The Intermucosal Connection between the Mouth and Gut in Commensal Pathobiont-Driven Colitis.

The precise mechanism by which oral infection contributes to the pathogenesis of extra-oral diseases remains unclear. Here, we report that periodontal inflammation exacerbates gut inflammation in vivo. Periodontitis leads to expansion of oral pathobionts, including Klebsiella and Enterobacter species, in the oral cavity. Amassed oral pathobionts are ingested and translocate to the gut, where they activate the inflammasome in colonic mononuclear phagocytes, triggering inflammation. In parallel, periodontitis results in generation of oral pathobiont-reactive Th17 cells in the oral cavity. Oral pathobiont-reactive Th17 cells are imprinted with gut tropism and migrate to the inflamed gut. When in the gut, Th17 cells of oral origin can be activated by translocated oral pathobionts and cause development of colitis, but they are not activated by gut-resident microbes. Thus, oral inflammation, such as periodontitis, exacerbates gut inflammation by supplying the gut with both colitogenic pathobionts and pathogenic T cells.

The NLRP6 Inflammasome Recognizes Lipoteichoic Acid and Regulates Gram-Positive Pathogen Infection.

The activator and composition of the NLRP6 inflammasome remain poorly understood. We find that lipoteichoic acid (LTA), a molecule produced by Gram-positive bacteria, binds and activates NLRP6. In response to cytosolic LTA or infection with Listeria monocytogenes, NLRP6 recruited caspase-11 and caspase-1 via the adaptor ASC. NLRP6 activation by LTA induced processing of caspase-11, which promoted caspase-1 activation and interleukin-1ß (IL-1ß)/IL-18 maturation in macrophages. Nlrp6-/- and Casp11-/- mice were less susceptible to L. monocytogenes infection, which was associated with reduced pathogen loads and impaired IL-18 production. Administration of IL-18 to Nlrp6-/- or Casp11-/- mice restored the susceptibility of mutant mice to L. monocytogenes infection. These results reveal a previously unrecognized innate immunity pathway triggered by cytosolic LTA that is sensed by NLRP6 and exacerbates systemic Gram-positive pathogen infection via the production of IL-18.

Control of pathogens and pathobionts by the gut microbiota.

A dense resident microbial community in the gut, referred as the commensal microbiota, coevolved with the host and is essential for many host physiological processes that include enhancement of the intestinal epithelial barrier, development of the immune system and acquisition of nutrients. A major function of the microbiota is protection against colonization by pathogens and overgrowth of indigenous pathobionts that can result from the disruption of the healthy microbial community. The mechanisms that regulate the ability of the microbiota to restrain pathogen growth are complex and include competitive metabolic interactions, localization to intestinal niches and induction of host immune responses. Pathogens, in turn, have evolved strategies to escape from commensal-mediated resistance to colonization. Thus, the interplay between commensals and pathogens or indigenous pathobionts is critical for controlling infection and disease. Understanding pathogen-commensal interactions may lead to new therapeutic approaches to treating infectious diseases.

Gut Microbiota-Induced Immunoglobulin G Controls Systemic Infection by Symbiotic Bacteria and Pathogens.

The gut microbiota is compartmentalized in the intestinal lumen and induces local immune responses, but it remains unknown whether the gut microbiota can induce systemic response and contribute to systemic immunity. We report that selective gut symbiotic gram-negative bacteria were able to disseminate systemically to induce immunoglobulin G (IgG) response, which primarily targeted gram-negative bacterial antigens and conferred protection against systemic infections by E. coli and Salmonella by directly coating bacteria to promote killing by phagocytes. T cells and Toll-like receptor 4 on B cells were important in the generation of microbiota-specific IgG. We identified murein lipoprotein (MLP), a highly conserved gram-negative outer membrane protein, as a major antigen that induced systemic IgG homeostatically in both mice and humans. Administration of anti-MLP IgG conferred crucial protection against systemic Salmonella infection. Thus, our findings reveal an important function for the gut microbiota in combating systemic infection through the induction of protective IgG.

Distinct Commensals Induce Interleukin-1ß via NLRP3 Inflammasome in Inflammatory Monocytes to Promote Intestinal Inflammation in Response to Injury.

