Mechanism Of STING Inhibitor In Alleviating Renal Interstitial Fibrosis By Inhibiting NK Cell Activation

Objective: Renal fibrosis is a final manifestation of chronic kidney disease(CKD).The morphological characteristics of renal fibrosis are tubulointerstitial fibrosis(TIF,also called renal interstitial fibrosis,RIF)and glomerulosclerosis.Generally,RIF is more serious than glomerulosclerosis and occurs earlier,which is an important factor to promote the progress of renal disease.Once kidney injury occurs,the tissue suffers ischemia and hypoxia,causing continuous activation of inflammation and inducing the release of a diverse range of fibroblast-promoting factors and inflammatory factors.At the same time,various immune cells are activated to further damage the renal tissue,leading to renal fibrosis and further end-stage renal disease(ESRD).A large number of epidemiological studies have shown that the prevalence of ESRD is increasing worldwide.Therefore,there remains a pressing unmet medical need for effective strategies to treat RIF.In the process of RIF,natural killer(NK)cells play an important role.NK cells,as innate immune cells,participate in the regulatory network of inflammatory response.It does not require specific adaptive immune response activation and promotes its immune activity by activating its own cell ligands.After activation,NK cells can directly release cytotoxic particles,cytokines such as interferon-γ(IFN-γ,a type II interferon)and tumor necrosis factor α(TNF-α)can also be synthesized and secreted and act on the epithelial cells of renal tubules and promote the progress of RIF and CKD.STING/TBK1/IRF3 pathway is one of the important pathways to activate innate immunity,which was first found in the cancer-immune environment.STING,as a key signal protein,can recognize non-self products from viruses,bacteria,and other pathogenic organisms,as well as damage-associated molecular patterns in vivo and activate downstream tank binding kinase 1(TBK1),interferon regulatory factor 3(IRF3),and nuclear factor kappa-B(NF-κB),promote type I interferon and activate NK cells and other immune responses.Some studies found that drug inhibition of STING can reduce the release of IFN-γ cytokines to alleviate RIF.The decreased release of IFN-γ is probably related to the activation of NK cells.This study focused on the effect of inhibition of STING on RIF and further studied the specific way STING/TBK1/IRF3 participates in the RIF process to explore the relationship between NK cell activation and this pathway in the RIF process.In order to improve the understanding of the potential pathogenesis of RIF and so as to provide new ideas and theoretical basis for clinical treatment and new drug research and development.Methods: 1.To detect the effect of STING inhibitor on RIF in mice.1.1 The renal fibrosis model of mice was established by intraperitoneal injection of folic acid(FA),and the STING inhibitor(FA+C176)treatment group was established.The serum urea nitrogen(BUN)and creatinine(Scr)were detected;1.2 The renal tissue of mice was stained with H&E,PAS,and Masson to evaluate the pathological renal damage;1.3 Western blot,q RT-PCR,and immunohistochemical were used to detect tissue smooth muscle markers(α-SMA),collagen type IV(Col-IV),and fibronectin(FN);1.4 The changes in inflammatory factors levels of TNF-α,IFN-γ,interleukin-6(IL-6),interleukin-10(IL-10),and chemokine 2(CCL2)in each group by ELISA and the m RNA levels were detected by q RT-PCR;2.Identify NK immune cells activation in RIF.2.1 Through bioinformatics analysis,the differential genes of transcriptome data of human renal fibrosis and normal kidney tissues were counted,and the differential genes were analyzed by Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),and Gene Set Enrichment Analysis(GSEA)and explore the regulatory mechanism in RIF process;2.2 Immunofluorescence assay was used to compare the expression of NK cell marker CD56,cell activation marker CD69,and IFN-γ released by NK cell activation spatial localization and expression between human renal fibrosis tissue samples and normal renal tissue samples and detect IFN-γ was the main source of NK cells activated in RIF;3.To explore the mechanism of STING inhibitors affecting NK cell activation and regulating renal tubular epithelial injury.3.1 Immunofluorescence assay was used to compare the activation of NK cells and the release of IFN-γ in the kidney of different groups of mice;3.2 To investigate the effect of injured renal tubular epithelial cells(HK-2)on NK cell activation.HK-2 cells were induced by TGF-β for 24 h to construct a cell fibrosis model,and their conditioned medium(CM)was collected.NK cells with different treatment conditions were exposed to CM.And the NK cell activation markers CD69 and IFN-γ were detected by Western blot and q RT-PCR;3.3 Western blot and q RT-PCR were used to detect the expression of STING,TBK1,and IRF3 proteins and genes in NK cells;Detection of the spatial localization and expression of NK cell marker CD161,cell activation marker CD69 and STING in different groups of mouse kidney tissues by immunofluorescence multiple staining;3.4 To explore the effect of NK cells on injured HK-2 cells.After TGF-β induction 24 h,HK-2 cells were exposed to CM of NK cells under different treatment conditions,and the changes in m RNA and proteins of α-SMA,Col-IV,and FN were detected by q RT-PCR and Western blot.Results: 1.STING inhibitors can mitigate RIF.1.1 STING inhibitor C176 can protect the renal function of RIF model mice induced by FA: compared with NC group,BUN and Scr in the