CD103hi Treg cells constrain lung fibrosis induced by CD103lo tissue-resident pathogenic CD4 T cells.

Tissue-resident memory T cells (TRM cells) are crucial mediators of adaptive immunity in nonlymphoid tissues. However, the functional heterogeneity and pathogenic roles of CD4+ TRM cells that reside within chronic inflammatory lesions remain unknown. We found that CD69hiCD103lo CD4+ TRM cells produced effector cytokines and promoted the inflammation and fibrotic responses induced by chronic exposure to Aspergillus fumigatus. Simultaneously, immunosuppressive CD69hiCD103hiFoxp3+ CD4+ regulatory T cells were induced and constrained the ability of pathogenic CD103lo TRM cells to cause fibrosis. Thus, lung tissue-resident CD4+ T cells play crucial roles in the pathology of chronic lung inflammation, and CD103 expression defines pathogenic effector and immunosuppressive tissue-resident cell subpopulations in the inflamed lung.

Epithelial glycosylation in gut homeostasis and inflammation.

Intestinal epithelial cells apically express glycans, especially α1,2-fucosyl linkages, which work as a biological interface for the host-microbe interaction. Emerging studies have shown that epithelial α1,2-fucosylation is regulated by microbes and by group 3 innate lymphoid cells (ILC3s). Dysregulation of the gene (FUT2) encoding fucosyltransferase 2, an enzyme governing epithelial α1,2-fucosylation, is associated with various human disorders, including infection and chronic inflammatory diseases. This suggests a critical role for an interaction between microbes, epithelial cells and ILC3s mediated via glycan residues. In this Review, using α1,2-fucose and Fut2 gene expression as an example, we describe how epithelial glycosylation is controlled by immune cells and luminal microbes. We also address the pathophysiological contribution of epithelial α1,2-fucosylation to pathogenic and commensal microbes as well as the potential of α1,2-fucose and its regulatory pathway as previously unexploited targets in the development of new therapeutic approaches for human diseases.

Intestinal commensal microbiota and cytokines regulate Fut2+ Paneth cells for gut defense.

Paneth cells are intestinal epithelial cells that release antimicrobial peptides, such as α-defensin as part of host defense. Together with mesenchymal cells, Paneth cells provide niche factors for epithelial stem cell homeostasis. Here, we report two subtypes of murine Paneth cells, differentiated by their production and utilization of fucosyltransferase 2 (Fut2), which regulates α(1,2)fucosylation to create cohabitation niches for commensal bacteria and prevent invasion of the intestine by pathogenic bacteria. The majority of Fut2- Paneth cells were localized in the duodenum, whereas the majority of Fut2+ Paneth cells were in the ileum. Fut2+ Paneth cells showed higher granularity and structural complexity than did Fut2- Paneth cells, suggesting that Fut2+ Paneth cells are involved in host defense. Signaling by the commensal bacteria, together with interleukin 22 (IL-22), induced the development of Fut2+ Paneth cells. IL-22 was found to affect the α-defensin secretion system via modulation of Fut2 expression, and IL-17a was found to increase the production of α-defensin in the intestinal tract. Thus, these intestinal cytokines regulate the development and function of Fut2+ Paneth cells as part of gut defense.

Segmented filamentous bacteria antigens presented by intestinal dendritic cells drive mucosal Th17 cell differentiation.

How commensal microbiota contributes to immune cell homeostasis at barrier surfaces is poorly understood. Lamina propria (LP) T helper 17 (Th17) cells participate in mucosal protection and are induced by commensal segmented filamentous bacteria (SFB). Here we show that MHCII-dependent antigen presentation of SFB antigens by intestinal dendritic cells (DCs) is crucial for Th17 cell induction. Expression of MHCII on CD11c(+) cells was necessary and sufficient for SFB-induced Th17 cell differentiation. Most SFB-induced Th17 cells recognized SFB in an MHCII-dependent manner. SFB primed and induced Th17 cells locally in the LP and Th17 cell induction occurred normally in mice lacking secondary lymphoid organs. The importance of other innate cells was unveiled by the finding that MHCII deficiency in group 3 innate lymphoid cells (ILCs) resulted in an increase in SFB-independent Th17 cell differentiation. Our results outline the complex role of DCs and ILCs in the regulation of intestinal Th17 cell homeostasis.

