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.

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.

Unique symbiont-derived sphingolipids: Dietary amino acids source branch formation.

Metabolites derived from symbionts have the potential to regulate host pathophysiological conditions, especially in the gut. In a recent issue of Nature, Oh et al. clarify unique molecular structures of α-galactosylceramides derived from B. fragilis and their immune-modulatory functions against host natural killer T (NKT) cells.

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.

Measurement of the Intestinal pH in Mice under Various Conditions Reveals Alkalization Induced by Antibiotics.

The intestinal pH can greatly influence the stability and absorption of oral drugs. Therefore, knowledge of intestinal pH is necessary to understand the conditions for drug delivery. This has previously been measured in humans and rats. However, information on intestinal pH in mice is insufficient despite these animals being used often in preclinical testing. In this study, 72 female ICR mice housed in SPF (specific pathogen-free) conditions were separated into nine groups to determine the intestinal pH under conditions that might cause pH fluctuations, including high-protein diet, ageing, proton pump inhibitor (PPI) treatment, several antibiotic treatment regimens and germ-free mice. pH was measured in samples collected from the ileum, cecum and colon, and compared to control animals. An electrode, 3 mm in diameter, enabled accurate pH measurements with a small amount of gastrointestinal content. Consequently, the pH values in the cecum and colon were increased by high-protein diet, and the pH in the ileum was decreased by PPI. Drastic alkalization was induced by antibiotics, especially in the cecum and colon. The alkalized pH values in germ-free mice suggested that the reduction in the intestinal bacteria caused by antibiotics led to alkalization. Alkalization of the intestinal pH caused by antibiotic treatment was verified in mice. We need further investigations in clinical settings to check whether the same phenomena occur in patients.

[Commensal bacteria prevent pathogenic bacterial infection by inducing of activation of host immune system].

Countless numbers of bacteria inhabit the intestinal tract. One of the important functions of gut microbiota is the "colonization resistance" against infection by pathogenic microorganisms. However, detailed mechanism of the colonization resistance of intestinal bacteria is still largely unknown. We tried to identify molecular and cellular mechanism of it and found that antigen presentation by dendritic cells is required for the induction of intestinal segmented filamentous bacteria (SFB)-induced T helper 17 (Th17) cells that contribute to the protection against infection by Citrobacter rodentium. We further identified that gut Th17 cells selectively recognize antigens derived from SFB. We also revealed that SFB induce α1,2-fucose, one of carbohydrate chains, expressed on the intestinal epithelial cells mediated by group 3 innate lymphoid cells. Epithelial α1,2-fucose protected against infection by pathogenic bacterium Salmonella typhimurium. Furthermore, it was found that intestinal bacteria inhibit colonization of the pathogenic fungus Candida albicans as well as pathogenic bacteria. From these studies, detailed mechanism of "colonization resistance" against pathogenic microorganisms by intestinal bacteria has been clarified.

Subtilase cytotoxin induces a novel form of Lipocalin 2, which promotes Shiga-toxigenic Escherichia coli survival.

Shiga-toxigenic Escherichia coli (STEC) infection causes severe bloody diarrhea, renal failure, and hemolytic uremic syndrome. Recent studies showed global increases in Locus for Enterocyte Effacement (LEE)-negative STEC infection. Some LEE-negative STEC produce Subtilase cytotoxin (SubAB), which cleaves endoplasmic reticulum (ER) chaperone protein BiP, inducing ER stress and apoptotic cell death. In this study, we report that SubAB induces expression of a novel form of Lipocalin-2 (LCN2), and describe its biological activity and effects on apoptotic cell death. SubAB induced expression of a novel LCN2, which was regulated by PRKR-like endoplasmic reticulum kinase via the C/EBP homologous protein pathway. SubAB-induced novel-sized LCN2 was not secreted into the culture supernatant. Increased intracellular iron level by addition of holo-transferrin or FeCl3 suppressed SubAB-induced PARP cleavage. Normal-sized FLAG-tagged LCN2 suppressed STEC growth, but this effect was not seen in the presence of SubAB- or tunicamycin-induced unglycosylated FLAG-tagged LCN2. Our study demonstrates that SubAB-induced novel-sized LCN2 does not have anti-STEC activity, suggesting that SubAB plays a crucial role in the survival of LEE-negative STEC as well as inducing apoptosis of the host cells.

Interleukin-22-mediated host glycosylation prevents Clostridioides difficile infection by modulating the metabolic activity of the gut microbiota.

