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Parasitic worms affect gut microbes Improved hygiene practices in high-income countries may come with an increased risk of developing inflammatory bowel disease (IBD) or other similar disorders. Ramanan et al. show that intestinal helminth infection, caused by parasitic worms, protects IBD-susceptible mice from developing the disease. The infection increases specific protective species and limits other inflammatory members of the microbiota. People from helminth-endemic regions harbored a similar protective microbiota, and their deworming led to an increase in inflammatory Bacteroidales species, similar to what the authors observed in the mice. Thus, a changing microbial environment may shape susceptibility to inflammatory disease. Science , this issue p. 608

Scardovia inopinata JCM 12537 T was isolated from human dental caries. Here, we report the complete genome sequence of this organism. This paper is the first report to demonstrate the fully sequenced and completely annotated genome of an S. inopinata strain.

Bifidobacterium angulatum JCM 7096(T) was isolated from human feces. Here we report the complete genome sequence of this organism. This paper is the first report demonstrating the fully sequenced and completely annotated genome of a B. angulatum strain.

Bifidobacterium pseudocatenulatum JCM 1200(T) was isolated from infant feces. This paper is the first report demonstrating the fully sequenced and completely annotated genome of a B. pseudocatenulatum strain.

Parascardovia denticolens JCM 12538 T was isolated from human dental caries. Here, we report the complete genome sequence of this organism. This paper is the first report demonstrating the completely sequenced and assembled genome of P. denticolens .

The recognition of microbial components by Toll-like receptors (TLRs) initiates signal transduction pathways, which trigger the expression of a series of target genes. It has been reported that TLR signaling is enhanced by cytokines such as IFN-γ, but the mechanisms underlying this enhancement remain unclear. The MyD88 adaptor, which is essential for signaling by many TLRs, recruits members of the IFN regulatory factor (IRF) family of transcription factors, such as IRF5 and IRF7, to evoke the activation of TLR target genes. In this study we demonstrate that IRF1, which is induced by IFN-γ, also interacts with and is activated by MyD88 upon TLR activation. We provide evidence that MyD88-associated IRF1 migrates into the nucleus more efficiently than non-MyD88-associated IRF1 and that this IRF1 selectively participates in the TLR-dependent gene induction program. The critical role of MyD88-dependent “IRF1 licensing” is underscored by the observation that the induction of a specific gene subset downstream of the TLR–MyD88 pathway, such as IFN-β, inducible NO synthase, and IL-12p35, are impaired inIrf1-deficient cells. Thus, our present study places IRF1 as an additional member participating in MyD88 signaling and provides a mechanistic insight into the enhancement of the TLR-dependent gene induction program by IFN-γ.

The type I interferon (IFN-α/β) gene family was identified about a quarter of century ago as the prototype of many cytokine gene families, and the gene induction mechanism for IFN-α/β was extensively studied in the context of host defense against viral infections. More recently, much attention has been focused on the induction and function of the IFN-α/β system regulated by Toll-like receptors (TLRs), which are critical for linking the innate and adaptive immunities. The understanding of mechanisms underlying IFN-α/β gene induction by TLRs is therefore an emerging theme, for which new insights have been gained over the past few years. We summarise recent progress on the role of interferon regulatory factors (IRFs) in the induction of IFN-α/β genes in normal cells and plasmacytoid dendritic cells (pDCs) that are specialized to produce IFN-α/β at very high levels upon stimulation by TLR9 and its subfamily members.

Robust type-I interferon (IFN-alpha/beta) induction in plasmacytoid dendritic cells, through the activation of Toll-like receptor 9 (TLR9), constitutes a critical aspect of immunity. It is absolutely dependent on the transcription factor IRF-7, which interacts with and is activated by the adaptor MyD88. How plasmacytoid dendritic cells, but not other cell types (such as conventional dendritic cells), are able to activate the MyD88-IRF-7-dependent IFN induction pathway remains unknown. Here we show that the spatiotemporal regulation of MyD88-IRF-7 signalling is critical for a high-level IFN induction in response to TLR9 activation. The IFN-inducing TLR9 ligand, A/D-type CpG oligodeoxynucleotide (CpG-A), is retained for long periods in the endosomal vesicles of plasmacytoid dendritic cells, together with the MyD88-IRF-7 complex. However, in conventional dendritic cells, CpG-A is rapidly transferred to lysosomal vesicles. We further show that conventional dendritic cells can also mount a robust IFN induction if CpG-A is manipulated for endosomal retention using a cationic lipid. This strategy also allows us to demonstrate endosomal activation of the IFN pathway by the otherwise inactive TLR9 ligand B/K-type oligodeoxynucleotide (CpG-B). Thus, our study offers insights into the regulation of TLR9 signalling in space, potentially suggesting a new avenue for therapeutic intervention.

