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Clostridioides difficile is endemic in the intestinal tract of healthy people. However, it is responsible for many healthcare-associated infections, such as nosocomial diarrhea following antibiotic treatment. Importantly, there have been cases of unsuccessful treatment and relapse related to the emergence of highly virulent strains of C. difficile and resistance to antimicrobial agents. Fecal microbiota transplantation (FMT) is considered an effective therapy for recurrent C. difficile infection. However, its safety is of concern because deaths caused by antibiotic-resistant bacterial infections after FMT were reported. Therefore, the development of effective C. difficile-specific treatments is urgently needed. In this review, we summarize the importance of phage therapy against C. difficile, and describe a novel next-generation phage therapy developed using metagenomic data.

Progress in genomic analysis has resulted in the proposal that the intestinal microbiota is a crucial environmental factor in the development of multifactorial diseases, such as obesity, diabetes, rheumatoid arthritis, and inflammatory bowel diseases represented by Crohn’s disease and ulcerative colitis. Dysregulated gut microbiome contributes to the pathogenesis of such disorders; however, there are few effective treatments for controlling only disease-mediating bacteria. Here, we review current knowledge about the intestinal microbiome in health and disease, and discuss a regulatory strategy using a parenteral vaccine with emulsified curdlan and CpG oligodeoxynucleotides, which we have recently developed. Unlike other conventional injectable immunizations, our vaccine contributes to the induction of antigen-specific systemic and mucosal immunity. This vaccine strategy can prevent infectious diseases such as Streptococcus pneumoniae infection, and control metabolic symptoms mediated by intestinal bacteria (e.g. Clostridium ramosum) by induction of high titers of antigen-specific IgA at target mucosal sites. In the future, our vaccination approach could be an effective therapy for common infectious diseases and dysbiosis-related disorders that have been difficult to control so far.

The intestinal microbiome is dominated by bacteria and plays a pivotal role in the occurrence and development of disease, including several metabolic and autoimmune disorders. While intestinal viral communities, primarily made up of bacteriophages, are also thought to play a role in disease pathogenesis in the gastrointestinal tract, they have received much less attention than intestinal bacteria. Thus, there is limited information about the relationship between bacteriophages and disease. This review explores a potential role for the intestinal viral microbiome in various metabolic and autoimmune diseases.

Our bodies are constantly exposed to a wide variety of pathogenic micro-organisms through mucosal sites. Therefore, effective vaccines that can protect at the mucosa are vital; however, only a few clinically established mucosal vaccines are available. Although conventional injectable vaccines can induce antigen-specific serum immunoglobulin G (IgG) and prevent severe infection, it is difficult to efficiently inhibit the invasion of pathogens at mucosal surfaces because of the inadequate ability to induce antigen-specific IgA. Recently, we have developed a parenteral vaccine with emulsified curdlan and CpG oligodeoxynucleotides and reported its application. Unlike other conventional injectable vaccines, this immunization contributes to the induction of antigen-specific mucosal and systemic immune responses. Even if antigen-specific IgA at the mucosa disappears, this immunization can induce high-titer IgA after boosting with a small amount of antigen on the target mucosal surface. Indeed, vaccination with Streptococcus pneumoniae antigen effectively prevented lung infection induced by this bacterium. In addition, vaccination with Clostridium ramosum, which is a representative pathobiont associated with obesity and diabetes in humans, reduced obesity in mice colonized with this microorganism. This immunization approach might be an effective treatment for intestinal bacteria-mediated diseases that have been difficult to regulate so far, as well as common infectious diseases.

The receptor activator of nuclear factor κB ligand (RANKL) is an important factor for osteoclastogenesis and contributes to the pathology of rheumatoid arthritis (RA); thus, the anti-RANKL antibody (Ab) has been expected to protect joint destruction in RA patients. IL-8 also has osteoclastogenic activity; however, the role of IL-8 in the bone pathology of RA as well as the relation between IL-8 and RANKL remain unclear. In the present study, clinical observation revealed serum IL-8 levels of 611 pg ml−1 in RA patients with anti-RANKL Ab and 266 pg ml−1 in the same patients without anti-RANKL Ab. In vitro assay showed that anti-RANKL Ab induced production of IL-8 from pre-osteoclast-like cells (OCLs), and IL-8 promoted the formation of OCLs from peripheral monocytes even without RANKL activity. We further showed that treatment with FK506 (tacrolimus) possibly inhibits the increase in IL-8 levels in RA patients with anti-RANKL Ab, and in vitro assay confirmed that FK506 suppressed IL-8 production in pre-OCLs. These results suggest that inhibition of RANKL induces the change in osteoclastogenesis-promoting factor from RANKL to IL-8, and FK506 may be a valuable combination drug to support the use of anti-RANKL Ab in treatment of RA., A therapeutic anti-RANKL Ab increases IL-8 production in RA

