Psoriatic Serum Inhibits The Immunomodulatory Function Of MSCs By Induction Of MiR-155 Expression

Background:Psoriasis is a common inflammatory skin disease.Its chronic course and repeated attacks seriously affect the physical and mental health and quality of life of patients.As an autoimmune disease,the pathogenesis of the disease is not clear.It is considered to be a disease mainly mediated by T cells and participated by a variety of immune cells.Mesenchymal stem cells（MSCs）have been widely used in cell therapy because they play an important role in many autoimmune diseases.MSCs can affect the function of immune cells by secretion of anti-inflammatory factors such as IL-10,PGE₂,and IDO.Besides,MSCs can also promote angiogenesis by inducing the expression of vascular endothelial growth factor（VEGF）,so we consider that MSCs may participate in the pathogenesis of psoriasis.It has been found that miR-155 can participate in cell development,autoimmune diseases and tumorigenesis by regulating the expression of downstream target genes.Recently,it has been proved that the expression of miR-155 can be significantly induced by TAB₂（TAK1 binding protein 2）in MSCs after stimulation with inflammatory factors,and the inhibition of MSCs on the proliferation of T cells after stimulation is reduced.At the same time,we found that miR-155 expression of dermal mesenchymal stem cells（DMSCs）in psoriasis patients was up-regulated,and its downstream target gene TAB₂ was down-regulated.Moreover,the expression of many immunoregulatory-related factors,chemokines and inflammatory factors were abnormal,the inhibition effect on T cell proliferation was weakened in psoriatic DMSCs.Based on the above research,we speculate that the abnormal expression of inflammatory factors in psoriatic lesions may induce the high expression of miR-155 in DMSCs.And reduce the expression levels of immunoregulatory factors in DMSCs by targeting TAB₂ or other target genes,so as to inhibit the immunosuppressive function of DMSCs,that is,the inhibitory effect on the proliferation of PBMCs and the secretion of inflammatory factors is weakened.To confirm this hypothesis,we plan to stimulate normal DMSCs with psoriatic serum and PBMCs,detect the expression levels of miR-155 and its target genes（TAB₂,SOCS-1,SMAD2）,the expression levels of IL-10,PGE₂,TLR4,and the inhibitory effect of DMSCs on the proliferation and cytokine expression of PBMCs.Methods:1.Normal DMSCs were isolated and cultured.The passage 3 DMSCs at 70%confluence were cultured in DMEM/F12 medium,containing 50%human serum（psoriatic patients and normal controls）for 24 hours.PBMCs were isolated from peripheral blood（psoriatic patients and normal controls）and co-cultured with 70%confluent DMSCs directly for 24 hours.Normal DMSCs treated with 10 ng/ml TNF-α+10 ng/ml IL-1βwere regarded as positive control,and untreated normal DMSCs as negative control;2.qRT-PCR was used to detect the expression of miR-155 in DMSCs.qRT-PCR and Western Blot were used to detect the expression of target genes TAB₂,SOCS-1,SMAD₂ and immunomodulatory factors IL-10,PGE₂ and TLR4;3.PBMCs and DMSCs were co-cultured in Transwell.DMSCs were added to the upper chamber at a cell density of 2×10⁵/well,and PBMCs stimulated by 50μg/m L Phytohemagglutinin were added to the lower chamber at a density of 1×10⁶/well.Experiment included the following five groups:a.Normal DMSCs group:n DMSCs（untreated DMSCs）+PBMCs;b.Normal serum group:HS-DMSCs（DMSCs treated with normal serum）+PBMCs;c.Psoriatic serum group:PS-DMSCs（DMSCs treated with psoriasis serum）+PBMCs;d.Cytokine group:Cy-DMSCs（DMSCs treated with TNF-α+IL-1β）+PBMCs,and e.Control:PBMCs cultured alone.All groups were co-cultured for 72h in 12-well Transwell plates with 0.4μm filter at 37°C.4.PBMC proliferation was measured using a CCK-8 assay kit and qRT-PCR was used to detect the levels of inflammatory factors IL-8,IL-17A and IL-23 in PBMCs.Results:1.Under the microscope,DMSCs were spindle shaped and adherent fibroblasts,and connected with each other.After 14 days of culture,the adherent cells were confluence 90%and arranged in a whirlpool shape.Although co-culture of n DMSCs with either psoriatic serum or psoriatic PBMCs did not induce noticeable changes in the morphology of DMSCs,the growth rate of the cells was accelerated.2.After DMSCs were treated with serum and PBMCs,qRT-PCR and Western Blot showed that psoriatic serum markedly up-regulated expression levels of miR-155（27.19±2.40 vs.3.51±1.19,P<0.001）,while down-regulating TAB₂ mRNA（0.28±0.04 vs.0.72±0.20,P<0.01）in comparison to HS-DMSCs.Psoriatic serum-induced down-regulation of TAB₂ mRNA was paralleled by decreased expression levels of TAB₂ protein（P<0.001）.And the mRNA and protein levels of PGE₂,TLR4 and IL-10 were significantly decreased（P<0.05）in PS-DMSCs.The results of Cy-DMSCs were consistent with those of PS-DMSCs.There was no significant difference in gene expression between psoriatic and normal PBMCs（P>0.05）.3.After co-culture of treated DMSCs and PBMCs for 72h,either n DMSC or HS-DMSCs inhibited PBMC proliferation（P<0.001 vs.PBMCs cultured alone）.In contrast,PS-DMSCs and Cy-DMSCs lost their ability to inhibit PBMC proliferation（P<0.001 vs.HS-DMSCs）.Similarly,they（PS-DMSCs and Cy-DMSCs）displayed less potency in inhibiting expression of mRNA for cytokines IL-8,IL-17A and IL-23 in PBMCs in comparison to HS-DMSCs（P<0.05 vs.HS-DMSCs）.Conclusion:1.Psoriatic serum up-regulates the expression of miR-155,and down-regulates the expression of IL-10,PGE₂,TLR4 in DMSCs;2.miR-155 regulates the gene levels of DMSCs by inhibiting the expression of target gene TAB₂;3.Psoriatic serum negatively regulates the immunosuppression of DMSCs on PBMCs proliferation and expression of inflammatory factors;4.The effect of TNF-α+IL-1βon DMSCs was consistent with that of psoriatic serum.