damage cancer cells and induce cell apoptosis. This role of AuNPs as radiosensitizers allows a reduction in the dose of X-rays used, with improved therapeutic results [START_REF] Setyawati | Phage Based Green Chemistry for Gold Ion Reduction and Gold Retrieval[END_REF]Zhang et al, 2015).

Drug delivery and vaccination

Targeted drug delivery is one of the most promising and actively emerging avenues for the therapeutic use of AuNPs [START_REF] Duncan | Gold nanoparticle platforms as drug and biomacromolecule delivery systems[END_REF][START_REF] Pissuwan | The forthcoming applications of gold nanoparticles in drug and gene delivery systems[END_REF] et al, 2015), plasmid DNAs (pDNAs), small interfering RNAs (siRNAs), and chemotherapeutic agents (Liu et al., 2015). AuNPs can be used as vaccine delivery vehicles because they are biocompatible and can be used as adjuvants to improve vaccine efficacy by stimulating antigen-presenting cells and ensuring the controlled release of antigens. AuNPs are used to develop HIV (Liu & Chen, 2016), Encephalitis [START_REF] Zhao | Inoculation of plasmids encoding Japanese encephalitis virus PrM-E proteins with colloidal gold elicits a protective immune response in BALB/c mice[END_REF], Hepatitis [START_REF] Pilling | The Assessment of Local Tolerance, Acute Toxicity, and DNA Biodistribution Following Particle-Mediated Delivery of a DNA Vaccine to Minipigs[END_REF], and other vaccines (Salazar-González et al, 2015; Sanchez-Villamil et al, 2019).

Nano-biosensor

One of the promising purposes of AuNPs is chemical and biological sensing. Inherent characteristics of AuNPs allow them the detection of different metal ions, anions, and molecules. Gold nanotubes attract much attention for biosensing among the various types of AuNPs due to its exceptionally unique optical properties.

Lipid nanoparticles (LNs)

Lipids are the inherent components of cell membranes where they provide a basic structural scaffold. Cell membranes are primarily composed of phospholipids and serve as a shield to protect the cell components from the external environment [START_REF] Simons | Cell membranes: A subjective perspective[END_REF].

In recent years, advances in the nanomedicine field demonstrated the utilization of lipid nanoparticles (LNs) in targeted delivery of therapeutic drugs. One of the advantages of LNs is the physiological properties that explain their biocompatibility and biodegradability in vivo. LNs are mainly composed of three major parts: a lipid part, a surfactant part, and water. [START_REF] Hentschel | Beta-carotene-loaded nanostructured lipid carriers[END_REF] Monostearin and caprylic and capric triglycerides Improved drug loading capacity and controlled release properties [START_REF] Hu | Preparation and characteristics of monostearin nanostructured lipid carriers[END_REF] Clozapine, triglycerides (trimyristin, tripalmitin and tristearin), soylecithin 95%, and poloxamer 188)

Improved bioavailability [START_REF] Venkateswarlu | Preparation, characterization and in vitro release kinetics of clozapine solid lipid nanoparticles[END_REF]) LNs such as liposomes and nanoemulsions are lymphotropic due to their lipid nature. Since last decades LNs loaded with a hydrophobic cyanine dye are used for in vivo fluorescence imaging. A modified formulation with human-use approved ingredients with broad absorption/emission spectra is designed by CEA-LETI in Grenoble. These newly formulated LNs with unique optical properties are termed as "Lipidots."

They are formulated by sonication between a lipid phase, containing glycerides and phospholipids (lecithin), and an aqueous phase containing a hydrophilic surfactant (Poly-Ethylene-Glycol (PEG) (Figure 19). These Lipidots are colloidal in nature and within a size between 50 and 200nm in diameter, depending on the lipid/surfactant ratio used. The viscosity of this nucleus can be adjusted according to its composition, which gives the particle variable rheological properties [START_REF] Delmas | Preparation and characterization of highly stable lipid nanoparticles with amorphous core of tuneable viscosity[END_REF]. These LNs were primarily used for imaging. These LNs can be labeled with a lipophilic fluorochrome, encapsulated in the core of the particle [START_REF] Texier | Cyanine-loaded lipid nanoparticles for improved in vivo fluorescence imaging[END_REF]. Furthermore, LNs are decorated with targeting ligands at their surface, like the graft of cRGD peptide targeting the αVβ1 integrins, known as markers of some tumors. The LNs have been used for in vivo imaging in mice to monitor targeted therapies [START_REF] Goutayer | Tumor targeting of functionalized lipid nanoparticles: assessment by in vivo fluorescence imaging[END_REF]. Interestingly, these NPs are detectable at very low doses, like 2pmol of particles that are enough to mark the lymph nodes of mice. Using this feature, LNs were used to mark biological fluids, the lymphatic system, and tumors [START_REF] Gravier | Lipidots: competitive organic alternative to quantum dots for in vivo fluorescence imaging[END_REF]Navarro et al, 2012a). This allowed non-invasive, realtime imaging of lymphatic vessels in mice in vivo. For instance, LNs (50nm) labeled with a hydrophobic cyanine DiD could image the development of lymphatic vessels to study the role of BMP-9 (bone morphogenetic protein 9) in this process [START_REF] Levet | Bone morphogenetic protein 9 (BMP9) controls lymphatic vessel maturation and valve formation[END_REF]. Recently, these LNs have been used for intraoperative fluorescence imaging during the excision of malignant tumors in dogs [START_REF] Cabon | Evaluation of intraoperative fluorescence imaging-guided surgery in cancer-bearing dogs: a prospective proof-of-concept phase II study in 9 cases[END_REF].

LNs are also used for the development of new vaccines to deliver protein antigens, including p24 of HIV [START_REF] Bayon | Overcoming immunogenicity issues of HIV p24 antigen by the use of innovative nanostructured lipid carriers as delivery systems: evidences in mice and non-human primates[END_REF][START_REF] Courant | Tailoring nanostructured lipid carriers for the delivery of protein antigens: Physicochemical properties versus immunogenicity studies[END_REF] 6.

Dendrimer

Dendrimers are unimolecular, monodisperse, micellar macromolecules with a highly branched threedimensional architecture. Dendrimer configurations are composed of three main components: the core, interior, and the surface (Figure 20). The core governs the 3D architecture of the dendrimer (i.e., spherical, ellipsoid, or cylindrical scaffolds). The interior influences the host-guest property of the dendrimer.

Dendrimer surface can be further polymerized or altered with peripheral functional groups. Both the core and the number/type of the internal branching units influence the overall dendrimer morphology.

Dendrimers are usually prepared using either a divergent approach or a converging one [START_REF] Hodge | Polymer science branches out[END_REF] with a structure like a tree branching out from the central point. Based on the dendrimers' growth process, these can be classified according to their generation numbers, such as G0.5, G1, G2, G3, G4, and G5.

Dendrimeric vectors are most frequently used as parenteral injections, either directly in the tumor tissue or intravenously for systemic delivery [START_REF] Tomalia | Dendrimers as multi-purpose nanodevices for oncology drug delivery and diagnostic imaging[END_REF]. showed the antimicrobial activity [START_REF] Chen | Quaternary Ammonium Functionalized Poly(propylene imine) Dendrimers as Effective Antimicrobials: Structure-Activity Studies[END_REF]. Although cationic dendrimer showed notable antimicrobial activity, they all suffer drawbacks due to Cytotoxicity towards eukaryotic cells. Recently, the Grinstaff group designed anionic amphiphilic dendrimers to remove the cationic charge and showed that elimination of positive charge from the dendrimer does not alter their antimicrobial activity (Meyers et al, 2008).

Dendrimers are also well studied for their antiviral effects, particularly against HIV-1. VivaGel, a sulfonated polysine dendrimer currently undergoing clinical trials (Mintzer & Grinstaff, 2011). VivaGel is one of the most effective anionic dendrimers used for antiviral purposes.

Dendrimers as MRI contrast agents

Magnetic resonance imaging has been a commonly used technique for the diagnosis of disease.

Dendrimer-based metal chelates function as contrast agents for magnetic resonance imaging. The unique properties of dendrimer make them extremely appropriate image contrast media for MRI (Mintzer & Grinstaff, 2011).

Dendrimers as transfecting agent

Viral vectors are efficient to induce long-term gene expression as they can be integrated into the cell genome. However, some of these vectors have been associated with a potential oncogenic activation and a significant infection of non-targeted cells hence making them a safety risk in clinical applications, thereby increasing the need for non-viral vectors. Among organic nanocarriers, dendrimer-based vectors have shown multiple advantages to others due to their biological and chemical properties, which provide a suitable and efficient nucleic acid delivery into targeted cells. Currently, several dendrimers polymers have been designed for siRNA delivery, for instance, PAMAM dendrimers, amphiphilic dendrimers (ADs), Poly PPI dendrimers, PLL dendrimers, and many more.

These dendrimers possess a positively charged amine domain at the surface, which facilitates the binding with siRNA molecules, which are negatively charged. Their interior structure keeps tertiary amines that promote the release of siRNA molecules into the cell cytoplasm (Figure 22). This nanovector-based technology showed excellent efficiency in delivering siRNA molecules in both in vitro and in vivo disease models [START_REF] Dong | A Dual Targeting Dendrimer-Mediated siRNA Delivery System for Effective Gene Silencing in Cancer Therapy[END_REF]Liu et al, 2014). activity [START_REF] Hayder | Phosphorus-Based Dendrimer ABP Treats Neuroinflammation by Promoting IL-10-Producing CD4+ T Cells[END_REF]. These ABP dendrimers are have shown immunomodulatory and antiinflammatory effects on the human immune system, which can be used to treat chronic inflammatory disorders.

Miscellaneous dendrimer applications

There are undoubtedly several other areas of biological chemistry where the application of dendrimer systems may be beneficial. Dendrimers can be used in burn treatment [START_REF] Halkes | Synthesis and biological activity of polygalloyl-dendrimers as stable tannic acid mimics[END_REF], prion research [START_REF] Supattapone | Pharmacological approaches to prion research[END_REF], and Photodynamic therapy [START_REF] Battah | Synthesis and Biological Studies of 5-Aminolevulinic Acid-Containing Dendrimers for Photodynamic Therapy[END_REF] are some of the diverse areas of fascinating ongoing dendrimer research, solubility enhancing agent, bio-sensors, tissue engineering and many more.

Immunotoxicity of nanoparticles

Nanotechnology is applied in many industrial applications and consumer products and is being used for several biomedical applications. In parallel to this technological development, Nanotoxicology has emerged to evaluate safety during industrial processes and the use of products. A functional understanding of the acute and chronic toxicological effects of engineered nanomaterials is currently lacking and may represent a significant limitation to the nanomaterial development and application.

Most of the research on the toxicology of NMs focused on the effects of nanoparticles that inadvertently infiltrate our body.

Immune and inflammatory responses are critical aspects of toxicology studies. Nanoparticles accumulate into cells due to their uptake properties and can provoke immunotoxicity due to increased interaction with immune components and the use of immunostimulatory materials in nano-formulations. For these reasons, Macrophages and Dendritic Cells appear as the most exposed immune cells to the exogenous nanomaterials due to their great capacity in foreign materials engulfment and in their important roles in the regulation of the inflammatory and immune responses.

In contrast to cytotoxicity, screening of immunotoxicity is not well established because of the problems in simulating the complexity of the immunological system in vitro, as well as in the extrapolation of in vitro and animal data to human reactions. On the other hand, different NMs exhibit different effects on the immune system, which cannot be generalized for all NMs.

Different agencies tried to give a brief guideline for immunotoxicity analysis. For example, according to the Agence Française de Sécurité Sanitaire des Produits de Santé (AFSSAPS), screening of NMs on the basis of immunotoxicity should focus on granulocytes, macrophages, and dendritic cells (DCs), and cytokine production should be considered as one of the readout parameters (Fröhlich, 2015a). Unfortunately, there are no harmonized internationally accepted guidelines until today for determining immunotoxicity.

Literature review showed different in vitro assays to ensure the safety of NMs [START_REF] Elsabahy | Cytokines as biomarkers of nanoparticle immunotoxicity[END_REF]Fröhlich, 2015a). Cytokine secretion, chemotaxis, phagocytosis, respiratory burst, Nitric Oxide (NO) generation, functions of dendritic cells, the release of elastase and myeloperoxidase can be assessed to get a preliminary idea about the biosafety of a particular NM.

Aim of the thesis

To investigate the immunotoxicity of different nanomaterials (NMs) in terms of their effect on cytokine, redox, and metabolic profile of primary immune cells.

8 Objectives of the thesis

• Toxicity evaluation of different NMs under investigation on primary macrophages (BMDMs) and dendritic cells (BMDCs).

• Analysis of the effect of different NMs on inflammatory responses on naïve and challenged states of BMDMs and BMDCs.

• Analysis of the effect of different NMs on metabolic profile of naïve and challenged state of BMDMs and BMDCs.

• Investigation on the effect of different NMs on antigen specific T cell response. Our investigation concluded AuNPs by themselves displayed no significant effect on primary macrophages (M) and DCs functions. However, when exposed to AuNPs, M and DCs responded differently to LPS or IL-4 stimulations. We showed AuNPs altered cytokine and ROS productions differently in M and DCs, whereas NO production by both cells remained unaffected. The metabolic profile underpins all functions of the immune cells and their polarisation. The analysis of the metabolic activity revealed that AuNPs significantly altered mitochondrial respiration and glycolysis of M, while only little effect was seen on DCs.

Furthermore, we showed that T cell responses increased when antigen was presented by AuNPs-exposed DCs, leading to stronger Th1, Th2, and Th17 responses.

Our data not only contributed to enrich the immunotoxicological effect of AuNPs but also, this kind of analysis can be extended to a set of NPs of different sizes and different natures.

A paper is published on Cells 2021, 10(1), 96; https://doi.org/10.3390/cells10010096. Although several drugs are available to treat IBD, but the major drawbacks of current therapies are (1)

Lack of Efficacy, (2) Tolerability, and (3) Convenience. In an attempt to address the demands of current IBD therapies, two approaches are addressed in this project 1. Specific inhibition of JAK1 by means of siRNA and RNAi interference.

2. Delivery of the siRNA by combining innovative nanostructured lipid carriers.

To achieve the goal of the project, NEWDEAL consortium is created with 11 partners, including academic laboratories and industries coming from 5 different countries. The entire "NEWDEAL" program is divided into 6 work packages (WPs) to execute the project (Figure 24).

• WP1 focuses on the screening, validation, and production of siRNA targeting JAK1

• WP2 focuses on product development & scale-up (WP2)

• WP3, WP4, WP5 focus on the efficacy and safety evaluation of the NEW DEAL therapeutic solution

• WP6 focuses on regulatory aspects, providing guidance, and collect all necessary data to constitute an IMPD file for phase 1 studies and generate a clinical development plan. As a part of the "NEWDEAL" project, our team participated in WP1, WP4, and WP5. The main objectives of the participating WP are

• WP1: to rapidly identify in-vitro, the best siRNA sequence that can efficiently and precisely (without off-target effects) downregulate JAK expression.

• WP4: to demonstrate the efficacy of JAK targeting using siRNA loaded NLCs in experimental models of intestinal inflammation.

• WP 5: to properly assess safety for correct validation of all products generated in WP1 and WP2, as well as to contribute to the standardization of nanotechnology, specifically in its application to healthcare and Pharma sectors.

I have been actively involved in these three WP, but for simplicity, only the data from WP5 concerning the toxicity evaluation of the lipid nanocarrier is exposed in this thesis.

Our lab had to test the immunotoxicity of the lipid nanocarriers used to deliver therapeutic siRNA.

Interestingly, previously works with neutral lipidic nanocarriers NLC have shown the absence of toxicity and pro-inflammatory effects on immune cells (Courand et al; Bayon et al). However, to deliver siRNA, cationic lipidic nanocarriers CLC) have been selected because of their property to associate tightly with nucleic acids. However, the introduction of charges on the lipidic carriers led to some effects on MDC.

Therefore, the accessibility to both NLC and CLC allowed us investigating how to reduce or abolish the effects on immunotoxicity of positive charge on the lipid carriers.

The study analysis was conducted using BMDCs and BMDMs as immune cell model system. We followed the same optimized scheme of experiments done for AuNPs. Finally, to establish the effect of charge, we reversed the positive charge on the CLC by using negatively charged siRNA. For this purpose, all-star negative control siRNA (siAS) was used at different N/P ratios (the ratio of positively-charged amine groups of dendrimer (N = nitrogen) to negatively-charged phosphate (P) groups of nucleic acid) and complexed with CLC. After complexation, the zeta potential of the CLC/siAS complex was measured in 1 mM NaCl. We also performed the analysis of different cellular functions such as cytokine productions, NO production, metabolic flux analysis on BMDMs after reversing the charge. This "charge reversal study" is presented in a format of an article, which will be submitted for publication

Introduction

In recent years, advances in nanotechnology field have demonstrated their potentials for precision medicine. For instance, lipid nanocarriers can be used for targeted delivery of therapeutic molecules, increasing their bioavailability and pharmacokinetic properties beyond the Lipinski rules [1]. Indeed, the development of nucleic acid therapeutics has long been hampered by the inherent hydrophilic nature, large size and poor membrane permeability of nucleic acids. Lipid nanocarriers can offer a very potent alternative to viral-mediated nucleic acid delivery, with a broad range of applications such as RNAi therapy or RNA-based vaccines, through the intracellular delivery respectively of short interfering RNA (siRNA) or messenger RNA (mRNA) [2].

One of the main advantages associated with lipid nanocarriers is their ease of manufacture and biocompatibility, enabling their use in vivo for human therapy [3]. Lipid nanocarriers are made of two major components: a lipid phase, and a water-phase containing surfactants. While the lipid phase can incorporate a variety of lipids such as phospholipids, free fatty acids, glycolipids or fatty alcohols, the surfactant phase plays a role of a stabilizing agent [4].

Lipid nanocarriers are generally divided into liposomes or nanoparticles, the latter which could be solid lipid nanoparticles (SLN) with a solid core and Nanostructured Lipid Carriers (NSLC) featuring a core with a mixture of solid and molten lipids. Over liposomes, NSLC offer the significant improvements in stability and allows better interaction with the immune system [5].

By incorporating cationic lipids in their formulation, NSLC are able to bind their negatively-charged nucleic acid cargo through electrostatic interactions. NSLC are able to protect bound nucleic acid from degradation and promote cellular uptake, as cytosolic availability is a prerequisite for therapeutic siRNA or mRNA biological activity.

However, nanoparticle based carriers, according to their characteristics (i.e. size and surface charge), can have immunomodulatory effects by interacting with APCs [START_REF] Liu | Effects of engineered nanoparticles on the innate immune system[END_REF]; especially dendritic cells and macrophages for which they can alter the secretion of pro-inflammatory cytokines [7]. In addition, integrating cues from the microenvironment, including exposure to nanoparticles, can drive distinct macrophage or dendritic cell fates and functions through the activation of different metabolic pathways. For example, while lipopolysaccharide (LPS)-stimulated classically activated proinflammatory macrophages (M1) rely on glycolysis, interleukin (IL)-4 stimulated alternatively activated anti-inflammatory macrophages (M2) mainly utilize fatty acid oxidation and oxidative phosphorylation (OXPHOS) [8]. Besides, exposition to cationic lipid particles has been shown previously to induce the expression of two co-stimulatory molecules, CD80 and CD86, in bone marrow derived dendritic cells (BMDCs) without inducing the secretion of pro-inflammatory cytokines [9]. Moreover, how the positive charge of lipid particles modulates the metabolic fitness of APCs and how this is related to the cellular function have not yet been elucidated. As the positive charge of lipid nanocarriers plays an important function both for nucleic acid delivery and immunogenicity [10], a precise tuning of the surface charge is required whether immunostimulation is required or to be avoided for a given therapeutic application. Therefore, understanding the impact of positively charged particles on immunogenicity and particularly on APCs metabolism, fate and cytokine secretion is crucial.

