The Role And Mechanism Of M1 Macrophages Polarization And Intestinal Inflammation Triggered By High Level Colonic Deoxycholic Acid Due To The High-fat-diet

Objective High-fat diet(HFD)could induce chronic systemic inflammation,and thus is closely associated with inflammatory bowel disease(IBD).High level colonic deoxycholic acid(DCA)may play an important role in the pathogenesis of inflammatory bowel disease,however,the detailed mechanism remains unclear.Recent evidences sμggest the essential role of M1 macrophage in the development of intestinal inflammation induced by a high-fat diet.In this study,we mainly try to investigate the role and mechanism of the involvement of high level colonic deoxycholic acid in the induction of M1 macrophage polarization and intestinal inflammation under the high-fat-diet settings.MethodsPartⅠ DCA,as a secondary bile acid,is mainly produced by the metabolism of specific gut bacteria(mostly Gram-positive bacteria),and high-fat diet could alter the gut microbes.To observe the changes of gut microbiota,deoxycholic acid level and intestinal inflammation under a high-fat diet,wild-type C57BL/6 mice were randomly divided into three groups and given a normal diet(ND),a high-fat diet(HFD)or high-fat diet plus vancomycin(for Gram-positive bacteria,HFD + VCM)respectively.The feces of three groups were collected to detect the alteration of gut microbiota by16 S r DNA microbial diversity analysis and DCA content by liquid chromatography-mass spectrometry(LC-MS)assay.Colon tissue sections were examined by HE staining and immunohistochemical staining to detect pathological damage and macrophage infiltration,and immunofluorescence staining was used to determine macrophage polarization.Then the HFD + VCM group and the HFD + VCM + DCA group(HFD supplemented with 0.2% DCA)were established to further explore whether DCA increment correlates with the colonic infiltration of proinflammatory macrophages.The weight changes of mice were dynamically recorded,colonic pathological damage,macrophage polarization and infiltration were observed accordingly.PartⅡ Different concentrations of DCA were used to stimulate murine macrophages(cell lines and bone marrow derived macrophages),DCA-untreated group was regarded as control group.Under the above conditions,m RNA expression levels of the M1 / M2 macrophage-related molecules involving IL-6,TNF-α,i NOS,CD206,and Arginase were detected by real-time PCR,the secretion of cytokine IL-6 and TNF-α in the culture supernatant were examined by ELISA and NO levels were measured via Griess reagent colorimetric method respectively.Furthermore,the mice were injected with DCA intraperitoneally,and the vehicle injection group was used as a control.Peritoneal macrophages were collected and counted,and then cultured for24 hours in vitro.Griess reagent colorimetric method was performed to detect the NO level and ELISA was used to detect the TNF-α secretion in the culture supernatants,and the macrophages polarization was evaluated by flow cytometry.In order to investigate the role of DCA in inducing chemokine monocyte chemotactic protein 1(MCP-1)production by intestinal epithelial cells,the murine intestinal epithelial cells were stimulated with different concentrations of DCA,and m RNA and protein expression levels of MCP-1 were detected by real-time PCR and ELISA respectively.PartⅢ To clarify the bile acid receptors involved in the induction of macrophage polarization by DCA,different bile acid receptors were separately blocked by specific inhibitors or si RNA,and then murine macrophages were stimulated with DCA followed by the measurement of NO,TNF-α,and IL-6 levels in the culture supernatants.To further explore the possible molecules participated in this process,murine bone marrow-derived macrophages were stimulated with DCA,and the untreated group was deemed as control.Protein mass spectrometry and bioinformatics were adapted to analyze key differentially expressed proteins.Luciferase Assay,Chromatin immunoprecipitation(Ch IP)and Western Blot were applied to explore the effects of DCA on the transcriptional regulation of selected target molecules as well as the activation of downstream signal pathways.Then the target molecules and signal pathways were selectively inhibited to verify their involvement in the induction of macrophage polarization by DCA.In addition,bile acid receptors antagonists and signaling pathways inhibitors were also used to investigate the possible mechanisms that DCA promotes MCP-1 production in murine intestinal epithelial cells.ResultsPartⅠ Compared with the normal diet,HFD could significantly increase the proportion of gram-positive bacteria(especially Clostridium)in feces of mice,accompanied by a significant increase of fecal DCA levels,and lead to colonic proinflammatory macrophage infiltration and tissue damage in mice.Fecal Gram-positive bacteria could be effectively eliminated by vancomycin(VCM),meanwhile,fecal DCA levels were greatly reduced and colonic M1 macrophage infiltration and intestinal tissue damage caused by HFD were significantly suppressed as well.However,DCA supplement to the diet in the VCM treatment group could induce colonic infiltration of massive pro-inflammatory macrophages,causing intense expression of proinflammatory factors(IL-6,TNF-α,IL-1β,i NOS)and colon tissue damage.PartⅡ DCA could induce M1 macrophage polarization and proinflammatory cytokine production(TNF-α,IL-6 and i NOS)time-and dose-dependently in vitro.And intraperitoneal injection of DCA in mice could also promote M1 polarization and intraperitoneal recruitment of macrophages.Furthermore,DCA could dose-dependently induce the expression of MCP-1 in murine intestinal epithelial cells.PartⅢ The role of DCA in promoting polarization of M1 macrophages was mainly mediated by the bile acid receptor M2-m Ach R.DCA stimulation could up-regulate the expression of M2-m Ach R in macrophages,while blocking M2-m Ach R could significantly inhibit DCA-induced TNF-α,IL-6,and NO production.Proteomic analysis showed that DCA stimulation of macrophages also potently increased the expression of TLR2 in macrophages.DCA enhanced TLR2 transcription mainly by targeting the transcription factor AP-1 via M2-m Ach R.Indeed,M2-m Ach R blockage effectively reduced DCA-induced TLR2 expression to the baseline level.Moreover,TLR2 and its downstream NF-κB / ERK / JNK signaling pathway were involved in the DCA-induced M1 macrophages polarization.Moreover,M2-m Ach R and NF-κB /ERK / JNK signalings also participated in the induction of MCP-1 secretion by DCA in murine intestinal epithelial cells.Conclusion Our study reveals that high level colonic DCA under HFD conditions could induce proinflammatory(M1)macrophages polarization and infiltration,which may serve as an important initiator in HFD-induced colonic inflammation.Regulation of gut microbiota or intervention of specific bile acid receptor signaling pathways could be potential therapeutic approaches for IBD or other HFD-related inflammatory diseases.