Role Of Macrophage AHR/TLR/STAT3 Signaling Axis In The Colitis Of Mice Induced By Aflatoxin B1

ObjectiveAflatoxin B1(AFB1)is one of the most common and toxic mycotoxin and is widely present in cereals and their food products.It is difficult to completely eliminate even after food processing due to its high chemical and thermal stability,thus it poses a potential harm to animal and human health.It is well known that AFB1 is a carcinogenic,mutagenic,teratogenic and immunosuppressive chemicals,and also could exert harmful effects on the liver,kidney and nervous system,thus large numbers of studies have been devoted to its related toxic mechanisms.Several studies indicated that sub-acute exposure to low doses of AFB1 was related to the intestinal inflammatory injury,but the molecular mechanism remains unclear.Recently,AFB1 has been identified to be a ligand of aryl hydrocarbon receptor(AHR),and the binding activity with AHR has been confirmed.It is accepted that the ligandreceptor binding is a sensitive and effective priming mechanism for cells to respond to external stimuli.Furthermore,AHR is regarded to attributed to the occurrence and development of immune or inflammatory diseases by targeting inflammation-related gene expression and regulating immune cell differentiation.Therefore,we speculated that AHR might be involved in intestinal inflammatory injury induced by AFB1.In order to further investigate the relationships between AFB1 and intestinal inflammatory injury,and confirm the mediating effect of AHR,the harmful effect on colonic tissue of mice induced by subacute treatment with AFB1 were demonstrated and its relationship with macrophages were evaluated.Furthermore,the potential mechanism of the macrophage mediated the damages of intestinal epithelial cells induced by AFB1 were illustrated by in vitro study.The multi-omics techniques were employed to reveal the changes of the AHR/TLR/STAT3-signaling axis in macrophage with the colitis of mice under AFB1 challenge,which will be beneficial to the prevention and treatment of colitis induced by AFB1 and other environmental pollutants.MethodsFirstly,BALB/c male mice were orally exposed to AFB1 for 30 days,and the systemic inflammatory status was evaluated by blood cell count and serum inflammatory lipidomics analysis,the colon damage was assessed by morphological observation,the infiltration of inflammatory cells and the recruitment of myeloid derived immune cells were observed respectively by HE staining and immunofluorescence staining of sections of colon.Secondly,human monocytic leukemia THP-1 cells were differentiated into macrophages(M0),followed by exposure to AFB1,then co-cultured with human colorectal adenocarcinoma Caco-2 cells in Transwell,the damages of intestinal epithelial cells mediated by macrophages treated with AFB1 were investigated.In addition,the effect of AFB1 on RNA and proteins of M0 macrophages differentiated from THP-1 were analyzed by Western blot,multi-omics and other experiments technical means respectively,which try to illustrate the mechanical effects of AHR/TLR/STAT signaling axis on AFB1-induced colorectal injury.ResultsThe results of in vivo experiments showed that mice were orally treated AFB1(5-50μg/kg.bw)resulted in the significant shortening of intestinal length,vague shape even disappearance of colonic crypts,mucin secretion increase of intestinal goblet cells and damages of epithelial cells,accompanied by significant inflammatory cell infiltration in colon.The results of serum inflammatory lipidomics showed the lipid compositions related to the inflammatory response obviously varied in mice with or without AFB1 treatment.The reactive oxygen species(ROS)and nitric oxide(NO)in colon of mice exposed to AFB1 increased in a dose-dependent manner.The immunofluorescence staining showed there was significant recruitment and accumulation of myeloid immune cell in colon of mice from AFB1 treated mice.The results of in vitro experiments showed that AFB1 exposure enhanced the phagocytic ability of M0 macrophages differentiated from THP-1,alone with the significant increase of ROS and malondialdehyde(MDA),as well as the aldehyde dehydrogenase(ALDH)protein levels.M0 macrophages were treated with AFB1 and followed by co-cultured with Caco-2 cells,the results of flow cytometry with 7-AAD/Annexin V staining demonstrated that the AFB1 prompted the demise of Caco-2,the levels of tumor necrosis factors-α(TNF-α)and interleukin-6(IL-6)in the culture medium were significant increased under AFB1 treatment.Transcriptome sequencing(RNA-seq)and proteomics analysis further proved that the inflammatory pathway-related mRNA and protein were significantly changed when macrophages were exposed to AFB1.Western blot also confirmed that AFB1 exposure induced increase of inflammation-related functional protein expression in macrophage,as well as the increase of key proteins of AHR and AHR/TLR signaling axis.Conclusions1.AFB1 sub-acute exposure resulted in the colitis in BALB/c mice,characterized by significant shortening of intestinal length,vague shape even disappearance of colonic crypts,mucin secretion increase of intestinal goblet cells and damages of epithelial cells,accompanied by significant inflammatory cell infiltration in colon.2.AFB1 could up-regulate the proteins expression of AHR,TLR2 and TLR4 in M0 macrophages via the AHR/TLR/STAT signaling axis,and down-regulate the expression of mitochondrial ROS regulatory factors STAT3 and phosphorylation of STAT3 at Ser727,which make it readily transform into inflammatory state and lead to the damages of intestinal epithelial.