In the present study, we analyzed the effect of NLCs surface charge on primary APCs using BMDCs and bone marrow derived macrophages (BMDMs), as cellular models. We evaluated the impact of neutral lipid carrier (NLCs) and cationic lipid carrier (CLCs) on the secretion of different signaling factors and on mitochondrial metabolism and glycolysis. Furthermore, after complexing CLCs with negatively charged siRNA we reversed the charge on CLCs and we evaluated the effect of different charges on cell function.

Materials and Methods

Cell culture

Murine macrophage cell line (J774.1A) was purchased from ATCC, cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillinstreptomycin.

As previously described [11], bone marrow derived dendritic cells (BMDCs) were generated from the bone marrow extracted from C57BL/6 mice (Charles River, l'Arbresle, France). Bone marrow cells were isolated by flushing from the tibia and femurs. Erythrocytes and GR1 positives cells were removed by incubating with Ly-6G/ Ly-6C (BD Pharmingen, #553125) and TER-119 (BD Pharmingen, #553672) antibodies, and the remaining negatively sorted cells were isolated using Dynabeads isolation kit (ThermoFisher, #11047) by magnetic cell sorting, then the remaining negatively sorted cells were resuspended at 5×10 5 cells/ml in complete Iscove's modified Dulbecco's medium (IMDM) supplemented with GM-CSF (Peprotech, #315-03), FLT-3L (Peprotech, #250-31L) and IL-6 (Peprotech, #216-16) according to Table 1. Transformation of the progenitors into fully active dendritic cells (DCs) was performed over a 10 days' timeframe.

Bone marrow derived macrophages (BMDMs) were also generated from bone marrow extracted from C57BL/6 mice. Erythrocytes were removed by RBC lysis buffer, and the remaining cells were cultured in complete DMEM medium with 20% L929 (Sigma, #85011425) conditioned medium (source of macrophage-colony stimulating factor (M-CSF)) for 7 days.

Cationic and neutral lipid nano carriers

NLCs and CLCs were prepared as previously described [12]. Briefly, for NLCs, a lipid phase was prepared containing triglycerides (Suppocire NB, Gattefossé; Super Refined soybean oil, Croda Uniqema), phospholipids (Lipoid S75-3, Lipoid), fusogenic lipid DOPE (1,2-dioleoyl-sn-glycero-3phosphoethanolamine, Avanti Polar Lipids). For CLCs, the same lipid phase supplemented with the cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammonium-propane chloride, Avanti Polar Lipids) was used. When indicated, Dil lipophilic dye (D282, Thermofisher) was added to the lipid phase to enable fluorescence detection of NLCs. A second aqueous phase containing the PEGylated surfactant PEG40-Stearate (Myrj S40, Croda Uniqema) was prepared in PBS (#806552, Sigma). Both lipid and aqueous phases were mixed together through high frequency sonication. Lipid nanoparticules are purified by dialysis in 100 volumes of LNP Buffer: 154 mM NaCl, 10 mM HEPES, pH7.4 using endotoxin-free ultra-pure water (TMS-011-A, Sigma) using 12-14 kDa MW cut-off membranes (ZelluTrans/Roth T3). Finally, the LNP solution was sterilized by filtration through a 0.22 µm Millipore membrane.

Nanoparticle uptake assay

For nanoparticle uptake assays, 0.5 * 10 5 cells/mL BMDCs and BMDMs were seeded in a 4-well Lab-Tek chambered coverslip. After 24 h of growth, cells were incubated with both CLCs and NLCs Dil-labeled nanocarriers for 24 h at 37°C with 5% CO2 and performed live imaging. Nanocarrier accumulation inside cells was monitored by microscopy using a spinning disk confocal microscope (Andromeda, TILL-FEI). Dillabeled nanocarriers were visualized using the lipophilic dye excitation wavelength of 514 nm while plasma membranes were labelled with FITC conjugated cholera toxin (Sigma, C1655) and visualized at the excitation wavelength of 488 nm. After acquisition images were processed in Icy 2.0.3.0 software and spectral deconvolution was performed using NIS 5.20.01 software.

there fluorescence was recorded at an excitation wavelength of 560 nm and an emission wavelength of 590 nm using a CLARIOstar® Microplate Reader (BMG LABTECH).

Phagocytosis assay

Nanocarrier-exposed macrophages (BMDMs and J774.1A) and BMDCs were incubated at a ratio of 10 microspheres per cell for 6 h with 1.0 µm FluoSpheres® Carboxylate-Modified Microspheres (ThermoFisher, F8851) labelled with a red fluorescent dye (580 nm excitation and 605 nm emission). Cells were analyzed by flow cytometry with an Accuri C6 instrument (Becton-Dickinson) and the analysis was performed by the FCS Express V5 software (De Novo Software).

Cell activation

Nanocarrier exposed BMDCs and BMDMs were stimulated for 24 h using 2 µg/mL Lipopolysaccharide (LPS) from E. Coli. Supernatants were collected for downstream cytokine immunoassay. The cells were labelled with antibodies specific for CD11b (Ozyme, BLE101226) and CD11c (Ozyme, BLE117318) or CD11b (Ozyme, BLE101216) and F4/80 (Ozyme, BLE123152) cell surface markers of BMDCs and BMDMs respectively after blocking the Fc receptor (BD Pharmingen, 553142) to reduce nonspecific binding. To evaluate the cell activation BMDCs and BMDMs were stained with anti-IAb (Ozyme, BLE116410) and CD86 (Ozyme, BLE105008) antibodies. In both cases, live cells were selected by negative 7-Aminoactinomycin D (7AAD) (BD Pharmingen, 559925) staining and analyzed by flow cytometry using a LSR II instrument (Becton-Dickinson). The proportion of activated cells was quantified using FCS Express V5 software.

Cytokine immunoassays

Cytokine production was measured from cell culture supernatants with Cytometric Bead Array (CBA) (BD Pharmingen, 552364) using mouse inflammation kit against IL-6, IL-12p70, MCP-1, TNFα, IL-10, and IFNγ.

Results were acquired by flow cytometry using a BD LSR II instrument and analyzed with FCAP Array Software v3.0 (BD Pharmingen, 652099).

NO and ROS Production

NO produced by BMDMs and BMDCs were determined by measuring nitrite concentration in cell culture media by Griess assay. Briefly, 50 µL of cell supernatant was transferred to a 96-well plate and incubated with equal volume of Sulphanilamide (Sigma, S9251) and N-alpha-naphthyl-ethylenediamine (NED) (Sigma, 222488) solutions respectively for 10 min each, protected from light. Optical density (OD) was measured at 540 nm using a CLARIOstar® Microplate Reader and sample nitrite concentration was determined using a a standard curve. ROS production by BMDMs and BMDCs was determined by ROS-Glo™ H2O2 Assay kit (Promega, G8821). The cells were cultured at 5 x 10 4 cell/mL concentration in a 96well plate, exposed to nanocarriers for 24 h and stimulated with 2 µg/mL of LPS. A volume of 20 µL of H2O2 substrate solution was added to each well 6 h prior ROS production measurement. ROS production measurement was perform by adding 100 µL of ROS-Glo™ Detection solution per well, before 20 min of incubation at 22°C followed by luminescence using a CLARIOstar® Microplate Reader.

Metabolic flux analysis

For mature BMDCs (on day 10) 1.5 x 10 5 cells per well were seeded in Seahorse culture plate (Agilent, 102416-100) precoated with Cell-Tak (Corning, 354240) to enable BMDCs adherence, in complete culture media supplemented with GM-CSF (5 ng /mL) and flt3L (25 ng/ mL). For mature BMDMs (on day 7) 0.8 x 10 5 cells per well were seeded in Seahorse culture plate. In the case of BMDCs at day 11 and for BMDMs, 1 h after plating (on day 7), cells were treated with nanocarriers. After 24 h of incubations with the nanocarriers, cells were washed and stimulated when indicated with LPS/IL-4. After 24 h of stimulation, cells were either washed and incubated at 37°C for 1 h without CO2 with either Glycolysis stress assay medium, composed of XF base medium supplemented (Agilent, 103575-100); with 1 mM glutamine(Agilent, 103579-100); or Mitochondrial stress assay medium, composed of XF base medium supplemented with 1 mM pyruvate (Agilent, 103578-100), 2 mM glutamine, and 10 mM glucose (Agilent, 103577-100). A Seahorse XFe96 instrument was used to measure the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) every 6minutes. Over the course of analysis, inhibitors were added as internal controls to determine which parameters in metabolism were affected from nanocarrier exposure. The inhibitors for Mitochondrial stress assay, added in the listed order, were Oligomycin (Sigma, #75351) at 1.5 µM, Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (Sigma, C2920) at 1.5, antimycin A (Sigma, A8674) and rotenone (Sigma, R8875) mixture at 1 µM each. In the case of Glycolytis stress assay glucose at 10 mM, Oligomycin at 1.5 µM, and 2-deoxy-D-glucose (Sigma, D8375) at 30 mM were injected sequentially. For the two assays, Hoechst 33342 (ThermoFisher, H21492) was added at the end of the experiment to normalize the data based on cell count. A graphical representation of the experiment design is presented in Supplementary Figure 3,7.

Statistical analysis

Results

Result

Neutral and Cationic lipid carriers do not induce cell toxicity and are efficiently internalized by

APCs

We first investigated whether exposure of NLCs and CLCs is toxic for APCs in vitro, using a macrophage cell line (J774.1A) or primary untransformed cells extracted from bone marrow: macrophages (BMDMs) and dendritic cells (BMDCs). Cells were exposed to NLCs and CLCs with concentrations ranging from 0 to 250 µg/mL and toxicity was measured using CytoTox-ONE Homogenous Membrane Integrity Assay kit (Figure 1A). Among all the tested cells, BMDCs was most susceptible to both NLCs and CLCs exposure, but none of the cells exhibited more than 20 percent cytotoxicity. Therefore, we chose for subsequent experiments 20 and 100 µg/mL as low and high standard doses without adverse effects, i.e. higher than 80% of cell viability after a 24 h incubation.

Next, we assayed the internalization and cellular localization of both NLCs and CLCs by BMDCs and BMDMs.

Both nanocarriers were internalized into the cytoplasm of BMDCs (Figure 1B) and BMDMs (Figure 1C) within a 24 h timeframe. Therefore, we can conclude from these first experiments on the absence of toxicity of these two nanocarriers up to a 260 µg/mL concentration, while they are both efficiently internalized by APCs.

NLCs and CLCs are internalized by APCs without affecting their phagocytic capacity

Accumulation of nanocarriers into phagocytic APC opens the question whether their functions could be altered, such as phagocytosis which is one of the main features of APC. The phagocytic capacity of BMDC or BMDM was assessed by counting the number of engulfed microspheres per cells by flow cytometry.

This parameter was not altered by either the neutral or the cationic nanocarrier supporting that the phagocytic capacity of both APC cells was not modified by any type of nanocarrier (Figure 2A to 2D).

Moreover, we noticed that the phagocytic capacity of BMDM was 20% higher than for BMDC (Figure 2B and 2D).

We also verified the impact of the nanocarriers on the phagocytic capacity of J774.1A, a well characterized macrophage cell line for phagocytosis analysis [13]. Similarly, we did not observe a significant change in phagocytic capacity between the nanocarrier treated cells or control cells. These results obtained with the J774.1A cell line were consistent with what we observed in the primary cells (Supplementary Figure 1A and B), although we observed a difference in the phagocytic capacity between J774.1A cell line and primary macrophage cells (BMDMs).

CLCs, but NLCs, can increase LPS activation of BMDMs

Next, we took advantage of the BMDCs and BMDMs models, which are primary non-transformed cells reproducing physiological immune cell responses ex vivo to study the impact of both the nanocarriers on APCs activation.

BMDCs were identified by CD11b and CD11c expression [14] whereas BMDMs were marked by CD11b and F4/80 expression [15] (see gating strategy in Supplementary Figure 2). Activation of BMDCs and BMDMs was evaluated by the frequency of CD86 and MHC-II double positive cells. After LPS stimulation, the frequency of CD86 + MHC-II + BMDCs increased from 27.83% to 75.9% (Figure 3A) while no significative changes were observed in BMDMs) (Figure 3B).

Exposure to increasing concentrations of NLCs or CLCs did not significantly alter LPS-induced double expression of CD86 and MHC-II in BMDCs. In case of BMDMs activation, CD86 and MHC-II double positive cell percentage was not altered when exposed to NLCs but significantly decreased when exposed the CLCs from 19.6% to 9.79%. In the case of unactivated BMDMs, the percentage of CD86 positive cell remains unaltered when exposed to NLCs (Supplementary table 2). Altogether, these data highlight that both nanocarriers do not activate BMDCs but the CLCs slightly alter the activation of BMDMs. BMDCs exposure to both nanocarriers, maintained their capacity to respond to LPS activation. However, in the case of BMDMs, CLCs exposure significantly increased the percentage of activated BMDMs from 14.69% to 29.76%, while it remained similar with the NLCs (Figure 3B). This suggests that exposure to nanocarriers alone is not sufficient to activate both BMDCs and BMDMs, however, in LPS-stimulated BMDMs, the exposure to CLCs increased the frequency of CD86+ MHCII+ activated cells. nanocarriers is not sufficient to activate both BMDCs and BMDMs although CLCs exposure enhanced the ability of BMDMs to respond to LPS stimulation.

CLCs and NLCs can alter the production of signal molecules of APCs

The capacity to produce different soluble factors, including signaling proteins like cytokines or chemokines and other small molecular mediators like NO and ROS is a hallmark of APCs activation.

Having demonstrated that exposure to a CLCs could alter the activation of BMDMs in response to LPS, we wondered what would be the impact of both nanocarriers on cytokine secretion. We observed that both nanocarriers did not induce cytokine secretion in unstimulated BMDCs and BMDMs (Figure 4A to 4D, left panel), although exposure to CLCs, but NLCs, significantly increased the production of the MCP-1 chemokine in both unstimulated BMDCs and BMDMs (Figure 4E and 4F, left panel).

Upon LPS stimulation of APCs, NLCs exposure did not alter IL-6 production by both BMDCs and BMDMs.

However, exposure to CLCs significantly increased IL-6 production by BMDMs (Figure 4B, right panel) but not by BMDCs (Figure 4A, right panel). In the case of BMDCs, both NLCs and CLCs increased TNF-α production at 100 µg/mL (Figure 4C, right panel). For BMDMs, TNF-α production was only increased at 100 µg/mL of CLCs but not for NLCs (Figure 4D, right panel). We also observed that treatment with CLCs but not NLCs significantly increased MCP-1 production in both stimulated BMDCs and BMDMs (Figure 4E and 4F, right panel).

Two other important secretory molecules, NO and ROS productions were evaluated from the culture supernatant of APCs by griess assay or H2O2 quantification respectively. In absence of LPS stimulation, we did not observe a production of NO by BMDCs and BMDMs in response to both nanocarriers (Figure 4G and 4H, left panel) although ROS production was detected by BMDCs at 100 µg/mL but and not in BMDMs Of note, both nanocarriers did not impaired the coupling efficiency of unstimulated or stimulated BMDMs (Supplementary Figure 5B).

As a whole, our results demonstrate that the CLCs has a more important effect on BMDMs metabolism compared to the NLCs, while both nanocarriers have very little effect on the metabolism of BMDCs.

NLCs and CLCs alter the glycolysis of BMDMs and not of BMDCs

Considering the alterations of the mitochondrial metabolism induced by the CLCs and to a lesser extent the NLCs, we sought to investigate their effects on the glycolytic profile of APCs as LPS stimulated cells are mostly dependent on glycolysis. To evaluate the different glycolytic parameters of BMDCs and BMDMs, cells were first pre-treated with different concentrations of both nanocarriers, and then stimulated with LPS or IL-4 for 24 hours. After stimulation, the extracellular acidification rate (ECAR) was measured using the glycolysis stress assay in order to assess glycolysis and glycolytic capacity.

Unlike for BMDCs that did not showed any alteration in glycolysis (Figure 5E) or glycolytic capacity (Supplementary Figure 6A), in BMDMs glycolysis (Figure 5F) and glycolytic capacity (Supplementary Figure 6B), were increased in both unstimulated and stimulated conditions when exposed to 100 µg/mL of CLCs.

However, exposition to the NLCs did not induce any alteration in glycolysis or glycolytic capacity in BMDMs, independently of stimulating conditions (Figure 5F and Supplementary Figure 6B).

The combination of these results revealed that the cationic but not the neutral lipid nanocarriers at the highest concentration alter the glycolytic profile in BMDMs. On the other hand, both nanocarriers have no effect on glycolysis in BMDCs.

Reversing the surface charge with a nucleic acid cargo prevent adverse effects of cationic lipid nanocarriers on APCs

As previous experiments combined pointed out that at 100 µg/mL CLCs had a more dramatic effect on BMDMs physiology, than a NLCs, we wondered if the surface charge alone could explain the differences observed.

and IL-4 stimulated BMDMs was not statistically significant for non-mitochondrial oxygen consumption and percentage of coupling efficiency (Supplementary Figure 8D and8E).

Altogether, these results revealed that decreasing zeta potential, hence the surface charge of the CLCs was able to reverse their effect on the different cellular functions of primary BMDMs upon both pro-and antiinflammatory stimulations. In addition, using a range of N/P ratios representing the surface charge of the nanocarriers, we demonstrated that the alteration of the BMDMs physiology was proportional to the overall surface charge of nucleic acid-loaded lipid nanoparticles.

Discussion

Lipid nanocarriers are promising nanodelivery systems for imaging [16], nucleic acids delivery [17], siRNA transfection [18], drug delivery [19], adjuvant delivery system [5] and other biomedical applications.

However, the potential reaction of the host immune system to those nanoparticles constitutes a major challenge in using different NLCs in biomedical science. Nanoparticles composed of cationic lipids have a strong capacity for binding and condensing nucleic acid by electrostatic interactions, and deliver the payload across cellular membranes within the target cell cytoplasm [20]. While siRNA off-target effects have been extensively studied, it is crucial to consider the side effects and toxicity of nanocarriers themselves, which remained to be more deeply characterized from immunological point of view.

Our current study revealed that BMDCs and BMDMs responded differently when treated with differently charged NLCs. In case of BMDMs we demonstrated that CLCs at high concentration provoked an enhanced immune response by increasing the production of different secretory molecules including IL-6, TNF-α, MCP-1, upon LPS stimulation while NLCs did not. However, we observe a reduction in TNF-α secretion by NLCs and CLCs exposed LPS-stimulated BMDCs.

To assess the influence of NLCs on the metabolism of BMDMs and BMDCs, we activated these cells with either LPS or IL-4. While LPS-activated pro-inflammatory cells undergoing a metabolic switch to enhanced glycolysis [21,22], IL-4 induced alternatively activated cells toward and anti-inflammatory response, which would then rely mostly on fatty acid oxidation (FAO) and mitochondrial oxidative phosphorylation (OXPHOS) [23]. As a result, altered metabolism is not only a characteristic of macrophages cell function, it is also a prerequisite for a proper response to an immune stimulus. In our study, we showed that both nanocarriers did not alter the basal mitochondrial respiration of BMDCs, but in the case of BMDMs basal respiration did increased when exposed to the highest concentration used (100 µg/mL) with the two NLCs.

