TLR2/4 Ligand-amplified Liver Inflammation Promotes Initiation Of Autoimmune Hepatitis

Objective:The formation and development of autoimmune hepatitis?AIH?is due to the induction of an autoimmune response by molecular mimicry in the absence of autoimmune tolerance,which results in chronic liver inflammation and fibrosis.In the course of AIH,the mechanism underlying the weakening of liver autoimmune tolerance is not clear.This study aimed to investigate whether non-AIH inflammation caused by adenovirus?Ad?and carbon tetrachloride?CCl₄?promotes self-mimicking antigen-inducedautoimmuneresponsesandAIH,andexplorerelated immunomodulatory mechanisms.Methods:?1?C57/BL6 mice received the i.v.injection of Ad?10⁹ pfu?,pCYP2D6plasmid?50?g per injection?or received the i.p.injection of CCl₄?1 ml/kg body weight?.The infiltration of inflammatory cells in liver tissue was detected by HE staining.The expression of inflammatory genes Il1b,Il6 and Tnfa in liver tissue was detected by real-time PCR to observe whether Ad,CCl₄ and CYP2D6 alone could cause liver non-AIH inflammation on d5,d10 and d15.Furthermore,it was observed whether Ad/CYP2D6 or CCl₄/CYP2D6 could induce autoantibody production on d28.Mice received the injection of adenovirus or CCl₄ with or without psTLR2 and psTLR4?psTLR2/4?during non-AIH inflammation.The expression of Il1b,Il6 and Tnfa genes in the liver was detected at mRNA level by real-time RT-PCR to observe whether the non-AIH inflammation was attenuated.Mice received the injection of Ad/pCYP2D6 or CCl₄/pCYP2D6 with or without psTLR2/4 and anti-CYP2D6 and anti-HP in the serum were detected by ELISA on d28 to determine whether psTLR2/4 treatment could decrease autoantibodies titer.?2?Mice were treated with different dosages of Ad and CCl₄.The serum level of ALT was detected,the release of HSP70 and HMGB1 was measured by Western blot and the expression of inflammatory genes Il1b,Il6 and Tnfa was detected by real-time RT-PCR to observe the effect of different doses of Ad and CCl₄ on hepatocyte injury.Mice received the injection of pCYP2D6 with the different dosages of adenovirus or CCl₄.On d28,anti-CYP2D6 and anti-HP in the serum were detected by ELISA.Mice received the injection of pCYP2D6 and/or CCl₄ once?on d0?or four times.The expression of Il1b and Il6 genes in the liver was detected at mRNA level by real-time RT-PCR.Anti-CYP2D6 and anti-HP in the serum were detected by ELISA on d28 to evaluate the relationship between inflammation intensity and autoantibody.?3?Mice were treated with Ad,CCl₄,pCYPD6,Ad/pCYP2D6 and CCl₄/pCYP2D6 respectively.HE staining was used to detect AIH inflammation at wk 5.Inflammatory scores were calculated at wk 5,wk 7 and wk 9.The ALT levels in the serum of mice were detected.Liver fibrosis was measured by Sirius red staining and fibrosis scores were calculated at wk 9.The gene expression of Col1a1 and Col1a2 in liver was detected by real-time PCR at wk 9 and wk 12.Mice received Ad/pCYP2D6,CCl₄/pCYP2D6 and psTLR2/4,HE staining and AIH inflammation scores were calculated at wk 5 and wk 8,and the gene expression of Col1a1 and Col1a2 in liver was detected by real-time PCR at wk 9 and wk 12.The AIH inflammation and liver fibrosis were observed.?4?When Ad/CYP2D6 or CCl₄/CYP2D6 triggered the autoimmune response in the mice,we continue to expand the time-frame of CYP2D6 expression.ELISA was used to detect changes in autoantibodies.HE staining and AIH inflammation scores were calculated to observe changes of AIH inflammation on d35 and d56.Liver fibrosis was observed with Sirius red staining and fibrosis scores were calculated on d63.?5?The mice received the injection of adenovirus?Ad?or CCl₄.The mice were sacrificed on d14.CD4⁺CD25⁺T cells were isolated from the spleen and liver of the mice.The suppressive function of the cells was tested in T cell proliferation assay.The expression of Foxp3 gene in the cells was detected by real-time RT-PCR and Western blot.The mice received the injection of adenovirus or CCl₄ with or without psTLR2/4.On d14,CD4⁺CD25⁺T cells were isolated from the liver for analyzing the function of the cells and detecting the expression of Foxp3 gene.The effect of Ad and CCl₄ on the