The Mechanism Of Neutrophils Modulating Intestinal Intraepithelial Lymphocyte Function And Intestinal Mucosal Homeostasis

1.IntroductionCirculating neutrophils as an early responder to inflammatory signals show the potent capabilities in immune regulation,pathogen clearance,and recruitment of immune cells in the gastrointestinal tract.Dysfunction of neutrophil contributes to impaired intestinal immune homeostasis and aggravated mucosal inflammation after pathogens invade,and the underlying mechanisms involved in the dysregulated immune response of neutrophils include proinflammatory cytokine burst,irreducible immune responses and discordant immune cell functions.Among the immune cells that contribute to the maintenance of intestinal homeostasis,intraepithelial lymphocytes(IELs)occupy a favorable terrain and play a key role in preserving the mucosal barrier intact,patrolling the epithelial layer,and synergizing with rushing neutrophils to initiate the timely primary defense against exogenous pathogens that first invade the gut tissue.Especially,TCRγδ+IELs are essential for limiting epithelial penetration of resident commensal microbiota through antimicrobial effectors(i.e.,RegⅢγ)which are dependent on intestinal epithelial cell-intrinsic MyD88 signaling following dextran sulfate sodium(DSS)-induced mucosal damage,indicative of a key role of proper TCRγδ+IEL functions in sustaining host-microbiota homeostasis following acute mucosal inflammation.Optimal spatial interactions and cooperation between neutrophils and IELs are essential for maintaining gut mucosal immune homeostasis in response to pathobiont challenges.Loss of the delicate immunoregulation of neutrophils and IELs compromises the intestinal barrier integrity and drives a series of pathogenic disorders in the gut such as neutropenic enterocolitis,infections,and inflammatory bowel diseases.Notably,accumulating activated CD8+IELs have been found to be correlated with gastrointestinal manifestations present Coronavirus Disease 2019(COVID-19)patients infected by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Significant progress has been made in better understanding neutrophil-mediated defense against intestinal pathogens.However,the underlying mechanisms whereby neutrophils establish contact with IELs and collectively preserve the intestinal homeostatic status are still not fully understood.Here,we unveiled an unanticipated mechanism whereby neutrophils fine-tune immunoregulation on IEL functions and provided novel insights into manipulating IEL activation as a potential therapeutic approach for patients suffering intestinal inflammatory disorders.2.Neutrophil orchestrate IEL function during gut inflammationAim:To investigate the role of neutrophils in modulating the IEL immune response.Methods:We established experimental models of acute colitis in wild-type(WT)and Cd177-/-mice induced by 2%DSS in drinking water and Citrobacter Rodentium(CR)orally infection,concomitantly treated intraperitoneally with anti-Ly6G to effectively deplete neutrophils in vivo,characteristics of acute colitis,including weight loss,diarrhea,bloody feces and survival rates,were observed daily.Then,we investigated the frequencies and functions of IELs in both small bowel(SB)and large bowel(LB)from indicated groups by flow cytometry.Results:Neutrophil-depleted WT mice and Cd177-/-mice developed more severe colitis compared with WT controls.The deficiency of neutrophils,or CD177+subset,did not affect the composition of IELs isolated from the SB and the LB at a steady state.However,TCRγδ+CD8αα+IELs were found to be increased in both the SB and the LB after DSS insults,and especially highly enriched in colitic PMN-depleted WT mice and Cd177-/-mice.Higher proportions of IFN-γ-,IL-17A-and TNF-α-expressing TCRγδ+CD8αα+IELs were detected in these mice compared to WT controls.In contrast,TCRαβ+CD4+IELs were significantly decreased during DSS-induced acute colitis in PMN-depleted WT mice and Cd177-/-mice.The