The Inflammatory Loop Formed By IL-36γ Is Based On TLR3 Regulating Liver Injury Via NLRP3 Inflammasome

OBJECTIVE:With the development of global liver disease,it has become one of the most leading causes of morbidity and mortality worldwide.Many studies have showed that the liver proposed as ’an immunological organ’ dominated by innate immunity.In homeostatic state,inflammatory cells infiltrate the liver and clear the pathogen.Once the inflammation is persistent or overwhelming,they in turn damage the hepatocytes and cause severe liver disease.Cytokines play a key role in regulating liver immune response.IL-1β is a multipotent cytokine in IL-1 family,and involved in the inflammatory response in various types of liver processes.Study has found that IL-1β can induce the production of interleukin-36y(IL-36y),as another member of the IL-1 family.Studies have demonstrated that IL-36γ paly an important role in skin diseases such as psoriasis,but its role in liver diseases is still unclear.Toll-like receptor 3(TLR3)is essential in liver injury induced by Polyinosinic acid-polycy tidy lic acid(Poly(I:C)),which can induce the expression of IL-36γ in keratinocytes.Therefore,in this study,we mainly discussed the specific mechanism of the interaction between IL-36y and IL-1β to mediated injury in liver diseases,which may provide new targets and ideas for the treatment of liver diseases.Methods:1.In vitro:1)The cell toxicity of recombinant IL-36y was tested in HepG2 cells by MTT assy;Recombinant IL-36y was used to stimulate HepG2,Hep3B and Murine Peritoneal Macrophages(MPM)cells,the expression of IL-1β was detected by ELISA and western blot.HepG2 and Hep3B cells were separately cultured with recombinant IL-1β,Tumor Necrosis Factor-α(TNF-α),IL-1β+TNF-α,IL-36y,Poly(I:C)and Imiquimod,the expression of IL-36yand IL-36β was detected by Western blot,and the cell toxicity of each group was tested by MTT.IL-1β,TNF-α,IL-1β+TNF-α,IL-36y,Poly(I:C),Imiquimod and ETOH50 stimulated Bone Marrow-derived Macrophage(BMDM),to detect the expression of IL-36y and IL-1β by Western blot,and to detect IL-1β release via ELISA.HepG2 and Hep3B cells were treated with IL-1 β,TNF-α,IL-β+TNF-α and IL-36y,and lipid deposition in hepatocytes was observed by Oil Red O staining,and the expression of P-AMPKα and AMPKa was detected through Western blot.IL-36y was used to stimulate HepG2 cell and to treat with IL-36Ra and Metformin simultaneously,and to detect the expression of P-AMPKα,AMPKα,P-ACC,ACC and IL-1β via Western blot,and to observe the expression of SRIT-1 via Immunofluorescence staining.HepG2 cells were stimulated to IL-36y in presence of IL-36Ra,A438079,CLI-095,PDTC and JSH23,to detect the expression of IL-1β by Western blot and ELISA.2)MPM cell was cultured in Lipopolysaccharide(LPS)combined with Adenosine-triphosphate(ATP),at same time was treated with TLR3 inhibitor,IL-36Ra and CLI-095,and to detect the expression of IL1β by Western blot and ELISA.MPM and BMDM cells were stimulated with LPS,IL-36γ,LPS/IL-36γ,LPS/ATP,IL-36γ/ATP and LPS/ATP/IL-36γ for 4 h or 24 h,Western blot and ELISA to observe the level of IL-1β.3)MPM and BMDM cells were treated with various times(0,10,30,60,120,180 min)Poly(I:C)and Poly(I:C)/ATP,the expression of IL-36y、IL-1β、Caspase-1 and HMGB1 were detected by Western blot and ELISA.Then,we established Poly(I:C)/ATP stimulated cells 60 min in vitro model,and A43 8079,TLR3 inhibitor,CLI-095,Caspase1 inhibitor,IL-36Ra and HMGB1 inhibitors were used to observe the expression of IL-36γ,IL-1β,Caspase-1 and HMGB1 through Western blot and Immunofluorescence staining,and the level of IL-1β and IL-36 yalso were detected by ELISA.4)Three different sequence P2X7R-SiRNA(64,65,66)and Con-SiRNA silenced BMDM cell for 48 h,Western blot and Real-time PCR(RT-PCR)to detect P2X7R protein and mRNA expression level.Then,select P2X7R-65-SiRNA and Con-SiRNA transfection BMDM cell for 48h,LPS/ATP stimulated for 4 h,the expression of IL-1 β was determined by Western blot and ELISA.BMDM cells was transfeccted with IL-36γ-ShRNA and Con-ShRNA for 72 h,and then treated with Poly(I:C)/ATP for 1h,the protein and mRNA level of IL-36γ and IL-1β was detected by Western blot,ELISA and