Nod1 promotes colorectal carcinogenesis by regulating the immunosuppressive functions of tumor-infiltrating myeloid cells.

Pioneering studies from the early 1980s suggested that bacterial peptidoglycan-derived muramyl peptides (MPs) could exert either stimulatory or immunosuppressive functions depending, in part, on chronicity of exposure. However, this Janus-faced property of MPs remains largely unexplored. Here, we demonstrate the immunosuppressive potential of Nod1, the bacterial sensor of diaminopimelic acid (DAP)-containing MPs. Using a model of self-limiting peritonitis, we show that systemic Nod1 activation promotes an autophagy-dependent reprogramming of macrophages toward an alternative phenotype. Moreover, Nod1 stimulation induces the expansion of myeloid-derived suppressor cells (MDSCs) and maintains their immunosuppressive potential via arginase-1 activity. Supporting the role of MDSCs and tumor-associated macrophages in cancer, we demonstrate that myeloid-intrinsic Nod1 expression sustains intra-tumoral arginase-1 levels to foster an immunosuppressive and tumor-permissive microenvironment during colorectal cancer (CRC) development. Our findings support the notion that bacterial products, via Nod1 detection, modulate the immunosuppressive activity of myeloid cells and fuel tumor progression in CRC.

Irreversible splenic atrophy following chronic LCMV infection is associated with compromised immunity in mice.

Lymphocytic choriomeningitis virus clone 13 (LCMV13) infection of mice is a widely used model for investigating the mechanisms driving persistent viral infection in humans. LCMV13 disrupts splenic architecture early during infection, but this returns to normal within a few weeks. However, the long-term effects of LCMV13 infection on splenic structure have not been reported. Here, we report that persistent infection with LCMV13 results in sustained splenic atrophy that persists for at least 500 days following infection, whereas infection with the acutely infecting LCMV Armstrong is associated with a return to preinfection spleen weights. Splenic atrophy is associated with loss of T, B, and non-B non-T cells, with B cells most significantly affected. These effects were partly ameliorated by anti-NK1.1 or anti-CD8 antibody treatment. Antigen presentation was detectable at the time of contraction of the spleen, but no longer detected at late time points, suggesting that continued antigen presentation is not required to maintain splenic atrophy. Immunity to Salmonella infection and influenza vaccination were decreased after the virus was no longer detected. Thus splenic atrophy following LCMV13 infection is irreversible and may contribute to impaired immunity following clearance of LCMV13.

Resilience of the intestinal microbiota following pathogenic bacterial infection is independent of innate immunity mediated by NOD1 or NOD2.

The innate immune receptors, NOD1 and NOD2, are key regulators of intestinal homeostasis. NOD2 deficiency is linked to increased risk for Crohn's disease, a type of inflammatory bowel disease characterized by chronic inflammatory pathology and dysbiosis within resident microbial communities. However, the relationship between NOD protein-regulated immune functions and dysbiosis remains unclear. We hypothesized that the relationship between NOD1 or NOD2 deficiency and altered community structure during chronic disease may arise via NOD-dependent impairment of community resilience over time. Using the Salmonella ΔaroA model of chronic colitis with littermate mice to control for environmental influences on the microbiota, we show that NOD proteins exert a relatively minor impact on the chronic inflammatory environment and do not significantly contribute to bacterial abundance or community resilience following infection. Rather, temporal shifts in relative abundance of targeted bacterial groups correlated with inflammatory phenotype driven by presence of the pathogen and the ensuing complex immune response.

Salmonella enterica serovar Typhimurium ΔmsbB triggers exacerbated inflammation in Nod2 deficient mice.

