Minocycline Induces Tolerance Of Dendritic Cells Via The LPS-TLR4/NF-?B Signaling Pathway

Objective:Dendritic cells(DC)are a bridge between congenital and adaptive immunity,in multiple sclerosis(MS)and animal models of experimental autoimmune encephalomyelitis(EAE)plays a key role in the development.In recent years,with the indepth study of DCs,DCs not only play a key role in initiating immune response,but also have the effect of maintaining peripheral immune tolerance and regulating immune response.Therefore,the concept of tolerogenic dendritic cells(tolDCs)has been proposed.Recent studies have found that mouse bone ma-rrowderived dendritic cells exhibit the characteristics of tolerant DCs in the presence of minocycline,and the number of dendritic cells is higher than that of the control group,and is not easily affected by lipopolysaccharide(lipopolysaccharide,LPS)induces maturation.At present,the mechanism of minocycline-induced tolerance of dendritic cells is unclear.In this study,tolDCs were induced by minocycline to detect wheth-er minocycline increased dendritic cell tolerance by blocking LPS-TLR4/NF-kappa B signal transduction pathway,providing more effective evide nce for minocycline in the treatment of multiple sclerosis.Methods:1.Mouse bone marrow-derived cells(BMDC)were cultured in vitro.Mouse bone marrow-derived dendritic cells(BMDC)were cultured in vitro according to the modified Son method and divided into three groups:Ctrl-DC:BMDCs were cultured until day 7,without drug intervention,and immature DCs were obtained on day.LPS-DC:The BMDC culture was stimulated with LPS(1?g/ml)for 24 h to induce maturation of immature dendritic cells on the sixth day.Mino-DC:BMDC was cultured in vitro on day 0,day 2,and day 4to add 5?m of minocycline.Stimulation of LPS(1?g/ml)for 24 h on the sixth day of culture to induce maturation of immature dendritic cells.The above three groups of cells were cultured on day 2 and day 4 with half and full volume changes,and supplemented with 10 ng/ml IL-4 and GM-CSF.2.One-way mixed lymphocyte reaction(MLR).Three groups of cells derived from C57BL/6 mice were stimulated cells.After separation and purification by CD11c MACS,30 mg/ml mitomycin C was added,incubated in a 37°C incubator for 30 min,and washed three times with PBS.Purified CD4~+T cells from Balb/c mice are responding to cells.According to the ratio of stimulating cells and reaction cells,1:5,1:10,1:20,1:40,the cells were mixed and cultured in 96-well plates.Each group consisted of 3 replicate wells,mixed culture for 72 h,before the end of the culture.4 h was added to CCK-8 20?L/well,and incubation was continued for 4 h.3.Cell morphology was observed by fluorescence inverted microscope and scanning electron microscope.The ultrastructure of cells was observed by transmission electron microscope.4.Flow cytometry On the 7th day,the cells in each group were collected from the above three groups,adjusted to a cell concentration of 2×10~6cells/mL,and added to CD11c,CD86,CD80,MHC-II,stained separately,and incubated at 4°C.After 30 min,0.01 M PBS was added after washing,and the sample was subjected to phenotypic analysis.5.Take the above groups of cells separately,extract the cell proteins after lysis by cell lysate,perform electrophoresis with 10%SDS polyacrylamide gel(50?g total protein per sample);transfer to PVDF membrane and concatenate under low temperature conditions.Incubate for 1 h at room temperature in blocking buffer and add a primary shaker at 4°C overnight.The next day,the secondary antibody was incubated for 1 h at room temperature,and the membrane was developed and fixed.6.The levels of IL-12p70,TNF-?,IL-10 in the supernatants of each group cultured to day 7 were measured by Enzyme Linked Immunosorbent Assay(ELISA).7.SPSS 21.0 was used for statistical analysis.Results:1.The Ctrl-DC group and the Mino-DC group were similar in morphology,rounded,with less wrinkles on the cell surface and sparse and short protrusions.The cells in the LPS-DC group were irregular in shape,and the surface folds were many and deep,and the protrusions were large,dense and long,and resembled dendritic.2.Compared with LPS-DC,there are fewer organelles such as Golgi,endoplasmic reticulum and mitochondria in the cytoplasm of Mino-DC group.3.Flow cytometry detection of cell surface molecules Mino-DC expressed CD80 and CD86 levels between immature Ctrl-DC and mature LPS-DC(P<0.01).Compared with LPS-DC,Mino-DC surface molecule MHC II expression was similar(P>0.05).4.Analyze the ability of Mino-DC to elicit allogeneic T cells using a mixed lymphocyte reaction assay.The results indicate that LPS-DC effectively induces allogeneic T cell proliferation.The maximum stimulatory activity was observed at a DC to T cell ratio of 1:5(P<0.01).However,the same stimulatory activity of Mino-DC was between Ctrl-DC and LPS-DC(P<0.05).5.The expression levels of TLR4(P<0.01)and NF-?B-p65(P<0.05)proteins in LPS-induced Mino-DC were lower than those of LPS-DC and Ctrl-DC.The expression level of I?B-?(P<0.01)was higher than the other two groups.6.The levels of cytokines secreted by three groups of cells were detected by ELISA.The levels of inflammatory cytokines such as IL-12p70 and TNF-?secreted by Mino-DC group were between LPS-DC group and Ctrl-DC group(P<0.01),and IL-10 was higher than those of the other two groups(P<0.01).Conclusions:1.Minocycline may inhibit the maturation of BMDC,making it semi-mature and inhibiting T cell proliferation.2.Minocycline increases dendritic cell tolerance by blocking the LPS-TLR4/NF-?B signaling pathway.