Dr Friederike Jönsson

Dr Véronique Wikto

Javier Pizarro-Cerda

Claudine Arnaud

Elodie Jean-Pierre Cyrielle

Manuel, Michel Nicole Patrick

Stéphane R Sylvain

Thibault… Je

Dr Remercie Le

Pascale Romby

Hippel - Lindau

Vesicle-associated membrane protein VHL

Keywords: Neutrophile, Hypoxie, Shigella, Dégranulation, Glycosaminoglycane, Antimicrobien Neutrophils Proteoglycofili (PGF) degrade Shigella virulence factors and inhibit bacterial growth Neutrophil, Hypoxia, Shigella, Degranulation, Glycosaminoglycan, Antimicrobial Dans un premier temps, je

épithéliales humaines (Hep-2). Nous avons démontré que l'hypoxie et l'infection à Shigella induisaient la libération de PGF in vitro et in vivo.

Titre : Les Proteoglycofili (PGF) des neutrophiles dégradent les facteurs de virulence de Shigella et inhibent la croissance Bactérienne

Résumé

Lors d'une infection par un pathogène, les neutrophiles sont les principales cellules immunitaires recrutées au niveau des foyers d'infection. L'activité antimicrobienne des neutrophiles repose sur leur capacité à phagocyter et à tuer les bactéries de manière intracellulaire, à produire des espèces réactives de l'oxygène (ROS) et à dégranuler des protéines ayant une activité antimicrobienne. Les neutrophiles sont aussi capables de libérer des fibres extracellulaires de chromatine (NETs) dans le but de cibler les pathogènes extracellulaires.

L'hypoxie est bien décrite lors d'événements inflammatoires infectieux notamment lors de certaines infections bactériennes. Shigella, une entérobactérie pathogène responsable de la dysenterie bacillaire due à l'invasion et à la destruction de la muqueuse colique, a été décrite comme formant des foyers d'infection fortement dépourvus en oxygène.

En étudiant les fonctions antimicrobiennes des neutrophiles dans des conditions pauvres en oxygène, nous avons identifié et caractérisé un nouveau mécanisme antimicrobien des neutrophiles, consistant en la libération de "fibres" polymériques nommées Proteoglycofili (PGF). Nous avons démontré que, contrairement aux NETs, les PGF ne sont pas composées d'ADN et sont libérées par des neutrophiles viables. Nous avons montré que les PGF sont composées de glycoprotéines granulaires telles que l'albumine, la lactoferrine, la myéloperoxydase (MPO), l'élastase (NE) et la cathélicidine (LL-37). Ces fibres contiennent également des glycosaminoglycanes, tels que le sulfate de chondroïtine qui semble jouer un rôle central dans l'oligomérisation des PGF. Nous avons montré que les PGF ont une activité bactériostatique (E. coli et Shigella spp.) et dégradent les facteurs de virulence de Shigella (Ipa A-D, IcsA, Pic et SepA). Outre ces activités antimicrobiennes, les PGF semblent avoir une activité cytotoxique sur les cellules Merci Matthieu pour ces deux années passées à tes côtés. En espérant te recroiser un matin pour la fameuse visite guidée de Kehl. J'imagine que tu auras toujours un lit (baignoire, paillasson, carrelage…) pour m'accueillir un week-end.

Merci à Adrien, Karl, La Sourbe, Ipoutx et Yadal. Ça a été un plaisir de passer ces années d'études à vos côtés sous les bannières du sérieux et de la sobriété.

Bien évidemment, je remercie ma famille et plus particulièrement mes parents pour m'avoir soutenu durant ces longues années d'études malgré l'abondance de nouvelles de ma part.

Zuretako, ez baitauzut aski erraiten « maite zaitut ».

Table of contents

INTRODUCTION Introduction

The immune system maintains the integrity of multicellular organisms by differentiating "self" from "non-self" components such as microorganisms or toxins.

The human immune system encompasses the innate and the adaptive immune systems. The innate immune system is the first line of defense against invading pathogens. It is composed of physical and chemical barriers (cell junction, low pH, mucus layer), soluble factors (cytokines, complement system…) and innate immune cells (neutrophils, macrophages, dendritic cells, …). The innate immune system mediates a rapid but non-specific response, based on the recognition of pathogenassociated molecular patterns (PAMPs), which are small and highly conserved molecules exposed on pathogen cell-surface. PAMPs are recognized by pattern recognition receptors (PRR) expressed by immune cells. This interaction induces their activation and leads to several antimicrobial host responses (cytokine release, ROS production or phagocytosis…). When the innate immune response is not sufficient to eliminate microbes, the adaptive immune system is triggered. Unlike the innate immune system the adaptive immune system takes a long time to be activated (6 days after infection) but this response is highly specific to the invading pathogen 1 .

Polymorphonuclear neutrophils are central players of the innate immune response. It has been more recently demonstrated that they may also be important in the modulation of the adaptive immune response, through direct interaction with lymphocytes (B & T) or with antigen presenting cells (APCs), or through the release of mediators (cytokines, proteases or reactive oxygen species) 2 . The study of neutrophils has been largely impaired due to their short lifespan in vitro. Their overall contribution to the defense against pathogens but also their function in inflammatory processes remains far from being fully understood.

I. Polymorphonuclear neutrophils

Polymorphonuclear neutrophils or neutrophils are the most abundant immune cells in the blood circulation (around 60% 3 ). They are morphologically characterized by a multilobed nucleus and the presence of abundant populations of cytoplasmic granules (Fig. 1A and1B). Due to their capacity to rapidly migrate within tissues (neutrophils are the fastest human cells 4 ), neutrophils are among the first cells to be recruited at inflammatory sites. The wide arsenal of neutrophil antimicrobials functions sustains their capacity to limit the dissemination of pathogens upon their invasion.

A. The myelopoiesis

Neutrophils are produced in the bone marrow from CD34+ hematopoietic stem cells (HSCs) during a process called hematopoiesis, which give rise to all circulating blood cells. This essential process involves series of maturational steps differentiating a pluripotent HSCs to a common myeloid progenitor to the granulocyte lineage (basophils, eosinophils and neutrophils) (Fig. 2). HSC differentiation in white blood cells is mediated by a cocktail of cytokines secreted by surrounding cells at the site of production. The exact cocktail of cytokines leading to the differentiation of a hematopoietic stem cell to a common myeloid progenitor to a myeloblast to a neutrophil is debated but the combination of interleukin-6 (IL-6), GM-CSF and G-CSF has been described to be essential for the formation of mature neutrophils [6][7][8] . The production of mature neutrophil from HSCs takes approximately 14 days 9 .

Throughout the duration of the process, neutrophils will express chemoreceptors, especially CXCR4, which are specific to chemokines (CXCL12) produced by bone marrow stromal cells 10 . This interaction maintains neutrophils in the bone marrow until their complete maturation. The expression of chemoreceptors targeting chemokines produced in the bone marrow will decrease during their maturation, thus favoring their release into the blood circulation. High neutrophil production rate is required to maintain their number in the bloodstream, due to their short lifespan and their terminally differentiated state. Thus, 10 11 neutrophils 11 are released each day into the blood circulation under basal conditions and this number can increase by a factor 10 upon infection 12 . Neutrophils are produced in the bone marrow during a process called hematopoiesis which gives rise to all blood cellular components. This essential process involves a series of maturational steps, drove by cytokines stimulation, differentiating a pluripotent hematopoietic stem into the different types of white blood cells.

B. Biogenesis and composition of neutrophil granules

1.

The granulopoiesis

Neutrophils are frequently characterized by their huge concentration of cytoplasmic granules. The production of neutrophil cytoplasmic granules (granulopoiesis) occurs between the myeloblast stage and the formation of fully differentiated neutrophils (Fig. 3). This phenomenon is controlled by specific transcription factors, such as the C/EBP receptor family (CCAAT/enhancer binding protein) 13,14,15 , Gfi-1 (Growth Factor Independent 1) 16,17 and PU.1 18,19 . These transcriptions factors are sequentially involved during the neutrophil maturation from myeloblasts to mature neutrophils.

The sequential activation of these transcription factors controls the sequential formation of the four types of neutrophil granules. C/EBP and Gfi-1 are the first transcription factors expressed at the myeloblast stage inducing the transition into a promyelocyte, associated with azurophil granules production. The expression of C/EBP and Gfi-1 slowly decreases and C/EBP sustains further neutrophil maturation, mediating the transition of a promyelocyte to myelocyte and metamyelocyte. This transition is associated with the production of specific granules.

The next transition consists of the passage into a band cell with the slow expression of C/EBPβ, C/EBPδ, C/EBPζ and PU.1. This transition is associated with the production of gelatinase granules. The final step is controlled by the increased expression of C/EBPβ, C/EBPδ, C/EBPζ and PU.1 which induces the formation of mature neutrophils, associated with the production of secretory vesicles 15,20 (Fig. 3).

The granular genesis occurs via the endoplasmic reticulum (ER)-golgi pathway 21,22 .

More precisely, granular proteins contain a signal peptide interacting with a signal peptide recognition receptor during their synthesis. This interaction triggers their delivery to the ER protein translocation channel, allowing their translation into the ER lumen. Granular proteins will then follow the classical ER-golgi pathway and will be stored in trans-golgi vesicles 23,24 . The question of whether granules of neutrophils are formed by fusion of units of trans-golgi vesicles has not been addressed but studies performed on horse eosinophils and rat basophils indicates that granules are formed by homotypic fusion of a finite but slightly varying number of smaller of transgolgi vesicles [25][26][27] . The production of cytoplasmic granules of neutrophils, named granulopoiesis, occurs during the transition between a myeloblast and a mature neutrophil. This maturation is drove by the sequential activation of transcription factors; the C/EBP family (CCAAT/enhancer binding protein), Gfi-1 (Growth Factor Independent 1) and PU.1.

During the granulopoiesis, azurophil granules are the first to be produced followed by specific and gelatinase granules. Secretory vesicles are the last type of granules to be produced. Modified from Borregaard et al 20 .

Neutrophil granule composition a) Azurophil granules

Azurophil granules (or primary granules, ) contain the myeloperoxidase (MPO; essential for the ROS production), proteases such as cathepsin G, proteinase 3 or elastase, and bacterial membrane targeting proteins such as the lysozyme or antimicrobial peptides, including defensins 28 (Table 1). Through membrane fusion, azurophil granules are involved in the formation of a phagolysosome and mediate the intracellular killing of phagocytized pathogens, via the release of its content at high concentration (as high as 500 mg/ml inside the lumen of the phagosome 29 ). It is well accepted that azurophil granules mobilization is difficult, limiting the release of its content extracellularly (see part I.D.4.b).

b) Specific granules

Specific granules (or secondary granules, 1) have less killing potential compared to azurophil granules due to a lower concentration of antibacterial components (Table 1). These granules are mainly composed of bacteriostatic compounds such as lactoferrin or NGAL (Neutrophil Gelatinase-Associated Lipocalin) and thus are involved in the control and the limitation of pathogens' growth within infectious foci.

This activity relies mainly on lactoferrin, which is stored at high concentration in specific granules 30,31 . Lactoferrin, a member of the transferrin family, sequesters free iron ions which are essential for bacterial growth. By preventing the access of bacteria to iron, lactoferrin has a powerful bacteriostatic activity. In addition, lactoferrin has a bactericidal activity relying on its capacity to interact with bacterial membranes (potentially via the formation of lactoferrin-derived peptides) 32 .

In addition to the release of soluble proteins, the fusion of granules with plasma membrane allows the display of numerous membrane-associated proteins. Thereby, specific granules possess numerous cluster of differentiation (CD) such as several integrins (CD11b, CD18) 33 . More importantly, the NADPH component cytb558 is present in the membrane of these granules, thus, by fusing specific granules with phagosome neutrophil will allow the association of a complete NADPH enzyme necessary for the ROS production 34 (See part I.D.2).

c) Gelatinase granules

Gelatinase granules (or 2) contain a high concentration of extracellular matrix degrading enzymes (Table 1). The most abundant one, Matrix Metalloproteinase-9 (MMP9), also named gelatinase B, is able to degrade a wide range of extracellular matrix components such as aggrecan, laminin, collagen, gelatin or elastin 35 . MMP9 mediates the degradation of the extracellular matrix, promoting neutrophil migration to inflamed/infected sites. Conversely, an excessive release of gelatinase granules content is deleterious and has been reported in various destructive tissues pathologies [36][37][START_REF] Garg | Matrix metalloproteinase-9-mediated tissue injury overrides the protective effect of matrix metalloproteinase-2 during colitis[END_REF] .

Similar to specific granules, gelatinase granules also contain important membranebounded proteins. The fusion of gelatinase granules with the plasma membrane leads to the cell-surface exposure of integrin receptors which are involved in the adhesion of neutrophils to the endothelium and allows their subsequent extravasation. Thereby, the stimulation of gelatinase degranulation with inflammatory signals (e.g. fMLP, Leukotriene B4 or C5a) strongly increases neutrophil adhesion (by a factor three) [START_REF] Jones | Characterization of a New Mobilizable Mac-1 (CD11b/CD18) Pool That Co-Localizes with Gelatinase in Human Neutrophils[END_REF] .

d) Secretory vesicles

In 1987 a new intracellular compartment (distinct from azurophil, specific and gelatinase granules) was described by Borregaard and colleagues, named secretory vesicles () 40 . These granules are distinguished from other granules by their origin and their composition. In contrast to azurophil, specific and gelatinase granules, secretory vesicles seem to be formed upon endocytosis of the plasma membrane [START_REF] Borregaard | Stimulus-dependent secretion of plasma proteins from human neutrophils[END_REF] , and their production has been shown to continue after neutrophil maturation even in the bloodstream 42 . Secretory vesicles mainly contain plasma proteins such as albumin, and the fact that neutrophils are devoid of plasma protein mRNA support the hypothesis of their endocytic origin [START_REF] Borregaard | Stimulus-dependent secretion of plasma proteins from human neutrophils[END_REF] . The contribution of matrix proteins from secretory vesicles to neutrophil function is not clear.

Despite of their different origins, secretory vesicles also have an important role in the regulation of transmembrane proteins. Indeed, these granules are described to be highly enriched in transmembrane receptors having an important role for the neutrophil adhesion and extravasation through the endothelium and for the recruitment to inflammatory sites 43,44 . Thus, we can detect the presence of numerous integrin (CD11b, CD18) and chemoattractant receptors such as fMLP receptor (Table 1). Neutrophil granules can be classified in four types depending on their protein composition. By fusion with the phagosome or the plasma membrane, each granule release proteins with specific activities such as pathogen killing, pathogenic growth control or neutrophil migration. The fusion of granules with the plasma membrane will also allow the display of important membrane proteins and receptors having a role in the recruitment and the extravasation of neutrophils.

C. Neutrophil recruitment to inflamed or infected sites

1.

Pathogen detection receptors

Mature neutrophils circulate in the bloodstream prior their recruitment to inflammatory or infectious sites. Pathogenic bacteria have developed many different mechanisms to invade and colonize a host. However, a wound constitutes one of the easiest ways of entry for bacteria. Indeed, epithelia are the first physical barrier to protect a host from pathogen entry. A wound destabilizes this barrier allowing the contact of host cells and bacteria, and thus starting an inflammatory event. An infection will be followed by an inflammatory response, characterized by changes of four parameters of the homeostasis of the infected area: pain, redness, heat and swelling.

The detection of invading bacteria by host cells is mostly due to the recognition of pathogen macromolecules, either secreted or cell-surface. These factors, called Pathogen-Associated Molecular Patterns (PAMPs), are molecules which are highly conserved among species allowing the non-specific detection of pathogens 45 .

Immune and epithelial cells are endowed with receptors specific to PAMPs [START_REF] Ferrandon | The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections[END_REF] glucan, a component of the cell wall of fungi) 47,[START_REF] Hardison | C-type lectin receptors orchestrate antifungal immunity[END_REF] . The detection of cytoplasmic PAMPs relies on the presence of cytoplasmic PRRs such as NOD-like receptors (NLR) or RIG-I-like Receptors (RLR) [START_REF] Takeuchi | Pattern Recognition Receptors and Inflammation[END_REF] . Neutrophils have been shown to express all of the aforementioned PRRs 50 . Depending on the ligand-receptor interaction, the immune response will be specialized against the invading pathogen 51 .

Neutrophil activation, diapedesis and migration

During a bacterial infection, the recognition of PAMPs by PRRs activates immune cells and induces the release of small proteins called cytokines. These released factors coordinate the immune response by stimulating surrounding cells (immune cells and others). Large quantity of pro-inflammatory cytokines are released inside inflammatory site, including prostaglandin (responsible of the pain) 52 , histamine [START_REF] Iuvone | Evidence that mast cell degranulation, histamine and tumour necrosis factor alpha release occur in LPS-induced plasma leakage in rat skin[END_REF] , leukotriene B4 54 or CXCL8 55 . Histamine and leukotriene B4 stimulate surrounding endothelial cells, inducing the dilatation of blood vessels and, consequently, reducing the speed of the blood flow 56,57 . This modification is responsible of the redness and the heat characteristic of an inflammatory site. More importantly, the reduction of the blood flow speed will greatly enhance the interaction of neutrophils with endothelial cells. This interaction allows neutrophils to cross the endothelium in a mechanism called leukocyte extravasation (Fig. 4).

The first step of extravasation consists of the activation of endothelial cells by proinflammatory effectors such as histamine 58 . These effectors induce the display of receptors (P and E-selectin) at the apical surface of the endothelium. These selectins are specific to sialyl-Lewis x , a ligand expressed on the surface of circulating neutrophils. This interaction will provoke the rolling of neutrophils over the endothelium. Subsequently, the step of rolling allows the interaction between CXCL8 and CXCR1 leading to the neutrophil activation. Indeed, in addition to selectins, activated endothelial cells display CXCL8 at their surface, a chemokine specific to CXCR1 expressed on the surface of neutrophils 59 . This interaction will trigger the conformational change and the activation of the membrane receptor LFA-1, which is specific to ligands of the ICAM family. The binding of LFA-1 and ICAMs, located at the surface of the endothelium, leads to the arrest of neutrophils and allows the extravasation. This final step involves PECAM (Platelet Endothelial Cell Adhesion Molecule), which is expressed both on the leukocyte surface and at the intercellular junctions of activated endothelial cells [START_REF] O'brien | PECAM-1-dependent neutrophil transmigration is independent of monolayer PECAM-1 signaling or localization[END_REF] . The PECAM-PECAM interaction mediates the diapedesis of neutrophils. Therefore, a large number of leukocytes will be recruited to inflammation sites, inducing the swelling of the infected area. The recruitment of neutrophils to infectious foci is mediated by a concentration gradient of chemokines (especially CXCL8) allowing the migration of neutrophils toward the inflammatory site [START_REF] Godaly | Role of epithelial interleukin-8 (IL-8) and neutrophil IL-8 receptor A in Escherichia coli-induced transuroepithelial neutrophil migration[END_REF]62 .

