The Inhibitory Effects And Related Mechanisms Of Iron Deprivation On T Helper 17 Cell Differentiation

Objective:T_H17 cells,characterized by the production of IL-17A and IL-17F,are critical participants in multiple autoimmune diseases,including rheumatoid arthritis（RA）,multiple sclerosis（MS）.Iron,as an essential microelement,plays a critical role in immune responses.However,its role in T helper cell differentiation and function remains poorly understood.We are intended to investigate the effect of blocking iron intake through anti-CD71 m Ab or DFO on CD4⁺T helper cell differentiation and the underlying molecular mechanism involved.Methods:1.Detection the effect of anti-CD71 m Ab on iron uptake by CD4⁺T cells（1）Expression of CD71 in CD4⁺T cell:na（?）ve CD4⁺T cell were stimulated with anti-CD3 m Ab and anti-CD28 m Ab for the indicated times and then the expression of CD71 was assessed by flow cytometry.C57BL/6 mice were immunized with MOG_35-55 peptide to induce EAE disease and the draining lymph node and CNS cells were harvested on day 0,5,10 after immunization.The expression of CD71,IFN-γand IL-17A in CD4⁺T cells was assessed by flow cytometry.（2）Effect of anti-CD71 m Ab on iron uptake by CD4⁺T cell:na（?）ve CD4⁺T cells were activated with anti-CD71 m Ab or with anti-CD71 m Ab plus FAC for 48 h,the cells were lysed in lysis buffer and then ferritin light chain was analyzed by western blotting.2.Detection the effect of anti-CD71 m Ab on CD4⁺T cell activation,function and proliferation.（1）Effect of anti-CD71 m Ab on CD4⁺T cell activation:na（?）ve CD4⁺T cells were activated in the presence or absence of anti-CD71 m Ab for 24 h,the expression of CD44,CD62L,CD25 and CD69 in CD4⁺T cells were detected by flow cytometry.（2）Effect of anti-CD71 m Ab on CD4⁺T cell function:na（?）ve CD4⁺T cells were activated in the presence or absence of anti-CD71 m Ab for indicated time.m RNA expression of Il2,Ifng and Tnfa in CD4⁺T cells were assayed by quantitative PCR.The expression of IL-2 in CD4⁺T cells were analyzed by flow cytometry.（3）Effect of anti-CD71 m Ab on CD4⁺T cell proliferation:na（?）ve CD4⁺T cells were activated in the presence or absence of anti-CD71 m Ab for 72 h,the expression of Ki67in CD4⁺T cells was measured by flow cytometry.3.Detection the effect of anti-CD71 m Ab on CD4⁺T helper cells differentiation in vitro（1）Effect of anti-CD71 m Ab on T_H17 cell differentiation:na（?）ve CD4⁺T cells were polarized under T_H17 cell conditions with or without anti-CD71 m Ab.The expression of IL-17A and IL-17F in CD4⁺T cells were analyzed by flow cytometry.Culture supernatant was collected and the concentration of IL-17A in culture supernatant was detected by ELISA.m RNA expression of T_H17-related genes in CD4⁺T cells were assayed by quantitative PCR.（2）Effect of anti-CD71 m Ab on T_H1,T_H2 and T_reg cell differentiation:na（?）ve CD4⁺T cells were polarized under T_H1,T_H2 and T_reg cell conditions with or without anti-CD71 m Ab,respectively,and the cells were analyzed in terms of IFN-γ（T_H1 cell）,IL-4（T_H2 cell）,and Foxp3(T_regcell)by flow cytometry.（3）Effect of anti-CD71 m Ab on pathogenic T_H17 cell differentiation:na（?）ve CD4⁺T cells were polarized under pathogenic T_H17 cell conditions with or without anti-CD71 m Ab.The expression of IL-17A and IL-17F were analyzed by flow cytometry.Culture supernatant was collected and the concentration of IL-17A in culture supernatant was detected by ELISA.