The activation of Toll-like receptors (TLRs) is central to innate and adaptive immunity. All TLRs use the adaptor MyD88 for signalling, but the mechanisms underlying the MyD88-mediated gene induction programme are as yet not fully understood. Here, we demonstrate that the transcription factor IRF-5 is generally involved downstream of the TLR-MyD88 signalling pathway for gene induction of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-12 and tumour-necrosis factor-alpha. In haematopoietic cells from mice deficient in the Irf5 gene (Irf5-/- mice), the induction of these cytokines by various TLR ligands is severely impaired, whereas interferon-alpha induction is normal. We also provide evidence that IRF-5 interacts with and is activated by MyD88 and TRAF6, and that TLR activation results in the nuclear translocation of IRF-5 to activate cytokine gene transcription. Consistently, Irf5-/- mice show resistance to lethal shock induced by either unmethylated DNA or lipopolysaccharide, which correlates with a marked decrease in the serum levels of proinflammatory cytokines. Thus, our study identifies IRF-5 as a new, principal downstream regulator of the TLR-MyD88 signalling pathway and a potential target of therapeutic intervention to control harmful immune responses.

Toll-like receptor (TLR) activation is central to immunity, wherein the activation of the TLR9 subfamily members TLR9 and TLR7 results in the robust induction of type I IFNs (IFN-α/β) by means of the MyD88 adaptor protein. However, it remains unknown how the TLR signal “input” can be processed through MyD88 to “output” the induction of the IFN genes. Here, we demonstrate that the transcription factor IRF-7 interacts with MyD88 to form a complex in the cytoplasm. We provide evidence that this complex also involves IRAK4 and TRAF6 and provides the foundation for the TLR9-dependent activation of the IFN genes. The complex defined in this study represents an example of how the coupling of the signaling adaptor and effector kinase molecules together with the transcription factor regulate the processing of an extracellular signal to evoke its versatile downstream transcriptional events in a cell. Thus, we propose that this molecular complex may function as a cytoplasmic transductional-transcriptional processor.

The discovery that lymphotoxin alpha (LTalpha) knockout mice lack peripheral lymphoid tissues reformed the study of organogenesis of peripheral lymphoid tissues from a research field that was solely descriptive and dependent on histological methods to one requiring all modern technologies. The concepts of inducer cells for organogenesis of peripheral lymphoid tissues as a separate hematopoietic lineage and of mesenchymal organizer cells have been established through this progress. These discoveries led to the comprehension of the basic framework of the events during organogenesis of peripheral lymphoid tissues. However, many important questions remain unanswered. This review discusses those questions which have arisen from our studies on the organogenesis of Peyer's patches.

A complex mechanism may be operational for dendritic cell (DC) maturation, wherein Toll-like receptor and other signaling pathways may be coordinated differently depending on the nature of the pathogens, in order for DC maturation to be most effective to a given threat. Here, we show that IFN-α/β signaling is selectively required for the maturation of DCs induced by double-stranded RNA or viral infection in vitro . Interestingly, the maturation is still observed in the absence of either of the two target genes of IFN-α/β, TLR3 and PKR (double-stranded-RNA-dependent protein kinase R), indicating the complexity of the IFN-α/β-induced transcriptional program in DCs. We also show that the DCs stimulated in vivo by these agents can migrate into the T cell zone of the spleen but fail to mature without the IFN signal. The immune system may have acquired the selective utilization of this cytokine system, which is essential for innate antiviral immunity, to effectively couple with the induction of adaptive immunity.

Type I interferons (IFN-α/β) affect many aspects of immune responses. Many pathogen-associated molecules, including bacterial lipopolysaccharide (LPS) and virus-associated double-stranded RNA, induce IFN gene expression through activation of distinct Toll-like receptors (TLRs). Although much has been studied about the activation of the transcription factor IRF-3 and induction of IFN - β gene by the LPS-mediated TLR4 signaling, definitive evidence is missing about the actual role of IRF-3 in LPS responses in vitro and in vivo. Using IRF-3 deficient mice, we show here that IRF-3 is indeed essential for the LPS-mediated IFN - β gene induction. Loss of IRF-3 also affects the expression of profile of other cytokine/chemokine genes. We also provide evidence that the LPS/TLR4 signaling activates IRF-7 to induce IFN - β , if IRF-7 is induced by IFNs prior to LPS simulation. Finally, the IRF-3 -deficient mice show resistance to LPS-induced endotoxin shock. These results place IRF-3 as a molecule central to LPS/TLR4 signaling.

Emerging evidence has suggested a potential impact of gut microbiota on the pathophysiology of heart failure (HF). However, it is still unknown whether HF is associated with dysbiosis in gut microbiota. We investigated the composition of gut microbiota in patients with HF to elucidate whether gut microbial dysbiosis is associated with HF. We performed 16S ribosomal RNA gene sequencing of fecal samples obtained from 12 HF patients and 12 age-matched healthy control (HC) subjects, and analyzed the differences in gut microbiota. We further compared the composition of gut microbiota of 12 HF patients younger than 60 years of age with that of 10 HF patients 60 years of age or older. The composition of gut microbial communities of HF patients was distinct from that of HC subjects in both unweighted and weighted UniFrac analyses. Eubacterium rectale and Dorea longicatena were less abundant in the gut microbiota of HF patients than in that of HC subjects. Compared to younger HF patients, older HF patients had diminished proportions of Bacteroidetes and larger quantities of Proteobacteria. The genus Faecalibacterium was depleted, while Lactobacillus was enriched in the gut microbiota of older HF patients. These results suggest that patients with HF harbor significantly altered gut microbiota, which varies further according to age. New concept of heart-gut axis has a great potential for breakthroughs in the development of novel diagnostic and therapeutic approach for HF.

