CD8??~+IELs Produce Antimicrobial Peptides And The Functional Investigation

The epithelium of the intestine functions as the primary "frontline" physical barrier between the host and a large number of bacteria in the lumen of the small intestine.Intestinal epithelial cells(IECs)are a single layer of cells covering the luminal side of the intestinal tract.These cells are confronted by trillions of resident commensal bacteria.Intraepithelial lymphocytes(IELs)are the lymphocytes located between enterocytes.IELs have a vital role in protective immunity against invading pathogens,as well as providing tolerance to commensal bacteria and maintaining intestinal homeostasis.These "frontline" IELs are exclusively T cells and are predominantly CD8+ and can be divided into two subsets based on the expression of T cell receptors(TCRs)and co-receptors:type A IELs and type B IELs.CD8??+ IELs are the major of type A IELs.They are the progeny of conventional CD8 T cells activated by peripheral antigens in Peyer's patches(PP)or mesenteric lymph nodes(MLNs)and accumulate with age in the epithelium of mice CD8??+ IELs are strongly cytotoxic,and are a subset of antigen-experienced cytotoxic T lymphocytes(CTLs).However,these IELs typically encounter a large number of commensal bacteria rather than invading pathogens,so two main questions arise:what is the effect of CD8??+ IELs on commensal bacteria?Do CD8??+IELs function in ways other than conventional CTLs when confronted by commensal bacteria?These questions and the relationship of these IELs to commensal bacteria have not been explored fully.We conducted our research according to these questions.Our results are summarized in four major parts as follows:1.Commensal bacteria specifically shape the number of CD8ap+ IELs in the intestinal epitheliumTo investigate further the crosstalk between CD8ap+ IELs and commensal bacteria,we first verified the positive relationship between them.A dramatic decrease in absolute numbers and proportions was shown in germ-free(GF)mice and Atb-treated mice,confirming that commensal bacteria could regulate the number of CD8aP+ IELs in the epithelium of the small intestine.A sharp decrease in the load of commensal bacteria was observed after antibiotic treatment,as assessed by the difference in global microbe DNA loads between Atb-treated and water-treated mice.This observation suggested that accumulation of CD8??+ IELs was influenced by the load of commensal microbes.Moreover,the intestines of Atb-treated mice had a markedly different microbial composition,especially in the proportion of Bacteroidetes and Actinobacteria groups.Individual antibiotic treatment also revealed that CD8aP+ IELs were influenced by some bacteria sensitive to ampicillin.To elucidate whether the gut microbiota eliminated by antibiotics,such as Bifidobacterium in phylum Bacteroidetes,could induce CD8??+ IELs,we carried out microbiota transplantation.After initial antibiotic treatment,Bifidobacterium species were removed completely.Then,Bifidobacterium species were transferred to these mice by gavage and,9 days later,the appearance of Bifidobacterium species was confirmed by qPCR of fecal material.Colonization by Bifidobacterium species induced a robust increase in the number of CD8??+IELs after 15 days.Therefore,we concluded that Bifidobacterium species were members of the commensal microbiota that induced accumulation of CD8??+ IELs in the small intestine.2.Toll-like receptor signaling is a requisite for commensal bacteria-dependent CD8??+ IELsTLRs are expressed by macrophages,dendritic cells(DCs)and IECs,and recognize various commensal-and pathogen-associated molecular patterns.To evaluate the impact of TLR signaling on type A IELs,CD8??+ IELs in various TLR-deficient mice(TLR2-deficient,TLR4-deficient,TLR9-deficient mice,and mice deficient in all three TLRs)were assessed.The absolute number of CD8??+ IELs in several TLR deficient mice exhibited a similar reduction to that in Atb-treated mice,suggesting that commensal bacteria maintained CD8??+ IELs indirectly via TLRs.3.Commensal bacteria-dependent CD8??+ IELs exhibit potent microbicidal activityOur data demonstrated an essential role for commensal bacteria in the maintenance of CD8??+ IELs in the epithelium of the small intestine.Hence,questions arose:why do the number of cytotoxic CD8??+ IELs correlate with commensal bacteria and,during homeostasis do cytotoxic CD8??+ IELs have other functions with regard to the commensal bacteria within the small intestine?To gain insight into these questions,CD8??+ IELs were compared with CD8a(3+ SPLs by microarray analysis.Without stimulation in vitro,CD8??+ IELs exhibited a 20-fold increase in the expression of a series of genes that mediate antibacterial processes(e.g.,Defal,Regllly,and lypd8).Among the genes whose expression was increased in CD8aP+ IELs were those for a family of a-defensins.This included mmp7,a member of a class of genes encoding matrix metalloproteinases known to process inactivated mouse a-defensins to mature-active peptides.RT-PCR,flow cytometry and immunofluorescence confirmed the expression of a-defensins in CD8??+ IELs.In GF and Atb-treated mice,there was a decrease in the number of defensin 1+ CD8??+ IELs in comparison with untreated SPF mice,indicating that these defensin 1+ CD8??+IELs were dependent upon commensal bacteria.In summary,CD8??+ IELs contained antimicrobial peptides and antimicrobial peptide-carrying CD8??+ IELs showed a positive relationship with commensal bacteria.4.Commensal bacteria-dependent CD8??+ IELs can directly inhibit the growth of bacteriaWe had shown expression of a-defensins in CD8??+ IELs.Defensins are broad-spectrum antimicrobial peptides that act against various bacteria.Hence,next we assessed the antimicrobial activity of CD8??+IELs ex vivo.Sorted CD8??+ IELs and CD8??+ SPLs were cultured for 24 h,and activated with PMA and ionomycin for the final 6 h,after which supernatants were collected,and antimicrobial activity determined.According to the antimicrobial assay,inhibition rings were observed around Oxford cups filled with supernatants from purified CD8??+ IELs.These data showed that secretion from stimulated CD8??+ IELs could inhibit the growth of bacteria,likely due to antimicrobial peptides such as defensins.Moreover,supernatants from purified CD8??+ IELs stimulated with IL-15 also produced inhibition rings with larger diameter,suggesteing that IL-15 promoted the antimicrobial activity of CD8??+ IELs.Conclusion:A large proportion of IELs in the epithelium of the intestine are CD8??+.However,as CD8??+ IELs are closely correlated with commensal bacteria,their function besides cytotoxicity is unclear.Nor is their role in homeostasis established.Herein,commensal bacteria were demonstrated to regulate the number of CD8??+ IELs along the intestinal epithelium via TLR and CD8??+ IELs can be induced by some specific members of commensal bacteria.Microarray analysis identified a series of genes that encode antibacterial peptides in commensal-dependent CD8??+IELs.The supernatants of these IELs were found to have direct antibacterial activity suggesting that commensal-dependent CD8??+ IELs can inhibit the growth of bacteria effectively.In conclusion,microbicidal CD8aP+ IELs are regulated by commensal bacteria which,in turn,secrete AMPs that have a vital role in maintaining the homeostasis of the small intestine.