Fig 4: Neutrophil recruitment and extravasation

Mature neutrophils circulate in the bloodstream waiting for their recruitment to an inflammatory site. Inflammatory site, such as bacterial infections, is characterized by the release of inflammatory signals by resident cells (chemokines or cytokines) or directly by invading pathogens (PAMP). These signals will activate surrounding endothelial cells inducing the display of receptors (selectins) on the apical surface of the endothelium. These selectins are specific to a ligand, sialyl-Lewis x , displayed on the surface of circulating neutrophils and this interaction will provoke the rolling of neutrophils over the endothelium. This rolling allows the interaction between CXCL8 and CXCR1 leading to the activation of the neutrophil and inducing a conformational change of a membrane receptor called LFA-1, specific to ligand of the family of ICAM displayed at the surface of activated endothelium. This interaction will arrest the neutrophil and the extravasation through the endothelium will be mediated thanks to adhesive molecules (PECAM) located both on the leukocyte and the intercellular junctions of activated endothelial cells. Once in the tissue, the specific recruitment of neutrophils to the infectious foci will be led by a concentration gradient of chemokines (especially CXCL8) inducing neutrophils to migrate toward the inflamed area. Modified from the Janeway Immunobiology book, 8 th edition 1 .

D. Neutrophil antimicrobial functions

1.

Pathogen phagocytosis and granule-phagosome fusion

Phagocytosis is an evolutionarily conserved mechanism consisting of the internalization of extracellular particles (Fig. 5). Neutrophils are professional phagocytic cells; they may internalize extracellular particles in less than 20 seconds 63 . Phagocytosis is initiated by the activation of phagocytic receptors located on the neutrophil surface, such as Fc receptors, complement receptors or PRRs.

The activation of a phagocytic receptors will lead to pseudopod extensions around the attached particle resulting in the formation of an intracellular compartment called phagosome 64 .

Fig 5: Neutrophil phagocytosis.

Immunofluorescence imaging of a human neutrophil phagocytosing Shigella flexneri (green) stained with DAPI (blue) and myelotracker-cy3 (red). Scale bar 10µm.

Phagocytosis is associated with intracellular pathogen killing mechanisms. Quickly after the internalization of the pathogen, cytoplasmic granules fuse with the membrane of the phagosome leading to the formation of a phagolysosome 63,[START_REF] Lancaster | Phagocytosis: what's on the menu? 1[END_REF]66 , mediating the contact of antimicrobials components with the engulfed pathogen.

Among these components, the most efficient are defensins or cathelicidin, which bind with high affinity into the bacterial membrane causing its disruption [START_REF] Dürr | LL-37, the only human member of the cathelicidin family of antimicrobial peptides[END_REF] , or the lysozyme which hydrolyzes the β (1-4) glycosidic linkages of bacterial peptidoglycan [START_REF] Baggiolini | RESOLUTION OF GRANULES FROM RABBIT HETEROPHIL LEUKOCYTES INTO DISTINCT POPULATIONS BY ZONAL SEDIMENTATION[END_REF] . Other antimicrobials components include the presence of proteases (e.g. elastase [START_REF] Pham | Neutrophil serine proteases: specific regulators of inflammation[END_REF] ) or metal chelating proteins (e.g. lactoferrin) which prevent the growth of pathogen [START_REF] Saito | Ultrastructural localization of lactoferrin in the granules other than typical secondary granules of human neutrophils[END_REF] .

More importantly, intracellular pathogen killing relies on another powerful antibacterial mechanism: the production of Reactive Oxygen Species (ROS).

Production of Reactive Oxygen Species (ROS)

ROS production is initiated by the stimulation of a specific class of membrane bounded receptors called G-Protein-Coupled-Receptors (GPCR) (Fig. 6). Neutrophils are endowed of different classes of GPCR such as Formyl-Peptide Receptors (e.g.

FPR1 which recognizes N-Formylmethionyl-leucyl-phenylalanine (fMLF) a tripeptide released by bacteria), complement receptors (e.g. C5aR) or chemokine receptors (e.g. CXCR1) 71 . These receptors are composed of an integral membrane protein which possesses seven transmembrane helices and a cytosolic part composed of a heterotrimeric G protein complex. The stimulation of one of these receptors induces the dissociation of the  and / subunits of the G-protein, and these subunits will activate the small GTPases Rac and Rho for the  subunit and the small GTPase Cdc42 for the / subunits 72 . The activation of these small GTPases induces an increase of the microbicidal activities of neutrophils. Indeed, the activation of the GTPases Rho, Rac and Cdc42 induces the migration of the neutrophil toward the stimulus (chemotaxis), the enhancement of the degranulation and the initiation of the oxidative burst responsible of the ROS production [73][74] . More precisely, the activation of a GPCR allows the association of the different subunits of the NADPH oxidase, an enzyme responsible of the ROS production. Under basal conditions, the NADPH oxidase is composed of 3 parts: 2 cytosolic components (Rac2 and the p47 phox /p40 phox /p67 phox complex) and the granular membrane associated component cytb558 (association of p22 phox and Nox2 located on the membrane of specific granules). The stimulation by a GPCR ligand activates GTPases, which will result in the activation of the different components of the NADPH oxidase: the phosphorylation of p47 phox /p40 phox /p67 phox complex and the activation of Rac2 via the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP). Once activated, the 2 cytosolic components will translocate simultaneously to the membrane to interact with cytb558, forming the NADPH oxidase complex [START_REF] Deleo | NADPH oxidase activation and assembly during phagocytosis[END_REF] . The complete NADPH oxidase will thus be present in the membrane of the phagolysosome and in the plasma membrane. NADPH oxidase reduces molecular oxygen (O2) to superoxide anion (O2• -) resulting in a transient increase of cell O2 consumption, known as the respiratory burst. Upon the phagosome-granule fusion, O2• -will be transformed by superoxide dismutase (SOD) into H2O2 which will be catalyzed by MPO to form the highly microbicidal molecule hypochlorous acid (HOCl) 76 . The presence of NADPH at the plasma membrane of activated neutrophils also allows the extracellular production of ROS 77 . ROS production is initiated by the stimulation of a specific class of membrane bounded receptors called G-Protein-Coupled-Receptors (GPCR). The stimulation of one of these receptors induces the dissociation of the  and / subunits of the Gprotein capable to activate GTPases such as Rho, Rac or Cdc42. These small GTPases are responsible of the activation of several neutrophils' microbicidal activities such as chemotaxis, degranulation and ROS production. Concerning ROS production, the stimulation of a GPCR receptor induces the activation of the different cytosolic components of the NADPH oxidase: the phosphorylation of p47 phox /p40 phox /p67 phox complex and the activation of Rac2 via the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP). Once activated, the 2 cytosolic components will translocate simultaneously to the membrane to interact with cytb558 (association of p22 phox and Nox2 located on the membrane of specific granules), forming the NADPH oxidase complex. The complete NADPH oxidase reduces molecular oxygen (O2) to superoxide anion (O2• -) which will be transformed by superoxide dismutase (SOD) into H2O2. The last step consists of the catalysis of H2O2 by MPO to form the highly microbicidal hypochlorous acid (HOCl). For that, the stimulation of neutrophils' receptors such as toll-like receptors (TLRs),

IgG receptors (FcR), complement receptors or cytokine receptors induce the production of these second messengers 78,80,81 . The use of PMA, an analogue of diacylglycerol, was shown to strongly activate NETosis and thus greatly facilitates the study of this phenomenon. The stimulation of neutrophils with PMA activates the PKC which will phosphorylate the granular membrane-associated component cytb558 of the NADPH oxidase which leads to the assembly of a functional NAPDH oxidase. The subsequent generation of ROS induces the rupture of granular and nuclear envelopes leading to the association of granular proteins with chromatin.

Among the large quantity of associated proteins, proteases, such as elastase, will play the important role of degrading the core histones, thereby inducing the chromatin decondensation. The final step consists of the rupture of the plasma membrane allowing the release of NETs fibers into the extracellular medium (Fig. 7) 80,82 .

NETs have been described to play essential roles in the defense against pathogens.

It has been reported that NETs kill microbes, although this point remains debated 83 . It has also been shown that NETs degrade extracellular virulence factors of bacteria (e.g. IcsA and IpaB of Shigella flexneri 78 ), due to the presence of various proteases such as elastase.

Neutrophil degranulation

The last well described antimicrobial mechanism of neutrophils consists of the extracellular release of compounds in a process known as degranulation. Neutrophils contain 4 four types of cytosolic pre-formed granules, as described above: azurophil (primary, ), specific (secondary, 1), gelatinase (tertiary, 2) and secretory granules (). These granules contain specific potent antimicrobial compounds and their mobilization upon stimulation is rapid and tightly controlled. Neutrophils may release granules' contents either inside the phagosome, upon phagosomal fusion, or extracellularly. The extracellular release of granular antimicrobial compounds allows the control of pathogen dissemination 84 , although adverse side effects are induced in surrounding tissues 85 . Thereby, this mechanism has to be tightly controlled.

a) Mechanism of degranulation

The process of degranulation can be divided in five different steps. This mechanism is initiated by the stimulation of cell surface receptors such as Fc receptors [86][START_REF] Kocher | Cross-linking of Fc gamma receptor IIa and Fc gamma receptor IIIb induces different proadhesive phenotypes on human neutrophils[END_REF][88] , GPCRs 89 and integrins 90,91 . The activation of one of these receptors induces the recruitment of cytoplasmic granules toward the targeted membrane, facilitated by the cytoskeleton remodeling (actin and microtubules). The rapprochement of granules and the targeted membrane is followed by the docking of granules with the inner surface of the targeted membrane in a step named priming. The priming of granules is followed by the fusion of the granules with the targeted membrane allowing the release of the contend of the granule 90,91 (Fig. 8).

Receptor stimulation. Different types of receptors (such as Fc receptors or integrins 86,90 ) have been described to be involved in the initiation of the process of degranulation but GPCRs are the most studied ones. Degranulation occurs after the stimulation of cell-surface GPCRs such as fMLP receptor or CXCR1 (IL-8 receptor) 92 (Fig. 8).

Granule translocation and docking. As described above, the stimulation of one of these receptors induces the dissociation of the 2 cytoplasmic subunits of the receptor ( and / subunits) which will activate important pathways allowing the translocation, the docking and the fusion of the granules with the plasma membrane. The first pathway activated after a GPCR stimulation is the activation of the GTPase Rac2, an important component for the antimicrobial activities of neutrophils [93][START_REF] Williams | Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency[END_REF][START_REF] Bokoch | Regulation of phagocyte function by low molecular weight GTPbinding proteins[END_REF] . One of its roles is the rearrangement of the actin and the microtubule cytoskeletons allowing the translocation of granules toward the targeted membrane. Several studies have shown the importance of Rac2 in the modulation of the cytoskeleton leading to the translocation and release of azurophil granules 73,[START_REF] Abdel-Latif | Neutrophil primary granule release and maximal superoxide generation depend on Rac2 in a common signalling pathway[END_REF][START_REF] Mitchell | Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling[END_REF] (Fig. 8). However, only azurophil granules exocytosis seems to be altered by Rac2 and SNAP-23 (Synaptosomal-Associated Protein) [START_REF] Martín-Martín | Co-expression of several human syntaxin genes in neutrophils and differentiating HL-60 cells: variant isoforms and detection of syntaxin 1[END_REF] . VAMP-2 and VAMP-7 are not located on the same granules, thus, VAMP-2 is known to be present at the surface of secretory vesicles, specific and gelatinase granules while VAMP-7 is present at the surface of azurophil granules [START_REF] Mollinedo | Role of vesicle-associated membrane protein-2, through Q-soluble N-ethylmaleimide-sensitive factor attachment protein receptor/R-soluble N-ethylmaleimide-sensitive factor attachment protein receptor interaction, in the exocytosis of specific and tertiary granules of human neutrophils[END_REF][100][101] . This difference could explain the fact that azurophil granules fuse more preferably with phagosome than others, as, in macrophages, VAMP-7 is known to play an essential role in the fusion between lysosomes and phagosomes 102 . Concerning the t-SNARE family, syntaxin 4, 6 and SNAP-23 have been described to be important for the degranulation of all types of neutrophils' granules 100,103 .

V-SNARE and t-SNARE are able to strongly interact together by protein-protein interactions in a model called "zippering" 104 which favor the close rapprochement of granules and the targeted membrane. This interaction will be stabilized by a protein called calmodulin, which binds to the v-SNARE/t-SNARE complex, inhibiting thereby the fusion of granules and membrane 105 (Fig. 8). "Primed" granules further require an additional signal to fuse with the target membrane. This signal consists of a strong increase of cytosolic calcium concentration, induced by the stimulation of GPCRs located at the surface of the neutrophils.

Granule fusion. Calcium has been described to play an important role in several neutrophil functions including superoxide production, adhesion, motility and degranulation [106][107][108][109][110] . Calcium is essential for the release of all types of neutrophil granules as calcium channel blocker inhibits the degranulation process 111 . Under basal conditions, cytosolic calcium concentration is maintained at a low level (100nM), which is 100 000 times lower than the extracellular concentration. This gradient is maintained by energy-driven pumps that extrude calcium ions out of the cell or sequester them intracellularly in organelles such as the endoplasmic reticulum 112,113 . Upon activation of GPCRs, an extremely rapid influx of cytosolic calcium ions into neutrophil cytosol (five to 10-fold increase 114 ) is induced and this event is correlated with the mobilization of neutrophil granules 115 . As seen before, GPCRs stimulation plays a role in early steps of the degranulation process (granule translocation and docking). Upon their activation, the released G-proteins will activate the membrane-associated phospholipase C (PLC) an enzyme capable to hydrolyze the inner membrane component phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG).

IP3 is a free second messenger which binds and activates IP3 receptors (IP3R) located in the membrane of the endoplasmic reticulum. IP3R is a Ca 2+ channel which, once activated, allows the passage of calcium from the lumen of the endoplasmic reticulum into the cytosol, leading to a modest increase of the cytosolic calcium concentration 116 . The decrease of the ER luminal calcium concentration is detected by calcium sensors STIM1 and 2 (Stromal Interaction Molecule) located in the membrane of the ER, which will activate Calcium Release-Activated Channels (CRAC) located in the plasma membrane 118 , leading to a strong influx of calcium into the cytosol 118 (Fig. 8).

The increase of the calcium concentration will be detected by two calcium-dependent proteins: calmodulin and synaptotagmin. Calmodulins prevent the fusion of primed granules with membranes and, conversely, synaptotagmins promote the fusion. The roles of calmodulins and synaptotagmins in neutrophil degranulation have not been well characterized, but have been extensively described in neurons. The increase of the calcium concentration induces the dissociation of calmodulin from SNAREs complexes 42 and the binding of synaptotagmins to SNARE complexes which mediate the rapprochement of the granules and the target membranes 119,120 . The interbilayer distance will reduce until a critical value of 9Å where the two opposing bilayers will fuse 121 . The content of the granule will thus be released in the extracellular medium (Fig. 8).

Fig 8: Neutrophil degranulation pathway

GPCRs activation induces the dissociation of the 2 cytoplasmic subunits of the receptor ( and / subunits) which activate different pathways essential for the degranulation. Firstly, the Rac2 pathway will be activated inducing the rearrangement of the cytoskeleton and favoring the translocation of granules toward the target membrane. The close rapprochement between granules and plasma membrane allows the priming of neutrophils granules through the interaction of v-SNAREs and t-SNAREs. This interaction is stabilized by calmodulin, inhibiting thereby the fusion of granules and the target membrane. Upon their activation, GPCRs will also activate the membrane-associated phospholipase C (PLC) which hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 binds and activates IP3 receptors (IP3R) located in the membrane of the endoplasmic reticulum. IP3R, a Ca 2+ channel, allows the passage of calcium from the lumen of the endoplasmic reticulum to the cytosol, leading to a modest increase of the cytosolic calcium concentration. The decrease of the ER luminal calcium concentration is detected by calcium sensors STIM1 and 2 located in the membrane of the ER, which activate Calcium Release-Activated Channels (CRAC) located in the plasma membrane, leading to a strong influx of calcium into the cytosol. The increase of the calcium concentration induces the dissociation of calmodulin from SNAREs complexes and the binding of synaptotagmins, thus mediating the rapprochement of the granules and the targeted membranes. The interbilayer distance will reduce until a critical distance where the two opposing bilayers will fuse, inducing the release of the granule content into the extracellular medium. Inspired from Hoenderdos et al 122 .

b) Sequential degranulation

Neutrophil degranulation has been associated with severe tissue damages on healthy areas [123][124][125] . Thus, this mechanism must be tightly regulated in order to prevent the release of cytotoxic component elsewhere. All types of granules rely on the cytosolic calcium concentration to fuse with the membrane. Thanks to this characteristic, neutrophils are able to selectively release each kind of granules separately. It has been described that each type of granules has a minimal calcium concentration threshold which has to be reach before their degranulation. Secretory vesicles have the lowest threshold stimulation followed by gelatinase, specific and azurophil granules which have the highest threshold. These different thresholds will allow neutrophils to release each granule at the right time. Secretory vesicles, thanks to their transmembrane receptors, have an important role for the adhesion and the extravasation of neutrophils through the vascular endothelium. It has been described

that the stimulation of neutrophils by cytokines present on the activated vascular endothelium was able to produce a calcium influx sufficient to release only the secretory vesicles. The firm contact between neutrophils and activated endothelium will increase the cytosolic calcium concentration, reaching the threshold of gelatinase which will induce the display of important surface integrin receptors having a role in the extravasation of the endothelium. This last kind of granule will also release matrix degrading enzyme which will facilitate the neutrophil migration toward the infectious site. The closer the neutrophil gets to the infectious site the more it will be stimulated with high concentration of inflammatory signals. This high level of stimulation will induce a strong calcium influx sufficient to release the two last kinds of neutrophil granules which contain the most cytotoxic component. Thus, the high calcium concentration threshold of specific and azurophil granules assures a release of their content in the close proximity of infectious foci and prevents a release in healthy area 106,107,126,127 .

E.

Neutrophil glycosaminoglycans

Neutrophil glycosaminoglycans (GAGs, or mucopolysaccharides) have been poorly investigated so far. However, it has been demonstrated that several proteins or cytokines released by neutrophils interact with exogenous GAGs present in the extracellular matrix or at the surface of endothelial cells. In addition, neutrophils express CD44 128 , which is the receptor of hyaluronic acid, belonging to the GAG family. After describing the GAG diversity, the interaction between neutrophil and extracellular GAGs will be described prior describing neutrophil GAGs and their subcellular localization.

The GAG family

GAGs are long linear polysaccharides consisting of repeating disaccharide units.

Members of the GAG family are hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate and keratan sulfate. At the exception of free hyaluronic acid, which is released through the membrane by transmembrane enzymes, GAGs are synthesized in the Golgi apparatus and are covalently linked to specific core proteins, forming proteoglycans such as aggrecan, syndecan, versican, biglycan or serglycin.

Proteoglycans are either secreted in the extracellular matrix, anchored to cell membranes or stored in secretory granules. GAG chains molecular weight may vary between 5 and 60 kDa, with the exception of hyaluronic acid chains which can reach very high molecular weight up to 20 000 kDa 129,130 .

GAGs are classified depending on the composition of their disaccharide unit. Thus, and D-N-acetylglucosamine (GlcNAc) 131 (Fig. 9).

An additional level of GAG diversity is associated with their level of sulfation, at the exception of hyaluronic acid which is a non-sulfated GAG. As an example, chondroitin sulfate may be non-sulfated, mono-sulfated (chondroitin-4-sulfate or chondroitin sulfate A and chondroitin-6-sulfate or chondroitin sulfate C), bi-sulfated Extracellular GAGs interact also with neutrophil granular proteins released upon neutrophil activation. Neutrophil elastase and cathepsin G have been shown to interact with heparan sulfate and chondroitin sulfate containing proteoglycan localized in neutrophil plasma membrane 143 , suggesting similar interaction may occur with extracellular GAGs. Neutrophil calprotectin (S100A8/A9) interacts with heparan sulfate and this interaction is required to bind to its receptor on endothelial cells 144 .

