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The impacts of individual commensal microbes on immunity and disease can differ dramatically depending on the surrounding microbial context; however, the specific bacterial combinations that dictate divergent immunological outcomes remain largely undefined. Here, we characterize an immunostimulatory Allobaculum species from an inflammatory bowel disease patient that exacerbates colitis in gnotobiotic mice. Allobaculum inversely associates with the taxonomically divergent immunostimulatory species Akkermansia muciniphila in human-microbiota-associated mice and human cohorts. Co-colonization with A. muciniphila ameliorates Allobaculum-induced intestinal epithelial cell activation and colitis in mice, whereas Allobaculum blunts the A.muciniphila-specific systemic antibody response and reprograms the immunological milieu in mesenteric lymph nodes by blocking A.muciniphila-induced dendritic cell activation and T cell expansion. These studies thus identify a pairwise reciprocal interaction between human gut bacteria that dictates divergent immunological outcomes. Furthermore, they establish a generalizable framework to define the contextual cues contributing to the "incomplete penetrance" of microbial impacts on human disease.

Background & aims: It is currently unclear whether reported changes in the gut microbiome are cause or consequence of inflammatory bowel disease (IBD). Therefore, we studied the gut microbiome of IBD-discordant and -concordant twin pairs, which offers the unique opportunity to assess individuals at increased risk of developing IBD, namely healthy cotwins from IBD-discordant twin pairs. Methods: Fecal samples were obtained from 99 twins (belonging to 51 twin pairs), 495 healthy age-, sex-, and body mass index–matched controls, and 99 unrelated patients with IBD. Whole-genome metagenomic shotgun sequencing was performed. Taxonomic and functional (pathways) composition was compared among healthy cotwins, IBD-twins, unrelated patients with IBD, and healthy controls with multivariable (ie, adjusted for potential confounding) generalized linear models. Results: No significant differences were observed in the relative abundance of species and pathways between healthy cotwins and their IBD-twins (false discovery rate

The immunopathogenesis of inflammatory bowel disease (IBD) has been attributed to a combination of host genetics and intestinal dysbiosis. Previous work in a small cohort of IBD patients suggested that pro-inflammatory bacterial taxa are highly coated with secretory immunoglobulin IgA. Using bacterial fluorescence-activated cell sorting coupled with 16S rRNA gene sequencing (IgA-SEQ), we profiled IgA coating of intestinal microbiota in a large cohort of IBD patients and identified bacteria associated with disease and treatment. Forty-three bacterial taxa displayed significantly higher IgA coating in IBD compared with controls, including 8 taxa exhibiting differential IgA coating but similar relative abundance. Patients treated with anti-TNF-α therapies exhibited dramatically altered microbiota-specific IgA responses compared with controls. Furthermore, increased IgA coating of Oscillospira was associated with a delay in time to surgery. These results demonstrate that investigating IgA responses to microbiota can uncover potential disease-modifying taxa and reveal improved biomarkers of clinical course in IBD.