Globally, in unstimulated BMDCs and BMDMs, nanocarriers had very few effects on cellular production of soluble factors in vitro. While no metabolic changes were observed in BMDCs; BMDMs showed a positive charge specific increase of glycolysis and mitochondrial respiration. Previous studies have shown a positive association between the glycolytic and the secretory activities in macrophages, however this was evaluated under LPS stimulation [22]. In unstimulated conditions with CLCs exposure, we do not observe this coupling, maybe because the CLCs-induced increase of glycolysis is not high enough to drive secretory adaptations as observed in CLCs-treated BMDMs under LPS stimulation. It is noteworthy that LPS activated BMDMs rely on mitochondrial respiration. These results obtained in vitro let us suppose that positively charged lipid carriers in vivo would not greatly affect the basal level of unstimulated DCs or macrophage secretory activity, hence avoiding unintended immune responses (suppression or activation) and subsequent harmful outcomes (cancer or autoimmunity).

Under LPS stimulation, CLCs exposed BMDCs and BMDMs increase their production of MCP-1. MCP-1 is responsible for monocytes/macrophages migration and their infiltration in tissues [24] . MCP-1 has been described as a pro-inflammatory chemokine associated with rheumatoid arthritis (RA) [25] or allergic asthma development [26]. Therefore, in vivo administration of CLCs might increase MCP-1 production facilitating the emigration of immature myeloid cells at the site of exposure.Then, immature myeloid cells may acquire pro-inflammatory or anti-inflammatory properties under the influence of the the inflammatory condition of the tissue. Pro-inflammatory behavior associated with M1 activation may lead to an imbalance in M1/M2 ratio promoting osteoclastogenesis in RA [27]. Anti-inflammatory behavior associated with M2 activation impair T-cell response [28], leading to neoangiogenesis [29,30]and stimulate tumor growth [31].

In our study, we have seen that the alteration of cellular function of APCs was predominantly linked with a CLCs (+45.8 mV). Therefore, we further studied the effect of the charge of the nanocarrier using BMDMs as cellular model since these cells appeared the most affect by the exposure to CLCs. We could modify the surface charge of the CLCs by the binding of negatively charged siRNA at different N/P ratios. We observed that the increase of the production of secretory molecules (IL-6, TNF-α, MCP-1 and NO) was proportional to the surface charge of the lipid nanocarriers. In parallel, metabolic parameters, including basal respiration, maximal respiration capacity, ATP production, spare respiratory capacity and proton leak, were also modulated accordingly to the charge of the lipid nanocarriers. These results show that the effects of positively charged nanocarriers, like CLCs, can be reversed by the complexation of negatively charged ligands, such as RNA, proportionally to the charge of resulting nanocarrier.

Several studies reported some effect of the charge of nanoparticles on cell behavior. For instance, N-Arginine-N-octyl chitosan (AOCS) is used to synthesize pH-sensitive charge-reversal lysosomolytic nanocarriers (ANLC), which could reduce the potential toxicity of the nanocarrier as well as the increase drug delivery efficiency [32]. In addition, Chen and coworkers showed that charge-reversal nanocarriers enhanced gene delivery to the tumor site [33]. Furthermore, Han and colleagues demonstrated that the use of chitosan and the pH-responsive charge-reversible polymer enhanced the siRNA delivery [34]. Here, our results highlight that fine tuning of the surface charge of cationic lipid nanocarriers with an oppositely charged biomaterial, for instance nucleic acid, could prevent immunostimulation properties of the cationic carrier and has to be kept in mind for the future use of such carriers for therapeutic applications. Overall, using the same cationic lipid nanocarrier with tunable surface charge, we propose that positive charge is one of the major factor responsible for the alteration of the immune response.

Conclusion

In conclusion, both BMDCs and BMDMs responded differently when exposed to the cationic or neutral variation of the same lipid nanocarrier formulation. Therefore, it is highly relevant to include both cell types in case of the analysis of immunotoxicity. We demonstrated that both nanocarriers, at low concentration, did not significantly alter several functions of both APCs. However, the cationic nanocarrier, at highest concentration, induced alterations of some functions of APCs. We demonstrated that this effect on APCs was dependent to the positive surface charge of the lipid carrier that could be offset by loading nucleic acid cargo that mediated reversal of charge. Finally, we propose that tuning the nucleic acid load, hence the surface charge of lipid nanocarriers is critical to their use for therapy, and prevent the alteration of immune cell response to stimuli.

Tables:

Table 1: Concentration of GM-CSF, FLT-3L and IL-6 for BMDCs culture. Reversing the surface charge with a nucleic acid cargo prevent adverse effects of cationic lipid nanocarrier on APCs

Expression of activation surface marker in APC Figure 3 Experimental design of metabolic flux analysis Effect of the net surface charge of cationic lipid nanocarriers on different cellular functions and metabolism of BMDMs

Impact of Amphiphilic dendrimers on the functions of macrophages and dendritic cells

In this project, several targeted drug delivery nanosystems (nDDSs) have been initially proposed to transport identified immunomodulators to the tumor in the attempt to reactivate innate immunity by stimulating various components of the immune system. This strategy of immunotherapy of cancers is promising; however, its efficacy has to be evaluated along with its safety as early as possible from the beginning of the development in order to select desired future candidates. The works presented below were achieved in the frame of an EuroNanoMed international program supporting the development of different nDDSs to modulate immune cells associated with brain tumors, including microglia/macrophage, lymphocytes, and natural killer (NK) cell Glioblastoma (GBM) is a hyper-aggressive brain tumor associated with short life expectancy, and current therapeutic means are poorly efficacious. Immunotherapy is considered the most promising approach to successfully cure and remove cancer due to a persistent and significant antitumor response mediated by effective immunomodulatory agents.

However, an advance in immunotherapy in the treatment of GBM involves a safe and convenient-to-use system that can deliver immunomodulators to tumor lesions. In order to address GBM with immunotherapy, the "NanoGlio" project was designed to develop nanotechnology-based systems for the targeted delivery of immunomodulating agents with an aim of developing and validating novel immunotherapeutic approaches to GBM.

To achieve this goal, different nDDSs were produced by partners using lipid, polymers, and dendrimers nanoformulations; then, the most promising candidates have been selected to be tested for anticancer activity in patient-derived xenograft models. In order to realize the project, NanoGlio project consortium has been built with 7 partners coming from 5 different countries. The work was subdivided into 6 WPs. As a part of the "NanoGlio project", I participated in WP 4 "Toxicity assay and cellular uptake mechanism."

WP 4 aimed to evaluate the toxicity and their potential immune-inflammatory effects of the loaded and empty nDDSs in order to decide, at an early stage, on whether a nDDSs is suitable for further cellular or animal studies. Specific objectives of this WP included:

1) in vitro evaluation of the cell toxicity,

2) determination of the immune-inflammatory responses of exposed cells and in vivo evaluation of toxicity.

These works were divided into two tasks, which altogether, gathered a panel of in vitro assays allowing rapid screening of nDDSs candidates for their nontoxicity and the absence of proinflammatory property in order to retain those presenting less risk for in vivo trials.

Task 4.1: In vitro toxicity assay of all the nDDSs has been performed using cells from various origins, including A549 (lung), Caco-2 (colon), and HepG2 (liver). In parallel, we used cell lines specialized in the internalization of foreign materials (nDDSs): THP-1 and U937 (human) and J774 (mice). Toxicity exhibited by different nDDSs on mentioned cell lines was monitored by LDH release assay and 7AAD/Annexin V. The aim of Task 4.1 is to choose the most suitable nDDSs for further experiments (Figure 25). For simplicity of the thesis, results from Task 4.1 is not shown here. But LDH assay and 7AAD/Annexin V assay suggested amphiphilic dendrimer designed by Dr. ling's laboratory in Aix-Marseille Université as the most suitable nDDSs for further toxicity analysis. encouraged us to write two manuscripts titled as 1) Synthesis and use of an amphiphilic dendrimer for siRNA delivery into primary immune cells.

2) Amphiphilic dendrimers: A potentially nontoxic siRNA delivery agent

The first article entitled "Synthesis and use of an amphiphilic dendrimer for siRNA delivery into primary immune cells" has already been accepted in "Nature Protocols" (NP-P200172B). This article aims to provide detailed protocols showing the high performance of amphiphilic dendrimer (AD) for siRNA delivery to a wide range of cell types, including highly challenging primary immune cells, such as human peripheral blood mononuclear cells (PBMCs), human B-and T-lymphocytes, NK cells (human and mouse), primary bone marrow-derived macrophages and primary microglial cells (rat and mouse). I contributed to this particular article in providing a proficient method for the transfection of primary mouse macrophages (BMDMs) by delivering ADs loaded with JAK1 siRNA as an example. 9.3.1 Synthesis and use of an amphiphilic dendrimer for siRNA delivery into primary immune cells siRNA mediated gene manipulation of immune cells is important for both basic immunological studies and therapeutic applications. However, siRNA delivery is challenging because primary immune cells are often sensitive to the standard transfection reagents and generate immune responses. To address these problems, Dr. Ling's team has developed an innovative amphiphilic dendrimer, which exhibits particularly high performance for siRNA delivery to a wide range of cell types. Notably, this dendrimer is able to form small and stable nanoparticles with siRNA, thus protecting the siRNA from degradation and facilitating cellular uptake of siRNA. The subsequent siRNA-mediated gene silencing is specific and effective at both the mRNA and protein levels, leading to consequential biological effects. Remarkably, this dendrimer does not induce apparent cellular toxicity or non-specific immune responses under experimental conditions.

This article provides a detailed protocol describing the amphiphilic dendrimer mediated siRNA delivery in human peripheral blood mononuclear cells (PBMCs), human B-and T-lymphocytes, NK cells (human and mouse), primary bone marrow-derived macrophages (BMDMs), and primary microglial cells (rat and mouse). The paper is already published on Nature Protocols volume 16, pages327-351(2021). DOI: https://doi.org/10.1038/s41596-020-00418-9

In the second manuscript entitled "Amphiphilic dendrimers: A potentially nontoxic siRNA delivery agent,"

I contributed to generate most of the immunotoxicity experiments except the western blotting and in vivo CAM experiment. This manuscript is still under process and waiting for the expert reviews of other participants in the project framework.

Introduction

Dendrimers are uprising polymeric nanostructured macromolecules with a well-defined structure. First discovered by Fritz Vogtle in 1978 and later by Donald Tomalia in 1980s (1,2), these macromolecules have shown their potential abilities in a lot of applications such as technology field where they can be used as sensors or contrast agents in magnetic resonance imaging. Moreover, dendrimers have shown a significant interest in biomedical field as they can be utilized in cancer treatment through improving pharmacokinetic properties of cancer drugs (3). In addition, their physical properties have shown a remarkable potential as efficient nanovectors for drug and gene delivery(4).

Dendrimers are highly tailored three-dimensional macromolecules with a globular shape. Their structure is comprised of three main parts: the inner core, end-groups also known as dendrons, and branched units linking the core and peripheral groups(4). Actually, the inner core is designated as generation 0 (G0) and the addition of another layer of branches will be G1, G2, G3 etc. (5). As the number of generations increases, the number of peripheral groups will also exponentially increase. These terminal groups can be modified so as to have various functionalities [START_REF] Liu | Effects of engineered nanoparticles on the innate immune system[END_REF]. In addition, the newly formed generation of dendrimer will have the double of the molecular weight of the previous generation, this can have an impact on dendrimer pharmacokinetics regarding in vivo application (7).

Amphiphilic dendrimers (ADs) are lipid/dendrimer hybrids that can undergo self-assemble in solution (8).

ADs are synthesized using hydrophobic and hydrophilic chemical structures and form self-assembled nanostructures. They present a high loading capacity for large panel of molecules, including proteins nucleic acids and various hydrophobic molecules (Liu, 2015;Torre, 2019). ADs are currently used to deliver a variety of RNA interference (RNAi) molecules to silence specifically gene expression to treat disease models including cancers (9,10) . RNAi is a natural process conserved in eukaryotes that involves in gene expression regulation (11). This mechanism is mediated by small non-coding RNA molecules divided into three classes: microRNA, short interfering RNA and short hairpin RNA. These molecules bind to a specific messenger RNA thereby suppressing its translation (12). Clinical application of RNAi in gene expression related diseases requires an efficient delivery vector (13,14). Other studies on dendrimers, such as polyamidoamine (PAMAM) have also shown an efficient delivery of small interfering RNA (siRNA) (15).

Owing to these biocompatible properties, dendrimers have promising therapeutic and biomedical applications including RNAi delivery (16). However, several studies have demonstrated that their use in biological systems could be limited by different challenges such as toxicity issues (17).

It is well known that dendrimer mediated toxicity is closely linked with the structure, surface charge and the generation of a particular dendrimer molecule (18). Nanoparticles (NPs) such as dendrimer have a molecular size similar to that of pathogens, this property allowing them to be uptake by the cell by mimicking the endocytosis of pathogens thus triggering the immune response through antigen presentation specifically by the help of dendritic cells (DCs) and macrophages (19). Therefore, dendrimers efficacy in the delivery of siRNA should be evaluated by understanding on how these nanoparticles target Antigen-presenting cells (APCs) but mostly the impact that it can have on cellular immune responses (20).

Antigen presenting cells (APCs) are immune cells that have the aptitude to express to their cell membrane the peptide antigen through surface markers such as Major Histocompatibility Complex (MHC) I or II (21).

The antigen recognition allows then the activation of the immune response by secreting signaling factors such as pro-inflammatory cytokines (22). Hence, while considering the application of dendrimer as carrier vectors it is important to evaluate the effect not only on APCs activation but also on the cell functions involving phagocytic capacity and the ability to secrete messenger factors that play a role in immune response.

Regarding the use of dendrimer as a delivery vector, it is essential to underline that the immunometabolism of macrophages and DCs is one of the mechanisms that controls these cells on how they carry out their functions. This process pathway consists in various metabolic processes including mitochondrial metabolism and glycolysis. Normally, cellular ATP energy is produced in mitochondria, this process entails the breakdown of macromolecules such as glucose and glutamine through a chain reaction.

For instance, fatty acid oxidation (FAO) and mitochondrial oxidative phosphorylation (OXPHOS) can be utilized to produce ATP in aerobic conditions whereas in hypoxia conditions ATP can then be produced through glycolysis pathway using glucose as a carbon source (23). Additionally, several studies have shown that stimulating cells by the help of antigens such as lipopolysaccharide (LPS), or interleukin-4 (IL-4) have different metabolic activities compared to unactivated cells (24,25). These observations emphasize on the fact that the use of dendrimers mediated delivery could dysregulate the overall cell metabolism hence impacting on the cellular immune response.

Here we analyzed amphiphilic dendrimer (ADs) toxicity and effect on immunometabolism in vitro in antigen presenting cells (APCs), using a macrophage cell line (J774.1A) or primary cells extracted from bone marrow: macrophages (BMDMs) and dendritic cells (BMDCs). In vivo dendrimer toxicity was also evaluated on embryonic development using White Leghorn eggs. Finally, we analyzed dendrimers as efficient siRNA delivery vectors by silencing JAK1 mRNA using BMDMs and BMDCs primary cells.

Materials and Methods

Cell culture

Murine macrophage cell line (J774.1A) was purchased from ATCC, cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillinstreptomycin.

BMDCs was generated from the bone marrow extracted from C57BL/6 mice (Charles River, l'Arbresle, France). As previously described ( 26), cells were isolated from bone marrow by flushing from the tibia and femurs. Erythrocytes and GR1 positives cells were removed by magnetic cell sorting using Dynabeads (ThermoFisher, cat.no: 11047) after incubation with Ly-6G/ Ly-6C (BD Pharmingen, cat.no: 553125) and TER-119(BD Pharmingen, cat.no: 553672) antibodies, and the remaining negatively sorted cells were isolated using Dynabeads isolation kit (ThermoFisher, cat.no: 11047) and resuspended at 5×10 5 cells/ml in complete Iscove's modified Dulbecco's medium (IMDM) (ThermoFisher, cat.no: 21980065) supplemented with GM-CSF (Peprotech, cat.no: 315-03), FLT-3L (Peprotech, cat.no: 250-31L) and according to Table 1. The transformation of the progenitors into fully active DC occurred after 10 days of culture.

BMDMs were also generated from bone marrow extracted from C57BL/6 mice. Erythrocytes were removed by RBC lysis buffer, and the remaining cells were cultured in complete DMEM (ThermoFisher, cat.no: 61965026) medium with 20% L929 conditioned medium (source of M-CSF) for 7 days.

Ovalbumin (OVA)-specific CD4 + T cells were obtained from OT II Mice (Charles River Laboratories). Briefly, mouse spleen was dissociated in RPMI medium, erythrocytes were lysed using Red Blood cell lysis buffer.

T cells were isolated by negative selection using Dynabeads® Untouched™ Mouse T Cell Kit (ThermoFisher, cat.no: 11413D) and resuspended in the culture medium of BMDCs.

ADs

ADs were provided by Aix-Marseille University in the framework of NanoGlio project (Euronanomed II).

ADs were synthesized via click chemistry using previously developed protocols developed by Dr. Ling

Peng's team (9,27). Mean size of these particles in complete DMEM media (10 % FBS,1 % Pen-strep) is 55.5 to 58.41 nm and a zeta potential of +40 mV.

Incubation with nanoparticles

The cells were plated either in 12, 24 or 96 wells plates from Falcon® or Seahorse XFe96 cell culture microplates at a concentration of 106cells/mL with ADs at 1.37, 2.75, 4 µM final concentration according to the different experimental setup and these cells were cultured for 24 h LPS (Sigma, cat.no: L2654) (2 µg/mL) or IL-4 (ThermoFisher, cat.no: 14-8041-80) (20 ng/mL) was used to stimulated the cells for 24 h. The impact of ADs on BMDMs and BMDCs were later measured for various parameters, such as viability, phagocytosis, activation, cytokine secretion, nitric oxide (NO) production, reactive oxygen species (ROS) production, glycolysis or mitochondrial metabolism.

Toxicity assessment

Different concentrations of ADs were used on cells while culturing in the in a 96 well plate for 24 h at 37°C, with 5% CO2. Cell viability was tested by CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega, cat.no: G7891) according to the manufacturer's optimized protocol. After 24 h lysis solution ( 2μl of Lysis Solution per 100 μl original volume) was used to generate a maximum LDH release as a positive control, later an equal volume of CytoTox-ONE™ Reagent (100 µL) was added to each well, and the plate was placed on a shaker for 30 seconds and then incubated for another 10 min in the dark. After that ½ of the volume of CytoTox-ONE™ Reagent (50 µL) was added to each well and the plate was placed on the shaker for another 10 seconds prior to record fluorescence with an excitation wavelength of 560 nm and an emission wavelength of 590 nm using CLARIOstar® Microplate Reader (BMG LABTECH).

Phagocytosis assay

ADs exposed J774 cell line was incubated with FluoSpheres® Carboxylate-Modified Microspheres (ThermoFisher, cat.no: F8851), 1.0 µm in diameter, crimson fluorescent at a ratio of 10 microspheres per cell for 6 h at 37°C in 5% CO2 incubator. Cells were analyzed for their fluorescence using Accuri C6 (Becton-Dickinson) and FCS Express V5 (De Novo Software).