Further investigations are required to fully understand the physiological role of GAG interaction with neutrophil degranulated proteins. In several studies, it has been reported that GAGs may block their enzymatic activity (heparin blocks neutrophil elastase and cathepsin activities) 145 . In other studies, the degradation of extracellular GAGs such as heparan sulfate by neutrophil elastase or MPO-derived ROS has been shown to be essential for neutrophil migration towards inflammatory sites 146 .

Neutrophil GAGs

Neutrophil GAGs have been poorly characterized so far. In several studies published in 1950s-1960s, the presence of chondroitin sulfate and hyaluronic acid was evidenced using indirect and potentially non-specific detection methods. Since, neutrophil GAGs diversity, overall subcellular localization, synthesis pathways and potential secretion remain largely unknown.

a) First characterization

In the 1950s, the presence of GAGs in neutrophils was assessed through paper electrophoresis followed by staining with alcian blue, using purified commercial GAGs as positive control. With this technical procedure, Bassiouni and colleagues first stated that "white blood cells contain a substance, or substances, with properties similar to chondroitin sulfate", and not heparin 147 . In 1955, this result was confirmed using a similar approach "In our laboratory leucocytes have been found to contain appreciable quantities of a substance resembling chondroitin sulfate", and not heparin 148 . Although the result are somehow difficult to interpret, the presence of chondroitin sulfate C and potentially of hyaluronic acid were further reported by infrared analysis of "isolated pure acid mucopolysaccharides from human leucocytes" 149 . The presence of sulfated mucopolysaccharides in certain granules of rabbit neutrophil was demonstrated combining alcian blue and PAS staining of purified granules with S 35 -sulfate incorporation assays. It was shown that these granules were similar histochemically to azurophil granules and that their abundance was increased upon injection of an LPS to animals 150 . Fedorko granules containing sulfated-GAGs (presumably chondroitin sulfates) were "similar histochemically to azurophil granules" 150 . In guinea pigs, it was reported that sulfated GAGs were stored in immature neutrophil azurophil granules using a high-resolution staining method (cationic colloidal gold, CCG). However, these compounds were no longer detected in mature neutrophils 153 . Last, in human neutrophils, using specific immunostaining methods, it has been reported that heparan sulfate accumulates in primary granules, while chondroitin sulfate AC accumulates in tertiary granules 152 .

These results have not been confirmed in other studies and need to be confirmed with more recent analytical methods.

Neutrophil proteoglycans

With the exception of hyaluronic acid, GAGs are linked to core proteins to form complexes known as proteoglycans (Fig. 9). The highly anionic state of GAGs allows proteoglycans to act as a scaffold for numerous proteins. This functionality has been well documented in the case of proteoglycan produced by immune cells such as serglycin. Seglycin has been described to be released in large quantities by mast cells and macrophages but more importantly released in complex with proteases 154- 156 . This ability to bind proteases has been hypothesized to protect the enzyme toward proteolytic attacks and to facilitate the presentation of substrates to serglycinbounded proteases 157 .

Concerning neutrophils, serglycin has been described to be present in immature granules, playing a role in the packaging of granular component [158][159][160] . However, its role in mature neutrophils has not been characterized so far but serglycin knockout mice are highly susceptible to Klebsiella pneumoniae infection, confirming its importance in neutrophil antimicrobial function 161 .

Only few studies have focused on the release of proteoglycans by neutrophils. Thus, neutrophils have been described to actively release GAGs and proteoglycans in the supernatant after in vitro adhesion 162 or when exposed to micro-organisms 163 but this domain remains largely unexplored. Shigella specifically infects and colonizes the colonic mucosa. This pathogen is transmitted by the fecal-oral route (ingestion of water or food contaminated by human infected stools). An American study on human voluntary inmates has estimated that an amount of the order of 10 bacteria is sufficient to induce shigellosis 169 . This low infective dose is hypothesized to be due to the high acid resistance of Shigella.

II. Shigella virulence A. Introduction

Indeed, in order to colonize the colonic mucosa bacteria must survive the acidic conditions faced in the stomach. Shigella has developed several mechanisms aiming to persist in acidic condition. Shigella flexneri has been described to increase the production of protons secreting enzymes and to decrease the expression of the ATP synthase in order to prevent the internal pH from decreasing to lethal levels 170 . After crossing the stomach, Shigella follows the intestinal tract until reaching the colonic epithelium.

Shigella virulence is mediated by the expression of several secretion systems involved in the different stages of infection.

B. Shigella secretion systems

To date, Shigella virulence has been shown to be mediated by the expression of three secretory apparatus: Type 3 (T3SS), Type 5 (T5SS) and Type 6 (T6SS). These systems correspond to membrane protein complexes mediating the secretion of virulence factors or effectors, which are involved in the adhesion, invasion and colonization of the colonic mucosa or the subversion of the host immune response 171 .

Shigella's T3SS is the best characterized of the secretion systems, T6SS has recently been identified in Shigella sonnei 172 and the contribution of the T5SS to Shigella virulence is still poorly described.

T6SS

T6SS, expressed only by Shigella sonnei, has been shown to mediate the killing of commensal bacteria, by injecting toxic effectors. Thus, the disruption of the flora facilitates colonization of the intestine by Shigella sonnei 172 . T6SS is a membrane complex crossing the envelope of bacteria. The cytoplasmic part of the system is formed by a protein sheath surrounding a protein tube in which effectors are found.

This sheath has the ability to contract to expel the tube, containing the toxins, through the host's double membrane into the cytoplasm of an adjacent target cell 171 .

T3SS

The invasion of colonic epithelial cells by Shigella is essential for its virulence and is mediated by the type III secretion system (T3SS), which is expressed from genes located on the virulence plasmid pINV. These different genes (encoding components of the secretion machinery and effectors) are mostly grouped together in pathogenicity islands (PAI), i.e. a region of the genome bringing together several genes associated with pathogenicity. The expression of genes present in this 30 kb region is activated by specific environmental signals such as a rise in temperature to 37 °C173 or changes in partial pressure of oxygen (pO2) 174 .

T3SS is formed by a protein complex crossing the bacterial envelope into which a molecular syringe protruding from the extracellular side is inserted. This syringe is able to penetrate the membrane of the target cell to form a channel between cytoplasms of bacteria and host cells 171 . The T3SS allows the injection of effectors essential for the invasion of non-phagocytic cells by Shigella. Among the multitude of injected effectors, one family has been well studied: the Invasion Plasmid Antigens proteins (IPAs). This family is composed of 5 proteins named IpaA, B, C, D and H and have been described to have numerous functions such as the lysis of the endocytosis vacuole, the blocking of exocytosis, and therefore the secretion of proinflammatory cytokines, but also in the inhibition of apoptosis, hence promoting the intracellular replication of Shigella 175 .

T5SS

During the infectious process, Shigella secretes additional virulence factors via the type 5 secretion system (T5SS). Five subtypes of T5SS have been described (Va-e).

They are one of the simplest bacterial secretion systems since the secretion pore is formed by a domain of the secreted protein. This secretion system is involved in various bacterial functions such as virulence, survival and adhesion 176 . Shigella expresses the type Va system to excrete factors in the extracellular environment.

Proteins secreted by type Va have a common organization to know; a Sec signal peptide located at the N-terminal of the protein, which allows the passage of the internal membrane, a central domain called "passenger" which is the functional and secreted domain of the protein and a "translocator" domain located at the C-terminal mediating the translocation of the passenger domain through the outer membrane (Fig. 11A). After its synthesis, the polypeptide is taken over by the Sec-translocon which allows its passage from the cytosol to the periplasm. The signal peptide that allowed this first step is then cleaved and the protein associates with chaperones to prevent its folding before secretion. The passage of the outer membrane is made possible by a signal sequence located at the C-terminal of the protein which is recognized by the Bam (β-barrel assembly machinery) complex, responsible for the insertion of proteins into the outer membrane.

This complex will then insert the "translocator" domain into the outer membrane which folds into a β-barrel; forming a pore through which the passenger domain will pass the membrane. Once exposed at the surface, the passenger domain may remain attached to the translocator domain or may be cleaved, leading to the release of the passenger domain into the extracellular environment 177 (Fig. 11B).

IcsA belongs to the T5SS; it is a cell-surface virulence factor expressed by all Shigella strains. IcsA passenger domain is not cleaved and remains attached to the external surface of the bacteria (Fig. 11B). Once in the cytosol of the host cell, IcsA recruits the actin regulator N-WASP, which in turn recruits Arp2/3 to subsequently induce the polymerization of actin filaments. The polymerization of actin filaments mediates the motility of Shigella through a phenomenon known as "actin comet tail" 178 (Fig. 10). This capacity of hijacking the actin machinery allows Shigella to move in infected cells and facilitates cell-to-cell spreading 179 . (A) Proteins secreted by the type Va have a common organization to know; a Sec signal peptide located at the N-terminal of the protein which allows the passage of the internal membrane, a central domain called "passenger" which is the functional and secreted part of the protein and a "translocator" domain located at the C-part terminal and which has the role of translocating the passenger domain through the outer membrane (B) After its synthesis, the polypeptide is took over by the Sectranslocon allowing its passage from the cytosol into the periplasm. The signal peptide is then cleaved and the protein associates with chaperones to prevent its folding before its secretion. The passage of the outer membrane is made possible by a signal sequence located at the C-terminal of the protein which is recognized by the Bam (β-barrel assembly machinery) complex, responsible for the insertion of proteins into this membrane. This complex will then insert the "translocator" domain into the outer membrane which folds into a β barrel forming a pore through which the passenger domain will be able to pass through the membrane. Once exposed at the surface, the passenger domain can either remain attached to the translocator domain (eg: IcsA) or be cleaved and released into the extracellular environment (eg: SPATEs). was injected intravenously to young guinea pigs prior Shigella intrarectal challenge.

C. Shigella induces hypoxia during infection

After 8 hours of infection, animals were sacrificed, and colons were collected for immunofluorescence analysis. This experiment confirmed the massive recruitment of neutrophils toward infectious foci of Shigella but also revealed an intense hypoxic signal within the infected colonic mucosa hence demonstrating that hypoxia was induced during Shigella infection 190 . To go further, our team focused on the causes of hypoxia. First, using the hypoxia marker EF5, we reported that hypoxia was induced in neutropenic guinea pigs, strongly suggesting that neutrophils were not essential for hypoxia induction within Shigella infectious foci. Conversely, we have shown that a Shigella mutant strain defective for the aerobic respiration (cydAB) was no longer able to induce hypoxia in vivo, confirming the central role of the bacterial aerobic respiration in this process 191 .

Shigella spp, like other facultative anaerobic enterobacteria, are well adapted to low oxygen environments as they express both an aerobic and an anaerobic respiratory chain. Shigella may then use alternative electron acceptors, such as nitrate, in the absence of oxygen to produce energy. Shigella has developed several regulatory pathways to sense the microenvironment oxygen availability such as ArcA (Aerobic Respiration Control protein A) and FNR (Fumarate and Nitrate Reductase) 192 . FNR is a well described oxygen sensor used by facultative anaerobic bacteria. FNR is monomeric in the presence of oxygen but dimerizes in the absence of oxygen. The dimeric form of FNR binds to DNA and regulates the expression of many operonic genes. Thus, FNR pathway has been described to activate the expression of genes necessary for the anaerobic metabolism and to repress the expression of genes for the aerobic respiration 193,194 . In Shigella spp, the activation of this pathway in the absence of oxygen has been described to also modulate the expression of virulence genes. Hence, FNR activates the expression of acid resistances system enzymes 195 and prevents the secretion of Ipa via the T3SS 174 . In a FNR-independent manner, the absence of oxygen has been shown to enhance the expression of ShiA, a gene encoding for a factor that attenuates the intestinal inflammatory response in shigellosis by decreasing the recruitment of neutrophils 196 .

During Shigella infection, the infected tissue pO2 will globally decrease and will become heterogeneous: low within foci of infection and higher elsewhere.

Consequently, the interaction between Shigella and immune cells, including neutrophils, may occur under various pO2. Neutrophil viability and antimicrobial functions, which are highly dependent on the oxygen availability, may be modulated in this pathophysiological environment shaped by Shigella.

III. Adaptation of neutrophils to hypoxia A. Tissue oxygenation under basal conditions

Normoxia is defined by a normal level of oxygen in an organ. The partial pressure of oxygen (pO2) corresponding to normoxic conditions is organ-specific, according to its level of perfusion and its oxygen consumption rate. Oxygen is an important chemical element for the physiology of human cells and is, notably, essential for the mitochondrial respiration. The oxidative phosphorylation pathway consists of the production of ATP, induced by a transfer of electrons through the respiratory chain complexes located within the mitochondrial inner membrane: oxygen is the final electron acceptor 197 . In physiological conditions, the distribution of oxygen is heterogeneous in the human body (fluid, tissue, organs…) and usually measured in gas tension expressed in millimeter of mercury (mmHg). The pO2 of the atmospheric air at sea level is 160 mmHg (corresponding to 21% O2) 198 . Thus, pulmonary alveolus (13.1% 199 ), arterial blood (~ 13% O2) 200,201 or kidneys (9.5% O2) 202 are among of the most oxygenated areas of the human body whereas the superficial region of the skin 203 or the colonic lumen are almost devoid of oxygen [204][205][206] (Fig. 13).

Figure 13: Oxygen level in different human organs under basal conditions.

In physiological condition, a wide range of oxygen concentration exists throughout the human body. Thus, pulmonary alveolus (13.1% 199 ), arterial blood (~ 13% O2) 200,201 or kidneys (9.5% O2) 202 are ones of the most oxygenated area of the human body whereas the superficial region of the skin (1%) 203 or the lumen of various zones of the intestines are almost devoid of oxygen [204][205][206] .

B. Different types of hypoxia

In certain pathophysiological conditions, the local oxygen consumption exceeds the oxygen supply, leading to a decrease of the pO2, defining hypoxic conditions.

Hypoxia may affect the whole body (generalized hypoxia) or being induced locally (local hypoxia) 207 . Hypoxia may have several causes such as abnormal pulmonary gas transfer (systemic hypoxaemia), sterile inflammation or pathogen infection associated with a damaged vasculature and/or an increased metabolic activity from pathogens or host cells (inflammatory hypoxia or infectious hypoxia) 208 .

The characterization of the hypoxia sensing machinery in eukaryotic cells by William Kaelin, Peter Ratcliffe and Gregg Semenza was awarded by the 2019 Nobel prize of Physiology of medicine. In immune cells, the modulation of their functions by hypoxia is also a growing field as observed by the increasing number of published studies on NCBI including the terms "Hypoxia" and "Immune cells" (Fig. 14). All immune cells, including neutrophils, sense variations of oxygen availability and adapt their metabolism and functions accordingly 209 (Fig. 16). The modulation of immune cells by hypoxia is a growing field of research. This can be observed by the increasing number of published studies on NCBI including the terms "Hypoxia" and "Immune cells".

C. The metabolic adaptation of neutrophils to hypoxia

During their overall life cycle, neutrophils face mainly low pO2 from the bone marrow, to the blood circulation, to hypoxic inflammatory or infectious sites. The bone marrow, has been described to contain a low amount of oxygen, reaching 1.3% O2 in some areas [210][211][212] . These conditions have been hypothesized to minimize ROS-dependent oxidative damages 213 . Following their production in the bone marrow, neutrophils are released in the plasma fraction of the blood. Oxygen is mainly transported by red blood cells and its solubility coefficient is very low, resulting in a low concentration of soluble oxygen in the plasma (0.1-0.4%) 214 . In addition, our team has recently described that the plasma was able to reduce oxygen, due to the presence of acid ascorbic 215 .

Hypoxia is induced at sterile or infectious inflammatory sites, where neutrophils are recruited. During sterile inflammation, hypoxia has been observed in ulcers 216 , in several chronic inflammatory diseases 217,218 and within tumors. Indeed, due the intense metabolism of cancer cells and a non-optimal perfusion system, the oxygen tension inside the tumor may reach 2% O2 219,220 . Hypoxia has been additionally observed in various infectious diseases caused by viruses 221 , fungi 222 , protozoan 223 or bacteria 224 . The first hypoxic foci of infection caused by bacteria was observed within Mycobacterium tuberculosis granulomas, which are essential for the longlasting persistence of the pathogen 225 . The lack of oxygen within Mycobacterium tuberculosis granulomas was confirming by PET scan combined with hypoxic probes (nitroimidazole derivatives) 226,227 . Hypoxic foci were further reported in models of bacterial infection such as Salmonella typhimurium 228 , Shigella flexneri 190,191 and Escherichia coli 229 . In addition to bacterial foci, oxygen depletion was observed within biofilms formed by Staphylococcus aureus 230,231 and Pseudomonas aeruginosa 232,233 (Fig. 15).

Neutrophils are the most abundant immune cells infiltrating hypoxic tumors and inflamed or infected tissues [234][235][236][237][238][239][240] . Thus, from their production in the bone marrow to their recruitment toward inflammatory events, neutrophils have to face poorly oxygenated microenvironments and need to adapt their metabolism and their antimicrobial functions accordingly. Neutrophils are well adapted to low oxygen microenvironments. Neutrophils contain few mitochondria; they produce energy mainly through glycolysis rather than through the oxidative phosphorylation [238][239][240] . Accordingly, the culture of neutrophils, but also eosinophils, in low oxygen conditions has been shown to promote their survival [244][245][246][247][248] (Fig. 16). How neutrophils antimicrobial functions are modulated in these conditions remains poorly described, although several of the main functions are dependent on the oxygen availability (ROS production, NETs formation).

D. Impact of hypoxia on neutrophils antimicrobial functions

1.

Limitation of Reactive Oxygen Species (ROS) production

The production of ROS by neutrophils under hypoxic conditions is not well documented but is expected to be limited by the lack of substrate (oxygen).

Neutrophils have been described to contribute to hypoxia during inflammation, through the production of ROS from molecular oxygen during the oxidative burst 246 .

However, this ability to produce ROS seems to be transitory as some studies conclude on an inhibition by hypoxia of the respiratory burst activity. To note that this inhibition was not due to a regulation in the NADPH components suggesting that the reduction is mediated by a lack of available molecular oxygen 247,248 (Fig. 16).

However, a recent study showed that hypoxia was able to increase the production and the release of neutrophil mitochondrial ROS associated with the stabilization of HIF-1  .

Phagocytosis

Several groups have challenged the phagocytosis activity of neutrophils in hypoxic conditions and their conclusions were consistent. The phagocytosis of heatinactivated FITC-labeled opsonized streptococci 247 or opsonized zymosan 250 was unchanged in hypoxia (respectively 0.8% and 3% O2) compared to normoxia (21% O2). In accordance with these results, macrophage phagocytosis was also not affected by oxygen levels as low as 1% 251 (Fig. 16).

Inhibition of neutrophils extracellular traps (NETs) release

The release of NETs by neutrophils has been described to be highly dependent on ROS produced by the respiratory burst machinery (NADPH, myeloperoxidase) [252][253][254] .

The importance of ROS in this process is confirmed by the fact that ROS neutralizing agents inhibit the formation of NETs.

Thus, taurine (an amino acid which neutralizes HOCl) was shown to significantly reduce the release of NETs 255 . In addition, the presence of exogenous catalase (an enzyme which neutralizes H2O2) or the inhibition of endogenous catalase was able to completely inhibit NETs formation. A similar result was obtained in the presence of serum, confirming its potential antioxidant activity 256 . As mentioned before, ROS production is impaired in hypoxic conditions and in accordance with the importance of ROS for the production of NETs, their release has been described to be drastically reduced under hypoxia (1% O2) 257,258 (Fig. 16).

