From pathogens to microbiota: How Drosophila intestinal stem cells react to gut microbes

The intestine acts as one of the interfaces between an organism and its external environment. As the primary digestive organ, it is constantly exposed to a multitude of stresses as it processes and absorbs nutrients. Among these is the recurring damage induced by ingested pathogenic and commensal microorganisms. Both the bacterial activity and immune response itself can result in the loss of epithelial cells, which subsequently requires replacement. In the Drosophila midgut, this regenerative role is fulfilled by intestinal stem cells (ISCs). Microbes not only trigger cell loss and replacement, but also modify intestinal and whole organism physiology, thus modulating ISC activity. Regulation of ISCs is integrated through a complex network of signaling pathways initiated by other gut cell populations, including enterocytes, enteroblasts, enteroendocrine and visceral muscles cells. The gut also receives signals from circulating immune cells, the hemocytes, to properly respond against infection. This review summarizes the types of gut microbes found in Drosophila, mechanisms for their elimination, and provides an integrated view of the signaling pathways that regulate tissue renewal in the midgut.     

肠道免疫系统与糖尿病关系的研究进展

正糖尿病(diabetes mellitus,DM)发病率呈稳定上升趋势,除基因突变、环境因素外,由于肠道免疫系统是接触饮食抗原的第一防护线,肠道病毒感染、口服牛乳或麸质蛋白抗原、肠道菌群等引起的肠道变化被证实与DM发病有关~[1]。一系列研究证据表明肠道免疫系统参与DM发病过程。首先,DM前期,鼠肠道组织学及免疫学已发生改变,DM易患鼠肠道渗透性增加,且肠道病变早于DM发生~[2],如黏膜隐     

On the road to tolerance — Generation and migration of gut regulatory T cells

The intestinal immune system potently supports the generation of induced Treg (iTreg) cells. Within intestinal lymphoid compartments iTreg cells receive homing cues, which direct these cells to the gut lamina propria where they expand and locally suppress immune responses. Yet iTreg cells are but one side of a coin, the other side of which comprises natural Treg (nTreg) cells generated in the thymus. nTreg cells, which act in concert with iTreg cells, also acquire a diversified pattern of homing receptors. Thus iTreg and nTreg cells can enter the gut, and draining lymph nodes to cooperatively ensure intestinal homeostasis.     

Plasmacytoid dendritic cells are dispensable for noninfectious intestinal IgA responses in vivo

Intestinal DCs orchestrate gut immune homeostasis by dampening proinflammatory T‐cell responses and inducing anti‐inflammatory IgA responses. Although no specific DC subset has been strictly assigned so far to govern IgA response, some candidate subsets emerge. In particular, plasmacytoid DCs (pDCs), which notoriously promote anti‐viral immunity and T‐cell tolerance to innocuous antigens (Ags), contribute to IgA induction in response to intestinal viral infection and promote T‐cell‐independent IgA responses in vitro. Here, using two transgenic mouse models, we show that neither short‐term nor long‐term pDC depletion alters IgA class switch recombination in Peyer's patches and frequency of IgA plasma cells in intestinal mucosa at steady state, even in the absence of T‐cell help. In addition, pDCs are dispensable for induction of intestinal IgA plasma cells in response to oral immunization with T‐cell‐dependent or T‐cell‐independent Ags, and are not required for proliferation and IgA switch of Ag‐specific B cells in GALT. These results show that pDCs are dispensable for noninfectious IgA responses, and suggest that various DC subsets may play redundant roles in the control of intestinal IgA responses.     

