Adélaïde Chesnay
email: adelaide.chesnay@univ-tours.fr

C Paget

N Heuzé-Vourc'h

T Baranek

G Desoubeaux

Pneumocystis pneumonia: pitfalls and hindrances to establish a reliable animal model

Keywords: Pneumocystis pneumonia, animal model, Pneumocystis spp., in vivo, infectious challenge

Pneumocystis pneumonia is a severe lung infection that occurs primarily in largely immunocompromised patients. Few treatment options exist and the mortality remains substantial. To develop new strategies in the fields of diagnosis and treatment, it appears critical to improve the scientific knowledge about the biology of the Pneumocystis agent and the course of the disease. In the absence of in vitro continuous culture system, in vivo animal studies represent a crucial cornerstone for addressing Pneumocystis pneumonia in laboratories. These models constitute an essential complement to clinical studies. Here, we provide an overview of the animal models of Pneumocystis pneumonia that were reported in the literature over the last 60 years. It summarizes the various technical parameters to consider in the preparation of the model and the interpretation of the limits / results of such studies.

Overall, this review highlights the great heterogeneity of the variables studied: the choice of the host species and its genetics, the different immunosuppressive regimens to render animal susceptible, the experimental challenge, and the different validation methods of the model.

INTRODUCTION

Pneumocystis pneumonia is a lung infection involving Pneumocystis jirovecii, an ubiquitous fungus with opportunistic behavior [START_REF] Salzer | Clinical, Diagnostic, and Treatment Disparities between HIV-Infected and Non-HIV-Infected Immunocompromised Patients with Pneumocystis jirovecii Pneumonia[END_REF]. First described in malnourished children during and after World War II (2), fatal Pneumocystis pneumonia was one of the first signals of the AIDS epidemic in the United States in the early 1980s [START_REF] Centers | A cluster of Kaposi's sarcoma and Pneumocystis carinii pneumonia among homosexual male residents of Los Angeles and Orange Counties, California[END_REF]. The advent of antiretroviral drugs has resulted in a significant decrease in the incidence of Pneumocystis pneumonia in Human Immunodeficiency Virus (HIV)-positive patients. Today, in regions where HIV testing and treatment are available without restrictions, Pneumocystis pneumonia primarily occurs in subjects undergoing non-viral sources of immunosuppression. This includes pathological conditions responsible for the decrease of blood leucocytes such as hematological malignancies, auto-immune diseases, and drug-induced immunosuppression such as corticosteroids, TNF-alpha inhibitors, alkylating agents [START_REF] Fillatre | Incidence of Pneumocystis jiroveci pneumonia among groups at risk in HIV-negative patients[END_REF][START_REF] Thomas | Pneumocystis pneumonia[END_REF]. Actually, Pneumocystis pneumonia occurs mainly when risk factors are cumulative, i.e. immunosuppressive therapeutic associated to a fragile medical condition. Altogether, Pneumocystis pneumonia affects each year more than 500,000 patients worldwide. After Candida spp, P. jirovecii is the second most common fungal agent among invasive fungal infections [START_REF] Bongomin | Global and Multi-National Prevalence of Fungal Diseases-Estimate Precision[END_REF]. Pneumocystis pneumonia mortality is significant and has been estimated at 10-20% in HIV-positive patients and 20-40% in HIV-negative patients [START_REF] Asai | Early diagnosis and treatment are crucial for the survival of Pneumocystis pneumonia patients without human immunodeficiency virus infection[END_REF][START_REF] Li | Pneumocystis jiroveci pneumonia in immunocompromised patients: Delayed diagnosis and poor outcomes in non-HIV-infected individuals[END_REF].

Two main forms co-exist during P. jirovecii life cycle: the asci and the trophic forms that are differentially involved. The transmission, which is human-to-human airborne, is ensured by the asci form, the only form capable of living transiently in the external environment [START_REF] Cushion | Echinocandin treatment of pneumocystis pneumonia in rodent models depletes cysts leaving trophic burdens that cannot transmit the infection[END_REF][START_REF] Martinez | Growth and airborne transmission of cell-sorted life cycle stages of Pneumocystis carinii[END_REF].

Next, the trophic forms thrive at the surface of type I-pneumocytes in the pulmonary alveoli. This leads to the generation of a local inflammation, while the infection remains extracellular and never becomes invasive in tissues [START_REF] Limper | Pneumocystis carinii: inhibition of lung cell growth mediated by parasite attachment[END_REF][START_REF] Ng | Extrapulmonary pneumocystosis[END_REF]. Clinically non-specific, Pneumocystis pneumonia can manifest with fever associated with non-productive cough and chest pain [START_REF] Salzer | Clinical, Diagnostic, and Treatment Disparities between HIV-Infected and Non-HIV-Infected Immunocompromised Patients with Pneumocystis jirovecii Pneumonia[END_REF][START_REF] Kovacs | Pneumocystis carinii pneumonia: a comparison between patients with the acquired immunodeficiency syndrome and patients with other immunodeficiencies[END_REF][START_REF] Roux | Pneumocystis jirovecii pneumonia in patients with or without AIDS, France[END_REF]. Chest scans usually show bilateral interstitial alveolar syndrome revealed by ground-glass findings, that are non-pathognomonic of Pneumocystis pneumonia [START_REF] Tasaka | Comparison of clinical and radiological features of pneumocystis pneumonia between malignancy cases and acquired immunodeficiency syndrome cases: a multicenter study[END_REF]. The specific biological diagnosis relies on microbiological identification of P. jirovecii in pulmonary secretions / lung tissues by microscopic examination and qPCR. Likewise, it can be indirectly suggested by measuring (1,3)-β-D-glucan, a polysaccharide component of the cell wall of P. jirovecii and other fungi, in the serum of patients [START_REF] Desoubeaux | Use of calcofluor-blue brightener for the diagnosis of Pneumocystis jirovecii pneumonia in bronchial-alveolar lavage fluids: A single-center prospective study[END_REF][START_REF] Desoubeaux | Combination of β-(1, 3)-D-glucan testing in serum and qPCR in nasopharyngeal aspirate for facilitated diagnosis of Pneumocystis jirovecii pneumonia[END_REF].

Despite some advance in the scientific knowledge, Pneumocystis pneumonia still contains many unknowns. The cycle of Pneumocystis is not fully elucidated yet, thus preventing from dispensing clear prevention guidelines. Concerning the pathophysiology, there is a critical need to investigate all the immune mechanisms integrated in the host response. Therefore, experimental models are essential to complete clinical studies. Although theoretically easier and sparing animal lives, in vitro models are unable to mimic the complexity of host-fungus interactions. Importantly, there is no in vitro continuous culture system for Pneumocystis spp., despite long research in this area [START_REF] Atzori | Current in vitro culture systems for Pneumocystis[END_REF][START_REF] Schildgen | Pneumocystis jirovecii can be productively cultured in differentiated CuFi-8 airway cells[END_REF]. Animal models can circumvent these limitations [START_REF] Walzer | Clinical relevance of animal models of Pneumocystis carinii pneumonia[END_REF][START_REF] Ceré | Animal pneumocystosis: a model for man[END_REF][START_REF] Elsegeiny | Murine models of Pneumocystis infection recapitulate human primary immune disorders[END_REF]. Therefore, various animal models of Pneumocystis pneumonia have been developed in attempt to address pathogenesis, virulence, immune response, diagnosis or therapy concerns. However, a single model cannot answer all the aforementioned questions, which explains in part the great multiplicity of supports that have been developed so far. This variability can hind the scientific comparisons, and each mammal species has its own Pneumocystis species (e.g. P. murina for the mouse or P. carini for the rat). Of all the animal model variables, the investigator has to question at the pivotal experimental parameters and major technical features that are assumed to likely influence the results according to the question asked.

Here, we conducted an extensive literature review of published reports related to animal models of Pneumocystis pneumonia using a search strategy in the PubMed database for articles published up to December 2020, based on MeSH terms. Our electronic request about animal models of Pneumocystis pneumonia retrieved 1,444 publications. Experimental animal studies were included when they met the following inclusion criteria: (a) the article was accessible and written in English; (b) the study was an original article, (c) the animal model was not exclusively used to produce Pneumocystis organisms for an in vitro study; (d) the study was not a post hoc analysis in laboratory or wild animals. After thorough reviewing, a total of 341 articles, corresponding to 749 distinct animal models, were finally retained for complete analysis (Figure 1). Initially, articles were mostly dedicated to the description of the implementation of animal models and to preclinical therapeutic studies (Figure 2). Then, at the beginning of the 2000s, pathophysiology studies became by far the largest area of experimentation. We now propose to the reader a progressive and in-depth review of the elements that we consider essentials in the design of an animal model of Pneumocystis pneumonia, i.e. the host species, the parameters inducing susceptibility to Pneumocystis pneumonia, , the implementation of the experimental infection (route of inoculation, fungal inoculum) and and the biological parameters to follow up to assert correct implementation of the disease the validation methods of the model.

