Attenuation of apoptotic cell detection triggers thymic regeneration after damage.

The thymus, which is the primary site of T cell development, is particularly sensitive to insult but also has a remarkable capacity for repair. However, the mechanisms orchestrating regeneration are poorly understood, and delayed repair is common after cytoreductive therapies. Here, we demonstrate a trigger of thymic regeneration, centered on detecting the loss of dying thymocytes that are abundant during steady-state T cell development. Specifically, apoptotic thymocytes suppressed production of the regenerative factors IL-23 and BMP4 via TAM receptor signaling and activation of the Rho-GTPase Rac1, the intracellular pattern recognition receptor NOD2, and micro-RNA-29c. However, after damage, when profound thymocyte depletion occurs, this TAM-Rac1-NOD2-miR29c pathway is attenuated, increasing production of IL-23 and BMP4. Notably, pharmacological inhibition of Rac1-GTPase enhanced thymic function after acute damage. These findings identify a complex trigger of tissue regeneration and offer a regenerative strategy for restoring immune competence in patients whose thymic function has been compromised.

NOD2 deficiency confers a pro-tumorigenic macrophage phenotype to promote lung adenocarcinoma progression.

Nucleotide-binding and oligomerization domain-containing protein 2 (NOD2) was a member of the NOD-like receptor family and played an important role in the innate immune response. Dysregulated NOD2 had been reported to contribute to tumorigenesis and progression. Here, we investigated that decreased NOD2 expressions could affect the phenotypic polarization of tumour-associated macrophages and thus lead to the poor prognosis of lung adenocarcinoma patients. We clustered the patients by the single-sample gene set enrichment analysis of tumour microenvironment and 13 prognostic differentially expressed immune-related genes (PDEIRGs) were obtained based on prognostic analyses. After multiple assessments on the 13 PDEIRGs, NOD2 was considered to be the central immune gene and had a strong effect on suppressing tumour progression. Decreased NOD2 expression could be induced by cancer cells and lead to the phenotypic polarization of macrophages from protective M1 phenotype to pro-tumorigenic M2 subtype which might be attributed to the down-regulating of NF-κB signalling pathway. This study draw attention to the role of inhibited innate immune function mediated by depletion of NOD2 in the TME. Our work also points to a potential strategy of NOD2-mediated TAM-targeted immunotherapy.

Intestinal lysozyme releases Nod2 ligand(s) to promote the intestinal mucosal adjuvant activity of cholera toxin.

Commensal bacteria boost serum IgG production in response to oral immunization with antigen and cholera toxin (CT) in a manner that depends on Nod2 (nucleotide-binding oligomerization domain-containing protein 2). In this study, we examined the role of intestinal lysozyme (Lyz1) in adjuvant activity of CT. We found that Lyz1 released Nod2 ligand(s) from bacteria. Lyz1 deficiency reduced the level of circulating Nod2 ligand in mice. Lyz1 deficiency also reduced the production of IgG and T-cellspecific cytokines after oral immunization in mice. Supplementing Lyz1-deficient mice with MDP restored IgG production. Furthermore, overexpression of Lyz1 in intestinal epithelium boosted the antigen-specific IgG response induced by CT. Collectively, our results indicate that Lyz1 plays an important role in mediating the immune regulatory effect of commensal bacteria through the release of Nod2 ligand(s).

NOD2 deficiency increases retrograde transport of secretory IgA complexes in Crohn's disease.

Intestinal microfold cells are the primary pathway for translocation of secretory IgA (SIgA)-pathogen complexes to gut-associated lymphoid tissue. Uptake of SIgA/commensals complexes is important for priming adaptive immunity in the mucosa. This study aims to explore the effect of SIgA retrograde transport of immune complexes in Crohn's disease (CD). Here we report a significant increase of SIgA transport in CD patients with NOD2-mutation compared to CD patients without NOD2 mutation and/or healthy individuals. NOD2 has an effect in the IgA transport through human and mouse M cells by downregulating Dectin-1 and Siglec-5 expression, two receptors involved in retrograde transport. These findings define a mechanism of NOD2-mediated regulation of mucosal responses to intestinal microbiota, which is involved in CD intestinal inflammation and dysbiosis.