（4）Na（?）ve CD4⁺T cells were polarized under T_H17 cell or pathogenic T_H17 cell conditions with anti-CD71 m Ab or with anti-CD71 m Ab plus FAC,IL-17A production by CD4⁺T cells was measured by flow cytometry.4.Detection the effect of anti-CD71 m Ab on CD4⁺T helper cells in mice with EAE（1）Effect of anti-CD71 m Ab on EAE in mice:C57BL/6 mice were immunized with MOG_35-55 peptide emulsified in CFA subcutaneously to induce EAE disease and then the mice were intrathecally injected with anti-CD71 m Ab or control as indicated.The severity of EAE were compared between the anti-CD71 m Ab treated and control mice.The frequency of IL-17A,GM-CSF,IFN-γand Foxp3 in CD4⁺T cells in the CNS and d LNs were measured by flow cytometry.The CNS cells were harvested and re-stimulated ex vivo with MOG_35–55 peptide under T_H17 condition,cytokine release of IL-17A and GM-CSF in culture supernatants measured by ELISA.（2）To investigate that the effect of anti-CD71 m Ab on EAE in mice was derived from the change of T_H17 cells,a passive transfer EAE was established.CD45.1⁺C57BL/6 mice were immunized with MOG_35-55 peptide emulsified in CFA subcutaneously to induce EAE disease and the draining lymph node and CNS cells were harvested on day 9 after immunization.The cells were restimulated ex vivo with MOG_35–55 peptide under T_H17 condition for 4 days,in the presence or absence of anti-CD71 m Ab.CD4⁺T cells were isolated and adoptively transferred into 4 Gy-irradiated CD45.2⁺mice recipient mice.The severity of EAE was monitored and the frequency of IL-17A in CD4⁺T cells in the CNS were measured by flow cytometry.5.Detection the effect of anti-CD71 m Ab on T_H17 cell pathogenic（1）Effect of anti-CD71 m Ab on gene expression of T_H17 cell:RNA sequencing was performed on CD4⁺T cells cultured under T_H17 cell condition with or without anti-CD71 m Ab for 3 days.The differentially expressed genes between control and anti-CD71 m Ab treated T_H17 cells was analyzed.（2）To confirm the effect of anti-CD71 m Ab on T_H17 cell pathogenic,a passive transfer EAE was established.2D2 na（?）ve CD4⁺T cells were activated under pathogenic T_H17 cell condition with or without anti-CD71 m Ab for 3 days and re-stimulated by TCR for another 2 days,and then adoptively transferred into Rag2^-/-mice.The severity of EAE was monitored and the frequency of IL-17A in CD4⁺T cells of in the d LN and CNS were measured by flow cytometry.6.Detection the effect of anti-CD71 m Ab on T_H17 cell differentiation by promoting IL-2 expression（1）Effect of anti-CD71 m Ab on IL-2 expression in T_H17 cell:na（?）ve CD4⁺T cells were polarized under T_H17 cell or pathogenic T_H17 cell conditions for 3 days with or without anti-CD71 m Ab,IL-2 production in CD4⁺T cell was measured by flow cytometry.（2）Effect of anti-CD71 m Ab on T_H17 cell differentiation by promoting IL-2expression:na（?）ve CD45.1⁺CD4⁺T cells were activated under T_H17 cell condition（with or without anti-IL2 m Ab）for 24 h.At the same time,na（?）ve CD45.2⁺CD4⁺T cells were activated under T_H17 cell condition（with or without anti-IL2 m Ab）in the presence or absence of anti-CD71 m Ab for 24 h.Cells were washed,mixed and then cocultured under the original T_H17 cell condition for an additional 48 h.IL-17A production by CD45.1⁺CD4⁺T cells was assayed by flow cytometry.Na（?）ve CD4⁺T cells were activated in the presence or absence of anti-CD71 m Ab under T_H17 cell culture condition for 3 days with anti-IL2 m Ab.The induction of IL-17A⁺in CD4⁺T cells was analyzed.7.The mechanism of anti-CD71 m Ab regulates T_H17 cell differentiation（1）Effect of anti-CD71 m Ab on IL-6 and TGF-βsignaling pathway:na（?）ve CD4⁺T cells were cultured under T_H17 conditions in the presence or absence of anti-CD71 m Ab.Phosphorylation of STAT3 and SMAD was measured by flow cytometry at the indicated time points.