Molecular crosstalk of probiotic bacteria with the intestinal immune system: Clinical relevance in the context of inflammatory bowel disease

It is current knowledge that the intestinal microbiota plays a major role in the development and maintenance of intestinal health. Intestinal epithelial cells (IEC) constitute the interface between the gut lumen and the innate and adaptive immune system. To maintain intestinal homeostasis, the organized and diffuse compartments of the gut-associated lymphoid tissue have to process the continuously varying information at the interface between the luminal side and the host. Dysregulated intestinal immune responses towards commensal bacteria are an important factor in the pathogenesis of inflammatory bowel diseases (IBD). In contrast to the colitogenic effects of enteric bacteria, clinical and experimental studies showed that specific probiotic strains are protective in the context of chronic intestinal inflammation. Although the molecular understanding of bacteria-host interaction is improving, the anti-inflammatory mechanisms induced by these probiotic bacteria are just starting to be unraveled. The present review is meant to summarize and discuss the clinical relevance of probiotics, but it also seeks to give an overview about currently known probiotic mechanisms in the context of chronic intestinal inflammation with a focus on IEC.     

Gut microbiota regulates NKG2D ligand expression on intestinal epithelial cells

Intestinal epithelial cells (IECs) are one of a few cell types in the body with constitutive surface expression of natural killer group 2 member D (NKG2D) ligands, although the magnitude of ligand expression by IECs varies. Here, we investigated whether the gut microbiota regulates the NKG2D ligand expression on small IECs. Germ‐free and ampicillin‐treated mice were shown to have a significant increase in NKG2D ligand expression. Interestingly, vancomycin treatment, which propagated the bacterium Akkermansia muciniphila and reduced the level of IFN‐γ and IL‐15 in the intestine, decreased the NKG2D ligand expression on IECs. In addition, a similar increase in A. muciniphila and a decreased NKG2D ligand expression was seen after feeding with dietary xylooligosaccharides. A pronounced increase in NKG2D ligand expression was furthermore observed in IL‐10‐deficient mice. In summary, our results suggest that the constitutive levels of NKG2D ligand expression on IECs are regulated by microbial signaling in the gut and further disfavor the intuitive notion that IEC NKG2D ligand expression is caused by low‐grade immune reaction against commensal bacteria. It is more likely that constitutively high IEC NKG2D ligand expression is kept in check by an intestinal regulatory immune milieu induced by members of the gut microbiota, for example A. muciniphila.     

Immunomodulatory dendritic cells in intestinal lamina propria

The lamina propria (LP) of the small intestine contains many dendritic cells (DC), which are likely to be in close contact with luminal antigens, but their role in intestinal immune responses has been overlooked. Here we show that after feeding mice ovalbumin (OVA), the majority of antigen uptake is associated with DC in the small intestinal LP, and we describe the isolation, purification and initial characterization of theses DC. We obtained >90% CD11c(+) DC using magnetic cell sorting, of which the majority were CD11b(+)CD8alpha(-), with smaller numbers of CD11b(-)CD8alpha(+) and CD11b(-)CD8alpha(-) DC as well as a distinct population of CD11c(int)class II MHC(lo) B220(+) DC. Freshly isolated LP DC expressed variable but generally low levels of CD40, CD80 and CD86, which were up-regulated by activation with LPS. LP DC were endocytic in vivo and in vitro and could present antigen to OVA-specific CD4(+) T cells in vitro. Antigen-loaded LP DC from OVA-fed mice also primed specific CD4(+) T cells in vivo and in vitro, but adoptive transfer of these DC into naive recipients induced hyporesponsiveness to subsequent challenge. LP DC also expressed significant levels of mRNA for IL-10 and type I IFN, but not IL-12, suggesting they may play a central and unique role in immune homeostasis in the gut.     

Depletion of Foxp3+ regulatory T cells is accompanied by an increase in the relative abundance of Firmicutes in the murine gut microbiome

A reciprocal interaction exists between the gut microbiota and the immune system. Regulatory T (Treg) cells are important for controlling immune responses and for maintaining the intestinal homeostasis but their precise influence on the gut microbiota is unclear. We studied the effects of Treg cell depletion on inflammation of the intestinal mucosa and analysed the gut microbiota before and after depletion of Treg cells using the DEpletion of REGulatory T cells (DEREG) mouse model. DNA was extracted from stool samples of DEREG mice and wild-type littermates at different time-points before and after diphtheria toxin application to deplete Treg cells in DEREG mice. The V3/V4 region of the 16S rRNA gene was used for studying the gut microbiota with Illumina MiSeq paired ends sequencing. Multidimensional scaling separated the majority of gut microbiota samples from late time-points after Treg cell depletion in DEREG mice from samples of early time-points before Treg cell depletion in these mice and from gut microbiota samples of wild-type mice. Treg cell depletion in DEREG mice was accompanied by an increase in the relative abundance of the phylum Firmicutes and by intestinal inflammation in DEREG mice 20 days after Treg cell depletion, indicating that Treg cells influence the gut microbiota composition. In addition, the variables cage, breeding and experiment number were associated with differences in the gut microbiota composition and these variables should be respected in murine studies.     