Periodontitis is linked to polymicrobial interactions and the presence of Porphyromonas gingivalis. In this issue of Cell Host & Microbe, Hajishengallis et al. (2011) demonstrate that P. gingivalis colonization in the oral cavity changes the composition of the oral commensal microbiota and accelerates microbiota-mediated bone-destructive periodontitis, indicating that this single, low-abundance species is a keystone in periodontal disease.

Mice deficient in lymphotoxin beta receptor (LTbetaR) or interleukin 7 receptor alpha (IL-7Ralpha) lack Peyer's patches (PPs). Deficiency in CXC chemokine receptor 5 (CXCR5) also severely affects the development of PPs. A molecular network involving these three signaling pathways has been implicated in PP organogenesis, but it remains unclear how they are connected during this process. We have shown that PP organogenesis is initiated at sites containing IL-7Ralpha(+) lymphoid cells and vascular cell adhesion molecule (VCAM)-1/intercellular adhesion molecule (ICAM)-1 expressing nonlymphoid elements. Here we characterize these lymphoid and nonlymphoid components in terms of chemokine signals. The lymphoid population expresses CXCR5 and has a strong chemotactic response to B lymphocyte chemoattractant (BLC). Importantly, chemokines produced by VCAM-1(+)ICAM-1(+) nonlymphoid cells mediate the recruitment of lymphoid cells. Furthermore, we show that these VCAM-1(+)ICAM-1(+) cells are mesenchymal cells that are activated by lymphoid cells through the LTbetaR to express adhesion molecules and chemokines. Thus, promotion of PP development relies on mutual interaction between mesenchymal and lymphoid cells.

Introduction In allogeneic stem cell transplantation (allo-SCT), recent reports have shown that post-transplant microbial diversity of the gastrointestinal tract is closely associated with clinical outcomes. Furthermore, pre-transplant microbial status has also been associated with the development of acute graft-versus-host disease (GVHD). These results indicate that pre-transplant microbial ecosystem may influence the immune system after allo-SCT, induce alloimmune response such as GVHD, and may finally affect the transplant outcome. Thus, we evaluate the association between pre-transplant microbial diversity and transplant outcomes. Patients and Methods Between April 2013 and March 2015, fecal samples were obtained from 107 patients in Komagome hospital. Fecal samples were collected within 2 weeks before conditioning. Microbial analysis was performed using 16S rRNA gene (hypervariable V1-2 region) sequencing. Operational taxonomic units (OTU)-based microbial diversity was estimated by calculating the Shannon index. Patients were classified into 3 groups based on the diversity index: low (3) diversity group, and we evaluated transplant outcomes such as survival, non-relapse mortality, and the cumulative incidence of GVHD, in these 3 groups. Results Among the 107 patients, there were 18 (16.8%) patients in the low diversity group, 48 (44.9%) in the intermediate diversity group, and 41 (38.3%) in the high diversity group. The median age of all patients was 49 (range: 16-72) years. The median follow-up period for survivors was 597 days (range, 23-1,046 days). We found no significant differences among 3 groups in terms of age, gender, underlying disease, disease status, performance status, hematopoietic cell transplantation comorbidity index, GVHD prophylaxis, conditioning regimen, stem cell sources, and human leukocyte antigen disparity. However, the patients in the low diversity group received intravenous antibiotics just before the conditioning more frequently compared to the patients in the intermediate /high diversity groups. The patients in the low diversity group showed poor survival (p=0.37, Figure 1a), higher non-relapse mortality (p=0.25, Figure 1b) and higher incidence of grade II-VI gastrointestinal GVHD (p=0.77, Fgiure 1c) compared with the patients in the intermediate /high diversity group although these results were not statistically significant. The cumulative incidence of relapse was similar between 3 groups. When compared between patients with and without acute GVHD, the composition of microbiota did not differ significantly between these patients. Discussion and conclusion Our results indicate that pre-transplant microbial diversity did not affect transplant outcomes. Furthermore, in contrast to previous report, the microbial composition was also irrelevant to the development of GVHD. Bacterial translocation after allo-SCT is a key trigger for the development of acute GVHD. Thus, microbial status in this period would be more important than that before allo-SCT. However, the patients in the low diversity group tended to show poor survival, higher incidence of non-relapse mortality and GVHD. Thus, further evaluation with larger sample size might be necessary to confirm the association between pre-transplant microbial ecosystem and transplant outcomes. Disclosures No relevant conflicts of interest to declare.

Th17 cells constitute a subset of activated CD4(+) T cells, characterized by their production of interleukin (IL)-17, IL-17F, and IL-22, that play a critical role in host defence against extracellular pathogens. An intriguing feature of these cells is their selective and constitutive presence in the intestinal lamina propria. The development of intestinal Th17 cells is controlled by intestinal commensal bacteria. Recently, segmented filamentous bacterium (SFB) was identified as a specific bacterial taxon that promotes Th17 differentiation in the small intestine of mice. We discuss the recent advances in our understanding of the mechanism of intestinal Th17 synthesis and its potential implications for the treatment of inflammatory bowel diseases.