similar characteristic changes in the composition of IELs were observed in mice following oral inoculation with CR.Conclusions:Neutrophils,especially CD177+neutrophils,modulate IEL activation and maintain gut homeostasis during intestinal mucosal inflammation.3.TCRγδ+CD8αα+IELs manifest with proinflammatory phenotype and hyper-responsiveness to microbiotaAim:To investigate the functional properties of TCRγδ+CD8αα+IELs on the induction of mucosal inflammation.Methods:We established an acute colitis model in WT mice by 2%DSS in drinking water and treated intraperitoneally with anti-TCRγδ mAb(Armenian hamster IgG,hIgG,served as a control)to deplete TCRγδ+CD8αα+IELs.Moreover,we performed single-cell RNA sequencing(scRNA-seq)analysis of IELs isolated from DSS-treated Cd177-/-mice and WT littermates.Results:Anti-TCRγδ-treated mice had mild architectural destruction in the colon,accompanied by rescued intestinal permeability,and reduced expression of proinflammatory cytokines(e.g.,IL-1β,IL-17A,IFN-γ,and TNF-α)at both mRNA and protein levels in the colon tissues.The scRNA-seq analysis revealed that TCRγδ+CD8αα+IELs appeared to be colitogenic with increased expression of proinflammatory genes(e.g.,Gzma,Gzmb,S100a8,S100a9,Fasl,and Klrb1b)and pyroptosis-related gene enrichment.We also observed a distinct gene expression profile of TCRγδ+CD8αα+IELs from Cd177-/-mice,involving bacterial invasion of epithelial cell pathway,defense response to Gram-negative bacteria,and response to bacteria compared to WT controls.Conclusions:TCRγδ+CD8αα+IELs contribute to the susceptibility to DSSinduced acute colitis under neutrophil dysfunctional conditions,and deletion of TCRγδ+CD8αα+IELs does mitigate the extent of the intestinal inflammation induced by DSS.Additionally,alternations of gut microbiota under colitic conditions may contribute to TCRγδ+CD8αα+IEL changes and exacerbate mucosal inflammation in Cd177-/-mice.4.Microbial dysbiosis orchestrates expansion of TCRγδ+CD8αα+IELAim:To investigate whether neutrophil dysfunction shapes TCRγδ+CD8αα+IEL function through changes in gut microbiota.Methods:We analyzed the microbial composition through 16S rDNA gene amplicon sequencing in the feces of Cd177-/-and WT mice on day 0 and day 10 during DSS treatment.Then,we conducted antibiotics treatment(including ampicillin,metronidazole,neomycin,and vancomycin)and co-housing procedure to eliminate the divergence in bacterial components between Cd177-/-and WT mice littermates before DSS modeling,characteristics of acute colitis,including weight loss,diarrhea,bloody feces and survival rates,were observed daily.The frequencies and functions of IELs in both small bowel(SB)and large bowel(LB)were detected by flow cytometry.Results:Distinct bacterial ingredient ratios in both phylum and genus categories and a decreased diversity denoted a dysbiosis of the gut microbiota in Cd177-/-mice.Analysis of microbiome composition further revealed a substantial decrease in protective bacteria,whereas the relative abundance in proinflammatory bacteria in Cd177-/-mice than in WT littermates.Flow cytometry analysis of IEL subsets revealed no difference in the number of TCRγδ+CD8αα+IELs between antibiotics-treated Cd177-/-and WT mice.Moreover,no differences were seen in the frequencies of TCRγδ+CD8αα+,TCRαβ+CD8αα+,TCRαβ+CD8αβ+,and TCRαβ+CD4+ IELs between co-housed Cd177-/-mice and WT littermates.Conclusions:Microbial dysbiosis orchestrates the proinflammatory remodeling of TCRγδ+CD8αα+IELs in Cd177-/-mice during colitis.5.Microbiota metabolite dimethyl fumarate restrains expansion of TCRγδ+CD8αα+ IELsAim:To investigate the role of microbiota metabolites in governing potential immune regulation of neutrophils on IEL functions.Methods:We performed an untargeted metabolomic analysis of the feces collected from Cd177-/-mice and WT littermates pre-and post-DSS insults.Results:Metabolomic analysis revealed a significant heterogeneity of metabolome in Cd177-/-mice.The functional analysis among