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.

Fiber-deficient diet inhibits colitis through the regulation of the niche and metabolism of a gut pathobiont.

Exclusive enteral nutrition (EEN) with fiber-free diets is an effective steroid-sparing treatment to induce clinical remission in children with Crohn's disease (CD). However, the mechanism underlying the beneficial effects of EEN remains obscure. Using a model of microbiota-dependent colitis with the hallmarks of CD, we find that the administration of a fiber-free diet prevents the development of colitis and inhibits intestinal inflammation in colitic animals. Remarkably, fiber-free diet alters the intestinal localization of Mucispirillum schaedleri, a mucus-dwelling pathobiont, which is required for triggering disease. Mechanistically, the absence of dietary fiber reduces nutrient availability and impairs the dissimilatory nitrate reduction to ammonia (DNRA) metabolic pathway of Mucispirillum, leading to its exclusion from the mucus layer and disease remission. Thus, appropriate localization of the specific pathobiont in the mucus layer is critical for disease development, which is disrupted by fiber exclusion. These results suggest strategies to treat CD by targeting the intestinal niche and metabolism of disease-causing microbes.

IκBζ controls IL-17-triggered gene expression program in intestinal epithelial cells that restricts colonization of SFB and prevents Th17-associated pathologies.

Control of gut microbes is crucial for not only local defense in the intestine but also proper systemic immune responses. Although intestinal epithelial cells (IECs) play important roles in cytokine-mediated control of enterobacteria, the underlying mechanisms are not fully understood. Here we show that deletion of IκBζ in IECs in mice leads to dysbiosis with marked expansion of segmented filamentous bacteria (SFB), thereby enhancing Th17 cell development and exacerbating inflammatory diseases. Mechanistically, the IκBζ deficiency results in decrease in the number of Paneth cells and impairment in expression of IL-17-inducible genes involved in IgA production. The decrease in Paneth cells is caused by aberrant activation of IFN-γ signaling and a failure of IL-17-dependent recovery from IFN-γ-induced damage. Thus, the IL-17R-IκBζ axis in IECs contributes to the maintenance of intestinal homeostasis by serving as a key component in a regulatory loop between the gut microbiota and immune cells.

Altering the Microbiome Inhibits Tumorigenesis in a Mouse Model of Oviductal High-Grade Serous Carcinoma.

Studies have shown bacteria influence the initiation and progression of cancers arising in sites that harbor rich microbial communities, such as the colon. Little is known about the potential for the microbiome to influence tumorigenesis at sites considered sterile, including the upper female genital tract. The recent identification of distinct bacterial signatures associated with ovarian carcinomas suggests microbiota in the gut, vagina, or elsewhere might contribute to ovarian cancer pathogenesis. Here, we tested whether altering the microbiome affects tumorigenesis in a mouse model of high-grade serous carcinoma (HGSC) based on conditional oviduct-specific inactivation of the Brca1, Trp53, Rb1, and Nf1 tumor suppressor genes. Cohorts of control (n = 20) and antibiotic-treated (n = 23) mice were treated with tamoxifen to induce tumor formation and then monitored for 12 months. The antibiotic cocktail was administered for the first 5 months of the monitoring period in the treatment group. Antibiotic-treated mice had significantly fewer and less advanced tumors than control mice at study endpoint. Antibiotics induced changes in the composition of the intestinal and vaginal microbiota, which were durable in the fecal samples. Clustering analysis showed particular groups of microbiota are associated with the development of HGSC in this model. These findings demonstrate the microbiome influences HGSC pathogenesis in an in vivo model that closely recapitulates the human disease. Because the microbiome can modulate efficacy of cancer chemo- and immunotherapy, our genetically engineered mouse model system may prove useful for testing whether altering the microbiota can improve the heretofore poor response of HGSC to immunotherapies. SIGNIFICANCE: This study provides strong in vivo evidence for a role of the microbiome in ovarian cancer pathogenesis.

Interleukin-11-expressing fibroblasts have a unique gene signature correlated with poor prognosis of colorectal cancer.

