The Effect Of Iguratimod On Differentiation And Maturation Of Human Monocyte-derived Dendritic Cell

Objective: Iguratimod（T-614）is a drug used for the treatment of adult patientswith active rheumatoid arthritis. It can inhibit the production of cytokines such asIFN-γ, IL-1, IL-6, TNF-a etc., as well as the generation of immunoglobulins(IgM, IgG). It is also reported to inhibit selectively the expression of COX-2.Iguratimod could inhibit inflammation through alterating the NF-κB signalpathway. Dendritic cells is an inportant target in the treatment of autoimmunediseases，in which play an inportant role in launching the immune response andinducing the immune tolerance. This work was mainly designed to explore theeffect of Iguratimod on the differentiation and maturation of humanmonocyte-derived dendritic cell, in order to further clarify the machanisms ofIguratimod in the modulating of autoimmune diseases.Methods: In this work, the density gradient centrifugation and adherent methodwere used to isolate DCs from human PBMCs and set an in vitro culture system.MTT is used to test the survival rate of the dendritic cells after iguratimodtreatment with different concentrations. Cell surface receptor expression wasdetected by flow cytometry to determine the matuaration status of dendritic cells.Functions of dendritic cells on lymphocyte proliferation was further detectedthrough mixed lymphocyte reaction. The secretion of IL-12by DCs which were treated by Iguratimod at different concentrations was detected using ELISAmethod.Results:1. Under an inverted phase contrast microscope, normal cultured human DCs,showed mostly round or oval, and many irregular dendritic projections on thesurface. Most of the suspension cells are typical mDC, while most iDCs adhere tothe bottom of the culture plates. iDC appear as low cell surface expression ofCD86and CD83, which are10.7%and17.2%respectively detected by flowcytometry, while the high expression for HLA-DR and CD8089.8%, and92.1%.After stimulation with LPS for24h, CD83and CD86positive cells reached to84.7%and78.7%.2. When Iguratimod with different concentrations (50ug/ml,100ug/ml,150ug/ml,200ug/ml,250ug/ml) were added into the culture system, the survival rate of DCsexceeded80%. With the increasing of the Iguratimod concentration, cells becomesmaller and round, comparing with the form of DCs in the matched control group.When DCs were treated for over3h, LPS was then added to cells to stimulat thematuration. With the concentration of Iguratimod at200ug/ml and250ug/ml,CD86positive cells decreased to19.3%and16.0%.3. The stimulation of allogeneic lymphocytes by Iguratimod treated DCs were thendetected using mixed lymphocytes culture method. Treatment of DCs withIguratimod at different concentration can significantly alter the DC inducedlymphocytes reactions (p <0.01). Iguratimod affected the poduction of IL-12in the supernatant of cultured DCs in a dose deppendent pattern, with a minimum valueof5.7pg/ml at250ug/ml of Iguratimod, and up to222.3pg/ml at50ug/ml ofIguratimod concentration.Conclusion:1. Iguratimod can alter the maturation of human monocyte-derived dendritic cellswith a dose deppendent pattern.2. Iguratimod concentration at200ug/ml and250ug/ml at3h time point, the CD86expression of treated cells decreased significantly..3. The initiation of lymphocytes proliferation by human matured dendritic cellswas prohibited by iguratimod when tested using allogeneic lymphocytes mixedculture. Proliferation of lymphocytes is inversely correlaed to the concentration ofiguratimod.4. Iguratimod inhibit the secretion of IL-12with a dose-dependently pattern byDCs.