Study On The Effect Of Lactobacillus Johnsonii On Maturation Of Chicken Bone Marrow-derived Dendritic Cells

Dendritic cells?DCs?are the most powerful professional antigen-presenting cells?APCs?,which only can directly activate primary T cells.DCs are widely present in the gastrointestinal tract.They are the main guardians of the intestinal mucosal immune system and play an important role in the regulation of immune responses and immune tolerance.In recent years,studies have found that a variety of microorganisms can affect the differentiation and maturation of DCs,including intestinal lactic acid bacteria.Lactic acid bacteria have immunomodulatory effect,and can regulate a variety of immune cells such as DCs and NK,promoting their growth and development.In this study,Lactobacillus johnsonii?L.johnsonii?of intestinal tract was studied for its effect on chicken bone marrow-derived dendritic cells?chBM-DCs?.The morphology of DCs,expression of cell surface molecules,phagocytosis,the mixed lymphocyte reaction?MLR?along with the transcription level of Toll like receptors?TLRs?,MyD88/NF-?B signaling pathway molecules and cytokine genes were evaluated.The experimental research contents and results are as follows:Extracted from femur and tibia using Histopaque-1119 lymphocyte separation solution,chicken bone marrow-derived mononuclear cells were cultured in six-well plates in pre-warmed RPMI-1640 complete medium for 7 days at 39?,5%CO₂.Recombinant chicken GM-CSF and IL-4 were added to the culture medium.After 4 days of inducing culture,a large number of colonies were observed by inverted microscope observation.When cultured for 6 days,cell colonies were loosely adhered and immature chBM-DCs were obtained.The cells cultured for 6days were divided into 3 groups:the negative control group,in which adds the same amount of RPMI-1640;the positive control group,including the lipopolysaccharide with a final mass of 200ng/mL;the experimental group added L.johnsonii with the concentration of 100?g/mL inactivated by UV,and continue to culture for 24 h.The expression of surface molecules of chicken bone marrow-derived mononuclear cells cultured for 1 and 7 days was detected by flow cytometry,and the following results were obtained:on the 1th day,the cells surface does not express CD11c and MHC-II molecules.In the non-stimulated group,LPS-stimulated group and L.johnsonii-stimulated group,the CD11c was highly expressed on the cell surface and the purity was over 70%.The unstimulated group chBM-DCs expressed CD40 and CD86 at low levels and the cells were immature.In the LPS-stimulated and L.johnsonii-stimulated groups the expression of CD40 and CD86 was up-regulated and the cells were stimulated to mature.RT-qPCR was used to detect the mRNA levels of CD80,CD86 and DEC-205 in chBM-DCs.Compared with the unstimulated group,the mRNA expression levels of CD80 CD83 and DEC-205 were up-regulated in the LPS-stimulated group and the L.johnsonii-stimulated group,among which the mRNA level of CD83 was significantly different?P<0.01?.Neutral red was used to examine phagocytic ability of non-stimulated cells induced for 2 d,4d,6 d and 7 d and the stimulated cells performed by LPS and L.johnsonii for 24 h.The results showed that the phagocytic ability of chicken bone marrow mononuclear cells induced by rcGM-CSF and rcIL-4 for 2 d,4 d,6 d and 7 d?without stimulation?was firstly enhanced and then decreased,and the phagocytic capacity reached the maximum on the 6th day.Compared with unstimulated chBM-DCs,the phagocytic ability of chBM-DCs stimulated by LPS and L.johnsonii decreased but the difference was not significant?P>0.05?.Immature chBM-DCs and mature chBM-DCs stimulated with LPS?200 ng/mL?and L.johnsonii?100?g/mL?were used as stimulator cells and allogeneic T lymphocytes were used as responder cells,mixed with 1:1,1:10,1:100 ratios,and CCK-8 kit was used to detect the proliferation of T cells.The results showed that the chBM-DCs stimulated by LPS and L.johnsonii could induce T cell proliferation and the difference was extremely significant compared with the control group?P<0.01?.The stimulus index decreases with the decline in the number of cells.ChBM-DCs were stimulated with LPS and L.johnsonii,and were harvested at 6 h,12 h and24 h after stimulation.The mRNA levels of TLR2/4/5/15 and MyD88/NF-?B signaling molecules were detected by RT-qPCR using unstimulated chBM-DCs as a control group to observe the effect of L.johnsonii on chBM-DCs at different times.The results showed that L.johnsonii increased the mRNA expression of TLR2,TLR4 and TLR5?fold change>1?,especially TLR5 and decreased the mRNA expression of TLR15 at each time period compared to the control group?fold change<1?.Compared with LPS group,the mRNA expression of TLR2 was significantly different?P<0.01?and significant?P<0.05?after L.johnsonii stimulation 6 h and 12 h.The mRNA expression of TLR4 was significantly different?P<0.05?after L.johnsonii stimulation for 6 h,while the mRNA expression of TLR5 was significantly different at each time point?P<0.01?.In the LPS and the L.johnsonii groups,the mRNA expression level of TLR15 was lower than that of the unstimulated group?fold change<1?.At the same time,L.johnsonii was able to increase the mRNA expression of MyD88/NF-?B signaling pathway at each time period?fold change>1?.ChBM-DCs were stimulated with different concentrations of L.johnsonii?1?g/mL,10?g/mL and 100?g/mL?and cells were harvested at 6 h,12 h,and 24 h after stimulation.The transcription levels of Th1 and Th2 cytokines,proinflammatory cytokines and chemokine genes were detected by RT-qPCR to analyze the effects of concentrations of L.johnsonii and stimulation times on the ChBM-DCs.The results showed that low concentrations of L.johnsonii?1?g/mL?decreased the relative expression of cytokines and chemokine mRNA at various time points?fold change<1?compared with the unstimulated group.The relative expression of Th1 cytokines,proinflammatory cytokines and chemokine?CXCLi1?mRNA was higher than that in the unstimulated group?fold change>1?in each time period after the stimulation of medium or high concentrations concentrations of L.johnsonii?10?g/mL and 100?g/mL?.However,only the high concentration L.johnsonii?100?g/mL?group could increase the relative mRNA expression of Th2-type cytokine?fold change>1?.What's more,the LPS group could not promote the relative mRNA expression of Th2-type cytokine?IL-4?at various times?fold change<1?.The mRNA relative expression of IL-12,IFN-?,IL-1?and IL-6 mRNA in the L.johnsonii group was higher than that in the LPS group?P<0.01?.In addition,the mRNA expression levels of Th1 cytokines?IFN-?,TNF-??,pro-inflammatory cytokines and chemokines decreased with increasing the stimulation time,while the mRNA expression level of Th2 cytokines increased.The above results show that L.johnsonii can stimulate the maturation of chBM-DCs,and influence the production of cytokines and chemokines of chBM-DCs in a dose-dependent and time-dependent manner.At the same time,it can regulate Th1/Th2 balance and maintain body homeostasis.