Study Of Tolerogenic Plasmacytoid Dendritic Cells Promote Chondrogenic Differentiation Of Synovial Fluid Mesenchymal Stem Cells

Objective:This study aims(1)to investigate whether indoleamine-2,3-dioxygenase 1(IDO1)has the potency to induce tolerogenic phenotype of plasmacytoid dendritic cells(p DCs);(2)to investigate the effect of tolerogenic p DCs on the chondrogenic differentiation of synovial fluid mesenchymal stem cells(MSCs).Methods:Synovial fluid(SF)and bone marrow were collected from patients with osteoarthritis(OA)during knee arthroplasty.The phenotype of p DCs in the SF and bone marrow of OA patients was tested by flowcytometer and q PCR after sorting p DCs.Then,bone marrow-derived DCs were isolated and cultured.The differentiation of p DCs was stimulated by TGF-β3(10 ng/m L)which was added to the culture of bone marrow-derived DCs.Further,p DCs were cultured with IDO1(10 ng/m L)and lipopolysaccharide(LPS)(10 ng/m L)to assess the stimulation of p DCs tolerogenic phenotype.Flowcytometry was used to detect the phenotype of p DCs in the p DCs+IDO1 and p DCs+LPS cell cultures.Then,q PCR was used to detect the m RNA expression of IFN-γ,TGF-β1,and IL-6 genes.Furthermore,mesenchymal stem cells(MSCs)were isolated and cultured from the SF of OA patients.Then,IDO-induced p DCs and LPS-induced p DCs were co-cultured with MSCs for 2 weeks compared to TGF-β3 as a positive control and PBS as a negative control for detection of chondrogenic potential differentiation.Later,m RNA expression of collagen II(COL2A1),Sox9,and aggrecan was detected in the induced MSCs.Mainwhile,the accumulation of chondrogenic proteoglycan in the SF-MSCs cultured with tp DCs was tested by alcian blue staining.To further clarify how tp DCs can promote chondrogenic differentiation of SF-MSCs,the expression of IFN-γ,TGF-β1,TGF-β3,and IL-1βgenes in tp DCs was assessed by q PCR.Results:1.The analysis of p DCs in the SF of OA patient’s showed low percentage(17.9%±2.3%),while the amount of c D11c~+CD123 in the bone marrow of OA patients were noticed significantly high 44.6%compared to normal control 13.7%(p<0.001).Also,the analysis of OA derived p DCs showed overexpression of IFN-γ,while the expression of TGF-β1,IDO1,and IL-6 genes was found significantly low,suggesting an inflammatory phenotype p DCs.Interestingly,IDO1 showed significant performance to induce tp DCs,while the LPS induced the inflammatory differentiation of p DCs.q PCR results showed that p DCs induced by LPS release high levels of IFN-γ(1.109±0.084,p<0.001),since p DCs induced by IDO1 showed high levels of TGF-β1(0.261±0.032)and low expression of IFN-γ(0.097±0.018),(p<0.01).2.MSCs that cultured with tp DCs showed significant expression of SOX9 and COL2A1 compared to MSCs that cultured with fp DCs(tp DCs:Sox9(2.612±0.215),COL2A1(2.308±0.132);fp DCs:Sox9(0.832±0.095),COL2A1(0.807±0.071),p<0.01).Indeed,the accumulation of proteoglycan in SF-MSCs stimulated with tp DCs was noticed high.These results indicate that IDO1-induced tp DCs has the potency to promote the expression of Sox9 and COL2A1 in SF-MSCs significantly,and subsequently chondrogenic differentiation.Conclusion:The percentage of p DCs increases in OA,particularly fp DCs which were found high in the bone marrow and SF samples of OA patients compared to non-OA patients.IDO1 showed a successful capability to alter the phenotype of preexisting p DCs towards rendered immature p DCs that exhibited tp DCs phenotype.Tp DCs exhibited significant release of TGF-β3,TGF-β1 which significantly promoted the expression of Sox9 and COL2A1 in SF-MSCs,and consequence chondrogenic differentiation.