Trace levels of peptidoglycan in serum underlie the NOD-dependent cytokine response to endoplasmic reticulum stress.

NOD1 and NOD2 are intracellular sensors of bacterial peptidoglycan that belong to the Nod-like receptor family of innate immune proteins. In addition to their role as direct bacterial sensors, it was proposed that the nucleotide-binding oligomerization domain (NOD) proteins could detect endoplasmic reticulum (ER) stress induced by thapsigargin, an inhibitor of the sarcoplasmic or endoplasmic reticulum calcium ATPase family that pumps Ca2+ into the ER, resulting in pro-inflammatory signaling. Here, we confirm that thapsigargin induces NOD-dependent pro-inflammatory signaling in epithelial cells. However, the effect was specific to thapsigargin, as tunicamycin and the subtilase cytotoxin SubAB from Shiga toxigenic Escherichia coli, which induce ER stress by other mechanisms, did not induce cytokine expression. The calcium ionophore A23187 also induced NOD-dependent signaling, and calcium chelators demonstrated a role for both intracellular and extracellular calcium in mediating thapsigargin-induced and NOD-dependent pro-inflammatory signaling, in part through the activation of plasma membrane-associated calcium release-activated channels. Moreover, our results demonstrate that both endocytosis and the addition of serum to the cell culture medium were required for thapsigargin-mediated NOD activation. Finally, we analyzed cell culture grade fetal calf serum as well as serum from laboratory mice using HPLC and MS identified the presence of various peptidoglycan fragments. We propose that cellular perturbations that affect intracellular Ca2+ can trigger internalization of peptidoglycan trace contaminants found in culture serum, thereby stimulating pro-inflammatory signaling. The presence of peptidoglycan in animal serum suggests that a homeostatic function of NOD signaling may have been previously overlooked.

Lack of Both Nucleotide-Binding Oligomerization Domain-Containing Proteins 1 and 2 Primes T Cells for Activation-Induced Cell Death.

Nucleotide-binding oligomerization domain (Nod)-containing proteins Nod1 and Nod2 play important roles in the innate immune response to pathogenic microbes, but mounting data suggest these pattern recognition receptors might also play key roles in adaptive immune responses. Targeting Nod1 and Nod2 signaling pathways in T cells is likely to provide a new strategy to modify inflammation in a variety of disease states, particularly those that depend on Ag-induced T cell activation. To better understand how Nod1 and Nod2 proteins contribute to adaptive immunity, this study investigated their role in alloantigen-induced T cell activation and asked whether their absence might impact in vivo alloresponses using a severe acute graft versus host disease model. The study provided several important observations. We found that the simultaneous absence of Nod1 and Nod2 primed T cells for activation-induced cell death. T cells from Nod1 × 2-/- mice rapidly underwent cell death upon exposure to alloantigen. The Nod1 × 2-/- T cells had sustained p53 expression that was associated with downregulation of its negative regulator MDM2. In vivo, mice transplanted with an inoculum containing Nod1 × 2-/- T cells were protected from severe graft versus host disease. The results show that the simultaneous absence of Nod1 and Nod2 is associated with accelerated T cell death upon alloantigen encounter, suggesting these proteins might provide new targets to ameliorate T cell responses in a variety of inflammatory states, including those associated with bone marrow or solid organ transplantation.

NOD1 deficiency impairs CD44a/Lck as well as PI3K/Akt pathway.

Pattern recognition receptors (PRRs) are crucial for host defense and tissue homeostasis against infecting pathogens. PRRs are highly conserved cross species, suggesting their key roles in fundamental biological processes. Though much have been learned for NOD1 receptor in the innate and adaptive immune responses, the roles of NOD1 during embryonic and larval stages remain poorly understood. Here, we report that NOD1 is necessary for the modulation of PI3K-Akt pathway and larval survival in zebrafish. Transcriptome analysis revealed that the significantly enriched pathways in NOD1 -/- zebrafish larvae were mainly involved in metabolism and immune system processes. Biochemical analysis demonstrated that NOD1 was required for the expression of CD44a that, in turn, activated the PI3K-Akt pathway during larval development. Conversely, over-expression of CD44a in NOD1-deficient zebrafish restored the modulation of the PI3K-Akt pathway and improved larval survival. Collectively, our work indicates that NOD1 plays a previously undetected protective role in larval survival through CD44a-mediated activation of the PI3K-Akt signaling.

