The STING-TBK1-IRF3 Pathway Regulates M1 Macrophage Effects On The Polarization And Renal Interstitial Fibrosis With HIF-PHI

Objective: Renal fibrosis leads to gradual deterioration of renal function and is a key cause of end-stage renal disease formation.Epidemiological surveys show that the prevalence of patients with chronic kidney disease in China is as high as 10.8%.The number of kidney replacement patients in China will increase in the future and is expected to exceed 1 million.China will have the largest number of kidney replacement therapy patients in the world.Therefore,a comprehensive and in-depth study of renal fibrosis is of great practical significance,and could provide innovative prevention and treatment strategies for chronic kidney disease,reduce the prevalence of end-stage renal disease,and improve the prognosis of patients.After inflammation,monocytes or macrophages entering the kidney are transformed into classically activated M1 macrophages and activated M2 macrophages,respectively.Activation of M1 macrophages is manifested by the increased expression of MHC-II antigen and the release of pro-inflammatory cytokines that aggravate the inflammatory response and promote renal fibrosis.The M1 macrophage subpopulation primarily mediates the inflammatory response and fibrosis in the kidney and is considered a target for elimination due to its pro-inflammatory and tissue-damaging effects.The STING-TBK1-IRF3-mediated type I interferon signaling pathway is an important natural immune pathway.It has been shown that in liver tissues of patients with nonalcoholic fatty liver disease(NAFLD),the STING-TBK1-IRF3 pathway can polarize M0 macrophages,transforming them into M1 macrophages,thus promoting liver fibrogenesis.Targeting STING-TBK1-IRF3 to inhibit macrophage pro-inflammatory activation is a feasible approach to treat or prevent liver tissue fibrosis.The antimicrobial defense of monocytes/macrophages during Mycobacterium pneumophila infection is dependent on the activation of the STING-TBK1-IRF3 pathway.The STING-TBK1-IRF3 pathway promotes inflammatory M1 macrophage infiltration during Borrelia burgdorferi infection and this metabolic reprogramming is regulated independently by hypoxia-inducible factor(HIF)-1α.However,the STING-TBK1-IRF3 signaling pathway has seldom been studied in the field of renal fibrosis.HIF-PHI(prolyl hydroxylase inhibitor,PHI)affects the STING-TBK1-IRF3 signaling pathway in M1 macrophages and renal fibrosis.The oral drug Roxadustat(FG-4592)is a small-molecule HIF-PHI class drug used for the treatment of renal anemia.Roxadustat inhibits the prolyl hydroxylase(PH)enzyme by mimicking ketoglutarate,one of the substrates of PH.This affects the role of the PH enzyme in maintaining the balance between the production and degradation rate of HIF,thereby correcting anemia.Although HIF-PHI is mainly used clinically for the treatment of renal anemia,Kim et al.in 2015 found that it also had anti-inflammatory involvement in tissue damage,and in the modulation of the immune response;HIF-PHI reduced mononuclear phagocyte-driven inflammation.In a model of adenine-induced chronic tubulointerstitial nephritis,HIF-PHI significantly attenuated renal insufficiency and tubulointerstitial injury in mice,suggesting that HIF-PHI had the potential to delay the progression of renal fibrosis.The renoprotective effect of HIF-PHI could be largely eliminated after the depletion of mononuclear macrophages.It indicated that mononuclear macrophages were important targets of HIF-PHI in chronic tubulointerstitial nephritis.However,whether HIF-PHI is involved in the macrophage pro-renal fibrosis process through the STING-TBK1-IRF3 pathway remains to be investigated.Therefore,in this study,we used a folic acid-induced kidney injury model,a TGF-β-induced renal tubular epithelial cell fibrosis model,and kidney puncture specimens from patients to comprehensively investigate the effects of HIF-PHI on STING-TBK1-IRF3 signaling and renal fibrosis in M1 macrophages.Methods: 1.Clinical trials section Blood specimens and kidney tissues of patients were obtained in accordance with relevant ethical regulations.The concentrations of serum MCP-1/CCL2,TNF-α,and i NOS were measured by ELISA in the control group and the group of patients with a definite diagnosis of interstitial renal fibrosis,and CD86 and STING proteins were detected in the kidney tissues of both groups by immunohistochemical staining,after confirming that the conditions of the two populations were balanced.2.Cellular experiments section(1)We observed the polarization of M1 macrophages by applying activators and inhibitors to regulate the STING-TBK1-IRF3 pathway and analyzed the protein expression changes of STING,p-IRF3,IRF3,CD86,and MHC-II using western blotting.The m RNA expression changes of TNF-α,IL-6,IFN-γ,i NOS,MHC-II,and Arg-1 were detected using q RT-PCR.(2)A TGF-β-induced renal tubular epithelial cell fibrosis model was established,and protein expression changes of α-SMA,COL-IV,and fibronectin(FN)were analyzed using western blotting.The m RNA expression changes of STING,IRF3,TNF-α,IL-6,IFN-γ,i NOS,MHC-II,and Arg-1 were detected using q RT-PCR.