GENERAL DESCRIPTION OF THE VARIOUS ANIMAL MODELS: HOST SPECIES AND STRAINS, SEX, WEIGHT AND AGE

The choice of the host species is critical to reproduce as faithfully as possible the pathology that develops in Humans, but also to ensure the best reproducibility. Indeed, as in Humans, animals need to be carriers of Pneumocystis and transmit it to their congeners by air. Also, as in Humans, depending on their immune status, they must be able to eliminate the fungus naturally without developing a disease if they are immunocompetent, or on the contrary, in case of immunosuppression. Overall so far, more than ten animal species have been used as host models to in vivo study Pneumocystis pneumonia (Table I). Not surprisingly, rodents, were extensively exploited (95.9%) compared to other orders of mammals. Mice were used in 74.8% of the selected studies, compared to 20.8% and 0.3% for rats and other rodents (e.g. Guinea pigs and hamsters), respectively. The mouse model was widely used for its well characterized physiology, as well as biochemical and genetic homologies with Humans (23), but also for the dedicated toolbox that has been developed. Rabbits were used in 1.3% of the studies. Nonetheless, rabbits usually display lower fungal loads than other animals, and few tools and products are adapted to rabbit's biology. In addition, they are more expensive and difficult to handle than rats and mice. In 1.3% of the models, non-human primates (NHP) were used, from two species belonging to the family of Cercopithecidae [START_REF] Furuta | Severe pulmonary pneumocystosis in simian acquired immunodeficiency syndrome induced by simian immunodeficiency virus: its characterization by the polymerase-chainreaction method and failure of experimental transmission to immunodeficient animals[END_REF][START_REF] Patil | Immune responses to Pneumocystis colonization and infection in a simian model of AIDS[END_REF][START_REF] Board | Experimental Pneumocystis carinii pneumonia in simian immunodeficiency virus-infected rhesus macaques[END_REF][START_REF] Kling | Pneumocystis colonization in immunocompetent and simian immunodeficiency virus-infected cynomolgus macaques[END_REF][START_REF] Kling | Relationship of Pneumocystis jiroveci humoral immunity to prevention of colonization and chronic obstructive pulmonary disease in a primate model of HIV infection[END_REF][START_REF] Kling | Trimethoprimsulfamethoxazole treatment does not reverse obstructive pulmonary changes in pneumocystiscolonized nonhuman primates with SHIV infection[END_REF][START_REF] Kling | Vaccine-Induced Immunogenicity and Protection Against Pneumocystis Pneumonia in a Nonhuman Primate Model of HIV and Pneumocystis Coinfection[END_REF][START_REF] Jiménez | Immunization with Pneumocystis recombinant KEX1 induces robust and durable humoral responses in immunocompromised non-human primates[END_REF]. The latter, thanks to their physiological similarities and evolutionary conservation with Humans, represented privileged models for studying Pneumocystis pneumonia in a viral immunodeficiency background. Nevertheless, even if Humans and NHP are closely related, it should be kept in mind that, in normal conditions, each is contaminated by its own species, P. jirovecii for Humans and P. macacae for macaques. Other mammals were rarely used such as ferrets [START_REF] Stokes | Experimental Pneumocystis carinii pneumonia in the ferret[END_REF][START_REF] Gigliotti | Pneumocystis carinii is not universally transmissible between mammalian species[END_REF][START_REF] Simpson-Haidaris | Induction of fibrinogen expression in the lung epithelium during Pneumocystis carinii pneumonia[END_REF], pigs [START_REF] Hong | Susceptibility of various animals to Pneumocystis carinii infection. Kisaengch'unghak chapchi The[END_REF][START_REF] Nielsen | Experimental corticosteroid induction of Pneumocystis carinii pneumonia in piglets[END_REF][START_REF] Nielsen | Pneumocystis carinii pneumonia--pig provocation model[END_REF], cats [START_REF] Hong | Susceptibility of various animals to Pneumocystis carinii infection. Kisaengch'unghak chapchi The[END_REF] and dogs [START_REF] Hong | Susceptibility of various animals to Pneumocystis carinii infection. Kisaengch'unghak chapchi The[END_REF]. Lastly, two arthropod-based studies, with Drosophila melanogaster and Galleria mellonella, assessed the non-susceptibility of non-mammalian species to Pneumocystis pneumonia [START_REF] Evans | Toll-deficient Drosophila are resistant to infection by Pneumocystis spp.: additional evidence of specificity to mammalian hosts[END_REF][START_REF] Fuchs | Galleria mellonella are resistant to Pneumocystis murina infection[END_REF]. Depending on the purpose/issue of the study, some animals were used more frequently than others (Figure 3).

Rabbits have most commonly been used to study the Pneumocystis agent and its transmission. Indeed, spontaneous Pneumocystis pneumonia is described in the absence of induced immunosuppression at the time of weaning, thus naturally facilitating its study [START_REF] Cere | In utero transmission of Pneumocystis carinii sp. f. oryctolagi[END_REF][START_REF] Sanchez | Exploring transplacental transmission of Pneumocystis oryctolagi in first-time pregnant and multiparous rabbit does[END_REF]. Mice and rats have also been used to study the transmission of Pneumocystis between the same or different host species. Mice have been are mostly used to study host-pathogen interactions and host immune response. Non-human primates have been little used, in part for ethical restrictions; but they have the great advantage to reproduce the development of Pneumocystis pneumonia in a context of virus-induced immunodepression. Finally, rats have been the preferred species for pre-clinical therapy studies (prophylactic, immunization, curative). The relative benefits and limitations of these models for the study of Pneumocystis pneumonia are summarized in the Table II.

The importance of an informed choice for the animals concerns not only the species, but also the strain. Focusing on mouse models, studies using inbred strains predominated: BALB/c and C57BL/6 were the more reported before C3H/HeN. Attention should be paid to the selection of strains, as highlighted in a study conducted by Swain et al, in which BALB/c and C57BL/6 mice have been shown to develop a specific early immune reaction after inoculation of P. murina [START_REF] Swain | Pneumocystis elicits a STAT6-dependent, strain-specific innate immune response and airway hyperresponsiveness[END_REF]. Strains also appeared to show a different permissiveness to Pneumocystis infection with variable lung burdens as shown by Tisdale et al. [START_REF] Tisdale | The Effects of Sex and Strain on Pneumocystis murina Fungal Burdens in Mice[END_REF]. Considering all animal models other than mice, outbred animals were used more frequently than inbred ones. For studies with outbred rats, Sprague-Dawley represented 64.1% of the rat models, while Wistar strain was associated with 14.7% reports. The data on susceptibility in different rat strains do not seem to be unanimous. Whereas Boylan et al. evaluated that Sprague-Dawley, Fisher 344, and Lewis rats immunosuppressed by steroid developed the same heavy infection six weeks after the inoculation, Hong et al. showed that Wistar rats developed an earlier and severer infection than Fisher and Sprague-Dawley rats under steroid immunosuppression [START_REF] Hong | Susceptibility of various animals to Pneumocystis carinii infection. Kisaengch'unghak chapchi The[END_REF][START_REF] Boylan | Improved rat model of Pneumocystis carinii pneumonia: induced laboratory infections in Pneumocystis-free animals[END_REF].

The sex of the animal chosen is also important although, in the majority of models (64.6%), it was not specified. When reported, they were females in 48.3%, males in 37.4%, and both genders in 14.3%. In a study comparing the progression of Pneumocystis pneumonia in male and female, Tisdale et al. showed that females of three distinct mouse strains had higher fungal burdens as compared to males after six weeks of infection [START_REF] Tisdale | The Effects of Sex and Strain on Pneumocystis murina Fungal Burdens in Mice[END_REF]. This contrasts with what is usually observed in Humans where men are the most affected by Pneumocystis pneumonia [START_REF] Bitar | Mycoses invasives en France métropolitaine, PMSI 2001-2010: incidence, létalité et tendances[END_REF][START_REF] Wickramasekaran | The changing trends and profile of pneumocystosis mortality in the United States, 1999-2014[END_REF]. Concerning the weight of animals used, when informed (14.2%), it was quite homogeneous and standard with 21.0 ± 4.5g and 189.4 ± 48.4g, for mice and rats respectively. In models of Pneumocystis pneumonia, weight loss is rarely reported and appears as a poor and irrelevant indicator of disease. Moreover, in human medicine, there are very few data on the importance of the initial weight of patients suffering from Pneumocystis pneumonia, only a few cases reported in a context of nutritional deprivation [START_REF] Halabieh | A Case of Pneumocystis jirovecii Pneumonia in a Severely Malnourished, HIV-Negative Patient[END_REF][START_REF] Hanachi | Negative role of malnutrition in cell-mediated immune response: Pneumocystis jirovecii pneumonia (PCP) in a severely malnourished, HIV-negative patient with anorexia nervosa[END_REF]. In contrast, the choice of life stage of the animals may be an important element, especially considering that the immune system is not fully developed during the first weeks of life and strongly evolves throughout aging [START_REF] Partridge | Facing up to the global challenges of ageing[END_REF]. Indeed, studies have compared the different life stages of mice in relation to the immune response: neonates showed a delay in the onset of the immune response due to an inadequate lung environment coupled with an inherent inability to develop a robust innate immune response to infection and an inexperienced adaptive immune system [START_REF] Garvy | Susceptibility to Pneumocystis carinii infection: host responses of neonatal mice from immune or naive mothers and of immune or naive adults[END_REF][START_REF] Garvy | Delayed inflammatory response to Pneumocystis carinii infection in neonatal mice is due to an inadequate lung environment[END_REF][START_REF] Kurkjian | Alveolar Macrophages in Neonatal Mice Are Inherently Unresponsive to Pneumocystis murina Infection[END_REF].

SELECTION OF THE REGIMEN INDUCING SUSCEPTIBILITY TO

PNEUMOCYSTIS PNEUMONIA

In the great majority of cases, tools to render animals susceptible to Pneumocystis pneumonia are an essential element to consider. Indeed, patients susceptible to Pneumocystis infection have the particularity of presenting pre-existing underlying conditions. Therefore, usage of a regimen inducing susceptibility to Pneumocystis pneumonia was reported in 663 animal models, i.e., 88.5% of those described. The advantages and disadvantages of the principal strategies to render animal susceptible to Pneumocystis pneumonia are summarized in Table III.

Based on analogy with other models of fungal infections of the respiratory tract (e.g. aspergillosis), anti-cancerous drugs like alkylating substances, and more specifically cyclophosphamide, were used to induce adequate immunocompromised conditions [START_REF] Weisbroth | Latent Pneumocystis carinii infection in commercial rat colonies: comparison of inductive immunosuppressants plus histopathology, PCR, and serology as detection methods[END_REF][START_REF] Desoubeaux | Rodent Models of Invasive Aspergillosis due to Aspergillus fumigatus: Still a Long Path toward Standardization[END_REF].

However, alkylating agents primarily target neutrophils, that are less involved in the response to Pneumocystis than T-lymphocytes and macrophages. The latter are rather targeted by steroids, recognized as major risk factor for the development of Pneumocystis pneumonia [START_REF] Yale | Pneumocystis carinii pneumonia in patients without acquired immunodeficiency syndrome: associated illness and prior corticosteroid therapy[END_REF][START_REF] Bienvenu | Pneumocystis pneumonia suspected cases in 604 non-HIV and HIV patients[END_REF][START_REF] Liu | Clinical characteristics, treatment outcomes, and prognostic factors of Pneumocystis pneumonia in non-HIV-infected patients[END_REF]. They have been largely used to induce immunosuppression in animal models of Pneumocystis pneumonia (30.8% of the animal models) [START_REF] Shellito | Murine CD4+ T lymphocyte subsets and host defense against Pneumocystis carinii[END_REF][START_REF] Swain | Neither neutrophils nor reactive oxygen species contribute to tissue damage during Pneumocystis pneumonia in mice[END_REF][START_REF] Lim | Glucocorticoids exert opposing effects on macrophage function dependent on their concentration[END_REF][START_REF] Eddens | Eosinophils Contribute to Early Clearance of Pneumocystis murina Infection[END_REF][START_REF] Nandakumar | Differential Macrophage Polarization from Pneumocystis in Immunocompetent and Immunosuppressed Hosts: Potential Adjunctive Therapy during Pneumonia[END_REF][START_REF] Cain | Immune regulation by glucocorticoids[END_REF][START_REF] Bhagwat | Intrinsic Programming of Alveolar Macrophages for Protective Antifungal Innate Immunity Against Pneumocystis Infection[END_REF]. Dexamethasone administered in drinking water at a concentration of 1 to 4 mg/L was the most commonly used (57.8% of steroids models), ahead of injectable cortisone acetate (23.9% of steroids models) and injectable methylprednisolone (15.2% of steroids models), both administered subcutaneously. Dexamethasone has the advantage of a longer duration of action, but also a higher anti-inflammatory potency than cortisone and methylprednisolone. Oral administration is convenient, relatively safe, economical and compatible with refinement of experimental procedures, although it does not possess the highest bioavailability compared to parenteral routes of administration [START_REF] Turner | Administration of substances to laboratory animals: routes of administration and factors to consider[END_REF]. In most models, steroid-dependent immunosuppression started one to two weeks prior experimental challenge, in order to reproduce a suitable condition for the development of Pneumocystis pneumonia (66), and was continuously pursued until the infection had been established [START_REF] Wolff | The development of Pneumocystis carinii pneumonia in germ-free rats requires immunosuppression and exposure to the Pneumocystis carinii organism[END_REF][START_REF] O'leary | Use of semiquantitative PCR to assess onset and treatment of Pneumocystis carinii infection in rat model[END_REF]. Other immunosuppressive drugs were alternatively used in rare models: dichloromethylene diphosphonate-containing liposomes or clodronateliposomes for the specific depletion of macrophages [START_REF] Limper | The role of alveolar macrophages in Pneumocystis carinii degradation and clearance from the lung[END_REF][START_REF] Lasbury | Suppression of alveolar macrophage apoptosis prolongs survival of rats and mice with pneumocystis pneumonia[END_REF][START_REF] Bhagwat | Intrinsic Programming of Alveolar Macrophages for Protective Antifungal Innate Immunity Against Pneumocystis Infection[END_REF], or more broad-spectrum medicines such as calcineurin inhibitors, tacrolimus and ciclosporin [START_REF] Hughes | Provocation of infection due to Pneumocystis carinii by cyclosporin A[END_REF], mTOR inhibitor, sirolimus [START_REF] Oz | Novel anti-Pneumocystis carinii effects of the immunosuppressant mycophenolate mofetil in contrast to provocative effects of tacrolimus, sirolimus, and dexamethasone[END_REF], or inhibitor of inosine-5'-monophosphate dehydrogenase, mycophenolate mofetil [START_REF] Oz | Novel anti-Pneumocystis carinii effects of the immunosuppressant mycophenolate mofetil in contrast to provocative effects of tacrolimus, sirolimus, and dexamethasone[END_REF].

