Toll-like Receptor 2 Regulates Myeloid-derived Suppressor Cell Differentiation And Dendritic Cell Maturation In Mice Comeal Transplantation

Background Corneal allograft rejection is the major reason for transplant failure,even though the cornea is an "immune priviledged" site,which is mainly mediated by CD4+T cell.Activation of CD4+ T cell mainly relies on the specific recognition of pathogens or antigens achieved by antigen-presenting cell(APC).Myeloid-derived suppressor cell(MDSC)is characterized by its early stage in differentiation,generally classified as immature myeloid cell that has not yet stopped its differentiation or myeloid progenitor that acts as precursor for APC,who secretes various molecules,the most effective one is inducible nitric oxide synthase(INOS),resulting in CD4+T cell function defece.The most potent APC is terminally differentiated myeloid cell known as dendritic cell(DC).Mature DC(mDC)is capable of up-regulating co-stimulatory maturation markers(CD86/CD80),there by moderating CD4+T cell activation and proliferation.Therefore,the process of MDSC differentiation into DC and the extent of DC maturation may play a key role in CD4+T cell-mediated immune responses.In vitro,the TLR2 is considered to moderate the process of MDSC differentiation into DC and the extent of DC maturation by many signaling pathways triggered by TLR2.Part Ⅰ TLR2 deficiency attenuates immune rejection in mice after corneal transplantationObjectiveTest the role of TLR2 in immune rejection after allogenetic corneal transplantation.MethodUnilateral orthotopic corneal transplantations were performed with BALB/c donor corneas in wild-type C57BL/6 mice(WT group)and B6.129-Tlr2tmlKir/J TLR2 gene knockout mice(TLR2 KO group)alongside corresponding normal mice(control).C57BL/6 mice served as host accepting isograft was termed as ISO group.The edema,transparency were observed twice per week under the slit lamp microscope after surgery,and rejection index(RI)and median survival time(MST)were scored based on the criteria of Sonoda.ResultWith the passage of time,edema and opacification of corneal graft occurred after the operation in three corneal transplantation groups,especially in WT groups.The RI scores were significantly lower in TLR2 KO group than those in the WT group 7,14,21,28,35,42,49,56 days after operation(all P<0.05).There was a significant difference of the MST between WT and KO group.The MST in TLR2 KO group was(35±3.8)d,while in WT group was(21±1.5)d(P<0.05).Significantly higher allograft survival was observed in TLR2 KO group(P<0.05),with 100%survival in TLR2 KO group compared to70%survival in WT group at 14 days after surgery.ConclusionTLR2 regulates immune rejection after allogenetic corneal transplantation.Part Ⅱ TLR2 regulates local immune responses of corneal graftObjectiveInvestigate the role of TLR2 in local immune responses of corneal graft.MethodBuild five groups as mentioned above.At 14 days,outcomes were recorded and tissues from graft were collected from euthanized mice for histological and qRT-pcr.Ipsilateral cervical draining lymph nodes(DLN)were detected for CD4+T cell(CD3+CD4+)by flow cytometry.ResultImmunohistochemistry staining and mRNA expression of TLR2 revealed that there was no expression of TLR2 could be detected in TLR2 KO group,while expressed weakly in ISO group,and increased in WT group.The HE staining revealed that corneal stromal edema,infiltration of inflammatory cells were more prominent 14 days after operation in WT group than those in TLR2 KO group or ISO group.Number of lymphoid CD4+T cells in WT group significantly increasing compared with TLR2 KO group(P<0.05).Immunohistochemistry staining and mRNA expression of IFN-γ revealed that there was a increasing of IFN-γ expression in WT group compared to TLR2 KO group(P<0.05).ConclusionTLR2 regulates immune rejection after allogenetic corneal transplantation.Part Ⅲ TLR2 regulates MDSC differentiation and DC matruationObjectiveInvestigate the role of TLR2 in MDSC differentiation and DC maturation in mice corneal transplantation.MethodBuild five groups as mentioned above.At 14 days,grafts were collected from euthanized mice for qRT-pcr and immunofluorescence assay.Ipsilateral cervical draining lymph nodes(DLN)were detected for MDSC(CD11b+GR-1+),DC(CD11c+)and mDC(CD11c+CD86 high/CD80high)by flow cytometry.ResultThe mRNA expression of INOS showed that no difference could be found between WT group and TLR2 KO group(P>0.05).Flow cytometry and immunofluorescence staining revealed a significant decreasing in DLN to MDSC in WT group compared to TLR2 KO group(P<0.05),while a increasing to DC(P<0.05).Flow cytometry and immunofluorescence staining revealed a increasing expression level of CD86 or CD80(both P<0.05)in WT group compared to TLR2 KO group(P<0.05).ConclusionTLR2 regulates MDSC differentiation and DC maturation in mice corneal transplantation.