The microbiota stimulates inflammation, but the signaling pathways and the members of the microbiota involved remain poorly understood. We found that the microbiota induces interleukin-1ß (IL-1ß) release upon intestinal injury and that this is mediated via the NLRP3 inflammasome. Enterobacteriaceae and in particular the pathobiont Proteus mirabilis, induced robust IL-1ß release that was comparable to that induced by the pathogen Salmonella. Upon epithelial injury, production of IL-1ß in the intestine was largely mediated by intestinal Ly6C(high) monocytes, required chemokine receptor CCR2 and was abolished by deletion of IL-1ß in CCR2(+) blood monocytes. Furthermore, colonization with P. mirabilis promoted intestinal inflammation upon intestinal injury via the production of hemolysin, which required NLRP3 and IL-1 receptor signaling in vivo. Thus, upon intestinal injury, selective members of the microbiota stimulate newly recruited monocytes to induce NLRP3-dependent IL-1ß release, which promotes inflammation in the intestine.

Epidermal clearance of Candida albicans is mediated by IL-17 but independent of fungal innate immune receptors.

IL-17 plays important roles in host defense against Candida albicans at barrier surfaces and during invasive infection. However, the role of IL-17 in host defense after colonization of the epidermis, a main site of C. albicans infection, remains poorly understood. Using a murine model of epicutaneous candidiasis without skin abrasion, we found that skin inflammation triggered by epidermal C. albicans colonization was self-limiting with fungal clearance completed by day 7 after inoculation in wild-type mice or animals deficient in IL-17A or IL-17F. In contrast, marked neutrophilic inflammation in the epidermis and impaired fungal clearance were observed in mice lacking both IL-17A and IL-17F. Clearance of C. albicans was independent of Dectin-1, Dectin-2, CARD9 (caspase-recruitment domain family, member 9), TLR2 (Toll-like receptor 2) and MyD88 in the epidermal colonization model. We found that group 3 innate lymphoid cells (ILC3s) and γδT cells were the major IL-17 producers in the epicutaneous candidiasis model. Analyses of Rag2-/- mice and Rag2-/-Il2rg-/- mice revealed that production of IL-17A and IL-17F by ILC3s was sufficient for C. albicans clearance. Finally, we found that depletion of neutrophils impaired C. albicans clearance in the epidermal colonization model. Taken together, these findings indicate a critical and redundant function of IL-17A and IL-17F produced by ILC3s in host defense against C. albicans in the epidermis. The results also suggest that epidermal C. albicans clearance is independent of innate immune receptors or that these receptors act redundantly in fungal recognition and clearance.

NOD1 and NOD2: signaling, host defense, and inflammatory disease.

The nucleotide-binding oligomerization domain (NOD) proteins NOD1 and NOD2, the founding members of the intracellular NOD-like receptor family, sense conserved motifs in bacterial peptidoglycan and induce proinflammatory and antimicrobial responses. Here, we discuss recent developments about the mechanisms by which NOD1 and NOD2 are activated by bacterial ligands, the regulation of their signaling pathways, and their role in host defense and inflammatory disease. Several routes for the entry of peptidoglycan ligands to the host cytosol to trigger activation of NOD1 and NOD2 have been elucidated. Furthermore, genetic screens and biochemical analyses have revealed mechanisms that regulate NOD1 and NOD2 signaling. Finally, recent studies have suggested several mechanisms to account for the link between NOD2 variants and susceptibility to Crohn's disease. Further understanding of NOD1 and NOD2 should provide new insight into the pathogenesis of disease and the development of new strategies to treat inflammatory and infectious disorders.

Interleukin-22 regulates the complement system to promote resistance against pathobionts after pathogen-induced intestinal damage.

Pathobionts play a critical role in disease development, but the immune mechanisms against pathobionts remain poorly understood. Here, we report a critical role for interleukin-22 (IL-22) in systemic protection against bacterial pathobionts that translocate into the circulation after infection with the pathogen Clostridium difficile. Infection with C. difficile induced IL-22, and infected Il22(-/-) mice harbored high numbers of pathobionts in extraintestinal organs despite comparable pathogen load and intestinal damage in mutant and wild-type mice. Pathobionts exhibited increased resistant against complement-mediated phagocytosis, and their intravenous administration resulted in high animal mortality. Selective removal of translocated commensals rescued Il22(-/-) mice, and IL-22 administration enhanced the elimination of pathobionts. Mechanistically, IL-22 augmented bacterial phagocytosis by increasing the expression and bacterial binding of complement C3. Our study demonstrates an unexpected role for IL-22 in controlling the elimination of pathobionts that enter the systemic circulation through the regulation of the complement system.