Interleukin (IL)-11 is a member of the IL-6 family of cytokines and is involved in multiple cellular responses, including tumor development. However, the origin and functions of IL-11-producing (IL-11+) cells are not fully understood. To characterize IL-11+ cells in vivo, we generate Il11 reporter mice. IL-11+ cells appear in the colon in murine tumor and acute colitis models. Il11ra1 or Il11 deletion attenuates the development of colitis-associated colorectal cancer. IL-11+ cells express fibroblast markers and genes associated with cell proliferation and tissue repair. IL-11 induces the activation of colonic fibroblasts and epithelial cells through phosphorylation of STAT3. Human cancer database analysis reveals that the expression of genes enriched in IL-11+ fibroblasts is elevated in human colorectal cancer and correlated with reduced recurrence-free survival. IL-11+ fibroblasts activate both tumor cells and fibroblasts via secretion of IL-11, thereby constituting a feed-forward loop between tumor cells and fibroblasts in the tumor microenvironment.

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.

Dietary L-serine confers a competitive fitness advantage to Enterobacteriaceae in the inflamed gut.

Metabolic reprogramming is associated with the adaptation of host cells to the disease environment, such as inflammation and cancer. However, little is known about microbial metabolic reprogramming or the role it plays in regulating the fitness of commensal and pathogenic bacteria in the gut. Here, we report that intestinal inflammation reprograms the metabolic pathways of Enterobacteriaceae, such as Escherichia coli LF82, in the gut to adapt to the inflammatory environment. We found that E. coli LF82 shifts its metabolism to catabolize L-serine in the inflamed gut in order to maximize its growth potential. However, L-serine catabolism has a minimal effect on its fitness in the healthy gut. In fact, the absence of genes involved in L-serine utilization reduces the competitive fitness of E. coli LF82 and Citrobacter rodentium only during inflammation. The concentration of luminal L-serine is largely dependent on dietary intake. Accordingly, withholding amino acids from the diet markedly reduces their availability in the gut lumen. Hence, inflammation-induced blooms of E. coli LF82 are significantly blunted when amino acids-particularly L-serine-are removed from the diet. Thus, the ability to catabolize L-serine increases bacterial fitness and provides Enterobacteriaceae with a growth advantage against competitors in the inflamed gut.

Microbial Metabolite Signaling Is Required for Systemic Iron Homeostasis.

Iron is a central micronutrient needed by all living organisms. Competition for iron in the intestinal tract is essential for the maintenance of indigenous microbial populations and for host health. How symbiotic relationships between hosts and native microbes persist during times of iron limitation is unclear. Here, we demonstrate that indigenous bacteria possess an iron-dependent mechanism that inhibits host iron transport and storage. Using a high-throughput screen of microbial metabolites, we found that gut microbiota produce metabolites that suppress hypoxia-inducible factor 2α (HIF-2α) a master transcription factor of intestinal iron absorption and increase the iron-storage protein ferritin, resulting in decreased intestinal iron absorption by the host. We identified 1,3-diaminopropane (DAP) and reuterin as inhibitors of HIF-2α via inhibition of heterodimerization. DAP and reuterin effectively ameliorated systemic iron overload. This work provides evidence of intestine-microbiota metabolic crosstalk that is essential for systemic iron homeostasis.

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.

Route Connection: Mouth to Intestine in Colitis.

In a recent study published in Science, Atarashi et al. (2017) showed that Klebsiella strains isolated from the saliva of Crohn's disease patients can induce Th1 cell responses to promote colitis. Their findings highlight the importance of the oral cavity as a potential reservoir for bacteria that can promote intestinal disease.

Staphylococcus aureus Virulent PSMα Peptides Induce Keratinocyte Alarmin Release to Orchestrate IL-17-Dependent Skin Inflammation.

Staphylococcus aureus commonly colonizes the epidermis, but the mechanisms by which the host senses virulent, but not commensal, S. aureus to trigger inflammation remain unclear. Using a murine epicutaneous infection model, we found that S. aureus-expressed phenol-soluble modulin (PSM)α, a group of secreted virulence peptides, is required to trigger cutaneous inflammation. PSMα induces the release of keratinocyte IL-1α and IL-36α, and signaling via IL-1R and IL-36R was required for induction of the pro-inflammatory cytokine IL-17. The levels of released IL-1α and IL-36α, as well as IL-17 production by γδ T cells and ILC3 and neutrophil infiltration to the site of infection, were greatly reduced in mice with total or keratinocyte-specific deletion of the IL-1R and IL-36R signaling adaptor Myd88. Further, Il17a-/-f-/- mice showed blunted S. aureus-induced inflammation. Thus, keratinocyte Myd88 signaling in response to S. aureus PSMα drives an IL-17-mediated skin inflammatory response to epicutaneous S. aureus infection.