The involvement of host immunity in the gut microbiota-mediated colonization resistance to Clostridioides difficile infection (CDI) is incompletely understood. Here, we show that interleukin (IL)-22, induced by colonization of the gut microbiota, is crucial for the prevention of CDI in human microbiota-associated (HMA) mice. IL-22 signaling in HMA mice regulated host glycosylation, which enabled the growth of succinate-consuming bacteria Phascolarctobacterium spp. within the gut microbiome. Phascolarctobacterium reduced the availability of luminal succinate, a crucial metabolite for the growth of C. difficile, and therefore prevented the growth of C. difficile. IL-22-mediated host N-glycosylation is likely impaired in patients with ulcerative colitis (UC) and renders UC-HMA mice more susceptible to CDI. Transplantation of healthy human-derived microbiota or Phascolarctobacterium reduced luminal succinate levels and restored colonization resistance in UC-HMA mice. IL-22-mediated host glycosylation thus fosters the growth of commensal bacteria that compete with C. difficile for the nutritional niche.

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.

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 Cells as a Transmitter of Signals From Commensal Bacteria and Host Immune Cells.

Intestinal epithelial cells (IECs) are non-hematopoietic cells that form a physical barrier against external antigens. Recent studies indicate that IECs have pleiotropic functions in the regulation of luminal microbiota and the host immune system. IECs produce various immune modulatory cytokines and chemokines in response to commensal bacteria and contribute to developing the intestinal immune system. In contrast, IECs receive cytokine signals from immune cells and produce various immunological factors against luminal bacteria. This bidirectional function of IECs is critical to regulate homeostasis of microbiota and the host immune system. Disruption of the epithelial barrier leads to detrimental host diseases such as inflammatory bowel disease, colonic cancer, and pathogenic infection. This review provides an overview of the functions and physiology of IECs and highlights their bidirectional functions against luminal bacteria and immune cells, which contribute to maintaining gut homeostasis.

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.

IL-22 induces Reg3γ and inhibits allergic inflammation in house dust mite-induced asthma models.

Previous studies have shown that IL-22, one of the Th17 cell-related cytokines, plays multiple roles in regulating allergic airway inflammation caused by antigen-specific Th2 cells; however, the underlying mechanism remains unclear. Here, we show that allergic airway inflammation and Th2 and Th17 cytokine production upon intratracheal administration of house dust mite (HDM) extract, a representative allergen, were exacerbated in IL-22-deficient mice. We also found that IL-22 induces Reg3γ production from lung epithelial cells through STAT3 activation and that neutralization of Reg3γ significantly exacerbates HDM-induced eosinophilic airway inflammation and Th2 cytokine induction. Moreover, exostatin-like 3 (EXTL3), a functional Reg3γ binding protein, is expressed in lung epithelial cells, and intratracheal administration of recombinant Reg3γ suppresses HDM-induced thymic stromal lymphopoietin and IL-33 expression and accumulation of type 2 innate lymphoid cells in the lung. Collectively, these results suggest that IL-22 induces Reg3γ production from lung epithelial cells and inhibits the development of HDM-induced allergic airway inflammation, possibly by inhibiting cytokine production from lung epithelial cells.

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.

IL-10-producing CD4(+) T cells negatively regulate fucosylation of epithelial cells in the gut.

Fucosylated glycans on the surface of epithelial cells (ECs) regulate intestinal homeostasis by serving as attachment receptors and a nutrient source for some species of bacteria. We show here that epithelial fucosylation in the ileum is negatively regulated by IL-10-producing CD4(+) T cells. The number of fucosylated ECs was increased in the ileum of mice lacking T cells, especially those expressing αß T cell receptor (TCR), CD4, and IL-10. No such effect was observed in mice lacking B cells. Adoptive transfer of αßTCR(+) CD4(+) T cells from normal mice, but not IL-10-deficient mice, normalized fucosylation of ECs. These findings suggest that IL-10-producing CD4(+) T cells contribute to the maintenance of the function of ECs by regulating their fucosylation.

[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.

Intestinal Monocyte-Derived Macrophages Control Commensal-Specific Th17 Responses.

Generation of different CD4 T cell responses to commensal and pathogenic bacteria is crucial for maintaining a healthy gut environment, but the associated cellular mechanisms are poorly understood. Dendritic cells (DCs) and macrophages (Mfs) integrate microbial signals and direct adaptive immunity. Although the role of DCs in initiating T cell responses is well appreciated, how Mfs contribute to the generation of CD4 T cell responses to intestinal microbes is unclear. Th17 cells are critical for mucosal immune protection and at steady state are induced by commensal bacteria, such as segmented filamentous bacteria (SFB). Here, we examined the roles of mucosal DCs and Mfs in Th17 induction by SFB in vivo. We show that Mfs, and not conventional CD103(+) DCs, are essential for the generation of SFB-specific Th17 responses. Thus, Mfs drive mucosal T cell responses to certain commensal bacteria.