Nod1 Limits Colitis-Associated Tumorigenesis by Regulating IFN-γ Production.

Chronic intestinal inflammation is a major risk factor for the development of colorectal cancer. Nod1, a member of the Nod-like receptor (NLR) family of pattern recognition receptors, is a bacterial sensor that has been previously demonstrated to reduce susceptibility of mice to chemically induced colitis and subsequent tumorigenesis, but the mechanism by which it mediates its protection has not been elucidated. In this study, we show that Nod1 expression in the hematopoietic cell compartment is critical for limiting inflammation-induced intestinal tumorigenesis. Specifically, Nod1-deficient T cells exhibit impaired IFN-γ production during dextran sulfate sodium (DSS)-induced acute inflammation in vivo, and administration of the Nod1 ligand KF1B enhances IFN-γ responses by anti-CD3-activated T cells in vitro. Absence of IFN-γ signaling results in increased inflammation-associated tumors in mice, and adoptive transfer of Nod1(-/-) or IFNγ(-/-) T cells into T cell-deficient mice results in increased tumorigenesis as compared with T cell-deficient mice that were adoptively transferred with wild-type T cells. Collectively, these results suggest a previously unappreciated role for the innate immune receptor Nod1 in suppressing colitis-associated tumorigenesis through a T cell-mediated mechanism.

Nucleotide-binding oligomerization domain 1 acts in concert with the cholecystokinin receptor agonist, cerulein, to induce IL-33-dependent chronic pancreatitis.

Nucleotide-binding oligomerization domain 1 (NOD1) fulfills important host-defense functions via its responses to a variety of gut pathogens. Recently, however, we showed that in acute pancreatitis caused by administration of cholecystokinin receptor (CCKR) agonist (cerulein) NOD1 also has a role in inflammation via its responses to gut commensal organisms. In the present study, we explored the long-term outcome of such NOD1 responsiveness in a new model of chronic pancreatitis induced by repeated administration of low doses of cerulein in combination with NOD1 ligand. We found that the development of chronic pancreatitis in this model requires intact NOD1 and type I IFN signaling and that such signaling mediates a macrophage-mediated inflammatory response that supports interleukin (IL)-33 production by acinar cells. The IL-33, in turn, has a necessary role in the induction of IL-13 and TGF-ß1, factors causing the fibrotic reaction characteristic of chronic pancreatitis. Interestingly, the Th2 effects of IL-33 were attenuated by the concomitant type I IFN response since the inflammation was marked by clear increases in IFN-γ and TNF-α production but only marginal increases in IL-4 production. These studies establish chronic pancreatitis as an IL-33-dependent inflammation resulting from synergistic interactions between the NOD1 and CCKR signaling pathways.

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).

The role of nucleotide-binding oligomerization domain 1 during cytokine production by macrophages in response to Mycobacterium tuberculosis infection.

Tuberculosis due to Mycobacterium tuberculosis infection is a leading cause of death worldwide. Recognition of this pathogen is crucial for the activation of innate and adaptive immune responses. Nucleotide-binding oligomerization domain (Nod)1 and Nod2 are cytoplasmic receptors that can detect unique muropeptides of bacterial peptidoglycan. Nod2 is critical for the initiation of the host immune response against M. tuberculosis infection, however the role of Nod1 remains largely unknown. We investigated the role of Nod1 with respect to cytokine production by bone marrow-derived macrophages (BMDMs) in response to M. tuberculosis infection. Production of proinflammatory cytokines, such as IL-6, TNF-α, and IL-1ß were induced in BMDMs; cytokine levels were not affected by a deficiency in Nod1. Activation of NF-κB and MAPKs was also comparable between wild-type and Nod1-deficient BMDMs. Levels of IL-6 and IL-1ß were reduced in Nod1/Nod2 double-deficient BMDMs to a greater extent than in Nod2-deficient cells. Furthermore, when signaling of Toll-like receptors (TLRs) was inhibited by lipopolysaccharide pre-treatment, cytokine production was diminished in Nod1-deficient BMDMs. Our results indicate that Nod1 cooperates with Nod2 or TLRs to produce cytokines in macrophages in response to M. tuberculosis infection.

Bacterial peptidoglycan stimulates adipocyte lipolysis via NOD1.