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.

IL-22 Controls Iron-Dependent Nutritional Immunity Against Systemic Bacterial Infections.

Host immunity limits iron availability to pathogenic bacteria, but whether immunity limits pathogenic bacteria from accessing host heme, the major source of iron in the body, remains unclear. Using Citrobacter rodentium, a mouse enteric pathogen and Escherichia coli, a major cause of sepsis in humans as models, we find that interleukin-22, a cytokine best known for its ability to promote epithelial barrier function, also suppresses the systemic growth of bacteria by limiting iron availability to the pathogen. Using an unbiased proteomic approach to understand the mechanistic basis of IL-22 dependent iron retention in the host, we have identified that IL-22 induces the production of the plasma hemoglobin scavenger haptoglobin and heme scavenger hemopexin. Moreover, the anti-microbial effect of IL-22 depends on the induction of hemopexin expression, while haptogloblin is dispensable. Impaired pathogen clearance in infected Il22-/- mice was restored by hemopexin administration and hemopexin-deficient mice had increased pathogen loads after infection. These studies reveal a previously unrecognized host defense mechanism regulated by IL-22 that relies on the induction of hemopexin to limit heme availability to bacteria leading to suppression of bacterial growth during systemic infections.

Nod2-mediated recognition of the microbiota is critical for mucosal adjuvant activity of cholera toxin.

Cholera toxin (CT) is a potent adjuvant for inducing mucosal immune responses. However, the mechanism by which CT induces adjuvant activity remains unclear. Here we show that the microbiota is critical for inducing antigen-specific IgG production after intranasal immunization. After mucosal vaccination with CT, both antibiotic-treated and germ-free (GF) mice had reduced amounts of antigen-specific IgG, smaller recall-stimulated cytokine responses, impaired follicular helper T (TFH) cell responses and reduced numbers of plasma cells. Recognition of symbiotic bacteria via the nucleotide-binding oligomerization domain containing 2 (Nod2) sensor in cells that express the integrin CD11c (encoded by Itgax) was required for the adjuvanticity of CT. Reconstitution of GF mice with a Nod2 agonist or monocolonization with Staphylococcus sciuri, which has high Nod2-stimulatory activity, was sufficient to promote robust CT adjuvant activity, whereas bacteria with low Nod2-stimulatory activity did not. Mechanistically, CT enhanced Nod2-mediated cytokine production in dendritic cells via intracellular cyclic AMP. These results show a role for the microbiota and the intracellular receptor Nod2 in promoting the mucosal adjuvant activity of CT.

TLR4, NOD1 and NOD2 mediate immune recognition of putative newly identified periodontal pathogens.

Periodontitis is a polymicrobial inflammatory disease that results from the interaction between the oral microbiota and the host immunity. Although the innate immune response is important for disease initiation and progression, the innate immune receptors that recognize both classical and putative periodontal pathogens that elicit an immune response have not been elucidated. By using the Human Oral Microbe Identification Microarray (HOMIM), we identified multiple predominant oral bacterial species in human plaque biofilm that strongly associate with severe periodontitis. Ten of the identified species were evaluated in greater depth, six being classical pathogens and four putative novel pathogens. Using human peripheral blood monocytes (HPBM) and murine bone-marrow-derived macrophages (BMDM) from wild-type (WT) and Toll-like receptor (TLR)-specific and MyD88 knockouts (KOs), we demonstrated that heat-killed Campylobacter concisus, Campylobacter rectus, Selenomonas infelix, Porphyromonas endodontalis, Porphyromonas gingivalis, and Tannerella forsythia mediate high immunostimulatory activity. Campylobacter concisus, C. rectus, and S. infelix exhibited robust TLR4 stimulatory activity. Studies using mesothelial cells from WT and NOD1-specific KOs and NOD2-expressing human embryonic kidney cells demonstrated that Eubacterium saphenum, Eubacterium nodatum and Filifactor alocis exhibit robust NOD1 stimulatory activity, and that Porphyromonas endodontalis and Parvimonas micra have the highest NOD2 stimulatory activity. These studies allowed us to provide important evidence on newly identified putative pathogens in periodontal disease pathogenesis showing that these bacteria exhibit different immunostimulatory activity via TLR4, NOD1, and NOD2 (Clinicaltrials.gov NCT01154855).

