Negative Regulation Of Immune Response By LRRFIP2,CD11b And The Underlying Mechanisms

Part Ⅰ Leucine-Rich Repeat Fli-Ⅰ-interacting Protein2(LRRFIP2) negatively regulates NLRP3inflammasome activity by facilitating inhibitory function of Flightliess-ⅠThe NLRP3inflammasome is the most characterized inflammasomes, which are platforms activated by cellular infection or stress and trigger the maturation of proinflammatory cytokines such as interleukin-1β (IL-1β). However, the molecular mechanism of NLRP3inflammasome activation, especially the negative regulation, remains to be further explored. Using Co-Immunoprecipitation and following LC-MS/MS, we investigated NLRP3inflammasome interacted proteins and found Leucine-Rich Repeat Fli-I-interacting Protein2(LRRFIP2) silencing promoted NLRP3inflammasome activity in mouse primary peritoneal macrophages. Co-Immunoprecipitation experiment indicated LRRFIP2could bind to NLRP3via its N-terminal motif upon NLRP3inflammasome related stimulation. Overexpression experiments in THP-1cells indicated that the N-terminal motif and Coil motif of LRRFIP2were required for its inhibitory function in NLRP3inflammasome. LRRFIP2could interact with Flightless-I via its Coil motif, and knocking down Flightless-I promoted inflammasome activation in mouse primary peritoneal macrophages. Co-Immunoprecipitation experiments suggested LRRFIP2might enhance the interaction between Flightless-I and NLRP3inflammasome, which facilitate the inhibitory function of Flightliess-Ⅰ on NLRP3inflammasome. Furthermore, the silencing of Flightless-Ⅰ abrogated the inhibitory function of LRRFIP2in NLRP3inflammasome. These data established a new regulation pathway that LRRFIP2helped Flightless-Ⅰ to interact with and to inhibit the activity of NLRP3inflammasome.Part Ⅱ Integrin CDllb negatively regulates inflammatory response via promoting the production of IL-10in macrophagesIntegrins play critical role in the migration and function of leukocytes in inflammation. However, the interaction between integrin aM (CD11b), which has high expression in monocytes and macrophages, and expression of IL-10triggered by Toll-like receptors (TLRs) remains unclear. Here we found that peritoneal macrophages from CDllb deficient mice produce less IL-10cytokine in response to TLR activation than the macrophages from its littermate control. CD11b promotes the TLR-triggered activation of tyrosine kinase Src. Src interacted with and induced phosphorylation of PI3K regulatory subunit p85, which led to its degradation by the E3ubiquitin ligase c-Cb1. The p85subunit degradation activates the PI3K/Akt signal pathway and triggers its downstream kinase GSK3α/β phosphorylation. The phosphorylated forms GSK3a/(3abrogate its inhibitory function on IL-10gene transcript factor CREB. Thus, CDllb integrin engages in crosstalk with the Src and PI3K pathways and subsequently promotes TLR signaling activation induced IL-10expression in macrophages. Furthermore, we found CDllb deficient mouse in DSS induced colitis disease model produce less IL-10in serum, have less regulatory T cell number in spleen and mesenteric lymph nodes, and exhibit more severe tissue damage in colon. These experiments suggest that CD11b promotes TLR triggered IL-10 expression via Src/PI3K/GSK3α/β pathway in macrophages, and negatively regulates DSS induced mouse colitis.