Cell activation

ADs exposed BMDMs and BMDCs were stimulated with 2 µg/mL Lipopolysaccharide (LPS) from E. Coli for 24 h at 37°C with 5% CO2. The supernatant was harvested for cytokine immunoassay, and cells were labelled with antibodies specific for CD11b (Ozyme, cat.no: BLE101226) and CD11c (Ozyme, cat.no: BLE117318) or CD11b (Ozyme, cat.no: BLE101216) and F4/80 (Ozyme, cat.no: BLE123152) cell surface markers of BMDCs and BMDMs respectively after blocking the Fc receptor (BD Pharmingen, cat.no: 553142) to reduce nonspecific binding. To evaluate the cell activation BMDCs and BMDMs were stained anti-IAb (Ozyme, cat.no: BLE116410) and CD86 (Ozyme, cat.no: BLE105008) antibodies. In both the cases, live cells were selected by 7AAD (7-Aminoactinomycin D) (BD Pharmingen, cat.no: 559925) staining and analyzed by flow cytometry using LSR II (Becton-Dickinson), and the proportion of activated cells was quantified using FCS Express V5 (De Novo Software)

Cytokine immunoassays

Cytokine production were measured in the supernatant of cell cultures using Cytometric Bead Array (CBA) (BD Pharmingen, cat.no: 552364) mouse inflammation kit against IL-6, IL-12p70, Monocyte chemoattractant protein-1 (MCP-1), Tumor necrosis factor-alpha (TNF-α), Interleukin 10 (IL-10), and

Interferon gamma (IFNγ). Results were acquired by flow cytometry using LSR II and analyzed with FCAP array software v3.0 (BD Pharmingen, cat.no: 652099).

NO production and ROS Production

Production of NO for BMDMs and BMDCs were determined by measuring nitrite concentration in cell culture media by Griess assay, 50 µL of cell supernatant was transferred to a 96-well plate and incubated with equal volume of Sulfanilamide (Sigma, cat.no: S9251) and N-alpha-naphthyl-ethylenediamine (NED) (Sigma, cat.no: 222488) solution respectively and were allowed to sit for 10 min each, in dark. Then the optical density (OD) was measured at 540 nm using CLARIOstar® Microplate Reader. A standard curve was used to obtain approximate concentration of nitrite in samples. Production of ROS on BMDMs and BMDCs were determined by ROS-Glo™ H2O2 Assay kit (Promega, cat.no: G8821). For this assay the cells were cultured at 5 x 10 4 cell/mL concentration in a 96-well plate and later they were exposed to ADs and stimulated with 2 µg/mL LPS. 6 h before the experiment, 20 µL of H2O2 substrate solution was added to each well. The plate was incubated for 20 min with 100 µL of ROS-Glo™ detection solution at 22°C before reading the luminescence using CLARIOstar® Microplate Reader.

Metabolic flux analysis

Mature BMDCs (on Day 10) were plated (1.5 x 10 5 cells per well) in 96-well Seahorse culture plate (Agilent, cat.no: 102416-100) precoated with Cell-Tak (Corning, cat.no: 354240) in complete culture media supplemented with GM-CSF (5 ng /mL) and FLT3L (25 ng/ mL). Mature BMDMs (on Day 7) were plated (0.8

x 10 5 cells per well) in 96-well Seahorse culture plate. 1 h after plating, cells were treated with ADs. After 24 h of culture, cells were washed and left unstimulated or stimulated with 1 ng/mL LPS or 20 ng/mL IL-4.

After 24 h of stimulation, cells were either washed with glycostress assay medium (XF base medium (Agilent, cat.no: 103575-100) supplemented with 1 mM glutamine (Agilent, cat.no : 103579-100) or

Mitostress assay medium (XF base medium supplemented with 1 mM pyruvate (Agilent, cat.no : 103578-100), 2 mM glutamine, and 10 mM glucose (Agilent, cat.no : 103577-100)) and replenished with the same medium (180 µL/well) and the cell culture plate was placed into a 37°C non-CO2 incubator for 45 min to 1 h before the assay. Seahorse XFe96 takes measurements of the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) every 6 to 7 min. Over the course of analysis, inhibitors were introduced to determine which parameters in metabolism were affected because of the ADs treatment by measuring the ECAR and OCR rates. The inhibitors for Mito stress assay, added in the listed order, were oligomycin (Sigma, cat.no: 75351) (1.5 µM -inhibits the F0/F1 ATPase), carbonyl cyanide-ptrifluoromethoxyphenylhydrazone (Sigma, cat.no: C2920) (1.5 µM -an uncoupling agent), antimycin A (Sigma, cat.no: A8674) and rotenone (Sigma, cat.no: R8875) mixture (1 µM -inhibits complex 3 and 1, respectively). In the case of Glycostress assay glucose (10 mM), Oligomycin (1.5 µM), and 2-deoxy-Dglucose (30 mM -inhibits glycolysis) were injected sequentially. In the case of both the assay, Hoechst 33342 (ThermoFisher, cat.no: H21492) was injected at the end to normalize the data based on cell count.

Antigen presentation assay

ADs exposed BMDCs were stimulated with 2 µg/mL LPS for 4 h and incubated with 25 µg/mL OVA for additional 4 h at 37°C and 5% CO2. 0.4 x 10 6 T cells (extracted from OT-II mice and resuspended in the culture medium of BMDCs) were added to 0.1 x 10 6 BMDCs, at a ratio of 1 BMDCs for 4 T cells. Co-cultures were incubated for 4 days, then supernatants were harvested for cytokine immunoassays to measure IFNγ and IL-17 by using TH1/TH2/TH17 CBA kit (BD Pharmingen, cat.no: 560485) and IL-13 (ThermoFisher, cat.no: 88-7137-77) by ELISA.

Western blot Analysis:

BMDCs and BMDMs resulting from primary mouse bone marrow cells were seeded in 12 well plates at a concentration of 10 6 cells per well. The cells were transfected using ADs at NP ratio 10 with siJAK1 (Sense Strand sequence: GAAUAAAUGCAGUAUCUAAAU; Anti-sense strand sequence:

UUAGAUACUGCAUUUAUUCGG) or siAllStars (Qiagen, cat.no: 1027281) for 48 h. 150 µL of RIPA lysis buffer was used per well for cell lysis and further 10 µg of protein load was used for western blot. In the western blot primary antibodies revealed the downregulation of JAK1 while βACTIN was used as housekeeping gene.

In vivo-CAM assay

Fertilized White Leghorn eggs were incubated at 37.5°C with 50% relative humidity for 9 days. At that moment (E9), the CAM was dropped down by drilling a small hole through the eggshell into the air sac, and a 1 cm² window was cut in the eggshell above the CAM. At least 20 eggs (depending on embryo surviving rate after 9 days of development, there could be more than 20 eggs per group) were used for each group. Because some deaths may occur during hours after the CAM dropping down (an invasive surgical act), data may be collected with less than 20 eggs per group (minimum of 15 eggs per group).

Before the first treatment, viability of each egg is checked and surviving eggs are randomized in groups.

All eggs of a group are treated with a volume of 100 μl of the working solution of compound. Embryonic viability was daily checked. The number of dead embryos was totally counted on E18. Besides, to evaluate treatment-induced embryo toxicity, the gross abnormalities on embryo (head formation, body development, limb evolution, skin aspect) and extra-embryonic structures were also estimated and recorded.

Statistical analyses

Results are expressed as mean values ± SD. Statistical analysis was performed using Graph Pad Prism

Results

ADs is not correlated with cell toxicity

We first investigate whether ADs exposure is toxic in vitro for phagocytic cells acting as antigen presenting cells (APCs), using a macrophage cell line (J774.1A), a commonly used model to analyse internalization processes and phagocytosis (28), or primary cells derived from bone marrow: macrophages (BMDMs) and dendritic cells (BMDCs). Cells were exposed to various ADs concentrations (0-4 µM) and cell toxicity was measured by the analysis of the cell membrane integrity as a marker of cell death (Figure 1A). For all the tested cells, no increase of cell death was observed after their exposure with in increasing concentrations of ADs up to 4 µM. Three subtoxic doses (1.37,2.75 and 4 μM) were defined where cell viability remains higher than 80% after 24 h incubation for the studies of the effect of ADs on specific cell functions.

ADs do not interfere with phagocytic capacity of J774 macrophages J774 is a macrophage cell line commonly used as a model to analyze internalization processes and phagocytosis (28).Phagocytosis capacity of the J774 cells can be evaluated with engulfment of polystyrene microspheres (1 µm) coupled to a fluorochrome. The engulfment of polystyrene microspheres by the cells can be measured using flow cytometry, where the fluorescence intensity reflects the number of internalized particles (Figure 2A). After exposure to ADs, this analysis was performed and the proportion of cells in each peak of fluorescence was evaluated (Figure 2B). The numbers of the cells which have up taken 2 or 1 or no beads are not modified by the exposure to ADs. Similarly, the numbers of cells which have internalized 3, or more, beads were not altered by the exposure to 1.37 µM of ADs, whereas the exposures to 2.75 µM and 4 µM led to a discrete, but significative increase of fluorescent cells (XX +-YY and XX +-YY) as compared to the unexposed cells (XX +-YY). In conclusion, ADs do not alter the phagocytic capacity macrophage cells which remained able to actively engulf exogenous materials.

ADs has no impact on antigen presenting cells activation by LPS

The impact of ADs on APCs activation was analysed using primary non-transformed BMDCs and BMDMs that better reproduce immune cell responses ex vivo than cell lines. Two scenarios of ADs exposure were investigated: firstly, the direct effects to evaluate putative induction of inflammatory responses; secondly, the indirect effects to evaluate the capacity of the APCs to respond to inflammatory stimulus of LPS. Cells were analysed by flow cytometry for the expression of surface markers differentiation for BMDCs (CD11b and CD11c) and BMDMs (CD11b and F4/80) (Figure 3A). For each cell preparation, double positive cells were gated to analyse the expression of the activation makers (Figure 3B). Activation of BMDCs and BMDMs was analysed by following the expression of CD86 and MHC-II double positive cell population.

Exposure to ascending concentrations of ADs increased the double positive cells of BMDCs from 28.85% to 43.74%, but we did not see any alteration in case of BMDMs (Figure 3b). These data highlight the capacity of high concentration of ADs to activate BMDCs but not BMDMs. When cells were exposed to ADs, their capacity to respond to LPS activation was maintained in the same amplitude, i.e. between 77.48% and 92.98% of BMDCs and 14.69 % and 10.93% for BMDMs which suggest a saturation in case of both APCs cell activation.

ADs modify the secretions of signaling factors by APCs

Important features of APCs responses to activation is their capacity to produce different soluble factors, including signalling proteins like cytokines or chemokines and small molecular mediators like NO and ROS.

To determine whether the exposure of APCs to ADs could modulate their capacity to secrete cytokines and chemokines, the supernatants of BMDCs and BMDMs cultures were collected and quantified by ELISA for their content in pro-inflammatory cytokines (IL-6, TNF-α), immuno-regulatory cytokines and chemokine (MCP-1). Interestingly, ADs did not induce cytokine secretion by themselves in both unstimulated BMDCs and BMDMs (Figure 4A-B-C). After LPS stimulation, moderate cumulative effects were observed for the production of IL-6 and TNF- in both BMDCs and BMDMs depending on the concentration of the ADs (Figure 4A,4B). On the other hand, the production of IL-10 was slightly decreased in BMDCs whereas not significantly altered in BMDMs (Figure 4C). In case of both BMDCs and BMDMs, production of IL-12 remained undetectable by CBA technique (Data not shown). Altogether, these data support that ADs have low if any effect on the levels of pro inflammatory or immuno-regulatory cytokines produced by APCs.

MCP-1 is one of the essential chemokines that governs the migration and infiltration of monocyte and macrophage. MCP-1 is secreted by a variety of cells such as endothelial, fibroblasts, epithelial, smooth muscle, mesangial, astrocyte, monocytes, and microglial cells (29). ADs are a potent siRNA delivery agent Efficient delivery of an active siRNA to different immune cells remains challenging (30). To demonstrate the validation and application of siRNA delivery into immune cells using the ADs as a transfecting agent, we have used BMDCs and BMDMs as a model system. BMDCs and BMDMs were transfected using ADs using NP ratio 10 with JAK1 targeting siRNA for 48 h. After 48 h western blot was performed to evaluate the knockdown efficiency of ADs mediated JAK1. Using ADs as the vector for the delivery of anti-JAK1 siRNA into primary BMDCs and BMDMs of mice, the expression JAK1 protein was decreased to 56% for BMDCs and 80% for BMDMs with an ADs N/P ratio 10/1 (Figure 9A). Neither ADs alone or AllStars negative control siRNA/ADs complex generated any JAK1 gene silencing. The capacity in the modulation of JAK1 activity in primary inflammatory cells such as macrophages opens a new window to intervene in treatment for inflammatory diseases.

ADs are not toxic during in vivo embryonic development

We then analyzed the possibility to use ADs in vivo to deliver siRNA. We first analyze the toxicity of the administration of siRNA loaded ADs in vivo. Among the in vivo model systems, the chorioallantoic membrane (CAM) assay has resurfaced as an adequate model system for the toxicity investigation. The chick embryos, which are in the process of development makes it a suitable model for in vivo toxicity assessment. To evaluate whether ADs mediated in vivo toxicity, fertilized White Leghorn eggs were used.

Embryonic viability was checked daily. The number of dead embryos was counted on E18 and represented in the terms of percentage in figure 10A. Besides, to evaluate treatment-induced embryo toxicity, the gross abnormalities on embryo (head formation, body development, limb evolution, skin aspect) and extraembryonic structures were also estimated and recorded (Figure 10B). Figure 10C depicts the final death ratio and a Kaplan-Meyer curve are provided for all groups.

Discussion

Dendrimers are a class of well-defined hyper branched polymers that can be used for developing antitumor system (31), biomimetic regeneration (32), drug delivery agents (33,34), contrast agent [START_REF] Kobayashi | Macromolecular MRI Contrast Agents with Small Dendrimers: Pharmacokinetic Differences between Sizes and Cores[END_REF], vaccine development [START_REF] Crespo | Peptide and Amide Bond-Containing Dendrimers[END_REF], delivery of nucleic acid [START_REF] Kanasty | Delivery materials for siRNA therapeutics[END_REF] and other biomedical applications. Despite of having multifunctional potentiality in applicative fields, the use of dendrimers is still limited due to their high cytotoxicity, immunogenicity [START_REF] Madaan | Dendrimers in drug delivery and targeting: Drug-dendrimer interactions and toxicity issues[END_REF]. In order to minimize these issues, a new amphiphilic dendrimer have been devised for different purposes specially for the efficient delivery of siRNA (9). Especially, this ADs is capable of forming small and stable siRNA nanoparticles, thus shielding the siRNA from degradation and facilitating the uptake of siRNA by cells.

APCs function screening should be subject to immunogenicity for nanoparticles as these phagocytic cells are part of the reticuloendothelial network, which is responsible for the elimination of foreign bodies [START_REF] Fröhlich | Value of phagocyte function screening for immunotoxicity of nanoparticles in vivo[END_REF].

The process for the recognition and clearance of nanoparticles relies on the interaction between opsonins and macrophages.

Although studies record the effect of dendrimer surface chemistry (cationic, neutral and hydrophobic/lipidated) on different cell types [START_REF] Albertazzi | Dendrimer Internalization and Intracellular Trafficking in Living Cells[END_REF], a detailed analysis of the effect of dendrimer on phagocytic cells is still lacking. The membrane affinity of the dendrimers was found to depend upon the amount of positive charges on their periphery. Increased membrane affinity was also linked to cytotoxicity as well as hindered different cellular function. The current work provides a study on the influence of ADs in three different subtoxic dosage on two primary phagocytic cells originating from mouse bone marrow, BMDCs and BMDMs on their phagocytic capacity, cytokine, redox and metabolic profiles.

Previous studies have demonstrated that positively charged dendrimer could cause cell death in RAW 264.7 murine macrophage-like cell line which was evident from the increased hypodiploid DNA population and a simultaneous decrease in diploid DNA content, indicating that DNA cleavage occurred after exposure of the cells to cationic dendrimers [START_REF] Kuo | Mechanism of cell death induced by cationic dendrimers in RAW 264.7 murine macrophage-like cells[END_REF]. Here we observed that BMDCs was most susceptible to ADs exposure, but none of the cells exhibited more than 20 percent cytotoxicity within the toxicity test range (0-4 µM).

Phagocytosis is one of the key functions of APCs in order to ingest foreign material for clearance, biodistribution and the tenuous balance between host tolerance and adverse nanotoxicity. Macrophages are demonstrated to be the most important cell type that process nanoparticles [START_REF] Gustafson | Nanoparticle Uptake: The Phagocyte Problem[END_REF]. It has been shown that different nanocarriers could prejudice the phagocytic capacity of macrophages [START_REF] Gustafson | Nanoparticle Uptake: The Phagocyte Problem[END_REF]. In the present study, we demonstrated that phagocytosis of polystyrene microspheres by macrophages was increased with increasing concentration of ADs which can be explained by high ADs-cell membrane interaction.

Accumulation of nanoparticles inside the APCs may trigger a major issue of cell activation status. Activation of both APCs can be identified by the expression of CD86 and MHC-II expression in case of BMDCs and CD86 expression in case of BMDMs. We found that high concentration of ADs activates BMDCs but do not alter their activation state upon LPS stimulation probably due to the saturation of cell activation.

Interestingly, BMDMs showed a different response when treated with ADs. We found that ADs neither activate BMDMs nor alter their activation state upon LPS stimulation.

Our current study revealed that BMDCs and BMDMs responded differently when treated with ADs. It's reported that PAMAM dendrimers induce IL-6, TNF-α and ROS in J774.1A cell line in time dependent, PAMAM generation and concentration dependent manner [START_REF] Naha | Reactive oxygen species (ROS) induced cytokine production and cytotoxicity of PAMAM dendrimers in J774A.1 cells[END_REF]. In our study we report that although ADs do not induce IL-6, TNF-α and ROS generation by themselves by both APCs but they responded differently when challenged with inflammatory signal by LPS. When ADs treated BMDMs challenged with LPS, IL-6

and TNF-α production increased but ROS production remain unchanged. Interestingly ADs treated BMDCs responded differently upon LPS stimulation. The alteration in IL-6 and TNF-α production by ADs treated LPS stimulated BMDCs was less evident while we observed an increase in ROS production. High ROS production could explain the susceptibility of BMDCs for ADs than BMDMs as reported by LDH mediated toxicity assay. Here we also highlighted that ADs did not induce any NO production by both APCs but ADs treated BMDCs produced less NO upon LPS treatment, while BMDMs showed an elevated level of NO production.

It has been reported that cationic PAMAM dendrimers entered the cell through endocytosis and were transported in endosomes and localized in mitochondria and produced ROS resulting in DNA damage and cell death [START_REF] Fox | PAMAM dendrimer -cell membrane interactions[END_REF]. Accumulation of ADs in mitochondria, classically appreciated for their role as the powerhouse of the cell, introduce the issue of effect of ADs on cell metabolism. To investigate the effect of ADs on the mitochondrial metabolism and glycolysis of BMDMs and BMDCs, we exposed them to different microenvironments to stimulate differently. Such stimulated cells exhibit a distinguishable regulation of their metabolism: LPS-activated proinflammatory cells undergoing a metabolic switch to enhance glycolysis [START_REF] Everts | Commitment to glycolysis sustains survival of NO-producing inflammatory dendritic cells[END_REF][START_REF] Van Den Bossche | Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis[END_REF]. Alternatively, IL-4 stimulated cells relay on both FAO and mitochondrial OXPHOS for sustained energy [START_REF] O'neill | A guide to immunometabolism for immunologists[END_REF]. Thus, altered metabolism is not only a key feature of stimulated cell function but also a prerequisite for a proper response to immune stimuli.