The gut is an extremely complicated ecosystem where micro-organisms, nutrients and host cells interact vigorously. Although the function of the intestine and its barrier system weakens with age, some probiotics can potentially prevent age-related intestinal dysfunction. Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131, which are the constituents of LB81 yogurt, are representative probiotics. However, it is unclear whether their long-term intake has a beneficial influence on systemic function. Here, we examined the gut microbiome, fecal metabolites and gene expression profiles of various organs in mice. Although age-related alterations were apparent in them, long-term LB81 yogurt intake led to an increased Bacteroidetes to Firmicutes ratio and elevated abundance of the bacterial family S24-7 (Bacteroidetes), which is known to be associated with butyrate and propanoate production. According to our fecal metabolite analysis to detect enrichment, long-term LB81 yogurt intake altered the intestinal metabolic pathways associated with propanoate and butanoate in the mice. Gene ontology analysis also revealed that long-term LB81 yogurt intake influenced many physiological functions related to the defense response. The profiles of various genes associated with antimicrobial peptides-, tight junctions-, adherens junctions- and mucus-associated intestinal barrier functions were also drastically altered in the LB81 yogurt-fed mice. Thus, long-term intake of LB81 yogurt has the potential to maintain systemic homeostasis, such as the gut barrier function, by controlling the intestinal microbiome and its metabolites.

Genome-wide association studies and subsequent deep sequencing analysis have identified susceptible loci for inflammatory bowel diseases (IBDs) including ulcerative colitis (UC). A gene encoding RING finger protein 186 (RNF186) is located within UC-susceptible loci. However, it is unclear whether RNF186 is involved in IBD pathogenesis. Here, we show that RNF186 controls protein homeostasis in colonic epithelia and regulates intestinal inflammation. RNF186, which was highly expressed in colonic epithelia, acted as an E3 ligase mediating polyubiquitination of its substrates. Permeability of small organic molecules was augmented in the intestine of Rnf186-/- mice. Increased expression of several RNF186 substrates, such as occludin, was found in Rnf186-/- colonic epithelia. The disturbed protein homeostasis in Rnf186-/- mice correlated with enhanced endoplasmic reticulum (ER) stress in colonic epithelia and increased sensitivity to intestinal inflammation after dextran sulfate sodium (DSS) treatment. Introduction of an UC-associated Rnf186 mutation led to impaired E3 ligase activity and increased sensitivity to DSS-induced intestinal inflammation in mice. Thus, RNF186 maintains gut homeostasis by controlling ER stress in colonic epithelia.

Mammalian mucosal surfaces form critical interfaces between the bodily tissues and foreign antigens such as commensal flora and ingested food. Innate mucosal immunity in the gut, as represented by intestinal barrier functions and germline-encoded pattern recognition receptor (PRR)-mediated immune responses, plays an important role in regulating intestinal health and in disease prevention. Intestinal epithelial cells not only function as physical barriers, but also participate in innate immune responses. Disrupted epithelial cell function in the gut can lead to intestinal disorders. Additionally, PRR gene defects, including those relating to toll-like receptors, C-type lectin receptors, retinoic-acid-inducible gene-I-like receptors, and NOD-like receptors, lead to mucosal dysfunction and the subsequent induction of intestinal inflammation. In this chapter, we focus primarily on the current understanding of the role played by innate immunity at mucosal surfaces as an intestinal barrier system and how PRRs relate to such immunity.

Background & Aims Dysregulation of the microbiome has been associated with development of complex diseases, such as obesity and diabetes. However, no method has been developed to control disease-associated commensal microbes. We investigated whether immunization with microbial antigens, using CpG oligodeoxynucleotides and/or curdlan as adjuvants, induces systemic antigen-specific IgA and IgG production and affects development of diseases in mice. Methods C57BL/6 mice were given intramuscular injections of antigens (ovalbumin, cholera toxin B-subunit, or pneumococcal surface protein A) combined with CpG oligodeoxynucleotides and/or curdlan. Blood and fecal samples were collected weekly and antigen-specific IgG and IgA titers were measured. Lymph nodes and spleens were collected and analyzed by enzyme-linked immunosorbent assay for antigen-specific splenic T-helper 1 cells, T-helper 17 cells, and memory B cells. Six weeks after primary immunization, mice were given a oral, nasal, or vaginal boost of ovalbumin; intestinal lamina propria, bronchial lavage, and vaginal swab samples were collected and antibodies and cytokines were measured. Some mice were also given oral cholera toxin or intranasal Streptococcus pneumoniae and the severity of diarrhea or pneumonia was analyzed. Gnotobiotic mice were gavaged with fecal material from obese individuals, which had a high abundance of Clostridium ramosum (a commensal microbe associated with obesity and diabetes), and were placed on a high-fat diet 2 weeks after immunization with C ramosum. Intestinal tissues were collected and analyzed by quantitative real-time polymerase chain reaction. Results Serum and fecal samples from mice given injections of antigens in combination with CpG oligodeoxynucleotides and curdlan for 3 weeks contained antigen-specific IgA and IgG, and splenocytes produced interferon-gamma and interleukin 17A. Lamina propria, bronchial, and vaginal samples contained antigen-specific IgA after the ovalbumin boost. This immunization regimen prevented development of diarrhea after injection of cholera toxin, and inhibited lung colonization by S pneumoniae. In gnotobiotic mice colonized with C ramosum and placed on a high-fat diet, the mice that had been immunized with C ramosum became less obese than the nonimmunized mice. Conclusions Injection of mice with microbial antigens and adjuvant induces antigen-specific mucosal and systemic immune responses. Immunization with S pneumoniae antigen prevented lung infection by this bacteria, and immunization with C ramosum reduced obesity in mice colonized with this microbe and placed on a high-fat diet. This immunization approach might be used to protect against microbe-associated disorders of intestine.