The intracellular pathogen Salmonella enterica serovar Typhimurium causes intestinal inflammation characterized by edema, neutrophil influx and increased pro-inflammatory cytokine expression. A major bacterial factor inducing pro-inflammatory host responses is lipopolysaccharide (LPS). S. Typhimurium ΔmsbB possesses a modified lipid A, has reduced virulence in mice, and is being considered as a potential anti-cancer vaccine strain. The lack of a late myristoyl transferase, encoded by MsbB leads to attenuated TLR4 stimulation. However, whether other host receptor pathways are also altered remains unclear. Nod1 and Nod2 are cytosolic pattern recognition receptors recognizing bacterial peptidoglycan. They play important roles in the host's immune response to enteric pathogens and in immune homeostasis. Here, we investigated how deletion of msbB affects Salmonella's interaction with Nod1 and Nod2. S. Typhimurium Δ msbB-induced inflammation was significantly exacerbated in Nod2-/- mice compared to C57Bl/6 mice. In addition, S. Typhimurium ΔmsbB maintained robust intestinal colonization in Nod2-/- mice from day 2 to day 7 p.i., whereas colonization levels significantly decreased in C57Bl/6 mice during this time. Similarly, infection of Nod1-/- and Nod1/Nod2 double-knockout mice revealed that both Nod1 and Nod2 play a protective role in S. Typhimurium ΔmsbB-induced colitis. To elucidate why S. Typhimurium ΔmsbB, but not wild-type S. Typhimurium, induced an exacerbated inflammatory response in Nod2-/- mice, we used HEK293 cells which were transiently transfected with pathogen recognition receptors. Stimulation of TLR2-transfected cells with S. Typhimurium ΔmsbB resulted in increased IL-8 production compared to wild-type S. Typhimurium. Our results indicate that S. Typhimurium ΔmsbB triggers exacerbated colitis in the absence of Nod1 and/or Nod2, which is likely due to increased TLR2 stimulation. How bacteria with "genetically detoxified" LPS stimulate various innate responses has important implications for the development of safe and effective bacterial vaccines and adjuvants.

Gut microbial metabolism drives transformation of MSH2-deficient colon epithelial cells.

The etiology of colorectal cancer (CRC) has been linked to deficiencies in mismatch repair and adenomatous polyposis coli (APC) proteins, diet, inflammatory processes, and gut microbiota. However, the mechanism through which the microbiota synergizes with these etiologic factors to promote CRC is not clear. We report that altering the microbiota composition reduces CRC in APC(Min/+)MSH2(-/-) mice, and that a diet reduced in carbohydrates phenocopies this effect. Gut microbes did not induce CRC in these mice through an inflammatory response or the production of DNA mutagens but rather by providing carbohydrate-derived metabolites such as butyrate that fuel hyperproliferation of MSH2(-/-) colon epithelial cells. Further, we provide evidence that the mismatch repair pathway has a role in regulating ß-catenin activity and modulating the differentiation of transit-amplifying cells in the colon. These data thereby provide an explanation for the interaction between microbiota, diet, and mismatch repair deficiency in CRC induction. PAPERCLIP:

Murine Plasmodium chabaudi malaria increases mucosal immune activation and the expression of putative HIV susceptibility markers in the gut and genital mucosae.

OBJECTIVE: To evaluate if systemic murine malarial infection enhances HIV susceptibility through parasite-induced mucosal immune alterations at sites of HIV sexual exposure. BACKGROUND: Malaria and HIV have a high degree of geographical overlap and interact substantially within coinfected individuals. We used a murine model to test the hypothesis that malaria might also enhance HIV susceptibility at mucosal sites of HIV sexual exposure. METHODS: Female C57/BL6 mice were infected with Plasmodium chabaudi malaria using a standardized protocol. Blood, gastrointestinal tissues, upper and lower genital tract tissues, and iliac lymph nodes were sampled 10 days postinfection, and the expression of putative HIV susceptibility and immune activation markers on T cells was assessed by flow cytometry. RESULTS: P. chabaudi malaria increased expression of mucosal homing integrin α4ß7 on blood CD4 and CD8 T cells, and these α4ß7 T cells had significantly increased co-expression of both CCR5 and CD38. In addition, malaria increased expression of the HIV co-receptor CCR5 on CD4 T cells from the genital tract and gut mucosa as well as mucosal T-cell expression of the immune activation markers CD38, Major Histocompatibility Complex -II (MHC-II) and CD69. CONCLUSIONS: Systemic murine malarial infection induced substantial upregulation of the mucosal homing integrin α4ß7 in blood as well as gut and genital mucosal T-cell immune activation and HIV co-receptor expression. Human studies are required to confirm these murine findings and to examine whether malarial infection enhances the sexual acquisition of HIV.

