Novel Insights Into The Blocking Of IL-6/IL-6 Receptor ?/STAT3 Pathway For Pig Organ Xenotransplantation In Baboons

Background:With the aging of the population,deterioration of the environment or other factors,the incidence of patients with organ failure in the world is increasing year by year.Allogeneic transplantation is the best cure for organ failure,but the shortage of donor organs severely restrict the development of transplantation.With the development of basic theory of xenotransplantation,the emergence of new gene editing techniques and immunosuppressive agents,xenotransplantation(such as pig-derived organs)will solve the problem of organ shortage.The survival rate and survival time of pig grafts have greatly increased in pig organ-primate xenotransplantation,and provided theoretical and practical supports for further clinical application.However,there are still many problems,such as inflammation,which led to grafts rejection,even grafts dysfunction.Interleukin-6(IL-6)is one of the most important pro-inflammatory cytokines involved in some inflammatory diseases.It have shown that IL-6 plays import roles in inflammation,cell proliferation,immune cell activation and rejection reaction,etc...The mechanism is that the IL-6 binds to IL-6 receptor ?(IL-6 R?)and IL-6 receptor ?(gp130),which in turn induces signal transduction and activates transcriptional protein 3 phosphorylation(p-STAT3)and transfers signal to the nucleus.tocilizumab(IL-6 R? monoclonal antibody)has been successfully used in the clinical treatment for some autoimmune diseases(such as autoimmune arthritis,multiple Castleman's disease).We have previously found that tocilizumab induces the level of IL-6increased in baboon recipients with a pig xenograft,and not show the benefits for the xenografts protection,and it was reported that tocilizumab can not suppress expression of p-STAT3 induced by IL-6 in pig arterial endothelial cells(PAECs)in vitro.Therefore,we hypothesized that there are species differences in IL-6 R?between pig xenografts and recipients and the tocilizumab can not cross with the pig IL-6 R?,and elevated IL-6 in the recipient will continue to activate IL-6/pig IL-6R?/STAT3 pathway in pig grafts and lead to grafts dysfunction.The aims of the present study were(I)In vivo study of the role of tocilizumab in blocking IL-6 R? and relative signal pathways in pig organ xenotransplantation,(II)In vitro study of the mechanism of tocilizumab in blocking IL-6/IL-6 R?/STAT3 pathway in pig organ xenotransplantation,(III)to find a strategy for inhibiting the human / pig IL-6 and human / pig IL-6 R? signal pathways in pig organ transplantation in nonhuman primates(NHP),and(IV)to study expression of genes induced by IL-6 in PAECs and inhibitory effects of related inhibitors.Methods:1.Detection of the level of baboon or pig-derived IL-6 in baboon serum.Total 23 baboons that received a pig graft(14 of which were pig kidney xenografts and 9 were pig heart xenografts),16 of these baboons received tocilizumab treatment(14 of which were pig kidney xenografts,and 2 were pig heart xenografts),and other 7baboons did not receive tocilizumab treatment.The cytometric bead assay(CBA)was used to detect the level of serum baboon-derived IL-6 in 23 baboons with a pig xenograft.ELISA was used to detect the level of serum pig-derived IL-6 in 14 baboons with a pig kidney xenograft and tocilizumab treatment.The relationship between IL-6 level and tocilizumab treatment was analyzed;we also observed the change of serum pig derived IL-6 level in the baboons with a pig kidney xenograft and tocilizumab treatment.2.Kidney graft(n=10)and liver tissue(n=7)from these baboons who received a pig kidney xenograft and tocilizumab treatment,2 negative controls(normal kidney or liver tissues)and 2 positive controls(1 baboon underwent hyperacute rejection after xenotransplantation and 1 baboon that experienced anaphylactic shock without xenotransplantation and tocilizumab).The positive rate of IL-6 and p-STAT3 in kidney xenografts and liver tissues were measured by immunohistochemistry(IHC),and the differences in the positive rate between IL-6 and p-STAT3 in pig kidney grafts or recipient liver tissues in these baboons with tocilizumab treatment were analyzed.3.We used different concentrations of recombinant human or pig IL-6,different time to stimulate PAECs or human arterial endothelial cells(HAECs),then the expression of p-STAT3 was measured by flow cytometry and the best IL-6stimulation concentration and time was identified by the expression of p-STAT3.4.Check the effect of tocilizumab suppresses IL-6/IL-6 R?