Negative Regulation Of TLRs Signaling In Macrophages By Rab7b And LAPF And The Underlying Mechanisms

PartⅠLate endosome/lysosome-localized Rab7b suppresses TLR9-initiated proinflammatory cytokine and typeⅠIFN production in macrophages and the underlying mechanismsInappropriate activation of TLR9 has been found to be involved in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus. TLR9 antagonists have been proposed to be therapeutic for some kinds of autoimmune disease. However, new negative regulators of TLR9 signal pathway need to be identified, and the mechanisms for the control of TLR9 response need to be fully investigated. It is well known that TLR9 will be finally transported to late endosome/lysosome once activated; however, the exact mechanism and the biological significance of the subcellular redistribution have not been fully elucidated. Ras related in brain (Rab) 7b is a small guanosine triphosphatase, identified by us before, which is mainly localized in late endosome/lysosome. Our previous study shows that Rab7b can negatively regulate TLR4 signaling by promoting lysosomal degradation of TLR4. In this study, we show that TLR9 ligation can inhibit Rab7b expression in macrophages via ERK and p38 activation, In turn, the late endosome/lysosome-localized Rab7b can colocalize with TLR9 in lysosomal-associated membrane protein 1-positive compartment and down-regulate the expression of the TLR9 in macrophages by promoting TLR9 degradation once TLR9 is activated. Accordingly, Rab7b can negatively regulate TLR9-triggered production of TNF-a, IL-6, and IFN-βin macrophages by impairing activation of MAPKs and NF-κB pathways. Our results suggest that the late endosome/lysosome-localized Rab7b can down-regulate TLR9-triggered proinflammatory cytokine and type I IFN production by impairing TLR9 signaling via promotion of TLR9 degradation.Part II Apoptosis-inducing protein LAPF negatively regulates TLR3-and TLR9-meidiated proinflammatroy cytokine and type I interferon production and the underlying mechanismsAccording to the subcellular localizations, the Toll-like receptors (TLR) could be divided into two categories, cell surface TLRs and intracelluar TLRs. The spatial differences of TLRs might be regulated by a new pattern related with their subcellular localization, which would provide a new mechanism for regulation of TLRs-triggered inflammatory innate response. Lysosome-associated apoptosis-inducing protein containing the plechstrin homology (PH) and FYVE domains, LAPF, has been first identified by us and found to be involved in the TNF-a-induced apoptosis of turner cells. The similar subcellular localization of LAPF and intracellular TLRs made us look for the possibility that LAPF might involve in the regulation of these intracellular TLRs signaling. As expected, we discovered that LAPF could negative regulate TLR3-and TLR9-initiated signaling, but not the cell surface-located TLR4, in macrophages. Overexpression of LAPF in primary peritoneal macrophages attenuated TLR3-and TLR9-trigged TNF-a, IL-6 and IFN-βproductions, and such a downregulation was partially dependent on its PH domain. Not only the intracellular TLRs signaling but also the intracellular dsRNA sensor RIG-I signaling could be regulated by LAPF. Sendaivirus-induced IFN-βproduction in macrophages, which was already domenstrated that this virus is recoginized by RIG-I, was inhibited by LAPF.2-D fluorescent difference gel electrophoresis and sequential protein spectrum indicated that overexpression of LAPF would decrease the total protein level of calmodulin in the cytoplasm. Calmodulin is the most important Ca2+binding protein and activates downstream CaMKII which has been demonstrated as a positive regulator in TLRs signaling. In addition, LAPF associated with phospho-Akt via its PH domain and ficilitied Akt's phosphorylation. Activated Akt downstream phosphorylated the N-terminal Ser residues of GSK-3 which inhibited its activity. The inhibition of GSK-3 competitively attenuated the NF-κB-dependent proinflammatory cytokine production. Thus, LAPF, the protein with the same subcellular localization of intracellular TLR3 and TLR9, negatively regulates TLR3 and TLR9 signaling via downregulating the calmodulin expression and activating Akt/GSK-3 pathway.