FA group were higher(P < 0.05),and C176 treatment improved renal function(P < 0.05).1.2 STING inhibitor can alleviate the pathological changes of injury in RIF model mice: the renal tissue was scored by H&E and PAS staining.No significant pathology was observed in the kidneys in the NC group.The FA group showed the highest renal injury score(P < 0.05),significantly higher than that of the FA+C176 group(P < 0.05).1.3 STING inhibitors can slow down the progression of RIF lesions in RIF model mice: Masson staining revealed that the degree of renal fibrosis in mice induced by FA was serious,which represented renal interstitial fibrosis.Expression levels of α-SMA,Col-IV,FN proteins,and genes were highly expressed(P < 0.05),and C176 reduced the expression level of these proteins and genes(P < 0.05).1.4 STING inhibitors can reduce the expression level of various inflammatory factors and chemokines in FA-induced RIF mice: the proteins and m RNA of expression levels of TNF-α,IFN-γ,IL-6,IL-10,and CCL2 in the FA group were significantly higher than those in NC group and FA+C176 group(P < 0.05).This suggested that C176 could reduce the expression levels of these proteins and m RNA and attenuate the release of inflammatory factors and chemokines(P < 0.05).2.NK cell activation participates in RIF.2.1 By analyzing the transcriptome of renal interstitial fibrosis,594 differential genes were screened,of which 134 were up-regulated,and 460 were down-regulated.The differential genes were mainly enriched in the immunity cell receptor signal pathway.Among them,IL2 RB was significantly down-regulated.The deletion of this gene can inhibit T cell activity but has little effect on NK cell activity.It suggested that the inhibition of T cell activity and an enhancement of NK cell activity in the course of RIF.With IFN-γ overexpression in RIF(mainly from activated NK and T cells)in RIF mouse model,NK cells may participate in the process of RIF.2.2 Co-immunofluorescence staining showed that higher expression of CD56 and CD69(co-localization)of NK cells were found in human renal fibrosis tissue biopsy specimens compared with normal renal tissue.The expression of CD69 and IFN-γ,the activated markers of NK cells,appeared co-localization.It suggested that the NK cell activation and the IFN-γ expression levels were significantly higher in fibrotic renal tissue than in normal renal tissue.3.STING inhibitors attenuate RIF by inhibiting NK cell activation.3.1 STING inhibitor can inhibit the activation of NK cells in RIF model mice: Immunofluorescence co-localization in the mouse kidney showed that the markers CD161 and CD69 of NK cells in the FA group were highly expressed,and the expression of CD161 and CD69 in FA+C176 group was lower than that in FA group.STING inhibitor reduced the activation of NK cells of RIF mouse model and reduced IFN-γ at the same time.3.2 In vitro,STING inhibitors can down-regulate NK cell activation: Under the conditions set by the institute,exposure of NK cells to the TGF-β induced HK-2 cells CM and cultured for 24 h.The results showed that the m RNA and protein expression level of NK cell activation markers CD69 and IFN-γ in the NK+CM group was significantly higher than in the NK group(P<0.05),while after adding H-151(NK+CM+H151 group),the m RNA and protein expression level CD69 and IFN-γ in NK+CM group were significantly lower than that in NK+CM group(P<0.05).It suggested that injured HK-2 cells can induce NK cell activation,while H151 can downregulate NK cell activation and the expression level of IFN-γ.3.3 STING inhibitors down-regulate NK cell activation by inhibiting STING/TBK1/IRF3 signaling pathway: The results showed that the expression level of STING and the phosphorylation levels of TBK1,IRF3 in the NK+CM group were significantly higher than those in NK group(P<0.05),while the corresponding index levels in NK+CM+H151 group were significantly lower than those in NK+CM group(P<0.05),suggesting that H151 could inhibit the activation of this pathway.In addition,triple fluorescent staining results of mice kidney tissue suggested the NK cell activation level in the NC group was lower,and the NK cell activation level in the FA group was significantly higher than that in the NC group,while the NK cell activation level in the FA+C176 group was significantly lower than that in the FA group of the mice kidney,and the expression of STING in NK cells was consistent with its activation trend.3.4 STING inhibitor alleviates the effect on fibrotic index of HK-2 cells by inhibiting NK cell activation: Firstly,used TGF-β induction of HK-2 cells for 24 hours to construct the fibrosis cell model,and re-exposure to NK cells CM for 24 h,the results showed that m RNA and protein levels of α-SMA,COL-IV,FN in HK-2+NK group were significantly higher than HK-2 group(P<0.05),and the corresponding index levels in HK-2+NK+H151 group were significantly lower than those in HK-2+NK group(P<0.05).It was suggested that NK cells might aggravate the degree of cellular fibrosis,while the STING inhibitor may slow down this pathological process.Conclusion: 1.STING inhibitors can protect the renal function of the RIF mice model induced by FA,alleviate renal injury and pathological changes of RIF,and reduce the expression of various inflammatory factors and chemokines.2.Activated NK cells and released the expression of IFN-γ and other inflammatory factors may participate in the RIF process.3.STING inhibitors can attenuate NK cell activation through STING/TBK1/IRF3 pathway and may subsequently reduce the damage of renal tubular epithelial cells and mitigate RIF.