NOD2 Deficiency Promotes Intestinal CD4+ T Lymphocyte Imbalance, Metainflammation, and Aggravates Type 2 Diabetes in Murine Model.

Type 2 diabetes (T2D) is a metabolic disease characterized by increased inflammation, NOD-like receptors (NLRs) activation and gut dysbiosis. Our research group has recently reported that intestinal Th17 response limits gut dysbiosis and LPS translocation to visceral adipose tissue (VAT), protecting against metabolic syndrome. However, whether NOD2 receptor contributes intestinal Th17 immunity, modulates dysbiosis-driven metabolic tissue inflammation, and obesity-induced T2D remain poorly understood. In this context, we observed that mice lacking NOD2 fed a high-fat diet (HFD) display severe obesity, exhibit greater adiposity, and more hepatic steatosis compared to HFD-fed wild-type (WT) mice. In addition, they develop increased hyperglycemia, worsening of glucose intolerance, and insulin resistance. Notably, the deficiency of NOD2 causes a deviation from M2 macrophage and regulatory T cells (Treg) to M1 macrophage and mast cells into VAT compared to WT mice fed HFD. An imbalance was also observed in Th17/Th1 cell populations, with reduced IL-17 and IL-22 gene expression in the mesenteric lymph nodes (MLNs) and ileum, respectively, of NOD2-deficient mice fed HFD. 16S rRNA sequencing indicates lower richness, alpha diversity, and a depletion of Allobaculum, Lactobacillus, and enrichment with Bacteroides genera in these mice compared to HFD-fed WT mice. These alterations were associated with disrupted tight-junctions expression, augmented serum LPS, and bacterial translocation into VAT. Overall, NOD2 activation is required for a protective Th17 over Th1 immunity in the gut, which seems to decrease gram-negative bacteria outgrowth in gut microbiota, attenuating the endotoxemia, metainflammation, and protecting against obesity-induced T2D.

NOD2 in hepatocytes engages a liver-gut axis to protect against steatosis, fibrosis, and gut dysbiosis during fatty liver disease in mice.

Obesity promotes nonalcoholic fatty liver disease (NAFLD). The intestinal microbiota contributes to NAFLD progression through a gut-to-liver pathway that promotes inflammation and fibrosis. Gut microbiota-derived factors can travel to the liver and activate immune responses in liver resident cells to promote inflammation and NAFLD. Little is known about bacterial sensors or immune responses that can protect against NAFLD. We tested whether the bacterial cell wall sensor nucleotide-binding oligomerization domain-containing (NOD)2 protects against diet-induced NAFLD in mice. Whole body deletion of NOD2 exacerbated liver steatosis and fibrosis in mice fed a NAFLD-promoting diet. Mice with a hepatocyte-specific deletion of NOD2 (Nod2-/-HKO) also had higher liver steatosis and fibrosis compared with littermate wild-type mice (WT) fed a NAFLD-promoting diet. Hepatocyte-specific NOD2 deletion altered the composition of the gut microbiome. Nod2-/-HKO mice had increased relative abundance of Clostridiales and lower Erysipelotrichaceae among other changes in cecal bacteria compared with littermate WT mice. Hepatocyte-specific NOD2 deletion altered a transcriptional program of liver inflammation, metabolism, and fibrosis. Nod2-/-HKO mice had higher levels of transcripts involved in lipid and cholesterol metabolism. Nod2-/-HKO mice had higher transcript levels of transforming growth factor-ß and collagen isoforms, which coincided with higher levels of liver collagen compared with WT mice. These data show that bacterial cell wall sensing within hepatocytes can engage retrograde cross-talk from the liver to the gut, where liver immunity communicates with the gut to influence the intestinal host-microbe relationship during diet-induced NAFLD, and NOD2 within the hepatocyte confers protection from liver steatosis and fibrosis.

Deficiency of Nucleotide-binding oligomerization domain-containing proteins (NOD) 1 and 2 reduces atherosclerosis.