（2）Effect of anti-CD71 m Ab on RORγt expression in CD4⁺T cell:na（?）ve CD4⁺T cells were activated in the presence or absence of anti-CD71 m Ab under T_H17 cell condition for 3 days.Expression of RORγt was analyzed by flow cytometry and western blotting.（3）Effect of anti-CD71 m Ab on the binding ability of RORγt to Il17a gene locus:na（?）ve CD4⁺T cells were activated in the presence or absence of anti-CD71 m Ab under T_H17 cell condition for 48 h.The CHIP assays were performed with anti-RORγt m Ab.Binding of antibodies to the Il17a promoter and CNS2 regions was detected by real-time PCR and was normalized according to the input control.（4）Effect of anti-CD71 m Ab on chromatin accessibility of Il17a gene locus:na（?）ve CD4⁺T cells were activated in the presence or absence of anti-CD71 m Ab under T_H17cell condition for 48 h.The CHIP assays were performed with the indicated antibodies:Anti-H3K4me3 m Ab,Anti-H3K27ac m Ab,Anti-H3K9me3 m Ab,Anti-RNA Pol II m Ab.Binding of antibodies to the Il17a promoter and CNS2 regions was detected by real-time PCR and was normalized according to the input control.8.Detection the effects of DFO on CD4⁺T helper cells in mice with EAE（1）Effect of DFO on T_H17 cell differentiation:na（?）ve CD4⁺T cells were polarized under T_H17 cell or pathogenic T_H17 cell conditions with or without DFO,IL-17A production by CD4⁺T cell was measured by flow cytometry.（2）Effect of DFO on EAE in mice:C57BL/6 mice were immunized with MOG_35-55 peptide emulsified in CFA subcutaneously to induce EAE disease and the mice were intraperitoneal injection with PBS or DFO（started 21 days before immunization）until the end of experiment.The severity of EAE were compared between the DFO treated and control mice.The frequency of IL-17A,GM-CSF,IFN-γand Foxp3 in CD4⁺T cells in the CNS were measured by flow cytometry.Results:1.Anti-CD71 m Ab inhibited iron uptake by CD4⁺T cells（1）Following TCR activation,the expression of CD71 was upregulated in CD4⁺T cell.CD4⁺T cells isolated from the spinal cords and draining lymph node of EAE mice expressed significantly higher CD71 along with IL-17A and IFN-γcompared with CD4⁺T cells from the naive mice.（3）Upon treatment with anti-CD71 m Ab,the intracellular ferritin light chain content was remarkably reduced in CD4⁺T cells,while this effect could be restored by supplementing the cellular iron with ferric ammonium citrate.2.Increase of IL-2 production in Anti-CD71 m Ab-treated CD4⁺T cells（1）The expression of CD44,CD62L,CD25 and CD69 were comparable between anti-CD71 m Ab treated and control CD4⁺T cell.（2）The percentages of Ifng and Tnfa were comparable between two group,while Il2 expression was increased in anti-CD71 m Ab treatment group.（3）Treatment with anti-CD71 m Ab led to inhibited the proliferation of CD4⁺T cell.3.Selective inhibition of T_H17 cell differentiation with anti-CD71 m Ab treatment in vitro（1）Flow cytometry results showed that the production of IL-17A and IL-17F were decreased in anti-CD71 m Ab treated CD4⁺T cells,compared to control treatment.The concentrations of IL-17A in supernatant of anti-CD71 m Ab treatment group were decreased.At the m RNA level,treatment of anti-CD71 m Ab significantly inhibited the expression of Il17a,Il17f and Il23r whereas the expression of Rorc and Rora were not significantly affected.（2）The percentages of T_H1,T_H2,or T_reg subsets were comparable between anti-CD71 m Ab treated and control CD4⁺T cell.（3）The results showed that the expression of IL-17A and IL-17F were decreased in anti-CD71 m Ab treated CD4⁺T cells,compared to control treatment.