Immunostimulating Commensal Bacteria and Their Potential Use as Therapeutics.

The gut microbiome is intimately intertwined with the host immune system, having effects on the systemic immune system. Dysbiosis of the gut microbiome has been linked not only to gastrointestinal disorders but also conditions of the skin, lungs, and brain. Commensal bacteria can affect the immune status of the host through a stimulation of the innate immune system, training of the adaptive immune system, and competitive exclusion of pathogens. Commensal bacteria improve immune response through the production of immunomodulating compounds such as microbe-associated molecular patterns (MAMPs), short-chain fatty acids (SCFAs), and secondary bile acids. The microbiome, especially when in dysbiosis, is plastic and can be manipulated through the introduction of beneficial bacteria or the adjustment of nutrients to stimulate the expansion of beneficial taxa. The complex nature of the gastrointestinal tract (GIT) ecosystem complicates the use of these methods, as similar treatments have various results in individuals with different residential microbiomes and differential health statuses. A more complete understanding of the interaction between commensal species, host genetics, and the host immune system is needed for effective microbiome interventions to be developed and implemented in a clinical setting.

Persistent colonization of non-lymphoid tissue-resident macrophages by Stenotrophomonas maltophilia.

Accumulating evidence has revealed that lymphoid tissue-resident commensal bacteria (e.g. Alcaligenes spp.) survive within dendritic cells. We extended our previous study by investigating microbes that persistently colonize colonic macrophages. 16S rRNA-based metagenome analysis using DNA purified from murine colonic macrophages revealed the presence of Stenotrophomonas maltophilia. The in situ intracellular colonization by S. maltophilia was recapitulated in vitro by using bone marrow-derived macrophages (BMDMs). Co-culture of BMDMs with clinically isolated S. maltophilia led to increased mitochondrial respiration and robust IL-10 production. We further identified a 25-kDa protein encoded by the gene assigned as smlt2713 (recently renamed as SMLT_RS12935) and secreted by S. maltophilia as the factor responsible for enhanced IL-10 production by BMDMs. IL-10 production is critical for maintenance of the symbiotic condition, because intracellular colonization by S. maltophilia was impaired in IL-10-deficient BMDMs, and smlt2713-deficient S. maltophilia failed to persistently colonize IL-10-competent BMDMs. These findings indicate a novel commensal network between colonic macrophages and S. maltophilia that is mediated by IL-10 and smlt2713.

Fucosyltransferase 2 induces lung epithelial fucosylation and exacerbates house dust mite-induced airway inflammation.

BACKGROUND: One of the pathognomonic features of asthma is epithelial hyperproduction of mucus, which is composed of a series of glycoproteins; however, it remains unclear how glycosylation is induced in lung epithelial cells from asthmatic patients and how glycan residues play a role in the pathogenesis of asthma. OBJECTIVE: The objective of this study was to explore comprehensive epithelial glycosylation status induced by allergic inflammation and reveal its possible role in the pathogenesis of asthma. METHODS: We evaluated the glycosylation status of lung epithelium using a lectin microarray. We next searched for molecular mechanisms underlying epithelial glycosylation. We also examined whether epithelial glycosylation is involved in induction of allergic inflammation. RESULTS: On allergen inhalation, lung epithelial cells were heavily α(1,2)fucosylated by fucosyltransferase 2 (Fut2), which was induced by the IL-13-signal transducer and activator of transcription 6 pathway. Importantly, Fut2-deficient (Fut2-/-) mice, which lacked lung epithelial fucosylation, showed significantly attenuated eosinophilic inflammation and airway hyperresponsiveness in house dust mite (HDM)-induced asthma models. Proteome analyses and immunostaining of the HDM-challenged lung identified that complement C3 was accumulated in fucosylated areas. Indeed, Fut2-/- mice showed significantly reduced levels of C3a and impaired accumulation of C3a receptor-expressing monocyte-derived dendritic cells in the lung on HDM challenge. CONCLUSION: Fut2 induces epithelial fucosylation and exacerbates airway inflammation in asthmatic patients in part through C3a production and monocyte-derived dendritic cell accumulation in the lung.

Fecal microbiota transplantation prevents Candida albicans from colonizing the gastrointestinal tract.