Atherosclerosis is crucially fueled by inflammatory pathways including pattern recognition receptor (PRR)-related signaling of the innate immune system. Currently, the impact of the cytoplasmic PRRs nucleotide-binding oligomerization domain-containing protein (NOD) 1 and 2 is incompletely characterized. We, therefore, generated Nod1/Nod2 double knockout mice on a low-density lipoprotein receptor (Ldlr)-deficient background (= Ldlr-/-Nod1/2-/-) which were subsequently analyzed regarding experimental atherosclerosis, lipid metabolism, insulin resistance and gut microbiota composition. Compared to Ldlr-/- mice, Ldlr-/-Nod1/2-/- mice showed reduced plasma lipids and increased hepatic expression of the scavenger receptor LDL receptor-related protein 1 after feeding a high-fat diet for 12 weeks. Furthermore, intestinal cholesterol and its bacterial degradation product coprostanol were elevated in Ldlr-/-Nod1/2-/- mice, correlating with the increased abundance of Eubacterium coprostanoligenes as assessed by 3rd generation sequencing of the gut microbiota. Atherosclerotic plaques of Ldlr-/-Nod1/2-/- mice exhibited less lipid deposition and macrophage accumulation. Moreover, macrophages from Ldlr-/-Nod1/2-/- mice showed higher expression of the cholesterol efflux transporters Abca1 and Abcg1 and accordingly reduced foam cell formation. Deficiency of Nod1 and Nod2 led to reduced plaque lipid deposition and inflammatory cell infiltration in atherosclerotic plaques. This might be explained by diminished plasma lipid levels and foam cell formation due to altered expression of key regulators of the hepatic cholesterol pathway as well as differential intestinal cholesterol metabolism and microbiota composition.

NOD2 Influences Trajectories of Intestinal Microbiota Recovery After Antibiotic Perturbation.

BACKGROUND & AIMS: Loss-of-function variants in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) impair the recognition of the bacterial cell wall component muramyl-dipeptide and are associated with an increased risk for developing Crohn's disease. Likewise, exposure to antibiotics increases the individual risk for developing inflammatory bowel disease. Here, we studied the long-term impact of NOD2 on the ability of the gut bacterial and fungal microbiota to recover after antibiotic treatment. METHODS: Two cohorts of 20-week-old and 52-week-old wild-type (WT) C57BL/6J and NOD2 knockout (Nod2-KO) mice were treated with broad-spectrum antibiotics and fecal samples were collected to investigate temporal dynamics of the intestinal microbiota (bacteria and fungi) using 16S ribosomal RNA and internal transcribed spacer 1 sequencing. In addition, 2 sets of germ-free WT mice were colonized with either WT or Nod2-KO after antibiotic donor microbiota and the severity of intestinal inflammation was monitored in the colonized mice. RESULTS: Antibiotic exposure caused long-term shifts in the bacterial and fungal community composition. Genetic ablation of NOD2 was associated with delayed body weight gain after antibiotic treatment and an impaired recovery of the bacterial gut microbiota. Transfer of the postantibiotic fecal microbiota of Nod2-KO mice induced an intestinal inflammatory response in the colons of germ-free recipient mice compared with respective microbiota from WT controls based on histopathology and gene expression analyses. CONCLUSIONS: Our data show that the bacterial sensor NOD2 contributes to intestinal microbial community composition after antibiotic treatment and may add to the explanation of how defects in the NOD2 signaling pathway are involved in the etiology of Crohn's disease.

NOD2 regulates microglial inflammation through the TAK1-NF-κB pathway and autophagy activation in murine pneumococcal meningitis.

Streptococcus pneumoniae is responsible for pneumococcal meningitis, with significant mortality and morbidity worldwide. Microglial inflammation plays a vital role in meningitis. The peptidoglycan sensor NOD2 (nucleotide-binding oligomerization domain 2) has been identified to promote microglia activation, but the role in autophagy following pneumococcal meningitis remains unclear. In the present study, we investigated the role of NOD2 in microglial inflammation and autophagy, as well as related signaling pathways, during S. pneumonia infection. NOD2 expression was knocked down by the injection of lentivirus-mediated short-hairpin RNA (shRNA). Our results revealed that NOD2 promotes microglial inflammation by increasing inflammatory mediators. We also showed that the TAK1-NF-κB pathway is involved in this process. In addition, NOD2 increased the expression of autophagy-related proteins and induced autophagosome formation. Rapamycin and 3-MA were utilized to assess the role of autophagy in microglial inflammation induced by S. pneumonia. We demonstrated that autophagy serves as a cellular defense mechanism to reduce inflammatory mediators. Similar to the in vitro results, NOD2 induced inflammation and autophagy in the brain in a mouse meningitis model. Moreover, NOD2 silencing significantly reduced brain edema and improved the neurological function of pneumococcal meningitis mice. Taken together, these data demonstrate that NOD2 promotes microglial inflammation and autophagy in murine pneumococcal meningitis, and the TAK1-NF-κB pathway is involved in microglial activation.