Summary Foxp3 + T cells play a critical role for the maintenance of immune tolerance. Here we show that in mice, Foxp3 + T cells contributed to diversification of gut microbiota, particularly of species belonging to Firmicutes. The control of indigenous bacteria by Foxp3 + T cells involved regulatory functions both outside and inside germinal centers (GCs), consisting of suppression of inflammation and regulation of immunoglobulin A (IgA) selection in Peyer's patches, respectively. Diversified and selected IgAs contributed to maintenance of diversified and balanced microbiota, which in turn facilitated the expansion of Foxp3 + T cells, induction of GCs, and IgA responses in the gut through a symbiotic regulatory loop. Thus, the adaptive immune system, through cellular and molecular components that are required for immune tolerance and through the diversification as well as selection of antibody repertoire, mediates host-microbial symbiosis by controlling the richness and balance of bacterial communities required for homeostasis.

Intestinal regulatory T cells (Treg cells) are necessary for the suppression of excessive immune responses to commensal bacteria. However, the molecular machinery that controls the homeostasis of intestinal Treg cells has remained largely unknown. Here we report that colonization of germ-free mice with gut microbiota upregulated expression of the DNA-methylation adaptor Uhrf1 in Treg cells. Mice with T cell-specific deficiency in Uhrf1 (Uhrf1(fl/fl)Cd4-Cre mice) showed defective proliferation and functional maturation of colonic Treg cells. Uhrf1 deficiency resulted in derepression of the gene (Cdkn1a) that encodes the cyclin-dependent kinase inhibitor p21 due to hypomethylation of its promoter region, which resulted in cell-cycle arrest of Treg cells. As a consequence, Uhrf1(fl/fl)Cd4-Cre mice spontaneously developed severe colitis. Thus, Uhrf1-dependent epigenetic silencing of Cdkn1a was required for the maintenance of gut immunological homeostasis. This mechanism enforces symbiotic host-microbe interactions without an inflammatory response.

Nitric oxide (NO) may either protect against or contribute to inflammatory lung injury. In this study we investigated whether inhalation of 20 ppm NO alters tyrosine nitration, and we assessed the degree of lung inflammation and edema in rats after lipopolysaccharide (LPS) instillation. The amount of nitrotyrosine relative to the total amount of tyrosine was measured in lung homogenates, and lung tissue sections were stained for nitrotyrosine and aminotyrosine (a reduced form of nitrotyrosine). Leukocytes in bronchoalveolar lavage fluid (BALF) were counted, and myeloperoxidase activity was measured in lung homogenate. Lung edema and inflammatory cell accumulation in lung tissue were estimated by extravascular lung water weight (EVLW) and extravascular dry lung weight (EVDW), respectively. LPS instillation caused increases in nitrotyrosine concentration and immunohistochemical staining of nitrotyrosine and aminotyrosine in the lungs. LPS instillation increased the BALF leukocyte count, myeloperoxidase activity in lung tissue, and both EVLW and EVDW. Inhalational exposure to 20 ppm NO induced nitrotyrosine and aminotyrosine formation only in bronchial epithelial cell surface of the lungs not instilled with LPS. NO inhalation reduced the increases in nitrotyrosine and aminotyrosine in LPS-instilled lung tissue as well as the leukocyte count in BALF and myeloperoxidase activity in lung tissue, but it did not significantly change EVLW or EVDW. Leukocyte depletion in LPS-instilled rats reduced interstitial inflammatory cells, which were stained with nitrotyrosine and aminotyrosine, and attenuated the nitrotyrosine staining of alveolar capillaries. These results suggest that inhalation of 20 ppm NO reduces leukocyte accumulation in the lungs and inhibits tyrosine nitration caused by LPS instillation.

Gene-knock-out studies implicate roles of lymphotoxin (LT) alphabeta and LT betaR in the initial phase of Peyer's patch (PP) organogenesis. We recently identified the requirement of IL-7R alpha/gamma c/Jak3 signal in LT alphabeta production of IL-7R alpha+ cells. These observations lead us to a hypothetical model for PP organogenesis with three cellular components. The first is the producer of the ligand for IL-7R alpha, which then stimulate the IL-7R alpha+ cells to produce LT alphabeta activating the LT betaR+ cells to form an organizing center for PP organogenesis. This model is similar to that of inflammation, suggesting that PP organogenesis is a programmed version of inflammation.

Bifidobacterium catenulatum JCM 1194(T) was isolated from human feces. This paper is the first report demonstrating the fully sequenced and completely annotated genome of a B. catenulatum strain.

Bifidobacterium bifidum JCM 1255 T was isolated from feces of a breast-fed infant. Here we report the complete genome sequence of this organism.

