The Induction Of Intestinal Th17 Cells By Flagellins From Segmented Filamentous Bacteria

BackgroundThe vertebrate intestine is inhabited by hundreds of distinct microbial species,which constitute a complex community,termed the “gut microbiota”.The gut microbiota and host maintain a highly coevolved relationship to achieve dynamic balance.On the one hand,gut microbiota can promote the the maturation of host immune system,on the other hand,the host senses and responds to the microorganisms through an immune response,which promote the colonization of beneficial microorganisms while resisting the invasion of pathogens,thus maintaining homeostasis between microbiota and host.It is now well-recognized that the composition of the microbiome can significantly influence the immune balance,and the host's perception of specific bacterial species trigger responses required for maintaining homeostasis between microbiota and host.Segmented filamentous bacteria(SFB),as a key member of the gut microbiota,drew the attention of researchers due to their unique ability to drive the accumulation of Th17 cells in the small intestinal lamina propria(SILP)of mice.SFB are spore-forming gram-positive bacteria with a segmented and filamentous morphology and widely colonize the distai ileum of vertebrate and invertebrate animals.These bacteria tightly adhere to small intestinal epithelial cells(SI ECs),influencing both innate and adaptive immune responses.In particular,SFB induces the differentiation of Th17 cells.Th17 cells are a subset of CD4+ T cells that are characterized by the production of the cytokines IL-17 A,IL-17 F and IL-22.These cytokines act on a variety of hematopoietic and non-hematopoietic cells to regulate host immunity,including granulopoiesis,neutrophil recruitment,and the induction of antimicrobial peptides.Therefore,Th17 cells play key roles in epithelial homeostasis and host defense against extracellular pathogens.At present,a large number of studies have proved that SFB could promote the differentiation of Th17 cells.But the major question still remained: How does SFB induce Th17 cells? And which components of SFB may be involved in this immune response process? Most reported microbiota-immune effects are mediated by the recognition of microbes by pattern recognition receptor(PRR)such as Toll-like receptors(TLRs).Such as,bacterial flagellin are recognized by Toll-like receptor 5(TLR5).It is well-known that the bacterial flagella gene is an important functional gene that affects bacterial colonization and host immune regulation.When flagellin adheres to the base of the intestinal epithelium,it initiates an innate immune response and the flagellin-mediated proinflammatory response.And the complete genome sequence of mouse SFB showed that SFB encoded more than 40(3% of total)putative chemotaxisand flagella-related proteins,and a complete set of genes for flagellar assembly was identified,although they have lost many enzymes for completing pathways essential for their growth and surviva.In addition,comparative genomic analysis showed that SFB genomes were similar to Clostridial genomes and several Clostridial genomes also encoded flagellar assembly proteins.Surprisingly,only SFB flagellin proteins have the TLR5 binding sites.In summary,it is well now known that SFB play key roles in promoting Th17 cells differerntiation in the small intestine.However,little information exists regarding the interactions between SFB and the host cells.Scanning electron microscopy results showed that SFB adhere to the surface of intestinal epithelium,but still unclear how the SFB were absorbed to this area.Although SFB flagellins were not detected by electron microscope analysis,given the proportion of flagella-related gene in the SFB genomes and the important functions of flagellin in other bacteria,we speculated that(1)SFB widely express the flagellin protein;(2)SFB flagellin protein might be the antigen that induce host cells reaction.Therefore,in this study,we used a variety of technologies to detect whether SFB express the flagellin protein and used recombinant SFB flagellin protein to investigate the interaction between SFB flagellin and host cells in vivo and in vitro.Part 1: The induction of Th17 cells by SFB flagellins in vitroAim: To investigate preliminarily whether SFB flagellins stimulate immune cells and participate in certain immune regulatory responses.Methods: 1)Lamina propria lymphocytes in mouse were isolated by mechanical grinding and density gradient centrifugation.Lamina propria CD4+ T cell subsets and CD11c+ cells were enriched by magnetic-activated cell sorting beads(MACS)and fluorescence-activated cell sorting(FACS).2)Mouse and rat SFB flagellin genes were subcloned into the p ET-28 a vector,and SFB flagellin proteins were then purified.3)Mouse lamina propria lymphocytes were stimulated ex vivo with SFB flagellin proteins.Enzyme-linked immunosorbent assay(ELISA),Enzyme-linked Immunospot Assay(ELISPOT)and quantitative real-time quantitative(q RT-PCR)were used to detect the immune difference of lamina propria lymphocytes and the expression levels of Th17 cell-related cytokines.Results:1)All of these SFB flagellins could induce IL-17 expression in a significant number of