Mastering gut permeability: New roles for old friends

Mast cells are innate immune cells that respond rapidly to infection in barrier tissues such as the skin and intestinal mucosa. Expulsion of parasitic worms in the gut involves a robust type 2 host response, and an acute mastocytosis is often generated at the site of infection. However, the role of mast cells in resistance to worm infections appears to be parasite specific. Mast cells are also involved in tissue repair, but the long‐term contribution of mast cell activation after worm expulsion has not been definitively studied. In this issue of European Journal of Immunology, Sorobetea et al. [Eur. J. Immunol. 2017. 47 : 257–268] demonstrate that activated mast cells persist in the large intestinal lamina propria and intraepithelial compartment long after worm expulsion, resulting in continued local and systemic presence of the mast cell protease mast cell protease 1 (MCPt‐1) and enhanced intestinal permeability. In this commentary, we discuss these findings in the wider context of mast cell function in health and disease.     

Mucosal innate immune cells regulate both gut homeostasis and intestinal inflammation

Continuous exposure of intestinal mucosal surfaces to diverse microorganisms and their metabolites reflects the biological necessity for a multifaceted, integrated epithelial and immune cell‐mediated regulatory system. The development and function of the host cells responsible for the barrier function of the intestinal surface (e.g., M cells, Paneth cells, goblet cells, and columnar epithelial cells) are strictly regulated through both positive and negative stimulation by the luminal microbiota. Stimulation by damage‐associated molecular patterns and commensal bacteria‐derived microbe‐associated molecular patterns provokes the assembly of inflammasomes, which are involved in maintaining the integrity of the intestinal epithelium. Mucosal immune cells located beneath the epithelium play critical roles in regulating both the mucosal barrier and the relative composition of the luminal microbiota. Innate lymphoid cells and mast cells, in particular, orchestrate the mucosal regulatory system to create a mutually beneficial environment for both the host and the microbiota. Disruption of mucosal homeostasis causes intestinal inflammation such as that seen in inflammatory bowel disease. Here, we review the recent research on the biological interplay among the luminal microbiota, epithelial cells, and mucosal innate immune cells in both healthy and pathological conditions.     

Human intestinal epithelial cells are susceptible to influenza virus subtype H9N2

Avian influenza viruses (AIV) replicate efficiently in guts of birds, and virus shedding is critical to viral transmission among birds and from birds to other species. In this study, we showed that an H9N2 viral strain, isolated from a human patient, caused typical influenza-like signs and illness including loss of body weight in Balb/c mice, and that viral RNA could be detected in intestinal tissues. We demonstrated that human intestinal epithelial cell line HT-29 was susceptible to the virus, and the infected cells went apoptotic at the early stage post infection. Compared to a pandemic (H1N1) 2009 influenza isolate, we found that the human H9N2 virus induced more severe apoptotic and stronger innate immune responses. Both extrinsic and intrinsic apoptotic pathways were activated in human intestinal epithelial cells, and the levels of FasL and TNF-α were induced up to hundreds-fold in response to the H9N2 infection. Interestingly, Bcl-2 family member Bid was cleaved during the course of infection, and the truncated Bid (tBid) appeared to play a role in the initiation of the intrinsic apoptosis with increased release of cytochrome c in cytosol. As for pro-inflammatory responses in H9N2-infected intestinal epithelial cells, RANTES and IP10 were induced significantly and may have played a major role in intestinal pathogenicity. Moreover, TLR-8, MyD88, and MDA-5 were all up-regulated in the infection, critical in the induction of IFN-β and host innate immunity against the H9N2 virus. Our findings have demonstrated a unique pattern of host responses in human gut in response to H9N2 subtype influenza viruses, which will broaden our understanding of the pathogenesis of AIV infection in both humans and animals.     