A relationship between the microbiota and GVHD has long been suspected but is still not well understood. Recently, several studies have indicated that obligately anaerobic commensal intestinal bacteria may be beneficial for intestinal health. Recent studies have found that obligate anaerobes in the intestine, in particular Clostridial species, are important mediators of intestinal homeostasis and prevent inflammation by upregulating intestinal regulatory T cells (Tregs). In this study we sought to further characterize the relationship between the microbiota and GVHD, focusing on the effects of antibiotics (ABX) with anaerobic coverage. First, we treated healthy 129S1 mice with either ABX that included significant activity against anaerobic bacteria (piperacillin-tazobactam; pip-tazo, imipenem-cilastatin; imipenem or ones with reduced activity (cefepime, aztreonam). Mice were treated by subcutaneous injections of each antibiotic twice a day for two days (500 mg/kg for pip-tazo and 100 mg/kg for others) and stool samples were collected, followed by 16S rRNA gene amplification and sequence analysis. We found that treatment with pip-tazo and imipenem significantly reduced the abundance of anaerobic Clostridial species and increased that of Enterococcus, while treatment with cefepime and aztreonam spared Clostridiales (Figure 1A). We next investigated the effects of antibiotic treatment in a clinically relevant MHC matched, minor antigen mismatched murine allogeneic BMT model. Lethally irradiated 129S1 recipients were transplanted with C57BL/6 T cell depleted bone marrow cells and 2 × 106 C57BL/6 T cells. Recipients were either treated with imipenem or aztreonam subcutaneously beginning on day +10 after BMT. We observed significantly worsened GVHD survival in imipenem-treated recipients (Figure 1B, P We have also retrospectively evaluated the impact of antibiotic spectrum of activity on clinical GVHD-related mortality. We examined a cohort of 546 adult patients transplanted at MSKCC from 1992 to 2013 who received conventional (non-T cell depleted) grafts and received treatment for peri-transplant fever with empiric ABX classified as either including anaerobic coverage or with reduced anaerobic activity. Of these, 156 patients who received ABX from both classifications were excluded, as were 99 patients exposed to other ABX with anaerobic coverage not routinely used to treat peri-transplant fever. We examined the impact of anaerobic coverage in the remaining 291 patients. Therapies for peri-transplant fever were divided into those that included significant activity against anaerobic bacteria (pip-tazo, ticarcillin-clavulanate, imipenem, meropenem), and those with reduced activity (ceftriaxone, ceftazidime, cefepime, aztreonam). Additional ABX that have anaerobic activity but were not routinely used to treat peri-transplant fever included metronidazole, oral vancomycin and clindamycin. We found that 60 patients that received ABX without anaerobic coverage had a significantly reduced incidence of GVHD-related mortality, compared to 231 patients treated with anaerobe-active ABX (Figure 2, P Taken together, our results suggest that a population of anaerobic commensals may mediate protection against life-threatening GVHD and that selecting ABX with a more limited spectrum of activity can prevent microbiota injury and reduce GVHD. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

We investigated the role of IL-7 receptor alpha (IL-7Ralpha) signal in the formation of Peyer's patch (PP) anlage. Although pan-lymphopenia is a common phenotype of rag2-/- and il7ralpha-/- mice, a close inspection revealed nodules corresponding to PP in the adult rag2-/- but not in the il7ralpha-/- mouse. In our previous study, three histologically distinct steps in the formation of PP were identified. The first is the appearance of VCAM-1 + spots in the intestine, which probably represents an initial stage of the formation of the PP anlage. Accumulation of cells bearing IL-7Ralpha, CD4 or Ia in this region then follows and eventually entry of mature lymphocytes expressing CD3 or B220 occurs just before birth. Based on this criterion, we next investigated which of these events is defective in mice with severe combined immunodeficiency. Formation of VCAM-1 + spots and cluster formation of IL-7Ralpha+ cells proceed normally in the rag2-/- mouse which completely lacks mature lymphocytes. In contrast, no VCAM-1+ spots were detected in the embryonic nor neonatal il7ralpha-/- mice, suggesting that IL-7Ralpha signal is involved in the early phase of PP anlage formation. The same defect was found in the jak3-/- mouse. In addition to the appearance of VCAM1+ spots, the clustering of IL-7Ralpha+ cells was absent in the jak3-/- mouse, though IL-7Ralpha+ cells are found to scatter over the intestine. These results indicate that IL-7Ralpha is an essential signal for an early step of PP anlage formation, without which the subsequent processes cannot be initiated.

Gut commensal microbes shape the mucosal immune system by regulating the differentiation and expansion of several types of T cell. Clostridia, a dominant class of commensal microbe, can induce colonic regulatory T (Treg) cells, which have a central role in the suppression of inflammatory and allergic responses. However, the molecular mechanisms by which commensal microbes induce colonic Treg cells have been unclear. Here we show that a large bowel microbial fermentation product, butyrate, induces the differentiation of colonic Treg cells in mice. A comparative NMR-based metabolome analysis suggests that the luminal concentrations of short-chain fatty acids positively correlates with the number of Treg cells in the colon. Among short-chain fatty acids, butyrate induced the differentiation of Treg cells in vitro and in vivo, and ameliorated the development of colitis induced by adoptive transfer of CD4(+) CD45RB(hi) T cells in Rag1(-/-) mice. Treatment of naive T cells under the Treg-cell-polarizing conditions with butyrate enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus, suggesting a possible mechanism for how microbial-derived butyrate regulates the differentiation of Treg cells. Our findings provide new insight into the mechanisms by which host-microbe interactions establish immunological homeostasis in the gut.