Background: In Behcet’s disease (BD), an auto-inflammatory vasculitis, an unbalanced gut microbiota can contribute to pro-inflammatory reactions. In separate studies, distinct pro- and anti-inflammatory bacteria associated with BD have been identified. Methods: To establish disease-associated determinants, we performed gut microbiome profiling in BD patients from the Netherlands (n = 19) and Italy (n = 13), matched healthy controls (HC) from the Netherlands (n = 17) and Italy (n = 15) and oral microbiome profiling in Dutch BD patients (n = 18) and HC (n = 15) by 16S rRNA gene sequencing. In addition, we used fecal IgA-SEQ analysis to identify specific IgA coated bacterial taxa in Dutch BD patients (n = 13) and HC (n = 8). Results: In BD stool samples alpha-diversity was conserved, whereas beta-diversity analysis showed no clustering based on disease, but a significant segregation by country of origin. Yet, a significant decrease of unclassified Barnesiellaceae and Lachnospira genera was associated with BD patients compared to HC. Subdivided by country, the Italian cohort displays a significant decrease of unclassified Barnesiellaceae and Lachnospira genera, in the Dutch cohort this decrease is only a trend. Increased IgA-coating of Bifidobacterium spp., Dorea spp. and Ruminococcus bromii species was found in stool from BD patients. Moreover, oral Dutch BD microbiome displayed increased abundance of Spirochaetaceae and Dethiosulfovibrionaceae families. Conclusions: BD patients show decreased fecal abundance of Barnesiellaceae and Lachnospira and increased oral abundance of Spirochaetaceae and Dethiosulfovibrionaceae. In addition, increased fecal IgA coating of Bifidobacterium, Ruminococcus bromii and Dorea may reflect retention of anti-inflammatory species and neutralization of pathosymbionts in BD, respectively. Additional studies are warranted to relate intestinal microbes with the significance of ethnicity, diet, medication and response with distinct pro- and inflammatory pathways in BD patients.

Anti-microbial signaling pathways are normally triggered by innate immune receptors when detecting pathogenic microbes to provide protective immunity. Here we show that the inflammasome sensor Nlrp1 aggravates DSS-induced experimental mouse colitis by limiting beneficial, butyrate-producing Clostridiales in the gut. The colitis-protective effects of Nlrp1 deficiency are thus reversed by vancomycin treatment, but recapitulated with butyrate supplementation in wild-type mice. Moreover, an activating mutation in Nlrp1a increases IL-18 and IFNγ production, and decreases colonic butyrate to exacerbate colitis. We also show that, in patients with ulcerative colitis, increased NLRP1 in inflamed regions of the colon is associated with increased IFN-γ. In this context, NLRP1, IL-18 or IFN-γ expression negatively correlates with the abundance of Clostridiales in human rectal mucosal biopsies. Our data identify the NLRP1 inflammasome to be a key negative regulator of protective, butyrate-producing commensals, which therefore promotes inflammatory bowel disease.

Stressed gut epithelium gets some relief Immunoglobulin A (IgA) is the most abundantly expressed antibody isotype and can be found at various mucosal surfaces in the body, including the gastrointestinal (GI) tract. IgA is polyreactive and can coat and restrain both commensal bacteria and enteric pathogens. Grootjans et al. found that endoplasmic reticulum (ER) stress in the intestinal epithelial cells of mice induced the T cell– and microbiota-independent expansion of peritoneal B1b cells, which secrete IgA. Similarly, human subjects homozygous for a variant of an autophagy gene ( ATG16L1 ) known to cause ER stress showed increased numbers of GI IgA + cells compared with controls. Thus, epithelial ER stress serves as an advantageous “eustress” response that can functionally antagonize its well-characterized role in promoting inflammation. Science , this issue p. 993

Immunodeficient mice reconstituted with a human immune system represent a promising tool for translational research as they may allow modeling and therapy of human diseases in vivo. However, insufficient development and function of human natural killer (NK) cells and T cell subsets limit the applicability of humanized mice for studying cancer biology and therapy. Here, we describe a human interleukin 15 (IL15) and human signal regulatory protein alpha (SIRPA) knock-in mouse on a Rag2-/- Il2rg-/- background (SRG-15). Transplantation of human hematopoietic stem and progenitor cells into SRG-15 mice dramatically improved the development and functional maturation of circulating and tissue-resident human NK and CD8+ T cells and promoted the development of tissue-resident innate lymphoid cell (ILC) subsets. Profiling of human NK cell subsets by mass cytometry revealed a highly similar expression pattern of killer inhibitory receptors and other candidate molecules in NK cell subpopulations between SRG-15 mice and humans. In contrast to nonobese diabetic severe combined immunodeficient Il2rg-/- (NSG) mice, human NK cells in SRG-15 mice did not require preactivation but infiltrated a Burkitt's lymphoma xenograft and efficiently inhibited tumor growth following treatment with the therapeutic antibody rituximab. Our humanized mouse model may thus be useful for preclinical testing of novel human NK cell-targeted and combinatory cancer immunotherapies and for studying how they elicit human antitumor immune responses in vivo.

