The considerable and rising impact of respiratory diseases worldwide, such as the COVID-19 pandemic, highlights the need to improve in vitro models to predict better and treat such diseases. The Print@Lung project aims to develop a physiologically relevant in vitro model of the human lung for studying host-pathogen interactions and validating therapeutic approaches. The human lung's intricate architecture and biological composition pose a substantial challenge for in vitro modelling. While current advancements in 3D culture techniques, like organoids, show promise, they still lack key elements such as a 3D architecture, vascular network, and immune component. The multidisciplinary Print@Lung consortium aims to recreate lung tissue's complex three-dimensional, multicellular structure using 3D bioprinting technology. The project seeks to accomplish three main objectives : 1) generate a fully functional, differentiated respiratory epithelium at a physiological scale, which 2) integrate vascularized connective tissue and cells specific to the lung immune system, while reproducing the architecture of the different zones of the respiratory tract (alveoli vs bronchioli), 3) utilize this model to investigate host responses to bacterial (S. aureus and P. aeruginosa) and viral (Sars-Cov2) infections, as well as evaluate the effectiveness of various antiviral strategies. Our model functionality will be assessed and compared with reference ALI cultures. The development of such a 3D physiological lung model will provide a valuable tool for understanding the mechanisms of infection, predicting the pathogenicity of emerging microbial strains, and validating the efficacy of treatments. In addition, this complex tissue model is in line with the 3Rs approach, offering an alternative to in vivo experimentation. And over the long term, PRINT@LUNG will forge new avenues for biomedical research, not only in infectious diseases but also in cancer, chronic respiratory diseases, and genetic disorders.

Therapeutic options in response to the SARS-Cov-2 outbreak are urgently needed and existing one are still limited. Facing pandemic development, actual strategy has been to repurpose some existing antiviral drugs used against respiratory diseases (SARS-Cov-1, MERS, Influenza) or chronic diseases (HIV-1, HCV, HBV, etc…). However, side effects are common and required short treatment mainly through intravenous injection, with a non optimal dose, and rarely through pulmonary route. A better formulation of such drug, adapted to this pulmonary disease, will allow an optimal efficacy of current drug or new candidates, such as protease inhibitors. Nanomedicine tools, such as used in HIV-1 treatment, could provide some solution by proposing innovative formulation, leading to the same efficacy with low dose of drugs and nasal/pulmonary delivery. Furthermore, nanoparticulate form favors drug stability, when present in aerosol via sprays or nebulizers. Thus, the CoviNanoMed project aims to formulate and test potential antiviral drugs or Host Targeted Agents using an existing robust nanoparticle platform in order to increase their potential efficacy, while diminishing their side effects. To this aim, we have identified six promising candidates, (non-exhaustive list which could be modulated according to ongoing clinical trials) and have gather four complementary research groups to: i) select the most promising for nanoformulation through in silico modeling and prepare reproducible and stable batch of nanodrugs ii) Assess their antiviral activities and toxicities using state of the art technique, adapted to their nanoparticulate form. It will allow us to classify them and to integrate in an iterative manner new ones according to collaborators iii) Compare nasal and pulmonary delivery in mice through devices used in clinical setting. We will use fluorescent particles and whole body imagine in mice and Non Human Primates to analyze the fate of particles in lung cells and tissue, and iv) Analyse drug release in bronchoalveolar fluids in mice and the most promising formulations in Non Human Primate. As this project concerns existing drugs, the analytical protocols and methods are already determined and will be quickly available. By this project, we expect to identify at least one promising candidate that could be quickly moved to clinical trial, as the biodegradable platform we propose will be considered as a new formulating excipient. Our system is solely based on poly–lactic acid (PLA), a biodegradable polymer approved by the FDA for medical devices such as sutures. It is therefore free of any controversial surfactant that frequently prevent the development of nanosystems to late phases. Additionally, the methodology we have set up could be adapted to new compounds that will emerge through ongoing clinical trials such as the multiple protease inhibitors that are undergoing studies.