Gut microbes symbiotically colonize the gastrointestinal (GI) tract, interacting with each other and their host to maintain GI tract homeostasis. Recent reports have shown that gut microbes help protect the gut from colonization by pathogenic microbes. Here, we report that commensal microbes prevent colonization of the GI tract by the pathogenic fungus, Candida albicans. Wild-type specific pathogen-free (SPF) mice are resistant to C. albicans colonization of the GI tract. However, administering certain antibiotics to SPF mice enables C. albicans colonization. Quantitative kinetics of commensal bacteria are inversely correlated with the number of C. albicans in the gut. Here, we provide further evidence that transplantation of fecal microbiota is effective in preventing Candida colonization of the GI tract. These data demonstrate the importance of commensal bacteria as a barrier for the GI tract surface and highlight the potential clinical applications of commensal bacteria in preventing pathogenic fungal infections.

Epithelial barrier: an interface for the cross-communication between gut flora and immune system.

Large numbers of environmental antigens, including commensal bacteria and food-derived antigens, constitutively interact with the epithelial layer of the gastrointestinal (GI) tract. Commensal bacteria peacefully cohabit with the host GI tract and exert multiple beneficial or destructive effects on their host. Intestinal epithelial cells (IECs) constitute the first physical and immunological protective wall against invasive pathogens and a cohabitation niche for commensal bacteria. As the physiological homeostasis of IECs is maintained by multiple biological processes such as apoptosis, autophagy, and the handling of endoplasmic reticulum stress, the aberrant kinetics of these biological events, which have genetic and environmental causes, leads to the development of host intestinal pathogenesis such as inflammatory bowel disease. In addition, IECs recognize and interact with commensal bacteria and give instructions to mucosal immune cells to initiate an immunological balance between active and quiescent conditions, eventually establishing intestinal homeostasis. The mucosal immune system regulates the homeostasis of gut microbiota by producing immunological molecules such as secretory immunoglobulin A, the production of which is mediated by IECs. IECs therefore play a central role in the creation and maintenance of a physiologically and immunologically stable intestinal environment.

The gut microbiota and inflammatory bowel disease.

PURPOSE OF REVIEW: Inflammatory bowel diseases (IBDs) reflect the cooperative influence of numerous host and environmental factors, including those of elements of the intestinal immune system, the gut microbiota, and dietary habits. This review focuses on features of the gut microbiota and mucosal immune system that are important in the development and control of IBDs. RECENT FINDINGS: Gut innate-type immune cells, including dendritic cells, innate lymphoid cells, and mast cells, educate acquired-type immune cells and intestinal epithelial cells to achieve a symbiotic relationship with commensal bacteria. However, perturbation of the number or type of commensal microorganisms and endogenous genetic polymorphisms that affect immune responses and epithelial barrier system can ultimately lead to IBDs. Providing beneficial bacteria or fecal microbiota transplants helps to reestablish the intestinal environment, maintain its homeostasis, and ameliorate IBDs. SUMMARY: The gut immune system participates in a symbiotic milieu that includes cohabiting commensal bacteria. However, dysbiotic conditions and aberrations in the epithelial barrier and gut immune system can disrupt the mutualistic relationship between the host and gut microbiota, leading to IBDs. Progress in our molecular and cellular understanding of this relationship has yielded numerous insights regarding clinical applications for the treatment of IBDs.

Critical role of dendritic cells in T cell retention in the interfollicular region of Peyer's patches.

Peyer's patches (PPs) simultaneously initiate active and quiescent immune responses in the gut. The immunological function is achieved by the rigid regulation of cell distribution and trafficking, but how the cell distribution is maintained remains to be elucidated. In this study, we show that binding of stromal cell-derived lymphoid chemokines to conventional dendritic cells (cDCs) is essential for the retention of naive CD4(+) T cells in the interfollicular region (IFR) of PPs. Transitory depletion of CD11c(high) cDCs in mice rapidly impaired the IFR structure in the PPs without affecting B cell follicles or germinal centers, lymphoid chemokine production from stromal cells, or the immigration of naive T cells into the IFRs of PPs. The cDC-orchestrated retention of naive T cells was mediated by heparinase-sensitive molecules that were expressed on cDCs and bound the lymphoid chemokine CCL21 produced from stromal cells. These data collectively reveal that interactions among cDCs, stromal cells, and naive T cells are necessary for the formation of IFRs in the PPs.