To analyse the effect of ADs on mitochondrial metabolism and glycolysis, mitostress and glycostress assays were performed using BMDCs and BMDMs.

We showed that although ADs were found not to alter the basal mitochondrial respiration of BMDCs they increase it for BMDMs. The possible explanation for this phenomenon is the different phagocytic capacity of these cells. Indeed it is established that the phagocytic index of BMDMs is higher than that of BMD [START_REF] Kiama | Evaluation of phagocytic activity in human monocyte-derived dendritic cells[END_REF] , thereby enabling accumulation of a large quantities of ADs in BMDMs, and increasing the basal respiration to meet the endogenous ATP demand of the cell.

Unaltered proton leakage in BMDCs and increased proton leak in BMDMs suggest that ROS generation is not solely governed by mitochondrial proton leak.

Measurement of ATP production showed that pre-treatment with ADs did not alter the ATP production of BMDCs but increased it in BMDMs, which is consistent with basal respiration.

The basal respiration rate does not accurately reflect the ability of cellular respiration to respond to increased energy demand. As such, estimating the maximum capacity of substrate oxidation can be extremely valuable for the discovery of mechanisms by which ADs could affect cell metabolism.

Conversely, spare respiratory capacity indicates the reserve capacity of a cell to respond to an increased ATP demand and withstand periods of stress. Here, we showed that pre-treatment with ADs did not alter the maximal and spare respiratory capacities of unstimulated and IL-4-stimulated BMDCs (cells that largely dependent on mitochondrial metabolism) but increased them significantly in unstimulated and IL-4stimulated BMDMs. The increase in these two parameters in BMDMs can be explained by the increase in basal respiration as well as ATP production due to the increase in cellular energy demand.

We also analysed the effect of ADs on glycolysis and glycolytic capacity of BMDCs and BMDMs in case of both unstimulated and LPS stimulated cells. Our results revealed that although ADs did not alter the glycolysis and glycolytic capacity in BMDCs, it increased them in BMDMs suggesting that ADs increased the energy demand in BMDMs.

To be activated, T cells have to recognize the antigen presented by MHC-II molecules and to be stimulated by CD86 accessory molecules, which are both expressed at the surface of BMDCs, in the context of inflammatory signal. We investigated the impact of BMDCs on T cell antigen responses by the analysis of the secreted cytokines. A significant decrease of IFN-γ was found, reflecting decreased Th1 cell polarisation while little or no alteration in IL-13, and IL-17 production suggested that ADs did not impact Th2 and Th17 cell polarization.

One of the main promising use of ADs is their capacity to deliver siRNA, and their safety profile while being incubated with primary immune cells opens a new avenue in immune-mediated inflammatory diseases (IMID) which include systemic and localized autoimmune diseases that can involve innate immunity or the adaptive immune system [START_REF] Sibilia | Comment définir et classer les maladies inflammatoires[END_REF]. As these diseases share similar alteration of cellular signaling, such as JAK/STAT signaling pathway, the same therapies are proposed to treat them [START_REF] Kuek | Immune-mediated inflammatory diseases (IMIDs) and biologic therapy: a medical revolution[END_REF][START_REF] David | Genetics of immune-mediated inflammatory diseases[END_REF]. Conventional therapies, such as 5-aminosalicylates, corticosteroids, immunosuppressants or monoclonal antibodies, have significant side effects and fail to cure patients. In this context, it is crucial to propose new and sustainable therapeutic strategies for long-term patient care [START_REF] Baumgart | Crohn's disease[END_REF][START_REF] Baker | Novel therapies for immune-mediated inflammatory diseases: What can we learn from their use in rheumatoid arthritis, spondyloarthritis, systemic lupus erythematosus, psoriasis, Crohn's disease and ulcerative colitis?[END_REF][START_REF] Sandborn | Vedolizumab as induction and maintenance therapy for Crohn's disease[END_REF][START_REF] Gecse | Optimizing biological therapy in Crohn's disease[END_REF]. The recent development of RNAi therapies opens the way of a more specific treatment, that could be also targeting immune cells to benefit to these IMID's patients. However, targeting these cells remains challenging, and the development of better vector is a key issue of this strategy. In this study, we demonstrated the potential of ADs as a very efficient vector of siRNA for primary immune cells targeting.

In vitro cytoxicity assay gives an insight about ADs mediated cellular function modulation that we further investigated using CAM, a widely used preclinical model for toxicity as analysis. Analysis of cytotoxicity in CAM model revealed that ADs did not impose any toxicity in in vivo CAM model which widens the preclinical use of ADs for different purposes.

Legends Tables Table 7:Concentration of GM-CSF, FLT-3L and IL-6 for BMDCs culture. Nanomaterials (NMs), with sizes ranging from 0.1 to 100 nm, are comparable to many biomolecules and organelles, which enable nanomaterials to interact closely with the biological systems and thereby be used in different biomedical applications. NMs can be of different shapes and chemical compositions that significantly defy their properties such as solubility and surface chemistry, which can be engineered to make them suitable for a particular biomedical application. Furthermore, because of their size, NMs have the ability to pass through biological membranes [START_REF] Clift | The impact of different nanoparticle surface chemistry and size on uptake and toxicity in a murine macrophage cell line[END_REF]; thus, they may be capable of influencing the physiological and biochemical functions of any cell in the body.

Immunotoxicity studies, such as effects on phagocytes, are the most suitable for screening for toxicological studies because these cells are involved in unspecific and specific immune responses.

Furthermore, they are present at epithelial barriers, in the blood, and in almost all organs. Since the last decade, several immunotoxicological studies have focused on NMs used for biomedical purposes, such as drug delivery or imaging. However, there is still a need to enrich our knowledge about the effect of these NMs. This is required to understand their toxicological effects and explore their potential beneficial uses that may propose new therapy based on actions on the immune system, such as anti-inflammatory drugs or vaccines.

Gold is one of the first materials developed for medical applications. Indeed, gold-based NPs (AuNPs) are extensively used for several biomedical applications because of their unique optical properties, making them excellent candidates for imaging applications and as a platform for drug delivery.

Two types of organic NMs present great interest in biomedical science: Lipid NMs such as LCs and

Dendrimers. On the one hand, LCs are promising multipurpose nano-agents for imaging (Navarro et al, 2012b), nucleic acids delivery [START_REF] Hibbitts | Co-delivery of free vancomycin and transcription factor decoy-nanostructured lipid carriers can enhance inhibition of methicillin resistant Staphylococcus aureus (MRSA)[END_REF], siRNA transfection [START_REF] Tezgel | Chitosan-lipid nanoparticles (CS-LNPs): Application to siRNA delivery[END_REF], drug delivery [START_REF] Hinger | Photoinduced effects of m-tetrahydroxyphenylchlorin loaded lipid nanoemulsions on multicellular tumor spheroids[END_REF], adjuvant and antigen delivery system [START_REF] Bayon | Overcoming immunogenicity issues of HIV p24 antigen by the use of innovative nanostructured lipid carriers as delivery systems: evidences in mice and non-human primates[END_REF] and probably many other applications. On the other hand, Dendrimers are broadly used for developing antitumor treatment (Kasai et al, 2002), biomimetic regeneration (Wu et al, 2013), drug delivery agents (El-Sayed et al, 2002;Wilbur et al, 1998), contrast agent (Kobayashi et al, 2003), vaccine development (Crespo et al, 2005), delivery of nucleic acid (Kanasty et al, 2013) and other biomedical applications.

Altogether, it is well established that different NMs represents a major stake for the future of the biomedical industry. However, the interaction of NPs with the immune system raises the question of the biological impact of these NPs on the functions of immune cells and, thus, on possible harmful actions on health.

Cell culture experiments provide a valuable alternative to in vivo experiments, allowing for more regulated manipulation of cell functions and processes. Although cell lines played a crucial role in scientific progress for decades, researchers are now increasingly skeptical when interpreting data generated from cell lines only. Factors such as misrepresented and contaminated cell lines have triggered a strong interest in primary cells (ATCC, 2010;Lorsch et al, 2014). In our study, to be closer to the physiological conditions, we conducted our experiments on bone marrow-derived primary macrophages (BMDMs) and dendritic cells (BMDCs). This enabled us to have a closer look in different immune cells function in response to NMs and allowed us to compare between effects of NMs on two important phagocytic cells of the immune system.

In this thesis, we summarized the effects of inorganic (AuNPs) and organic (NLCs, CLCs, and ADs) NMs on BMDMs and BMDCs.

In contrast to cytotoxicity, the importance of in vitro immunotoxicity testing is not well established. The reason is mainly because of the complexity of the immunological system that makes it difficult to extrapolate in vitro and animal data to human reactions in establishing NP-specific immunotoxicity. For this purpose, we have designed a panel of ex vivo tests to assess the impact of the different NMs on the main functions of primary phagocytic immune cells (Figure 26). Several tests are available to identify the effect of a particular molecule, such as acute toxicity, subchronic toxicity, chronic toxicity, carcinogenicity, reproductive toxicity, developmental toxicity, and dermal toxicity, ocular toxicity, neurotoxicity, genotoxicity, immunotoxicity. Before moving to immunotoxicity assays, it is essential to define the subtoxic dose of the NMs. There are several techniques available addressing the cytotoxicity. When treated with a particular molecule, living cells face one of two fates: they could either stop growing and dividing; alternatively, they could die. The most frequently used method to evaluate cell death is based on analyzing the loss of membrane integrity. We selected "lactate dehydrogenase (LDH) assay" as a rapid and quantitative cytotoxicity assay, which relies on the release of LDH, a stable intracellular enzyme found inside every living cell, into the surrounding extracellular space when cell membrane is disrupted. Since this is only possible when cell membrane integrity is compromised, the presence of this enzyme in the culture medium acts as an indicator of cell death marker. LDH assay of AuNPs, NLCs, CLCs, and ADs with varying concentrations revealed that BMDCs are more susceptible than BMDMs to all these NMs. Besides, we noted that the different NMs showed various levels of toxicity to macrophages (BMDMs and J774.1A) and dendritic cells (BMDCs). Finally, by this assay, we have defined the subtoxic doses for AuNPs (10 and 50 µg/mL), NLCs (20 and 100 µg/mL), CLCs (20 and 100 µg/mL), and ADs (1.37, 2.75, and 4 µM) to be used in further immunotoxicity assays.

After defining the subtoxic doses of each NMs, we analyzed one of the most important function, which is shared by the BMDMs and BMDCs: phagocytosis. Phagocytosis is a key mechanism of the immune defense system against an alien agent. It ensures the elimination of pathogens, debris, and apoptotic bodies.

Phagocytosis is also necessary to capture antigens in the cellular environment. These antigens are then presented to T cells to induce an adaptive response or to guide tolerance to self (Aderem & Underhill, 1999). Previous studies have demonstrated that phagocytosis can be altered by carbon NPs, metal NPs and metal oxides (Fröhlich, 2015a). J774 is a macrophage cell line commonly used as a model to analyze internalization processes and phagocytosis [START_REF] Luo | Phagocytic efficacy of macrophage-like cells as a function of cell cycle and Fcgamma receptors (FcgammaR) and complement receptor (CR)3 expression[END_REF]. To define whether the NMs lead to the alteration of the phagocytic capacity, we analyzed the phagocytic capacity of J774.1A to engulf fluorescently labeled polystyrene microspheres after exposure to NMs. The advantage of using these microspheres is 1) they are inert to phagocytic cells, 2) their phagocytosis by cells can be easily monitored by fluorescence measurement. The data revealed that AuNPs, CLCs, and NLCs did not alter the phagocytic capacity of J774.1A cells, but AD slightly increased the phagocytic capacity only at high concentration. Thus altogether, phagocytosis capacity of macrophage remains unaffected by the exposure to NMs.

To investigate the effect of different NMs on the functions of BMDCs and BMDMs, we opted for two approaches. In the first approach, we investigated the direct effect of exposure to NMs. In the second approach, we studied the indirect effect by activating the NMs exposed cells with different activators .

To evaluate the direct effects (Figure 27), we exposed BMDCs and BMDMs to NMs; then, we evaluate their 'Neill, 2016). In the case of LPS mediated metabolic reprogramming, during the early stage of activation, cells initiate an enhancement of glycolysis meditated by TBK1/IKKε/Akt signaling, which is independent of mTOR/HIF1α/iNOS signaling pathway and does not rely on NO (Everts et al, 2014a). During the late stage of LPS, mediated cell activation mTOR/HIF1α/iNOS plays a crucial role in governing glycolysis in a NO-dependent manner [START_REF] Amiel | Mechanistic Target of Rapamycin Inhibition Extends Cellular Lifespan in Dendritic Cells by Preserving Mitochondrial Function[END_REF]Everts et al, 2012b). NO produced by inflammatory cells break the Krebs' cycle at two stages. The first break inhibits the SDH enzyme, responsible for converting succinate to fumarate (Jha et al., 2015b).

This break leads to the accumulation of succinate, which is involved in the biosynthesis of proinflammatory molecules such as IL-6 and prostaglandins. The second break occurs because of the transcriptional repression of isocitrate dehydrogenase (Idh), the enzyme responsible for converting isocitrate to alpha-ketoglutarate (α-KG). This results in the accumulation of citrate, which takes part in fatty biosynthesis and serves as a precursor for NO and ROS (O 'Neill, 2011). In turn, NO production ensures the commitment of proinflammatory cells towards glycolysis (Everts et al., 2012b), thus leading to a positive loop maintaining the glycolysis fueling the inflammation. Our data about the effects of NMs exposure revealed that direct exposure to AuNPs, NLCs, CLCs, and ADs did not alter overall mitochondrial metabolism of BMDCs based on Basal respiration, proton leak, ATP production, and non-mitochondrial respiration. However, an in-depth investigation showed an indirect effect of AuNPs on mitochondrial metabolism of BMDCs depicted by reduced the Maximal respiration capacity (MRC), Spare respiratory capacity (SRC). The evaluation of MRC and SRC is important because MRC tells us about the maximum respiration rate that the cell can achieve in response to increased energy demand, while SRC gives insight about the ability of a cell to produce energy in stressed conditions. In other words, we can say that SRC allows a cell appropriately responding to an increased ATP demand and withstand periods of stress.

Although the precise reason of AuNPs mediated MRC diminution is not known yet, additional investigations are necessary for a better understanding, including analysis of the content of mitochondria and their cristae density, the specificity of permeabilization for substrates and the respiratory chain complex activity [START_REF] Divakaruni | Analysis and interpretation of microplate-based oxygen consumption and pH data[END_REF].

Further analysis of cellular metabolism showed AuNPs slightly increased the glycolysis of BMDCs while other NMs (NLCs, CLCs, and ADs) did not alter the glycolytic profile of these cells. Interestingly, AuNPs, CLCs, and ADs significantly increased the mitochondrial respiration of BMDMs, whereas NLCs did not. A similar observation was noted in the case of glycolysis. Altogether, these data showed that the NMs, except for NLCs, increased by themselves the cellular energy demand of BMDMs. The possible explanation could be that exposure to NMs initiate several responses such as, MAPK/ERK pathway (Glista-Baker et al, 2012) and cellular stress, which require more energy to tackle the situation. This increase of the energy demand in BMDMs leads to rising their mitochondrial respiration and glycolysis. Our study of LPS activated BMDCs demonstrated that ADs slightly increased the IL-6 production while other NMs (AuNPs, NLCs, and CLCs) did not. The TNF- production was differently affected by NM exposures: no effect with AuNPs, a slight increase with ADs, and a significant decrease with CLCs and NLCs.

On the other side, the MCP-1 production was not affected by AuNPs and NLCs but significantly increased with CLCs and ADs. These data indicate that activation of BMDCs is not correlated for all NMs with the production of cytokines such as MCP-1, IL-6, and TNF-.

Interestingly, in case of LPS activated BMDMs, we noticed CLCs and ADs significantly increased the IL-6

production while AuNPs and NLCs did not. The production of TNF- and MCP-1 was differently affected by NMs exposures: no effect with NLCs and a significant increase with AuNPs, CLCs, and ADs. Just like observed for BMDCs, we found that activation of NMs exposed BMDMs is not associated with the production of different cytokines and chemokines such as IL-6, TNF- and MCP-1.

Overall observation on cytokine and chemokine productions by NMs exposed cells revealed that MCP-1 is the most sensitive indicator of NMs exposure. Upregulation of MCP-1 production is not only related to NMs exposed BMDMs and BMDCs, since even mesothelial 2 (NRM2) cells have been shown upregulating MCP-1 production upon exposure of nickel NPs (Glista- [START_REF] Glista-Baker | Nickel nanoparticles enhance platelet-derived growth factor-induced chemokine expression by mesothelial cells via prolonged mitogen-activated protein kinase activation[END_REF], thus further supporting MCP-1

as an important responder to NMs exposure.

In addition to cytokines and chemokines, NO is an essential secretory molecule when cells are activated by LPS, a TLR4 agonist. NO also plays direct or indirect role in the production of other pro-inflammatory secretory molecules as well as cellular metabolism. TLR-mediated NO production occurs in two distinct phases. In the early phase of TLR driven activation relies on urea cycle as a major source of NO, generated from extracellular arginine, which is imported following activation [START_REF] Qualls | Sustained generation of nitric oxide and control of mycobacterial infection requires argininosuccinate synthase 1[END_REF]. When extracellular arginine levels are depleted, citrulline is imported and converted to arginine to support NO production later in the late phase of activation [START_REF] Qualls | Sustained generation of nitric oxide and control of mycobacterial infection requires argininosuccinate synthase 1[END_REF]. Investigation on NO production revealed that, except AuNPs, all thee NMs (NLCs, CLCs, ADs) significantly downregulated NO production by LPS activated BMDCs. Notably, CLCs and ADs increased NO production in LPS activated BMDMs while AuNPs and NLCs did not.

One of the important factors closely related to NO production is the expression level of iNOS. It is well known that the ability to express iNOS greatly varies between different subsets of splenic DCs (Everts et al., 2012b). Here, we hypothesize that iNOS expression level also differs between BMDCs vs. BMDMs.

Different levels of iNOS expression lead to different behaviors of NMs exposed BMDCs and BMDMs in NO production.

In response to LPS, ROS is secreted to influence the production of pro-inflammatory cytokines such as IL-6 and TNF- [START_REF] Bulua | Mitochondrial reactive oxygen species promote production of proinflammatory cytokines and are elevated in TNFR1-associated periodic syndrome (TRAPS)[END_REF]. TLR mediated cell activation leads to the initiation of PPP [START_REF] Tannahill | Succinate is an inflammatory signal that induces IL-1β through HIF-1α[END_REF]. One of the important outcomes of PPP is NADPH. NADPH is used by NADPH oxidase to generate ROS as well as a counterbalance of ROS, which is glutathione and other antioxidants. The effects of NMs exposure on cell response to LPS are summarized in Table 8 for BMDCs and in Table 9 for BMDMs. During the analysis, we noticed, in many cases, NMs mediated alteration is statistically significant, but the amplitude of alteration is low. In other cases, we observed a major alteration of different secretory molecule production. To better understand the indirect effect of NMs on BMDCs and BMDMs, alteration in the production of secretory molecules by these cells is scored in the form of numerical values. Scoring is mainly based on the p-value. A minor increase (p > 0.05) is scored as +1, major increase (p ≤ 0.05) is scored as +2, minor decrease (p > 0.05) is scored as -1, major decrease (p ≤ 0.05) is scored as -2. In the end, an overall score is calculated; a positive score indicates an overall increase of proinflammatory molecule production, while a negative score indicates a reduction of pro-inflammatory molecule production. This score helps us to understand the pro-inflammatory or anti-inflammatory effects of NMs.