Extracellular adenosine 5'-triphosphate (ATP) performs multiple functions including activation and induction of apoptosis of many cell types. The ATP-hydrolyzing ectoenzyme ecto-nucleotide pyrophosphatase/phosphodiesterase 3 (E-NPP3) regulates ATP-dependent chronic allergic responses by mast cells and basophils. However, E-NPP3 is also highly expressed on epithelial cells of the small intestine. In this study, we showed that E-NPP3 controls plasmacytoid dendritic cell (pDC) numbers in the intestine through regulation of intestinal extracellular ATP. In Enpp3-/- mice, ATP concentrations were increased in the intestinal lumen. pDC numbers were remarkably decreased in the small intestinal lamina propria and Peyer's patches. Intestinal pDCs of Enpp3-/- mice showed enhanced cell death as characterized by increases in annexin V binding and expression of cleaved caspase-3. pDCs were highly sensitive to ATP-induced cell death compared with conventional DCs. ATP-induced cell death was abrogated in P2rx7-/- pDCs. Accordingly, the number of intestinal pDCs was restored in Enpp3-/- P2rx7-/- mice. These findings demonstrate that E-NPP3 regulates ATP concentration and thereby prevents the decrease of pDCs in the small intestine.

The innate immune system provides the first line of host defense against invading pathogens. Innate immune responses are initiated by germline-encoded PRR, which recognize specific structures expressed by microorganisms. TLR are a family of PRR which sense a wide range of microorganisms, including bacteria, fungi, protozoa and viruses. TLR are also expressed in the intestine and are critical for intestinal homeostasis. Recently, cytoplasmic PRR, such as NLR and RLR, have been shown to detect pathogens that have invaded the cytosol. One of the NLR, NOD2, is thought to be involved in the pathogenesis of Crohn's disease. This review focuses on the innate immune responses triggered by PRR in the intestine.

Clowse et al 1 have reported minimal to no transfer of certolizumab pegol (CZP) into breast milk, and their findings have supported the continuation of CZP treatment during breast milk feeding. Rheumatoid arthritis (RA) often develops in women of childbearing age. It is generally difficult to treat these patients with methotrexate, which is the anchor drug for RA. Therefore, biologics, such as tumour necrosis factor (TNF) inhibitors, are often considered for active RA during pregnancy. However, the biologics cross the placenta from mother to fetus and transfer into breast milk during lactation. Although a meta-analysis report indicated that anti-TNF-α therapy did not increase the risks, such as congenital malformation or abortion during pregnancy, in patients with inflammatory …

The intestinal cell types responsible for defense against pathogenic organisms remain incompletely characterized. Here we identify a subset of CD11c(hi)CD11b(hi) lamina propria dendritic cells (LPDCs) that expressed Toll-like receptor 5 (TLR5) in the small intestine. When stimulated by the TLR5 ligand flagellin, TLR5(+) LPDCs induced the differentiation of naive B cells into immunoglobulin A-producing plasma cells by a mechanism independent of gut-associated lymphoid tissue. In addition, by a mechanism dependent on TLR5 stimulation, these LPDCs promoted the differentiation of antigen-specific interleukin 17-producing T helper cells and type 1 T helper cells. Unlike spleen DCs, the LPDCs specifically produced retinoic acid, which, in a dose-dependent way, supported the generation and retention of immunoglobulin A-producing cells in the lamina propria and positively regulated the differentiation interleukin 17-producing T helper cells. Our findings demonstrate unique properties of LPDCs and the importance of TLR5 for adaptive immunity in the intestine.