Inflammasomes are large cytosolic protein complexes that detect infection and stress-associated signals and promote immediate inflammatory responses. In the intestine, activation of the inflammasome leads to an inflammatory response that is important for controlling enteric infections but can also result in pathological tissue damage. Recent studies have suggested that the inflammasome also regulates intestinal homeostasis through its effects on the intestinal microbiota. Notably, many conflicting studies have been published regarding the effect of inflammasome deficiencies on intestinal homeostasis. Here, we attempt to reconcile these contrasting data by highlighting the many ways that the inflammasome contributes to intestinal homeostasis and pathology and exploring the potential role of alterations in the microbiota in these conflicting studies.

The ligand specificity of human TLR (hTLR) 2 is determined through the formation of functional heterodimers with either hTLR1 or hTLR6. The chicken carries two TLR (chTLR) 2 isoforms, type 1 and type 2 (chTLR2t1 and chTLR2t2), and one putative TLR1/6/10 homologue (chTLR16) of unknown function. In this study, we report that transfection of HeLa cells with the various chicken receptors yields potent NF-κB activation for the receptor combination of chTLR2t2 and chTLR16 only. The sensitivity of this complex was strongly enhanced by human CD14. The functional chTLR16/chTLR2t2 complex responded toward both the hTLR2/6-specific diacylated peptide S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) and the hTLR2/1 specific triacylated peptide tripalmitoyl-S-(bis(palmitoyloxy)propyl)-Cys-Ser-(Lys)3-Lys (Pam3CSK4), indicating that chTLR16 covers the functions of both mammalian TLR1 and TLR6. Dissection of the species specificity of TLR2 and its coreceptors showed functional chTLR16 complex formation with chTLR2t2 but not hTLR2. Conversely, chTLR2t2 did not function in combination with hTLR1 or hTLR6. The use of constructed chimeric receptors in which the defined domains of chTLR16 and hTLR1 or hTLR6 had been exchanged revealed that the transfer of leucine-rich repeats (LRR) 6–16 of chTLR16 into hTLR6 was sufficient to confer dual ligand specificity to the human receptor and to establish species-specific interaction with chTLR2t2. Collectively, our data indicate that diversification of the central LRR region of the TLR2 coreceptors during evolution has put constraints on both their ligand specificity and their ability to form functional complexes with TLR2.

SummarySpecific members of the intestinal microbiota dramatically affect inflammatory bowel disease (IBD) in mice. In humans, however, identifying bacteria that preferentially affect disease susceptibility and severity remains a major challenge. Here, we used flow-cytometry-based bacterial cell sorting and 16S sequencing to characterize taxa-specific coating of the intestinal microbiota with immunoglobulin A (IgA-SEQ) and show that high IgA coating uniquely identifies colitogenic intestinal bacteria in a mouse model of microbiota-driven colitis. We then used IgA-SEQ and extensive anaerobic culturing of fecal bacteria from IBD patients to create personalized disease-associated gut microbiota culture collections with predefined levels of IgA coating. Using these collections, we found that intestinal bacteria selected on the basis of high coating with IgA conferred dramatic susceptibility to colitis in germ-free mice. Thus, our studies suggest that IgA coating identifies inflammatory commensals that preferentially drive intestinal disease. Targeted elimination of such bacteria may reduce, reverse, or even prevent disease development.