Clostridium species belonging to clusters XIVa and IV are predominant organisms in the gut microbiota. They are known to affect various aspects of host biology, including the optimal breakdown of foods, competition with pathogenic microorganisms, maintenance of the intestinal epithelial barrier, and immune system development. We have recently shown that a defined mixture of 46 strains of mouse-derived Clostridium species belonging to clusters XIVa and IV affects the development and function of colonic Foxp3+ CD4+ regulatory T (Treg) cells in mice. Clostridium-induced Treg cells are likely to play a role in the maintenance of immune homeostasis in the gut.

Interleukin-22 (IL-22) is central to host protection against bacterial infections at barrier sites. Both innate lymphoid cells (ILCs) and T cells produce IL-22. However, the specific contributions of CD4+ T cells and their developmental origins are unclear. We found that the enteric pathogen Citrobacter rodentium induced sequential waves of IL-22 producing ILCs and CD4+ T cells that were each critical to host defense during a primary infection. Whereas IL-22 production by ILCs was strictly IL-23–dependent, development of IL-22 producing CD4+ T cells occurred via an IL-6–dependent mechanism that was augmented by, but not dependent on, IL-23, and was dependent on both transcription factors T-bet and AhR. Transfer of CD4+ T cells differentiated with IL-6 in the absence of TGF-β (“Th22” cells) conferred protection of infected IL-22-deficient mice whereas transferred Th17 cells did not. These findings establish Th22 cells as an important component of mucosal anti-microbial host defense.

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.

Summary Mouse and human dendritic cells (DCs) are composed of functionally specialized subsets, but precise interspecies correlation is currently incomplete. Here, we showed that murine lung and gut lamina propria CD11b+ DC populations were comprised of two subsets: FLT3- and IRF4-dependent CD24+CD64− DCs and contaminating CSF-1R-dependent CD24−CD64+ macrophages. Functionally, loss of CD24+CD11b+ DCs abrogated CD4+ T cell-mediated interleukin-17 (IL-17) production in steady state and after Aspergillus fumigatus challenge. Human CD1c+ DCs, the equivalent of murine CD24+CD11b+ DCs, also expressed IRF4, secreted IL-23, and promoted T helper 17 cell responses. Our data revealed heterogeneity in the mouse CD11b+ DC compartment and identifed mucosal tissues IRF4-expressing DCs specialized in instructing IL-17 responses in both mouse and human. The demonstration of mouse and human DC subsets specialized in driving IL-17 responses highlights the conservation of key immune functions across species and will facilitate the translation of mouse in vivo findings to advance DC-based clinical therapies., Highlights • Mucosal CD11b+ DCs consist of CD24+CD64− DCs and CD24−CD64+ macrophages • Mucosal CD24+CD11b+ DCs are IRF4-dependent • IRF4-dependent CD24+CD11b+ DCs secrete IL-23α and control mucosal IL-17 responses • Human CD1c+CD11b+ DCs are functional homologs of murine CD24+CD11b+ DCs

Obesity has become more prevalent in most developed countries over the past few decades, and is increasingly recognized as a major risk factor for several common types of cancer. As the worldwide obesity epidemic has shown no signs of abating, better understanding of the mechanisms underlying obesity-associated cancer is urgently needed. Although several events were proposed to be involved in obesity-associated cancer, the exact molecular mechanisms that integrate these events have remained largely unclear. Here we show that senescence-associated secretory phenotype (SASP) has crucial roles in promoting obesity-associated hepatocellular carcinoma (HCC) development in mice. Dietary or genetic obesity induces alterations of gut microbiota, thereby increasing the levels of deoxycholic acid (DCA), a gut bacterial metabolite known to cause DNA damage. The enterohepatic circulation of DCA provokes SASP phenotype in hepatic stellate cells (HSCs), which in turn secretes various inflammatory and tumour-promoting factors in the liver, thus facilitating HCC development in mice after exposure to chemical carcinogen. Notably, blocking DCA production or reducing gut bacteria efficiently prevents HCC development in obese mice. Similar results were also observed in mice lacking an SASP inducer or depleted of senescent HSCs, indicating that the DCA-SASP axis in HSCs has key roles in obesity-associated HCC development. Moreover, signs of SASP were also observed in the HSCs in the area of HCC arising in patients with non-alcoholic steatohepatitis, indicating that a similar pathway may contribute to at least certain aspects of obesity-associated HCC development in humans as well. These findings provide valuable new insights into the development of obesity-associated cancer and open up new possibilities for its control.

The large intestinal epithelial cells and immune cells are exposed to a variety of molecules derived from commensal microbiota that can activate innate receptors, such as Toll-like receptors (TLRs) and retinoic acid-inducible gene-I-like receptors (RLRs). Although the activation of these receptors is known to be critical for homeostasis of the large intestine, the underlying gene regulatory mechanisms are not well understood. Here, we show that IFN regulatory factor (IRF)3 is critical for the suppression of dextran sulfate sodium-induced colitis. IRF3-deficient mice exhibited lethal defects in the inflammatory and recovery phases of the colitis, accompanied by marked defects in the gene induction for thymic stromal lymphopoietin (TSLP), a cytokine known to be essential for protection of the large intestine. We further provide evidence that DNA and RNA of the large intestinal contents are critical for Tslp gene induction via IRF3 activation by cytosolic nucleic acid receptors. We also demonstrate that IRF3 indeed activates the gene promoter of Tslp via IRF-binding sequences. This newly identified intestinal gene regulatory mechanism, wherein IRF3 activated by microbiota-derived nucleic acids plays a critical role in intestinal homeostasis, may have clinical implication in colonic inflammatory disorders.