/STAT3 pathway in vitro.To evaluate the effect and species differences of tocilizumab bind to IL-6 R? in peripheral blood mononuclear cells(PBMCs),we used different concentrations of tocilizumab to block the IL-6 R? in human,baboon and pig PBMCs and the expression of IL-6 R? was measured by flow cytometry.To evaluate effect of the tocilizumab suppresses recombinant human or pig IL-6(20ng/mL,15min)activated the STAT3 pathway in PAECs or HAECs and was assessed by the expression of p-STAT3 that measured by the flow cytometry.Further evaluate the effect of tocilizumab suppresses STAT3 pathway in different species of immune cells,the different concentrations of tocilizumab were used to suppress recombinant human or pig IL-6(20ng/mL,15min)activated the STAT3 pathway in human,baboon,pig PBMCs and the expression of p-STAT3 was measured by flow cytometry.5.Check the effect of siltuximab suppresses IL-6/IL-6 R?/STAT3 pathway in vitro.Different concentrations of siltuximab were used to neutralize recombinant human or pig IL-6 and suppress their activation of STAT3 pathway in PAECs or HAECs and the effect and species differences of siltuximab neutralize recombinant human or pig IL-6 were evaluated by the expression of p-STAT3.The baboon derived IL-6 was checked whether can activate IL-6/IL-6 R?/STAT3 pathway in PAECs,and also checked whether siltuxiamb can neutralize baboon derived IL-6 and neutralization effect in vitro.Different concentrations of siltuximab were used to suppress recombinant human or pig IL-6(20 ng/mL,15 min)activates the STAT3 pathway in baboon PBMCs,and the effect and species differences of siltuximab neutralize recombinant human or pig IL-6 were evaluated in baboon immune cells by the expression of p-STAT3.6.Check the effect of rapamycin suppresses IL-6/IL-6 R?/STAT3 pathway in vitro.Firstly,The PAECs or HAECs were treated by five concentrations of rapamycin for 24 hours,followed by recombinant human or pig IL-6(20ng/mL,15min)activated STAT3 pathway in PAECs or HAECs,and finally the flow cytometry was used to measure expression of p-STAT3.7.The qPCR was used to detect the inhibitory effect of siltuximab or rapamycin on related genes in PAECs induced by recombinant human or pig IL-6.Result:1.The level of serum baboon derived IL-6 was gradually increased in 23 baboons with pig kidney or heart xenotransplantation after immunosuppression induction therapy.The data showed that the level of serum baboon derived IL-6 in baboons who treated with tocilizumab at 0 day(before transplantation,but after immunosuppression induction therapy)was significantly higher than level of baboon derived IL-6 in these without tocilizumab treatment(255 vs 35 pg/mL,p< 0.05).Due to no control group in the pig kidney xenotransplantation group on 1 day after surgery,we only analyzed the pig heart transplantation group and the level of baboon derived IL-6 in baboons who treated with tocilizumab at one day after surgery was significantly higher than those without the tocilizumab treatment(183 vs 23 pg/mL,p<0.01);the data also showed that the level of baboon derived IL-6 in the baboons with a pig kidney xenograft and tocilizumab treatment was significantly higher than these baboons with a pig heart xenograft and tocilizumab treatment(p<0.05).At the time of euthanasia,the level of baboon derived IL-6 in these baboons with pig kidney xenograft and tocilizumab treatment was significantly increased,but there was no significantly different from the time of 1 day after surgery.In