Lipopolysaccharide O structure of adherent and invasive Escherichia coli regulates intestinal inflammation via complement C3.

Gut dysbiosis associated with intestinal inflammation is characterized by the blooming of particular bacteria such as adherent-invasive E. coli (AIEC). However, the precise mechanisms by which AIEC impact on colitis remain largely unknown. Here we show that antibiotic-induced dysbiosis worsened chemically-induced colitis in IL-22-deficient mice, but not in wild-type mice. The increase in intestinal inflammation was associated with the expansion of E. coli strains with genetic and functional features of AIEC. These E. coli isolates exhibited high ability to out compete related bacteria via colicins and resistance to the host complement system in vitro. Mutation of wzy, the lipopolysaccharide O polymerase gene, rendered AIEC more sensitive to the complement system and more susceptible to engulfment and killing by phagocytes while retaining its ability to outcompete related bacteria in vitro. The wzy AIEC mutant showed impaired fitness to colonize the intestine under colitic conditions, but protected mice from chemically-induced colitis. Importantly, the ability of the wzy mutant to protect from colitis was blocked by depletion of complement C3 which was associated with impaired intestinal eradication of AIEC in colitic mice. These studies link surface lipopolysaccharide O-antigen structure to the regulation of colitic activity in commensal AIEC via interactions with the complement system.

Impact of dietary manganese on experimental colitis in mice.

Diet plays a significant role in the pathogenesis of inflammatory bowel disease (IBD). A recent epidemiological study has shown an inverse relationship between nutritional manganese (Mn) status and IBD patients. Mn is an essential micronutrient required for normal cell function and physiological processes. To date, the roles of Mn in intestinal homeostasis remain unknown and the contribution of Mn to IBD has yet to be explored. Here, we provide evidence that Mn is critical for the maintenance of the intestinal barrier and that Mn deficiency exacerbates dextran sulfate sodium (DSS)-induced colitis in mice. Specifically, when treated with DSS, Mn-deficient mice showed increased morbidity, weight loss, and colon injury, with a concomitant increase in inflammatory cytokine levels and oxidative and DNA damage. Even without DSS treatment, dietary Mn deficiency alone increased intestinal permeability by impairing intestinal tight junctions. In contrast, mice fed a Mn-supplemented diet showed slightly increased tolerance to DSS-induced experimental colitis, as judged by the colon length. Despite the well-appreciated roles of intestinal microbiota in driving inflammation in IBD, the gut microbiome composition was not altered by changes in dietary Mn. We conclude that Mn is necessary for proper maintenance of the intestinal barrier and provides protection against DSS-induced colon injury.

Neutrophils Restrict Tumor-Associated Microbiota to Reduce Growth and Invasion of Colon Tumors in Mice.