（4）Flow cytometry results showed that the expression of IL-17A could be partially restored by supplementing the cellular iron with ferric ammonium citrate.4.Amelioration of EAE development with anti-CD71 m Ab administration in vivo（1）Mice treated with the anti-CD71 m Ab exhibited a much-reduced severity of symptoms in comparison to the control group.Anti-CD71 m Ab decreased IL-17A and production in the CNS but the percentage of IFN-γand Foxp3 remained unaffected.（2）The recipients of anti-CD71 m Ab-treated T cells showed a lower clinical score compared with the recipients of T cells in the control group.The frequencies of IL-17A⁺CD4⁺T cells in CNS of recipients were decreased.5.Repression of the pathogenic signature of T_H17 cell with anti-CD71 m Ab treatment（1）The genes in relation to the differentiation and pathogenicity of T_H17 cell were down-regulated in anti-CD71 m Ab treated T_H17 cells.（2）The recipients of anti-CD71 m Ab-treated 2D2 T cells showed a lower clinical score compared with the recipients of 2D2 T cells in the control group and was associated with decreased frequencies of IL-17A⁺CD4⁺T cells in CNS of recipients.6.Anti-CD71 m Ab treatment enhance the expression of IL-2 in T cells,which in turn suppress the differentiation of T_H17 cells（1）In anti-CD71 m Ab-treated T_H17 cells,frequencies of IL-2 were significant increased compared with non-anti-CD71-treated T_H17 cells.（2）We found that CD45.1⁺CD4⁺T cells co-cultured with anti-CD71 m Ab-treated T cells displayed less IL-17A production than the cells co-cultured with non-anti-CD71m Ab-treated T cells.When the coculture experiment was performed in the presence of anti-IL2 antibodies,the percentages of IL-17A expressed by CD45.1⁺CD4⁺T cells in the control and anti-CD71 m Ab treated groups were at a similar level.IL-2 blockade by anti-IL2 m Ab could not reverse IL-17A production directly in anti-CD71 m Ab-treated T cells.7.Modulation of histone modifications in T_H17 cells with anti-CD71 m Ab treatment（1）The phosphorylation activation status of STAT3 and SMAD were comparable between anti-CD71 m Ab treated and control CD4⁺T cell.（2）Flow cytometry and immunoblotting assay results indicated that the expression of RORγt were comparable between anti-CD71 m Ab treated and control CD4⁺T cell.（3）By using CHIP-qPCR,we found that the binding of RORγt to the Il17a locus was reduced in anti-CD71 m Ab treated CD4⁺T cell.（4）By using CHIP-qPCR,we found that anti-CD71 m Ab treatment led to reduced H3K4me3 and H3K27acenrichment at Il17a gene locus,whereas the modifications of repressive H3K9me3 were comparable between anti-CD71 m Ab and control CD4⁺T cell.8.Amelioration of EAE development with DFO administration in vivo（1）The results showed that the expression of IL-17A and IL-17F were decreased in DFO treated CD4⁺T cells,compared to control treatment.（2）Mice treated with the DFO exhibited a much-reduced severity of symptoms in comparison to the control group in EAE,reduced frequencies of IL-17A-producing CD4⁺T cell in the CNS while the percentage of IFN-γand Foxp3 were comparable between two groups.Conclusion:1.Anti-CD71m Ab treatment restrained iron availability of T cells and suppressed the differentiation of T_H17 cells,significantly relieved the development of EAE diseases.2.Iron deprivation by targeting of CD71 enhanced IL-2 production,which inhibited T_H17 cell differentiation in a paracrine manner;Iron deprivation by targeting of CD71impaired chromatin accessibility at the Il17a locus and reduced the binding of RORγt.3.DFO suppressed the differentiation of T_H17 cells and relieved the development of EAE diseases.