The objective of our project is on the one hand to characterise, in a SARS-CoV-2 infected human pulmonary organoids, the molecular determinants involved in the pro-inflammatory response, including rate and kinetics of IFN type-I and type III production on the other hand to understand the influence of age and sex on SARS-CoV-2-induced inflammatory response. To this end we will use primary human bronchial organoid designed to mimic the essential of lung structures and the physiological environment conditions. In our preliminary results, we have validated the production methodology and the permissivity of bronchial organoïds to SARS-CoV-2. Using this model, we plan to address important questions in the first task including: to characterize the profile, rate and kinetics of pro-inflammatory and anti-inflammatory cytokine production during infection with the SARS-CoV-2 virus. Because our pulmonary organoid model contains essentially epithelial cells, we will focus essentially on the study of the rate and kinetics of IFN-III and pro-inflammatory cytokines / chemokines including IL-6, TNF-a, IL-8 and IP-10. By using lung organoids prepared from different age (young 60) or gender (male or female) we will measure if there is a relation between the age and gender for the rate and kinetics of infection-induced pro-inflammatory cytokine production that can help to understand protection or susceptibility markers to develop severe form of COVID-19 ? Other questions will be to study the effect of infection-induced cytokine production (including IFN-III) on the control of SARS-CoV-2 replication, IFNAR (IFN-I receptor) and IFNLR (IFN-III receptor), expression level of ACE2/TMPRSSII viral receptors, innate immune viral sensor (TLRs, RLRs) expression and tissue damage. In the second task, we will analyze the innate immune receptors involved in SARS-CoV-2 sensing and the subsequent pro-inflammatory response. This will be investigated in A549 pulmonary cell line by both pharmacological and genetic approaches using CRISPR/Cas9 system. In the third task, the identified candidate sensors of SARS-CoV-2 (TLR, RLR e.g.) will be challenged in the more relevant physiological model of pulmonary organoid and their involvement in infection-induced inflammation confirmed after their invalidation by CRISPR/Cas9 approach. In parallel, we will test the effect of CPG-52364, a phase-I approved TLR8 inhibitor, and Salazopyrine and Leflunomide, two inhibitors of NF-kB approved by the FDA and currently used in the treatment of Arthritis, for their action on the SARS-CoV-2-induced pro-inflammatory cytokines. Ultimately, this project by using SARS-CoV-2/human pulmonary organoid model will help to better understand the molecular and cellular mechanisms involved in the induction of the hyper-inflammation state, including IFN-III, a deleterious response associated with COVID-19 disease and could contribute to the development of new therapeutic strategies to treat, in particular, against the most severe forms of the disease.