Complement Component C3 Regulates Th17 Cells Response In Allograft Rejection

?Background?Transplantation is an efficient treatment method for organ failure,and rejection is the main cause of allografts loss.Studies have shown that CD4+T cells play an essential role in allograft rejection,and each T cell subset performs different function.In contrast to Th2 and Treg cells inhibit allograft rejection,Th1 and Th17 cells promote this process.The widely used immunosuppressive agents,such as CsA,FK506 and Rapamycin,promote allograft tolerance by inhibiting the whole T cell subsets response,which has obvious shortages during this process.Novel immunosuppressive agents that could selectively inhibit Th1/17 response while enhance Treg cells expansion are the future of anti-rejection drugs.The research on the mechanism of T cells differentiation in allograft rejection is great significance,which will strengthen the knowledge of rejection pathophysiology and improve the development of new immunosuppressive drugs targeting on T cells.Th17,characterized by massive IL-17 production,is an important subset of CD4+T cells.IL-17 could recruit a mass of neutrophils and mononuclears or sensitize other cytokines in regulating inflammatory response.Th17 subset plays an essential role in allergic asthma,diabetes,psoriasis,SLE as well as allograft rejections.It was shown that Th17 cells infiltration was closely related with renal graft function and tissue damage.Blocking IL-17 signaling with specific antibodies significantly attenuated the bronchiolitis obliterans with clear decreased lymphocytes and IFN-? positive cells infiltration in lung allografts.Exploring the underlying mechanism of Th17 cells proliferation and differentiation is great importance for specific Th17-targeted drugs in the future.Complement,which is proved to be involved in the regulation of T cells response,is a critical component of innate immune system.After binding to complement component C3 a or C5 a,complement receptors C3 aR or C5 aR,expressed on CD4+T cells,initiate the downstream G-protein signaling and enhance T cells proliferation and differentiation,Th1 response as well as IFN-? production.C5 a R deficiency remarkably prolonged renal allograft survival with significantly alleviated alloreactive T cell response,which suggested that complement regulates T cells proliferation and differentiation in allograft rejection.However,whether the complement activation related and further regulated Th17 response remains unclear.Complement component C3 is a pivot molecule in complement system.Complement component C3 deficiency attenuates Th1/Th17 cells response and regulates Treg expansion and function simultaneously,which indicates that complement C3 play a vital role in regulating allograft rejection by impacting on Treg/Th17 response.?Objectives?1.To elucidate the relations of complement activation and Th17 cells response in allograft rejection;2.To estimate the essential role of complement component C3 in allograft rejection and Th17 response;3.To explore the underlying mechanisms that complement component C3 impacts on Th17 cells response in allograft rejection.?Methods?1.The complement activation and Th17 cells response during allograft rejection.1)Periphery blood from renal transplant patients was collected and PBMCs was isolated.Th17 cells response and complement activation components,named C3 a and C5 a,were tested by FCM as well as ELISA;2)Rejected renal samples or normal renal tissue were obtained,and then the complement activation,IL-17 expression as well as Th17 cells infiltration were examined by IHC and IF;3)Following stimulated with complement component C3 a,the expression of IL-17 in HK2 cells was tested by IHC;4)After stimulated with complement component C5 a,the expression of IL-17 in HK2 cells was analyzed by FCM and IHC.2.The role of complement component C3 in allograft rejection and Th17 response.1)Tail grafts from Bm12,C3+/+ and C3-/-were transplanted to the back of Bm12 recipients.7 days later,the rejections of allografts were monitored and recorded;2)Tail allografts Bm12 were transplanted to the back of C3+/+ and C3-/-recipients.After 7 days,the rejections of allografts were monitored;3)Neutrophils,macrophage,Th17 cells and DCs infiltration in allografts were tested by HE,IHC and IF.And the expression of cytokines and chemokines was examined by q PCR;4)The draining lymphocytes and spleen cells of Bm12 recipients were collected and Th1/17 response was tested by FCM at indicated time points.3.The underlying mechanisms that complement C3 