Considering that CD4 + T-lymphocytes count is a reliable predictor of opportunistic Pneumocystis pneumonia during HIV infection (66), a more specific treatment of this lineage has also been tested. Depleting monoclonal antibodies (mAbs) targeting CD4 + T-lymphocytes (clone GK1.5) were widely used (81.7% of these models based on immunotherapy) alone or in combination with other T-cell depleting mAbs such as anti-CD8 (clone 2.43) or anti-Thy1.2 (clone 30H12) mAbs in mice. Some other antibodies were given, such as anti-CD20 mAb (clone 5D2 or 18B12) allowing B cell depletion [START_REF] Elsegeiny | Anti-CD20 Antibody Therapy and Susceptibility to Pneumocystis Pneumonia[END_REF][START_REF] Opata | B Lymphocytes Are Required during the Early Priming of CD4+ T Cells for Clearance of Pneumocystis Infection in Mice[END_REF]. mAbs could be administered either once, before or just after the experimental infection, or several times throughout the course of infection. Immunotherapy was most often administered by intraperitoneal injection and almost exclusively in mice. Unfortunately, the risk of hypersensitivity reaction or cytokine release-associated acute reactions, and the multiplication of parenteral injections constitute major drawbacks [START_REF] Hansel | The safety and side effects of monoclonal antibodies[END_REF][START_REF] Akarsu | Hypersensitivity Reactions to Biologicals: from Bench to Bedside[END_REF].

Genetically modified mice also offer interesting advantages for the development of Pneumocystis pneumonia and have been widely used (56.4% of the studies in mouse models).

They can be grossly divided into two groups: 1) models displaying a general immunodeficiency such as SCID or RAG1 -/-mice that lack functional T-cells and/or B-cells, or 2) more refined models that target a specific gene implicated in the host response. The first ones aforementioned were primarily used to study Pneumocystis biology including its life cycle and efficiency of anti-Pneumocystis curative drugs. In 1993, the study by Chen et al. used CB17/scid (SCID) mice to support the concept that Pneumocystis pneumonia develops in immunocompromised patients because of recent exposure to an exogenous source and not necessarily because of reactivation of latent infection [START_REF] Chen | Latency is not an inevitable outcome of infection with Pneumocystis carinii[END_REF]. The second ones were exploited to study and identify cellular and molecular entities involved in the innate and adaptive anti-Pneumocystis immune responses. For example, the involvement of the surfactant proteins A and D in fighting against Pneumocystis was highlighted by the generation of deficient mice that were knocked out for the relative encoding genes [START_REF] Linke | Immunosuppressed surfactant protein A-deficient mice have increased susceptibility to Pneumocystis carinii infection[END_REF][START_REF] Atochina | Enhanced lung injury and delayed clearance of Pneumocystis carinii in surfactant protein A-deficient mice: attenuation of cytokine responses and reactive oxygen-nitrogen species[END_REF][START_REF] Atochina | Delayed clearance of pneumocystis carinii infection, increased inflammation, and altered nitric oxide metabolism in lungs of surfactant protein-D knockout mice[END_REF][START_REF] Linke | Surfactant protein A limits Pneumocystis murina infection in immunosuppressed C3H/HeN mice and modulates host response during infection[END_REF][START_REF] Linke | Efficient resolution of Pneumocystis murina infection in surfactant protein A-deficient mice following withdrawal of corticosteroid-induced immunosuppression[END_REF][START_REF] Linke | Effects of surfactant protein-A on the interaction of Pneumocystis murina with its host at different stages of the infection in mice[END_REF][START_REF] Linke | Pneumocystis murina colonization in immunocompetent surfactant protein A deficient mice following environmental exposure[END_REF]. Later in 2018, Elsegeiny et al.

used several mouse models to recapitulate human primary immune disorders, enabling them to understand which types of CD4 T-cells were involved or relevant to mediate the clearance of Pneumocystis [START_REF] Elsegeiny | Murine models of Pneumocystis infection recapitulate human primary immune disorders[END_REF]. However, care should be taken when interpreting outcomes in these models because of redundancy in the immune system and/or compensatory hyperactivity that can lead to confounding effects [START_REF] Rudmann | Susceptibility to Pneumocystis carinii in mice is dependent on simultaneous deletion of IFN-gamma and type 1 and 2 TNF receptor genes[END_REF]. In addition, scientists have to keep in mind that the use of such genetically modified or defined mice under standardized environmental conditions may influence host immunity and inflammation [START_REF] Enriquez | Genomic, microbial and environmental standardization in animal experimentation limiting immunological discovery[END_REF]. While the generation of such mice still remains complicated, expensive and time consuming, they represent very useful biological tools for studying the host immune response to Pneumocystis.

Alternative immunosuppression procedures have also been implemented. This was the case for the majority of Pneumocystis pneumonia models in NHP. In order to reproduce as closely as possible the immunosuppression that affects AIDS patients, NHP were infected intravenously with Simian Immunodeficiency Virus (SIV) [START_REF] Furuta | Severe pulmonary pneumocystosis in simian acquired immunodeficiency syndrome induced by simian immunodeficiency virus: its characterization by the polymerase-chainreaction method and failure of experimental transmission to immunodeficient animals[END_REF][START_REF] Patil | Immune responses to Pneumocystis colonization and infection in a simian model of AIDS[END_REF][START_REF] Board | Experimental Pneumocystis carinii pneumonia in simian immunodeficiency virus-infected rhesus macaques[END_REF][START_REF] Kling | Pneumocystis colonization in immunocompetent and simian immunodeficiency virus-infected cynomolgus macaques[END_REF][START_REF] Kling | Relationship of Pneumocystis jiroveci humoral immunity to prevention of colonization and chronic obstructive pulmonary disease in a primate model of HIV infection[END_REF][START_REF] Kling | Trimethoprimsulfamethoxazole treatment does not reverse obstructive pulmonary changes in pneumocystiscolonized nonhuman primates with SHIV infection[END_REF][START_REF] Kling | Vaccine-Induced Immunogenicity and Protection Against Pneumocystis Pneumonia in a Nonhuman Primate Model of HIV and Pneumocystis Coinfection[END_REF].

To enhance the magnitude of Pneumocystis infection, low protein diet was used in 7.9% of the models (87). This particular diet, harmful to longevity/metabolic health, was set up to reproduce the malnutrition status observed in some patients suffering from Pneumocystis pneumonia. However, it was quite expensive and barely used after the 2000s.

Since the models are mostly immunocompromised, it is important to use antibiotic prophylactic strategy to prevent from the occurrence of opportunistic bacterial infection, that would occur more quickly than the Pneumocystis pneumonia. Antibiotics were used in 23.0% of the models. The molecules used belonged to a broad spectrum of antibiotic families. Cyclins were the most widely used, in 70.9% of the models using antibiotics.

Tetracycline was administered in drinking water at a concentration between 0.5 and 1 mg/mL, and doxycycline, by far less used, was administrated by subcutaneous injection. Betalactamins were used in 26.2% of the models, along with ampicillin, cephadrin, penicillin G, amoxicillin with or without clavulanic acid. They were mostly administered in drinking water. Other antibiotics were less used, such as quinolones with ciprofloxacin (69), aminosides with streptomycin and gentamicin [START_REF] Sheldon | Experimental pulmonary Pneumocystis carinii infection in rabbits[END_REF][START_REF] Walzer | Clinically used antimicrobial drugs against experimental pneumocystosis, singly and in combination: analysis of drug interactions and efficacies[END_REF], or sulfamides with sulfadiazine [START_REF] Hussain | Efficacy of tetroxoprim/sulphadiazine in the treatment of Pneumocystis carinii pneumonitis in rats[END_REF]. Anecdotally, 2.4% of the models used polyenes, nystatin or amphotericin B to prevent for other fungal diseases. The antibiotic prophylaxis strategy based on the use of cyclins, especially tetracycline, which is widely used, inexpensive and easily administered in drinking water, is to be preferred. Concerning the use of antibiotic prophylactic strategy, the parallel with what can be observed in human medicine is complicated to establish. Indeed, while most cases of Pneumocystis pneumonia occur in immunocompromised patients, little or no retrospective data are available on the use of antibiotics concomitant with the development and/or diagnosis of Pneumocystis pneumonia. Such information could be of interest in assessing the impact of such treatment might have on the pathophysiology of the disease.

IMPLEMENTATION OF THE EXPERIMENTAL INFECTION

Setting up a relevant animal model of fungal infection first requires considering the route of infection. Three main methods of experimental challenge have been proposed in the literature for generating Pneumocystis pneumonia.

A first passive strategy was based on the presumed latency of Pneumocystis within the lung alveoli and its subsequent reactivation following the induction of immunosuppression. This strategy was adopted in 20.7% of the models, especially in the pioneer reports. With respect to the recent evidences rather in favor of a de novo infection, this protocol seemed clearly inadequate and moreover insufficient to ensure a methodologically strict and reproducible study. Indeed, in most of these ancient reports, animals were kept under unspecified exposure conditions, and the occurrence of Pneumocystis pneumonia was quite random and most likely due to the transmission of Pneumocystis organisms by the other animals housed in the same facilities. Nowadays, one acknowledges that it is essential to use animals with the SOPF (Specific and Opportunistic Pathogen Free) certification in housing conditions such as microisolator/filtered cages that eliminate the risk of transmission from other animals.