NOD2 Supports Crypt Survival and Epithelial Regeneration after Radiation-Induced Injury.

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) affords stem cell protection and links microbes to intestinal epithelial regeneration. We investigated whether NOD2 status is associated with crypt survival and intestinal epithelial regeneration independent of microbiota-derived molecules. To assess crypt survival, a clonogenic microcolony assay was performed with 15 Gy of X-ray irradiation. The fractional crypt survival rate (46.0 ± 15.5% vs. 24.7 ± 9.2%, p < 0.01) and fractional EdU-positive crypt survival rate (29.8 ± 14.5% vs. 9.79 ± 4.37%, p = 0.015) were significantly decreased in the NOD2-/- mice compared with the wild-type (WT) mice at 3.5 days after irradiation. To evaluate intestinal epithelial regeneration capability, organoid reconstitution assays were performed. Small bowel crypts of the WT and NOD2-/- mice were isolated and seeded into Matrigel for 3D culture. In the organoid reconstitution assays, the number of organoids formed did not differ between the NOD2-/- and WT mice. Organoid formation ability was also assessed after exposure to 5 Gy irradiation. Organoid formation ability was significantly decreased in the NOD2-/- mice compared with the WT ones after exposure to 5 Gy irradiation (33.2 ± 5.9 vs. 19.7 ± 8.8/well, p < 0.01). NOD2 supports crypt survival after potentially lethal irradiation damage and is associated with intestinal epithelial regeneration.

Nod2 deficiency functionally impairs adaptation to short bowel syndrome via alterations of the epithelial barrier function.

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) gene mutations are a risk factor for Crohn's disease and also associated with worse outcome in short bowel syndrome (SBS) patients independent of the underlying disease. The aim of this study was to analyze the effect of Nod2 deficiency on barrier function and stool microbiome after extensive ileocecal resection in mice. Male C57BL6/J wild-type (WT) and Nod2-knockout (KO) mice underwent 40% ileocecal resection. Sham control mice received simple transection of the ileum. Clinical outcome was monitored daily. Barrier function was measured with Ussing chambers using FITC-4-kDa-Dextran flux, transmucosal electrical resistance, and dilution potentials. Immunofluorescence of claudin-2 was studied. Composition of the stool microbiome was assessed by 16S rRNA gene sequencing. Resected Nod2-KO mice had impaired clinical outcome compared with resected WT mice. This was accompanied by increased stool water contents and increased plasma aldosterone. Histomorphological adaptation was independent of Nod2. Barrier function studies revealed impaired sodium to chloride permeability and altered claudin-2 localization in the absence of Nod2. Resection induced decreases of bacterial diversity and a shift of bacteriodetes-to-firmicutes ratios. Ileum and cecum resection-induced increase in proteobacteria was absent in Nod2-deficient mice. Verrucomicrobia were temporarily increased in Nod2-KO mice. Nod2 deficiency functionally impairs adaptation to short bowel syndrome via a lesser increase of epithelial sodium pore permeability, altered epithelial barrier function, and the microbiome.NEW & NOTEWORTHYNOD2 gene mutations are associated with the development of severe short bowel syndrome and intestinal failure. The influence of Nod2 mutations on intestinal adaptation in experimental short bowel syndrome has not been studied yet. Here, we provide data that Nod2 deficiency worsens clinical outcome and functional adaptation under SBS conditions in mice, indicating that NOD2 is required for successful adaptation after ileocecal resection.

Innate lymphoid cells support regulatory T cells in the intestine through interleukin-2.