BACKGROUND & AIMS: Neutrophils are among the most prevalent immune cells in the microenvironment of colon tumors; they are believed to promote growth of colon tumors, and their numbers correlate with outcomes of patients with colon cancer. Trials of inhibitors of neutrophil trafficking are underway in patients with cancer, but it is not clear how neutrophils contribute to colon tumorigenesis. METHODS: Colitis-associated colon cancer was induced in mice with conditional deletion of neutrophils (LysMCre;Mcl1fl/fl) and wild-type littermates (LysMCre;Mcl1wt/wt, control mice) by administration of azoxythmethane and/or dextran sulfate sodium. Sporadic colon tumorigenesis was assessed in neutrophil-deficient and neutrophil-replete mice with conditional deletion of colon epithelial Apc (Cdx2-CreERT2;Apcfl/fl). Primary colon tumor tissues from these mice were assessed by histology, RNA sequencing, quantitative polymerase chain reaction, and fluorescence in situ hybridization analyses. Fecal and tumor-associated microbiota were assessed by 16s ribosomal RNA sequencing. RESULTS: In mice with inflammation-induced and sporadic colon tumors, depletion of neutrophils increased the growth, proliferation, and invasiveness of the tumors. RNA sequencing analysis identified genes that regulate antimicrobial and inflammatory processes that were dysregulated in neutrophil-deficient colon tumors compared with colon tumors from control mice. Neutrophil depletion correlated with increased numbers of bacteria in tumors and proliferation of tumor cells, tumor-cell DNA damage, and an inflammatory response mediated by interleukin 17 (IL17). The 16s ribosomal RNA sequencing identified significant differences in the composition of the microbiota between colon tumors from neutrophil-deficient vs control mice. Administration of antibiotics or a neutralizing antibody against IL17 to neutrophil-deficient mice resulted in development of less-invasive tumors compared with mice given vehicle. We found bacteria in tumors to induce production of IL17, which promotes influx of intratumor B cells that promote tumor growth and progression. CONCLUSIONS: In comparisons of mice with vs without neutrophils, we found neutrophils to slow colon tumor growth and progression by restricting numbers of bacteria and tumor-associated inflammatory responses.

Mesenchymal Cell-Specific MyD88 Signaling Promotes Systemic Dissemination of Salmonella Typhimurium via Inflammatory Monocytes.

Enteric pathogens including Salmonella enteric serovar Typhimurium can breach the epithelial barrier of the host and spread to systemic tissues. In response to infection, the host activates innate immune receptors via the signaling molecule MyD88, which induces protective inflammatory and antimicrobial responses. Most of these innate immune responses have been studied in hematopoietic cells, but the role of MyD88 signaling in other cell types remains poorly understood. Surprisingly, we found that Dermo1-Cre;Myd88fl/fl mice with mesenchymal cell-specific deficiency of MyD88 were less susceptible to orogastric and i.p. STyphimurium infection than their Myd88fl/fl littermates. The reduced susceptibility of Dermo1-Cre;Myd88fl/fl mice to infection was associated with lower loads of S. Typhimurium in the liver and spleen. Mutant analyses revealed that S. Typhimurium employs its virulence type III secretion system 2 to promote its growth through MyD88 signaling pathways in mesenchymal cells. Inflammatory monocytes function as a major cell population for systemic dissemination of S. Typhimurium Mechanistically, mesenchymal cell-specific MyD88 signaling promoted CCL2 production in the liver and spleen and recruitment of inflammatory monocytes to systemic organs in response to STyphimurium infection. Consistently, MyD88 signaling in mesenchymal cells enhanced the number of phagocytes including Ly6ChiLy6G- inflammatory monocytes harboring STyphimurium in the liver. These results suggest that S. Typhimurium promotes its systemic growth and dissemination through MyD88 signaling pathways in mesenchymal cells.

Staphylococcus δ-toxin induces allergic skin disease by activating mast cells.

Atopic dermatitis is a chronic inflammatory skin disease that affects 15-30% of children and approximately 5% of adults in industrialized countries. Although the pathogenesis of atopic dermatitis is not fully understood, the disease is mediated by an abnormal immunoglobulin-E immune response in the setting of skin barrier dysfunction. Mast cells contribute to immunoglobulin-E-mediated allergic disorders including atopic dermatitis. Upon activation, mast cells release their membrane-bound cytosolic granules leading to the release of several molecules that are important in the pathogenesis of atopic dermatitis and host defence. More than 90% of patients with atopic dermatitis are colonized with Staphylococcus aureus in the lesional skin whereas most healthy individuals do not harbour the pathogen. Several staphylococcal exotoxins can act as superantigens and/or antigens in models of atopic dermatitis. However, the role of these staphylococcal exotoxins in disease pathogenesis remains unclear. Here we report that culture supernatants of S. aureus contain potent mast-cell degranulation activity. Biochemical analysis identified δ-toxin as the mast cell degranulation-inducing factor produced by S. aureus. Mast cell degranulation induced by δ-toxin depended on phosphoinositide 3-kinase and calcium (Ca(2+)) influx; however, unlike that mediated by immunoglobulin-E crosslinking, it did not require the spleen tyrosine kinase. In addition, immunoglobulin-E enhanced δ-toxin-induced mast cell degranulation in the absence of antigen. Furthermore, S. aureus isolates recovered from patients with atopic dermatitis produced large amounts of δ-toxin. Skin colonization with S. aureus, but not a mutant deficient in δ-toxin, promoted immunoglobulin-E and interleukin-4 production, as well as inflammatory skin disease. Furthermore, enhancement of immunoglobulin-E production and dermatitis by δ-toxin was abrogated in Kit(W-sh/W-sh) mast-cell-deficient mice and restored by mast cell reconstitution. These studies identify δ-toxin as a potent inducer of mast cell degranulation and suggest a mechanistic link between S. aureus colonization and allergic skin disease.