Nat. Immunol. 17, 505–513 (2016); published online 21 March 2016; corrected after print 26 August 2016 In the version of this article initially published, the middle initial (C) for the sixteenth author was incorrect. The correct author name should be Thomas M. Schmidt (and the corresponding initials in the Author Contributions section should be T.

IL-21 is the signature cytokine produced by T follicular helper (Tfh) cells and is vital in driving both the germinal centre (GC) reaction and formation of high affinity antibodies. However, the degree of Tfh cell heterogeneity and function is not fully understood. By utilising a novel IL-21eGFP reporter mouse carrying a diphtheria toxin receptor (DTR)-enhanced green fluorescent protein (eGFP) fusion gene, we identified a subpopulation of highly differentiated Tfh cells within Peyer’s Patches (PPs). This subpopulation demonstrated highest expression of the key Tfh molecules - Bcl6, ICOS and IL-21, and was found within germinal centres and surrounding B cell areas. TCRb repertoire analysis of GFP+Tfh cells revealed that these were polyclonal and closely related to GFP− Tfh cells, indicating selection by common antigens. In vitro stimulation of IL-21eGFP CD4+ cells in the presence of IL-6, TGFb and retinoic acid led to the induction of GFP+ cells that did not co-express IL-17 or Foxp3. Treatment of IL-21eGFP mice with antibiotics led to an ablation of PP GFP+CD4+ cells meaning that the presence of these cells relies on an intact bacterial microbiota. Furthermore, both polyclonal CD4+ T cell and B cell activation is necessary to support the differentiation of GFP+Tfh cells. Finally, GFP+ cell depletion resulted in a reduced frequency of PP GC B cells and IgG1+ cells and small but significant shifts in gut microbiome composition. Therefore, using a new IL-21-reporter mouse we have identified a subpopulation of Tfh cells induced by the gut microbiota and necessary for optimal PP GC and IgG1 responses.

TCRαβ thymocytes differentiate into either CD8αβ(+) cytotoxic T lymphocytes or CD4(+) helper T cells. This functional dichotomy is controlled by key transcription factors, including the helper T cell master regulator ThPOK, which suppresses the cytolytic program in major histocompatibility complex (MHC) class II-restricted CD4(+) thymocytes. ThPOK continues to repress genes of the CD8 lineage in mature CD4(+) T cells, even as they differentiate into effector helper T cell subsets. Here we found that the helper T cell fate was not fixed and that mature, antigen-stimulated CD4(+) T cells terminated expression of the gene encoding ThPOK and reactivated genes of the CD8 lineage. This unexpected plasticity resulted in the post-thymic termination of the helper T cell program and the functional differentiation of distinct MHC class II-restricted CD4(+) cytotoxic T lymphocytes.

Manipulation of the gut microbiota holds great promise for the treatment of inflammatory and allergic diseases. Although numerous probiotic microorganisms have been identified, there remains a compelling need to discover organisms that elicit more robust therapeutic responses, are compatible with the host, and can affect a specific arm of the host immune system in a well-controlled, physiological manner. Here we use a rational approach to isolate CD4(+)FOXP3(+) regulatory T (Treg)-cell-inducing bacterial strains from the human indigenous microbiota. Starting with a healthy human faecal sample, a sequence of selection steps was applied to obtain mice colonized with human microbiota enriched in Treg-cell-inducing species. From these mice, we isolated and selected 17 strains of bacteria on the basis of their high potency in enhancing Treg cell abundance and inducing important anti-inflammatory molecules--including interleukin-10 (IL-) and inducible T-cell co-stimulator (ICOS)--in Treg cells upon inoculation into germ-free mice. Genome sequencing revealed that the 17 strains fall within clusters IV, XIVa and XVIII of Clostridia, which lack prominent toxins and virulence factors. The 17 strains act as a community to provide bacterial antigens and a TGF-β-rich environment to help expansion and differentiation of Treg cells. Oral administration of the combination of 17 strains to adult mice attenuated disease in models of colitis and allergic diarrhoea. Use of the isolated strains may allow for tailored therapeutic manipulation of human immune disorders.

Foxp3+ CD4+ cells are prominent immune regulatory T (Treg) cells that are most abundant in the intestine. Recent studies have suggested that intestinal Treg cells consist of thymically and extrathymically developed cells that have unique characteristics. A fraction of intestinal Treg cells express T cell receptors that recognize antigens that are derived from the gut microbiota. The presence of the gut microbiota, particularly the Clostridium species, affects the development and function of Treg cells. These intestinal bacteria-induced Treg cells are likely to play a role in the tolerance toward the gut microbiota. These recent advances provide new insight into how T cells are educated in the intestine to maintain homeostasis with the gut microbiota.