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.

Infection mobilizes hematopoietic stem cells through cooperative NOD-like receptor and Toll-like receptor signaling.

Adult hematopoietic stem cells (HSCs) are maintained in specialized niches within the bone marrow under steady-state conditions and mobilize for extramedullary hematopoiesis during periods of stress such as bacterial infections. However, the underlying mechanisms are unclear. We show that systemic infection of mice with Escherichia coli, commonly associated with bacteremia in humans, mobilizes functional HSCs to the spleen. Accumulation of splenic HSCs (CD150+CD48-Lin(-/low)Sca1+cKit+) was diminished in TLR4-deficient and RIPK2-deficient mice, implicating TLRs and cytosolic NOD1/NOD2 signaling in the process. Accordingly, dual stimulation of NOD1 and TLR4 in radio-resistant cells alone was sufficient to mobilize HSCs, while TLR4 expression on HSCs was dispensable. Mechanistically, TLR4 and NOD1 synergistically induced granulocyte colony-stimulating factor (G-CSF), which was required for extramedullary HSC accumulation. Mobilized HSCs and progenitor cells gave rise to neutrophils and monocytes and contributed to limiting secondary infection.

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.

Tumor necrosis factor receptor-associated factor (TRAF) 2 controls homeostasis of the colon to prevent spontaneous development of murine inflammatory bowel disease.

Fine-tuning of host cell responses to commensal bacteria plays a crucial role in maintaining homeostasis of the gut. Here, we show that tumor necrosis factor receptor-associated factor (Traf)2(-/-) mice spontaneously developed severe colitis and succumbed within 3 weeks after birth. Histological analysis revealed that apoptosis of colonic epithelial cells was enhanced, and B cells diffusely infiltrated into the submucosal layer of the colon of Traf2(-/-) mice. Expression of proinflammatory cytokines, including Tnfa, Il17a, and Ifng, was up-regulated, whereas expression of antimicrobial peptides was down-regulated in the colon of Traf2(-/-) mice. Moreover, a number of IL-17-producing helper T cells were increased in the colonic lamina propria of Traf2(-/-) mice. These cellular alterations resulted in drastic changes in the colonic microbiota of Traf2(-/-) mice compared with Traf2(+/+) mice. Treatment of Traf2(-/-) mice with antibiotics ameliorated colitis along with down-regulation of proinflammatory cytokines and prolonged survival, suggesting that the altered colonic microbiota might contribute to exacerbation of colitis. Finally, deletion of Tnfr1, but not Il17a, dramatically ameliorated colitis in Traf2(-/-) mice by preventing apoptosis of colonic epithelial cells, down-regulation of proinflammatory cytokines, and restoration of wild-type commensal bacteria. Together, TRAF2 plays a crucial role in controlling homeostasis of the colon.

Nucleotide-binding oligomerization domain 1 mediates recognition of Clostridium difficile and induces neutrophil recruitment and protection against the pathogen.

Clostridium difficile is a Gram-positive obligate anaerobic pathogen that causes pseudomembranous colitis in antibiotics-treated individuals. However, host immune protective mechanisms against C. difficile are largely unknown. In this study, we show that C. difficile possesses potent stimulatory activity for nucleotide-binding oligomerization domain 1 (Nod1), an intracellular pattern recognition molecule that senses bacterial peptidoglycan-related molecules. Nod1(-/-), but not Nod2(-/-), mice exhibited increased lethality in response to C. difficile intestinal infection despite comparable levels of intestinal damage and epithelial permeability in Nod1(-/-) and control mice. The enhanced lethality was accompanied by impaired C. difficile clearance, increased bacterial translocation, and elevated levels of endotoxin and IL-1ß in the serum of Nod1(-/-) mice. Histological and flow cytometric analyses revealed that Nod1(-/-) mice had defective recruitment of neutrophils, but not macrophages, to the intestine after C. difficile infection. The reduced recruitment of neutrophils correlated with impaired production of CXCL1, but not CCL2, XCL1, and other cytokines/chemokines, in infected Nod1(-/-) mice. The influx of neutrophils also was reduced when C. difficile was administered i.p., suggesting that Nod1 directly recognizes C. difficile to induce the recruitment of neutrophils to the infected site. These results indicate that Nod1 regulates host susceptibility to C. difficile and suggest that Nod1-mediated neutrophil recruitment is an important immune response against the enteric pathogen.