Synthesis of characteristic Mycobacterium peptidoglycan (PGN) fragments utilizing with chemoenzymatic preparation of meso-diaminopimelic acid (DAP), and their modulation of innate immune responses.

Peptidoglycan (PGN) is a major component of bacterial cell wall and is recognized as a potent immunostimulant. The PGN in the cell envelope of Mycobacterium Tuberculosis has been shown to possess several unique characteristics including the presence of N-glycolyl groups (in addition to N-acetyl groups) in the muramic acid residues, and amidation of the free carboxylic acid of d-Glu or of meso-DAP in the peptide chains. Using a newly developed, highly stereoselective, chemoenzymatic approach for the synthesis of meso-DAP in peptide stems, we successfully synthesized for the first time, a series of Mycobacterium PGN fragments that include both mono- and disaccharides of MurNGlyc or 1,6-anhydro-MurNGlyc, as well as peptide-amidated variants. The ability of these PGN fragments to stimulate the immune system through activation of human Nod1 and Nod2 was examined. The PGN fragments were found to modulate immune stimulation, specifically, amidation at the d-Glu and meso-DAP in the peptide stem strongly reduced hNod1 activation. This effect was dependent on modification position. Additionally, N-glycolyl (instead of acetyl) of muramic acid was associated with slightly reduced human Nod1 and Nod2 stimulatory capabilities.

Regulation of the gut microbiota by the mucosal immune system in mice.

The benefits of commensal bacteria to the health of the host have been well documented, such as providing stimulation to potentiate host immune responses, generation of useful metabolites, and direct competition with pathogens. However, the ability of the host immune system to control the microbiota remains less well understood. Recent microbiota analyses in mouse models have revealed detailed structures and diversities of microbiota at different sites of the digestive tract in mouse populations. The contradictory findings of previous studies on the role of host immune responses in overall microbiota composition are likely attributable to the high ß-diversity in mouse populations as well as technical limitations of the methods to analyze microbiota. The host employs multiple systems to strictly regulate their interactions with the microbiota. A spatial segregation between the host and microbiota is achieved with the mucosal epithelium, which is further fortified with a mucus layer on the luminal side and Paneth cells that produce antimicrobial peptides. When commensal bacteria or pathogens breach the epithelial barrier and translocate to peripheral tissues, the host immune system is activated to eliminate them. Defective segregation and tissue elimination of commensals result in exaggerated inflammatory responses and possibly death of the host. In this review, we discuss the current understanding of mouse microbiota, its common features with human microbiota, the technologies utilized to analyze microbiota, and finally the challenges faced to delineate the role of host immune responses in the composition of the luminal microbiota.

NODs: intracellular proteins involved in inflammation and apoptosis.

NOD (nucleotide-binding oligomerization domain) proteins are members of a family that includes the apoptosis regulator APAF1 (apoptotic protease activating factor 1), mammalian NOD-LRR (leucine-rich repeat) proteins and plant disease-resistance gene products. Several NOD proteins have been implicated in the induction of nuclear factor-kappaB (NF-kappaB) activity and in the activation of caspases. Two members of the NOD family, NOD1 and NOD2, mediate the recognition of specific bacterial components. Notably, genetic variation in the genes encoding the NOD proteins NOD2, cryopyrin and CIITA (MHC class II transactivator) in humans and Naip5 (neuronal apoptosis inhibitory protein 5) in mice is associated with inflammatory disease or increased susceptibility to bacterial infections. Mammalian NOD proteins seem to function as cytosolic sensors for the induction of apoptosis, as well as for innate recognition of microorganisms and regulation of inflammatory responses.