Obesity is associated with inflammation that can drive metabolic defects such as hyperlipidemia and insulin resistance. Specific metabolites can contribute to inflammation, but nutrient intake and obesity are also associated with altered bacterial load in metabolic tissues (i.e. metabolic endotoxemia). These bacterial cues can contribute to obesity-induced inflammation. The specific bacterial components and host receptors that underpin altered metabolic responses are emerging. We previously showed that Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) activation with bacterial peptidoglycan (PGN) caused insulin resistance in mice. We now show that PGN induces cell-autonomous lipolysis in adipocytes via NOD1. Specific bacterial PGN motifs stimulated lipolysis in white adipose tissue (WAT) explants from WT, but not NOD1⁻/⁻mice. NOD1-activating PGN stimulated mitogen activated protein kinases (MAPK),protein kinase A (PKA), and NF-κB in 3T3-L1 adipocytes. The NOD1-mediated lipolysis response was partially reduced by inhibition of ERK1/2 or PKA alone, but not c-Jun N-terminal kinase (JNK). NOD1-stimulated lipolysis was partially dependent on NF-κB and was completely suppressed by inhibiting ERK1/2 and PKA simultaneously or hormone sensitive lipase (HSL). Our results demonstrate that bacterial PGN stimulates lipolysis in adipocytes by engaging a stress kinase, PKA, NF-κB-dependent lipolytic program. Bacterial NOD1 activation is positioned as a component of metabolic endotoxemia that can contribute to hyperlipidemia, systemic inflammation and insulin resistance by acting directly on adipocytes.

Nod1/Nod2-mediated recognition plays a critical role in induction of adaptive immunity to anthrax after aerosol exposure.

Toll-like receptors and Nod-like receptors (NLR) play an important role in sensing invading microorganisms for pathogen clearance and eliciting adaptive immunity for protection against rechallenge. Nod1 and Nod2, members of the NLR family, are capable of detecting bacterial peptidoglycan motifs in the host cytosol for triggering proinflammatory cytokine production. In the current study, we sought to determine if Nod1/Nod2 are involved in sensing Bacillus anthracis infection and eliciting protective immune responses. Using mice deficient in both Nod1 and Nod2 proteins, we showed that Nod1/Nod2 are involved in detecting B. anthracis for production of tumor necrosis factor alpha, interleukin-1 alpha (IL-1 alpha), IL-1 beta, CCL5, IL-6, and KC. Proinflammatory responses were higher when cells were exposed to viable spores than when they were exposed to irradiated spores, indicating that recognition of vegetative bacilli through Nod1/Nod2 is significant. We also identify a critical role for Nod1/Nod2 in priming responses after B. anthracis aerosol exposure, as mice deficient in Nod1/Nod2 were impaired in their ability to mount an anamnestic antibody response and were more susceptible to secondary lethal challenge than wild-type mice.

Nonhematopoietic cells control the outcome of infection with Listeria monocytogenes in a nucleotide oligomerization domain 1-dependent manner.

We analyzed the defensive role of the cytosolic innate recognition receptor nucleotide oligomerization domain 1 (NOD1) during infection with Listeria monocytogenes. Mice lacking NOD1 showed increased susceptibility to systemic intraperitoneal and intravenous infection with high or low doses of L. monocytogenes, as measured by the bacterial load and survival. NOD1 also controlled dissemination of L. monocytogenes into the brain. The increased susceptibility to reinfection of NOD1(-/-) mice was not associated with impaired triggering of listeria-specific T cells, and similar levels of costimulatory molecules or activation of dendritic cells was observed. Higher numbers of F480(+) Gr1(+) inflammatory monocytes and lower numbers of F480(-) Gr1(+) neutrophils were recruited into the peritoneum of infected WT mice than into the peritoneum of infected NOD1(-/-) mice. We determined that nonhematopoietic cells accounted for NOD1-mediated resistance to L. monocytogenes in bone marrow radiation chimeras. The levels of NOD1 mRNA in fibroblasts and bone marrow-derived macrophages (BMM) were upregulated after infection with L. monocytogenes or stimulation with different Toll-like receptor ligands. NOD1(-/-) BMM, astrocytes, and fibroblasts all showed enhanced intracellular growth of L monocytogenes compared to WT controls. Gamma interferon-mediated nitric oxide production and inhibition of L. monocytogenes growth were hampered in NOD1(-/-) BMM. Thus, NOD1 confers nonhematopoietic cell-mediated resistance to infection with L. monocytogenes and controls intracellular bacterial growth in different cell populations in vitro.

The innate immune receptor Nod1 protects the intestine from inflammation-induced tumorigenesis.