Stimulation of neutrophil degranulation

Hypoxia has been shown to stimulate the secretion of proteins in various cell types (immune or non-immune). Steiner et al. demonstrated that when rats were exposed to hypoxia, the degranulation of mast cells was stimulated 259 . It was confirmed in vitro that mast cells exposed to hypoxia (1% O2) degranulated higher amounts of IL-6 260 .

Similar results were obtained on eosinophils; hypoxia (0.8-3% O2) increased their capacity to degranulate IL-8 244,245 . In addition, the secretion of cytokines such as TNF-, IL-6 and IL-8 by macrophages is increased under hypoxia 261-263 (Fig. 16).

Concerning non-immune cells and more precisely epithelial cells or goblet cells, hypoxia has been shown to induce the secretion of many compounds such as interleukins, mucins or proteoglycans [264][265][266][267][268][269] .

The modulation of neutrophils degranulation by hypoxia has been investigated by several groups. Firstly, Tamura and colleagues focused on the impact of acute hypoxic events caused by Acute Respiratory Distress Syndrome (ARDS) on different functionalities of circulating neutrophils. Healthy subjects were desaturated until their blood O2 saturation reached 68%. Neutrophils were then isolated and stimulated with an inflammatory signal (fMLP) and the degranulation, as measured by elastase release, was challenged. In this study, the authors were able to conclude that the degranulation was enhanced in purified neutrophils following the hypoxemic event, however, cytokines secretions was unchanged 270 . This was thus confirmed by others studies; McGovern and colleagues were able to show a strong increase of the degranulation from primed and activated neutrophils cultured for 4 hours in hypoxia (0.8% O2) 247 . This observation was also described by Hoenderdos and colleagues.

The authors showed that hypoxia (0.8% O2) increases neutrophil degranulation regardless of the granule types. Thus, the authors showed an augmentation (two to six-fold) of the release of azurophil (elastase, myeloperoxidase), specific (lactoferrin)

and gelatinase (MMP-9) granule contents. When neutrophils were cultured in hypoxia, the neutrophil culture supernatants induced extensive airway epithelial cells detachment and death, as compared to supernatant from control neutrophils.

Importantly, the authors demonstrated that the effect of hypoxia on neutrophil degranulation is independent from the HIF pathway, which mediates most of signaling events induced by hypoxia 124 . Ong and colleagues investigated the relationship between hypoxia and neutrophil-driven pathology in tuberculosis infection (associated with the formation of highly hypoxic foci of infection). The authors showed that hypoxia increased the secretion of MMP-8 and elastase from neutrophils stimulated with supernatants of monocytes infected with Mycobacterium tuberculosis. These enzymes degrade important components of the extracellular matrix and the authors showed that the degradation of these components (collagen and elastin) was increased by hypoxia 257 .

The modulation of neutrophil extracellular degranulation by hypoxia has been reported in various models of study. However, a potential modulation of the intraphagosomal fusion has not been investigated and is currently unknown. Some teams have set up innovative approaches to quantify the intracellular fusion of granules with the phagosome, which could be useful to address this question and to study this phenomenon in relation with oxygen availability 271,272 . Hypoxia has been shown to modify myeloid cell antimicrobials functions. Hypoxia increases lifespan and the degranulation activity of neutrophils but decreases ROS production and inhibits NETs process. In the same way, hypoxia increases lifespan and degranulation of eosinophils. For macrophages, hypoxia has been shown to stabilize HIF-1 but phagocytosis was also affected. Hypoxia also increases the degranulation activity of mast cells. Among all myeloid cells, basophils functions under hypoxic conditions are the less studied and no modifications are yet determined.

a) Molecular basis of hypoxia enhanced degranulation of neutrophils (1)

The Hypoxia-Inducible Factor (HIF) pathway Hypoxia-Inducible factors (HIFs)-dependent pathways are the most described pathways involved in the sensing and response to variation of the oxygen availability.

HIFs are composed of two subunits: a cytosolic HIF-α subunit (HIF-1α, HIF-2α or HIF-3α) and a nuclear HIF-β subunit. In normoxia, HIF-α (which is continuously produced) is hydroxylated by oxygen-dependent enzymes named prolyl-4hydroxylases (PHDs). Hydroxylated HIF binds to the Von Hippel Lindau (VHL)

protein, leading to the linkage of the E3 ubiquitin ligase complex. HIF is then polyubiquinated and is directed to the proteasome for degradation. Thus, in normoxia the half-life of HIF-α is around 5 minutes 273 . Under hypoxia, HIF-α is not hydroxylated and is stabilized. HIF-α will then translocate into the nucleus where it will form a heterodimer with its sibling HIF-β. This heterodimerization results in an active Moreover, hypoxic incubation of neutrophils was not able to increase the content of granule proteins either at the mRNA or protein level, while the transcription of BNIP-2 (a known target of HIF pathway) was enhanced, confirming the engagement of HIF pathway in these experimental conditions 124 . Hence, the role of HIF in the hypoxiaenhanced neutrophil degranulation remains controversial and the involvement of other pathways need to be considered.

(

) 2
The PhosphoInositide 3-Kinase (PI3K) pathway

The PI3K (PhosphoInositide 3-Kinase) pathway is an intracellular signal transduction pathway implicated in numerous cell functions such as metabolism, growth or cell survival. This pathway is activated following the stimulation of cell surface receptors belonging to the RTK (Receptor Tyrosine Kinase) family. The activation of a RTK by its ligand (including growth factors, cytokines and hormones) leads to the recruitment of the PI3K to the intracellular domain of the receptor. The recruited PI3K phosphorylates PIP2 (phosphatidylinositol-4,5-bisphosphate) a specific phospholipid of the membrane. The PIP3 formed will then serve as a docking platform for the recruitment of downstream proteins of the pathway. Thus, this pathway participates to the recruitment, among others, of AKT, an important enzyme well described for its role in the promotion of the cell survival 276 .

The PI3K pathway has been described to play an essential role in most of neutrophil antimicrobial functions including chemotaxis, phagocytosis, ROS production and degranulation [277][278][279][280] . However, the link between hypoxia-enhanced degranulation and the PI3K pathway is poorly described. Confirming the fact that hypoxic upregulation of neutrophil degranulation was not dependent on HIF pathway, Hoenderdos and colleagues demonstrated that in the presence of a PI3K inhibitor (LY294002), the degranulation was completely abrogated, confirming the key role of the PI3K pathway in the regulation of the neutrophil degranulation. However, how hypoxia might modulate PI3K activity and thus neutrophil degranulation is currently unknown and need to be further investigated 124 .

Thesis objectives

The modulation of cells metabolism and functions by hypoxia is a growing field especially in the case of immune cells where hypoxia has been shown to affect the majority of all of them. Polymorphonuclear neutrophils are well adapted to hypoxic condition, indeed, these cells remain in poor oxygen environments from their production site to their recruitment at an inflammatory site. Hypoxia is well described during infectious inflammatory events notably in the case of several bacterial infections. Shigella, a human pathogenic Enterobacteriaceae responsible of bacillary dysentery due to the invasion and the destruction of the colonic mucosa, has been described to form foci of infection highly depleted of oxygen. The immune response following an infection by Shigella consists of a quick and strong influx of neutrophils, thus, the interaction between Shigella and neutrophils is characterized to happen in a very low oxygen concentration environment.

Neutrophils are specialized in the defense against invading bacteria and for that they are endowed of powerful antimicrobial mechanisms such as phagocytosis, ROS production, NETs release and degranulation of antimicrobial compounds. Set apart phagocytosis that doesn't seem to be affected by hypoxia, others neutrophil antimicrobial mechanisms tend to be modulated by the absence of oxygen. Thus, hypoxia has been described to decrease the production of ROS and to inhibit the mechanism of NETosis but, conversely, the absence of oxygen has been shown to stimulate the activity of degranulation. More precisely, several groups were able to show that hypoxia, in association with inflammatory signals, greatly enhances the release of proteins stored in different type of granules.

During my PhD, I investigated the interaction between polymorphonuclear neutrophils and the pathogenic enterobacteria Shigella. Our team has previously demonstrated that Shigella infection leads to the formation of hypoxic foci of infection 190,191 , raising the question of the adaptation of neutrophils to this pathophysiological microenvironment. We and others have confirmed that neutrophils survival is maintained in the absence of oxygen [241][242][243] , probably in relation to their glycolytic capacity [238][239][240] .

Introduction

Neutrophils are the first and most abundant immune cells recruited upon Shigella infection. Our group previously demonstrated that hypoxia is induced within Shigella foci of infection, raising the question of the adaptation of immune cells, including neutrophils, to these pathophysiological conditions. We and others have revealed that neutrophils are well-adapted to hypoxia and that excessive oxygen-exposure is consistently inducing neutrophil cell-death. Beyond these metabolic aspects, we aimed at better understanding which antimicrobial strategies are deployed by neutrophils in the absence of oxygen to control Shigella colonization and dissemination.

Since ROS production and NETs formation rely on the oxygen availability, we investigated the capacity of neutrophils to degranulate antimicrobial compounds. We demonstrated that neutrophil degranulation occurred in the absence of oxygen and was even favored, through a mechanism which remains elusive. We demonstrated that neutrophils release polymeric compounds named proteoglycofili (PGF) under anoxia. The first observation of PGF secretion was made possible by the development of innovative methods to manipulate neutrophils under controlled pO2 and the use of adapted methods for PGF imaging and biochemical characterization.

We have shown that, as opposed to NETs, PGF are DNA-free and are released by viable neutrophils. We demonstrated that the protein moiety of PGF is mainly composed of granular proteins (MPO, elastase, lactoferrin, cathelicidin, albumin).

PGF oligomerized structure is sustained by its saccharide moiety, which is mainly composed of glycosaminoglycans (GAGs), and more precisely chondroitin sulfate. It has been suggested in old article that GAGs were expressed by neutrophils although no further investigation was performed.

In Article I, we demonstrated that PGF release is induced under hypoxia and that PGF are released by neutrophils infected by Shigella. PGF degrade Shigella virulence factors (Ipa A-D, IcsA, Pic and SepA) and block its growth. Besides these antimicrobial activities, PGF appear to have a cytotoxic activity on human epithelial cells (Hep-2). We provided an in-depth characterization of neutrophil chondroitin sulfate and demonstrated its importance in PGF structure and antimicrobial activity.

Neutrophils degranulate GAG-containing proteoglycofili, which block Shigella growth and degrade virulence factors

Antonin C André

Results

Neutrophil degranulation is induced by the mobilization of azurophil (), specific (1), gelatinase (2) granules and secretory vesicles (). It was long considered that the mobilization of different types of granules was differential and that degranulated proteins diffused in the extracellular microenvironment.

Neutrophils are mainly glycolytic cells [4] and are well-adapted to low-oxygen microenvironments [5]. We previously demonstrated that neutrophil viability was maintained when cells were purified and cultured under anoxia [6,7], allowing the study of their physiology in vitro during extended periods of time. Applying this new method (Fig. 1a), we observed that a dense oligomeric material composed of polysaccharides or glycoproteins was released by neutrophils under anoxia, as revealed by alcian blue and periodic acid-Schiff (PAS) staining (Fig. 1b). According to its composition and ultrastructure, this extracellular network was named neutrophil proteoglycofili (PGF). The integrity of PGF, like other mucoidal biomaterials, was only preserved upon fixation with a Carnoy's solution, not with alternative fixative solutions, such as paraformaldehyde (Extended Data Fig. 1a). By SEM, we determined that the mean diameter of individual polymers composing PGF was 43.9

 20.3 nm (Fig. 1c). We confirmed in a kinetic study, that neutrophils secreting PGF under anoxic conditions remained viable (Extended Data Fig. 1b, until t=18h, p>0.5).

Interestingly, neutrophil PGF were labeled with the Myelotracker peptide, a specific marker of lactoferrin [8], both in vitro (Fig. 1d) and in vivo (Fig. 1e). Under basal conditions, lactoferrin is stored in neutrophil secondary (1) and specific granules (2): its detection within PGF strongly suggests that neutrophil degranulation sustains PGF formation. These results are consistent with previous reports showing that neutrophil degranulation is promoted under low-oxygen conditions [9]. In addition, PGF were detected in vivo within hypoxic foci of infection formed by the pathogenic enterobacteria Shigella within the guinea pig colonic mucosa [10] (Fig. 1e and Extended Data Fig. 1d). Our aim was to define the PGF biochemical composition and to investigate its structural organization and function. PGF were purified from neutrophils in anoxic cultures by differential centrifugations. The oligomeric organization of purified PGF was confirmed by TEM (Fig. 2a) and we demonstrated by mass spectrometry that PGF were mainly composed of neutrophil granular proteins (Extended Data Fig. 2a).

The most abundant PGF proteins were myeloperoxidase, elastase, lactoferrin, cathelicidin and albumin (Fig. 2b): their presence within PGF was confirmed by western blot analysis (using SDS-Agarose gel instead of SDS-Page gel electrophoresis, due to the PGF high molecular-weight, Extended Data Fig. 2b) and by immunofluorescence imaging in vitro (Fig. 2c). PGF proteins were found to be accumulated within Shigella infectious sites in vivo (Extended Data Fig. 2c). No modulation of the expression of the corresponding genes was observed in neutrophils secreting PGF (RNAseq analysis, Extended Data Fig. 2d). These results demonstrated that all types of neutrophil granules were mobilized during PGF formation, regardless to the hierarchy of the mobilization of granules [11].

Neutrophil PGF are released by viable neutrophils (Extended Data Fig. 1b), as opposed to Neutrophil Extracellular Traps (NETs) formation, which is associated with cell death, cell disintegration and the release of the nuclear chromatin [12]. The cell integrity of neutrophils secreting PGF was confirmed by SEM (Extended Data Fig. 3a). We demonstrated by immunofluorescence imaging that neutrophils secreting PGF have multilobulated shape nuclei (Fig. 1d). As opposed to PMA-induced NETs, no extracellular DNA was detected within PGF (Fig. 2d). This result was confirmed by quantitative imaging analysis, co-staining DNA with Dapi and PGF with Myelotracker (Fig. 2e, p=0.0000). PGF were consistently not susceptible to DNaseI but were degraded by Proteinase K (Extended Data Fig. 3b). To investigate the PGF antimicrobial activity, we studied its impact on the virulence and the growth of Shigella. The invasion of the epithelium by Shigella is mediated by an oxygen-dependent Type 3 secretion system (T3SS) [13]. Shigella exports additional virulence factors belonging to the Type 5 secretion system (T5SS) (SepA, Pic and IcsA) to colonize and disseminate within infected tissues [14,15].

Accumulation of PGF was observed in vivo within Shigella infectious foci (Fig. 1e)

and we demonstrated that PGF release was consistently induced in neutrophils infected by Shigella in vitro, regardless of the oxygen availability (Fig. 3a and Extended Data Fig. 4a). In more details, the interaction between Shigella and PGF was confirmed by 3D-reconstitution imaging, using the Imaris software (Fig. 3b)

We demonstrated that PGF degrade Shigella virulence factors secreted through the T3SS (IpaB, IpaC and IpaD) or belonging to the T5SS (IcsA, SepA and Pic) (Fig. 3c).

PGF proteolytic activity is inhibited in the presence of a protease inhibitor cocktail or upon heat inactivation (Extended Data Fig. 4b). It has been previously reported that Shigella virulence factors were degraded by elastase [3], which may be responsible of this PGF activity. We confirmed that, as control, PGF did not degrade important human proteins involved in the host antimicrobial response such as lactoferrin and the heavy and light chains of IgG and IgA (Extended Data Fig. 4c).

We demonstrated that PGF blocked Shigella flexneri 5a growth in vitro, in the presence of oxygen (Fig. 3d, 18h p<0.01). The integrity of bacteria appeared to be altered by PGF, as observed by phase contrast microcopy. PGF also blocked to the same extent E. coli K12, Shigella flexneri 2a and Shigella sonnei growth (Extended Data Fig. 5a, 18h p<0.001). We confirmed that the antimicrobial activity of PGF was maintained in the absence of oxygen, using Shigella flexneri 5a as a target (Extended Data Fig. 5b, +O2 p<0.001 vs -O2 p<0.01). The antimicrobial activity of PGF was not altered by heat-inactivation or by the addition of a cocktail of protease inhibitors (Extended Data Fig. 6, p>0.05). Further investigations will be required to explain these results. It must be noticed that PGF elicited adverse effects on human epithelial cells in culture. We demonstrated that when incubated at high yield (300 g/mL), PGF drastically changed the shape and integrity of epithelial cells in vitro (Extended Data Fig. 7a) and decreased cell adhesion (Extended Data Fig. 7b, p<0.001). As anticipated, based on these results, we demonstrated that epithelial cell death was consistently induced by PGF after 6h and 24h incubation (Extended Data Fig. 7c and7d, p<0.01). We further aimed at identifying PGF components which sustain their fibrillar ultrastructure, which cannot be supported by any protein composing PGF. It has been previously demonstrated in several past studies that neutrophils may produce glycosaminoglycans (GAGs), which are known to form long linear polysaccharides. It was reported using paper electrophoresis that chondroitin sulfate-like substances were detected in neutrophil total cell extracts [16-18]. It was further reported that chondroitin sulfate represents more than 60% of total neutrophil GAGs while heparan sulfate (30%) and hyaluronic acid (10%) represent minor GAGs fractions [19]. We hypothesized that GAGs may be present in PGF and may support their fibrillar ultrastructure. We confirmed this hypothesis by demonstrating, using RPIP-HPLC, that purified PGF contain chondroitin sulfate, not heparan sulfate (Fig. 4a). This result was confirmed by western blot analysis (Extended Data Fig. 8a). In more details only non-sulfated (non-S) and mono-sulfated (4S/6S) chondroitin were identified in PGF (Fig. 4a). It has to be noticed that chondroitin sulfate (non-sulfated or mono-sulfated) was also identified in NETs (Extended Data Fig. 8b). Under basal conditions, we observed that chondroitin sulfate was accumulated in the perinuclear region and in some vesicles (Extended Data Fig. 9a). The secretion of chondroitin sulfate by neutrophils in culture was confirmed in vitro by immunofluorescence imaging (Fig. 4b). We could reveal an accumulation of chondroitin sulfate in vivo, within Shigella foci of infection (Fig. 4c).

We further aimed to determine the contribution of chondroitin sulfate in PGF structure and antimicrobial functions (degradation of virulence factors and inhibition of bacterial growth). To proceed, PGF were treated with hyaluronidase type I-S from bovine testes, which cleaves β-N-acetylhexosamine-[1→4] glycosidic bonds of chondroitin sulfate and hyaluronic acid. We demonstrated that the oligomeric structure of PGF released by neutrophils in culture was altered by a hyaluronidase type I-S treatment (Fig. 4d), not by DNAse I (Extended data Fig. 9b). In addition, the treatment of purified PGF with this enzyme reverses its capacity to block S. flexneri 5a growth (Fig. 4e and f, p<0.001). Taken together, these results confirm the essential role of chondroitin sulfate in PGF structural organization and antimicrobial function. Since hyaluronidase type I-S targets also hyaluronidase, further experiments will have to be conducted with a hyaluronidase type IV, which cleaves specifically chondroitin sulfate, and the potential presence of hyaluronic acid within PGF will have to be determined. Together, these data suggest that neutrophil degranulation product defined as PGF are a well-structured compound composed of chondroitin sulfate and granular antimicrobial proteins. PGF are composed of a cocktail of granular proteins stored in all types of granules (, 1, 2 and ) suggesting that, during PGF release, the mobilization of neutrophil granules is not sequential but appeared to be simultaneous. The diffusion of the soluble content of mobilized granules may be limited and is anticipated to accumulate in the close vicinity of activated neutrophils within inflammatory sites. Chondroitin sulfate has a strong affinity for cytokines such as IL-8, CXCL1, CXCL5 [20,21] suggesting that PGF may play a central role in the formation of cytokines concentration gradients in vivo, favoring the chemoattraction of immune cells to inflammatory sites. PGF are an additional degranulation compounds which will have to be investigated together with granular proteins to better understand the immunomodulatory role of neutrophil degranulation [22].