Interleukin (IL)-2 is a pleiotropic cytokine that is necessary to prevent chronic inflammation in the gastrointestinal tract1-4. The protective effects of IL-2 involve the generation, maintenance and function of regulatory T (Treg) cells4-8, and the use of low doses of IL-2 has emerged as a potential therapeutic strategy for patients with inflammatory bowel disease9. However, the cellular and molecular pathways that control the production of IL-2 in the context of intestinal health are undefined. Here we show, in a mouse model, that IL-2 is acutely required to maintain Treg cells and immunological homeostasis throughout the gastrointestinal tract. Notably, lineage-specific deletion of IL-2 in T cells did not reduce Treg cells in the small intestine. Unbiased analyses revealed that, in the small intestine, group-3 innate lymphoid cells (ILC3s) are the dominant cellular source of IL-2, which is induced selectively by IL-1ß. Macrophages in the small intestine produce IL-1ß, and activation of this pathway involves MYD88- and NOD2-dependent sensing of the microbiota. Our loss-of-function studies show that ILC3-derived IL-2 is essential for maintaining Treg cells, immunological homeostasis and oral tolerance to dietary antigens in the small intestine. Furthermore, production of IL-2 by ILC3s was significantly reduced in the small intestine of patients with Crohn's disease, and this correlated with lower frequencies of Treg cells. Our results reveal a previously unappreciated pathway in which a microbiota- and IL-1ß-dependent axis promotes the production of IL-2 by ILC3s to orchestrate immune regulation in the intestine.

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.

B Cell Defects Observed in Nod2 Knockout Mice Are a Consequence of a Dock2 Mutation Frequently Found in Inbred Strains.

Phenotypic differences among substrains of laboratory mice due to spontaneous mutations or pre-existing genetic variation confound the interpretation of targeted mutagenesis experiments and contribute to challenges with reproducibility across institutions. Notably, C57BL/6 Hsd mice and gene-targeted mice that have been backcrossed to this substrain have been reported to harbor a duplication in exons 28 and 29 of Dock2 In this study, we demonstrate the presence of this Dock2 variant in the widely used Nod2-/- mice. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is a cytosolic innate immune receptor associated with inflammatory bowel disease susceptibility. Consistent with a role of NOD2 in an immunological disorder, Nod2-/- mice bred at our institution displayed multiple B cell defects including deficiencies in recirculating B cells, marginal zone B cells, and B1a cells in vivo, as well as defects in class switch recombination in vitro. However, we found that these effects are due to the Dock2 variant and are independent of Nod2 deletion. Despite originating from the same gene-targeted founder mice, Nod2-/- mice from another source did not harbor the Dock2 variant or B cell defects. Finally, we show that Dock2-/- mice display the same B cell defects as mice harboring the Dock2 variant, confirming that the variant is a loss-of-function mutation and is sufficient to explain the alterations to the B cell compartment observed in Nod2-/- mice. Our findings highlight the effects of confounding mutations from widely used inbred strains on gene-targeted mice and reveal new functions of DOCK2 in B cells.

Genetic deficiency of NOD2 confers resistance to invasive aspergillosis.

Invasive aspergillosis (IA) is a severe infection that can occur in severely immunocompromised patients. Efficient immune recognition of Aspergillus is crucial to protect against infection, and previous studies suggested a role for NOD2 in this process. However, thorough investigation of the impact of NOD2 on susceptibility to aspergillosis is lacking. Common genetic variations in NOD2 has been associated with Crohn's disease and here we investigated the influence of these  genetic variations on the anti-Aspergillus host response. A NOD2 polymorphism reduced the risk of IA after hematopoietic stem-cell transplantation. Mechanistically, absence of NOD2 in monocytes and macrophages increases phagocytosis leading to enhanced fungal killing, conversely, NOD2 activation reduces the antifungal potential of these cells. Crucially, Nod2 deficiency results in resistance to Aspergillus infection in an in vivo model of pulmonary aspergillosis. Collectively, our data demonstrate that genetic deficiency of NOD2 plays a protective role during Aspergillus infection.

No difference in renal injury and fibrosis between wild-type and NOD1/NOD2 double knockout mice with chronic kidney disease induced by ureteral obstruction.

