The Effects Of Butyrate On The Differentiation And Functions Of Bone Marrow Derived Dendritic Cells

Objective: In human and animal intestines, butyrate acid is one of the most important short chain fatty acids(SCFAs), which generate from indigestible carbohydrates in the food by anaerobic bacteria in the colon lumen. SCFAs build up into the intestinal region microenvironment together with the gut microbes, maintaining the intestinal immune steady state. So far, the differentiation, function and potential mechanism of immune cells in mucosa regulated by SCFAs are still not well demonstrated. This study explored the regulation of butyrate on both bone marrow derived dendritic cells and retinoic acid induced mucosal dendritic cells in vitro.Methods: 1. Regulation of butyrate on bone marrow derived dendritic cells.(1) Bone marrow precursor cells were obtained and erythrocytes were lysed. After total number was counted, cells suspension was adjusted and seeded into 6-well plate.(2) BMDCs were culturing for 7 days and then harvested and seeded into 24-well plate with LPS and butyrate treatment. The morphology of DCs were observed under microscope.(3) The expressionof CD80, CD86, MHC-Ⅱand B7-DC on BMDCs was analyzed by the flow cytometry.(4) The m RNA levels of IL-6 and IL-12 was analyzed by Q-PCR.(5) NO production in the supernatant was detected by Griess reaction.(6) The capability in priming antigen specific CD8+ T cells proliferation was detected by using flow cytometry.(7) After being treated with Butyrate at 0min, 15 min, 30 min, 60 min, the levels of p-ERK in cells were detected by using Westernblot analysis. 2. Effects of butyrate on the differentiation and functions of mucosal dendritic cells.(1) Bone marrow precursor cells were obtained and erythrocytes were lysed. After total number was counted, cells suspension was adjusted and seeded into 6-well plate.(2) Cells were culturing for 7 days, treated with RA and butyrate on day 3. Then DCs were harvested and seeded into 24-well plate with LPS stimulation. The morphology of DCs were observed under microscope.(3) The number of CD11b+CD11c+ cells was analyzed by the flow cytometry.(4) FSC, SSC, CFSE fluorescene intensity of DCs was analyzed by the flow cytometry.(5) The expression of CCR9, α4β7, CD103, CD80, CD86, MHC-Ⅱand B7-DC was analyzed by the flow cytometry.(6) ALDH activity was assessed by flow cytometry.(7) The m RNA levels of IL-6, IL-12, TNF-α and Nos2 were analyzed by Q-PCR. IL-12p70 protein levels in the supernatant were detected by ELISA.(8) The capability in priming OVA specific CD4+ T cells proliferation was detected by using flow cytometry.(9) The capability in inducing CD4+CD25+Foxp3+ Tregs was detected by using flow cytometry.Results:(1) Butyrate decreases the expression of CD80, CD86, MHC- Ⅱ and B7-DC, and down regulates the capability of BMDCs in priming the proliferation of CD8+T cells. Furthermore, Butyrate suppresses the secretion of IL-6, IL-12, NO2- and the phosphorylation of ERK in BMDCs.(2) Butyrate inhibits the CD11 c expression and the ALDH activity of retinoic acid induced mucosal DCs, but upregulates the expression of gut-homing receptors CCR9, α4β7 and CD103. The ability of DCs in priming the antigen specific T cells proliferation is impaired by butyrate. Furthermore, retinoic acid induced mucosal DCs induce more Tregs than BMDCs with no obvious downregulation when treated with butyrate.Conclusion: Our results demonstrated that butyrate downregulates the immune functions of bone marrow derived dendritic cells via inhibition of ERK phosphorylation in TLR4 signaling pathway. Interestingly, bone marrow derived mucosal DCs induced by retinoic acid perform the characteristics of gut-associated DCs in the intestinal tract including the expression of homing receptors and CD103, which are upregulated by butyrate. However, butyrate impairs the ability of mucosal DCs in priming OVA specific CD4+T cell proliferation but not the capacity of inducing Tregs, which is important to the gut immune tolerance. These data demonstrated that butyrate plays an important role in the gut homing of bone marrow derived DCs and the intestinal low immune responsiveness.