Functional Impairment Of MSCs Mediated By IL-34 Stimulation In Regulating Rheumatoid Arthritis Peripheral T Cells

Objective:Mesenchymal stem cells(MSCs)in rheumatoid arthritis(RA)have been reported to display impaired immunoregualtory effect on T cells.However the mechanism is not clear.Leukin-34(IL-34)plays an important role in regulating the immune system.In this study,we aimed to investigate the effect of il-34 on the immunomodulatory function of MSCs.Method:(1)CD4+T cells of RA patients were isolated and activated by anti-cd3/CD28 antibody(anti-cd3 antibody:3 g/ml).Anti-cd28 antibody:2 g/ml),which was co-cultured with or without IL-34 umbilical cord MSCs(MSCs:CD4+T cells=1:10)for 72 hours,suspension cells were collected,percentage of CD4+CXCR5+ICOS+T cells and Treg cells were detected by flow cytometry,and mRNA expression levels of IL-21 and Bcl-6 in CD4+T cells were detected by RT-PCR,The PEDF protein level in the supernatant was detected by ELISA,MSCs in the co-culture system were collected,and the PEDF mRNA expression level in MSCs was detected by RT-PCR.(2)Added anti-CSF-1R antibody to the co-culture system,suspension cells were collected,percentage of CD4+CXCR5+ICOS+T cells and Treg cells were detected by flow cytometry,and mRNA expression levels of IL-21 and Bcl-6 in CD4+T cells were detected by RT-PCR,The PEDF protein level in the supernatant was detected by ELISA,MSCs in the co-culture system were collected,and the PEDF mRNA expression level in MSCs was detected by RT-PCR.(3)The expression of CSF-1R on MSCs was detected by flow cytometry.Anti-CSF-1R antibody was added or not added to the MSCs stimulated by IL-34,and the MSCs were cultured for 0h,24h,48h and 72h,respectively.The mRNA expression level in MSCs was detected by rt-pcr,the supernatant was collected,and the PEDF protein level was detected by ELISA.(4)MSCs,transfected with empty pEX-4 or PEDF-pEX-4 plasmid for 24h,then co-cultured with activated CD4+T cells for 72h in the presence or absence of IL-34.The percentages of CD4+CXCR5+ICOS+T cells were analyzed by using flow cytometry,the mRNA expressions of IL-21 and Bcl-6 in CD4+T cells were tested with RT–PCR.The PEDF level in the supernatants was measured with ELISA and the mRNA expressions of PEDF in cultured MSCs were assessed with RT–PCR,The mRNA expresions of IL-10,IL-6,IDO,TGF-β,HGF and COX-2 in PEDF-MSCs or Neo cocultured with activated T cells in the presence or absence of IL-34 were analyzed with qPCR.(5)MSCs were pretreated with or without anti-CSF-1R antibody(25ng/ml)for 1 h and then stimulated by IL-34 for 15 or 30 min.Protein was acquired in whole cell lysis buffer;phosphorylations of P38,NF-κB,ERK1/2,and JNK were analyzed with Western blotting by using anti-phospho-specific antibody.Total P38,NF-κB,ERK1/2,and JNK were determined with Western blotting by using corresponding antibodies,respectively.The expression ratios of p-P38 to P38,p-NF-κB to NF-κB,p-ERK1/2 to ERK1/2,and p-JNK to JNK were represented in a bar graph.MSCs were pretreated with inhibitors of signaling molecules for 1 h,and then incubated with IL-34 for 24 h.The PEDF level in the supernatants was measured by ELISA.Results:(1)The results showed that percentage of CD4+CXCR5+ICOS+T cells was obviously increased and Treg was significantly reduced in the activated RA CD4+T cells when co-cultured with IL-34-treated MSCs as compared with those co-cultured with un-treated MSCs.There were higher expression of both IL-21 and Bcl-6 on activated CD4+T cells co-cultured with IL-34-treated MSCs.MSCs express CSF-1R,the frequency of peripheral CD4~+CXCR5~+ICOS~+T cells was substantially reduced and Treg was significantly increased along with lower mRNA expression of IL-21 and Bcl-6,when anti-CSF-1R antibody was added into the co-culture system of IL-34-treated MSCs and activated T cells.(2)the levels of PEDF were lower in those culture systems with IL-34 or without IL-34,and the decrease of PEDF was in time-depended manner.when anti-CSF-1R antibody was added into those culture systems,significant increased PEDF was observed.While PEDF were not been detected both in CD4+T cells stimulated with IL-34 and without IL-34(data not show).(3)In the presence of IL-34,MSCs transfected with PEDF downregulated peripheral CD4+CXCR5+ICOS+T cells,as well as mRNA expression of IL-21 and Bcl-6,more obviously than Neo.Compared with Neo,MSCs transfected with PEDF expressed extremely higher levels of IL-6 mRNA and lower mRNA expressions of COX-2 in the co-culture system of IL-34-treated MSCs and activated CD4+T cells,accompanied with over-expressed PEDF.(4)The phosphorylation of P38,NF-κB and JNK but not ERK1/2 in the cytoplasm of MSCs was significantly increased after the cells were stimulated with IL-34 for 15 min and 30 min,respectively.Furthermore,when anti-CSF-1R antibody was pretreated into IL-34-stimulted MSCs,the phosphorylation of P38 and NF-κB was suppressed apparently.PEDF levels were increased when IKK-16 and SB203580 were added into MSCs.However the addition of FR180204 and SP600125 had no effect on the production of PEDF.Conclusion:In this study,we found that IL-34 stimulation could weaken the immunomodulatory function of MSCs in RA follicular CD4+CXCR5+ICOS+T cells by decreased PEDF expression,and decreased PEDF significantly up-regualted mRNA expression of IL-6 and reduced COX-2 in IL-34-treated MSCs.Moreover,IL-34inhibited PEDF secretion of MSCs possibly via the activation of NF-κB and p38MAPK signaling pathways.These findings deeply revealed the pathogenesis of RA,providing a new theoretical and experimental basis for clinical application of allogeneic MSCs transplantation in the treatment of RA.