Nod2 activates NF-kB in CD4+ T cells but its expression is dispensable for T cell-induced colitis.

Although the etiology of Crohn's disease (CD) remains elusive this disease is characterized by T cell activation that leads to chronic inflammation and mucosal damage. A potential role for maladaptation between the intestinal microbiota and the mucosal immune response is suggested by the fact that mutations in the pattern recognition receptor Nod2 are associated with higher risks for developing CD. Although Nod2 deletion in CD4(+) T cells has been shown to impair the induction of colitis in the murine T cell transfer model, the analysis of T cell intrinsic Nod2 function in T cell differentiation and T cell-mediated immunity is inconsistent between several studies. In addition, the role of T cell intrinsic Nod2 in regulatory T cell (Treg) development and function during colitis remain to be analyzed. In this study, we show that Nod2 expression is higher in activated/memory CD4(+) T cells and its expression was inducible after T cell receptor (TCR) ligation. Nod2 stimulation with muramyl dipeptide (MDP) led to a nuclear accumulation of c-Rel NF-kB subunit. Although functionally active in CD4(+) T cells, the deletion of Nod2 did not impair the induction and the prevention of colitis in the T cell transfer model. Moreover, Nod2 deletion did not affect the development of Foxp3(+) Treg cells in the spleen of recipient mice and Nod2 deficient CD4 T cells expressing the OVA specific transgenic TCR were able to differentiate in Foxp3(+) Treg cells after OVA feeding. In vitro, CD25(+) Nod2 deficient T cells suppressed T cell proliferation as well as wild type counter parts and T cell stimulation with MDP did not affect the proliferation and the cytokine secretion of T cells. In conclusion, our data indicate that Nod2 is functional in murine CD4(+) T cells but its expression is dispensable for the T cell regulation of colitis.

Constitutive induction of intestinal Tc17 cells in the absence of hematopoietic cell-specific MHC class II expression.

The enteric pathogen Citrobacter rodentium induces a mucosal IL-17 response in CD4(+) T helper (Th17) cells that is dependent on the Nod-like receptors Nod1 and Nod2. Here, we sought to determine whether this early Th17 response required antigen presentation by major histocompatibility complex class II (MHCII) for full induction. At early phases of C. rodentium infection, we observed that the intestinal mucosal Th17 response was fully blunted in irradiated mice reconstituted with MHCII-deficient (MHCII(-/-) →WT) hematopoietic cells. Surprisingly, we also observed a substantial increase in the relative frequency of IL-17(+) CD8(+) CD4(-) TCR-ß(+) cells (Tc17 cells) and FOXP3(+) CD8(+) CD4(-) TCR-ß(+) cells in the lamina propria and intraepithelial lymphocyte compartment of MHCII(-/-) →WT mice compared with that in WT→WT counterparts. Moreover, MHCII(-/-) →WT mice displayed increased susceptibility, increased bacterial translocation to deeper organs, and more severe colonic histopathology after infection with C. rodentium. Finally, a similar phenotype was observed in mice deficient for CIITA, a transcriptional regulator of MHCII expression. Together, these results indicate that MHCII is required to mount early mucosal Th17 responses to an enteric pathogen, and that MHCII regulates the induction of atypical CD8(+) T-cell subsets, such as Tc17 cells and FOXP3(+) CD8(+) cells, in vivo.

Nod1 and Nod2 signaling does not alter the composition of intestinal bacterial communities at homeostasis.