QPCR,Immunofluorescence staining observe the expression of Caspase-1 and HMGB1.5)Kupffer cells(KCs)were cultured with LPS.ATP,LPS/ATP,Poly(I:C).Poly(I:C)/ATP,and to detectd the expression of HMGB1,IL-1β and Caspase-1 via Western blot,ELISA and immunofluorescence staining.2.In vivo methods:1)Poly(I:C)/D-GalN model.C57BL/6 nice were randomly divided into 4 groups(n=6):① Normal group:intraperitoneal injection with the same amount normal saline;②Poly(I:C)(1μg/mouse)group:intraperitoneal injection with Poly(I:C)(1 μg/mouse);③ Poly(I:C)/D-GalN group:intraperitoneal injection with D-GalN(10 mg/mouse)and Poly(I:C)(1 μg/mouse);④ Poly(I:C)group:intraperitoneal injection with Poly(I:C)(20 g/g body weight).After18 h,all mice were sacrificed and serum levels of ALT and IL-36y,and Hematoxylin-eosin Staining(HE)and Immunofluorescence Staining(IF)were utilized to observed the pathological changes of liver and the expression of F4/80.The expression of IL-36y and neutrophil elastase(NE)was detected by Immunohistochemistry(IHC),and mRNA levels of IL-36y and CXCL1 were detected by QPCR2)Poly(I:C)/D-GalN plus various inhibitors model.Intraperitoneally injected with TLR3 Complex inhibitor(1mg/mouse),IL-36Ra(6μg/mouse),CLI-095(3 mg/mouse),Ly6G antibody(200μg/mouse)and subcutaneous injection of A438079(30 mg/kg).Except for the normal group,mice were intraperitoneal injection with D-GalN(10 mg/mouse)and Poly(I:C)(1 μg/mouse).Dnase-I group was intraperitoneally injected 1 h after Poly(I:C)/D-GalN inject,and the normal group was intraperitoneally injected with the same amount of normal saline.All mice were sacrificed 18 h later and serum and liver were collected.Serum levels of ALT and IL-36γ were detected,and the pathological changes of liver and the expression of IL-36γ were observed by HE and IHC separately.The expression of IL-36γ,IL-1β and Caspase-1 was detected by Western blot,and mRNA level of IL-36y was detected by QPCR.Results:1.In vitro results:1)Various of Recombinant IL-36y,IL-1β,TNF-α and Poly(I:C),Imiquimod have been found no toxicity on hepatocytes,and increased the expression of IL-36y,IL-36βand IL-1β in hepatocytes and macrophages,and IL-36γ induced the production of IL-1βin hepatocytes and macrophages.IL-1β,TNF-α,IL-1 β+TNF-α and IL-36y also induced lipid deposition and inhibited AMPK activity,however IL-36Ra and Metformin inhibited lipid deposition,and IL-36Ra and other inhibitors such as A438079 reversed synthesis of mature IL-1β.2)TLR3 inhibitor、IL-36Ra and CLI-095 reduced the production of IL-1βinduced by LPS/ATP in MPM cells,compared with the LPS/ATP,LPS/ATP/IL-36y increased the secretion of IL-1β in macrophages.3)Poly(I:C)/ATP stimulation caused the secretion of IL-36y、IL-1 β、Caspase-1 and HMGB1 compared with Poly(I:C),and the highest expression levels of this 4 proteins appeared 60 min after administration,A438079,TLR3 inhibitor,CLI-095,Caspase-1 inhibitor and IL-36Ra reduced their secretion.4)Silencing P2X7R by P2X7R-SiRNA transfection decreased the level of P2X7R,and also reduced the release of IL-1β induced by LPS/ATP;While similarly transfection IL-36γ-ShRNA down-regulated the expression of IL-36γ and IL-1β in protein and mRNA5)Treatment with LPS/ATP increased the expression of HMGB1,IL-1β and Caspase-1 in KCs,and Poly(I:C)/ATP up-regulated the expression of HMGB1,IL-1β.2.In vivo:1)Mice were treated with various of inhibitors and depletion neutrophils,significantly ameliorated the liver damage induced by Poly(I:C)/D-GaIN,reversed the increase of serum ALT and IL-36γ,and relieved inflammatory cells infiltration in liver tissue,down-regulate the expression of IL-36y,and reduced the expression level of IL-36γ,and down-regulated the expression of inflammation-related proteins IL-1β and Caspase-1;In addition,TLR3 inhibitor,IL-36Ra,A438079 and CLI-095 also reduced P2X7R expression in liver macrophages,Ly6G antibody and Dnase-I depleted the neutrophils in liver,and A438079 and CLI-095 inhibited neutrophils recruitment.Conclusion:Interaction between IL-36y and IL-1β can induced lipid deposition in hepatocytes.TLR3 mediates IL-36ysecretion and activatesd NLRP3 inflammasome Blocking IL-36y and NLRP3 activation ameliorated Poly(I:C)-induced liver injury.