Research On The Regulation And Mechanism Of Rab7 On TypeⅠInterferon Expression In TLR9 Activated Macrophages

TLR9 is a member of the TLR family of pattern recognition receptors involved in the initiation of the innate immune response, which recognizes the CpG ODN that contains CpG motif of oligonucleotides. But the abnormal or excessive activation of TLR9 can lead to the disorder of the physiological function and the occurrence of the disease. Rab7 is a small GTPase protein which localized in the late endosomal/lysosomal structure. Studies have shown that Rab7 is involved in a variety of signaling processes. But research on the regulation of the TLR9 signaling pathway by Rab7 is relatively sparse.Purpose:To study the effects of Rab7 on the type I IFN and other cytokines activated by TLR9 and effects on the MAPK signaling pathway, and to explore its mechanism.Methods:(1) Silencing the Rab7 gene in RAW264.7 cells using siRNA interference technology, then detect the expression level changes of TLR9 mRNA and protein by real time PCR and western blot; and its effects on the expression of TNF-α, IL-1β, IL-10, IFN-α, IFN-β, IP-10, iNOS mRNA by real time PCR; Detect the changes in the level of ERK, JNK and p38 phosphorylation downstream of the TLR9 signaling pathway by western blot. (2) The Rab7 eukaryotic expression plasmid (pcDNA3.1/mRab7), C terminal deletion plasmid (pcDNA3.1/Rab7AC), and T22N mutant plasmid (pcDNA3.1/ Rab7T22N) transfection of mouse macrophage RAW264.7 were done by lipofectamine, and G418 screening was used to establish the stable expression of Rab7 and its deletion and mutant cell lines. TLR9 mRNA and protein levels, TNF-a, IL-1β, IFN-a, IFN-p mRNA levels and its effect on MAPK signal pathway were detected by real time PCR and western blot in these transfected cell after CpG stimulation. (3) Several cell lines were pretreated with ERK, JNK, p38, and NF-κB inhibitor PD98059, SP600125, SB203580, PDTC for 30min, and changes in IFN-P and IL-10 in the cell culture supernatant were subsequently detected by ELISA.Results:(1) Rab7 gene silencing inhibited the expression of TLR9 mRNA and protein; Overexpression of Rab7 significantly promoted the expression of TLR9 mRNA and protein while the expression of TLR9 mRNA and protein was significantly inhibited after Rab7 mutation. (2)The expression of TNF-a, IL-1β, IL-10, IFN-a, IFN-p, IP-10 and iNOS mRNA was promoted after Rab7 gene silencing; Overexpression of Rab7 significantly decreased the expression level of TNF-α, IL-1β, IFN-a, IFN-P, whereas the expression level of TNF-α, IL-1β, IFN-α, IFN-β was significantly increased after Rab7 mutation. (3) Rab7 gene silencing significantly promoted the phosphorylation of p38 and ERK, and inhibited the phosphorylation of JNK; the phosphorylation levels of ERK and p38 were significantly decreased after Rab7 overexpression, the phosphorylation level of ERK and p38 was significantly increased after Rab7 C deletion, the phosphorylation level of ERK and p38 were not significantly changed after Rab7T22N mutation. (4) CpG stimulation could not promote the secretion of IFN-β after treated with SP60015 compared with the DMSO pretreatment control group after Rab7 mutation, while there did not have a similar effect when inhibitor SB203580, PD98059 and PDTC pretreatment; The inhibitor PDTC and SB203580 pretreatment could not promote the secretion of IL-10 after CpG stimulated for 8 hours and there did not have a similar effect after pretreatmented by inhibitor PD98059 and SP60015.Conclusion:(1) Rab7 promotes the expression of TLR9. (2) Rab7 inhibits the ERK and p38 MAPK signaling pathways in the TLR9 signal transduction pathway in a C-dependent manner. (3)Rab7 inhibits the expression of TNF-α、IL-1β、IFN-a and IFN-β mRNA. (4) Rab7 regulates the secretion of IFN-β through the JNK MAPK pathway, and regulates the secretion of IL-10 by the p38 MAPK and NF-κB signaling pathway.