Patients with inflammatory bowel diseases (IBD) harbour intestinal bacterial communities with altered composition compared with healthy counterparts; however, it is unknown whether changes in the microbiota are associated with genetic susceptibility of individuals for developing disease or instead reflect other changes in the intestinal environment related to the disease itself. Since deficiencies in the innate immune receptors Nod1 and Nod2 are linked to IBD, we tested the hypothesis that Nod-signaling alters intestinal immune profiles and subsequently alters bacterial community structure. We used qPCR to analyze expression patterns of selected immune mediators in the ileum and cecum of Nod-deficient mice compared with their Nod-sufficient littermates and assessed the relative abundance of major bacterial groups sampled from the ileum, cecum and colon. The Nod1-deficient ileum exhibited significantly lower expression of Nod2, Muc2, α- and ß-defensins and keratinocyte-derived chemokine (KC), suggesting a weakened epithelial barrier compared with WT littermates; however, there were no significant differences in the relative abundance of targeted bacterial groups, indicating that Nod1-associated immune differences alone do not promote dysbiosis. Furthermore, Nod2-deficient mice did not display any changes in the expression of immune markers or bacterial communities. Shifts in bacterial communities that were observed in this study correlated with housing conditions and were independent of genotype. These findings emphasize the importance of using F2 littermate controls to minimize environmental sources of variation in microbial analyses, to establish baseline conditions for host-microbe homeostasis in Nod-deficient mice and to strengthen models for testing factors contributing to microbial dysbiosis associated with IBD.

Role of mouse peptidoglycan recognition protein PGLYRP2 in the innate immune response to Salmonella enterica serovar Typhimurium infection in vivo.

Peptidoglycan recognition proteins (PGRPs) are a family of innate pattern recognition molecules that bind bacterial peptidoglycan. While the role of PGRPs in Drosophila innate immunity has been extensively studied, how the four mammalian PGRP proteins (PGLYRP1 to PGLYRP4) contribute to host defense against bacterial pathogens in vivo remains poorly understood. PGLYRP1, PGLYRP3, and PGLYRP4 are directly bactericidal in vitro, whereas PGLYRP2 is an N-acetylmuramyl-L-alanine amidase that cleaves peptidoglycan between the sugar backbone and the peptide stem. Because PGLYRP2 cleaves muramyl peptides detected by host peptidoglycan sensors Nod1 and Nod2, we speculated that PGLYRP2 may act as a modifier of Nod1/Nod2-dependent innate immune responses. We investigated the role of PGLYRP2 in Salmonella enterica serovar Typhimurium-induced colitis, which is regulated by Nod1/Nod2 through the induction of an early Th17 response. PGLYRP2 did not contribute to expression of Th17-associated cytokines, interleukin-22 (IL-22)-dependent antimicrobial proteins, or inflammatory cytokines. However, we found that Pglyrp2-deficient mice displayed significantly enhanced inflammation in the cecum at 72 h postinfection, reflected by increased polymorphonuclear leukocyte (PMN) infiltration and goblet cell depletion. Pglyrp2 expression was also induced in the cecum of Salmonella-infected mice, and expression of green fluorescent protein under control of the Pglyrp2 promoter was increased in discrete populations of intraepithelial lymphocytes. Lastly, Nod2(-/-) Pglyrp2(-/-) mice displayed increased susceptibility to infection at 24 h postinfection compared to Pglyrp2(-/-) mice, which correlated with increased PMN infiltration and submucosal edema. Thus, PGLYRP2 plays a protective role in vivo in the control of S. Typhimurium infection through a Nod1/Nod2-independent mechanism.

Examining host-microbial interactions through the lens of NOD: From plants to mammals.

Nod-like receptors (NLRs) for detecting microbial invaders are features of many plant and animal families. Although broadly similar in form and function, intimate co-evolutionary events with environmental microbes have shaped specific classes of NLRs in different types of hosts. Details of the roles of different NLRs in signaling cellular immune responses to invading microbes are only beginning to emerge. This review will discuss the current understanding of NLRs in plants, invertebrates, and mammals, with emphasis on their role in regulating NF-κB and inflammasome activity in mammals.