There is growing evidence that the host innate immune system has a critical role in regulating carcinogenesis, but the specific receptors involved and the importance of their interaction with commensal bacteria need to be elucidated. Two major classes of innate immune receptors, the Toll-like receptors and Nod-like receptors, many of which are upstream of nuclear factor-kappaB, are involved in the detection of intestinal bacteria. The Toll-like receptors have been implicated in promoting colon tumorigenesis, but the role of Nod-like receptors in regulating tumorigenesis remains unclear. Using an established mouse model system of colitis-associated colon tumorigenesis, we show that Nod1 deficiency results in the increased development of both colitis-associated and Apc tumor suppressor-related colon tumors. In the absence of Nod1 signaling, there is a greater disruption of the intestinal epithelial cell barrier due to chemically induced injury as manifested by increased surface epithelial apoptosis early on during chemically induced colitis and increased intestinal permeability. The increased intestinal permeability is associated with enhanced inflammatory cytokine production and epithelial cell proliferation in Nod1-deficient mice as compared with wild-type mice. Depletion of the gut microbiota suppressed tumor development in Nod1-deficient mice, thus highlighting a link between the commensal bacteria within the intestine and the host innate immune Nod1 signaling pathway in the regulation inflammation-mediated colon cancer development.

Cross-tolerization between Nod1 and Nod2 signaling results in reduced refractoriness to bacterial infection in Nod2-deficient macrophages.

Nod2 is an intracellular innate immune receptor that plays a role in host defense and susceptibility to inflammatory disease. We show in this study that macrophages rendered refractory to TLR4 and Nod2 signaling by exposure to LPS and muramyl dipeptide (MDP) exhibit impaired TNF-alpha and IL-6 production in response to pathogenic Listeria monocytogenes and Yersinia pseudotuberculosis as well as commensal bacteria including Escherichia coli and Bacteroides fragilis. Surprisingly, Nod2 deficiency was associated with impaired tolerization in response to pathogenic and commensal bacteria. Mechanistically, reduced tolerization of Nod2-null macrophages was mediated by recognition of bacteria through Nod1 because it was abolished in macrophages deficient in Nod1 and Nod2. Consistently, Nod2-null macrophages tolerant to LPS and MDP showed enhanced production of TNF-alpha and IL-6 as well as increased NF-kappaB and MAPK activation in response to the dipeptide KF1B, the Nod1 agonist. Furthermore, reduced tolerization of Nod2-deficient macrophages in response to bacteria was abolished when mutant macrophages were also rendered tolerant to the Nod1 ligand. Finally, MDP stimulation induced refractoriness not only to MDP, but also to iE-DAP stimulation, providing a mechanism to explain the reduced tolerization of Nod2-deficient macrophages infected with bacteria. These results demonstrate that cross-tolerization between Nod1 and Nod2 leads to increase recognition of both pathogenic and commensal bacteria in Nod2-deficient macrophages pre-exposed to microbial ligands.

Differential function of the NACHT-LRR (NLR) members Nod1 and Nod2 in arthritis.

The pathogenesis of chronic joint inflammation remains unclear, although the involvement of pathogen recognition receptors has been suggested recently. In the present article, we describe the role of two members of the NACHT-LRR (NLR) family, Nod1 (nucleotide-binding oligomerization domain) and Nod2 in a model of acute joint inflammation induced by intraarticular injection of Streptococcus pyogenes cell wall fragments. Here, we show that Nod2 deficiency resulted in reduced joint inflammation and protection against early cartilage damage. In contrast, Nod1 gene-deficient mice developed enhanced joint inflammation with concomitant elevated levels of proinflammatory cytokines and cartilage damage, consistent with a model in which Nod1 controls the inflammatory reaction. To explore whether the different function of Nod1 and Nod2 occurs also in humans, we exposed peripheral blood mononuclear cells (PBMCs) carrying either Nod1ins/del or Nod2fs mutation with SCW fragments in vitro. Production of both TNFalpha and IL-1beta was clearly impaired in PBMCs carrying the Nod2fs compared with PBMCs isolated from healthy controls. In line with results in Nod1 gene-deficient mice, PBMCs from individuals bearing a newly described Nod1 mutation produced enhanced levels of proinflammatory cytokines after 24-h stimulation with SCW fragments. These data indicate that the NLR family members Nod1 and Nod2 have different functions in controlling inflammation, and that intracellular Nod1-Nod2 interactions may determine the severity of arthritis in this experimental model. Whether a distorted balance between the function of Nod1 and/or Nod2 is involved in the pathogenesis of human autoinflammatory or autoimmune disease, such as rheumatoid arthritis, remains to be elucidated.