function of Treg was studied.?6?The mice received the injection of adenovirus?Ad?or CCl_4.IL-6 and IL-12 in the liver were detected by ELISA.On d14,CD4⁺CD25⁺T cells were isolated from the liver.The phosphorylation of STAT3 and STAT4 was detected by Western blot.The mice were untreated or treated with anti-IL-6 and/or anti-IL-12antibodies to neutralize IL-6 and/or IL-12 in vivo.On d14,the expression of Foxp3 gene in hepatic CD4⁺CD25⁺T cells was detected at mRNA level by real-time RT-PCR.The mice received the injection of CCl₄ or plasmids pIL6 and pIL12.The mice were sacrificed on d14.CD4⁺CD25⁺T cells were isolated from the liver of the mice.The expression of Foxp3 gene was detected by real-time RT-PCR and Western blot.The suppressive function of the cells was tested in T cell proliferation assay.?7?The mice received the injection of pCYP2D6 and/or adenovirus?Ad?or CCl₄?The expression of Il2 and Ifng genes in the liver was detected at mRNA level by real-time RT-PCR on d28.The mice received the injection of pCYP2D6 with different dosages of adenovirus or CCl₄.The expression of Il2 and Ifng genes was detected by real-time RT-PCR on d28.The mice received the injection of pCYP2D6 and adenovirus or CCl₄ with or without psTLR2 and psTLR4.On d28,the expression of Il2 and Ifng genes in the liver was detected by real-time RT-PCR.The effects of Ad/CYP2D6,CCl₄/CYP2D6 and sTLR2/4on the Th1 immune response were observed.?8?The mice received the injection of pCYP2D6 and CCl₄ or plasmids pIL6/pIL12.The expression of Il4 and Il13 genes in the liver was detected at mRNA level by real-time RT-PCR on d28.The antibodies against hepatic proteins of naive mice?anti-HP?in the serum were detected by ELISA at the indicated time points.The mice received the injection of Ad and CCl₄ and pIL6/pIL12 or plasmids psTLR2/4.IL-4,IL-25,and IL-33 in the liver were detected by ELISA.The mice received the injection of Ad/pCYP2D6 and CCl₄/pCYP2D6 with or without plasmids psTLR2/4.The expression of Il4 and Il13 genes in the liver was detected by real-time RT-PCR.The mice received the injection of pCYP2D6 and CCl₄.The mice were untreated or treated with the i.p.injection of anti-IL-4,anti-IL-25,or IL-33antibodies to neutralize IL-4,IL-25,or IL-33 in vivo.The expression of Il4 and Il13genes in the liver was detected by real-time RT-PCR.Results:?1?Both adenovirus and CCl₄,but not the i.v.injection of pCYP2D6plasmid,could efficiently induce non-AIH hepatic inflammation.The long-lasting inflammatory response,evaluated by the expression of Il1b,Il6 and Tnfa genes in the liver,was induced by either adenovirus or the repeated CCl₄ injection,which did not influence the transgene expression of CYP2D6 in the liver,but promoted the production of antibodies against CYP2D6 and normal hepatic proteins.Blocking TLR2/4 signaling suppressed the adenovirus-and CCl₄-induced inflammatory response.Accordingly,sTLR2/sTLR4 suppressed the induction of antibodies against human CYP2D6 and the hepatic proteins of mice.?2?Either adenovirus or CCl₄ could induce the injury of the liver,evaluated by ALT levels in serum,which was related to the dosage of virus and CCl₄.Higher dosage of adenovirus and CCl₄ resulted in the increased release of TLR2/4ligands,and induced stronger inflammatory response in the liver.Intriguingly,the stronger inflammation?non-AIH inflammation?resulted in stronger immune response to CYP2D6,and was required for CYP2D6 to induce autoimmune antibodies against the hepatic proteins from normal liver.On the other hand,we also used CCl₄ to induce the inflammatory response within shorter time-frame,which was not sufficient to promote the autoimmune response induced by CYP2D6.?3?Five weeks after the first injection of adenovirus or CCl₄,the adenovirus-and CCl₄-induced liver inflammation?non-AIH inflammtion?and injury were no longer existent.However,in the mice treated with Ad/CYP2D6 and CCl₄/CYP2D6,but not CYP2D6 alone,the lobular inflammation and portal inflammation?AIH inflammation?were observed in the liver.AIH inflammation and liver injury were persistently detected.Moreover,the sustained AIH inflammation in Ad/CYP2D6 and CCl₄/CYP2D6 groups resulted in the fibrosis in the