Protective role of commensals against Clostridium difficile infection via an IL-1ß-mediated positive-feedback loop.

Clostridium difficile is a Gram-positive obligate anaerobic pathogen that causes pseudomembranous colitis in antibiotic-treated individuals. Commensal bacteria are known to have a significant role in the intestinal accumulation of C. difficile after antibiotic treatment, but little is known about how they affect host immunity during C. difficile infection. In this article, we report that C. difficile infection results in translocation of commensals across the intestinal epithelial barrier that is critical for neutrophil recruitment through the induction of an IL-1ß-mediated positive-feedback loop. Mice lacking ASC, an essential mediator of IL-1ß and IL-18 processing and secretion, were highly susceptible to C. difficile infection. ASC(-/-) mice exhibited enhanced translocation of commensals to multiple organs after C. difficile infection. Notably, ASC(-/-) mice exhibited impaired CXCL1 production and neutrophil influx into intestinal tissues in response to C. difficile infection. The impairment in neutrophil recruitment resulted in reduced production of IL-1ß and CXCL1 but not IL-18. Importantly, translocated commensals were required for ASC/Nlrp3-dependent IL-1ß secretion by neutrophils. Mice lacking IL-1ß were deficient in inducing CXCL1 secretion, suggesting that IL-1ß is the dominant inducer of ASC-mediated CXCL1 production during C. difficile infection. These results indicate that translocated commensals play a crucial role in CXCL1-dependent recruitment of neutrophils to the intestine through an IL-1ß/NLRP3/ASC-mediated positive-feedback mechanism that is important for host survival and clearance of translocated commensals during C. difficile infection.

Oral pathobiont Klebsiella chaperon usher pili provide site-specific adaptation for the inflamed gut mucosa.

The ectopic gut colonization by orally derived pathobionts has been implicated in the pathogenesis of various gastrointestinal diseases, including inflammatory bowel disease (IBD). For example, gut colonization by orally derived Klebsiella spp. has been linked to IBD in mice and humans. However, the mechanisms whereby oral pathobionts colonize extra-oral niches, such as the gut mucosa, remain largely unknown. Here, we performed a high-density transposon (Tn) screening to identify genes required for the adaptation of an oral Klebsiella strain to different mucosal sites - the oral and gut mucosae - at the steady state and during inflammation. We find that K. aerogenes, an oral pathobiont associated with both oral and gut inflammation in mice, harbors a newly identified genomic locus named "locus of colonization in the inflamed gut (LIG)" that encodes genes related to iron acquisition (Sit and Chu) and host adhesion (chaperon usher pili [CUP] system). The LIG locus is highly conserved among K. aerogenes strains, and these genes are also present in several other Klebsiella species. The Tn screening revealed that the LIG locus is required for the adaptation of K. aerogenes in its ectopic niche. In particular, we determined K. aerogenes employs a CUP system (CUP1) present in the LIG locus for colonization in the inflamed gut, but not in the oral mucosa. Thus, oral pathobionts likely exploit distinct adaptation mechanisms in their ectopically colonized intestinal niche compared to their native niche.

Microbiota-dependent activation of CD4+ T cells induces CTLA-4 blockade-associated colitis via Fcγ receptors.

Immune checkpoint inhibitors can stimulate antitumor immunity but can also induce toxicities termed immune-related adverse events (irAEs). Colitis is a common and severe irAE that can lead to treatment discontinuation. Mechanistic understanding of gut irAEs has been hampered because robust colitis is not observed in laboratory mice treated with checkpoint inhibitors. We report here that this limitation can be overcome by using mice harboring the microbiota of wild-caught mice, which develop overt colitis following treatment with anti-CTLA-4 antibodies. Intestinal inflammation is driven by unrestrained activation of IFNγ-producing CD4+ T cells and depletion of peripherally induced regulatory T cells through Fcγ receptor signaling. Accordingly, anti-CTLA-4 nanobodies that lack an Fc domain can promote antitumor responses without triggering colitis. This work suggests a strategy for mitigating gut irAEs while preserving antitumor stimulating effects of CTLA-4 blockade.