Nod1 and Nod2 induce CCL5/RANTES through the NF-kappaB pathway.

The Nod-like receptor proteins Nod1 and Nod2 participate in innate immune responses against bacteria through intracellular detection of peptidoglycan, a component of bacterial cell wall. Recent evidence has demonstrated that Nod1 stimulates the release of chemokines that attract neutrophils at the site of infection, such as CXCL8/IL-8 in humans, and CXCL1/keratinocyte-derived chemokine and CXCL2/MIP-2 in mice. We aimed to determine whether Nod proteins could trigger the release of CCL5/RANTES, a chemokine known to attract a number of immune cells, but not neutrophils. Our results demonstrate that activation of both Nod1 and Nod2 results in substantial secretion of CCL5 by murine macrophages. Moreover, in vivo, the intraperitoneal injection of murine Nod1 or Nod2 agonists resulted in a rapid secretion of CCL5 into the bloodstream. We also observed that Nod-dependent secretion of CCL5 did not correlate with the induction of the interferon-beta pathway, a major signaling cascade for the activation of CCL5 by viruses. In contrast, we identified a key role of the NF-kappaB pathway in Nod-dependent stimulation of the CCL5 promoter. Together, these results identify a novel target downstream of Nod1 and Nod2, which is likely to play a key role in orchestrating the global Nod-dependent immune defense during bacterial infections.

Nod1/RICK and TLR signaling regulate chemokine and antimicrobial innate immune responses in mesothelial cells.

Mesothelial cells that line the serous cavities and outer surface of internal organs are involved in inflammatory responses induced by microbial stimuli and bacterial infection. Upon exposure to bacterial products, mesothelial cells secrete chemokines, but the signaling pathways by which these cells recognize bacteria to mediate innate immune responses remain largely unknown. We report that stimulation of primary peritoneal mesothelial cells via nucleotide-binding oligomerization domain (Nod)1, a member of the intracytoplasmic Nod-like receptor family, induced potent secretion of the chemokines CXCL1 and CCL2 as well as expression of inducible NO synthase and such responses required the kinase RICK. Mesothelial cells also produced chemokines in response to TLR2, TLR3, TLR4, and TLR5 agonists, but unlike that induced by Nod1 stimulation, the TLR-mediated responses were independent of RICK. Yet, Nod1 stimulation of mesothelial cells via RICK enhanced chemokine secretion induced by LPS or IFN-gamma and cooperated with IFN-gamma in the production of NO. The i.p. administration of KF1B, a synthetic Nod1 agonist, elicited chemokine production in the serum and peritoneal fluid as well as the recruitment of neutrophils into the peritoneal cavity of wild-type mice, but not RICK-deficient mice. Finally, infection of mesothelial cells with Listeria monocytogenes induced production of CXCL1 and this response was significantly reduced in Nod1- or RICK-deficient cells. These results define mesothelial cells as microbial sensors through TLRs and Nod-like receptors and identify Nod1 and RICK as important mediators of chemokine and antimicrobial responses in mesothelial cells.

RICK/RIP2 mediates innate immune responses induced through Nod1 and Nod2 but not TLRs.

RICK is a kinase that has been implicated in Nod1 and Nod2 signaling. In addition, RICK has been proposed to mediate TLR signaling in that its absence confers reduced responses to certain bacterial products such as LPS. We show here that macrophages and mice lacking RICK are defective in their responses to Nod1 and Nod2 agonists but exhibit unimpaired responses to synthetic and highly purified TLR agonists. Furthermore, production of chemokines induced by the bacterial dipeptide gamma-d-glutamyl-meso-diaminopimelic acid was intact in MyD88 deficient mice but abolished in RICK-null mice. Stimulation of macrophages with muramyl dipeptide, the Nod2 activator, enhanced immune responses induced by LPS, IFN-gamma, and heat-killed Listeria in wild-type but not in RICK- or Nod2-deficient macrophages. Finally, we show that the absence of RICK or double deficiency of Nod1 and Nod2 was associated with reduced cytokine production in Listeria-infected macrophages. These results demonstrate that RICK functions in innate immunity by mediating Nod1 and Nod2 signaling but not TLR-mediated immune responses.