We observed that PGF and NETs contain granular proteins and chondroitin sulfate at similar yields, although PGF do not contain DNA (Fig. 2d-e and Extended Data Fig.

3

) and are released by viable cells, as opposed to NETs formation which is associated with neutrophil cell-death. Further investigations are required to determine whether PGF release is a pre-requisite for NETs formation, considering that NET inducers such as PMA are well-known inducers of neutrophil degranulation [23]. The respective contribution of the chromatin and PGF in NETs structural organization will have to be additionally clarified. In addition, the presence of PGF in NETs raises the question of their contribution to important immunomodulatory functions of NETs, such as the promotion of metastases in cancer through their interaction with tumor cells [24,25], the stimulation of macrophage activation including cytokine production in atherosclerosis [26] or cyclic GMP-AMP synthase (cGAS)-dependent stimulation of an interferon response, since elastase has been identified as the main inducer of this pathway [27]. Finally, the protein composition of activated neutrophil exosomes is similar to PGF (elastase, lactoferrin, myeloperoxidase and MMP-9). Further investigations are required to determine the presence of chondroitin sulfate in these released particles and its potential contribution to their extracellular matrix degradation activity [28].

Our findings highlight the unrecognized and potentially underappreciated role of GAGs in association with granular proteins in neutrophil antimicrobial activity or in their capacity to stimulate other immune cells. Our work provides information for understanding the overall function of the neutrophil degranulation and provides original directions for the design of innovative compounds to target neutrophil in inflammatory diseases [29].

Methods

Human cell culture

Blood collection and neutrophil purification. Human blood samples were collected from the antecubital vein into tubes containing sodium citrate (3,8% final) as an anticoagulant.

Blood samples were rapidly transferred into an anoxic chamber (DG250, Don Whitley) for human neutrophil anoxic purification, using oxygen-free media, as described previously [7]. Briefly, whole blood samples were centrifuged at 600 x g for 20 minutes. Platelet rich plasma (PRP) was collected and centrifuged at 2650 x g for 20 min to form platelet poor plasma (PPP). Blood cells were resuspended in NaCl 0.9% and dextran sulfate (0.72%). After 30 min sedimentation, the neutrophilcontaining upper layer was centrifuged at 300 x g for 10 min. Pelleted cells were resuspended in 1 mL PPP and were subsequently separated on a 42% Percollplasma (GE Healthcare) gradient by centrifugation at 800 x g for 20 min. Neutrophils were collected from the pellet with remaining red blood cells, which were removed using CD235a (glycophorin) microbeads (Miltenyi Biotec). Purified neutrophils were centrifuged at 300 x g for 10 minutes. Pelleted cells were resuspended in PPP or RMPI 1640 culture medium supplemented with 10 mM Hepes and 3mM glucose [2],

according to experimental settings.

(03115879001, Merck) or hyaluronidase type I-S from bovine testes at 840U/mL (H3506, Merck) were added for 1h at 37°C prior fixation in a Carnoy's solution.

For high yield production of PGF, neutrophils were cultured in 75 cm 2 polystyrene culture flasks (Thermofisher Scientific) under anoxic conditions (DG250 workstation, Don Withley). After indicated time, neutrophil cultures were collected and centrifuged at 300 x g for 5 minutes to pellet cells. PGF were collected from culture supernatant by ultracentrifugation (46 500 x g for 50 min). Pelleted PGF were resuspended in 1mL PBS, as previously described for NETs purification [31]. Samples were sonicated before storage at -20°C.

For imaging analysis, neutrophils were allowed to secrete PGF onto glass coverslips ProLongGold® (Invitrogen) and allowed to dry overnight at RT. For electron microscopy imaging, fixed samples were rinsed twice in 0.1M phosphate buffer and dehydrated in a series of increasing ethanol concentrations (30 to 100%). Samples were dried via critical point drying (Leica EM CPD300, Austria). Afterwards cover glasses were mounted onto specifics stubs and coated with platinum, using a sputter coater (Q150T, Quorum Technologies, Kent, UK).

NETs. Neutrophil Extracellular Traps (NETs) formation was induced in RPMI 1640 medium (supplemented with 10mM Hepes, 3mM Glucose) in the presence of PMA (Phorbol 12-myristate 13-acetate, ref. P1585, Sigma-Amldrich) (0.1μM) during 4 hours at 37°C under atmospheric conditions (21%), as described previously [30,31].

Samples were processed similarly to PGF (for high yield production and for immunofluorescence/electron microscopy imaging).

Mass spectrometry

Prior to mass spectrometry, samples were prepared via reduction, alkylation, and trypsin digestion, as previously described [8]. After treatment with trypsin, digested peptides were transferred to a 10 kDa MWCO Vivaspin centrifugal concentrating column and centrifuged at 4.500 x g until the whole sample volume had been recovered. Digested tryptic peptides were then acidified to pH 2.0 using 1 % (v/v) trifluoroacetic acid, and subsequently purified using C18 ZipTips (Millipore, Durham, UK). Using an UltiMate 3000 Rapid Separation LC coupled to a LTQ Velos Pro mass spectrometer liquid chromatography tandem mass spectrometry (LC-MS/MS) (Thermo Fisher Scientific, Waltham, USA) was performed on peptide containing samples. Firstly, a pre-column of 20 mm x 180 μm was used to concentrate sample peptides. A 75 mm x 250 mm 1.7 μM ethylene bridged hybrid C18 analytical column (Waters, Manchester, UK) was then used to separate peptides in a gradient from 99% solution A (0.1% formic acid in ddH2O) and 1% solution B (0.1% formic acid in acetonitrile) to 25% solution B. Peptides were then automatically selected for fragmentation via a process of data dependent analysis.

The MASCOT search engine was used to analyze the processed mass spectrometry data. Data were searched against the SwissProt database using the following parameters: 1.2 Da mass accuracy for parent ions, 0.6 Da mass accuracy for fragment ions, one missed cleavage site was allowed for, 2 + and 3 + ions were selected, carbamidomethyl-cysteine was selected as a fixed modification, and methionine oxidation as a variable modification. The data generated from MASCOT was then analyzed in a semi-quantitative manner using Scaffold Proteome software (Proteome Software, Portland, USA), allowing for unique peptide counts of identified proteins to be determined.

Neutrophil glycosaminoglycans (GAG) analysis

GAG extraction and compositional analysis was performed as described previously [32]. Here, PGF and NETs were digested with 0.2mg/mL proteinase K (Merck) for 1h at 37°C. Potential GAG/protein complexes were dissociated by incubation with 2M NaCl (30 min at room temperature); protein denaturation was achieved by heating samples (100°C for 10 min). After centrifugation, supernatants were recovered, and concentrated to 50-100 µL in 50 mM Tris-HCl pH 7,5 supplemented with 50 mM NaCl and 2 mM CaCl2 (GAG digestion buffer) using a 3kDa cutoff Centricon® (Merck).

GAGs were then exhaustively digested into disaccharides by incubating samples for 48 h at 37°C, with either Chondroitinase ABC (Sigma-Aldrich, 100 mU) for CS/DS analysis, or with a mix of Heparinase I, II and III (Grampian enzymes, 10 mU each) for HS analysis. Compositional analyses were performed by RPIP-HPLC, as previously described 9 . Samples were applied to a C18 reversed phase column equilibrated in a H2O/acetonitrile (8.5%) buffer supplemented with 1.2 mM of ionpairing tetra-N-butylammonium hydrogen sulphate (TBA), then resolved using a multi-step NaCl gradient calibrated with authentic GAG disaccharide standards (Iduron). On-line post-column disaccharide derivatization was achieved by the addition of 2-cyanoacetamide (0.25%) in NaOH (0.5%), followed by fluorescence detection (excitation 346 nm, emission 410 nm).

Antibodies and fluorescent dyes

Lactoferrin was labelled with a rabbit polyclonal antibody (ref.

Flow cytometry

Cell viability was determined by staining with 0.01% propidium iodide (PI, ref. P4170, Marck) in PBS + 2 mM EDTA for 15 min at RT. PI only penetrates into and stains the DNA of non-viable cells. Fluorescence was measured using a FACSCalibur (BD Bioscience) flow cytometer, recording at least 10,000 events. Data were analysed with CellQuest Pro software (BD Biosciences) and were quantified using the FlowJo software (FlowJo, LLC).

Electrophoresis and western blot

SDS-agarose gel. To separate high molecular-weight protein complexes (as described previously [35]), samples were loaded in 0.8% agarose gel (containing 0.1% SDS). Sample migration occurred at 60V for 3h. The gel was then reduced for

RNAseq analysis

Neutrophil mRNA were purified from 5.10 7 neutrophils either freshly purified or cultured for 18h in anoxia in RPMI 1640 medium supplemented with 10 mM Hepes and 3 mM glucose. Cells were washed once with PBS and centrifuged for 5 minutes at 300 x g. Pelleted cells were lysed in 350 µL RLT buffer (Qiagen). Lysates were resuspended with a needle to increase the lysis efficiency. TRIzol (3 volumes) was added to samples followed by vortex and short spin. 1/5 of chloroform was added and samples were vortexed and incubated for 3 minutes at room temperature prior transferring into PLG heavy tubes. Samples were centrifuged for 15 min at 12.000 x g (4°C) and RNA-containing aqueous solution was transferred into a new tube. RNAs were purified using the RNeasy Mini Kit (Qiagen) and quantified with nanodrop and the quality was assessed with Tapestation. RNA samples were analyzed and sequenced by the GENOM'IC platform (Institut Cochin, Paris, France); RNA sequences were analyzed with the Deseq2 method. Results are averaged from three biological triplicates for each condition.

Guinea pig model of shigellosis

Young guinea pigs (Hartley, <150g) were previously infected intrarectally with 10 10

CFU exponentially grown S. flexneri 5a pGFP to evaluate acute infection (8h p.i) [10].

In this study, ascorbate-deficient guinea pigs were challenged intrarectally with 10 10 CFU exponentially grown S. flexneri 5a pGFP and animals were sacrificed 48h p.i.. In this new animal model of shigellosis, infection symptoms are more severe and the colonization of the colonic mucosa by Shigella is more prolonged [36]. After sacrifice, infected colons were collected and fixed in 3.3% paraformaldehyde (PFA) for 2 hours. Fixed colon samples were washed in PBS, incubated at 4ºC in PBS containing 15% sucrose for 90 min, followed by incubation in PBS with 30% sucrose overnight.

Samples were frozen in OCT (Sakura) on dry ice. 7 m sections were obtained using a cryostat CM-3050 (Leica). For immunofluorescent labelling, tissues were washed and permeabilized with PBS-Triton 0.1% and stained with primary antibodies in the presence of 1% BSA overnight at 4°C, washed three times in PBS-Triton 0.1%, prior incubation with secondary antibodies for 1h at RT. After three washes in PBS-Triton 0.1%, three washes in PBS and three washes in water, tissues were mounted with 10μL ProLongGold® (Invitrogen) and allowed to dry overnight at RT.

Neutrophil infection with Shigella

Neutrophils were seeded (2.10 6 cells/well) onto coverslips in RPMI 1640 medium supplemented with 10 mM Hepes and 3 mM glucose in 24-well plates. Cells were infected at a multiplicity of infection (MOI) of 20 with S. flexneri 5a pGFP grown until exponentially phase. Bacteria were spun onto cells by centrifugation at 300 x g for 10 min. After 1h, 2h and 4h incubation at 37°C, samples were fixed with Carnoy's solution for 2h at room temperature. For immunofluorescent labelling, coverslips were washed and permeabilized with PBS-Triton 0.1% and stained with primary antibodies for 1h at room temperature (RT), washed three times in PBS-Triton 0.1%, prior incubation with secondary antibodies for 1h at RT. After three washes in PBS-Triton 0.1%, three washes in PBS and three washes in water, coverslips were mounted with 10μL ProLongGold® (Invitrogen) and allowed to dry overnight at RT.

Epithelial cell viability assay

HEp-2 cells were seeded in 24-well plates onto coverslips at 5.10 5 cell/mL in 1 mL DMEM supplemented with 10 mM Hepes. After overnight growth, cells were washed with PBS and incubated with purified PGF diluted in a RPMI + 10 mM Hepes medium at two concentrations: low PGF corresponds to 30 µg/mL PGF and high [PGF] corresponding to 300 µg/mL PGF. For immunofluorescence imaging, treated cells were washed in PBS and fixed in PFA 3.3%; for flow cytometry analysis, cells were trypsinized and resuspended 300 µL PBS supplemented with 2mM EDTA and propidium iodide.

Bacterial growth assay

Overnight bacterial cultures were subcultured in fresh M9 medium (1:100 dilution) at 37°C until a OD600 of 0.3 was reached. 1 mL bacterial suspension was centrifuged at 8.500 x g for 3 minutes; pelleted bacteria were resuspended in 500 µL PBS. 1 L bacterial suspension was deposited onto a 1% M9-agar pad and covered with a square coverslip. Agar pads were incubated at 37°C to allow bacterial growth, which was assessed by widefield microscopy at t=0, t=4h and t=18h. When indicated, bacterial growth was performed in an anoxic chamber (-O2 condition). To evaluate the impact of PGF on bacterial growth, when indicated, 5 µg of purified PGF were deposited onto the agar pad prior to depositing the bacterial suspension. When indicated, PGF heat-inactivation may be performed (80°C, 10 min); the addition of a protease inhibitor cocktail may also be done following manufacturer instructions (Merck™ Complete™, Mini Protease Inhibitor Cocktail). Purified PGF were treated reached. The bacterial culture was centrifuged at 17.000 x g for 20 min and pellets were resuspended in 1mL PBS. Samples were sonicated (4 cycles of 30 seconds) and ultracentrifuged at 120.000 x g for 1h30 at 4°C. IcsA-containing pelleted membranes were resuspended in 1mL PBS and stored at -20°C.

PGF proteolytic activity

To assess PGF proteolytic activity, purified PGF (10μg

)

PGF vs NETs comparison by quantitative imaging analysis

In order to make a distinction between PGF and NETs by an imaging quantitative analysis, fixed samples were stained with Myelotracker-Cy5 and DAPI and imaged with a confocal microscope. Both Myelotracker and DAPI signals were normalized using the 1st and 99.9th percentile as minimum and maximum limits. The cell body region of interest (ROI cell) is computed using the Myelotracker channel. First, a

Difference of Gaussian filter with corresponding sigma 1=4 and  2=16 for PGF and  1=5 and 2=20 for NETs. Then, a threshold using Otsu approach is applied, followed by a morphological binary dilation with a structural element of radius r=10 for In Review I, we deciphered how pathogenic bacteria cope with exposure to various levels of oxygen when infecting human cells and tissues. This review highlights the selective advantage of facultative anaerobes. These pathogens have the capacity to grow in the presence of oxygen, leading to its local depletion: these pathogens have the capacity to grow and survive to this new microenvironment they contributed to shape. We reported that the induction of hypoxia during bacterial infection is observed in a wide range of infection models, both extracellularly and intracellularly.

We discussed additional bacteria classification criteria. In particular, we highlighted the importance of the aero-and anaerotolerance in addition to the capacity of pathogenic bacteria to use or not oxygen for growth.

Impact of pathogenic infections on tissue microenvironment and nutrient availability

Following infections, tissue microenvironments are largely modified, due to nutrient consumption by pathogens and to the release of catabolic products. Pathogenic infections may lead to a decrease in pH (acidosis), an increase in the partial pressure of carbon dioxide (pCO2) (hypercapnia), a glucose depletion or a decrease in the partial pressure of oxygen (pO2) (hypoxia). The development of metabolomic studies allowed the characterization of common features observed during bacterial or fungal infections, but also led to the identification of specific "metabolic signatures" [1]. In this review, we will focus on hypoxia, which is a pathophysiological condition in which the partial pressure of oxygen is lower than the one encountered in basal conditions (defined as normoxia). Infectious hypoxia is induced when pathogens colonize and disseminate within body compartments; either within biofilms or within foci of infections.

We will review current knowledge on metabolic adaptation of pathogens to changes in oxygen availability, which is required to survive and colonize host tissues. Then, we will describe the related causes of hypoxia and its impact on pathogen virulence and on the efficacy of the immune response.

In particular, the antimicrobial activity of macrophages and neutrophils requires oxygen (including reactive oxygen species (ROS) production). Host immune cells are able to sense and respond to hypoxia at a transcriptional level. Hypoxia inducing factors (HIF) are the main dimeric transcriptional regulators mediating adaptation of eukaryotic cells to poorly oxygenated microenvironments. In the presence of oxygen, the  cytosolic subunit of HIF is hydroxylated (by prolyl hydroxylases, PHDs), and subsequently degraded through the ubiquitin-proteasome pathway (involving the von Hippel-Lindau factor, VHL) (reviewed in [2]).

Under hypoxic conditions,  cytosolic subunits of HIFs are stabilized and translocate into the nucleus to dimerize with the  subunit forming a transcription factor regulating the expression of various HIF-dependent genes [2]. It should be highlighted that, in the presence of oxygen, several molecules such as lipopolysaccharide-induced succinate [3] or the transcriptional regulator GATA3 [4] may stabilize the  cytosolic subunit of HIFs. On the other hand, there are various cellular pathways, which mediate the adaptation of eukaryotic cells to hypoxia, that do not rely on HIF-dependent transcriptional regulation. These include processes such as mRNA stabilization [5] and cell receptor trafficking [6]. Furthermore, it has been reported that the HIF-dependent response to hypoxia may be impaired by other environmental cues such as hypercapnia [7].

Pathogens have been characterized and classified in regard to their ability to grow and survive in the presence and absence of oxygen. Facultative anaerobic bacteria express functional aerobic and anaerobic respiratory chains, supporting their unique ability to consume oxygen and to grow in its presence or absence. This dual respiratory capacity provides a selective advantage to these pathogens, since it allows them to evolve with changing oxygen levels encountered during infectious processes (reviewed in [8]). This selective advantage of facultative anaerobes is illustrated by their overrepresentation among antibiotic-resistant "priority pathogens" reported by the World Health Organization (including 8 among 12 pathogens: Pseudomonas aeruginosa, Enterobacteriaceae, Enterococcus faecium, Staphylococcus aureus, Salmonellae, Streptococcus pneumoniae, Haemophilus influenzae, Shigella spp.) [9].

Other pathogens, including fungi, have been shown to adapt to changes in oxygen availability during host colonization. Fungal infectious diseases may be caused by a wide variety of species, including Aspergillus fumigatus and Candida albicans, which represent a major threat to immunocompromised patients. In the case of Aspergillus fumigatus, germinated forms are obligate aerobes, while conidia can survive in the absence of oxygen.

