Role Of IL-17in The Therapeutic Effects Of Bone Marrow-derived Stem Cells On Liver Injury

【Background】Hepatic cirrhosis presents the final common pathway of virtual all chronic liverinjury. Traditional routine medical treatments cannot prolong the survival time of patientswith decompensated liver cirrhosis. Although liver transplantation provides the onlyeffective treatment, it is largely limited by such shortcomings as lack of donor organ,high cost and so on. More recently, advances in the development of researches aboutstem cells have promoted the use of bone marrow-derived stem cells （BMSCs） onvarious human diseases. Accumulating clinical studies have investigated anddemonstrated that transplantation of autologous BMSCs could improve liver function ofpatients with decompensated liver cirrhosis. Most researchers believed that BMSCscontribute to clinical improvement of patients with liver disease through immunoregulation of microenvironment in vivo, besides transdifferentiation intohepatocytes or fusion with hepatocytes. However, there is no report about how BMSCsregulate immune microenvironment of patients in vivo.Previously, we have investigated the therapeutic effects of autologous stem cells onpatients with decompensated liver cirrhosis. In our clinical study, we transplantedautologous stem cells, which were mobilized by granulocyte-colony stimulating factor（G-CSF） from bone marrow into peripheral blood, into decompensated liver cirrhoticpatients via hepatic artery. And the results of clinical randomized controlled trial showedthat liver function of patients received stem cell transplantation was significantlyimproved compared with those who received only G-CSF. Up to December of2012,416decompensated liver cirrhotic patients in our hospital have received the treatment of stemcell transplantation. The results showed that stem cell transplantation markedly increasedthree-year survival rate of patients （87%vs29%of control） during their follow-up. Weanalyzed the changes of immune cells and their related cytokines in42patients receivedstem cell transplantation. And we found that during their follow-up at1,3,6,9, and12months after transplantation, serum IL-17levels decreased gradually, which was closelyrelated to the improvement of liver function. These suggest that IL-17might play acritical role in the therapeutic effects of BMSCs on liver disease.In this study, we adopted the mouse model of carbon tetrachloride （CCl₄）-inducedliver injury, which was treated by transplantation of homologous BMSCs. We aimed toclarify the roles of IL-17in the pathogenesis of liver injury and in the therapeutic effectsof BMSCs on liver injury using this model. This will deepen our understanding of themechanisms for BMSCs-mediated improvement of liver diseases.【Objectives】1. To clarify the role of IL-17in the pathogenesis of liver injury induced by CCl₄administration in mice;2. To investigate the implication of IL-17in the process of BMSCs-mediatedamelioration in liver injury. 【Methods】1. Mouse model of liver injury was induced by CCl₄injected intraperitoneally.During the development of liver injury, H&E and Sirius red staining was to analyze liverinflammation and fibrosis, serum biochemistry of alanine aminotransferase （ALT） andalbumin （ALB） were used to monitor liver function. Meanwhile, ELISA was used tomeasure serum IL-17levels, real-time PCR to measure hepatic mRNA expression ofproinflammatory cytokines IL-6, IL-8, TNF-, IFN-γ, IL-17A, IL-17F, IL-17RA, andIL-17RC, flow cytometry to detect the percentage of Th17cells from hepatic monocytes;2. We changed IL-17levels to intervene the development of liver injury induced byCCl₄in mice by blockade of endogenous IL-17with neutralizing IL-17-specific antibodyor administration of exogenous recombinant mouse （rm） IL-17. H&E and Sirius redstaining was to analyze liver inflammation and fibrosis, serum biochemistry of ALT andALB were used to monitor liver function. The hepatic mRNA expression of fibrosisrelated cytokines matrix metalloproteinase3（MMP3）, tissue inhibitor ofmetalloproteinase1（TIMP1）, and TGF-β1were analyzed by real-time PCR. And theexpression of-SMA was measured by immunohistochemistry, real-time PCR, andWestern blot;3. Mouse model of liver injury induced by CCl₄injection were treated by a tailvein injection of homologous BMSCs （5×106cells/mouse）. ELISA was used to measureserum IL-17levels, real-time PCR was to measure hepatic mRNA expression of IL-17A,IL-17F, IL-17RA, IL-17RC, and IL-23p19, and flow cytometry was to analyze thepercentage of Th17cells from hepatic monocytes;4. We changed IL-17levels to intervene the therapeutic effects of BMSCs onCCl₄-induced mouse liver injury by injecting anti-IL-17mAb or rmIL-17. Liverinflammation and fibrosis were determined by H&E and Sirius red staining. Real-timePCR was to measure hepatic collagen-1deposit. And serum biochemistry of ALT andALB was to detect liver function. 【Results】1. IL-17is closely related with the severity of liver injuryH&E and Sirius red staining, serum levels of ALT and ALB showed visible liverinjury in mice after repeatedly6-week CCl₄treatment, displaying infiltration of immunecells, formation of fibrosis, increase of ALT, and decrease of ALB. The results of ELISA,real-time PCR, and flow cytometry showed that serum IL-17levels and hepatic mRNAexpression of IL-17A, IL-17F, IL-17RA, IL-17RC, IL-6, IL-8, and TNF-weresignificantly increased after6-week CCl₄injection, but the changes of IFN-γ and Th17cells in liver tissue were not significant during the development of liver injury.Furthermore, the aforementioned changes gradually reversed to nearly normal levelsafter CCl₄withdrawal.2. IL-17exacerbates the severity of liver injury induced by CCl₄injectionDuring the development of liver injury induced by CCl₄injection, liver injury wassignificantly exacerbated by plus rmIL-17injection, but ameliorated by plus neutralizinganti-IL-17mAb compared with only CCl₄injection. The results of real-time PCR,immunohistochemistry, and Western blot displayed that hepatic mRNA expression offibrosis related cytokines MMP3, TIMP1, TGF-β1and-SMA were significantlyincreased in group of CCl₄plus rmIL-17injections, but decreased in group of CCl₄plusanti-IL-17mAb compared to only CCl₄injection group.3. Homologous BMSCs down-regulate IL-17levelTransplantation of homologous BMSCs to CCl₄-treated mice significantlyameliorated liver injury. Meanwhile, serum IL-17level was markedly decreased even tonormal level. And hepatic expressions of IL-23p19, IL-17A, IL-17F, IL-17RA, andIL-17RC were also decreased as the improvement of liver injury after BMSCstransplantation. However, there was no significant change of the percentage Th17cells inliver tissue.4. IL-17plays a critical role in the therapeutic benefits of BMSCs on mouse liverinjury induced by CCl₄injectionThe results demonstrated that exogenous rmIL-17partly abolished the therapeutic benefits of BMSCs on liver injury induced by CCl₄injection. The severity of liverinflammation and fibrosis in the group of BMSCs plus rmIL-17injections were increasedcompared with that in only BMSCs group. However, liver inflammation and fibrosiswere significantly ameliorated by neutralizing anti-IL-17mAb, similar to the therapeuticeffect of BMSCs transplantation.【Conclusion】These data suggest that IL-17participate in liver injury induced by CCl₄injection inmice, and BMSCs could ameliorate liver injury through down-regulating IL-17. However,the underlying mechanism is worth to be further investigated.