From Table 8, Table 9, we noticed that positively charged CLCs (+45. 10) of BMDMs. Thus, the output of this experiment supports that the positive charge of an NM can be reversed by using negatively charged siRNA. Several studies reported some effect of the charge of nanoparticles on cell behavior. For instance, N-Arginine-N-octyl chitosan (AOCS) is used to synthesize pH-sensitive charge-reversal lysosomolytic nanocarriers (ANLC), which could reduce the potential toxicity of the nanocarrier as well as the increase drug delivery efficiency [START_REF] Sun | Arginine-Modified Nanostructured Lipid Carriers with Charge-Reversal and pH-Sensitive Membranolytic Properties for Anticancer Drug Delivery[END_REF]. In addition, Chen and coworkers showed that charge-reversal nanocarriers enhanced gene delivery to the tumor site [START_REF] Chen | Charge-reversal nanoparticles: novel targeted drug delivery carriers[END_REF]. Furthermore, Han and colleagues demonstrated that the use of chitosan and the pH-responsive charge-reversible polymer enhanced the siRNA delivery [START_REF] Han | Enhanced siRNA delivery and silencing gold-chitosan nanosystem with surface charge-reversal polymer assembly and good biocompatibility[END_REF]. Here, we provide evidence that the level of the charge proportionally modifies several functions and metabolism activity of BMDMs. Therefore, we can conclude that the reversal of the effects of a charged NMs helps to design efficient NMs to treat a particular disease condition, especially for the delivery of siRNA.

CLC

Effect of charge reversal on the different cellular function of BMDMs

Finally, we conclude that different NMs differently effect functions of a particular immune cell type; thus, an NMs specific detailed immunotoxicity assay is much required, not only to prevent the unexpected toxic

  de la Santé et de la Recherche Médicale (INSERM) for providing financial support to conduct experiments. I would also like to extend my thanks to University Grenoble Alpes for the IDEX Mobility fellowship to conduct a part of my Ph.D. research at Institut d'Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), Barcelona.

  become plasma cells to attack the invader. 9 Antigen processing and presentation by MHC Class I and MHC Class II. 10 Schematic representation of the T-cell receptor-CD3 complex. 11 B cell antigen receptor signal transduction cascade.

  vivo tests assesses the impact of different NMs.27Evaluation of the direct effect of different NMs on APCs.28Evaluation of indirect effect of different NMs on APCs.

Figure 2 :

 2 Figure2: The components of the innate and adaptive immune system. The innate immune system act as the first line of defense against infections. It consists of soluble factors such as complement system and various cellular components, including granulocytes (basophils, eosinophils, and neutrophils), mast cells, macrophages, dendritic cells, and natural killer cells. The adaptive immune system takes time to respond but manifests as increased antigen specificity and memory. It consists of antibodies, B cells, and CD4+ and CD8+ T lymphocytes. Natural killer T cells and γδ T cells are cytotoxic lymphocytes that come at the interface of innate and adaptive immunity. (Adapted from[START_REF] Dranoff | Cytokines in cancer pathogenesis and cancer therapy[END_REF]

  Mechanical barrier retards entry of microbes • Acidic environment (pH 3-5) retards growth of microbes Mucous membrane • Normal flora competes with microbes for attachment sites • Mucous entraps foreign microbes Cilia propel microbes out of body Temperature • Body temperature/fever response inhibits growth of some pathogens Low pH • Acidic pH of stomach kills most undigested microbes Chemical mediators • Lysozyme cleaves bacterial cell wall Interferon induces antiviral defences in uninfected cells Complement lyses microbes or facilitates phagocytosis Phagocytic and endocytic barriers • Tissue damage and infection induce leakage of vascular fluid containing serum protein with antibacterial activity, leading to influx of phagocytic cells into the affected area • Various cells internalize (endocytosis) and break down foreign macromolecules • Specialized cells (blood monocytes, neutrophils, tissue macrophages) internalize (phagocytose), kill and digest whole organisms

Figure 3 :

 3 Figure 3: The polarization of macrophages depends on the factors present in their environment. M1/M2 paradigm of microglia and their immunoregulatory functions. Microglia are resident macrophage cells responsible for primary active immune defense in the central nervous system (CNS). PAMPS or DAMPS stimulates resting microglial cells through TLR or ATP receptors. In the presence of LPS and/or IFNγ, microglial cells polarize to the M1 phenotype and produce a variety of pro-inflammatorycytokines/mediators like IL-1β, IL-6, TNF-5-007, CCL2, ROS, and NO. Conversely, IL-4 and IL-13 induce alternatively activated microglia, known as M2 ('M2a') phenotype, play a role in the downregulation of M1 functions by anti-inflammatory cytokine, IL-10. (Adapted from[START_REF] Nakagawa | Role of microglial m1/m2 polarization in relapse and remission of psychiatric disorders and diseases[END_REF]

  . Among them, IL-12, IL-10, IL-23, ICOS-1, and OX-40 are involved in the T cells' polarization in Th1, Th2, Th17, or T reg. DCs originate from the hematopoietic stem cells of bone marrow. Three DCs precursors are recognized in humans: granulocyte, monocyte, and DCs progenitors (GMDPs), monocyte and DCs progenitors (MDPs), and common DCs progenitors (CDPs). When cells differentiate, they acquire various phenotypes. It is known that they originate from CDPs in the presence of Flt3-L, cDC1, cDC2, and pDCs. It has been established that there are many precursors in the mouse: CMPs, MDPs, and CDPs. CDPs divide into pre-cDCs and pre-pDCs. Pre-cDCs give rise to pre-cDC1 and pre-cDC2, which later differentiate into cDC1 and cDC2, respectively. Pre-pDCs develop into pDCs (Figure 4) (Castell-Rodríguez et al, 2017). To simplify, a few examples of DCs subtypes are explained below.

Figure 4 :

 4 Figure 4: Classification of DCs in humans and mice. Three DCs precursors are recognized in humans: GMDPs, MDPs, and CDPs.In mice, there are many precursors, namely CMPs, MDPs, and CDPs. CDPs divide into pre-cDCs and pre-pDCs. Pre-cDCs give rise to pre-cDC1 and pre-cDC2, which later differentiate into cDC1 and cDC2, respectively. Pre-pDCs develop into pDCs. Phenotypes of each cell and cytokines involved in the development process include ell myeloid progenitors (CMPs), macrophages and dendritic cells progenitors (MDPs), common myeloid progenitors (CDPs), granulocytes, macrophages and DCs progenitors (GMDPs), preconventional dendritic cells (pre-cDCs), pre-plasmacytoid dendritic cells (pre-pDCs), pre-conventional dendritic cells 1 (pre-cDC1), pre-conventional dendritic cells 2 (pre-cDC2), conventional dendritic cells 1 (cDC1), conventional dendritic cells 2 (cDC2), plasmacytoid dendritic cells (pDCs). (Adapted from[START_REF] Castell-Rodríguez | Dendritic Cells: Location, Function, and Clinical Implications[END_REF]

Figure 5 :

 5 Figure 5: Differentiation of mDCs to acquire pro-inflammatory or regulatory phenotype depends on the surrounding microenvironment. The pro-inflammatory function of mDCs involves the production of inflammatory molecules and prime the proinflammatory subsets of effector T cells. On the other hand, tolerogenic mDCs, counterbalance inflammatory signals by regulating the activity of pro-inflammatory T cells, macrophages, and by activating immunosuppressive regulatory T cells (Adapted from[START_REF] Chistiakov | Myeloid dendritic cells: Development, functions, and role in atherosclerotic inflammation[END_REF].

Figure 6 :

 6 Figure 6: Story of Adaptive immune system (Adapted from "Dr. Biology. (2011, February 16))

  developing T cells obey a pattern of development in which they first express neither CD4 nor CD8 (double negative) in the cortex and then express both CD4 and CD8 (double-positive [DP])(von Boehmer et al, 1989)(Figure 7). DP cells are screened in the thymic cortex through positive selection, and those that are selected on the basis of MHC-I molecules become CD4-CD8 +, and those selected on the basis of MHC-II molecules become CD4+CD8-. The fact that the CD4 molecule facilitates a stable interaction between the developing T cell and the MHC-II molecules on the selecting APCS while the CD8 molecule establishes communication between T cells and MHC-II molecules is fundamental to the association of CD4 with MHC-II restricted antigen recognition and of CD8 with MHC-I restricted antigen recognition. Cells that survive positive selection migrate to the thymic medulla for negative selection and move to the periphery. 60-70% of T cells in the blood and secondary lymphoid organs are CD4+CD8-(CD4 +) and 30-40% are CD4-CD8 + (CD8 +). The largest T cell population in the body is the CD4+  TCR population. During their thymic development, αβ T cells divide into distinct subpopulations, each with specified effector function repertoires. They can function as helper cells, delivering cognate (including direct cell contact) or cytokine signals to maximize both B-and T-cell responses, as well as triggering mononuclear phagocytes.

Figure 7 :

 7 Figure 7: Overview of the development of thymocytes, emphasizing the role of key transcription factors and signaling molecules involved in the developmental process. Different thymocyte development stages are illustrated in the figure, with transcription factors and thymic stromal cells signaling. Receptor-ligand interactions are shown separately in the bottom. To determine the thymic seeding progenitor cells (TSPs) on the T cell lineage and to instruct transcription factors to adopt and commit to the T cell pathway at specific stages during differentiation, notch -Dll4 signaling is essential. Thymic epithelial signals, from Hh-(Shh and Ihh), Wnt-, and IL-7-signaling pathways, aid in the engagement with the T cell lineage and continued proliferation and survival of developing thymocytes. (Adapted from (Shah & Zúñiga-Pflücker, 2014)).Th1 cells were characterized by their capacity to produce IL-2, IFN-γ, and lymphotoxin and their expression of the transcription factor t-bet. While, Th2 cells secret IL-4, IL-5, IL-10, and IL-13, and their development is influenced by IL-4 and the transcription factor GATA-3 and Th17 cells secret IL-6 and IL-17 and express the transcription factor RORC2.

Figure 8 :

 8 Figure 8: B-cells become plasma cells to attack the invader (Adapted from "Dr. Biology. (2011, February 16)) 5.4 Linking Innate and Adaptive immunity Innate and adaptive immune responses together build the host defense system that ensures the safety of the host organism. Communication between innate and adaptive immunity requires correct and efficient translation of innate signals of danger to the adaptive system.

Figure 9 :

 9 Figure 9: Antigen processing and presentation by MHC Class I and MHC Class II. The immunoproteasome, which comprises several subunits, including LMP2, processes intracellular antigens, such as virus or tumor antigens, into peptides. Transportation of peptide to the endoplasmic reticulum ( ER) leads to the peptide loading into the MHC I complex groove, consisting of a heavy chain and β2 microglobulin (β2 m). MHC class I complex present antigens on the cell surface to CD8+ T cells. Antigens derived from extracellular sources such as bacterial antigens are transformed into peptides by endolysosomal enzymes. These peptides bind to the groove of the MHC class II complex by expelling class II-related invariant chain peptide (CLIP), derived from the invariant chain (Ii) of MHC class II molecule. HLA-DO and HLA-DM control the antigen-loading process. The MHC class II complex presents antigens to CD4+ T cells. Essential proteins involved in the MHC class II pathway are Transactivator-regulated genes (CIITA), MHC class II compartment (MIIC), T cell receptor (TCR).(Adapted from[START_REF] Kobayashi | NLRC5: a key regulator of MHC class I-dependent immune responses[END_REF]).

Figure 10 :

 10 Figure 10: Schematic representation of the T-cell receptor-CD3 complex. The heterocomplex is formed by variable TCR-α and TCRβ chains coupled to three dimeric signaling transduction modules CD3δ/ε, CD3γ/ε, and CD3ζ/ζ or CD247. CD3, Cluster of differentiation 3; CD247, cluster of differentiation 247 or CD3ζ/ζ; ITAM, immunoreceptor tyrosine-based activation motif; TCR, Tcell receptor (Adapted from[START_REF] Franco | Basic Pharmacological and Structural Evidence for Class A G-Protein-Coupled Receptor Heteromerization[END_REF]).

Figure 11 :

 11 Figure 11: B cell antigen receptor signal transduction cascade. Signal transduction initiates at the cell membrane following ligandinduced aggregation of the membrane immunoglobulin (mIg) and associated signal transducing elements Igα and Igβ (Adapted from[START_REF] Dal Porto | B cell antigen receptor signaling 101[END_REF]).

Figure 12 :

 12 Figure 12: Six major metabolic pathways. The conversion of glucose in pyruvate by glycolysis is further converted into lactate or acetyl-CoA and enters into the tricarboxylic acid (TCA) cycle where it generates NADH and FADH2 for the electron transport chain, leading to the production of ATP. Glycolysis also generates glucose-6-phosphate, which enters the pentose phosphate pathway (PPP) to produce ribose for nucleotides, amino acids, and NADPH. NADH is utilized for fatty acid synthesis, which uses citrate produced from the TCA cycle. Fatty acids can also be oxidized, leading to the production of NADH and FADH2, which drive the electron transport chain to produce ATP. In the end, amino acid metabolism can feed the TCA cycle, which is also crucial for cell growth and protein biosynthesis. In this diagram, the pathways requiring oxygen are shown in green boxes, and oxygenindependent pathways are shown in blue boxes. Additionally, inhibitors of different metabolic pathways are indicated in grey boxes. For example, BPTES, bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulphide. (Adapted from (O'Neill et al., 2016))

Figure 13 :

 13 Figure13: Glycolysis is the predominant source of energy in M1 macrophages because of hypoxia-inducible factor 1α (HIF1α) activation. HIF1α is responsible for initiating glycolysis and induces several genes that encode inflammatory cytokines, especially interleukin-1β (IL-1β). Another glycolytic enzyme, hexokinase 1, has also been involved in NLRP3 inflammasomes activation, which leads to caspase 1 activation and the processing of pro-IL-1 β. The pentose phosphate pathway(PPP) is fed by glycolysis and generates ribose for nucleotides for DNA and RNA biosynthesis, but generates NADPH for NADPH oxidase glutathione biosynthesis, promoting an antioxidant response. (Adapted from[START_REF] O'neill | A guide to immunometabolism for immunologists[END_REF]

Figure 14 :

 14 Figure 14: In LPS activated M1-like macrophages, TCA cycle is broken in two places -after citrate and after succinate. This leads to the accumulation of citrate, which is later used to generate fatty acids and prostaglandin productions. Fatty acid takes part in membrane biogenesis. The broken TCA cycle also leads to the generation of itaconic acid via the enzyme immune-responsive gene 1 (IRG1). Itaconic acid shows antimicrobial activity against Mycobacterium tuberculosis and Salmonella sp. (Adapted from (O'Neill et al., 2016))

Figure 15 :

 15 Figure 15: The role of nanoscience and nanotechnology in science and engineering (Adapted from[START_REF] Kolahalam | Review on nanomaterials: Synthesis and applications[END_REF]).

Figure 16 :

 16 Figure 16 : Carbon-based nanomaterials (Adapted from[START_REF] Rahmati | Biological Response to Carbon-Family Nanomaterials: Interactions at the Nano-Bio Interface[END_REF]

Figure 17 :

 17 Figure 17: Organic nanoparticles: a -Dendrimers, b -Liposomes, and c -micelles.6.3 Biomedical usage of nanoparticlesThe advancement of biomedical research has been based on two dogmas. First, assessment of biological behaviors over large populations. Second, improvement of ill-behaviors by systemic administration of therapeutic treatments. Interestingly nanotechnology significantly contributed to the advancement of both dogmas. In the framework of biology and medicine, nanotechnology underpins materials, devices, and systems whose structure and function are relevant to small scales, from nanometers (10-9 m) to microns(10-6 m) (Whitesides 2003). Living organisms are made up of cells that are usually ten μm in size.

Figure 18 :

 18 Figure 18: Biomedical applications of AuNPs (Adapted from (Liu et al, 2015)) Photodynamic therapy

Figure 19 :

 19 Figure 19: Lipidot structure. (a) Lipidots are dye-loaded oily droplets dispersed in aqueous buffer, whose diameter can be adjusted between 30 and 120 nm. Their fluorescent properties are conferred by lipophilic or amphiphilic cyanine dyes (b), encapsulated in the lipid core, and/or the surfactant layer.

Figure 20

 20 Figure 20 Three dimensional projection of dendrimer core-shell architecture for G=4.5 PAMAM dendrimer with principal architectural components (I) core, (II) interior & (III) surface. (Adapted from[START_REF] Tripathy | Dendrimers and their Applications as Novel Drug Delivery Carriers[END_REF]

Figure 21 :

 21 Figure 21: Chemical structures of several commonly used dendrimer (Adapted from (Mintzer & Grinstaff, 2011))Altogether, dendrimer generation, composition of the core, and peripheral groups play a crucial role in defining the physicochemical properties of dendrimer, which substantially encompasses the biomedical applications of a particular dendrimer molecule.

Figure 22 :

 22 Figure22:Representation of interaction between amphiphilic dendrimer and siRNA for siRNA delivery (Adapted from(Liu et al., 2014)) Dendrimer as an immunomodulatory agent

Figure 23 : 1

 231 Figure 23: Objectives of the thesis (Created with BioRender.com)

9. 2

 2 Impact of Lipid nanoparticles on the functions of macrophages and dendritic CellsMany innovations for drug delivery have been generated by nanotechnology, thus paving the way for the proposals of new therapies. Lipid Nano Carriers attracted a lot of interest in the development of potent delivery systems. In this context, I was engaged in the 'NEWDEAL' European project, funded by the "Horizon 2020 research and innovation program" of the European Union (agreement No 720905). The purpose of this project was to develop innovative therapy against chronic disease in the call that excluded cancers and infectious diseases. NEWDEAL was proposing to develop lipid nanocarriers to deliver new antiinflammatory agents, consisting of siRNA interfering with the expression of the JAK kinases, as new treatments for inflammatory bowel disease (IBD). IBD is an immune-mediated chronic intestinal condition that involves chronic inflammation of all or part of the digestive tract. The two conditions of IBD, Crohn's disease and ulcerative colitis are characterized by chronic inflammation of the gastrointestinal (GI) tract.

Figure 24 :

 24 Figure 24: NEW DEAL global methodology (Reused from "NEWDEAL" project proposal)

  entitled "Tuning the immunostimulation properties of cationic lipid nanocarriers for nucleic acid delivery." In this manuscript, I actively participated in producing all the data related to cellular function analysis and writing the original draft of the manuscript. The study was achieved in collaborating with CEA-LETI at Grenoble with Dr. A Nougarede, M Escudé, Dr. D Jary, Dr. F. Navarro) as partners who produced and fully characterized NLC and CLC. Dr. F Clément provides advice is siRNA handling, Dr. C Fournier in metabolism analysis, and Dr. E Jouvin-Marche in immunology. The study was designed by PN Marche. The outcome of this "charge reversing experiment" improve the knowledge about the effect of the positive charge in the course of different drug designing experiments.