(3)To assess the effects of HIF-PHI levels on the polarization of each macrophage type,the expression of each macrophage type marker CD86,MHC-II,CD163,and Arg-1 protein was analyzed by western blotting,then the effects on cell viability were measured using CCK-8.3.Animal experiments section(1)To establish a model of folic acid-induced kidney injury,this study was conducted in 8-week-old male C57BL/6 mice using STING-TBK1-IRF3 pathway inhibitor C176 intraperitoneally as a positive control for the drug HIF-PHI.This verified the effects of HIF-PHI on the STING-TBK1-IRF3 signaling pathway,M1 macrophages,and kidney fibrosis by inhibiting the STING-TBK1-IRF3 pathway in mice.(2)Examination of the model and assessment of renal function: HE and PAS staining were used to assess renal histopathological damage,and blood creatinine and urea nitrogen kits were used to detect renal function in each group of mice.(3)Masson assessment of renal interstitial fibrosis indicated that folic acid(FA)injection for 14 days successfully constructed a mouse renal fibrosis model.Immunohistochemical staining for α-SMA and western blotting analysis of protein expression changes of α-SMA,COL-IV,and FN were performed.(4)Tissue immunofluorescence was used to detect the M1 macrophage marker CD86 in each group,and ELISAs were performed to detect the downstream secretory factor IL-1β in each group of macrophages.(5)We analyzed the protein expression changes of STING,p-IRF3,IRF3,CD86,MHC-II,α-SMA,COL-IV,FN,and HIF-1α in each group of mice by western blotting.RNA expression changes of STING,IRF3,i NOS,MHC-II,Arg-1,TNF-α,IL-6,and IFN-γ were detected using q RT-PCR.Results: 1.Clinical trials section Serum MCP-1/CCL2,TNF-α,and i NOS concentrations were increased in patients with interstitial fibrosis;STING-TBK1-IRF3 pathway proteins and M1 macrophage cell marker CD86 were elevated in the renal tissues of patients.The results suggested the presence of M1 macrophage infiltration and STING-TBK1-IRF3 pathway activation in interstitial fibrosis kidney tissues.2.Cellular experiments section(1)By regulating the STING-TBK1-IRF3 pathway,we found that its activation increased the expression of specific markers CD86 and MHC-II in M1 macrophages,along with the release of cytokines such as TNF-α,IL-6,and IFN-γ;inhibition of the STING-TBK1-IRF3 pathway effectively reduced this trend.This indicated that the activation of the STING-TBK1-IRF3 pathway led to the increased polarization of M1 macrophages and increased secretion of corresponding cytokines.There was a significant elevation in the agonist group compared with the M0+LPS group,and the expression level was basically the same in the inhibitor group compared with the M0+LPS group,indicating that the inhibition of the STING-TBK1-IRF3 pathway could more thoroughly attenuate the polarization of M1 macrophages.(2)The expression levels of α-SMA,COL-Ⅳ and FN were compared between multiple groups using co-culture of HK-2 and M1 macrophages,and low expression in the HK-2 group were seen.Compared with the HK-2 group alone,α-SMA,COL-Ⅳ and FN expression were significantly upregulated in the M1 group after 48 hours of co-culture,verifying the pro-fibrotic effect of M1 macrophages,which was slightly reduced compared with the TGF-β-model group.However,the difference was not significant,and the pro-fibrotic effect was more pronounced when the two effectors were used.When the STING-TBK1-IRF3 pathway was inhibited with H151,most of the elevation trend was reversed.Therefore,we clarified the pro-fibrotic effect of M1 macrophages on renal interstitial fibrosis,and this effect was largely influenced by the STING-TBK1-IRF3 pathway.(3)To investigate the target of HIF-PHI action,the effect of HIF-PHI on the polarization state of M1 and M2 macrophages was explored under similar conditions.We found that HIF-PHI had a more prominent effect on the polarization of M1 macrophages,while it had little effect on the polarization of M2 macrophages.3.Animal experiments section An 8-week-old male group of C57BL/6 mice was used to successfully construct a mouse kidney fibrosis model using FA intraperitoneal injection.Histopathology of kidney injury was assessed by HE and PAS staining.Blood creatinine and urea nitrogen kits were used to detect the kidney function in each group of mice.Renal fibrosis was assessed with Masson staining.It was confirmed that FA injection for 14 days could successfully construct a mouse renal fibrosis model.Immunohistochemistry,immunofluorescence,western blot analysis,and q RT-PCR were used to detect STING-TBK1-IRF3 pathway proteins,M1 macrophage markers,and inflammatory factors.HIF-PHI was found to reduce glomerulosclerosis and interstitial fibrosis while decreasing renal injury by inhibiting the STING-TBK1-IRF3 pathway.At least part of the inhibition was mediated by the STING-TBK1-IRF3 pathway.Conclusion: 1.STING-TBK1-IRF3 pathway activation and M1 macrophage infiltration occurred during renal fibrosis.2.The STING-TBK1-IRF3 signaling pathway regulated M1 macrophage polarization and renal interstitial fibrosis.3.HIF-PHI attenuated M1 macrophage polarization and renal interstitial fibrosis by inhibiting the STING-TBK1-IRF3 pathway.