The Nipah and Hendra viruses (NiV & HeV) (Henipavirus genus) are zoonotic pathogens responsible for severe encephalitis in humans. Their natural reservoir are fruit bats. Inter-human transmission of NiV and circulation of Henipaviruses in bat species from a growing number of countries, constitute a serious threat to human health. Although an efficient vaccine against HeV in horses is available, neither vaccines nor therapeutic treatments are available in humans against Henipaviruses. The high pathogenicity, wide host range and interspecies transmission of NiV & HeV led to their classification as BSL4 pathogens and potential bio-terrorism agents. The prevention and/or the containment of present and future epidemics will depend on our capacity to conceive effective strategies to combat these viruses. The long-term goal of this project is to shed light onto the molecular mechanisms of Henipavirus pathogenesis as a prerequisite for the rational design of future therapeutic approaches. Their V and W proteins are key players in the evasion of the host innate immune and inflammatory response. V and W share an N-terminal intrinsically disordered (ID) domain (NTD) and have distinct C-terminal domains. A region of HeV NTD was found to confer to V the ability to undergo a liquid to gel phase transition accompanied by the formation of amyloid-like fibrils. Congo red staining of transfected or infected cells suggests fibril formation also in cellula, and fluorescence microscopy showed that W forms condensates in the nuclei of transfected cells. Our hypothesis is that the phase-separated condensates formed by the V/W proteins may sequester key cell proteins involved in the cell innate immune and inflammatory response thereby contributing to the high pathogenicity of these viruses. Our aim is to further investigate the abilities of these proteins to phase separate and fibrillate and to shed light onto their functional implications. We will combine in vitro (Partner 1) and in cellula (Partners 2 & 3) studies. In vitro studies will use purified wt and mutated proteins and will enable deciphering the molecular and sequence determinants governing the ability of V/W proteins to phase separate and fibrillate. In cellula studies will imply V/W transient expression and infection experiments followed by quantitative cell imaging, interaction studies with cellular proteins of the innate immune pathway and measurements of IFN-stimulated genes & chemokines responses. V/W transient expression and infection studies will also use bat cells, which will enable unveiling possible differences between human and bat cells, thus potentially providing hints on the mechanisms by which bats efficiently control Henipavirus infection. We will also investigate the ability of well-known inhibitors of protein fibrillation to block Henipavirus V and W fibrillation and possibly hamper the adverse effects of V/W proteins on cell functions. Partner 1 is an expert in the structure-function relationships of ID regions from paramyxoviral proteins, Partner 2 is an expert of Henipavirus infection, bat cells engineering & innate immune response, and Partner 3 developed innovative approaches to image and quantify viral amyloids in infected cells. The strength of this project lies in (i) its feasibility (availability of multiple preliminary data & tools already generated by the partners), (ii) the expertise and complementarity of the partners, (iii) its originality (very few studies have investigated the impact of phase separation by viral proteins on host functions and even fewer have described amyloidogenic viral proteins), (iv) its potential to unveil a new molecular mechanism underlying Henipavirus pathogenesis, (v) to shed light on the molecular basis by which bats control Henipavirus infection, (vi) to illuminate the relationships between intrinsic disorder, phase transitions and amyloid formation and (vii) to set the stage for the development of new antiviral strategies.

Lungs are constantly exposed to the external environment, the infectious and toxic agents present in the air. Both viral and bacterial pathogens trigger damage to the lung epithelial cells leading to the alteration of the respiratory efficiency and in some cases to severe illness of the affected person or animal. Understanding the physio-pathological processes of infection remains one of the main goals to control and treat the diseases. Those threats represent major health and economic issues for cattle and among them, bovine tuberculosis and bronchopneumonia are two major pathologies. These bovine diseases have their human counterparts, namely bronchiolitis in infants and human tuberculosis. Being able to predict the pathogenicity of different circulating or emerging strains of virus or bacteria, could help to better apprehend the disease and develop preventive or curative measures. To better understand the determinants of the infectious process, to study the cell-pathogen interactions and to be able to predict the virulence of respiratory pathogens, it is necessary to have relevant and innovative study models. Indeed, very few reliable in vitro models mimic the lower respiratory tract and more precisely the alveolar compartment, the offensive site of these pathogens. In order to specifically study the reactivity of pulmonary epithelial cells to respiratory bacteria and virus and their involvement in the infectious process, we propose to develop alternative in vitro models using the stem cell plasticity to the in vivo experimental approaches. The EpiLungCell main aims are: i) to develop new in vitro cellular models mimicking the different stages of lung epithelial state of differentiation, from iPS (induced Pluripotent Stem) and eIP (induced Epithelial Progenitor) cells to mature bronchiolar and alveolar epithelial cells. ii) to challenge these cells with Respiratory Syncytial Virus (RSV), and Mycobacterium bovis (Mb) pathogens and to investigate the functional consequences of the infection on their physiology. iii) to propose those substrates as tools to evaluate the pathogenicity of new viral or bacterial respiratory strains. The EpiLungCell project takes place over 3 years and is divided into four work packages (WP). The first WP aims to establish new cellular substrates with original epithelial phenotypes. The physiological response of these substrates to infection by bacteria such as Mb (second WP) or by the RSV virus (third WP) will be studied and compared at the molecular and cellular levels with what is observed in cells directly derived from animal models. Finally, in a fourth and final WP, these new substrates will be developed and used for diagnostic purposes to identify and predict the pathogenicity of new strains identified and isolated directly from the field. The development and the use of these cellular substrates also aim to reduce the use of animal models in the study of these pathogens. The main results of the proposal will be the production of new cell lines, in particular lung epithelial cells, a better understanding of the mechanisms of infection by these pathogens and the development of new diagnostic tools. The two aspects of basic knowledge with cell response to infection and of applied science with the diagnostic tool will thus be satisfied. Together, it will provide a better understanding of the physiology of the pulmonary epithelium, its characterization, its susceptibility to infectious agents and its alterations during the infectious processes.

Pulmonary hypertension (PH) is a deadly enigmatic disease with substantial unmet medical needs. PH is characterized by the dysregulation of multiple vascular cell types and is accompanied by inflammation and fibrosis throughout the vasculature. We reported that activation of resident adventitial fibroblasts drives extracellular matrix (ECM) remodeling and vascular stiffness, leading to pulmonary vascular dysfunction. Beyond collagen and elastin production, activated fibroblasts produce and secrete hundreds of ECM proteins. Whether and how these proteins affect vascular wall structure and thus reprogram vascular cells to promote PH remains unknown. Using a quantitative label-free proteomics screening, we identified fibulin-2 (FBLN2) as an essential ECM protein modulated in multiple PH models and human subjects. We also made the unexpected discovery that FBLN2 modulation does not affect matrix stiffness, but reducing the FBLN2 protein level in the ECM broadly rewires the metabolism of vascular cells. Therefore, using a unique combination of primary pulmonary vascular cells, gold-standard preclinical models of PH, and human tissues, we propose to comprehensively decipher the role of FBLN2 in PH and associated metabolic disorders.