Cytosolic inflammasome complexes mediated by a pattern recognition receptor (PRR) defend against pathogen infection by activating caspase 1. Pyrin, a candidate PRR, can bind to the inflammasome adaptor ASC to form a caspase 1-activating complex. Mutations in the Pyrin-encoding gene, MEFV, cause a human autoinflammatory disease known as familial Mediterranean fever. Despite important roles in immunity and disease, the physiological function of Pyrin remains unknown. Here we show that Pyrin mediates caspase 1 inflammasome activation in response to Rho-glucosylation activity of cytotoxin TcdB, a major virulence factor of Clostridium difficile, which causes most cases of nosocomial diarrhoea. The glucosyltransferase-inactive TcdB mutant loses the inflammasome-stimulating activity. Other Rho-inactivating toxins, including FIC-domain adenylyltransferases (Vibrio parahaemolyticus VopS and Histophilus somni IbpA) and Clostridium botulinum ADP-ribosylating C3 toxin, can also biochemically activate the Pyrin inflammasome in their enzymatic activity-dependent manner. These toxins all target the Rho subfamily and modify a switch-I residue. We further demonstrate that Burkholderia cenocepacia inactivates RHOA by deamidating Asn 41, also in the switch-I region, and thereby triggers Pyrin inflammasome activation, both of which require the bacterial type VI secretion system (T6SS). Loss of the Pyrin inflammasome causes elevated intra-macrophage growth of B. cenocepacia and diminished lung inflammation in mice. Thus, Pyrin functions to sense pathogen modification and inactivation of Rho GTPases, representing a new paradigm in mammalian innate immunity.

Campylobacter infection in humans is accompanied by severe inflammation of the intestinal mucosa, in contrast to colonization of chicken. The basis for the differential host response is unknown. Toll-like receptors (TLRs) sense and respond to microbes in the body and participate in the induction of an inflammatory response. Thus far, the interaction of Campylobacter with chicken TLRs has not been studied. Here, we investigated the potential of four Campylobacter strains to activate human TLR1/2/6, TLR4, TLR5, and TLR9 and chicken TLR2t2/16, TLR4, TLR5, and TLR21. Live bacteria showed no or very limited potential to activate TLR2, TLR4, and TLR5 of both the human and chicken species, with minor but significant differences between Campylobacter strains. In contrast, lysed bacteria induced strong NF-κB activation through human TLR1/2/6 and TLR4 and chicken TLR2t2/16 and TLR4 but not via TLR5 of either species. Interestingly, C. jejuni induced TLR4-mediated beta interferon in human but not chicken cells. Furthermore, isolated chromosomal Campylobacter DNA was unable to activate human TLR9 in our system, whereas chicken TLR21 was activated by DNA from all of the campylobacters tested. Our data are the first comparison of TLR-induced immune responses in humans and chickens. The results suggest that differences in bacterial cell wall integrity and in TLR responses to Campylobacter LOS and/or DNA may contribute to the distinct clinical manifestation between the species.

Campylobacter jejuni is the principal bacterial foodborne pathogen. A major challenge still is to identify the virulence strategies exploited by C. jejuni. Recent genomics, proteomics, and metabolomics approaches indicate that C. jejuni displays extensive inter- and intrastrain variation. The diverse behavior enables bacterial adaptation to different environmental conditions and directs interactions with the gut mucosa. Here, we report recent progress in understanding the molecular mechanisms and functional consequences of the phenotype diversity. The results suggest that C. jejuni actively penetrates the intestinal mucus layer, secretes proteins mainly via its flagellar apparatus, is engulfed by intestinal cells, and can disrupt the integrity of the epithelial lining. C. jejuni stimulates the proinflammatory pathway and the production of a large repertoire of cytokines, chemokines, and innate effector molecules. Novel experimental infection models suggest that the activation of the innate immune response is important for the development of intestinal pathology.