impacts on Th17 cells response.1)The draining lymphocytes of Bm12 recipients were collected and the proliferation of lymphocytes was tested by MLR 10 days after transplantation;2)The draining lymphocytes and spleen cells of Bm12 recipients were collected and Treg cells response was tested by FCM at indicated time points;3)Treg cells were eliminated by anti-CD25 m Ab application,and the C3-/-allografts survival was monitored;4)The purified na?ve CD4+T cells were cocultured with irradiated BMDCs for up to 12 days.The concentration of IL-10 and Treg cells differentiations were analyzed by ELISA,PCR as well as FCM.?Results?1.Enhanced complement activation and Th17 cells response in allograft rejection.1)The concentrations of C3 a and C5 a after renal allograft transplant were significantly increased,compared with that of before the transplant surgery;dramatically increased C3,C5 a R as well as C5b-9 expression were detected in rejected allografts;2)The frequency of Th17 cells in PBMCs after renal allograft transplant was remarkably enhanced,compared with that of before the transplant surgery;clear increased Th17 cell infiltrations were detected in rejected renal allografts;3)Following stimulated with C3 a and C5 a,the expression of IL-17 was significantly enhanced in HK2 cells.2.C3 deficency significantly prolonged the survival of allografts and dramatically attenuated Th17 response.1)In contrast to C3+/+ allografts were rejected within 17 days,it was shown that sixty percent of C3-/-allografts survived until day 30,which indicated that C3 deficiency could prolong MHC-?molecular disparate allograft survival;2)No difference in survival for Bm12 allografts were observed in C3+/+ or C3-/-recipients,which suggested that systemic C3 deficency could not prolong the allograft survival;3)Compared with the extensive necrosis and massive mononuclear infiltrations in C3+/+ allografts,it was observed that C3-/-allografts were intact and only few mononuclear cells infiltrations,which were detected by HE staining.Agree with that,it was shown that neutrophils,macrophages,T cells in C3-/-allografts were significantly lower than that of C3+/+ allografts.Furthermore,IF staining revered that Th17 cells in C3-/-allografts were remarkably decreased;4)C3-/-allografts performed clearly decreased the mRNA expression of critical cytokines(IFN-?,IL-17 and IL-23)and chemokine(CXCL-9),which are related with Th1/17 cells responses.Similarly,C3-/-allografts also performed significantly reduced m RNA levels of inflammatory cytokines(IL-1?,IL-6 and TNF-?).These observations indicated that C3 deficiency in allografts leads to decreased expression of inflammatory cytokines and chemokines;5)C3 deficiency remarkably reduced Th1/17 frequency,which suggests that complement C3 enhances Th1/17 cells response.3.Complement component C3 promotes Th17 cells response by attenuated Treg cells population,which promotes the allograft rejection.1)C3 deficiency significantly attenuated lymphocytes proliferation;2)It was shown that Foxp3 expression was significantly increased in C3-/-allograft,which indicates that the prolonged C3-/-allografts survival could be related with Treg cells;3)C3 deficiency remarkably increased Treg cells expansion,which indicates that the prolonged C3-/-allografts survival might be dependent on Treg cells;4)Following Treg cells elimination by applying anti-CD25 mAb,it was shown that Th17 cells response and allograft rejection were enhanced,which leads to impaired C3-/-allografts survival,indicating a critical role of Treg cells expansion in the prolonged C3-/-allografts survival;5)Decreased DCs infiltration and attenuated CD80 expression were detected in C3-/-allografts by IF staining.Compare with C3+/+DC,C3-/-DC significantly increased IL-10 production as well as Treg cells expansion.?Conclusions?In this study,we first collected renal allograft samples and tested complement activation and Th17 cells expansion;the impacts of complement components on IL-17 expression was further analyzed in vitro;at last,the effects of complement component C3 on allograft survival and the underlying mechanism that complement component C3 regulates Th17 response were explored.The conclusions as follow:1)Abundant complement activation and enhanced Th17 cells response performs a close correlation in allograft rejection.2)C3 deficiency dramatically prolonged the survival of allografts and remarkably attenuated Th17 response.3)Complement component C3 promotes Th17 cells response by decreased Treg cells population,which strengthens the allograft rejection.