| Objective:Diverse physiological and pathophysiological conditions that lead to the accumulation of misfolded/unfolded proteins in the lumen of the ER cause a burden on the ER that is generally defined as ER stress(ERS).Increasing studies have found that the proportion of CD4~+CXCR5~+ICOS~+T cells were upregulated in patients with rheumatoid arthritis(RA).In recent years,a large number of researches have shown that the allogenic mesenchymal stem cells(MSC)can significantly inhibit the generation of T-follicular helper cells(Tfh)in RA.However,the effects of ER stressed MSC on RA peripheral CD4~+CXCR5~+ICOS~+T cells are still unclear.The aim of this research was to explore whether ER-stressed MSC possessed better regulatory effect on RA peripheral CD4~+CXCR5~+ICOS~+T cells.Methods:(1)Tissue mass adherent culture method is adopted for separating and culturing MSC that were obtained from healthy mothers'umbilical cord after normal deliveries.The ERS phenomenon of MSC was induced by thapsigargin(Tg),MSC was stimulated by Tg at different concentrations(0.1?M,0.25?M,0.5?M,1?M)for 4 h,cell viability was detected by cell counting kit-8(CCK-8).(2)The mRNA and protein levels of ERS marker gene glucose-regulated protein78(GRP78)were checked by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and western immunoblotting(WB),respectively.The levers of X-box binding protein-1(XBP-1)were measured by reverse transcription-polymerase chain reaction(RT-PCR).(3)The peripheral blood mononuclear cells(PBMC)were separated from RA patients by density gradient centrifugation and CD4~+T cells were isolated by magnetic beads separation.Tg-stimulated or unstimulated MSC were co-cultured with or without RA CD4~+T cells activated by anti-CD3/CD28(3?g/m L and 2?g/m L)antibodies,the percentage of CD4~+CXCR5~+ICOS~+T cells were analyzed by flow cytometry.(4)MSC were treated with Tg for 4 h,major soluble factors secreted by MSC in the supernatants and co-cultured system,including cyclooxygenase-2(COX-2)/prostaglandin E2(PGE2),interleukin-6(IL-6),indoleamine 2,3-dioxygenase(IDO),hepatocyte growth factor(HGF)and transforming growth factor-?(TGF-?),were detected by qRT-PCR and enzyme-linked immunosorbent assay(ELISA).(5)The mRNA levels of PGE2 and IL-6 of Tg-treatment or Tg-untreatment MSC in the co-cultrue system were detected by qRT-PCR and the protein levels were checked by ELISA.(6)The expression of PGE2 receptors,EP1-4,in CD4~+T cells were measured by RT-PCR and flow cytometry.(7)EP2/EP4 antagonists and IL-6R antagonists were added to co-culture system for 72 h,respectively.And the proportion of CD4~+CXCR5~+ICOS~+T cells was analysed by flow cytometry.Results:(1)Compared with unstimulated MSC,Tg-treated(0.1?M~1?M)MSC showed no cytotoxicity and the expression of ERS marker,GRP78,was significantly increased in a dose-dependent manner(P<0.05,P<0.01);Another ERS marker gene,XBP-1,was driven the unconventional splicing of X-binding protein 1(XBP-1u)to produce spliced XBP1(XBP1s).(2)Compared with CD4~+T cells alone,the percentage of CD4~+CXCR5~+ICOS~+T cells were decreased significantly after MSC co-cultured with CD4~+T cells for 72 hours in RA patients(P<0.01);while comparisons to MSC co-cultured with CD4~+T cells,the rates of CD4~+CXCR5~+ICOS~+T cells significantly lower than that in the co-culture group of Tg-treatment MSC co-cultured with CD4~+T cells(P<0.01).(3)ELISA and qRT-PCR results showed that the expression of PGE2 and IL-6 in Tg-stimulated MSC was notablely higher than that in Tg-untreated MSC(P<0.05,P<0.05)and there was no significant difference in the expression of IDO,TGF-?and HGF(P>0.05,P>0.05,P>0.05).In addition,PGE2 and IL-6 levers in the co-culture systems that Tg-treatment MSC co-cultured with CD4~+T cells were slgnIficantly higher compared to that in MSC cultured with CD4~+T cells(P<0.05).(4)RT-PCR and flow cytometry results showed that EP2 and EP4 were expressed in CD4+T cells but EP1/EP3 not.(5)Adding EP2/EP4 antagonist and IL-6R antagonist to the co-culture system,respectively,the rates of CD4~+CXCR5~+ICOS~+T cells in the group of Tg-stimulatment MSC co-curtured with CD4~+T cells were significantly higher than the co-culture group that MSC cultured with CD4~+T cells(P<0.01;P<0.01).However,the co-culture group with the IL-6R antagonist did not change significantly(P>0.05).Conclusion:ER-stressed MSC produced higher levels PGE2,which further down-regulated RA peripheral CD4~+CXCR5~+ICOS~+T cells by binding with EP2 or EP4.Our study highlighted about how the immunomodulatory effect of MSC can be ameliorated,which will provide supports for MSC application in RA treatment. |
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