A second passive strategy, used in 17.0% of the studied models, was implemented by cohousing healthy animals with Pneumocystis-pre-infected seeder mate-fellows. Indeed, the airborne route was clearly established in the early 1980's in germ-free immunocompromised rats that had been exposed to potential sources of Pneumocystis carinii (i.e. natural Pneumocystis species in rats) [START_REF] Hughes | Natural mode of acquisition for de novo infection with Pneumocystis carinii[END_REF]. In isolators, animals exposed to filtered sterile air and unsterile water and food did not acquire P. carinii, while rats exposed in open cages to room air but maintained on sterile diet acquired the infection. Thus thanks to this model, it has been demonstrated that Pneumocystis was naturally acquired by horizontal transmission as an airborne organism in a de novo infection [START_REF] Wolff | The development of Pneumocystis carinii pneumonia in germ-free rats requires immunosuppression and exposure to the Pneumocystis carinii organism[END_REF][START_REF] Hughes | Natural mode of acquisition for de novo infection with Pneumocystis carinii[END_REF][START_REF] An | Exposure of immunocompetent adult mice to Pneumocystis carinii f. sp. muris by cohousing: growth of P. carinii f. sp. muris and host immune response[END_REF]. In the same vein, healthy immunocompromised animals were co-housed with fellows of the same species infected with Pneumocystis, for a time varying from one day to several weeks [START_REF] Roths | Both immunity and hyperresponsiveness to Pneumocystis carinii result from transfer of CD4+ but not CD8+ T cells into severe combined immunodeficiency mice[END_REF][START_REF] Yasuoka | Successful treatment of Pneumocystis carinii Pneumonia in mice with benanomicin A (ME1451)[END_REF][START_REF] Gigliotti | Antibody-mediated shift in the profile of glycoprotein A phenotypes observed in a mouse model of Pneumocystis carinii pneumonia[END_REF][START_REF] Wright | Pulmonary inflammation disrupts surfactant function during Pneumocystis carinii pneumonia[END_REF][START_REF] Dumoulin | Transmission of Pneumocystis carinii disease from immunocompetent contacts of infected hosts to susceptible hosts[END_REF][START_REF] Chabé | Immunocompetent hosts as a reservoir of pneumocystis organisms: histological and rt-PCR data demonstrate active replication[END_REF]. It appeared that inoculum or dose effect determined the rate at infection progression [START_REF] Gigliotti | Characterization of transmission of Pneumocystis carinii f. sp. muris through immunocompetent BALB/c mice[END_REF]. Although this kind of strategy replicates the natural transmission of Pneumocystis in mammals, it could lack control and reproducibility.

In order to control these points, a third experimental infection strategy was developed through the direct inoculation of Pneumocystis organisms into the animals' respiratory tract. Various modes of administration have been developed. Most of the time, animals were sedated or anesthetized prior delivery, in order to minimize struggling and sneezing. The anesthesia procedure and the operator skills were critical steps to achieve a robust and reliable infection (100). Inoculation of Pneumocystis organisms could be achieved by intranasal, oropharyngeal, intratracheal instillation or by transtracheal deposition. The intranasal instillation, consisting in the deposition of droplets of a Pneumocystis suspension close to the nostrils, appears as the softest methods (easiest and the least invasive technique). At the opposite, the transtracheal alternative requires to expose the trachea surgically to a direct injection of organisms inside. Intratracheal delivery of Pneumocystis via blunted -needle or feeding cannula allows refining of the procedure by getting rid of the surgical incision.

Overall, the direct inoculation strategy was the most common method used in mouse models, with a majority of administration based on intratracheal instillation (Table I). The frequency of Pneumocystis inoculation was generally based on a single administration, except for some specific studies that completed two or three successive inoculations, separated by two to twenty days (50, 101-104). Garvy et al. performed several inoculations to induce immunization (50), whereas Vuk et al. used a second inoculation to be certain that mice strains used, known to exhibit low levels of Pneumocystis infection according to them, were sufficiently exposed to P. murina organisms (104). None of the studies compared multiple inoculations vs. a single one; thus, it is difficult to appreciate whether this resulted in a greater infection. However, the time until the onset of Pneumocystis pneumonia was similar whatever the number of inoculations used. The advantages and disadvantages of each strategy to implement Pneumocystis pneumonia are summarized in Table III.

Other concerns raised from the variability of the composition and the size of the Pneumocystis inoculum. Because, so far, in vitro production of Pneumocystis has not been successful, Pneumocystis were extracted, and mostly purified, from fresh or frozen pulmonary grindings of previously-infected animals. Extraction could be based on different methods, such as stomacher blending, ultrasonication or magnetic stirring (10, 105-107). Because Pneumocystis organisms can only be partially purified, inoculum will contained immune cells, cytokines, or other immune stimulators that may affect the host's pulmonary immune response. Thus, a control with lungs from healthy animals having undergone the same purification process seems essential.. In some rare publications, the animal received Pneumocystis asci from another animal species [START_REF] Evans | Toll-deficient Drosophila are resistant to infection by Pneumocystis spp.: additional evidence of specificity to mammalian hosts[END_REF][START_REF] Fuchs | Galleria mellonella are resistant to Pneumocystis murina infection[END_REF]108,109). Although Walzer et al. initially showed that the sporadic transmission of Pneumocystis was possible between rats and mice (108), the opposite was subsequently demonstrated and definitively admitted (109).

Furthermore, there was a great diversity in the way to count the number of Pneumocystis organisms in order to prepare the infectious suspension for the experimental challenge. When some counted only the asci through microscopic observation, others counted in addition the trophic forms [START_REF] Atochina | Delayed clearance of pneumocystis carinii infection, increased inflammation, and altered nitric oxide metabolism in lungs of surfactant protein-D knockout mice[END_REF](110)(111)(112). Noteworthy, counting of trophic forms is a tedious task and requires a great deal of experience on the part of the microscopist, and taking trophic forms in account is also quite sensitive, since they were shown as insufficient to induce Pneumocystis pneumonia [START_REF] Cushion | Echinocandin treatment of pneumocystis pneumonia in rodent models depletes cysts leaving trophic burdens that cannot transmit the infection[END_REF][START_REF] Martinez | Growth and airborne transmission of cell-sorted life cycle stages of Pneumocystis carinii[END_REF]111,113). In a concern of homogeneity and scientific relevance, it seems more appropriate to consider and count only the asci for the inoculum. Large variations in the inoculum size, defined by the prior numbering of Pneumocystis forms, were observed, from 1.0x10 4 to 1.0x10 8 Pneumocystis forms, with an average around 1.0x10 6 to 1.0x10 7 Pneumocystis organisms. Thereafter, the experimentalist should be aware that the establishment of the clinical Pneumocystis pneumonia is a long process requiring four to 356 seven weeks after the inoculation 357

VALIDATION OF THE MODEL AND OUTCOME PARAMETERS TO FOLLOW UP

In all the infectious animal models, it is essential to verify the effective infection or colonization and quantify the microorganism load. Since the clinical and radiological signatures of Pneumocystis are not specific, the use of histological / biological techniques was almost systematic, although none of these methods provided actual information about the viability of the fungal elements. Overall, 98.4% of the articles reported at least one histological or biological test (including microscopic approaches) to confirm that the experimental infection has been correctly implemented in the exposed animals or to assess the fungal burden. However, most of the models exploited only one technique (78.6%).

Microscopic observations of pulmonary secretions/lung sections/lung grindings slides, longtime considered as the reference standard to prove Pneumocystis pneumonia or colonization, have been largely described, in 81.8% of all models. These direct methods used different types of staining like Diff quick, Giemsa, Grocott methanamine silver nitrate (GMS), and toluidine blue O or calcofluor-blue brightener to demonstrate the presence of discoid Pneumocystis asci and/or ascospores and/or trophic forms. Microscopic approaches require substantial microscopic expertise, but they seem essential because they allow to distinguish quickly the asci forms, while being easy-to-implement and inexpensive methods.

Methods based on molecular biology like nucleic acid amplification by qPCR or fluorescence in situ hybridization (FISH) are more sensitive techniques. They are more refined to determine the fungal load (asci and trophic forms included), and could be used in various kinds of samples (lung tissues, bronchial-alveolar lavage fluids (BALF), or oral swab samples). They were widely used, in 31.8% of the models, with the following targets: mitochondrial large subunit (mtLSU) rRNA gene, 5.8 S rRNA gene or dihydrofolate reductase (DHFR) gene. As for other molecular biology methods, qPCR requires specialized costly equipment and reagents, which are now available in a large number of laboratories. It should be noted that the primers used for Pneumocystis jirovecii usually do not overlap with those of other Pneumocystis spp., like P. jirovecii.

Other tools were used, such as detection of anti-Pneumocystis antibodies, performed in 6.9% of the models, or the blood / BALF detection of (1,3)-β-D-glucan [START_REF] Desoubeaux | Combination of β-(1, 3)-D-glucan testing in serum and qPCR in nasopharyngeal aspirate for facilitated diagnosis of Pneumocystis jirovecii pneumonia[END_REF]112,(114)(115)(116). The serology, never used alone, was attended by huge difficulties involving potential falsenegative test results and is questionable with regard to the production of antibodies in immunocompromised animals. In Humans, its use is restricted to epidemiological questions (117). Detection of (1,3)-β-D-glucan is not specific to Pneumocystis pneumonia and is quite costly.

In general, and whatever the type of study, to assess Pneumocystis presence, identify its forms, and ensure the most accurate quantification possible, the combination of a microscopic and a molecular biology technique appears the most suitable.

CONCLUSION

Pneumocystis pneumonia is a severe respiratory disease that occurs especially in immunocompromised patients. Worldwide, the number of deaths due to Pneumocystis spp. is estimated at almost 250,000 (Gaffi data, 2017). In absence of models of continuous in vitro culture, in vivo animal studies represent a crucial cornerstone for the study of Pneumocystis pneumonia. However, it is important to keep in mind that Pneumocystis species are hostspecific [START_REF] Gigliotti | Pneumocystis carinii is not universally transmissible between mammalian species[END_REF]; they have progressively diverged several tens of millions years ago and coevolved with their hosts, thus defining their host obligate nature (118, 119). Therefore, these models are imperfect, and we can wonder about the extrapolation of the results obtained with models using microorganisms genetically different from those infecting Humans.

Ethical considerations are important when planning the use of an animal model and should be governed by the "3 R's" rule: Replacement, Reduction, and Refinement (120). Animal experiments should be designed in such a way that they allow statistically significant results with the smallest possible number of animals, while being robust and reproducible. In such a manner, the choice of the animal species and strains to study Pneumocystis pneumonia is decisive. As seen previously, the mouse seems to be the most suitable species. Refinement in animal models of Pneumocystis pneumonia can be achieved by choosing a mean of immunosuppression that avoids parenteral administration (same comment for the choice of antibiotics prophylaxis), and by using parameters other than the overall mortality to assess the disease progression.