Innate IL-17 and IL-22 responses to enteric bacterial pathogens.

With the identification of T helper (Th)17 cells, a specific subset of CD4 T cells expressing interleukin (IL)-17 and IL-22, research on the function of these cytokines initially largely focused on traditional adaptive immune responses. However, IL-17 and IL-22 enhance basic innate barrier defenses at mucosal surfaces, such as antimicrobial peptide production and neutrophil recruitment; both events that occur rapidly and precede adaptive phase immunity. At the intestinal mucosal surface, it is now clear that innate lymphoid cells are also important sources of IL-17 and IL-22 during early phases of infection. Here, we discuss the function of innate IL-17- and IL-22-producing lymphocytes during enteric bacterial infection and their regulation by the intestinal microbiota, Toll-like receptors (TLRs) and Nod-like receptors (NLRs).

Nucleotide oligomerization domain-containing proteins instruct T cell helper type 2 immunity through stromal activation.

Although a number of studies have examined the development of T-helper cell type 2 (Th2) immunity in different settings, the mechanisms underlying the initiation of this arm of adaptive immunity are not well understood. We exploited the fact that immunization with antigen plus either nucleotide-binding oligomerization domain-containing proteins 1 (Nod1) or 2 (Nod2) agonists drives Th2 induction to understand how these pattern-recognition receptors mediate the development of systemic Th2 immune responses. Here, we show in bone-marrow chimeric mice that Nod1 and Nod2 expression within the stromal compartment is necessary for priming of effector CD4(+) Th2 responses and specific IgG1 antibodies. In contrast, sensing of these ligands by dendritic cells was not sufficient to induce Th2 immunity, although these cells contribute to the response. Moreover, we determined that CD11c(+) cells were the critical antigen-presenting cells, whereas basophils and B cells did not affect the capacity of Nod ligands to induce CD4(+) Th2 effector function. Finally, we found that full Th2 induction upon Nod1 and Nod2 activation was dependent on both thymic stromal lymphopoietin production by the stromal cells and the up-regulation of the costimulatory molecule, OX40 ligand, on dendritic cells. This study provides in vivo evidence of how systemic Th2 immunity is induced in the context of Nod stimulation. Such understanding will influence the rational design of therapeutics that could reprogram the immune system during an active Th1-mediated disease, such as Crohn's disease.

Identification of an innate T helper type 17 response to intestinal bacterial pathogens.

Interleukin 17 (IL-17) is a central cytokine implicated in inflammation and antimicrobial defense. After infection, both innate and adaptive IL-17 responses have been reported, but the type of cells involved in innate IL-17 induction, as well as their contribution to in vivo responses, are poorly understood. Here we found that Citrobacter and Salmonella infection triggered early IL-17 production, which was crucial for host defense and was mediated by CD4(+) T helper cells. Enteric innate T helper type 17 (iT(H)17) responses occurred principally in the cecum, were dependent on the Nod-like receptors Nod1 and Nod2, required IL-6 induction and were associated with a decrease in mucosal CD103(+) dendritic cells. Moreover, imprinting by the intestinal microbiota was fully required for the generation of iT(H)17 responses. Together, these results identify the Nod-iT(H)17 axis as a central element in controlling enteric pathogens, which may implicate Nod-driven iT(H)17 responses in the development of inflammatory bowel diseases.

Essential role of Rip2 in the modulation of innate and adaptive immunity triggered by Nod1 and Nod2 ligands.