BACKGROUND: Chronic kidney disease (CKD) is characterized by sustained tissue damage and ongoing tubulo-interstitial inflammation and fibrosis. Pattern recognition receptors (PRRs) including Toll-like receptors (TLRs) and NOD-like receptors (NLRs) can sense endogenous ligands released upon tissue damage, leading to sterile inflammation and eventually irreversible kidney disease. It is known that NOD1 and NOD2 contribute to the pathogenesis of various inflammatory diseases, including acute kidney injury. However their role in chronic kidney disease is largely unknown. The aim of this study was therefore to investigate the contribution of NOD1 and NOD2 in renal interstitial fibrosis and obstructive nephropathy. METHODS: To do so, we performed unilateral ureteral obstruction (UUO) in wild type (WT) and NOD1/NOD2 double deficient (DKO) mice and analysed renal damage, fibrosis and inflammation. Data were analysed using the non-parametric Mann-Whitney U-test. RESULTS: Minor changes in inflammatory response were observed in NOD1/2 DKO mice, while no effects were observed on renal injury and the development of fibrosis. CONCLUSION: No difference in renal injury and fibrosis between WT and NOD1/NOD2 DKO mice following obstructive nephropathy induced by ureteral obstruction.

LipL21 lipoprotein binding to peptidoglycan enables Leptospira interrogans to escape NOD1 and NOD2 recognition.

Leptospirosis is a widespread zoonosis, potentially severe in humans, caused by spirochetal bacteria, Leptospira interrogans (L. interrogans). Host defense mechanisms involved in leptospirosis are poorly understood. Recognition of lipopolysaccharide (LPS) and lipoproteins by Toll-Like Receptors (TLR)4 and TLR2 is crucial for clearance of leptospires in mice, yet the role of Nucleotide Oligomerization Domain (NOD)-like receptors (NOD)1 and NOD2, recognizing peptidoglycan (PG) fragments has not previously been examined. Here, we show that pathogenic leptospires escape from NOD1 and NOD2 recognition both in vitro and in vivo, in mice. We found that leptospiral PG is resistant to digestion by certain hydrolases and that a conserved outer membrane lipoprotein of unknown function, LipL21, specific for pathogenic leptospires, is tightly bound to the PG. Leptospiral PG prepared from a mutant not expressing LipL21 (lipl21-) was more readily digested than the parental or complemented strains. Muropeptides released from the PG of the lipl21- mutant, or prepared using a procedure to eliminate the LipL21 protein from the PG of the parental strain, were recognized in vitro by the human NOD1 (hNOD1) and NOD2 (hNOD2) receptors, suggesting that LipL21 protects PG from degradation into muropeptides. LipL21 expressed in E. coli also resulted in impaired PG digestion and NOD signaling. We found that murine NOD1 (mNOD1) did not recognize PG of L. interrogans. This result was confirmed by mass spectrometry showing that leptospiral PG was primarily composed of MurTriDAP, the natural agonist of hNOD1, and contained only trace amounts of the tetra muropeptide, the mNOD1 agonist. Finally, in transgenic mice expressing human NOD1 and deficient for the murine NOD1, we showed enhanced clearance of a lipl21- mutant compared to the complemented strain, or to what was observed in NOD1KO mice, suggesting that LipL21 facilitates escape from immune surveillance in humans. These novel mechanisms allowing L. interrogans to escape recognition by the NOD receptors may be important in circumventing innate host responses.

NOD2 (Nucleotide-Binding Oligomerization Domain 2) Is a Major Pathogenic Mediator of Coxsackievirus B3-Induced Myocarditis.