order to check whether pig kidney xenografts produce pig derived IL-6,we tested the level of pig derived IL-6 in these baboons with a pig kidney xenograft and tocilizumab treatment on 0 day,1 day after surgery,and euthanasia.Compared to at 0 day,the data showed that pig derived IL-6 can be detected in baboon serum at 1 day after surgery and euthanasia(p<0.05,p<0.05).IHC was used to detect IL-6 and p-STAT3 in pig kidney grafts and baboon liver tissues.The negative controls showed low or no expression of IL-6 and p-STAT3.In positive controls,IL-6 and p-STAT3 in kidney and liver tissues showed strongly positive.There were no differences in the positive rates between IL-6 and p-STAT3 in 10 pig kidney xenografts in these baboons with tocilizumab treatment(positive rate of IL-6was 90%,positive rate of p-STAT3 was 60%);The data showed that there were significant differences in the positive rate between IL-6 and p-STAT3 in the liver tissues in 7 baboons with pig kidney xenograft and tocilizumab treatment(P<0.001)(positive rate of IL-6 was 85.7%;positive rate of p-STAT3 was 28.6%).2.Tocilizumab blocks IL-6 R?(mainly T cells and monocytes)in human andbaboon PBMCs,but can not block IL-6 R? in pig PBMCs.We identified that recombinant human or pig IL-6 optimally activates the IL-6/IL-6 R?/STAT3 pathway at a concentration and time(20 ng/mL,15 min).Tocilizumab completely suppresses recombinant human or pig IL-6 activated STAT3 pathway in HAECs(optimal concentration 12.5 ?g/mL),but can not suppress IL-6-activated STAT3 pathway in PAECs(whether wild-type or GTKO/CD46)(even the highest concentration of 312.5?g/mL).Tocilizumab can totally suppress recombinant human or pig IL-6 activated STAT3 pathway(concentration 12.5 ?g/mL)in human and baboon PBMCs,except in pig PBMCs(even the highest concentration of 312.5 ?g/mL).3.Siltuximab completely neutralizes recombinant human IL-6 and suppresses the p-STAT3 expression in PAECs or HAECs(optimal concentration of 12.5 ?g/mL),but not recombinant pig IL-6(even at the highest concentration of 312.5 ?g/mL).The data showed that siltuximab significantly suppresses p-STAT3 expression induced by the baboon derived IL-6 in PAECs,although neutralization assays showed that siltuximab could not completely neutralize baboon derived IL-6 in vitro.Siltuximab also showed that it can totally suppress p-STAT3 expression induced by recombinant human IL-6(concentration 12.5 ?g/mL)in baboon PBMCs,but can not suppress p-STAT3 expression induced by recombinant pig IL-6.4.Rapamycin(40ng/mL,24h)not only suppresses p-STAT3 expression induced by recombinant human IL-6 in PAECs(p<0.01),and also suppresses p-STAT3 expression induced by recombinant pig IL-6 in PAECs(p<0.01).The data also showed that rapamycin suppresses p-STAT3 expression induced by recombinant human or pig IL-6 in HAECs(p<0.05,p<0.01).5.Recombinant human or pig IL-6 can up-regulate the level of E-selectin,ICAM-1,VEGF and FGF2 mRNA in PAECs.Siltuximab can effectively suppress the up-regulation of the four genes activated by recombinant human IL-6 in PAECs.Rapamycin can significantly suppress up-regulation of VEGF,FGF2 mRNA induced by recombinant human or pig IL-6,but can not or mildly suppress up-regulation of E-selectin,ICAM-1 induced by recombinant human or pig IL-6 in PAECs.Conclusion:`1.There are big differences in IL-6/IL-6 R?/STAT3 pathway in xenotran-splantation due to the species differences in IL-6 and IL-6 R?.2.Tocilizumab can elevate level of serum IL-6 in recipients,while tocilizumab does not cross with IL-6 R? in pig xenografts.Elevated IL-6 continuously activates IL-6/IL-6 R?/STAT3 pathway in pig xenografts and leads to graft dysfunction.3.The administration of a combination of siltuximab and rapamycin might suppress human or pig IL-6/human or pig IL-6 R? pathways activation in pig-to-primate xenotransplantation.4.There are species differences in IL-6 and IL-6 R? in pig organ to human or baboon xenotransplantation,and developing monoclonal antibodies target for human,baboon and pig IL-6 or IL-6 R? have big great prospects.