CD4+ cells.And the concentration of IL-17 A gradually increased with time and the activation effect of CD4+ T cells was the best at about 72 h.2)SFB flagellins promoted Th17 cells response and significantly promoted the expression levels of Th17 cell-related cytokines.Part 2: SFB flagellins promote the Th17 cells differention in the ileum of mouse.Aim: To further demonstrate that SFB flagellins could induce the differentions of small intestinal Th17 cells in vivo.Methods: 1)Rat derived and mouse derived purified SFB flagellins were intraperitoneally injected into C57BL/6 mice.After 2 h,the ileal tissues and serum were collected for subsequent experiments.2)The ileum tissues(0.5 cm anterior to the ileocecal junction)were collected,and the proteins of samples were lysed.ELISA was used to detect the cytokine concentrations of ileum tissues and serum.3)Total RNA was isolated from ileum tissues,and q RT-PCR were used to detect the expression levels of mouse ileum related cytokines.Results: 1)We found that when mice were intraperitoneally injected with SFB flagellins for 2 h,Th17 cells had significant changes,mainly manifested by significant increased concentration and transcription levels of Th17 cells related cytokines,such as IL-17 A and IL-6,in both ileum and serum of mice.2)However,the concentration and transcription levels of Th17 cells related cytokines all returned to normal level in the ileum and serum of mice with flagellin administration for 24 h.Part 3: Influence of SFB flagellins on the intestinal gene expression profiles of miceAim: To futher determine the influence of SFB flagellins host gene expression,we compared the gene expression profiles in the ileum of C57BL/6 mice after administration of SFB flagellins with control.Methods: 1)Rat and mouse derived purified SFB flagellins were intraperitoneally injected into C57BL/6 mice.After 2 h,the ileum tissues were collected and total RNA was isolated for RNA sequencing(RNA-Seq).2)Total RNA was isolated from spleens,and q RT-PCR were used to detect the expression levels of mouse ileum related cytokines.Results: 1)We found that administration of mice with SFB flagellins induced a significant change in the expression of vast genes.In addition,these four gene sets showed fraction overlap,which contained 594 genes.2)GO and KEGG enrichment analyse showed that SFB-m Fli C3 significantly induced the immune system pathways.For example,the chemokine signaling pathway,Toll-like receptor signaling pathway,and NF-kappa B signaling pathway had significantly changed.3)Gene profiling indicated that the expression of genes specific to IL-17-mediated signaling,including Th17-promoting cytokines,chemokines,and antimicrobial molecules were significantly enhanced.4)The transcription levels of Th17 cells related cytokines were not significantly enhanced in the spleen of mice with flagellin administration.Part 4: SFB flagellins interact with host intestinal epithelial cells directly.Aim: To preliminarily explore the possible mechanism of SFB flagellins promoting Th17 cell differentiation,laying a foundation for understanding of the molecular and cellular mechanisms that SFB regulates in intestinal immunity.Methods: 1)The ileum tissues were collected and total RNA was isolated for RNA sequencing(RNA-Seq).we compared the expression profiles of intestinal epithelial related genes.2)Total RNA was isolated from ileum tissues,and q RT-PCR were used to detect the expression levels of intestinal epithelial related cytokines in mice.3)The mouse SI EC line(MODE-K)were cocultured with SFB flagellins for 24 h in vitro and total RNA was isolated.q RT-PCR were used to detect the transcription level of specific genes in MODE-K cells.4)An anti-TLR5 monoclonal antibody was introduced into the cocultures of CD4+ T and CD11c+ cells to neutralize TLR5.Cells were then treated with SFB flagellin proteins for 72 h.ELISA was used to detect the expression levels of Th17 cell-related cytokine.Results: 1)The expression profiles of intestinal epithelial related genes shown that the expression of some genes in SI ECs of mice intraperitoneally injected with SFB flagellins were highly upregulated.In addition,the transcription level of these genes was verified by q RT-PCR.2)In the experiment of co-incubation of intestinal epithelial cell lines with SFB flagellins,it was proved that SFB flagellins could induce SI EC to produce SAAs and then evoke the induction of IL-17.3)The effect of T cell activation was not significantly attenuated when the anti-TLR5 blocking antibody was added.These findings suggest that SFB flagellins promote T cell activation and this effect may not or at least not only correlate with TLR5 activation.Conclusion: In this study,we found that SFB flagellins have the similar immune function as SFB,which as a previously unappreciated component,may be one of the key protein components of SFB in regulating intestinal immunity.The main performance is that SFB flagellins alone can induce the production of Th17 cells in the small intestine,and interact with host intestinal epithelial cells directly.The present study provides new ideas and possibilities for revealing the molecular and cellular mechanisms involved in SFB-induced Th17 cell differentiation.