Candida albicans may grow in the presence or absence of oxygen [10], suggesting that fungi may disseminate within hypoxic microenvironments.

Adaptation of pathogens to infectious hypoxia

Bacteria. Bacteria colonize host tissues through the formation of foci of infection or biofilms.

Pathogens form organized and heterogeneous communities in this context [11]. Hypoxia was reported within foci of infection formed in host tissues by various pathogenic bacteria such as Salmonella typhimurium [12], Shigella flexneri [13][14], Escherichia coli [15], and

Staphylococcus aureus [16] [17] [18] (Fig. 1). Most of pathogenic bacteria which have been shown to evolve within hypoxic foci of infection are facultative anaerobes. Until now, the formation of such foci by strict aerobes (e.g. Neisseria, Acinetobacter) has not been reported, with the exception of Mycobacterium tuberculosis. These bacteria forms specific foci of infection named granulomas, which are characterized by inflammatory mononuclear cell infiltrates. Hypoxia has been shown to contribute to bacterial persistence, which may be considered as a metabolic adaptation [19] [20] (Fig. 1). It has been shown that the virulence regulator PhoP, together with the hypoxia regulator DosR, mediate the hypoxia-inducible expression of genes involved in nitrogen metabolism [21]. This observation was, to some extent, intriguing since Mycobacterium tuberculosis is a strict aerobe.

Invasive bacteria form foci of infection to successfully colonize host tissues. Conversely, noninvasive bacteria remain on host organ surfaces and may form biofilms. In biofilms, a gradient of oxygen is established from the center to the edge (pO2=0 mmHg in the center) [22]. It was first shown in cystic fibrosis patients that hypoxia was induced within mucus layer of lungs, upon Pseudomonas aeruginosa colonization [23] (Fig. 1); the regulation of the expression of respiratory complexes in this context was further described (e.g. overexpression of cytochrome cbb3 oxidases, among other proteins) [24]. Direct measurements within Pseudomonas aeruginosa biofilms using microsensors have revealed that anoxia was induced in the center of the structure. Modulations in the expression of hypoxia-inducible genes were reported by transcriptomic analyses in Pseudomonas aeruginosa and Escherichia coli K12 [25]. Oxygenation heterogeneity in biofilms is a key parameter that has been incorporated in vitro in most reconstituted biofilm models (Staphylococcus aureus and Pseudomonas aeruginosa) [26] [27] (Fig. 1), which have been developed to better mimic pathophysiological conditions encountered in vivo [28] [29]. In response to hypoxia, the expansion of biofilms is enhanced (UPEC (Uropathogenic E. coli)

and Staphylococcus aureus [30] [31]). The adaptation of all major pathogenic facultative anaerobes to hypoxia is mainly driven by transcriptional regulators such as FNR (fumarate-nitrate reductase regulator) and the twocomponent system ArcAB (Aerobic Respiratory Control). FNR is transcriptionally active only in the absence of oxygen (through the stabilization of a [4Fe-4S]2+ cluster and its dimerization) and induces the expression of anaerobic respiration and fermentation enzymes, while causing repression of aerobic respiration enzymes. The ArcAB twocomponent system is also essential for the adaptation of bacteria to anaerobic growth. In the absence of oxygen, the kinase ArcB is activated by autophosphorylation and subsequently activates the response regulator ArcA by phosphorylation. Upon activation, ArcA represses the expression of genes encoding enzymes involved in the citric acid cycle (TCA) and the βoxidation of fatty acids. ArcA induces the expression of several enzymes that are important for bacterial adaptation to microaerobic or anaerobic environments (e.g. cytochrome bd oxidase (cydAB), pyruvate formate lyase (focA-pflB) and hydrogenase 1 (hya) (as reviewed elsewhere [32]).

Fungi. While fungi are generally considered obligate aerobes, there are certain interesting exceptions. For instance, it has been shown that Aspergillus fumigatus can tolerate oxygen levels as low as 0.1% [33]. It has also been shown that Candida albicans can grow anaerobically [10]. Candida albicans colonizes human epithelial surfaces, including the skin.

Under certain conditions, such as immunosuppression, invasion of epithelia occurs. It has been shown in vivo that Candida albicans can induce hypoxia in subdermal abscesses [34].

It has been similarly demonstrated that oxygen depletion occurs during Aspergillus fumigatus infection, together with an increase of ethanol production within infected tissues. Ethanol fermentation has been shown to be a key mechanism for Aspergillus fumigatus hypoxia adaptation and virulence. Notably, an alcohol dehydrogenase null mutant is less virulent in vivo [35]. Hypoxia sensing, which is facilitated by transcriptional regulators such as Upc2, Efg1 or Snf5 in Candida albicans, mediates the repression of the conidia to hyphae transition under low-oxygen conditions [36] [37]. In Aspergillus fumigatus, this transition is repressed by transcriptional regulators such as SrbA and SrbB under low-oxygen conditions [38]. In addition, it has been shown that non-coding RNAs (ncRNAs) such as Afu-182 [39] may also repress germination of Aspergillus fumigatus conidia.

Furthermore, a lack of oxygen blocks the germination of conidia and the formation of hyphae (given that this process is dependent on oxygen and water availability) (Candida albicans [40], Aspergillus fumigatus [41]). In Aspergillus fumigatus, mutations in genes encoding for components of the mitochondrial respiratory chain such as the alternative oxidase (aoxA) and cytochrome C (cycA) abolishes this transition, confirming the importance of the mitochondrial respiration for germination of conidia [42]. This metabolic adaption is essential for survival of aerobic fungi within oxygen-deprived infectious sites.

In conclusion, hypoxia is widely observed during bacterial and fungal infections, leading to a range of metabolic adaptations of pathogens to this pathophysiological condition. We will further describe the main causes of infectious hypoxia.

Pathogenic aerobic respiration is the main cause of hypoxia

Hypoxia can be detected using dedicated reporters (e.g. nitroimidazole derivatives) and was previously reported in a mouse colitis model [43]. In this model, hypoxia was shown to be promoted by neutrophils, which consume oxygen to produce ROS [44]. Both facultative anaerobic bacteria and fungi consume oxygen through the aerobic respiratory chain and mitochondria respectively, which are anticipated to sustain hypoxia.

Bacteria. The causes of hypoxia during bacterial infection are believed to be diverse and potentially bacteria specific. In several studies, bacterial aerobic respiration has been identified as a main cause of hypoxia, by inducing oxygen depletion locally, as described during Shigella flexneri infection [14]. It has been shown in Pseudomonas aeruginosa that impairing aerobic respiration limits biofilm growth (e.g. cbb3-type cytochrome c oxidases defective mutants [45]). Using sophisticated and powerful reaction-diffusion mathematical models, hypoxia was shown to be caused in vitro by both bacterial respiration and neutrophil activation within biofilms formed by Pseudomonas aeruginosa and Klebsiella pneumoniae [46] [47]. Further investigations are required to confirm the contribution of bacterial aerobic respiration in other models.

Fungi. In Candida albicans, mitochondrial respiration was shown to be the main cause of oxygen depletion in vitro [48], although this direct relationship has not yet been confirmed in vivo. In the case of Aspergillus fumigatus, a significant reduction in oxygen consumption was reported in vitro in the presence of KCN (inhibitor of the respiratory chain complex IV), confirming the importance of mitochondrial respiration in oxygen depletion in vitro [42].

Mitochondrial activity has been detected both in the conidia and hyphae of Aspergillus fumigatus [41], but their respective impact to oxygen depletion in vivo remains to be elucidated.

In conclusion, a wide range of virulent bacteria and fungi have the capacity to survive and grow in low oxygen conditions. Pathogens contribute to excessive oxygen consumption, mainly associated with the aerobic respiration, which contributes to the establishment of these pathophysiological conditions. In addition, hypoxia may also be promoted by a decrease of tissue oxygenation, due to blood vessel occlusion or coagulation [46]. We will further review how hypoxia sustains pathogen virulence, growth and spreading within host tissues in various models of hypoxic infections [49].

Hypoxia promotes pathogen virulence, tissue colonization and evasion of host immune response

Most life-threatening pathogens are facultative anaerobic bacteria or fungi, which can adapt to hypoxic microenvironments, via the formation of foci of infection, biofilms, or granulomas.

The heterogeneity of pathogen populations contributes to their ability to evade host immune response and to successfully colonize changing microenvironments (Pseudomonas aeruginosa [50] Yersinia pseudotuberculosis [11], E. coli [51], Aspergillus fumigatus [52]).

Conversely, current investigations aim at defining how the host immune response may induce heterogeneity in pathogen populations, by exerting a selective pressure (reviewed and discussed in [53]).

Bacteria.

The assumption that hypoxia promotes bacterial infection is supported by the fact that mutation of individual genes encoding for transcriptional regulators, such as FNR or ArcA, which mediate adaptation to hypoxia, abolishes bacteria virulence (fnr in Shigella, Salmonella, and pathogenic E. coli (ETEC), among others [54] [55] and arcA in Shigella and Vibrio cholerae [56] [57]).

Bacterial virulence relies on the ability of the bacterium to reach and subsequently, adhere to the host epithelial barrier (skin, lung or intestine). Subsequently, the capacity of bacteria to subvert important antimicrobial functions of immune cells is essential for their replication and propagation within the host. Invasive bacteria have the ability to cross epithelial barriers and disseminate within targeted organs, allowing the formation of foci of infection.

Bacteria can express various secretion systems -Type 1-9 secretion systems [58] -to export virulence factors. In addition, macromolecular complexes may be expressed to mediate bacterial adherence (pili or fimbriae) or motility (flagellum), allowing bacteria to reach and to colonize their specific niches. For instance, the type IV pili of Neisseria meningitidis are essential for the formation of microcolonies in blood vessels (reviewed in [59]). The importance of the flagellum can be seen in the case of Salmonella typhimurium, whereby it's essential for its motility on epithelial cell surfaces and for its virulence [60]. Host immune cells may additionally detect the presence of pathogenic bacteria by binding to components of the lipopolysaccharide (LPS) via TLR4 receptor and the peptidoglycan via peptidoglycan recognition proteins (PGRPs) or cytosolic Nucleotide-binding oligomerization domaincontaining protein (NODs) (reviewed in [61] and [62]).

The impact of oxygen level variation on the composition and function of bacterial virulence systems has not been properly investigated so far. Only few reports have described the oxygen-dependent regulation of bacterial secretion systems. We have previously shown that in the absence of oxygen, Shigella FNR represses the expression of minor components (Spa32 and Spa33) of the Type III secretion system (T3SS) which becomes inactive in these conditions in vitro [54] and in vivo, within hypoxic infectious sites [14] (Table 1). Salmonella expresses two T3SS: SPI-1 T3SS mediates bacterial invasion in non-phagocytic cells and SPI-2 T3SS is essential for Salmonella replication within vacuoles. It has been reported that the SPI-1 T3SS of Salmonella typhimurium is inactive in the absence of oxygen (Table 1)

and is under the control of alternative regulatory pathways involving small RNAs (FnrS and ArcZ) [63]. The oxygen-dependent regulation of Salmonella SPI-2 T3SS has not been investigated so far. T3SS inactivation under hypoxia suggests an essential role at early infection stages when bacteria have not yet formed foci of infection and when oxygen is still available.

More recently, it has been reported that the expression of the Type VI secretion system (T6SS) of Klebsiella pneumoniae is increased under low-oxygen conditions [64] (Table 1).

Further investigations are required to confirm this result under tightly controlled pO2 and in other pathogenic bacteria which express T6SS.

Proteins released through the Type V secretion system (T5SS) are either cell-surface exposed or secreted. They mainly belong to the autotransporter family and encompass proteases, lipases or non-fimbrial adhesins (> 40 proteins) [65]. Only few reports describe a putative oxygen-dependent regulation of T5SS. EibG is a member of T5SS in Shiga toxinproducing E. coli (STEC), which belongs to the immunoglobulin binding protein family and interact with and neutralize secreted IgA and IgG. The expression of EibG has been shown to be repressed in the presence of oxygen (shaking vs. static cultures) [66] (Table 1).

Conversely, the oxygen level does not seem to modulate the expression of Pic and Pet, which belong to the secreted serine protease autotransporters of Enterobacteriaceae (SPATE) family in Enteroaggregative E. coli (EAEC) [67] (Table 1). The presence of oxygen has been shown to be required for T3SS activation in Shigella (14) (54) and Salmonella (SPI-1 ( 63)). The expression of EibG, an adhesin belonging to the T5SS, in pathogenic E. coli STEC is activated in low-oxygen conditions (66). The expression of Pic and Pet (T5SS) of EAEC are not regulated by oxygen (67). It has been demonstrated that K. pneumoniae T6SS expression is repressed in the presence of oxygen (64). The impact of oxygen level variations on the expression of T1SS, T2SS, T4SS, T7SS, T8SS and T9SS has not yet been determined (N.D.).

In addition to secretion systems, oxygen may modulate the expression and the function of macromolecular complexes involved in bacterial virulence such as fimbria (or pili) and flagella. Bacterial adhesion to the host epithelial or endothelial cells is mediated by fimbrial adhesins such as Chaperone-Usher pili (CUP), Alternative Chaperone-Usher pili, Type I pili, Type IV pili or Nucleation-Precipitation pili [68].

It has been shown that hypoxia, encountered in the intestinal lumen, mediates a decrease in the expression of the colonization factor antigen I (CFA/I) adhesin genes (clustered in an operon), in an FNR-dependent manner. This repression is relieved in the presence of oxygen, leading to FNR inactivation [55]. Oxygen also induces the expression of the aggregative adherence fimbriae (AggR) of Enteroaggregative E. coli (EAEC) [67]. Similarly, expression of the UPEC type1 pili (FimA) is repressed under low-oxygen conditions [69].

Conversely, another study describes how anaerobic conditions induce the expression of the Sfp fimbriae in Enterohaemorrhagic E. coli (EHEC), speculating on the fact that low-oxygen conditions are encountered in vivo at the epithelium surface [70].

Bacterial adhesion and motility are also mediated by Type IV pili. It was demonstrated that in the absence of oxygen, the Type IV pili of Neisseria gonorrhoeae switch to a low-speed mode, limiting bacterial motility within hypoxic microenvironments [71]. The motility of Pseudomonas aeruginosa was also affected in the absence of oxygen, although the precise molecular mechanism involved in this process has not been identified [72]. To our knowledge, the impact of oxygen on flagellar expression or dynamics has not been studied.

Fungi. Humans are constantly exposed to fungal pathogens such as Candida albicans and

Aspergillus fumigatus, which may cause infection when immune defenses are weakened (immunocompromised patients). The underlying molecular mechanisms mediating the early steps of fungal infection remain elusive. Virulence of both Candida albicans and Aspergillus fumigatus relies on their ability to subvert host immune responses, which are mainly mediated by macrophages and neutrophils. β-glucans are the most abundant polysaccharides found in the fungal cell wall and are detected as a pathogen-associated molecular pattern (PAMP) by the immune cell receptor dectin-1, expressed on myeloid cells (macrophages, neutrophils and dendritic cells). The interaction between β-glucans and dectin-1 mediates phagocytosis, fungal killing and cytokine production [73]. The most abundant fungal β-glucan found in Candida albicans and Aspergillus fumigatus is composed of a β-1,3 glycosidic chain, with β-1,6 branches, hereafter named β-glucan (74). Significant changes in the cell wall structure of Candida albicans have been observed under hypoxia, including a reduction of β-glucan cell-surface exposure. Hypoxia-induced β-glucan masking impairs dectin-1-dependent myeloid cells anti-fungal activity and was shown to dampen macrophage and neutrophil response, promoting fungal colonization [34]. β-glucan masking was shown to be dependent upon both mitochondrial function and the activity of the cAMPprotein kinase A (PKA) [74]. Hypoxia blocks the germination of Aspergillus fumigatus conidia, limiting the formation of immunogenic hyphae during colonization. The low immunogenicity of Aspergillus fumigatus conidia was shown to be associated with cell-surface coating by hydrophobin, an immunologically inert protein. Hydrophobin forms a so-called "rodlet layer", masks β-glucan, and limits conidia detection by immune cells [75], which is anticipated to promote Aspergillus fumigatus capacity to colonize tissues.

Taken together, these studies highlight the diversity of strategies used by bacteria and fungi to optimize their capacity to colonize host tissues under low-oxygen conditions. We will further describe how bacteria dampen the antimicrobial activity of immune cells, by depriving them of oxygen within infectious sites.

Adaptation of immune cells to hypoxia

Under basal conditions, immune cells circulate under low oxygen conditions (pO2<1.2%) in the blood plasma [76] [77]. Subsequently, immune cells face various oxygen levels upon transmigration in tissues upon pathogen infection, depending on the organs; elevated oxygen level will be encountered in efficiently perfused organs (e.g. 13% O2 in pulmonary alveoli) compared to poorly oxygenated ones (e.g. 0% O2 in the colonic lumen) (43).

Under hypoxia, HIF-1 has been shown to maintain the survival of myeloid cells (neutrophil, basophils, eosinophils and macrophages) and to sustain their antimicrobial activity. As a consequence, mouse hif1 -/-neutrophils are defective for Streptococcus pneumoniae, Pseudomonas aeruginosa or Helicobacter pylori killing [78] [79] and hif1 -/-macrophages are defective for Streptococcus pneumoniae or Pseudomonas aeruginosa killing [80]. HIF-1 promotes macrophage control of Mycobacterium tuberculosis infection [81]. Interestingly, it has been shown that some pathogens target immune cell HIF-1 as a defense mechanism.

For instance, Pseudomonas aeruginosa alkyl quinolones promote HIF-1 degradation to survive in macrophages [82], similarly to Chlamydia pneumoniae [83].

Neutrophil viability is maintained under low-oxygen conditions [84]. While molecular mechanisms remain elusive, it has also been shown that hypoxia increases degranulation and the lifespan of both eosinophils and mast cells [85] (Fig. 2), facilitates their proliferation Intestinal epithelial cells are exposed to hypoxia under basal conditions (physiological hypoxia), leading to stabilization of the transcriptional regulator HIF-1 (43). When pathogens manage to cross the epithelial barrier, hypoxic foci of infection are formed, resulting in modulation of the function of host immune cells. HIF-1 stabilization mediates an increase of macrophage phagocytic capacity (80) (81). Some studies show an increase in the proliferation of mast cells and IL-6 secretion under hypoxia, although these results remain debated (86) (87). Neutrophil viability and phagocytosis capacity are maintained under hypoxia (78) (84) (93), probably in relation to their glycolytic activity. NETs release is impaired under hypoxia (92) or anoxia (93), Until now, the impact of hypoxia on basophil physiology has not be determined (N.D.); while it has been shown that similarly to neutrophils, the survival and degranulation capacity of eosinophils is promoted under hypoxia (85).

One important pending question is whether chondroitin sulfate production is constitutive or stimulated, and in the latter case, by which stimuli.

We could define that only mono-sulfated chondroitin sulfates were identified in neutrophil PGF. It will be important to determine the respective proportions of 4S and 6S forms and the importance of chondroitin sulfatation in its structural organization (chain length, interaction with granular proteins, etc..).

GAGs bind cytokines with a high affinity. Until now, this interaction has been reported between GAGs from the extracellular matrix (including heparan sulfate) and cytokines released by epithelial or immune cells. Our results strongly suggest that neutrophils may secrete both GAGs and cytokines upon activation at infectious sites.