Ecto-nucleoside triphosphate diphosphohydrolase 7 controls Th17 cell responses through regulation of luminal ATP in the small intestine.

Extracellular ATP is released from live cells in controlled conditions, as well as dying cells in inflammatory conditions, and, thereby, regulates T cell responses, including Th17 cell induction. The level of extracellular ATP is closely regulated by ATP hydrolyzing enzymes, such as ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases). ENTPDase1/CD39, which is expressed in immune cells, was shown to regulate immune responses by downregulating the ATP level. In this study, we analyzed the immunomodulatory function of ENTPDase7, which is preferentially expressed in epithelial cells in the small intestine. The targeted deletion of Entpd7 encoding ENTPDase7 in mice resulted in increased ATP levels in the small intestinal lumen. The number of Th17 cells was selectively increased in the small intestinal lamina propria in Entpd7(-/-) mice. Th17 cells were decreased by oral administration of antibiotics or the ATP antagonist in Entpd7(-/-) mice, indicating that commensal microbiota-dependent ATP release mediates the enhanced Th17 cell development in the small intestinal lamina propria of Entpd7(-/-) mice. In accordance with the increased number of small intestinal Th17 cells, Entpd7(-/-) mice were resistant to oral infection with Citrobacter rodentium. Entpd7(-/-) mice suffered from severe experimental autoimmune encephalomyelitis, which was associated with increased numbers of CD4(+) T cells producing both IL-17 and IFN-γ. Taken together, these findings demonstrate that ENTPDase7 controls the luminal ATP level and, thereby, regulates Th17 cell development in the small intestine.

[Roles of the gut mucosal immune system in symbiosis and immunity].

The intestine is a unique organ which is continuously exposed to various antigens such as food-derived antigens, as well as both commensal and pathogenic bacteria, under physiological conditions. Intestinal epithelial cells constitute both a physical and an immunological barrier system against this vast array of antigens. The α1,2-fucose-conjugated carbohydrate chains expressed on intestinal epithelial cells are physiologically and immunologically important and are regulated by type III innate lymphoid cells (ILC3). IL-22-producing ILC3 induce anti-microbial molecules such as RegIIIγ, contributing to the formation of a safeguard system for homeostasis of commensal flora in the intestinal lumen, containment of Alcaligenes in Peyer's patches, and establishment of a defensive platform against infection by pathogenic bacteria. The other intestinal innate immune cell type, the mast cell, is also a critical player. Mast cells are activated by ATP produced in host cells and commensal flora, predisposing to the development of inflammatory bowel diseases. Furthermore, mucosal mast cells regulate the differentiation of follicular helper T cells through ATP signals and contribute to subsequent IgA affinity maturation and regulating the homeostasis of commensal microflora.

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.

Soy sauce-like seasoning enhances the growth of Agathobacter rectalis and the production of butyrate, propionate, and lactate.

The short-chain fatty acids responsible for gut homeostasis are volatile fatty acids produced by commensal bacteria in the gut as fermentation products from undigested food components. Among the short-chain fatty acids, butyrate is important for maintaining intestinal tract anaerobic conditions, promoting epithelial barrier functions, and inducing regulatory T cells that suppress inflammatory bowel disease and allergic diarrhea. However, the type of food-derived molecular components and mechanisms by which they regulate the growth and butyrate production of butyrate-producing bacteria are not clearly understood. Agathobacter rectalis is a butyrate-producing bacterium highly colonized in the gut of the Japanese population. In this study, we investigated the effects on A. rectalis of a soy sauce-like seasoning made by brewing with a low salt concentration. The soy sauce-like seasoning promoted the growth of A. rectalis 2.6-fold. An ethanol precipitate prepared from the soy sauce-like seasoning was critical for promoting the growth of A. rectalis and the production of butyrate, propionate, and lactate. Fourier transform infrared spectroscopy (FT-IR) analysis suggested that polysaccharides were active ingredients in the ethanol precipitate of the soy sauce-like seasoning. Inulin, a representative prebiotic with effects against butyrate-producing bacteria, had a limited effect on the growth of A. rectalis compared with the soy sauce-like seasoning. Our results indicate that polysaccharides in a soy sauce-like seasoning contributed to the growth of A. rectalis and enhanced production of butyrate, propionate, and lactate.