Abnormal prion protein (PrP Sc) generated from the cellular isoform of PrP (PrP C) is assumed to be the main or sole component of the pathogen, called prion, of transmissible spongiform encephalopathies (TSE). Because PrP is a host-encoded protein, acquired immune responses are not induced in TSE. Meanwhile, activation of the innate immune system has been suggested to partially block the progression of TSE; however, the mechanism is not well understood. To further elucidate the role of the innate immune system in prion infection, we investigated the function of interferon regulatory factor 3 (IRF3), a key transcription factor of the MyD88-independent type I interferon (IFN) production pathway. We found that IRF3-deficient mice exhibited significantly earlier onset with three murine TSE strains, namely, 22L, FK-1, and murine bovine spongiform encephalopathy (mBSE), following intraperitoneal transmission, than with wild-type controls. Moreover, overexpression of IRF3 attenuated prion infection in the cell culture system, while PrP Sc was increased in prion-infected cells treated with small interfering RNAs (siRNAs) against IRF3, suggesting that IRF3 negatively regulates PrP Sc formation. Our findings provide new insight into the role of the host innate immune system in the pathogenesis of prion diseases., Journal of Virology, 86(9), pp.4947-4955; 2012

The mammalian alimentary tract harbors hundreds of species of commensal microorganisms (microbiota) that intimately interact with the host and provide it with genetic, metabolic, and immunological attributes. Recent reports have indicated that the microbiota composition and its collective genomes (microbiome) are major factors in predetermining the type and robustness of mucosal immune responses. In this review, we discuss the recent advances in our understanding of host-microbiota interactions and their effect on the health and disease susceptibility of the host.

The human intestinal mucosa is constantly exposed to commensal microbiota. Since the gut microbiota is beneficial to the host, hosts have evolved intestine-specific immune systems to co-exist with the microbiota. On the other hand, the intestinal microbiota actively regulates the host's immune system, and recent studies have revealed that specific commensal bacterial species induce the accumulation of specific immune cell populations. For instance, segmented filamentous bacteria and Clostridium species belonging to clusters XIVa and IV induce the accumulation of Th17 cells in the small intestine and Foxp3(+) regulatory T cells in the large intestine, respectively. The immune cells induced by the gut microbiota likely contribute to intestinal homeostasis and influence systemic immunity in the host.

The addition of KO2 to ferrous cytochrome P450cam in the presence of d-camphor under anaerobic condition resulted in the formation of exo- and endo-5-hydroxycamphor. The product formation was inhibited by the addition of superoxide dismutase, not catalase. The ferrous form of P450cam reacts with O−2 to form an intermediate species of the enzyme, of which spectrum is closely similar to that of the oxygenated form of P450cam. Subsequently, the intermediate was converted to native ferric P450cam. In contrast, ferrous form of horseradish peroxidase reacted with O−2 to form Compound I of the enzyme without detection of intermediates.

The gastrointestinal tract represents a major gateway for infection by potential microbial pathogens, and, at the same time, contains hundreds of species of indigenous microbes. Thus, the intestinal immune system should actively react with potentially pathogenic microbes, while simultaneously remaining tolerant to or unaffected by the vast majority of commensal microbes. The recognition, and perhaps the discrimination, of pathogens and commensal microbes is primarily mediated by pathogen-associated molecular pattern (PAMP) receptors, including Toll-like receptors (TLRs) and nucleotide-binding domain (NOD)-like receptor (NLR) family proteins. The regulation of PAMP receptor signaling is critical in the maintenance of immune homeostasis in the intestine, and an aberrant signaling may result in inflammatory bowel diseases.

The activation of innate immune responses by nucleic acids is central to the generation of host responses against pathogens; however, nucleic acids can also trigger the development and/or exacerbation of pathogenic responses such as autoimmunity. We previously demonstrated that the selective activation of nucleic acid-sensing cytosolic and Toll-like receptors is contingent on the promiscuous sensing of nucleic acids by high-mobility group box proteins (HMGBs). From this, we reasoned that nonimmunogenic nucleotides with high-affinity HMGB binding may function as suppressing agents for HMGB-mediated diseases, particularly those initiated and/or exacerbated by nucleic acids. Here we characterize an array of HMGB-binding, nonimmunogenic oligodeoxynucleotides (ni-ODNs). Interestingly, we find that binding affinity is rather independent of nucleotide sequence, but is instead dependent on length and structure of the deoxyribose backbone. We further show that these ni-ODNs can strongly suppress the activation of innate immune responses induced by both classes of nucleic acid-sensing receptors. We also provide evidence for the suppressive effect of an ni-ODN, termed ISM ODN, on the induction of adaptive immune responses and in mouse models of sepsis and autoimmunity. We discuss our findings in relation to the critical role of HMGBs in initiating immune responses and the possible use of these ni-ODNs in therapeutic interventions.

CD4(+) T regulatory cells (T(regs)), which express the Foxp3 transcription factor, play a critical role in the maintenance of immune homeostasis. Here, we show that in mice, T(regs) were most abundant in the colonic mucosa. The spore-forming component of indigenous intestinal microbiota, particularly clusters IV and XIVa of the genus Clostridium, promoted T(reg) cell accumulation. Colonization of mice by a defined mix of Clostridium strains provided an environment rich in transforming growth factor-β and affected Foxp3(+) T(reg) number and function in the colon. Oral inoculation of Clostridium during the early life of conventionally reared mice resulted in resistance to colitis and systemic immunoglobulin E responses in adult mice, suggesting a new therapeutic approach to autoimmunity and allergy.