these differentially expressed metabolites revealed that 17 upregulated metabolites were imprinted proinflammatory properties and that 22 downregulated metabolites were related to immunoregulation in Cd177-/-mice.Notably,dimethyl fumarate(DMF)with broad anti-inflammatory effect was found to be markedly decreased in Cd177-/-mice.Consistently,fumarate-produced bacteria(including Akkermansia,Coriobacteriaceae,Erysipelotrichaceae,and Peptostreptococcus)were also decreased,while fumarateconsumed bacteria(including Bacteroides,Clostridium,Acinetobacter,Vibrio,Propionibacterium,Prevotella,Clostridiales,Ruminococcus,Geobacter,Desulfovibrio and Pseudomonas)were increased in the feces of Cd177-/-mice.Conclusions:DMF may suppress IEL activation and maintain intestinal mucosal homeostasis,and that a deficiency of DMF may profoundly trigger intestinal mucosal inflammation and induce fundamental activation of pathogenic TCRγδ+CD8αα+IELs.6.Microbiota-derived fumarate decreases in IBD patientsAim:To investigate whether microbiota-derived fumarate is also declined in IBD patients.Methods:We detected the relative quantification of energy metabolismassociated metabolites in the stool samples from patients with Crohn’s disease(CD)and ulcerative colitis(UC)without any antibiotic therapy,combined with 16S rDNA microbial analysis of the same samples.We also performed 16S rDNA microbialanalysis utilizing biopsy specimens of inflamed colon isolated from patients with CD and uninflamed mucosal biopsies isolated from non-IBD patients(i.e.,intestinal polyposis)during endoscopic examination as controls.Results:The relative abundance of fumarate was significantly decreased in both CD and UC patients compared with healthy controls.Consistently,proportions of fumarate-produced microbiota were decreased in stool samples of IBD patient relative to healthy controls,while the level of fumarate-consumed microbiota was elevated.Microbial producers of fumarate were found to be significantly decreased in inflamed mucosa from CD patients compared with uninflamed controls.Moreover,an increase of microbiota that perform fumarate respiration were found in inflamed mucosa from CD patients.Conclusions:Microbiota-derived fumarate decreases under inflammatory or neutrophil dysfunction conditions,contributing to intestinal inflammation.7.DMF supplementation suppresses TCRγδ+CD8αα+IEL expansion and ameliorates intestinal mucosal inflammationAim:To determine whether DMF intake prevent the expansion of colitogenic TCRγδ+CD8αα+IELs and associated disease phenotypes.Methods:We designed DSS-induced colitis in Cd177-/-and WT mice with DMF replenishment by oral gavage,characteristics of acute colitis,including weight loss,diarrhea,bloody feces and survival rates,were observed daily.The frequencies and functions of IELs in both small bowel(SB)and large bowel(LB)were detected by flow cytometry.Results:Administration of DMF substantially ameliorated colitis development in Cd177-/-mice.Moreover,administration of DMF also restricted TCRγδ+CD8αα+IEL expansion in Cd177-/-mice and counteracted the discrepancy in the frequencies of TCRαβ+CD4+IELs between Cd177-/-and WT mice.Notably,we observed that a significant increase of Gsdmd expression in TCRγδ+CD8αα+IELs of Cd177-/-mice through scRNA-seq.Consistently,the level of pore-forming GSDMD-N was markedly increased in TCRγδ+CD8αα+IELs of Cd177-/-mice through Western blot experiment,but significantly reduced after DMF administration.High levels of GSDMD-N were also detected in IECs and colon tissues of Cd177-/-mice,but concomitantly blocked by DMF.Conclusions:The expansion of TCRγδ+CD8αα+IELs is mediated by a decrease of microbial metabolite DMF,which is ascribed to the dysregulated immunoregulation of neutrophils on gut microbiota.Importantly,supplementation of DMF restricts the expansion of TCRγδ+CD8αα+IELs in Cd177-/-mice,suggesting that the microbial metabolite DMF is sufficient for restraining the overactivation of TCRγδ+CD8αα+IELs in the gut mucosa.