Eosinophils Contribute to Oral Tolerance via Induction of RORγt-Positive Antigen-Presenting Cells and RORγt-Positive Regulatory T Cells.

Oral tolerance has been defined as the specific suppression of immune responses to an antigen by prior oral administration of the antigen. It has been thought to serve to suppress food allergy. Previous studies have shown that dendritic cells (DCs) and regulatory T cells (Tregs) are involved in the induction of oral tolerance. However, the detailed mechanisms of Treg induction in oral tolerance remain largely unknown. Eosinophils have been recognized as effector cells in allergic diseases, but in recent years, the diverse functions of tissue-resident eosinophils have been reported. Eosinophils in the intestine have been reported to induce Tregs by releasing TGF-ß, but the role of eosinophils in oral tolerance is still controversial. In this study, we analyzed the roles of eosinophils in oral tolerance using eosinophil-deficient ΔdblGATA mice (mice lacking a high-affinity GATA-binding site in the GATA1 promoter). ΔdblGATA mice showed impaired antigen-induced oral tolerance compared to wild-type mice. The induction of RORγt+ Tregs in mesenteric lymph nodes (MLNs) by oral tolerance induction was impaired in ΔdblGATA mice compared to wild-type mice. An increase in RORγt+ antigen-presenting cells (APCs), which are involved in RORγt+ Treg differentiation, in the intestine and MLNs was not seen in ΔdblGATA mice. Notably, the expansion of group 3 innate lymphoid cells (ILC3s), a subset of RORγt+ APCs, by oral tolerance induction was seen in wild-type mice but not ΔdblGATA mice. These results suggest that eosinophils are crucial in the induction of oral tolerance, possibly via the induction of RORγt+ APCs and RORγt+ Tregs.

Intestinal epithelial cells as mediators of the commensal-host immune crosstalk.

Commensal bacteria regulate the homeostasis of host effector immune cell subsets. The mechanisms involved in this commensal-host crosstalk are not well understood. Intestinal epithelial cells (IECs) not only create a physical barrier between the commensals and immune cells in host tissues, but also facilitate interactions between them. Perturbations of epithelial homeostasis or function lead to the development of intestinal disorders such as inflammatory bowel diseases (IBD) and intestinal cancer. IECs receive signals from commensals and produce effector immune molecules. IECs also affect the function of immune cells in the lamina propria. Here we discuss some of these properties of IECs that define them as innate immune cells. We focus on how IECs may integrate and transmit signals from individual commensal bacteria to mucosal innate and adaptive immune cells for the establishment of the unique mucosal immunological equilibrium.

PRAT4A-dependent expression of cell surface TLR5 on neutrophils, classical monocytes and dendritic cells.

AbstractToll-like receptor 5 (TLR5), a sensor for bacterial flagellin, mounts innate and adaptive immune responses, and has been implicated in infectious diseases, colitis and metabolic syndromes. Although TLR5 is believed to belong to cell surface TLRs, cell surface expression has never been verified. Moreover, it has remained unclear which types of immune cells express TLR5 and contribute to flagellin-dependent responses. In this study we established an anti-mouse TLR5 monoclonal antibody and studied the cell surface expression of TLR5 on immune cells. The macrophage cell line J774 expressed endogenous TLR5 on the cell surface and produced IL-6 and G-CSF in response to flagellin. Cell surface expression of TLR5 and flagellin-induced responses were completely abolished by silencing a TLR-specific chaperone protein associated with TLR4 A (PRAT4A), demonstrating that TLR5 is another client of PRAT4A. In the in vivo immune cells, cell surface TLR5 was mainly found on neutrophils and CD11b (hi) Ly6C (hi) classical monocytes in the bone marrow, circulation, spleen and inflammatory lesions. Ly6C (hi) classical monocytes, but not neutrophils, produced cytokines in response to flagellin. Splenic CD8 (-) CD4 (+) conventional dendritic cells and CD11c (hi) CD11b (hi) lamina propria DCs, also clearly expressed cell surface TLR5. Collectively, cell surface expression of TLR5 is dependent on PRAT4A and restricted to neutrophils, classical monocytes and specific DC subsets.