Their interaction will have to be clarified to determine how neutrophils may contribute to the formation of cytokine concentration gradients on their own (see Article 1 discussion for more specific details).

PGF are composed of granular proteins pre-stored in all types of granules (, 1, 2 and ). First, this result challenges the well-accepted concept of the sequential granule mobilization, which is not observed during PGF secretion. In addition, the secretion of MPO (stored in granules) was thought to be barely secreted, circumventing ROS production in the phagolysosome. Here, we report that MPO is one of the most abundant granular proteins forming PGF. Further investigations will be required to define how extracellular MPO and membrane-bound NADPH oxidase work together to produce extracellular ROS, and their contribution to bacterial clearance.

We described a powerful bacteriostatic activity of PGF over gram negative bacteria.

The characterization of this activity over gram positive bacteria or bacteria capable to release extracellular hyaluronidases (Staphylococcus aureus, Streptococcus pneumoniae, Clostridium perfringens 281 ) could be interesting. Moreover, some pathogens, such as helminth parasites, are too large to be phagocytized 282 . Thus, the extracellular mechanism of PGF could be a powerful response to deal against large extracellular pathogens.

Neutrophils antimicrobial functions are frequently associated with side effects over host healthy areas. More precisely, neutrophils degranulation is responsible of strong tissue damages in several chronic inflammatory diseases. Cystic fibrosis (CF), a progressive genetic disease that causes persistent lung infections, is characterized by the presence of hypoxic mucopurulent masses where bacteria such as Pseudomonas aeruginosa can develop 233 . CF patients show a peribronchial neutrophil infiltration 283 and the presence of sputum highly depleted of oxygen can potentially induce the release PGF. It might be important to study the possible presence and role of cytotoxic PGF in the case of cystic fibrosis.

12 I

 12 Abbrevations

Fig 1 :

 1 Fig 1: Immunofluorescence and transmission electron microscopy images of human neutrophils. (A) Transmission electron microscopy image of a human neutrophil. Scale bar 5µm. (B) Immunofluorescence image of a human neutrophil: DNA is stained with DAPI (blue) and lactoferrin is stained with Myelotracker-Cy3 5 . Scale bar 5µm.

Fig 2 :

 2 Fig 2: Neutrophil production from hematopoiesis.

Fig 3 :

 3 Fig 3: Sequential formation of neutrophil granules during granulopoiesis.

  . These receptors, called Pattern Recognition Receptors (PRR), are classified in different types depending on their location (cytosolic or membrane-bounded) or their ligand specificity. For example, receptors belonging to the Toll-Like Receptors family (TLRs) are membrane-bounded receptors which recognize a broad range of conserved pathogen ligands. Thus TLR-2 recognizes peptidoglycan, TLR-4 recognizes LPS of gram-negative bacteria or TLR-7 is specific to single-stranded RNA of viruses. C-type Lectin Receptors (CLR) belong to another class of membrane-bounded PRRs which are specialized into the recognition of fungal pathogens (e.g. Dectin-1 will binds to β-

Fig 6 :

 6 Fig 6: Neutrophil phagosomal ROS production.

Figure 5 :

 5 Figure 5: Neutrophil Extracellular Traps (NETs). Human neutrophils stimulated for 4 hours with 0.1µM of PMA observed by (A) scanning electron microscopy and (B) confocal microscopy. For confocal microscopy imaging, DNA was detected with DAPI (blue) and lactoferrin (a protein linked to NETs) with myelotracker-cy3 (red). Scale bars 10µm.

  hyaluronic acid (Hyaluronan, HA) disaccharides are composed of D-glucuronic acid (GlcA) and D-N-acetylglucosamine (GlcNAc). Chondroitin sulfate (CS) disaccharides are composed of D-glucuronic acid and N-acetylgalactosamine (GalNAc). Dermatan sulfate (previously named chondroitin sulfate B, DS) disaccharides are composed of L-iduronic acid (IdoA) and N-acetylgalactosamine (GalNAc). Heparan sulfate (HS) disaccharides are composed of L-iduronic acid (IdoA) and D-N-acetylglucosamine (GlcNAc). Keratan sulfate (KS) disaccharides are composed of D-galactose (Gal)

(chondroitin- 2 , 6 -Fig 9 :

 269 Fig 9: Structural composition of glycosaminoglycans.

  Bacteria of the genus Shigella are strict human pathogenic Enterobacteriaceae. Shigella is the causative agent of the bacillary dysentery or shigellosis, characterized by hemorrhagic diarrhea caused by the invasion and the destruction of the colonic mucosa. Discovered in 1897 by Kiyoshi Shiga, Shigella are responsible of 188 million shigellosis cases annually, leading to 600,000 deaths 164 . Shigella are Gram-negative facultative anaerobes and non-sporulating bacteria. The genus Shigella comprises 4 species classified according to their serotype (defined by the LPS O-antigen structure): S. dysenteriae (15 serotypes), S. flexneri (19 serotypes), S. boydii (19 serotypes) and S. sonnei (1 serotype) 165 . These bacteria are closely related toEscherichia coli to such an extent that the genus Shigella forms a paraphyletic group in which certain strains of Escherichia coli are found. These two species would have diverged following the acquisition of a virulence plasmid pINV by Shigella 166 . It is a non-conjugative plasmid found in a single copy in all species of Shigella. Most virulence genes are located on the virulence plasmid; its loss induces Shigella avirulence167,168 .

Fig 10 :

 10 Fig 10: Immunofluorescence image of actin tails polymerized by Shigella. Immunofluorescence imaging of actin comets tail (indicated by white arrow) formed by Shigella. F-actin in red and Shigella in green. Scale bar 10µm. Image is extracted from the review of Welch et al 178 .

Fig 11 :

 11 Fig 11: Structure and secretion model of T5SS (type Va) through the bacterial envelope.

  Our team has investigated the impact of oxygen on Shigella virulence and on the host immune cell function. The immune response following colonization of the intestinal mucosa by Shigella results in a large influx of immune cells, mainly neutrophils, attracted to the infectious site due to the release of interleukin-8 from infected epithelial cells, forming a chemokine concentration gradient toward the infectious foci188 . The study of the interaction between Shigella and neutrophils in vivo has been impaired by the lack of a suitable animal model of shigellosis. Mice are not susceptible to Shigella infection, regardless of the inoculation route. Young guinea pigs develop shigellosis symptoms transiently upon intrarectal (not oral) inoculation of Shigella; this animal is now used by our team and others as an animal model of shigellosis 189 . This model greatly helped to characterize the infectious context of shigellosis and allowed to demonstrate that hypoxia is induced within Shigella foci of infection. The first observation of oxygen depletion following Shigella infection has been done by our team. In this study, nitroimidazole (a hypoxia marker)

Fig 12 :

 12 Fig 12: Colonic mucosa hypoxia induction during Shigella flexneri infection. Guinea pigs were injected with the hypoxia marker EF5 (nitroimidazole). (A) Uninfected or (B) infected colonic mucosa (Shigella flexneri pGFP, green) were labeled with an -EF5-cy3 antibody (red) and MUB40-Alexa405 (blue).Figure has been extracted from Arena et al 190 .

Figure 14 :

 14 Figure 14: Number of published studies per year including the terms "Hypoxia" and "Immune cells".

Figure 15 .

 15 Figure 15. Infectious hypoxia induction within bacterial foci of infections, granulomas, and biofilms. Hypoxia was induced by a wide range of pathogenic bacteria, during the formation of foci of infection (pathogenic E. coli, Salmonella, Shigella), granulomas (Mycobacterium. tuberculosis) or biofilms (Pseudomonas aeruginosa and Staphylococcus aureus). Targeted organs are indicated in the figure for each pathogenic bacteria infectious site.

Figure 16 .

 16 Figure 16. Hypoxia modulates immune cells viability and antimicrobial functions.

  transcription factor which binds to specific DNA sites named Hypoxic Response Elements (HRE) and induces the transcription of the downstream genes.In neutrophils, HIFs pathways have been shown to play an essential role in several responses to changing oxygen conditions. Notably, the deletion of HIF-1α in mouse neutrophils has been shown to drastically decrease the production of ATP from glycolysis274 . The same model has been later used to determine the role of HIF-1α in the survival of neutrophils under reduced oxygen tensions. In this study, the authors concluded that the presence of HIF-1α was essential for neutrophils to survive in the absence of oxygen 242 . The role of HIF-1α in the regulation of the degranulation process is more debated. Ong and colleagues demonstrated an increase of MMP-8 and MMP-9 secretion from neutrophils cultured for 24h in hypoxia and treated with DMOG, an inhibitor of PHD favorizing the stabilization of HIF-1α 257 . Conversely, Berger and colleagues reported a reduction of several antimicrobial peptides in the supernatant of neutrophil treated with 17-DMAG, a drug which inhibits the expression of HIF-1α 275 . However, Hoenderdos and colleagues bring a contrary idea about the role of HIF in the hypoxia-enhanced degranulation of neutrophils. In their study, the treatment of neutrophils with HIF stabilizers (DMOG and desferrioxamine) did not recapitulate the effect of hypoxia on the release of elastase or other granule proteins.

7

  Center for Microbes, Development and Health, Institut Pasteur of Shanghai/Chinese Academy of Sciences, Shanghai, China 8 University of Strasbourg Institute for Advanced Study (USIAS), Strasbourg, France 9 Institut Pasteur, Université de Paris, Inserm 1225 Unité de Pathogenèse des Infections Vasculaires, F-75015 Paris, unistra.fr or marteyn@pasteur.fr) Institut de Biologie Moléculaire et Cellulaire -Université de Strasbourg & Institut Pasteur, Paris (France) Summary paragraph Neutrophil degranulation plays a central role in their ability to kill pathogens but also to stimulate other immune cells [1-3]. Here we show that neutrophil degranulation, induced in hypoxia or upon Shigella infection in vitro and in vivo, leads to the release of polymers called neutrophil Proteoglycofili (PGF). PGF are mainly composed of granular proteins (myeloperoxidase, elastase, lactoferrin, cathelicidin, albumin) pre-stored in various types of granules, and chondroitin sulfate. PGF individual fibers have a diameter of 43.9  20.3 nm. They are secreted by viable neutrophils and do not contain DNA, as opposed to NETs which also contain granular proteins, chondroitin sulfate in addition to chromatin released upon neutrophil disintegration and cell death. We demonstrated that PGF block the growth of Shigella and other bacteria and degrade Shigella virulence factors. The degradation of the chondroitin sulfate polymers with hyaluronidases destabilizes PGF ultrastructure and abolishes its antimicrobial activity. Our results provide novel insights in the neutrophil degranulation process and open new doors for the investigation of PGF contribution to cytokines concentration gradient formation and adaptive immune cells activation. Further investigations are required to better appreciate the importance of this mechanism in infectious or inflammatory diseases.

Figure 1 .

 1 Figure 1. Neutrophil extracellular proteoglycofili (PGF) are released by viable cells and contain lactoferrin. (a) PGF are released by neutrophils purified and cultured for 18h, in an optimized culture medium (RPMI 1640 + 10 mM Hepes + 3 mM glucose), under anoxic conditions to maintain their viability [6]. (b) PGF are composed of neutrophil secreted glycoproteins, as revealed upon fixation in Carnoy's solution and staining with PAS and Alcian Blue (18h culture, -O2). Bars, 100 m. (c) Neutrophils secreting PGF were fixed and imaged by scanning electron microscopy (SEM). Bar, 300 nm. (See additional PGF and NETs SEM imaging in Extended Data Fig. 3a.). PGF diameter was measured by quantitative image analysis, using the Fiji Software (n=377). (d) Neutrophils secreting PGF (4h culture, -O2) were fixed in a Carnoy's solution and stained with Myelotracker-Cy5 (1 g/mL, magenta), a specific lactoferrin marker [8], and DNA was stained with DAPI (blue). Bars, 40 m. (e) PGF were detected in vivo with Myelotracker-Cy3 (red) within the guinea pig colonic mucosa infected by Shigella pGFP (green). DNA was stained with DAPI (blue). Bars, 40 m.

Figure 2 .

 2 Figure 2. PGF are composed of neutrophil granular proteins. (a) PGF were purified from neutrophil cultures by differential centrifugation. PGF ultrastructure was determined by TEM. Bar, 400 nm. (b) The most abundant proteins in PGF are lactoferrin, myeloperoxidase (MPO), elastase, cathelicidin (LL-37) and albumin, as determined by MS (n=3, see also Extended Data Fig. 2a-c). The sub-cellular localization of each protein is indicated (, 1, 2 or  granules). A representative PGF protein profile is shown on the left-hand panel (4-12% SDS Page gel, Coomassie staining). (c) MPO, cathelicidin, lactoferrin and elastase were immunodetected with specific antibodies within PGF secreted by neutrophils (4h culture, -O2, Carnoy's fixation). This result was confirmed by western blot (see Extended Data Fig. 2b) and in vivo upon Shigella infection (see Extended Data 2c). DNA was stained with DAPI (blue). Bars, 30 m. (d-e) PGF differ from NETs. PGF do not contain DNA, as shown by detecting extracellular DNA in neutrophil cultures (supernatant, sup.) upon PGF secretion (4h, -O2) as compared to NETs release (4h, +O2, +PMA). This result was confirmed by immunofluorescence imaging and quantitative image analysis. In both conditions, neutrophil cultures were fixed in a Carnoy's solution and stained with Myelotracker-Cy5 (magenta) and DAPI (blue). Bars, 40 m. Only extracellular NETs were co-stained with DAPI and Myelotracker-Cy5, as opposed to PGF (p=0.0000).

Fig. 3 .

 3 Fig.3. Shigella infection stimulates the secretion of neutrophil PGF, which bind to bacteria, degrade virulence factors and block bacterial growth. (a) Shigella infection induces neutrophil PGF secretion in -O2 conditions, as revealed by immunostaining. Similar results were obtained in +O2 conditions (see Extended Data Fig. 4a). Shigella flexneri 5a was labelled with a specific -LPS antibody (green), PGF were stained with Myelotracker-Cy3 (red) and DNA was stained with DAPI. Bars, 50 m. (b) 3D segmentation of DAPI, Shigella and PGF using the surface tool of Imaris 9.6 (BitPlane), and a blow-up with an oblique slicer on the PGF surface to observe Shigella located within PGF filaments. Bars, 30 m (top) and 10 m (bottom). (c) The degradation by PGF of Shigella virulence factors (IpaB, IpaC, IpaD, IcsA, SepA) or Shigella flexneri 2a (Pic) were observed after 1h incubation at 37°C, as revealed by Coomassie staining (SepA and Pic) or by western blot (IpaB, IpaC, IpaD and IcsA) (d) S. flexneri 5a growth inhibition by PGF was demonstrated by culturing bacteria on agar pads in the presence of PGF (37°C, 4h and 18h) Bar, 5µm. The confluence of bacterial cultures was quantified using the Fiji software (n=3, * indicates p < 0.05 and ** indicates p < 0.01(Student's T-test).

Fig. 4 .

 4 Fig.4. PGF oligomeric structure is sustained by chondroitin sulfate, which is essential for the bacteriostatic activity. (a) The presence of chondroitin/chondroitin sulfate in PGF was confirmed by RPIP-HPLC. Heparan sulfate, chondroitin di-or tri-sulfate were not detected. Results are expressed as percentage of total PGF GAGs (n=3). This result was confirmed by western blot (see Extended Data Fig. 8a) (b) Chondroitin sulfate was immunodetected with a specific antibody within PGF secreted by neutrophils (4h culture, -O2, Carnoy's fixation). PGF were stained with Myelotracker-Cy3 (white) and with an anti-chondroitin sulfate antibody (magenta); DNA was stained with DAPI (blue). Bars, 50 m. (c) PGF were immunodetected within the guinea pig colonic mucosa infected by Shigella flexneri 5a pGFP (green). PGF were stained with Myelotracker-Cy3 (white) and with an anti-chondroitin sulfate antibody (magenta); DNA was stained with DAPI (blue). Bar, 100 m. (d) PGF secreted by neutrophils were treated with hyaluronidase type I-S. (4h culture, -O2, Carnoy's fixation). PGF were stained with Myelotracker-Cy3 (white) and with an anti-chondroitin sulfate antibody (magenta); DNA was stained with DAPI. Bars, 50 m. (e) Shigella flexneri 5a was grown on agar pad with purified PGF treated or not with hyaluronidase type I-S. (37°C, 4h and 18h) Bar, 5µm. (f) The confluence of bacterial cultures was quantified using the Fiji software (n=3, * indicates p < 0.05 and ** indicates p < 0.01(Student's T-test).

  All participants gave written, informed consent in accordance with the Declaration of Helsinki principles. Peripheral human blood was collected from healthy patients at the ICAReB service of the Pasteur Institute (authorization DC No.2008-68), the Etablissement Français du Sang (EFS) Aquitaine-Limousin (authorization DC No. AC 2013-2025) or the Etablissement Français du Sang (EFS) of Strasbourg (authorization n°ALC/PIL/DIR/AJR/FO/606).

in polypropylene 24 -

 24 well plates (ref. 290-8324-03F, Evergreen Scientific). At each indicated time point, neutrophils were fixed in Carnoy's solution (60% ethanol, 30% chloroform and 10% glacial acetic acid) for 1 hour at room temperature. Sample fixation in PFA 3.3% was tested but did not allow the maintenance of the structural integrity of PGF. Samples were subsequently washed in PBS prior staining with alcian blue (ref. B8438, Merck) or Periodic Acid-Schiff (PAS) (refs. P7875 and 3952016, Merck). For immunofluorescent labelling, coverslips were carefully washed and permeabilized with PBS-Triton 0.1% and stained with primary antibodies for 1h at room temperature (RT), washed three times in PBS-Triton 0.1%, prior incubation with secondary antibodies for 1h at RT. After three washes in PBS-Triton 0.1%, three washes in PBS and three washes in water, coverslips were mounted with 10μL

  ab77780, Abcam)(1:500 dilution for WB and 1:100 for IF) or with RI-Myelotracker-cy3 or RI-Myelotracker-Cy5, as indicated, used at 1 μg.mL -1[8,33] (see www.myelotracker.com for more technical details). DNA was labeled with 4′,6-Diamidino-2-phenylindole (Dapi, ref. D9542, Merck) and was used at 1 μg.mL -1 . Actin was labeled with an Alexa Fluor™ 488 Phalloidin (ref. A12379, ThermoFisher Scientific) (1:40 final dilution for IF). Elastase was detected with a rabbit polyclonal antibody (ref. ab68672, Abcam) (1:500 final dilution for WB and 1:100 final dilution for IF). Cathelicidin was detected with a rabbit polyclonal antibody (ref. ab93357, Abcam) (1:1000 final dilution for WB and 1:200 for IF). Myeloperoxidase was detected with a rabbit monoclonal antibody (ref. A0398, Dako) (1:500 final dilution for WB and 1:200 final dilution for IF). Chondroitin sulfate was detected with a mouse monoclonal antibody (ref. C8035, Merck) (1:200 final dilution for WB and 1:100 for IF). Heparan sulfate was detected with a mouse monoclonal antibody (clone F58-10E4, ref: 370255-1, Amsbio) (1:100 final dilution for WB). Keratan sulfate was detected with a mouse monoclonal antibody (clone 373E1, ref: AMS.PRPG-KS-M01, Amsbio) (1:100 final dilution for WB). Murine anti-Shigella 5a lipopolysaccharide polyclonal antibody was used for immunofluorescent labelling (1:500 final dilution) [34]. IpaB was detected with a mouse monoclonal antibody (clone H16) kindly supplied by Dr. Armelle Phalipon, Institut Pasteur) (1:1.000 final dilution for WB). IpaC was detected with a mouse monoclonal antibody (clone K24) kindly supplied by Dr. Armelle Phalipon, Institut Pasteur) (1:1.000 final dilution for WB). IpaD and IcsA were detected with rabbit polyclonal antibodies kindly supplied by Dr. Claude Parsot, Institut Pasteur (1:10.000 final dilutions for WB). For immunofluorescence staining, rabbit and mouse secondary antibodies conjugated with an Alexa-568 fluorophore were used (refs. A11036 and A11031, ThermoFisher Scientific) (1:1.000 final dilution). For westernblot analyses, rabbit and mouse secondary antibodies conjugated with horseradish peroxidase were used (refs. 115-035-003 and 111-035-003, Jackson Immuno Research) (1:10.000 final dilution).