Mammalian Toll-like receptor 5 (TLR5) senses flagellin of several bacterial species and activates the innate immune system. The avian TLR repertoire exhibits considerable functional diversity compared to mammalian TLRs and evidence of a functional TLR5 in the avian species is lacking. In the present study we cloned and successfully expressed chicken TLR5 (chTLR5) in HeLa cells, as indicated by laser confocal microscopy. Infection of chTLR5 transfected cells with Salmonella enterica serovar Enteritidis activated NF-kappaB in a dose- and flagellin-dependent fashion. Similar NF-kappaB activation was observed with recombinant bacterial flagellin. Targeted mutagenesis of the proline residue at position 737 in the chTLR5-TIR domain was detrimental to chTLR5 function, confirming that the observed effects were conferred via chTLR5 and the MyD88 signaling pathway. Comparison of human, mouse and chicken TLR5 activation by flagellin of S. enterica serovar Typhimurium revealed that chTLR5 consistently yielded stronger responses than human but not mouse TLR5. This species-specific reactivity was not observed with flagellin of serovar Enteritidis. The species-specific TLR5 response was nullified after targeted mutagenesis of a single amino acid (Q89A) in serovar Typhimurium flagellin, while L415A and N100A substitutions had no effect. These results show that chickens express a functional TLR5 albeit with different flagellin sensing qualities compared to human TLR5. The finding that single amino acid substitutions in bacterial flagellin can alter the species-specific TLR5 response may influence the host range and susceptibility of infection.

Introduction of Salmonella enterica serotype Typhimurium into food products results from its ability to persist in the intestine of healthy livestock by mechanisms that are poorly understood. The non-fimbrial adhesin ShdA is a fibronectin binding protein required for persistent intestinal carriage of S. Typhimurium. We further investigated the molecular mechanism of ShdA-mediated intestinal persistence by determining the binding-site of this receptor in fibronectin. Analysis of ShdA binding to fibronectin proteolytic fragments and to recombinant fibronectin fusion proteins identified the (13)FnIII repeat module of the Hep-2 domain as the primary binding site for this adhesin. The (13)FnIII repeat module of fibronectin contains a cationic cradle formed by six basic residues (R6, R7, R9, R23, K25 and R54) that is a high affinity heparin-binding site conserved among fibronectin sequences from frogs to man. Binding of ShdA to the (13)FnIII repeat module of fibronectin and to a second extracellular matrix protein, Collagen I, could be inhibited by heparin. Furthermore, binding of ShdA to the Hep-2 domain was sensitive to the ionic buffer strength, suggesting that binding involved ionic interactions. We therefore determined whether amino acid substitutions of basic residues in the cationic cradle of the Hep-2 domain that inhibit heparin binding also abrogate binding of ShdA. Combined substitution of R6S and R7S strongly reduced ShdA binding to (13)FnIII. These data suggest that ShdA binds the Hep-2 domain of fibronectin by a mechanism that may mimic binding of the host polysaccharide heparin.

Most Helicobacter pylori strains are susceptible to tetracycline, an antibiotic commonly used for the eradication of H. pylori . However, an increase in incidence of tetracycline resistance in H. pylori has recently been reported. Here the mechanism of tetracycline resistance of the first Dutch tetracycline-resistant (Tet r ) H. pylori isolate (strain 181) is investigated. Twelve genes were selected from the genome sequences of H. pylori strains 26695 and J99 as potential candidate genes, based on their homology with tetracycline resistance genes in other bacteria. With the exception of the two 16S rRNA genes, none of the other putative tetracycline resistance genes was able to transfer tetracycline resistance. Genetic transformation of the Tet s strain 26695 with smaller overlapping PCR fragments of the 16S rRNA genes of strain 181, revealed that a 361-bp fragment that spanned nucleotides 711 to 1071 was sufficient to transfer resistance. Sequence analysis of the 16S rRNA genes of the Tet r strain 181, the Tet s strain 26695, and four Tet r 26695 transformants showed that a single triple-base-pair substitution, AGA 926-928 →TTC, was present within this 361-bp fragment. This triple-base-pair substitution, present in both copies of the 16S rRNA gene of all our Tet r H. pylori transformants, resulted in an increased MIC of tetracycline that was identical to that for the Tet r strain 181.