liver,evaluated by Picrosirius staining and the expression of Col1a1 and Col1a2 genes.Intriguingly,when Ad/CYP2D6 or CCl₄/CYP2D6 was used to trigger AIH,the AIH inflammation and the up-regulation of Col1a1 and Col1a2 genes were prevented by expressing sTLR2 and sTLR4 in the liver to block TLR2/4 signaling during early non-AIH inflammation.?4?If Ad/CYP2D6 or CCl₄/CYP2D6 triggered the autoimmune response in the mice,expanding the time-frame of CYP2D6 expression resulted in the increase of autoimmune response,the higher titers of autoantibodies,and more severe AIH inflammation and fibrosis in the liver.However,Simply expressing CYP2D6 in the liver could not induce AIH in the mice,even if the time-frame of CYP2D6 expression was expanded.?5?When adenovirus and CCl₄ were used to induced the sustained non-AIH liver inflammation,the suppressive function of hepatic,but not splenic,Treg cells was significantly reduced.Consistently,the expression of Foxp3 was significantly reduced in hepatic Treg cells.The down-regulation of Foxp3 gene expression was positively related to not only the dosage of adenovirus and CCl₄ but also the repeated injection of CCl₄,thus relating to the amplified?stronger and long-lasting?liver inflammation.The negative effect of the amplified liver inflammation on the function and Foxp3 gene expression of hepatic Treg cells was suppressed by expressing sTLR2/4 in the liver.?6?The amplified inflammation in the liver resulted in the sustained increase of the hepatic levels of IL-6 and IL-12.Accordingly,the activation of STAT3 and STAT4 in hepatic CD4⁺CD25⁺T cells was increased.When IL-6 and IL-12 were simultaneously blocked in vivo by using neutralizing antibody,the effect of inflammation on the expression of Foxp3 gene was abrogated,while blocking either IL-6 or IL-12 alone could partially attenuate the down-regulation of Foxp3 gene.When IL-6 and IL-12 were expressed in the liver by using expressing vectors,IL-6/IL-12 could efficiently suppress Foxp3 gene expression and the function of hepatic CD4⁺CD25⁺T cells.?7?When Ad/CYP2D6 or CCl₄/CYP2D6was used to induce autoimmune hepatitis,Th1 response was induced.Th1 response was also related to the dosage of adenovirus and CCl₄.The expression of Il2 and Ifng genes was suppressed by blocking TLR2/4 signaling with sTLR2/4 in vivo during early non-AIH inflammation.?8?The hepatic expression of IL-6/IL-12/CYP2D6 was not efficient in inducing Th2 response,evaluated by the expression of Il4 and Il13 genes in the liver.Accordingly,IL-6/IL-12/CYP2D6 was not efficient in inducing the production of autoimmune antibodies.Differently,CCl₄/CYP2D6 was much more efficient in inducing Th2 response and the production of autoantibodies.In the absence of self-mimicking antigen,the injection of adenovirus or CCl₄,but not simply expressing IL-6/IL-12,resulted in the increase of IL-4,IL-25 and IL-33 in the liver.The increase of these cytokines was abrogated by blocking TLR2/4 signaling in vivo.CCl₄/CYP2D6-and Ad/CYP2D6-induced Th2 response was abrogated by blocking TLR2/4 signaling.Interestingly,blocking IL-4 or IL-25,but not IL-33 in vivo could abrogate Th2 response,but had little influence on Th1 response.Conclusion:The liver injury resulting from virus?adenovirus?or chemicals?CCl₄?could induce an amplified?stronger/long-lasting?hepatic inflammation by releasing danger signals such as the ligands for TLR2/TLR4.The amplified inflammation resulted in the impairment of hepatic Treg function due to the sustained increase of IL-6 and IL-12 in the liver.IL-6/IL-12 increased the activation of STAT3 and STAT4 in hepatic CD4⁺CD25⁺Treg cells,thus suppressing Foxp3 gene expression to reduce their suppressive function.The increase of IL-12 and the impairment of Treg function promoted Th1 response in presence of self-mimicking antigen?human CYP2D6?.The amplified inflammation resulted in the increase of IL-4 and IL-25 in the liver.The moderate increase of IL-4 was sufficient for cooperating with IL-25 to initiate Th2response,but inefficient in suppressing Th1 response,favoring the balanced Th1/Th2responses.Consequently,either adenovirus/CYP2D6 or CCl₄/CYP2D6 could induce AIH in the mice,leading to hepatic fibrosis.