Studying articles published for the last 60 years has enabled us to establish a wide range of criteria and factors to be considered for implementing an animal model to address Pneumocystis pneumonia. It requires to make choices to best answer the question posed and includes many elements such as permissiveness to infection, homology, analogy, and fidelity with Humans, reproducibility, ease of handling, safety, and of course cost. Thus, if one wonders about the cycle of Pneumocystis, it seems more relevant to replicate the natural transmission of Pneumocystis in mammals by using co-housing of healthy animals with infected fellows, whether rodents with which we have the most experience, or NHP, whose Pneumocystis species is the closest phylogenetically to that of Humans. In contrast, in preclinical therapeutic studies that require rigorous design to obtain homogenous population, a model with an implementation of the infection by direct inoculation of Pneumocystis organisms allows necessary reproducibility and high control. For studies focusing on the understanding of the pathophysiology and particularly the host immune response, several types of models can be suggested. The first ones use refined genetically modified mice with a very specific immunodeficiency to study its specific involvement in the host response. The other ones study the immune response more generally, using models displaying general immunodeficiency such as genetically modified SCID or RAG1-/-mice, or animals immunosuppressed by the use of corticosteroids, the major iatrogenic risk factor of Pneumocystis pneumonia in Humans [START_REF] Yale | Pneumocystis carinii pneumonia in patients without acquired immunodeficiency syndrome: associated illness and prior corticosteroid therapy[END_REF][START_REF] Bienvenu | Pneumocystis pneumonia suspected cases in 604 non-HIV and HIV patients[END_REF][START_REF] Liu | Clinical characteristics, treatment outcomes, and prognostic factors of Pneumocystis pneumonia in non-HIV-infected patients[END_REF].

Finally according to our experience, we can propose a relevant example of an animal model to study the immune response that uses genetically modified or not and steroid immunosuppressed rodents (Figure 4), challenged by intranasal inoculation of Pneumocystis murina and validated by a microscopic and a molecular biology technique. However, the scientific debate is not close to be shut.


Sandrine Le Guellec

Laurine Allimonnier

Nathalie Heuzé-Vourc'h

Maria Cabrera

Frédéric Ossant

Jérémie Pourchez

Laurent Vecellio

Laurent Plantier
email: laurent.plantier@univ-tours.fr

Laurent Pr

Plantier

Low-frequency Intrapulmonary Percussive Ventilation Increases Aerosol Penetration in a 2-Compartment Physical Model of Fibrotic Lung Disease

Keywords: Pulmonary fibrosis, nebulization, physiology, compliance, aerosol, inhalation

In patients with fibrotic pulmonary disease such as idiopathic pulmonary fibrosis (IPF), inhaled aerosols deposit mostly in the less affected region of the lungs, resulting in suboptimal pharmacokinetics of airway-delivered treatments. Refinement of aerosol delivery technique requires new models to simulate the major alterations of lung physiology associated with IPF, i.e. heterogeneously reduced lung compliance and increased airway caliber. A novel physical model of the respiratory system was constructed to simulate aerosol drug delivery in spontaneously breathing (negative pressure ventilation) IPF patients. The model comprises upper (Alberta ideal throat) and lower airway (plastic tubing) models and branches into two compartments (Michigan lung models) which differ in compliance and caliber of conducting airway. The model was able to reproduce the heterogeneous, compliance-dependent reduction in ventilation and aerosol penetration (using NaF as a model aerosol) seen in fibrotic lung regions in IPF. Of note, intrapulmonary percussive ventilation induced a 2 to 3-fold increase in aerosol penetration in the low-compliance/high airway caliber compartment of the model, demonstrating the responsiveness of the model to therapeutic intervention.

Introduction

Fibrotic interstitial lung diseases (ILD) are a group of severe chronic non-communicable lung diseases where excessive deposition of abnormal extracellular matrix, in association with epithelial and/or endothelial lesions, results in progressive respiratory failure. Idiopathic pulmonary fibrosis (IPF) is the most common and the most severe idiopathic ILD [START_REF] King | Idiopathic Pulmonary Fibrosis[END_REF]. In IPF, fibrotic lesions follow the heterogeneous "usual interstitial pneumonia" pattern where fibrotic lesions alternate with preserved regions, and predominate in peripheral (supleural) areas.

Current treatments for IPF are unsatisfactory. Although two therapies, pirfenidone and nintedanib, both slow the decline of lung function [START_REF] Noble | Pirfenidone in Patients with Idiopathic Pulmonary Fibrosis (CAPACITY): Two Randomised Trials[END_REF][START_REF] King | A Phase 3 Trial of Pirfenidone in Patients with Idiopathic Pulmonary Fibrosis[END_REF]Richeldi et al. 2014) and may increase survival [START_REF] Canestaro | Drug Treatment of Idiopathic Pulmonary Fibrosis: Systematic Review and Network Meta-Analysis[END_REF], neither drug blocks or reverses the progress of disease and their tolerance is fair at best [START_REF] Fletcher | The Safety of New Drug Treatments for Idiopathic Pulmonary Fibrosis[END_REF]. There is a need for new drugs and treatment delivery routes for IPF and other ILDs.

IPF is spatially restricted to the lung, making it an ideal candidate disease for the use of topical, i.e. inhaled drugs. Aerosolized delivery through the airways has key advantages over the oral or injected routes for the administration of drugs to the lungs. Aerosolization is non-invasive and provides high therapeutic index. Aerosolized drug delivery is used routinely in patients with airway diseases. By contrast, the inhaled route is not currently used for the treatment of IPF, owing in part to low aerosol deposition in the lung.

IPF is associated with multiple alterations in lung structure with potential to hinder penetration of inhaled aerosols [START_REF] Plantier | Physiology of the Lung in Idiopathic Pulmonary Fibrosis[END_REF]). Fibrosis leads to major reductions in lung compliance (Zielonka et al. 2010) which are presumed heterogeneous given the spatial heterogeneity of lesions, and which result in reduced ventilation of the affected regions. Lung compliance is defined by the ratio of change in lung volume (V) over change in the transpulmonary pressure (Ptp). Besides fibrosis of alveolar regions, bronchial lesions may be observed whose nature is debated. Although the total number of airways in reduced in end-stage IPF (Verleden et al. 2020), the number of visible distal airways in increased in lungs with severe IPF (Verleden et al. 2020), while conducting airway volume is increased by 32% in patients with moderate IPF in comparison with control subjects (Plantier et al. 2016) and expiratory airflow is increased [START_REF] Plantier | Physiology of the Lung in Idiopathic Pulmonary Fibrosis[END_REF].

Anatomical and physiological alterations of the respiratory system in IPF may result in altered deposition of inhaled aerosols. Similar total lung deposition, as measured by concentrations in bronchoalveolar lavage fluid [START_REF] Khoo | A Randomized, Double-Blinded, Placebo-Controlled, Dose-Escalation Phase 1 Study of Aerosolized Pirfenidone Delivered via the PARI Investigational EFlow Nebulizer in Volunteers and Patients with Idiopathic Pulmonary Fibrosis[END_REF] or planar scintigraphy (Samuel and Smaldone 2019), can be achieved in IPF patients as compared to healthy subjects. However, planar scintigraphy images suggest that deposition of micrometric aerosols is not homogenous in some IPF patients, in contrast with healthy subjects [START_REF] Kanazawa | Assessment of pulmonary aerosol deposition and epithelial permeability in 99mTc-DTPA inhalation scintigram[END_REF][START_REF] Khoo | A Randomized, Double-Blinded, Placebo-Controlled, Dose-Escalation Phase 1 Study of Aerosolized Pirfenidone Delivered via the PARI Investigational EFlow Nebulizer in Volunteers and Patients with Idiopathic Pulmonary Fibrosis[END_REF]. Similarly, deposition of the nanometric aerosol Technegas was patchy and predominated in the upper regions of the lungs, which are typically the less damaged areas in 2 IPF patients (Watanabe et al. 1995). We hypothesize that reduced lung compliance results in reduced penetration and subsequent heterogeneous deposition of inhaled aerosols in IPF.

At present, inhalation devices and protocols allow high aerosol penetration and deposition in ventilated regions, but poor penetration and deposition in the regions of the respiratory system receiving little or no ventilation. The preferential deposition of aerosols in the less damaged areas needs to be overcome for satisfactory spatial targeting of inhaled therapeutics in IPF. It is therefore important to develop optimized aerosolization protocols allowing to target the low compliance alveolar regions in IPF. Models able to simulate IPF lungs under spontaneous ventilation are required to test the efficacy of such protocols.

Acoustic/pulse pressure waves hold potential to increase aerosol delivery in low compliance lungs. Indeed, acoustic waves increase aerosol penetration and deposition in the maxillary sinuses [START_REF] Durand | Sonic Aerosol Therapy to Target Maxillary Sinuses[END_REF][START_REF] Möller | Drug Delivery to Paranasal Sinuses Using Pulsating Aerosols[END_REF][START_REF] Leclerc | Impact of Acoustic Airflow on Intrasinus Drug Deposition: New Insights into the Vibrating Mode and the Optimal Acoustic Frequency to Enhance the Delivery of Nebulized Antibiotic[END_REF][START_REF] Moghadam | Toward Smart Nebulization: Engineering Acoustic Airflow to Penetrate Maxillary Sinuses in Chronic Rhinosinusitis[END_REF]. Maxillary sinuses are bone structures, which have near-null compliance and thus receive no ventilation during the respiratory cycle. Multiple experiments established that acoustic waves were most effective in the 100-300Hz frequency range, close to the resonance frequency of the maxillary sinuses [START_REF] Durand | Sonic Aerosol Therapy to Target Maxillary Sinuses[END_REF][START_REF] Möller | Drug Delivery to Paranasal Sinuses Using Pulsating Aerosols[END_REF][START_REF] Leclerc | Impact of Acoustic Airflow on Intrasinus Drug Deposition: New Insights into the Vibrating Mode and the Optimal Acoustic Frequency to Enhance the Delivery of Nebulized Antibiotic[END_REF][START_REF] Moghadam | Toward Smart Nebulization: Engineering Acoustic Airflow to Penetrate Maxillary Sinuses in Chronic Rhinosinusitis[END_REF].

In light of these elements, we hypothesized 1) that poor penetration of inhaled aerosols in low compliance lung regions could be reproduced in a two-compartment mechanical model of fibrotic lungs under negative-pressure (spontaneous) ventilation and 2) that acoustic pressure waves near the resonance frequency of the model would increase aerosol penetration in the low compliance compartment.

Materials and Methods

Construction of a two-compartment model of the respiratory system

A two-compartment model of the entire respiratory system (schematic in Figure 1) was constructed.

