GIT2 Inhibits TLR-induced Inflammatory Response

Toll-like receptors (TLRs) signaling pathway is crucial for host innate immune responses by sensing the components of pathogens, such as bacterial DNA, Lipopolysaccharide (LPS) and viral double-stranded RNA. The uncontrolled activation of TLR-triggered signaling pathway promotes chronic inflammation and autoimmune diseases. Therefore, negative regulation of TLRs signaling is crucial to maintain immune homeostasis.G-coupled receptor kinase-interactor 2 (GIT2) belongs to the group of ADP-ribosylation factor (Arf)-directed GTPase activating proteins (GAPs). To our knowledge, none of these proteins has been reported to directly regulate TLR signaling. GIT2 with multidomains involved in diverse biological processes, such as regulating the dynamics of cytoskeleton, receptor internalization and functioning as scaffold protein in signal transduction. The deficiency of GIT2 leads to spontaneous splenomegaly, impaired directional chemotaxis and increased oxygen anion production in neutrophils as well as impaired positive selection of CD4 single positive thymocytes in thymics. The results above indicate the important roles of GIT2 in immunity. We identified that GIT2 interacts with the components of NF-κB signaling pathway by a large-scale yeast two-hybrid screening, which suggested the possible relationship of GIT2 and NF-κB signaling pathway. However, whether GIT2 directly regulates the activity of NF-κB triggered by TLRs and their physiological roles remain unknown.NF-κB activation is a regulator of inflammation. Therefore, we investigated the roles of GIT2 in Lipopolysaccharide (LPS)-induced endotoxic shock. Compared with wild-type (WT) mice, Git2-deficient mice were more susceptible to LPS-induced systemic inflammation characterized with accelerated lethality, elevated the levels of proinflammatory cytokines, splenomegaly as well as aggravated lung injury. We also examined the roles of GIT2 played in Escherichia coli-induced septic peritonitis. In addition to similar pathological features, Git2-deficient mice infected with Escherichia coli showed reduced the ability of bacterial clearance. The results above indicated that GIT2 protects against LPS-induced endotoxic shock and septic peritonitis.Having shown that GIT2 regulated endotoxin-induced inflammatory responses and the important roles of GIT2 played in inflammatory responses and Escherichia coli-induced septic peritonitis in vivo, we next focused our attention on bone-marrow derived macrophages (BMDMs) to identify the mechanisms by which GIT2 control TLR-induced inflammation. The levels of Tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were greater in LPS or Pam3CSK4-stimulated Git2-/-BMDMs than in stimulated WT BMDMs. The similar results also obtained using mouse embryonic fibroblasts (MEFs) stimulated with LPS or Pam3CSK4. When the BMDMs preincubated with inhibitors of TLRs (TAK-242 for TLR4 and OxPAPC for TLR2/4), the levels of TNF-αand IL-6 in LPS or Pam3CSK4-stimulated Git2-/-BMDMs were similar with the levels in LPS or Pam3CSK4-stimulated WT BMDMs. Thses results confirmed that GIT2 negatively regulated TLR-induced NF-κB activity.Our previous results indicated that GIT2 negatively regulates TLR-induced NF-κB signaling through recruitment of CYLD to inhibit the ubiquitination of TRAF6. In order to elucidate the roles of GIT2 in inflammatory disease, we applied the dextran sulfate sodium (DSS)-induced colitis models. Compared with WT mice, Git2-/- mice are more hypersuceptible to DSS-induced colitis, featured with accelerated lethality, weight loss, shorten colon length, elevated the levels of proinflammatory cytokines as well as severe colon injury. When treated with selective antibiotics, there is no difference in the severity of colon injury between Git2-/- mice and WT mice. Git2 deficeincy also contributes to development of chronic colitis.In conclusion, GIT2 is an essential terminator of TLR signaling and that loss of GIT2 leads to uncontrolled inflammation, such as LPS-induced systemic inflammatory responses, Escherichia coli-induced septic peritonitis and DSS-induced colitis. GIT2 may be a useful target for drug discovery.