肠道菌群在高原低氧肠道损伤中的作用研究

目的　探讨肠道菌群在高原低氧环境中参与肠道损伤的作用机制。 方法　将20只C57 BL/6小鼠按1：1比例随机分为对照组和暴露组，建立6000 m高原低氧模型，造模成功后收集两组小鼠粪便、血液和近端结肠组织。采用16S rDNA测定粪便中肠道菌群结构；检测小鼠血液生化指标；HE和PAS染色观察结肠肠道黏膜结构的改变；RT-qPCR测定结肠组织ZO-1、Occludin、IL-6和TNFα的mRNA表达水平。 结果　与对照组相比，暴露组小鼠血细胞、血红蛋白和红细胞压积值显著升高，高原低氧模型建模成功；16 SrDNA结果显示肠道菌群紊乱、多样性下降，黏蛋白降解菌艾克曼菌，普雷沃氏菌、梭状芽胞杆菌XVIII等致病菌含量上升，短链脂肪酸产生菌罗斯氏菌、Odoribacter菌、Lachnospiracea菌、Butyricicoccus菌和欧氏菌等益生菌含量下降；HE和PAS染色结果显示结肠组织上皮连续性中断、腺体萎缩、隐窝变短、杯状细胞数量减少，提示肠道结构损伤且黏膜屏障破坏；结肠组织紧密连接蛋白Occludin和 ZO-1 mRNA表达水平下降，进一步暴露组小鼠肠道黏膜受损，炎症因子IL-6和TNFα 的mRNA表达量上升，可能与肠道炎症反应有关。 结论　高原低氧环境导致的肠道损伤可能与肠道菌群改变有关。肠道菌群紊乱、多样性下降，致病菌相对丰度上升，益生菌相对丰度下降，菌群的这些改变造成肠道黏膜损伤，引起肠道炎症，进而出现肠道损伤，最终导致高原肠道相关疾病。     

An epithelial armamentarium to sense the microbiota

Intestinal epithelial cells were once thought to be inert, non-responsive cells that simply acted as a physical barrier that prevents the contents of the intestinal lumen from accessing the underlying tissue. However, it is now clear that these cells express a full repertoire of Toll- and Nod-like receptors, and that their activation by components of the microbiota is vital for the development of a functional epithelium, maintenance of barrier integrity, and defense against pathogenic organisms. Additionally, mounting evidence suggests that epithelial sensing of bacteria plays a significant role in the management of the numbers and types of microbes present in the gut microbiota via the production of antimicrobial peptides and other microbe-modulatory products. This is a critical process, as it is now becoming apparent that alterations in the composition of the microbiota can predispose an individual to a wide variety of chronic diseases. In this review, we will discuss the bacterial pattern recognition receptors that are known to be expressed by the intestinal epithelium, and how each of them individually contributes to these vital protective functions. Moreover, we will review what is known about the communication between epithelial cells and various classes of underlying leukocytes, and discuss how they interact with the microbiota to form a three-part relationship that maintains homeostasis in the gut.     

Effect of gut flora mediated-bile acid metabolism on intestinal immune microenvironment

According to reports, gut microbiota and metabolites regulate the intestinal immune microenvironment. In recent years, an increasing number of studies reported that bile acids (BAs) of intestinal flora origin affect T helper cells and regulatory T cells (Treg cells). Th17 cells play a pro-inflammatory role and Treg cells usually act in an immunosuppressive role. In this review, we emphatically summarised the influence and corresponding mechanism of different configurations of lithocholic acid (LCA) and deoxycholic acid (DCA) on intestinal Th17 cells, Treg cells and intestinal immune microenvironment. The regulation of BAs receptors G protein-coupled bile acid receptor 1 (GPBAR1/TGR5) and farnesoid X receptor (FXR) on immune cells and intestinal environment are elaborated. Furthermore, the potential clinical applications above were also concluded in three aspects. The above will help researchers better understand the effects of gut flora on the intestinal immune microenvironment via BAs and contribute to the development of new targeted drugs.     