This model comprised a physical model of the upper airways (Alberta ldealised throat, Copley Scientific, UK), a plastic tube modeling the subglottic airways down to the 4 th bronchial division, and a Y-piece connecting each lung compartment. Each lung compartment included a plastic tube modeling the distal airways, and a mechanical lung model (Michigan Lung Model, Michigan Instruments, USA) modeling the alveolar regions. Each mechanical lung model comprised 1) a first bellows connected to the airway model described above ("active bellows") and 2) a second bellows ("passive bellows"), which was mechanically coupled to the first bellows, and was ventilated using a clinical ventilator (Servo 300, Siemens, Germany). Thus, the first bellows was ventilated under negative pressure, allowing to simulate physiological spontaneous breathing. Airway diameter and bellows compliance could be individually adjusted. To simulate normal lung, in the "Control compartment" airway diameter was 17 mm, and lung compliance was 100 ml/cm H 2 O. To simulate fibrotic lung, in the "Experimental compartment" airway diameter was 22 mm, and lung compliance was ≤100 ml/cm H 2 O. Overall, the volumes corresponded to those observed in an adult man. Filters (Anest-Guard, Teleflex, France) were inserted in each compartment, at the junction of the tube modeling the airways and the bellows modeling the alveolar regions.

Distribution of ventilation in the model

For characterization of ventilation distribution, the model was actuated under the following ventilator settings: Frequency=10/min, inspiratory pressure between 5 and 18 cm H 2 O, inspiratory time = 2 seconds. These settings simulate spontaneous breathing in an adult subject. Ventilation of each compartment was measured with spirometers (300 L/min, AD Instruments, Dunedin, New Zealand) inserted in place of the filters (Figure 1). In addition, the resonance frequency of the model were measured using a clinical forced oscillations device (Tremoflo, EMKA, France).

NaF aerosol delivery and quantitation of aerosol deposition

The model was connected to a jet nebulizer aerosol generator (Atomisor AOHBOX/ NL9M, DTF medical, France) at the upper airway opening. This generator delivers an aerosol with 4-5 µm mass median aerodynamic diameter. Inspiratory and expiratory valves were used. For all aerosol delivery experiments, the pulmonary model was actuated with the following ventilator settings to simulate tidal ventilation: Frequency = 10/min, inspiratory time = 2 sec, inspiratory pressure = 5 cm H2O.

Three ml of a 2.5% sodium fluoride (NaF) solution were nebulized over 10 to 15 min. After each nebulization session, the filters were dismounted. The filters were flushed with 30 ml of 2% vol./vol. aqueous total ionic strength adjustment buffer (TISAB) solution (Sigma-Aldrich, Saint Louis, USA) and incubated for 3 hours at room temperature. NaF was then assayed by electrochemical analysis (SevenGo Pro, Mettler Toledo, USA). The quantity of NaF aerosol deposited in each filter (Control or Experimental) was expressed as a fraction of the initial NaF dose that was loaded in the nebulizer.

Aerosol distribution in the model

Aerosol distribution was controlled for each nebulization experiments by dosing all parts of the model. Aerosol deposition was quantitated at the level of the nebulizer chamber, expiratory filter, construct, upper airway (Alberta throat model, trachea tube and Y-piece), and each lung compartment (tubing and filter). Each section was washed with 30-250 ml TISAB and NaF was assayed as described above.

Delivery of acoustic/pulse pressure waves

To generate acoustic pressure waves in the 20-500 Hz frequency range, sinusoidal signals were generated with VB Generator freeware (Https://Www.vb-Audio.Com/, n.d.), amplified (t.amp PM40C, Thomann, Germany), and transmitted to a vibration exciter (S-50009, Tira GmbH, Germany).

Amplifier gain was set so that acoustic pressure was identical across the frequency range, using a sonometer; 80 dB acoustic waves could be generated at all frequencies. To generate pulse pressure waves in the 1-10 Hz frequency range, a Pegaso (Dima Italia, Italy) intrapulmonary percussion ventilation device was used. IPV devices deliver small volumes of air; the shape of the signal is not sinusoidal (Toussaint et al. 2012). IPV pulse pressure was set at 40 cmH 2 0 and the inspiratory/expiratory ration was set to 1. In a first set of experiments, the vibration exciter and the IPV device were connected to the nebulizer/valves construct as shown in Figure 2A. In a second set of experiments, the IPV device was connected as shown In Figure 2B following optimization experiments. Both high-frequency acoustic waves and low-frequency IPV pulses were delivered to the model for the duration of nebulization.

Statistical analysis

All data were expressed as means and standard deviations. Group by group comparisons were performed with Student's t-test (2 groups) or ANOVA (more than 2 groups). Paired or unpaired analyses were done as appropriate. Relationships between variables were assessed by linear regression. Prism v5.0 (Graphpad, San Diego, USA) was used for all analyses.

Results

Ventilation and aerosol deposition in the model

Firstly, distribution of ventilation (tidal volume) between the two compartments of the models was assessed. As expected, tidal volume in both compartments of the model was a linear function of the pressure setting on the ventilator, which was applied to the passive bellows of the lung model, and of the compliance setting of the lung model (supplementary figure S1). For a given level of ventilation pressure and compliance, tidal volume was not significantly different between compartments.

Then, a NaF aerosol was delivered into the model while ventilated, and deposition onto the compartment filters, which assesses aerosol penetration into the respective compartment, was measured. When the compliance of both compartments was set at 100 ml/cmH 2 O, aerosol deposition in the Experimental compartment (then characterized only by 33% larger airway) trended to be slightly reduced in comparison with the Control compartment of the model (3.9+/-0.5% of the loaded dose versus 3.0+/-0.3%, p=0.06 by paired analysis -Figure 3A).

Then, aerosolization experiments were repeated when compliance of the Experimental compartment was decreased to 70, 50 and 30 ml/cmH 2 O. As shown in figure 3A, decreasing compliance of the Experimental compartment had two effects. First, an increase in aerosol penetration was observed in the Control compartment; this increase correlated negatively with compliance in the Experimental compartment (R 2 =0.48, p=0.012). Second, aerosol penetration decreased in the Experimental condition; this decrease followed a strong linear relationship with compliance (R 2 =0.96, p<0.0001).

Thus, these experiments demonstrated that decreasing lung compliance in one compartment of the system shifted penetration of an inhaled aerosol away from the low compliance compartment and into the compliant compartment. As shown in figure 1B, aerosol penetration in the Experimental compartment was strongly (R 2 =0.95, p<0.0001) associated with tidal volume, supporting the hypothesis that regional reduction in lung compliance and subsequent reduction in local ventilation are key drivers in reducing aerosol deposition in fibrotic lungs.

For further experiments, the compliance of the Experimental compartment was set at 50 ml/cmH 2 O, so that the model could detect both increases and decreases in aerosol penetration following delivery of acoustic/pulse pressure waves. Oscillometry parameters were determined under this setting. Under these conditions, the resonance frequency of the system was 5Hz.

20-500 Hz acoustic waves failed to increase aerosol penetration in the low compliance compartment

Audible acoustic waves are known to increase aerosol penetration in the maxillary sinuses [START_REF] Leclerc | Impact of Acoustic Airflow on Intrasinus Drug Deposition: New Insights into the Vibrating Mode and the Optimal Acoustic Frequency to Enhance the Delivery of Nebulized Antibiotic[END_REF]. Thus, in a first set of experiments, 80 dB/20-500 Hz acoustic waves were delivered to the nebulizer construct for the duration of nebulization using the construct shown in Figure 2A. As shown in Figure 4A, audible acoustic waves had no effect on aerosol penetration either in the Control or in the Experimental compartment, and the Experimental/control deposition ratio was unchanged.

In maxillary sinuses, matching acoustic wave frequency with the resonance frequency of the target structures results in increased aerosol deposition [START_REF] Durand | Sonic Aerosol Therapy to Target Maxillary Sinuses[END_REF]. Because the measured resonance frequency of the model (5 Hz) was much lower than 20 Hz, we raised the hypothesis that low-frequency pressure waves were required to increase aerosol deposition. To deliver such pressure waves, an intra pulmonary percussion ventilation (IPV) device was used. IPV devices offer important advantages in the context of aerosol deposition in ILD as 1) they are already certified for clinical use in humans including in association with inhaled aerosols, and 2) they have been shown to increase aerosol penetration in a simple one-compartment model independent of compliance [START_REF] Karashima | Variations in the Efficiency of Albuterol Delivery and Intrapulmonary Effects With Differential Parameter Settings on Intrapulmonary Percussive Ventilation[END_REF].

Low-frequency IPV pressure waves increase aerosol penetration 2-fold in the low compliance compartment

Initial experiments using IPV (3Hz, 40 cm H 2 O) delivered through the same nebulizer construct as the one used for audible acoustic wave delivery showed, in comparison with absence of pulse pressure waves, 1) that aerosol deposition in both the Control and Experimental compartments was decreased but 2) that the ratio of aerosol deposited in the Experimental / Control compartments increased from 0.16 to 0.41 (Supplementary Figure 2). Additional experiments were then conducted to evaluate the effect of IPV (3 Hz) delivered through different nebulizer constructs (supplementary Figure 3). As shown in Supplementary Figure 4A, aerosol deposition in the Experimental compartment was increased by IPV (3 Hz) in 2 out of 6 constructs. To verify that the increase in aerosol penetration was IPV-dependent and not only construct-dependent, aerosol penetration was measured in constructs 3 and 4 both with and without IPV (3 Hz). IPV increased aerosol penetration in the Experimental compartment in both constructs (supplementary Figure 4B). Construct 3 was retained for further experiments where the effect of IPV frequency on aerosol penetration was tested. As shown in Figure 4B, low-frequency IPV pressure waves in the 1-3 Hz frequency range increased aerosol penetration in the Experimental compartment. The increase was maximal (3-fold) for 1 Hz IPV. By contrast, 10 Hz IPV decreased aerosol penetration in the Experimental compartment. IPV did not significantly increase aerosol penetration in the Control compartment of the model although a trend was present (+29%, p=0.08).

Because low-frequency, high pressure IPV may be uncomfortable in man, an additional experiment was conducted using low-pressure (10 cmH 2 O) IPV. As shown in Figure 5, 1 Hz, 10 cmH 2 O IPV increased aerosol penetration in the Experimental compartment by 121% in comparison with no IPV.

Aerosol distribution in the model

In the normal lung model, 97.8+/-12.1% of aerosol was recovered (Table 1). 12+/-1.7% of aerosol deposited in the respiratory system (including upper airways and lung models) while 85.8+/-10.6%

were recovered outside the model (including nebulizer, expiratory filter and construct). When compliance was reduced in the experimental compartment to simulate IPF, 90.7+/-7.8% was recovered owing to reduced deposition in both lung and delivery apparatus. Under 1Hz-40 cmH 2 O IPV conditions, 95.5+/-17.2% of total aerosol was recovered. In addition to a 3-fold increase of aerosol deposition in the experimental compartment's filter, IPV induced increases in the construct and the throat model, as well as a ≈50% decrease in the expiratory filter.