Muramyl peptides are the building blocks of bacterial peptidoglycan, and their biological functions in mammals have been extensively studied. In particular, muramyl peptides trigger inflammation, contribute to host defense against microbial infections, and modulate the adaptive immune response to antigens. These bacterial molecules are detected by nucleotide oligomerization domain 1 (Nod1) and Nod2, and recent evidence suggests that muramyl dipeptide also activates NLRP3 and NLRP1 inflammasomes. Here, we investigated the role of Rip2, the adaptor for Nod1- and Nod2-dependent signaling, in multiple aspects of the host response to muramyl peptides in vivo, such as inflammatory cytokine secretion, activation and recruitment of macrophages and neutrophils to the site of injection, systemic activation of myeloid, T and B cells in the spleen, adjuvanticity and capacity to polarize the adaptive response to ovalbumin. Our results demonstrate that Rip2 was crucial for all the biological functions studied. We also identified CD11c(int) CD11b(+) inflammatory dendritic cells as a major myeloid cell population responding to Nod stimulation in vivo. Together, our results highlight the importance of Rip2 for Nod-dependent induction of innate and adaptive immunity.

Nod1 and Nod2 regulation of inflammation in the Salmonella colitis model.

The pattern recognition molecules Nod1 and Nod2 play important roles in intestinal homeostasis; however, how these proteins impact on the development of inflammation during bacterial colitis has not been examined. In the streptomycin-treated mouse model of Salmonella colitis, we found that mice deficient for both Nod1 and Nod2 had attenuated inflammatory pathology, reduced levels of inflammatory cytokines, and increased colonization of the mucosal tissue. Nod1 and Nod2 from both hematopoietic and nonhematopoietic sources contributed to the pathology, and all phenotypes were recapitulated in mice deficient for the signaling adaptor protein Rip2. However, the influence of Rip2 was strictly dependent on infection conditions that favored expression of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS), as Rip2 was dispensable for inflammation when mice were infected with bacteria grown under conditions that promoted expression of the SPI-1 TTSS. Thus, Nod1 and Nod2 can modulate inflammation and mediate efficient clearance of bacteria from the mucosal tissue during Salmonella colitis, but their role is dependent on the expression of the SPI-2 TTSS.

Role of Nod1 in mucosal dendritic cells during Salmonella pathogenicity island 1-independent Salmonella enterica serovar Typhimurium infection.

Recent advances in immunology have highlighted the critical function of pattern-recognition molecules (PRMs) in generating the innate immune response to effectively target pathogens. Nod1 and Nod2 are intracellular PRMs that detect peptidoglycan motifs from the cell walls of bacteria once they gain access to the cytosol. Salmonella enterica serovar Typhimurium is an enteric intracellular pathogen that causes a severe disease in the mouse model. This pathogen resides within vacuoles inside the cell, but the question of whether cytosolic PRMs such as Nod1 and Nod2 could have an impact on the course of S. Typhimurium infection in vivo has not been addressed. Here, we show that deficiency in the PRM Nod1, but not Nod2, resulted in increased susceptibility toward a mutant strain of S. Typhimurium that targets directly lamina propria dendritic cells (DCs) for its entry into the host. Using this bacterium and bone marrow chimeras, we uncovered a surprising role for Nod1 in myeloid cells controlling bacterial infection at the level of the intestinal lamina propria. Indeed, DCs deficient for Nod1 exhibited impaired clearance of the bacteria, both in vitro and in vivo, leading to increased organ colonization and decreased host survival after oral infection. Taken together, these findings demonstrate a key role for Nod1 in the host response to an enteric bacterial pathogen through the modulation of intestinal lamina propria DCs.

Unleashing the therapeutic potential of NOD-like receptors.

Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) are a family of intracellular sensors that have key roles in innate immunity and inflammation. Whereas some NLRs - including NOD1, NOD2, NAIP (NLR family, apoptosis inhibitory protein) and NLRC4 - detect conserved bacterial molecular signatures within the host cytosol, other members of this family sense 'danger signals', that is, xenocompounds or molecules that when recognized alert the immune system of hazardous environments, perhaps independently of a microbial trigger. In the past few years, remarkable progress has been made towards deciphering the role and the biology of NLRs, which has shown that these innate immune sensors have pivotal roles in providing immunity to infection, adjuvanticity and inflammation. Furthermore, several inflammatory disorders have been associated with mutations in human NLRgenes. Here, we discuss the effect that research on NLRs will have on vaccination, treatment of chronic inflammatory disorders and acute bacterial infections.