(

  Figure 4I and 4J, left panel). In LPS stimulated conditions, both NLCs and CLCs decreased NO production by BMDCs at 100 µg/mL (Figure4G, right panel), while CLCs at 100 µg/mL was responsible for increasing NO production in BMDMs (Figure4H, right panel). After stimulation by LPS, both APCs produced increased only slightly increase basal respiration and non-mitochondrial oxygen consumption (Supplementary Figure4A and 5A).

Figure 1 -

 1 Figure 1 -Neutral and Cationic lipid nanocarriers do not induce cell toxicity but are efficiently internalized by APC.

Figure 2 -

 2 Figure 2 -Phagocytic capacity of APCs exposed to neutral or cationic lipid nanocarriers

Figure 3 -

 3 Figure 3 -Expression of activation surface marker in APCs following exposure to neutral or cationic lipid nanocarriers

Figure 4 -

 4 Figure 4 -Secretions of signalling factors by APCs in response to neutral or cationic lipid nanocarriers

Figure 5 -

 5 Figure 5 -Mitochondrial metabolism in naïve, pro-inflammatory or anti-inflammatory activated APCs in response to neutral or cationic lipid nanocarriers

Figure 6 -

 6 Figure 6 -Reversing the surface charge with a nucleic acid cargo prevent adverse effects of cationic lipid nanocarrier on APCs

Figures Figure 1 2 3 4 5

 12345 Figures

Figure 4 6 7

 467 Figure 4 Basal respiration, Maximal respiration capacity, and Spare respiratory capacity of naïve, classically activated or alternatively activated APCs in response to neutral or cationic lipid nanocarriers

Figure 25 :

 25 Figure 25:Identification of nDDSs without cytotoxicity.Task 4.2: In task 4.2, we analysed the immunotoxicity exhibited by dendrimer in detail using the same experimental schemed developed during the immunotoxicity analysis of AuNPs. The outcome of task 4.2

version 8 . 4 . 2 (

 842 679). Statistically significant differences were assessed by ordinary one-way ANOVA or repeated measures ANOVA with Tukey's multiple comparisons test; p-value below 0.05 was considered as statistically significant. Significance of the results is indicated according to p-values *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). p-values below 0.05 were considered as statistically significant.

  Interestingly we noticed direct exposure to ADs resulted increase of MCP-1 production by both unstimulated BMDCs (Figure 4D, left) and BMDMs (Figure 4D, right). Furthermore, when ADs exposed cells are stimulated by LPS, MCP-1 production is increased 3.46 folds by BMDCs and 8.95-fold by BMDMs at highest concentration of ADs (4µM).The NO and ROS productions were evaluated in the culture supernatant of APCs cultures respectively with Griess assay or H2O2 quantification. The exposure to ADs of both unstimulated APCs did not provoke the production of either NO nor ROS (figure 5A and 5B respectively). After stimulation by LPS, both APCs produced significant quantities of mediators. ADs significantly decreased NO production by BMDCs while increasing it by BMDMs (Figure5A). ROS production was significantly increased by LPS stimulated BMDCs whereas ROS production remained unchanged in stimulated BMDMs (Figure5B). These data indicate that BMDCs and BMDMs are differently affected by ADs in their capacity of NO and ROS productions after their activation by LPS.ADs influence the mitochondrial metabolism of BMDMs and not BMDCsCell metabolism plays a key role in different function of a particular cell type. Polarized cells display a distinct regulation of cellular metabolism, with LPS-activated proinflammatory cells undergoing a metabolic switch to enhanced glycolysis. Conversely, enhanced fatty acid oxidation (FAO) and mitochondrial oxidative phosphorylation (OXPHOS) provides sustained energy in IL-4-induced antiinflammatory cells. To analyze the impact of ADs on mitochondrial metabolism, mito-stress testing was performed on both BMDCs and BMDMs. The exposure to ADs showed no alterations in the basal mitochondrial respiratory capacity of unpolarized or polarized BMDCs (Figure 6A, left panel, black points), although ADs exposure significantly increased basal mitochondrial respiratory capacity of untreated or IL-4 treated BMDMs in a dose dependent effect, without showing substantial change in LPS treated BMDMs (Figure 6A, right panel, black points). We saw no variations in the proton leak in the case of BMDCs (Figure 6B, left), but the exposure of ADs increased significantly the proton leak in untreated or IL-4 treated BMDMs in a dose dependent effect, although no major changes were observed in LPS activated BMDMs (Figure 6B, right). No significant reduction in maximum respiration was observed in the case of unpolarized or polarized BMDCs (Figure 6C, left), whereas an increase in the maximum respiration capacity of untreated or IL-4 treated BMDMs was observed in a concentration-dependent manner, without significant change was observed in the case of LPS activated BMDMs (Figure 6C, right). We found that ADs did not alter ATP production by either polarized or unpolarized BMDCs (Figure 6D, left), whereas ATP production increased significantly in case of untreated or IL-4 treated BMDMs without alteration in the case of LPS treated BMDMs (Figure 6C, right).Non-mitochondrial respiratory ability wasn't altered after ADs exposure in either polarized or unpolarized BMDCs (Figure6E, let) whereas it significantly increased the respiratory capacity of both the stimulated and unstimulated BMDMs in a concentration dependent manner(Figure 6E, right). Altogether these data revealed that ADs influence the mitochondrial metabolism of untreated or IL4 polarized BMDMs, without impacting BMDCs.ADs alter the glycolysis of BMDMs and not of BMDCsAlteration of the mitochondrial metabolism by ADs leads us to investigate their effects on the glycolytic profile of APCs. In order to evaluate the different glycolytic parameters of BMDCs and BMDMs after ADs exposure, the cells were first pre-treated with different ADs concentrations, then stimulated with LPS or IL-4 or remained unstimulated for 24 hours. After stimulation, the extracellular acidification rate (ECAR) was measured. Both unpolarized or polarized BMDCs exposed to ADs did not show an increase in glycolysis (Figure7A, left), whereas glycolysis of BMDMs was increased in unstimulated and IL-4 cells and not in LPS polarized BMDMs (Figure7A, right). The pre-treatment of ADs did not alter the glycolytic reserve of both polarized and non-polarized BMDCs (Figure7B, left), but the it significantly increased the concentrationdependent glycolytic reserve all polarized BMDMs (Figure7B, right). The combination of the result revealed that ADs alter the mitochondrial metabolism of BMDMs and not that of BMDCs.ADs exposure modifies the lymphocyte T polarizing effect of BMDCsTo explore whether alteration of metabolic profile of BMDCs impair activation of T cell response, we performed an in vitro model of antigen presentation. Briefly, ADs exposed BMDCs were activated with LPS and a model antigen, OVA, were then co-cultured with lymphocytes T (L purified from the spleen of OT-II transgenic mice. OT-II mice exhibit a transgenic T cell receptor (TCR), which recognizes OVA peptide presented by the CPA on MHC-II IAb; all LT from these mice are specific to OVA. After 4 days, the supernatant of the co-cultures was collected and the cell fate of LT was evaluated according to the cytokines they have secreted: gamma interferon (IFNγ) is the major effector of Th1 response, IL-13 and IL-4 are involved in Th2 response and IL-17 is representative of Th17 response. These cytokines secretions were analyzed by CBA. Exposure of ADs and OVA decreased the secretion of both IFNγ in a dose dependent manner, but the alteration was less evident in case of IL-17 and IL-13 production by LT (Figure8).

Figure 1 :

 1 Figure 1: Definition of sub-toxic ADs exposure of APCs. [A] Cell mortality (LDH Assay) of BMDCs, BMDMs and J774 was analysed after exposure to different concentration of ADs for 24hData is normalized to the untreated cells (No ADs), considering as 100% live. The results are the mean and standard deviation of 3 independent experiments.

Figure 2 :

 2 Figure 2: Phagocytosis capacity of macrophages. [A] J774 were exposed (gray) or not (black) to 100µg/ml ADs for 24 h then incubated with fluorescent microspheres Fluospheres® for 6h, and analysed by flow cytometry. Overlaid histograms are shown. [B] After exposure to different concentrations of ADs, the proportion of cells in each peak was analysed. The results are the mean +/-SD of 3 independent experiments.

Figure 3 :

 3 Figure 3: Expression of activation surface marker of APCs. [A] CD11b and CD11c expression of BMDCs and CD11b and F4/80 expression of BMDMs. [B] Expression of activation marker of BMDCs and BMDMs after exposure to ADs for 24 h, followed by LPS stimulation for additional 24h. Percentage of Double positive (CD86 and MHC-II) BMDCs and CD86 positive BMDMs are counted gated on CD11b and Cd11c positive cells for BMDCs and CD11b and F4/80 positive cells for BMDMs and plotted in a bar graph. The results are representative one of the three independent experiment. Results are mean +/-SD from 3 experiments.

Figure 4 :Figure 5 :

 45 Figure 4: Cytokine and chemokine productions by activated APCs. [A, B, C and D] Relative cytokine and chemokine concentration in the supernatant of BMDCs and BMDMs exposed to ADs and activated by LPS. Results are mean +/-SD from 5 experiments. Paired student test was performed ns P > 0.05* P ≤ 0.05 **P ≤ 0.01

Figure 6 :

 6 Figure 6: Mitochondrial metabolism of activated or un activated APCs. [A] Basal respiration, [B] H+ (Proton) leak, [C] Maximal respiration Production, [D] ATP production, [E] Non-mitochondrial respiration of BMDCs and BMDMs were measured after been exposed to ADs or not for 24h and activated by LPS or IL-4 for another 24 h or not as indicated below the graphs. After measuring Oxygen consumption rate (OCR) by using Seahorse XF analyser, the data were normalized on the basis of cell number by using Hoechst 33342 staining. Results are mean +/-SD from 5 independent experiments. Paired student test was performed *P ≤ 0.05**P ≤ 0.01***P ≤ 0.001.

Figure 7 :

 7 Figure 7: Glycolysis of activated or unactivated BMDCs and BMDMs. [A] Glycolysis, [B] Glycolytic reserve of BMDCs and BMDMs were evaluated after exposed to ADs or not for 24h and after activation or not by LPS or IL-4 for another 24 h, as indicated below the graphs. After measuring Extracellular acidification rate (ECAR) using Seahorse XF analyser, the data were normalized on the basis of cell number by using Hoechst 33342 staining. Results are mean +/-SD from 5 independent experiments. Paired student test was performed *P ≤ 0.05**P ≤ 0.01***P ≤ 0.001.

Figure 8 :Figure 9 :

 89 Figure 8: Antigen specific T-cell responses. [A] OT-II T cells were co-cultured with ADs exposed/OVA loaded/LPS activated BMDCs for 4 days. T-cell cytokine production is shown. Results are mean +/-SD from 3 experiments. Paired student test was performed *P ≤ 0.05**P ≤ 0.01***P ≤ 0.001****P ≤ 0.0001

Figure 10 :

 10 Figure 10: JAK1 knockdown in BMDCs and BMDMs. Primary BMDCs and BMDMs were obtained from 5-8weeks old C57BL/6 mice. After 11 and 7 days of BMDCs and BMDMs culture, 1x10 6 cells per condition in 12 well plate were transfected using ADs at NP ratio 10 with siJAK1 or siAS for 48 h. Protein lysis was performed using 150 µL of RIPA lysis buffer. Western blotting was performed using 10 µg of protein load, and primary antibodies revealed the presence of JAK1 and βACTIN as housekeeping gene.

Figure 26 :

 26 Figure 26: Panel of ex vivo tests assesses the impact of different NMs ( Created with BioRender.com).

  fate and functions. First, we checked for effect on cell activation; for this, we analyzed the expression of major histocompatibility class II proteins (MHC-II) and the costimulatory molecule CD86 on cell surface.The function of the MHC II molecules is to present processed antigens, derived essentially from exogenous sources to CD4 T lymphocytes. Thus, MHC-II is critical to antigen-specific immune responses. On the other hand, CD86 provides during antigen presentation costimulatory signals necessary for T cell activation and survival. Our data revealed that exposure to AuNPs, CLCs, NLCs and ADs did not yield to the activation of BMDCs. Interestingly in the case of BMDMs, we observed a drop in the number of activated cells when directly exposed to AuNPs, CLCs, and ADs, whereas NLCs did not alter the number of activated cells. This shows that although NMs did not remarkably influence the number of activated BMDCs but decreased the number of activated BMDMs. BMDMs and BMDCs produce soluble factors to achieve some of their functions, including cytokines and chemokines, NO and ROS. The analysis of the cytokine productions showed that none of the NMs by themselves induced the inflammatory cytokines IL-6 and TNF- production by either BMDCs or BMDMs. However, the production of MCP-1 by both BMDCs and BMDMs was significantly increased by CLCs and ADs, while AuNPs and NLCs did not. The investigation on NO production revealed that none of the NMs by themselves induced NO production by both BMDCs and BMDMs. The analysis of another important signaling molecule, ROS, showed that only lipid-based NMs, i.e., NLCs and CLCs, induced ROS production in BMDCs, whereas AuNPs and ADs did not. None of the NMs by themselves provoked ROS production by BMDMs. The main effect of NMs exposure on the production of soluble factors resides in the induction of MCP-1 chemokine. As immunometabolism governs immune cell function (Mathis & Shoelson, 2011), we further investigated the effect of the NMs on BMDCs and BMDMs fate by the evaluation of their cellular metabolism based on mitochondrial metabolism and glycolysis. It is well known that LPS and IL-4 create different microenvironments for the cells, which affect cellular functions leading to the polarization of the BMDCs and BMDMs. LPS is known to activate classically pro-inflammatory cells that facilitate inflammation and participate in the host defense against various kinds of microbial threats. On the other hand, IL-4 dependent alternatively activated anti-inflammatory cells act as potent suppressors and controllers of ongoing immune responses. Once stimulated, BMDCs and BMDMs, exhibit a distinguishable regulation of their metabolism: LPS-activated pro-inflammatory cells undergoing a metabolic switch to enhance glycolysis (Everts et al, 2012a; Van den Bossche et al, 2015) while IL-4 stimulated anti-inflammatory cells mainly depends on Krebs' cycle and oxidative phosphorylation (Mills & O

Figure 27 :

 27 Figure 27: Evaluation of the direct effect of different NMs on APCs. BMDCs and BMDMs were cultured from mouse bone marrow for 11 and 7 days, respectively. After harvesting, the cells were seeded either in 12, 24, or 96 wells plates from Falcon® or Seahorse XFe96 cell culture with different concentrations of AuNPs (10 and or 50 μg/mL), NLCs (20 and or 100 μg/mL), CLCs (20 and or 100 μg/mL), and Ads(1.37, 2.75 and 4 μM). After 24 h of cell culture, cells were washed and remained unstimulated for another 24 h. At the end of 24 h, different downstream experiments were conducted according to the protocol.

  During infection, proinflammatory cells go for rapid ROS production to clear the infectious agent, followed by the induction of antioxidants to prevent excessive tissue damage. Study of ROS production by LPS activated BMDCs depicted AuNPs significantly reduced while ADs significantly increased, and the other two NMs (CLCs and NLCs) did not alter ROS production. Investigation on BMDMs revealed all four NMs (AuNPs, NLCs, CLCs, and ADs) did not alter the ROS production by these cells. All together, we can conclude that NMs do not interface with the ROS production of LPS activated BMDMs. Experiments on metabolic profile showed that only exposure to AuNPs indirectly affected mitochondrial respiration of activated BMDCs by reducing MRC and SRC. The exposure to the other three NMs (NLCs, CLCs, and ADs) did not alter the mitochondrial metabolic profile of BMDCs upon activation by either LPS or IL-4. Strikingly, we noticed in case of BMDMs, all three NMs (AuNPs, CLCs, ADs) significantly enhanced mitochondria respiration of activated cells, but NLCs did not. Data on glycolytic profile of BMDCs showed only AuNPs slightly increased glycolysis in IL-4 activated BMDCs while the other three NMs (NLCs, CLCs, and ADs) did not. In the case of BMDMs, we observed all three NMs (AuNPs, CLCs, ADs) significantly increased glycolysis in activated BMDMs except NLCs. All together, we can conclude that exposure of NMs remarkably increased energy flux in activated BMDMs.One of the most important functions of BMDCs is to activate the naïve T Cell. To be activated, T cells have to recognize the antigen under the form of a peptide presented by MHC molecules and be stimulated by CD86 accessory molecules, which are both expressed at the surface of DCs in the context of the inflammatory signal. We studied the cytokine production by T cells when activated by NMs exposed BMDCs. Different NMs showed a different effect on T cell response when NMs exposed BMDCs presented an antigen to T cells. In the case of AuNPs, we observed a significant increase of IFN-y, IL-13, and IL-17 productions, reflecting Th1, Th2, and Th17 cell responses, which could be correlated with the activation of the BMDCs seen by high CD86 and MHC-II expression levels. In the case of CLCs, we observed a significant decrease of IFN-y, IL-13, and IL-17 productions. For ADs, only IFN-y production is dropped, but IL-13 and IL-17 production is not or discreetly altered. Therefore, we can conclude that the effect of all the NMs on T cell response is not universal.To further understand the mechanism of action of NMs on T cell responses, we need to consider several issues: 1) the direct effect of NMs on T cell response; 2) the effect of NMs on the peptide processing and antigen presentation by BMDCs. The direct effect of NMs on T cell response could be evaluated by exposing the T cells to different NMs, followed by activation with anti CD3/CD28. The effect of NMs on the antigen processing by BMDCs could be addressed in comparing T cell responses obtained with whole OVA protein with those obtained with already processed OVA antigenic peptides.Indirect effect of NMs on the production of secretory molecules by

  8 mV) and ADs (+35.9mV) exposed BMDCs and BMDMs remarkably show an increased overall score of pro-inflammatory molecule production than AuNPs (+19.573mV) and NLCs (-16.7mV). This score suggests that positively charged NMs promote proinflammatory behavior of BMDCs and BMDMs upon infection. Altogether, the data of secretory molecule production and cell metabolism showed BMDMs upon activation (LPS or IL4) remarkably enhanced IL-6, TNF-, MCP-1, NO, Glycolysis, and Mitochondrial activity by positively charged CLCs and ADs. This observation leads us to hypothesize that the positive charge of CLCs and ADs is responsible for enhancing different functions in BMDMs. To establish our hypothesis, we used CLCs as a representative of positively charged NMs and reversed the charge of CLCs by combining with negatively charged siRNA. Our experiments showed that by reversing the charge, we could reverse the effect of positively charged CLCs on IL-6, TNF-, MCP-1, and NO production as well as cellular metabolism (glycolysis and mitochondrial activity) (Table

Table 10 :

 10 Zeta potential measurement of CLC with siRNA at different N/P ratios was performed on a zeta sizer. IL-6, TNFα, MCP-1, NO production was quantified from the supernatant of BMDMs exposed to 100 µg/mL of CLC or CLC +siRNA complexes at different N/P ratios and activated by LPS. Glycolysis in BMDMs exposed to 100 µg/mL of CLC or CLC +siRNA complexes at different N/P ratios and activated by LPS was determined by ECAR. Basal respiration, ATP production in BMDMs exposed to 100 µg/mL of CLC or CLC +siRNA complexes at different N/P ratios and activated or not by IL-4 was determined by OCR.

