Abstract:
NLR family proteins are cytoplasmic receptors responsible for the regulation of inflammation. Whereas some NLRs such as Cryopyrin form protein complexes recognizing pathogens and activating inflammation called the inflammasome; the function of other NLRs is still to be characterized. NLRC3, one of the less studied members of this family, was found to be a negative regulator of T-cell activation, NFκB pathway and STING-dependent cytokine secretion. In this thesis, we investigate the role of NLRC3 in the regulation of the Cryopyrin inflammasome, in cell proliferation and its possible involvement in immune tolerance mechanisms. By overexpression and knock-down analyses, we identified whether NLRC3 inhibits Cryopyrin-induced IL-1β cleavage and secretion in response to different stimuli. We determined that NLRC3 exerts its suppressive effect downstream of Cryopyrin by interacting with diffused ASC protein and pro-Caspase-1 via their respective CARD domains and thus may disrupt complex assembly. Secondly, potential regulation of stem cell proliferation by NLRC3 was clarified. Nlrc3 was gradually silenced in more proliferative Apc and Apc Kras organoids. Nlrc3 KO primary tissues were generated using CRISPR/Cas9 techniques and organoid formation capacity of Nlrc3 KO, KD and overexpressing cells were measured. Suppression of Nlrc3 expression increased cell proliferation and organoid formation whereas the opposite phenotype was observed in NLRC3 overexpressing cells. We showed that Nlrc3 KO increases the expression of stem cell (Lgr5) and cell cycle (CyclinD1) markers. Finally, we showed that NLRC3 could be involved in immune tolerance mechanisms since it is expressed in cell lines and tissues from these sites. Furthermore, NLRC3 localized in the nucleus, suggesting that it could act as a transcription factor.