Discussion

In this study, we developed a novel 2-compartment model of the respiratory system simulating fibrotic lung disease, and we showed that heterogeneous reductions in lung compliance resulted in 1) an increase of aerosol penetration in the preserved lung regions and 2) proportionate decreases in tidal volume and aerosol penetration in the less-compliant region. The strength of the associations between lung compliance, tidal volume and aerosol penetration support the hypothesis that reduced lung compliance is the main determinant of reduced aerosol deposition in fibrotic lung regions. Then, we used this model to explore whether acoustic/pulse pressure waves modulated aerosol penetration, and showed that low-frequency intrapulmonary pressure ventilation pulses reliably increased aerosol penetration in the low compliance compartment.

Although the total amount of aerosol-delivered drugs may be similar in patients with fibrotic lung disease and healthy controls at the level of the whole organ [START_REF] Diaz | Delivery and Safety of Inhaled Interferon-γ in Idiopathic Pulmonary Fibrosis[END_REF][START_REF] Khoo | A Randomized, Double-Blinded, Placebo-Controlled, Dose-Escalation Phase 1 Study of Aerosolized Pirfenidone Delivered via the PARI Investigational EFlow Nebulizer in Volunteers and Patients with Idiopathic Pulmonary Fibrosis[END_REF], imaging studies suggest that inhaled aerosols may mainly deposit into the less-affected regions of the lungs and thus presumably miss their cellular targets [START_REF] Kanazawa | Assessment of pulmonary aerosol deposition and epithelial permeability in 99mTc-DTPA inhalation scintigram[END_REF]Watanabe et al. 1995). Despite recent demonstration that small aerosol particles (1.5 µm diameter) and polydisperse aerosols should be preferred for IPF patients ("The Topical Study of Inhaled Drug (Salbutamol) Delivery in Idiopathic Pulmonary Fibrosis. -PubMed -NCBI" n.d.), preferential deposition of aerosols in the less damaged regions needs to be overcome. Optimizing spatial targeting is a major challenge to develop inhaled therapies for diseases which, like IPF, are characterized by heterogeneously distributed fibrotic lung lesions.

Experimental models are required to understand the mechanisms driving aerosol penetration and deposition in fibrotic lung disease. To this day, few studies were conducted in the aim of simulating both disease mechanics and aerosol deposition in fibrotic lungs under spontaneous ventilation. Montigaud et al. developed an ex vivo model combining heat-treated porcine lungs and a 3D-printed replica of the human airways [START_REF] Montigaud | Development of an Ex Vivo Preclinical Respiratory Model of Idiopathic Pulmonary Fibrosis for Aerosol Regional Studies[END_REF]. Although this model better simulates a branched airway anatomy, allows to replicate reductions in lung compliance predominating in subpleural regions, and can be used for aerosol deposition studies, it is unwieldy and cannot be reused for multiple experiments. To our knowledge, the mechanical model described herein is the first to offer the possibility to simulate the geometrical and physical characteristics of either mild or severe, restrictive or obstructive (using airflow resistances) lung disease in vitro, and to conduct replicated experiments of inhaled drug delivery. Moreover, the nebulized aerosol deposited in the whole of lower airways of the Normal lung model (10% of the nominal dose in trachea, Y-piece, both bronchus & compartments) was in accordance with most of clinical data's obtained in vivo, in healthy volunteers or in patients during inhalation experiments with jet nebulizer devices (Rau, 2005;dugermier, 2016). This suggests that this new in vitro model presents a good level of in vivo representiveness. However, the main limitation of the mechanical model lies in the fact that it is not clear whether a separate low compliance lung compartment would act the same as isolated low compliance hotspots embedded in otherwise normal lung parenchyma.

Our experiments support that heterogeneous reductions in lung compliance could play a role in the distribution of aerosol penetration in fibrotic lung disease. Indeed, we observed both increased aerosol penetration in the normal-compliance compartment, and reduced aerosol penetration in the reduced-compliance compartment. Although the clinical relevance of this finding remains to be defined, preferential deposition of inhaled drugs to preserved lung regions may result in a reduced therapeutic index and a requirement for higher doses. There is thus a need for inhaled drug delivery methods allowing to increase aerosol penetration and deposition in fibrotic, low compliance lung regions.

The requirement to deliver drugs to low compliance regions of the respiratory system led to the discovery that audible acoustic waves enhance deposition of nasally-delivered aerosolized drugs into maxillary sinuses [START_REF] Möller | Drug Delivery to Paranasal Sinuses Using Pulsating Aerosols[END_REF]. By analogy, we hypothesized that acoustic/pressure waves may increase aerosol penetration in the fibrotic lung model. Interestingly, although audible acoustic waves had no effect, contrary to sinus models, low-frequency IPV pulses had a marked effect and increased aerosol penetration 3-fold. Although the high variance in total mass balance mandates caution in the interpretation of aerosol deposition experiments, the effect of IPV on aerosol deposition in the experimental compartment was highly reproducible. This result is in line with the observation by others that IPV increases penetration of therapeutic aerosols in a single compartment mechanical model of the respiratory system, independent of model compliance [START_REF] Karashima | Variations in the Efficiency of Albuterol Delivery and Intrapulmonary Effects With Differential Parameter Settings on Intrapulmonary Percussive Ventilation[END_REF].

The mechanisms by which pressure pulses, such as those delivered by IPV, increase aerosol penetration remain unknown. These mechanisms can be discussed by analogy with acoustic waveenhanced delivery of aerosols from the nose to maxillary sinuses, which, like distal airspaces in fibrotic lungs, can be considered a low compliance cavity. A hypothesis is periodic compression of air by acoustic waves, generating bidirectional airflow (Xi et al. 2017). Under this hypothesis, a maximal effect is expected when transmission of the input pressure to the target cavity is maximal, that is, when the frequency of acoustic waves equals the resonance frequency of the target cavity. Indeed, matching soundwave frequency to resonance frequency of the maxillary sinuses (estimated with the Helmhotz resonator model) increases aerosol delivery [START_REF] Leclerc | Impact of Acoustic Airflow on Intrasinus Drug Deposition: New Insights into the Vibrating Mode and the Optimal Acoustic Frequency to Enhance the Delivery of Nebulized Antibiotic[END_REF][START_REF] Durand | Sonic Aerosol Therapy to Target Maxillary Sinuses[END_REF], while frequency sweeps bracketing the estimated resonance frequency of the sinus yield optimal results [START_REF] Moghadam | Toward Smart Nebulization: Engineering Acoustic Airflow to Penetrate Maxillary Sinuses in Chronic Rhinosinusitis[END_REF]. Thus, we used the Helmhotz resonator model to estimate the resonance frequency of fibrotic lungs.

The Helmhotz resonator is defined by the volume of its cavity (V), and the length (l) and area (a) of the tube that connects it to outside air. The resonance frequency of the system can be calculated from V, l [START_REF] Durand | Sonic Aerosol Therapy to Target Maxillary Sinuses[END_REF]. When using this model to describe fibrotic lung regions in IPF, we assumed V to be within 1 and 3 liters, l as equal to 27 cm based on the sum of the average lengths of the upper airways (7 cm) and of the bronchial tree down to segmental bronchi (20 cm), and a as equal to 1.65 cm2 based on 1) morphometric data of normal lungs (Crystal, West, and Weibel, n.d.) , 2) the 1.32 increase in airway volume observed in IPF (Plantier et al. 2016) and 3) the assumption that fibrotic lung regions comprise half of the lungs. These assumptions lead to estimation that the resonance frequency of fibrotic lung may range from 23.8 Hz to 41.3 Hz.

At variance with the hypothesis that acoustic waves emitted at the resonance frequency of the target may increase aerosol penetration, we observed that audible acoustic waves had no effect. Rather, increased aerosol penetration was observed using low-frequency (≤3 Hz) IPV. Whether it is important that acoustic/pressure pulse frequency is inferior to the resonance frequency of the system (here, 5 Hz) remains to be explored. Interestingly, in a 2-bottle physical model, maximal aerosol penetration aerosol is observed with 100 Hz acoustic waves, while the estimated resonance frequency of the model cavity is 303 Hz (Xi et al. 2017). The mechanisms contributing to IPV-increased aerosol penetration thus remain to fully elucidate. Acoustic radiation pressure, which is well described in the ultrasonic frequency range [START_REF] Foresti | Acoustophoretic Contactless Transport and Handling of Matter in Air[END_REF] and is also physically relevant for low-frequency (trending towards 0 Hz) acoustic waves [START_REF] Löfstedt | Theory of Long Wavelength Acoustic Radiation Pressure[END_REF], may be a candidate mechanism. Importantly, it is unclear how the frequency of the pressure waves that dissipate in the airway relates to the frequency IPV volume/pressure pulses.

There is currently great interest in the development of inhaled therapies for IPF and other ILD.

Inhaled antifibrotic treatments would represent a key advance in IPF. Although several inhaled drugs were or are currently being evaluated in clinical trials in IPF, such as glutathione [START_REF] Borok | Effect of Glutathione Aerosol on Oxidant-Antioxidant Imbalance in Idiopathic Pulmonary Fibrosis[END_REF], heparin [START_REF] Markart | Safety and Tolerability of Inhaled Heparin in Idiopathic Pulmonary Fibrosis[END_REF], interferon- [START_REF] Diaz | Delivery and Safety of Inhaled Interferon-γ in Idiopathic Pulmonary Fibrosis[END_REF]) and pirfenidone [START_REF] Khoo | A Randomized, Double-Blinded, Placebo-Controlled, Dose-Escalation Phase 1 Study of Aerosolized Pirfenidone Delivered via the PARI Investigational EFlow Nebulizer in Volunteers and Patients with Idiopathic Pulmonary Fibrosis[END_REF], none has been successful so far.

IPV is a respiratory physiotherapy technique that consists in delivery of small volumes of air into the airways, typically in the 1-10Hz frequency and 10-50 cmH 2 O pressure ranges (Reychler et al. 2018). It is currently used to mobilize secretions, promote lung recruitment and improve gas exchange in chronic conditions such as neuromuscular diseases, cystic fibrosis, and chronic obstructive pulmonary disease. Although previous studies into IPV-coupled nebulized delivery were disappointing (Reychler et al. 2004), our results suggest that IPV may be repurposed to improve aerosolized drug delivery in the clinic.