BACKGROUND: The cytoplasmatic pattern recognition receptor, NOD2 (nucleotide-binding oligomerization domain 2), belongs to the innate immune system and is among others responsible for the recognition of single-stranded RNA. With Coxsackievirus B3 (CVB3) being a single-stranded RNA virus, and the recent evidence that the NOD2 target, NLRP3 (NOD-like receptor family, pyrin domain containing 3) is of importance in the pathogenesis of CVB3-induced myocarditis, we aimed to unravel the role of NOD2 in CVB3-induced myocarditis. METHODS AND RESULTS: Endomyocardial biopsy NOD2 mRNA expression was higher in CVB3-positive patients compared with patients with myocarditis but without evidence of persistent CVB3 infection. Left ventricular NOD2 mRNA expression was also induced in CVB3-induced myocarditis versus healthy control mice. NOD2 knockdown(-/-) mice were rescued from the detrimental CVB3-mediated effects as shown by a reduced cardiac inflammation (less cardiac infiltrates and suppression of proinflammatory cytokines), cardiac fibrosis, apoptosis, lower CAR (Coxsackievirus and adenovirus receptor) expression and CVB3 copy number, and an improved left ventricular function in NOD2-/- CVB3 mice compared with wild-type CVB3 mice. In agreement, NOD2-/- decreased the CVB3-induced inflammatory response, CVB3 copy number, and apoptosis in vitro. NOD2-/- was further associated with a reduction in CVB3-induced NLRP3 expression and activity as evidenced by lower ASC (apoptosis-associated speck-like protein containing a CARD) expression, caspase 1 activity, or IL-1ß (interleukin-1ß) protein expression under in vivo and in vitro CVB3 conditions. CONCLUSIONS: NOD2 is an important mediator in the viral uptake and inflammatory response during the pathogenesis of CVB3 myocarditis.

Absence of Nucleotide-Oligomerization-Domain-2 Is Associated with Less Distinct Disease in Campylobacter jejuni Infected Secondary Abiotic IL-10 Deficient Mice.

Human Campylobacter jejuni-infections are progressively increasing worldwide. Despite their high prevalence and socioeconomic impact the underlying mechanisms of pathogen-host-interactions are only incompletely understood. Given that the innate immune receptor nucleotide-oligomerization-domain-2 (Nod2) is involved in clearance of enteropathogens, we here evaluated its role in murine campylobacteriosis. To address this, we applied Nod2-deficient IL-10-/- (Nod2-/- IL-10-/-) mice and IL-10-/- counterparts both with a depleted intestinal microbiota to warrant pathogen-induced enterocolitis. At day 7 following peroral C. jejuni strain 81-176 infection, Nod2 mRNA was down-regulated in the colon of secondary abiotic IL-10-/- and wildtype mice. Nod2-deficiency did neither affect gastrointestinal colonization nor extra-intestinal and systemic translocation properties of C. jejuni. Colonic mucin-2 mRNA was, however, down-regulated upon C. jejuni-infection of both Nod2-/- IL-10-/- and IL-10-/- mice, whereas expression levels were lower in infected, but also naive Nod2-/- IL-10-/- mice as compared to respective IL-10-/- controls. Remarkably, C. jejuni-infected Nod2-/- IL-10-/- mice were less compromised than IL-10-/- counterparts and displayed less distinct apoptotic, but higher regenerative cell responses in colonic epithelia. Conversely, innate as well as adaptive immune cells such as macrophages and monocytes as well as T lymphocytes and regulatory T-cells, respectively, were even more abundant in large intestines of Nod2-/- IL-10-/- as compared to IL-10-/- mice at day 7 post-infection. Furthermore, IFN-γ concentrations were higher in ex vivo biopsies derived from intestinal compartments including colon and mesenteric lymph nodes as well as in systemic tissue sites such as the spleen of C. jejuni infected Nod2-/- IL-10-/- as compared to IL10-/- counterparts. Whereas, at day 7 postinfection anti-inflammatory IL-22 mRNA levels were up-regulated, IL-18 mRNA was down-regulated in large intestines of Nod2-/- IL-10-/- vs. IL-10-/- mice. In summary, C. jejuni-infection induced less clinical signs and apoptosis, but more distinct colonic pro- and (of note) anti-inflammatory immune as well as regenerative cell responses in Nod2 deficient IL-10-/- as compared to IL-10-/- control mice. We conclude that, even though colonic Nod2 mRNA was down-regulated upon pathogenic challenge, Nod2-signaling is essentially involved in the well-balanced innate and adaptive immune responses upon C. jejuni-infection of secondary abiotic IL-10-/- mice, but does neither impact pathogenic colonization nor translocation.

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.