Table 1 :

 1

Defensive barriers of the innate immune system (Adapted from

(Marshall et al., 2018)

Table 2 :

 2 Common Innate Immune Recognition Strategies. (Adapted from

Table 3 (

 3 [START_REF] Robbins | Novel insights into the relationships between dendritic cell subsets in human and mouse revealed by genome-wide expression profiling[END_REF].

	Dendritic cell subset	Surface markers
		Human	Mouse
	cDC1	CD1c/BDCA-1 +++ CD11c ++ TLR2 +++ TLR4 +++	CD11b +++ CD209a + Tlr7 + Tlr9 + Tlr12 +++ Tlr13 +++
	cDC2	CD141/BDCA-3 +++ CD11b -CD11c ++ CLEC9A + XCR1 + TLR3 +++ TLR10 +++	CD8α +++ Clec9a +++ Xcr1 + Ly75 +++ Tlr3 ++ Tlr11 +++

Table 3 :

 3 Surface marker expression of cDCs subsets in human and mouse (Adapted from

Table 4 (

 4 [START_REF] Robbins | Novel insights into the relationships between dendritic cell subsets in human and mouse revealed by genome-wide expression profiling[END_REF].

	Dendritic cell subset	Surface markers
		Human	Mouse
	pDC	CD11c + CD123 + BDCA-2 +++ BDCA-4 +++ TLR7 +++ TLR9+++ CD1c low CD141c low	CD11c + B220 + mPDCA -1 +++ CD11b -Clec12a +++ SiglecH +++ Tlr7 +++ Tlr9 +++

Table 4 :

 4 Surface marker expression of pDCs subsets in human and mouse(Adapted from

  . Biocompatibility, unique optical, physicochemical properties, low toxicity, controlled disparity, tunable monolayers, functional flexibility, high surface area for loading the density of drugs, and stability make AuNPs an efficient nanocarrier in drug delivery systems (nDDSs). AuNPs are capable of transferring various drugs such as peptides(Lu et al, 2013), proteins(Love

Table 5 .

 5 The lipid part can have different forms, such as phospholipids, free fatty acids, glycolipids, or fatty alcohols. The surfactant part plays a role of a stabilizing agent.Lipid nanocarrier is generally divided into two types: Solid lipid nanoparticles (SLNs) and Nanostructured Lipid Carriers (NSLC). Solid lipid nanoparticles (SLNs) are solid core lipid nanocarriers that can accommodate both hydrophilic and hydrophobic drugs[START_REF] Paliwal | Solid lipid nanoparticles: a review on recent perspectives and patents[END_REF]. SLNs act as an alternate solution to conventional colloidal carriers such as polymeric microparticles, nanoparticles, liposomes, and emulsions. The biocompatibility of the lipid matrix is a crucial factor that enables SLNs to be used as drug carriers. SLNs can be manufactured using homogenization techniques, spray congealing methods, and microemulsions. Large surface area, Small in size, High stability than biological liposomes, High drug loading capacity, Less toxic than ceramic or polymer nanoparticles, Readily biodegradable make SLN an excellent drug carrier system. Biomedical applications of some of the SLNs are listed in

	SLN composition	Drug	Application
	Stearic acid	Rifampicin, isoniazid,	Mycobacterium
		pyrazinamide	Tuberculosis (Pandey &
			Khuller, 2005)
	Stearic acid, soya phosphatidylcholine, and	Ciprofloxacin hydrochloride,	Gram-negative
	Sodium taurocholate	tobramycin	bacteria,
			Gram-positive bacteria
			and mycoplasma (Jain
			& Banerjee, 2008)
	Soyabean-oil	Doxorubicin Breast cancer	Breast cancer (Wong et
			al, 2006)
	SLN	Tamoxifen, Methotrexate,	Breast cancer (Fontana
		and camptothecin	et al, 2005; Ruckmani et
			al, 2006)

Table 5 :

 5 Biomedical application of solid lipid nanoparticles (SLN)NSLC is formulated from a mixture of solid and liquid lipids, but the particles are in solid-state at body temperature. NSLC manufacturing is mainly based on solidified emulsion (dispersed phase) technology.Distorted lipid structures allow NSLCs to accommodate drug molecules. NSLCs of certain structures can be used for burst drug release by applying the trigger impulse to the matrix to convert in a more ordered structure.(Radtke and Mu¨ ller, 2001). NSLCs can be used in pharmaceutics for topical drug delivery, oral and parenteral (subcutaneous or intramuscular, and intravenous) routes. Some examples of biomedical applications of NLCs are listed in

Table 6 .

 6

	Nanostructured lipid carrier's composition	Application
	Phosphatidylcholine, dynasan and flurbiprofen	Sustained release of the anti-inflammatory drug
		(Bhaskar et al, 2009)
	Stearic acid, oleic acid, carbapol and minoxidil	Pharmaceutical, cosmetic and biochemical
		purposes (Silva et al, 2009)
	Fluticasone propionate, glyceryl palmito-stearate,	Topical corticotherapy (Doktorovová et al, 2010)
	and PEG
	Beta-carotene loaded Propylene glycol	Evaluate the feasibility
	monostearate

Table 6 :

 6 Biomedical application of nonstructured lipid carriers (NLC).

Table 2 :

 2 Percentage of Activated APCs with or without NLCs treatment.

	Cells are cultured 100 mm TC-treated Cell Culture Dish with 15 mL culture media
			Day 0	Day 3		Day 5		Day 7	Day 10
	Cell Concentration	0.6 * 106/mL	0.5 * 106 /mL	0.5 * 106 /mL	0.5 * 106 /mL	According to cell plating
	Supplement	IL-6 FLT-3 GM-CSF	5 ng /mL 50 ng /mL	2.5 ng /mL 40 ng /mL	2.5 ng /mL 30 ng /mL 5 ng /mL	-25 ng /mL	-25 ng /mL
		Double positive (CD86 and MHC-II) cells population percentage (mean ± SD)
				BMDCs			BMDMs
			Unstimulated	LPS stimulated	Unstimulated	LPS stimulated
		Cells	27.83 ± 8.58	75.9 ± 1.62	19.6 ± 2.13	14.69 ± 0.93
	Cells + NLCs 20	28.61 ± 12.22	80.51 ± 2.97	19.98 ± 1.92	16.32 ± 2.35
		ug/mL
	Cells + NLCs 100	29.3 ± 11.21	71.38 ± 4.85	16.3 ± 1.90	18.1 ± 1.05
		ug/mL
		Cells + CLCs 20	28.97 ± 7.79	79.57 ± 4.27	20.61 ± 3.39	25.84 ± 0.98
		ug/mL
	Cells + CLCs 100	27.74 ± 6.37	79.91 ± 2.39	9.79 ± 3.07	29.76 ± 2.45
		ug/mL

Table 2 :

 2 Concentration of GM-CSF, FLT-3L and IL-6 for BMDCs culture.

		Cells are cultured 100 mm TC-treated Cell Culture Dish with 15 mL culture media
				Day 0	Day 3	Day 5	Day 7	Day 10
	Cell Concentration	0.6 * 10 6 /mL 0.5 * 10 6 /mL 0.5 * 10 6 /mL 0.5 * 10 6 /mL	According to cell plating
	Supplement	IL-6 FLT-3 GM-CSF		5 ng /mL 50 ng /mL	2.5 ng /mL 40 ng /mL	2.5 ng /mL 30 ng /mL 5 ng /mL	-25 ng /mL	-25 ng /mL
		Double positive (CD86 and MHC-II) cells population in percentage ± SD
					BMDCs		BMDMs
				Unstimulated	LPS stimulated	Unstimulated	LPS stimulated
		Cells		28.85 ± 10.02	77.48 ± 3.39	19.6 ± 2.13	14.69 ± 0.93
	Cells + Den1.37 uM	24.81 ± 4.87	83.82 ± 2.63	12.2 ± 6.69	18.48 ± 4.9
	Cells + Den2.75 uM	28.95 ± 3.59	90.36 ± 1.051	2.91 ± 1.72	18.4 ± 7.46
	Cells + Den4.00 uM	43.74 ± 10.61	92.98 ± 0.76	5.43 ± 2.52	10.93 ± 3.43

Table 8 :

 8 Changes in different secretory molecule production by NMs exposed BMDCs after 24 h of LPS stimulation. IL-6, TNF-  MCP-1, NO and ROS are key proinflammatory molecules produced after TLR mediated cell activation. In the table. Alteration in different proinflammatory molecule production is recorded accordingly: minor increase (p > 0.05) = +1, major increase (p ≤ 0.05) = +2, minor decrease (p > 0.05) = -1, major decrease (p ≤ 0.05) = -2 Indirect effect of NMs on the production of secretory molecules by BMDMs

		AuNPs	NLCs	CLCs	ADs
	IL-6	0	0	+2	+1
	TNF-	+1	0	-2	+2
	MCP-1	+2	0	+2	+2
	NO	0	0	+2	+2
	ROS	0	0	0	0
	Total Score	+3	0	+4	+7

Table 9 :

 9 Changes in different secretory molecule production by NMs exposed BMDMs after 24 h of LPS stimulation. IL-6, TNF-  MCP-1, NO and ROS are key proinflammatory molecules produced after TLR mediated cell activation. Alteration in different

	proinflammatory molecule production is recorded accordingly: minor increase (p > 0.05) = +1, major increase (p ≤ 0.05) = +2, minor
	decrease (p > 0.05) = -1, major decrease (p ≤ 0.05) = -2.

(IRG1). Itaconic acid shows antimicrobial activity against Mycobacterium tuberculosis and Salmonella sp.(Adapted from (O'Neill et al.,

2016))

Acknowledgements

We thank Bertin Ndayishimiye for helping to build a library of published studies.

Funding Statement

This project has received funding from the European Union's Horizon 2020 research and innovation program H2020 "NEWDEAL" (grant agreement No. 720905). AD, AN and FC were supported by a fellowship from H2020 NEWDEAL project.

Disclaimer

This publication reflects only the author's view and the Commission is not responsible for any use that may be made of the information it contains.

Funding Statement

This project has received funding from the European Union's Horizon 2020 research and innovation program H2020 "NEWDEAL" (grant agreement No. 720905). AKD and FC were supported by a fellowship from H2020 NEWDEAL project.

 1. Hydrodynamic diameter of AuNPs. The hydrodynamic diameter and polydispersity index (PdI) of the AuNPs were measured by dynamic light scattering (DLS) with a 1 µg/mL AuNPs dispersion in complete DMEM media (In presence of 10% FBS) and H20. Each measurement was performed in three replications at 25°C. Supplementary Figure 1. Experimental scheme of AuNPs Supplementary Figure 1. Experimental scheme of AuNPs: BMDCs and BMDMs were cultured from mouse bone marrow for 11 and 7 days respectively. After harvesting, the cells were seeded either in 12, 24 or 96 wells plates from Falcon® or Seahorse XFe96 cell culture with AuNPs at 10 and or 50 µg/mL final concentrations. After 24 h of culture, cells were washed and stimulated with LPS or IL-4 for 24 h and downstream experiments were conducted according to the protocol.

Supplementary Figure 2.: Experimental scheme for metabolic flux analysis.

Supplementary Figure 2.: Experimental design of metabolic flux analysis: Mature BMDCs and BMDMs were seeded in Seahorse culture plate. 1 h after plating, cells were treated with AuNPs. After 24 h of culture, cells were washed and left unstimulated or stimulated with LPS/IL-4 for 24 h and the metabolic analysis is done using a Seahorse bio analyzer using the mitostress and glycostress assay protocol.

Supplementary Figure 3. Schematic representation of Antigen presentation assay.

Supplementary Figure 3. Schematic representation of Antigen presentation assay: AuNPs exposed BMDCs were stimulated with 2µg/mL LPS for 4h and incubated with 25µg/mL OVA for additional 4 h at 37°C and 5% CO2. 0.4 * 10 6 T cells were added to 0.1 * 10 6 BMDCs, at a ratio of 1 BMDCs for 4 T cells. Co-cultures were incubated for 4 days, and then supernatants were harvested for cytokine immunoassays.

Physical characterisation of NLCs

The hydrodynamic diameter and polydispersity index (PDI) of the NLCs were determined by dynamic light scattering (DLS) and the zeta potential was determined by electrophoretic light scattering (ELS) using a Zeta Sizer Nano ZS instrument (Malvern). The hydrodynamic diameter and PDI were measured with a 1 mg/mL NLCs dispersion in PBS while Zeta potential was measured with a 1 mg/mL NLCs dispersion in 1 mM NaCl. Each assay was performed in three replications at 25°C.

Complexation of CLCs nanocarrier with Nucleic acid (NA)

Complexation of CLCs with model NA, all-star negative control siRNA (siAS) was carried out in PBS. The required volume for siAS was calculated according to the desired N/P ratios at a constant concentration of the CLCs nanocarrier (100 µg/mL). CLCs carrier and diluted siAS were gently homogenized by pipetting and kept for 10 minutes at room temperature before immediate use for downstream experiments.

Incubation with nanoparticles

For cell culture 12, 24 and 96 cell culture microplates manufactured by Falcon® or Seahorse XFe96 were used. Cells were seeded at a concentration of 10 6 cells/mL and cultured for 24 h. An incubation of 24 h

with NLCs or CLCs at concentration ranging from 20 to 100 µg/mL was performed. Cells were subsequently washed and stimulated with LPS (2 µg/mL) or IL-4 (20 ng/mL) for another 24 h. Finally, the impact of the two nanocarriers on BMDMs and BMDCs was assayed using various parameter, such as: viability, phagocytosis, activation, cytokine secretion, nitric oxide (NO) production, reactive oxygen species (ROS) production and glycolysis or mitochondrial metabolism.

Toxicity assessment

Toxicity was measured by quantifying the cell viability using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay kit (Promega, G7891) according to the manufacturer protocol. Briefly, lysis solution (2 μl of Lysis Solution per 100 μl original volume) as a positive control for lactate dehydrogenase (LDH) release. A volume of 100 µL of CytoTox-ONE™ reagent was added to each well, before homogeneization on a shaker for 30 seconds and followed by an incubation for another 10 minutes in the dark. After that stop solution (50 µL) was added to each well and the plate was placed on the shaker for another 10 second. Finally, quantities of mediators, but ROS production which was not significantly altered by exposure of BMDCs and BMDMs to both nanocarriers (Figure 4I and 4J,right panel). These results indicate that BMDCs and BMDMs are differently affected by neutral or cationic nanocarriers regarding their capacity to produce NO and ROS, depending on activation stimuli.

Overall, both nanocarriers have an influence on signal molecule secretion, although limited to a slight increase in MCP-1 secretion (CLCs only) for both unstimulated APCs and in ROS production (CLCs and NLCs)

for unstimulated BMDCs only. In activated BMDMs, CLCs increased IL-6, TNF-α, MCP-1 secretion and NO production. We can therefore conclude that the cationic charge of CLCs might be responsible for increased adverse effects on APCs physiology and the secretion of signaling molecules.

NLCs and CLCs have a significant impact on the mitochondrial metabolism of BMDMs but BMDCs

As cellular metabolism plays a key role in different functions of APC, we sought to determine the effect of differentially charged lipid nanocarriers on mitochondrial metabolism. For instance, pro-inflammatory stimuli by LPS is known to triggers a metabolic switch that would enhance glycolysis, whereas enhanced fatty acid oxidation (FAO) and mitochondrial oxidative phosphorylation (OXPHOS) is a hallmark of IL-4induced anti-inflammatory activity in immune cells. To analyze the impact of neutral and cationic nanocarriers on mitochondrial metabolism, APCs cells were first treated with different concentrations of both nanocarriers for 24 h, then stimulated with either LPS or IL-4 for 24 h before measuring the oxygen consumption rate (OCR), using the experimental setup detailed in Supplementary Figure 3.

Upon exposition to both nanocarriers, no alteration in the basal respiration, maximal respiration capacity, spare respiratory capacity, non-mitochondrial oxygen consumption and coupling efficiency (Supplementary Figure 4A,4C,4E, 5A, 5C) proton leak or ATP production (Figure 5A and 5C) in unstimulated or stimulated BMDCs were found.

In BMDMs, exposure to both nanocarriers increased basal respiration and non-mitochondrial oxygen consumption of unstimulated cells at 100 µg/mL, as well as the non-mitochondrial oxygen consumption of LPS-stimulated cells treated with the NLCs (Supplementary Figure 4B,5B). Treatment with 100 µg/mL of CLCs increased significantly the proton leak, ATP production, Basal respiration, maximal respiration capacity, spare respiratory capacity and non-mitochondrial oxygen consumption (Figure 5B and 5D, Supplementary Figure 4B, 4D, 4F, 5B) in unstimulated or IL-4 stimulated BMDMs whereas the NLCs did This led us to investigate whether we could reverse the phenotype observed on APCs by reversing the surface charge of the CLCs with a nucleic acid cargo, here a negative control siRNA (siAS). We used different surface charges by finely tuning the ratio of the positively-charged amine groups of CLCs nanocarriers (N = NH 3+ group) relative to the negatively-charged phosphate groups (P) from each phosphodiester bonds within the nucleic acid sequence, hence called N/P ratio. After complexation between siRNA and CLCs nanocarrier, the zeta potential and hydrodynamic diameter these nanocomplexes was measured. Naked

CLCs showed a zeta potential of 45.80 ± 3.8 mV while increasing amounts of the nucleic acid cargo and thus decreasing the N/P ratio lead to lower the zeta potential values down to -9.97 ± 0.94 mV, while naked

NLCs was measured at -16.50 ± 0.53 mV (Figure 6A). Of note, the complexation of CLCs with different quantities of siRNA did not altered significantly the size of the nanocomplexes (Figure 6B).

Using different N/P ratios, we generated nanocarriers with different zeta potentials that we used to investigate their effects on BMDMs functions. BMDMs were exposed to 100 µg/mL of CLCs nanocarrier, CLCs-siRNA nanocomplexes at N/P 8 to N/P 1 or NLCs nanocarrier. The culture supernatants were collected and the secretion of pro-inflammatory cytokines (IL-6, TNFα) or chemokine (MCP-1) was quantified by immunoassay. IL-6 and TNFα productions by LPS-stimulated BMDMs were correlated to the zeta potential of the nanocarriers they were exposed to (Figure 6C and 6D), i.e. the productions were maximum with CLCs and decreased when CLCs are complexed to siRNA reaching at N/P ratio 1 a similar level than the one obtained with NLCs. The production of NO and MCP-1 by LPS-activated BMDMs did also decrease with lower N/P ratios but to a lesser extent than for IL-6 and TNFα (Figure 6E and Supplementary Figure 8A).

To analyze the effect of the surface charge on glycolysis, we measured ECAR in BMDMs exposed to nanocomplexes at different N/P ratios and then stimulated or not with LPS. Both unstimulated and LPSstimulated BMDMs showed decrease in both glycolysis and glycolytic capacities with decreasing zeta potential, and almost down to the same values than that of the NLCs for the unstimulated cells (Figure 6F and Supplementary figure 8B).

Next, we analyzed the effect of the surface charge on the mitochondrial metabolism of BMDMs, by measuring the OCR in BMDMs exposed to nanocomplexes at different N/P ratios and then stimulated or not with IL-4. The exposure to differently charged nanocarrier showed a decrease in basal respiration, maximal respiration capacity, ATP production, spare respiratory capacity, and proton leak correlated with a decrease in zeta potential in both unstimulated and IL-4 stimulated BMDMs (Figure 6G to 6J and Supplementary Figure 8C). However, the effect of differently charged nanocarriers on both unstimulated

Supplementary figures

Phagocytosis capacity of macrophage cell line J774.1A

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