Whether low-frequency (≤3Hz) IPV is a feasible option to improve aerosol delivery to patients with IPF depends on a number of issues, including 1) whether it does improve aerosol delivery to low compliance lung regions in vivo, 2) whether it can be tolerated by IPF patients and 3) whether it is innocuous. Although IPV appears to be well tolerated at frequencies in the order of 4-6 Hz [START_REF] Nava | Physiological Response to Intrapulmonary Percussive Ventilation in Stable COPD Patients[END_REF][START_REF] Paneroni | Safety and Efficacy of Short-Term Intrapulmonary Percussive Ventilation in Patients with Bronchiectasis[END_REF], whether low-frequency is tolerated for the duration required for nebulized drug delivery remains to explore. Since patient discomfort is associated with IPV pressure, our observation that 10cmH 2 O did increase aerosol penetration in the low-compliance compartment suggests that this method may be usable in patients. Although IPV appears to be generally safe [START_REF] Paneroni | Safety and Efficacy of Short-Term Intrapulmonary Percussive Ventilation in Patients with Bronchiectasis[END_REF], it is essential to assess whether IPV-associated volutrauma or barotrauma is deleterious in IPF patients. Notably, whether IPV can induce IPF exacerbations will need to be ruled out before clinical application. IPF exacerbations are life-threatening events which can be triggered by medical procedures such as mechanical ventilation and lung surgery (Ryerson et al. 2015). Adverse effects of VPIP are rare in healthy subjects and patients with obstructive lung disease, with cases of pneumothorax and proximal airway obstruction attributed to handling error [START_REF] Flurin | Interest of Percussionator in neuromuscular patients. Eight years experience in three quadriplegic ventilator-dependant children[END_REF].

Conclusion

Using a mechanical model of fibrotic lung disease, we observed that reduced compliance of target lung regions is a critical determinant of aerosol penetration, and thus presumably of aerosol deposition. This finding underscores the need for techniques specifically tailored to enhance delivery of inhaled aerosols to low compliance regions of the lungs, if fibrotic lung disease is to be successfully treated with inhaled therapies. Low-frequency (≤3 Hz) IPV pulses may represent an attractive option to increase aerosol deposition in fibrotic lung regions. 1,7 ± 0,3 % 0,9 ± 0,3 % 0.009 4,0 ± 1,1 % 0.002 Trachea and Y-piece 0,6 ± 0,3 % 0,6 ± 0,3 % 0.910 0,5 ± 0,1 % 0.352 Control bronchus 0,4 ± 0,3 % 0,4 ± 0,1 % 0.580 0,3 ± 0,2 % 0.253 Control compartment 4,6 ± 1,2 % 5,5 ± 0,6 % 0.578 7,4 ± 1,9 % 1.000 Experimental bronchus 0,4 ± 0,3 % 0,2 ± 0,1 % 0.219 0,2 ± 0,2 % 0.079 Experimental compartment 4,4 ± 1,6 % 0,7 ± 0,2 % 0.020 2,2 ± 0,5 % 0.002 TOTAL 97,8 ± 12,1 % 90,7 ± 7,8 % 0.899 95,5 ± 17,2 % 0.626

  alterations of lung geometry (increased volume of conducting airways) and physiology (reduced compliance) of fibrotic lungs. The model reproduced the reduced penetration of model aerosols observed in man. The model was then used to demonstrate how low-frequency intrapulmonary percussive ventilation, a physiotherapy technique used for airway clearance in chronic respiratory disease, increased aerosol penetration in low compliance regions of the respiratory system.

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Constructs used for acoustic/pulse pressure wave delivery during nebulization. All constructs are connected to the throat model (right). A: Construct used for acoustic wave delivery and initial IPV delivery. B: Construct used for multiple-frequency IPV delivery.

Figure 3 .

 3 Figure 3. Aerosol deposition in the Control (open circles) and Experimental (filled circles) as a function of A: Compliance of the Experimental compartment and B: Tidal volume in the Experimental compartment. Compliance was set at 100 ml/cmH2O in the Control compartment). *: p<0.05. Regression lines are shown. N=3. Means and SD.

Figure 4 .

 4 Figure 4. Aerosol deposition in the Control (100 ml/cmH 2 O compliance, open circles) and Experimental (50 ml/cmH 2 O compliance, filled circles) compartments of the model. A: 20-500 Hz audible acoustic waves (n=3) and B: 1-10 Hz IPV pressure pulses (n=6) were delivered during nebulization. Dotted lines show the control condition (no sound or no IPV). *: p<0.05 versus no IPV. Means and SD.

Figure 5 .

 5 Figure 5. Aerosol deposition in the Control (100 ml/cmH 2 O compliance, open circles) and Experimental (50 ml/cmH 2 O compliance, filled circles) compartments of the model. Aerosol was delivered with either no IPV or 1 Hz IPV at different pressure levels. *: p<0.05 vs no IPV.

Table 1 .

 1 Mass balance of aerosol deposition experiments. Deposition in the normal lung model (compliance of both compartments set at 100 ml/cmH 2 O) was compared with the fibrotic lung model (compliance of the Experimental compartment set at 50 ml/cmH 2 O) without IPV. Deposition in the fibrotic model with 1Hz-40cmH 2 O IPV was compared with the fibrotic lung model without IPV.Aerosol was expressed as % of the dose loaded in the nebulizer.

		Normal lung	Fibrotic lung model, no IPV	Fibrotic lung model with1Hz-
		model (n=3)	(n=6)		40cmH 2 O IPV (n=6)
	Deposition site	Deposition	Deposition	p value vs Normal	Deposition	p value versus Fibrotic
	Expiratory filter	27,9 ± 6,7 %	32,2 ± 4,4 %	0.651	17,8 ± 5,3 %	0.001
	Construct	7,1 ± 1,6 %	4,8 ± 1,5 %	0.530	14,1 ± 4,2 %	0.009
	Nebulizer	50,7 ± 8,0 %	45,5 ± 5,9 %	0.896	49,1 ± 7,8 %	0.509
	Alberta throat

Acknowledgments

The authors thank LVL medical for providing home ventilators used in preliminary experiments as a loan, and Resmed France for providing the IPV generator as a loan.

Funding

This work was funded by a grant from Hôpitaux Universitaires du Grand Ouest (HUGO) and Fondation Maladies Rares. European Respiratory Review: An Official Journal of the European Respiratory Society 27 (147). https://doi.org/10.1183/16000617.0062-2017. Plantier, Laurent, Marie-Pierre Debray, Candice Estellat, Martin Flamant, Carine Roy, Catherine Bancal, Raphaël Borie, et al. 2016. "Increased Volume of Conducting Airways in Idiopathic Pulmonary Fibrosis Is Independent of Disease Severity: A Volumetric Capnography Study." Journal of Breath Research 10 (1): 016005. https://doi.org/10.1088/1752-7155/10/1/016005. Reychler, Gregory, Emilie Debier, Olivier Contal, and Nicolas Audag. 2018. "Intrapulmonary Percussive Ventilation as an Airway Clearance Technique in Subjects With Chronic Obstructive Airway Diseases." Respiratory Care 63 (5): 620-31. https://doi.org/10.4187/respcare.05876. Reychler, Gregory, André Keyeux, Caroline Cremers, Claude Veriter, Daniel O. Rodenstein, and Giuseppe Liistro. 2004. "Comparison of Lung Deposition in Two Types of Nebulization: Intrapulmonary Percussive Ventilation vs Jet Nebulization." Chest 125 (2): 502-8. https://doi.org/10.1378/chest.125.2.502. Richeldi, Luca, Roland M. du Bois, Ganesh Raghu, Arata Azuma, Kevin K. Brown, Ulrich Costabel, Vincent Cottin, et al. 2014. "Efficacy and Safety of Nintedanib in Idiopathic Pulmonary Fibrosis." The New England Journal of Medicine 370 (22): 2071-82. https://doi.org/10.1056/NEJMoa1402584. Ryerson, Christopher J., Vincent Cottin, Kevin K. Brown, and Harold R. Collard. 2015. "Acute Exacerbation of Idiopathic Pulmonary Fibrosis: Shifting the Paradigm." The European Respiratory Journal 46 (2): 512-20. https://doi.org/10.1183/13993003.00419-2015. Samuel, Joshua, and Gerald C. Smaldone. 2019. "Maximizing Deep Lung Deposition in Healthy and Fibrotic Subjects During Jet Nebulization." Journal of Aerosol Medicine and Pulmonary Drug Delivery, December. https://doi.org/10.1089/jamp.2019.1552. "The Topical Study of Inhaled Drug (Salbutamol) Delivery in Idiopathic Pulmonary Fibrosis. -PubMed -NCBI." n.d. Accessed October 19, 2019. https://www-ncbi-nlm-nihgov.gate2.inist.fr/pubmed/?term=29409488. Toussaint, Michel, Marie-Charlotte Guillet, Stéphanie Paternotte, Philippe Soudon, and Jurn Haan. 2012. "Intrapulmonary Effects of Setting Parameters in Portable Intrapulmonary Percussive Ventilation Devices." Respiratory Care 57 (5): 735-42. https://doi.org/10.4187/respcare.01441. Verleden, Stijn E., Naoya Tanabe, John E. McDonough, Dragoş M. Vasilescu, Feng Xu, Wim A. Wuyts, Davide Piloni, et al. 2020. "Small Airways Pathology in Idiopathic Pulmonary Fibrosis: A Retrospective Cohort Study." The Lancet. Respiratory Medicine 8 (6): 573-84. https://doi.org/10.1016/S2213-2600(19)30356-X. Watanabe, N., T. Inoue, S. Tomioka, T. Yamaji, and K. Endo. 1995. "Discordant Findings between Krypton-81m Gas and Tc-99m Labeled Ultrafine Aerosol Lung Ventilation SPECT in Two Patients with Idiopathic Pulmonary Fibrosis." Clinical Nuclear Medicine 20 (4): 315-17. https://doi.org/10.1097/00003072-199504000-00006. Xi, Jinxiang, Xiuhua April Si, Shannon Peters, Dannielle Nevorski, Tianshu Wen, and Mark Lehman. 2017. "Understanding the Mechanisms Underlying Pulsating Aerosol Delivery to the Maxillary Sinus: In Vitro Tests and Computational Simulations." International Journal of Pharmaceutics 520 (1-2): 254-66. https://doi.org/10.1016/j.ijpharm.2017.02.017. Zielonka, Tadeusz M., U. Demkow, E. Radzikowska, B. Bialas, M. Filewska, K. Zycinska, M. H. Obrowski, J. Kowalski, K. A. Wardyn, and E. Skopinska-Rozewska. 2010. "Angiogenic Activity of Sera from Interstitial Lung Disease Patients in Relation to Pulmonary Function." European Journal of Medical Research 15 Suppl 2 (November): 229-34.

Conflicts of interest

SLG is an employee of DTF medical. LV is currently an employee of Nemera.

Authors' contribution

LA, SLG, and MC conducted experiments and analyzed results. LV, FO, SLG, NHV, JP provided scientific input into acoustic/pulse wave physics and contributed to experiment design. LP coordinated the overall project, designed and analyzed experiments, and drafted the manuscript. All authors reviewed and approved the manuscript.

Contribution to the field statement

Inhaled drug delivery is a mainstay of treatment for obstructive lung disease but is not currently used in the context of fibrotic interstitial lung disease, due to insufficient deposition of inhaled aerosols in diseased lung regions. In this work, we report the development of a novel two-compartment mechanical model of the respiratory system that allows the simulation of the heterogeneous