Homeostasis and function of goblet cells during rotavirus infection in mice

Rotaviruses are the leading cause of severe viral gastroenteritis in young children. To gain insight in goblet cell homeostasis and intestinal mucin expression during rotavirus infection, 6-day-old mice were inoculated with murine rotavirus. To determine epithelial cell migration, mice were injected with BrdU just before inoculation. Small intestines were isolated at different days postinfection (dpi) and evaluated for rotavirus and goblet cell-specific gene expression. Small intestinal mucins of control and infected animals at 1, 2, and 4 dpi were isolated and tested for their capability to neutralize rotavirus infection in vitro. After inoculation, two peaks of viral replication were observed at 1 and 4 dpi. During infection, the number of goblet cells in infected mice was decreased in duodenum and jejunum, but was unaffected in the ileum. Goblet cells in infected animals accumulated at the tips of the villi. Muc2 mRNA levels were increased during the peak of viral replication at 1 dpi, whereas at other time points Muc2 and Tff3 mRNA levels were maintained at control levels. Muc2 protein levels in the tissue were also maintained, however Tff3 protein levels were strongly decreased. The number of goblet cells containing sulfated mucins was reduced during the two peaks of infection. Mucins isolated at 1 and 2 dpi from control and infected mice efficiently neutralized rotavirus infection in vitro. Moreover, mucins isolated from infected mice at 4 dpi were more potent in inhibiting rotavirus infection than mucins from control mice at 4 dpi. In conclusion, these data show that during rotavirus infection, goblet cells, in contrast to enterocytes, are relatively spared from apoptosis especially in the ileum. Goblet cell-specific Muc2 expression is increased and mucin structure is modified in the course of infection. This suggests that goblet cells and mucins play a role in the active defense against rotavirus infection and that age-dependent differences in mucin quantities, composition, and/or structure alter the anti-viral capabilities of small intestinal mucins.     

IgA production by peritoneal cavity B cells is IL-6 independent: Implications for intestinal IgA response

We have shown previously both in vitro and in vivo that IL-6 is an important factor for the development of IgA-producing B cells. However, despite the lack of this cytokine in mice with targeted disruption of the interleukin (IL)-6 gene (gene knockout mice), a substantial number of IgA-producing plasma cells occur in their intestinal mucosa. The experiments reported here indicate that there is a population of IgA-producing B cell precursors originating from the peritoneal cavity, distinguished from conventional Peyer's patch-derived precursors by their expression of CD5, and that IgA secretion by these cells is IL-6-independent. Further, there is an increase in CD5 expression among brightly staining IgA-producing cells obtained from the intestinal lamina propria of IL-6 gene-disrupted mice compared to normal controls. These data suggest an explanation for the persistence of IgA-producing plasma cells in the intestinal mucosa of IL-6-depleted mice and indicate the importance of IL-6 for development of conventional precursors of IgA-producing B cells, but not those derived from the peritoneal cavity pool.     

Mucosal T cells in gut homeostasis and inflammation

The antigen-rich environment of the gut interacts with a highly integrated and specialized mucosal immune system that has the challenging task of preventing invasion and the systemic spread of microbes, while avoiding excessive or unnecessary immune responses to innocuous antigens. Disruption of the mucosal barrier and/or defects in gut immune regulatory networks may lead to chronic intestinal inflammation as seen in inflammatory bowel disease. The T-cell populations of the intestine play a critical role in controlling intestinal homeostasis, and their unique phenotypes and diversities reflect the sophisticated mechanisms that have evolved to maintain the delicate balance between immune activation and tolerance at mucosal sites. In this article, we will discuss the specialized properties of mucosal T cells in the context of immune homeostasis and inflammation.     

Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent infections in human intestinal cells

Immunodeficient patients whose gut is chronically infected by vaccine-derived poliovirus (VDPV) may excrete large amounts of virus for years. To investigate how poliovirus (PV) establishes chronic infections in the gut, we tested whether it is possible to establish persistent VDPV infections in human intestinal Caco-2 cells. Four type 3 VDPV mutants, representative of the viral evolution in the gut of a hypogammaglobulinemic patient over almost 2 years [J. Virol. 74 (2000) 3001], were used to infect both undifferentiated, dividing cells, and differentiated, polarized enterocytes. A VDPV mutant excreted 36 days postvaccination by the patient was lytic in both types of intestinal cell cultures, like the parental Sabin 3 (S3) strain. In contrast, three VDPVs excreted 136, 442, and 637 days postvaccination, established persistent infections both in undifferentiated cells and in enterocytes. Thus, viral determinants selected between day 36 and 136 conferred on VDPV mutants the capacity to infect intestinal cells persistently. The percentage of persistently VDPV-infected cultures was higher in enterocytes than in undifferentiated cells, implicating cellular determinants involved in the differentiation of enterocytes in persistent VDPV infections. The establishment of persistent infections in enterocytes was not due to poor replication of VDPVs in these cells, but was associated with reduced viral adsorption to the cell surface.     

Glicentin-containing cells in intestinal metaplasia,adenoma and carcinoma of the stomach

Summary Glicentin-containing cells (Glic. cells) in intestinal metaplasia, adenoma and carcinoma of the stomach were examined using immunohistochemical techniques. Glic. cells first occurred in the gastric mucosa of the transitional area between metaplastic and intact gastric glands. They frequently showed hyperplasia or micronoduli in the budding area of the deeper metaplastic glands, but in completely intestinalized mucosa these endocrine cells decreased remarkably. Gastric adenomas with mild dysplasia had a good number of glicentin-immunoreactive cells which were located in the deeper adenoma glands. Gastrin- and somatostatin-positive cells were also detected in the adenomas. The incidence of glicentin-positive tumor cells was significantly higher in well differentiated adenocarcinoma than in poorly differentiated adenocarcinoma. Among the seven cases of scirrhous argyrophil cell carcinoma, three showed glicentin- and glucagon-immunoreactivity in the same area of the tumor. These findings suggest that the selective increase of Glic. cells in intestinal metaplasia may be closely related to the development of gastric adenoma. Glicentin positive tumor cells in gastric carcinomas can be regarded to be an expression of intestinal or fetal markers.     

Oral tolerance to haptens: intestinal epithelial cells from 2,4-dinitrochlorobenzene-fed mice inhibit hapten-specific T cell activation in vitro

The mechanisms underlying the induction of immunological tolerance after feeding soluble exogenous antigens, including proteins and haptens, are still unclear. Using a model of oral tolerance to the contact-sensitizing hapten 2,4-dinitrochlorobenzene (DNCB), we have compared the ability of intestinal epithelial cells and of Peyer's patch APC to present DNCB in vitro or ex vivo after oral feeding, to specific peripheral lymph node T cells from DNCB-sensitized mice. In contrast to Peyer's patch APC, which induce efficient hapten-specific T cell activation upon exposure to the hapten either in vitro or in vivo, mature MHC class-II-positive intestinal epithelial cells were unable to induce T cell activation in either case. Interestingly, enterocytes from DNCB-fed mice exerted a dramatic inhibitory effect on the proliferative response of hapten-primed T cells in response to dinitrobenzene sulfonate presented by syngeneic spleen cells. This inhibitory effect, which was also observed with supernatant of intestinal epithelial cells from DNCB-fed mice, could be reversed by neutralizing anti-transforming growth factor (TGF)-β antibodies. In addition, pre-incubation of hapten-sensitized T cells with enterocytes from DNCB-fed mice induced T cell anergy, which could be reversed by exogenous interleukin-2 or interleukin-4. These data demonstrate that intestinal epithelial cells activated in vivo by oral administration of DNCB are able to block proliferation of activated T cells through secretion of immunosuppressive cytokines such as TGF-β. It is proposed that intestinal epithelial cells may play a significant role in oral tolerance by limiting T cell-mediated hypersensitivity responses.