  15 min in a SSC (Saline-Sodium Citrate) buffer containing 10 mM DTT and washed in a SSC buffer without DTT. Protein transfer onto a PVDF membrane was achieved under vacuum (GE Healthcare™, VacuGene XL Vacuum Blotting Unit) at 50 mbar for 1h30. SDS-Page gel. 4-12% SDS-PAGE gradient gel (NuPAGE; ref NO0322PK2, ThermoFischer Scientific). Migration occurred at 200V for 25 minutes. When indicated, gels were stained with InstantBlue TM Coomassie (ref. ab119211, Abcam) or protein were transferred onto a PVDF membrane (at 350 mA for 180 min) (Mini TransBlot Electrophoretic Transfer Cell, Biorad). Agarose gel. The presence of DNA in cells and in purified PGF and NETs was determined by migrating samples in 1% agarose gel in the presence of propidium iodide, followed by UV revelation (Molecular Imager®, Biorad). Western blot. PVDF membranes were saturated with a solution of Tris buffered saline with tween 20 (TBST) solution (ref. 91414, Merck) containing 5% milk for 1h and incubated overnight with primary antibodies in TBST. Membranes were washed with TBST three times (5 min) and incubated with a secondary antibody for 1h in TBST containing 1% milk. After three additional washes (5 min) antibody binding was detected with chemiluminescence (ECL™ Prime Western Blotting Detection Reagent, GE Healthcare).

Extended Data Fig. 1 (Extended Data Figure 4 ( a )Extended Data Figure 5 (Extended Data Figure 6 Extended Data Figure 7 ( a )Extended Data Figure 8 ( a )Extended Data Figure 9 ( a )

 14a567a8a9a PGF and r=2 for NETs. The region of interest is then used to remove any cell related intensity for both Myelotracker and DAPI channels. The remaining structure in the image, corresponding to the extracellular polymeric structures, is then detected by thresholding the Myelotracker channel minus the cell areas with the Otsu method.Both Myelotracker and DAPI intensities are collected for each pixel belonging to the polymeric structures. The distribution of Myelotracker and DAPI signal intensities was displayed for both PGF and NETs. A linear regression was used to compare the relationship between Myelotracker and DAPI signal intensities on both samples. A significant difference between PGF (0.0751 ± 0.009) and NETs (0.361 ± 0.009) was observed.Statistical analysisAllstatistical analyses have been performed with the Prism 9 software (GraphPad software, USA). Comparisons with two groups were supported with Student paired or unpaired-t tests, as indicated. When p-values for the significance of pairwise comparisons were calculated and displayed on figures, we used the following convention: ns not significant; p ≥ 0.05;*p < 0.05 & p ≥ 0.01; ** p < 0.01 & p ≥0.001; *** p < 0.001 a) Neutrophils cultured for 4h in anoxia were fixed either with Carnoy's solution or PFA 3.3%. Samples were labeled with Myelotracker-Cy3 (red) and DAPI (blue) and imaged by confocal microscopy. Bars, 10 µm. (b) Kinetic study of PGF secretion by neutrophils (0, 4h and 18h, -O2 conditions) in RPMI 1640 medium supplemented with 10 mM Hepes and 3 mM glucose. Samples were stained with alcian blue. Bars, 80 µm. (c) The viability of neutrophils secreting PGF (see (b)) was assessed by flow cytometry using propidium iodide (PI) staining. 'ns' indicates p>0.05 (Student's T-test, n=3) (d) As a control of Fig. 1e, non-infected guinea pig colonic mucosa was stained with DAPI (blue) and Myelotracker-Cy3 (red) and imaged by confocal microscopy. Extended list of proteins identified in purified PGF by mass spectrometry analysis, in complement to data presented in Fig. 2b. The subcellular localization of each protein is indicated (, 1, 2 or  granules) in addition to the number of unique peptide (1, 2-3 or >4) identified in each biological replicate (n=3) (b) PGF were separated on a SDS-PAGE gel (4-12%) or on an agarose gel (0.8%) and stained with Coomassie or transfer onto a PVDF membrane for western blot analysis, as indicated. The presence of myeloperoxidase, elastase, lactoferrin, cathelicidin (LL-37) and albumin in PGF was confirmed by western blot. (c) The accumulation of PGF most abundant proteins in vivo within Shigella foci of infection was confirmed by immunodetecting cathelicidin, elastase or myeloperoxidase (MPO) with specific antibodies (magenta) in the guinea pig colonic mucosa infected with Shigella flexneri 5a pGFP (green). Samples were additionally stained with DAPI (blue) and Myelotracker-Cy3 (red). Images were acquired by confocal microscopy. Bars, 20 m. (d) The mRNA expression profiles of naïve neutrophils and neutrophils secreting PGF (18h culture under -O2 conditions) were compared by RNAseq analysis (n=3). Results for lactoferrin, lysozyme, cathelicidin, elastase, cathepsin G and myeloperoxidase are highlighted in yellow. Extended Data figure 3. (a) The morphology of neutrophils secreting PGF (4h culture, -O2 condition) and neutrophils releasing NETs (4h culture, +O2 condition, +PMA) were compared by scanning electron microscopy (SEM) at x4.000, x10.000 and x25.000 magnifications. Bars, 1 m. (b) The susceptibility of PGF to proteinase K treatment and not DNAseI was confirmed by immunofluorescence. Neutrophils were cultured for 4h in -O2 condition and further treated with proteinase K or DNaseI for 1h at 37°C. Samples were fixed with a Carnoy's solution and labeled with DAPI (blue) and Myelotracker-Cy5 (magenta). Samples were imaged by confocal microscopy. Bars, 20µm. Shigella infection induces neutrophil PGF secretion in +O2 condition, as revealed by immunostaining. Similar results were obtained in -O2 condition (see Fig. 3a). Shigella flexneri 5a was labeled with a specific -LPS antibody (green), PGF were stained with Myelotracker-Cy3 (red) and DNA was stained with DAPI (blue). Bars, 50 m. (b) PGF proteolytic activity was abolished in the presence of a cocktail of protease inhibitors or upon heat-inactivation (control of data presented in Fig. 3c). Treated PGF were incubated for 1h at 37°C with SepA or Pic, which were separated on SDS-Page gel and stained with Coomassie. (c) The stability of human lactoferrin and immunoglobulins (IgG and IgA heavy/light chains) in the presence of PGF was confirmed after 1h co-incubation at 37°C. Proteins were separated on SDS-Page gel (4-12%) and stained with Coomassie. a) E. coli K12, Shigella flexneri 2a and Shigella sonnei growth inhibition by purified PGF was demonstrated by culturing bacteria on agar pads in the presence of PGF (37°C, 4h and 18h). Bars, 5 µm. The confluence of bacterial cultures was quantified using the Fiji software (n=3, * indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001, Student's T-test). (b) The inhibition of Shigella flexneri 5a growth by PGF is comparable in the presence or in the absence of O2. Bacteria were cultured with PGF on agar pads (37°C, 4h and 18h). Bars, 5 µm. The confluence of bacterial cultures was quantified using the Fiji software (n=3, * indicates p < 0.05, and *** indicates p < 0.001, Student's T-test). PGF heat-inactivation or the addition of a cocktail of protease inhibitors did not affect PGF inhibition of E. coli K12 growth. Bacteria were cultured with pre-treated PGF on agar pads (37°C, 4h and 18h). Bars, 10 µm. The confluence of bacterial cultures was quantified using the Fiji software (n=3, 'ns' indicates p > 0.05, Student's T-test). The cytotoxic activity of purified PGF was assessed by incubating human epithelial HEp-2 cells with purified PGF for 6h or 24h. Low [PGF] corresponds to 30 µg purified PGF/mL and High [PGF] corresponds to 300 µg purified PGF/mL. The impact of PGF on Hep-2 cell morphology was assessed by immunofluorescence. Treated cells were fixed in PFA 3.3%. Actin was stained with phalloidin-FITC (green) and DNA was stained with DAPI (blue). (b) The adherence of treated cell was assessed by quantifying the confluence of cell cultures using the Fiji software (n=3, *** indicates p < 0.001, Student's T-test). (c) The viability of Hep-2 cells upon PGF treatment was assessed by flow cytometry using a PI staining. Cells were treated as indicated in (a) and were trypsinized prior analysis. (d) Percentage (%) of cell death corresponding to the proportion of PI-positive cells in (c) (n=3, ** indicates p < 0.01, Student's T-test). The presence of chondroitin sulfate or heparan sulfate in purified PGF was analyzed by western blot using specific antibodies, after electrophoresis on 0.8% agarose gel or 4-12% SDS-Page gel. (b) The presence of chondroitin/chondroitin sulfate in NETs was confirmed by RPIP-HPLC. Heparan sulfate, chondroitin di-or trisulfate were not detected. Results are expressed as percentage of total NETs GAGs (n=3). Freshly purified naïve neutrophils were fixed with PFA 3.3%. Chondroitin sulfate (magenta) and lactoferrin (white) were immunodetected with specific antibodies. DNA was stained with DAPI (blue). Bars, 20 m. (b) PGF secreted by neutrophils was treated with DNAseI. (4h culture, -O2, Carnoy's fixation). PGF were stained with Myelotracker-Cy3 (white) and with an anti-chondroitin sulfate antibody (magenta); DNA was stained with DAPI. Bars, 50 m. Title: The selective advantage of facultative anaerobes relies on their unique ability to cope with changing oxygen levels during infection Introduction Bacteria are classified based on their ability to survive and/or grow in the presence or in the absence of oxygen. Using this criteria, bacteria were classified in five different groups: strict aerobes which require oxygen to grow (e.g. Neisseria), strict anaerobes which do not use oxygen and do not grow in its presence (e.g. Clostridia), aerotolerant anaerobes which do not use oxygen to grow but are insensitive to oxygen exposure (e.g. Lactobacilli), microaerophillic bacteria which grow only in low levels of oxygen (e.g. Helicobacter pylori or Campylobacter jejuni) and facultative anaerobes which can grow either in the presence or the absence of oxygen (e.g. Shigella or Escherichia). The unique capacity of facultative anaerobes to grow in changing oxygen conditions relies on the expression of an aerobic respiratory chain and an anaerobic respiratory chain, providing to these bacteria an underestimated selective advantage during infection. Facultative anaerobes are consistently overrepresented in the World Health Organization (WHO) antibiotic-resistant priority pathogens list (8/12) and most bacterial infections are caused by facultative anaerobes. Facultative anaerobes are well adapted to conditions encountered in the host and they may colonize a wide range of microenvironments.

Title:

  The battle for oxygen during bacterial and fungal infectionsIntroductionBacterial and fungal infections are associated with deep modification of the tissue microenvironments due to the consumption of important nutrients and to the release of catabolic products by pathogens. These modifications include the decrease of pH (acidosis), the increase of the partial pressure of carbon dioxide (pCO2) (hypercapnia), the glucose depletion or the decrease of the partial pressure of oxygen (pO2) (hypoxia).In Review II, we focused on both bacteria and fungi strategies to induce metabolic adaptation mediating their capacity to survive in the absence of oxygen. In addition, we reviewed the consequences of hypoxia on the modulation of their virulence mechanisms and strategies. It appears that transcriptional regulation plays a key role in bacteria and fungi adaptation to hypoxia. Fungi have an additional capacity to modulate the conidia to hyphae transition in response to changes in oxygen availability. Persistence of fungi in such hostile microenvironment is often associated with a repression of conidia germination (which requires water and oxygen).Comparing bacteria and fungi provides to the reader a broader view of adaptive responses of pathogens following hypoxia. Review II also focuses on the hypoxic modulation of the host immune response which have developed alternative strategies to circumvent pathogen dissemination in the absence of oxygen. Important antimicrobial responses are oxygen-dependent such as neutrophil ROS production or NETs formation. How the immune response remains efficient in the absence of oxygen is an important unanswered question, which has been investigated in Article I.

Figure 1 .

 1 Figure 1. Infectious hypoxia within bacterial foci of infections, granulomas and biofilms Hypoxia is induced by a wide range of pathogens (8), during the formation of foci of infection (Salmonella, Shigella, pathogenic E. coli) (12) (13) (14) (15), granulomas (Mycobacterium. tuberculosis) (20) (21) and biofilms formed by bacteria (Pseudomonas aeruginosa and Staphylococcus aureus) (16) (17) (23) or fungi (Aspergillus fumigatus, Candida albicans (10) (34) (35). Targeted organs are indicated in the figure for each pathogenic infectious site.

Figure 2 .

 2 Figure 2. Hypoxia modulates the host cell's viability and antimicrobial functions during intestinal bacterial infections

Table 1 : Protein composition of neutrophil granules.

 1

  1,2 , Mélina Siegwald 1 , Vanessa Paul 1 , Caroline Ridley 3 , Isabelle

	Svahn 4 , Stéphane Rigaud 5 , Jean-Yves Tinevez 5 , Romain R Vivès 6 , Philippe J
	Sansonetti 7 , David J Thornton 3 , Benoit S Marteyn 1,8,9 *
	1 Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire, Architecture
	et Réactivité de l'ARN, CNRS UPR9002, F-67000 Strasbourg, France

2 Université de Paris, 5 Rue Marie-Andrée Lagroua Weill-Hallé, 75013 Paris, France 3 Wellcome Trust Centre for Cell-Matrix Research and the Lydia Becker Institute of Immunology and Inflammation, School of Biological Sciences, The University of Manchester, Manchester, UK. 4 University of Bordeaux, CNRS, INSERM, Bordeaux Imaging Center, BIC, UMS 3420, US 4, F-33000 Bordeaux, France 5 Institut Pasteur, Imagopole 28 rue du Dr Roux, 75724 Paris Cedex 15, France 6 Institut de Biologie Structurale, CEA Grenoble

Table 1 : Oxygen-dependent regulation of the activity of secretion systems of Gram- negative bacteria

 1

	Secretion	T1SS	T2SS	T3SS	T4SS		T5SS		T6SS	T7SS	T8SS	T9SS
	system					Proteases	Lipases	Adhesins
	O2-	N.D.	N.D.	activation	N.D.	No	N.D.	repression	repression	N.D.	N.D.	N.D.
	dependent					regulation
	regulation
	Bacteria			S. flexneri		EAEC		STEC	K.
				S. typhimurium		Pic, Pet		EibG	pneumoniae
				(SPI-1)

Acknowledgments

Acknowledgements A.C.A. was granted with a PhD fellowship from the University of Paris. We acknowledge support from the Biological Mass Spectrometry core facility at the University of Manchester. IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA). This work was supported by the Agence Nationale de la Recherche ANR JCJC grant [grant number ANR-17-CE15-0012] and has benefitted from the support provided by the University for Advanced Study (USIAS) for a fellowship, within the French national program "Investment for the future" (IdEx-UNISTRA) (B.S.M.). This work was also supported by a grant from Medical Research Council [grant number MR/R002800/1] (D.J.T.). The Wellcome Trust Centre for Cell-Matrix Research, University of Manchester (D.J.T.), is supported by core funding from the Wellcome Trust [grant number 088785/Z/09/Z]. This work was also supported by the CNRS and the GDR GAG [GDR 3739], the "Investissements d'avenir" program Glyco@Alps [grant number ANR-15-IDEX-02], by grants from the Agence Nationale de la Recherche [grant number ANR-17-CE11-0040]. Electron microscopy imaging was performed on the Bordeaux Imaging Center, member of the FranceBioImaging national infrastructure, funded by the ANR [grant number ANR-10-INBS-04].

In memory of Dr. Fabrizia Stavru (1978-2021).

Acknowledgments

The authors thank Ellen Arena, Jean-Yves Tinevez, Thierry Fontaine, Yanis Ben Amor and Guillaume Dumenil for their thoughtful comments, text editing and scientific discussions. This work was supported by the ANR JCJC grant (ANR-17-CE15-0012) and has benefitted from the support provided by the University for Advanced Study (USIAS) for a fellowship, within the French national program "Investment for the future" (IdEx-UNISTRA) (B.S.M.).

In memory of Dr. Fabrizia Stavru (1978-2021).

Data availability

The data that support the findings of this study are available from the corresponding author upon request.

RESULTS

Competing interests

The authors declare no competing interests

Concluding remarks

Infectious hypoxia has been reported in a wide range of bacterial and fungal infections. The ability of the pathogen to consume oxygen through respiration plays a key role in local oxygen depletion and the subsequent formation of hypoxic biofilms or foci of infection. It has to be noticed that the ability of pathogens to induce hypoxia might depend on the basal level of available oxygen, which is dependent on the efficiency of organ perfusion. Nevertheless, it seems that this "battle for oxygen" is won by pathogens, impairing important antimicrobial activities of immune cells, such as ROS production and NETs formation. The capacity of facultative anaerobes and fungi to evolve under changing oxygen conditions is essential for their survival and dissemination in host.

The impact of hypoxia during the course of infection (from early infection, to tissue colonization, to infection resolution) is an active field of research. Deciphering how oxygen depletion impacts infectious processes will be essential to better understand bacterial and fungal virulence and to further direct the development of new antimicrobial molecules and vaccines. Important technological developments are urgently needed to study the impact of oxygen in host-pathogen interaction in vivo, in situ, and potentially at a single-cell level.

Discussion and perspectives

Discussion and perspectives

During my PhD project I investigated the impact of low oxygen level on Shigella virulence modulation and on the adaptation of neutrophil antimicrobial functions. This question is relevant since oxygen is poorly abundant within Shigella foci of infection and probably heterogeneously distributed in surrounding tissues. Shigella is a facultative anaerobe which is well adapted to changing pO2 during its virulence cycle.

Neutrophils evolve mainly under low oxygen conditions during their own lifecycle from the bone marrow to the blood plasma fraction, to infected tissues. This is the reason why I focused on the study of their behavior under anoxic conditions. All experimental strategies have been developed in the laboratory to tightly control neutrophils oxygen-exposure, from their purification to their culture in vitro. In particular, we exploited the fact that neutrophils are mainly glycolytic cells, which are well adapted to poorly oxygenated microenvironments.

The first observation of GAG-containing PGF secretion by neutrophil was made possible by the application of these innovative strategies and concepts. This If we determined that chondroitin sulfate is produced by neutrophils, further work is required to define whether additional GAGs are produced by neutrophils, especially hyaluronic acid. Chondroitin sulfate belongs to the family of GAGs linked to proteoglycans such as serglycin. Serglycin has been described to be stored and released by certain immune cells. In neutrophils these proteins have been poorly investigated.

Chondroitin sulfate perinuclear and vesicular localization was intriguing: its localization in the Golgi compartment (which is abundant in the perinuclear zone) will have to be investigated. Its secretion pathway will need to be clarified as well.