Nod1, a CARD protein,enhances pro-interleukin-1beta processing through the interaction with pro-caspase-1

The production of bioactive interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, is mediated by activated caspase-1. One of the known molecular mechanisms underlying pro-caspase-1 processing and activation involves interaction between the caspase recruit domains (CARDs) of caspase-1 and a serine/threonine kinase RIP2. While the association of Nod1 with both caspase-1 and RIP2 is already known, the consequences of these interactions are poorly understood. Because Nod1 also binds to RIP2, we hypothesized that Nod1 plays a role in pro-caspase-1 activation and IL-1beta processing. We show here that Nod1 binds to both RIP2 and caspase-1 by CARD interactions. Nod1 enhances pro-caspase-1 oligomerization and pro-caspase-1 processing. Nod1 enhances caspase-1-induced IL-1beta secretion, as well as lipopolysaccharide (LPS)-induced IL-1beta secretion in transfected cells. Moreover, HT1080 cells stably transfected with Nod1 showed higher LPS-induced IL-1beta secretion than non-transfected cells, suggesting a role of Nod1 in LPS-induced responses. Our data indicate that Nod1 can regulate IL-1beta secretion, implying that Nod1 may play a role in inflammatory responses to bacterial LPS.     

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.     

Cutting edge: Crohn's disease-associated Nod2 mutation limits production of proinflammatory cytokines to protect the host from Enterococcus faecalis-induced lethality

Nucleotide-binding oligomerization domain 2 (Nod2) mutations including L1007fsinsC are associated with the development of Crohn's disease (CD). These CD-associated Nod2 mutations are common in healthy white populations, suggesting that they may confer some protective function, but experimental evidence is lacking. Using a mouse strain that expresses Nod2(2939iCstop), the equivalent of the L1007fsinsC mutation, we found that macrophages homozygous for Nod2(2939iCstop) are impaired in the recognition of muramyl dipeptide and Enterococcus faecalis, a commensal bacterium that is a common cause of sepsis-associated lethality in humans. Notably, Nod2 deficiency and homozygocity for Nod2(2939iCstop) were associated with reduced production of TNF-α and IL-6 and lethality after systemic infection with E. faecalis despite normal bacteria loads. Consistently, inhibition of TNF-α signaling protected wild-type mice from E. faecalis-induced lethality. These results suggest that the same Nod2 mutation can increase the susceptibility to CD, but also protect the host from systemic infection by a common enteric bacterium.     

Murine splenocytes produce inflammatory cytokines in a MyD88-dependent response to Bacillus anthracis spores

Bacillus anthracis is a sporulating Gram-positive bacterium that causes the disease anthrax. The highly stable spore is the infectious form of the bacterium that first interacts with the prospective host, and thus the interaction between the host and spore is vital to the development of disease. We focused our study on the response of murine splenocytes to the B. anthracis spore by using paraformaldehyde-inactivated spores (FIS), a treatment that prevents germination and production of products associated with vegetative bacilli. We found that murine splenocytes produce IL-12 and IFN-gamma in response to FIS. The IL-12 was secreted by CD11b cells, which functioned to induce the production of IFN-gamma by CD49b (DX5) NK cells. The production of these cytokines by splenocytes was not dependent on TLR2, TLR4, TLR9, Nod1, or Nod2; however, it was dependent on the signalling adapter protein MyD88. Unlike splenocytes, Nod1- and Nod2-transfected HEK cells were activated by FIS. Both IL-12 and IFN-gamma secretion were inhibited by treatment with B. anthracis lethal toxin. These observations suggest that the innate immune system recognizes spores with a MyD88-dependent receptor (or receptors) and responds by secreting inflammatory cytokines, which may ultimately aid in resisting infection.     

NOD2-deficient mice have impaired resistance to Mycobacterium tuberculosis infection through defective innate and adaptive immunity

NOD2/CARD15 mediates innate immune responses to mycobacterial infection. However, its role in the regulation of adaptive immunity has remained unknown. In this study, we examined host defense, T cell responses, and tissue pathology in two models of pulmonary mycobacterial infection, using wild-type and Nod2-deficient mice. During the early phase of aerosol infection with Mycobacterium tuberculosis, Nod2(-/-) mice had similar bacterial counts but reduced inflammatory response on histopathology at 4 and 8 wk postchallenge compared with wild-type animals. These findings were confirmed upon intratracheal infection of mice with attenuated Mycobacterium bovis bacillus Calmette-Guérin. Analysis of the lungs 4 wk after bacillus Calmette-Guérin infection demonstrated that Nod2(-/-) mice had decreased production of type 1 cytokines and reduced recruitment of CD8(+) and CD4(+) T cells. Ag-specific T cell responses in both the spleens and thoracic lymph nodes were diminished in Nod2(-/-) mice, indicating impaired adaptive antimycobacterial immunity. The immune regulatory role of NOD2 was not restricted to the lung since Nod2 disruption also led to reduced type 1 T cell activation following i.m. bacillus Calmette-Guérin infection. To determine the importance of diminished innate and adaptive immunity, we measured bacterial burden 6 mo after aerosol infection with M. tuberculosis and followed a second infected group for assessment of survival. Nod2(-/-) mice had a higher bacterial burden in the lungs 6 mo after infection and succumbed sooner than did wild-type controls. Taken together, these data indicate that NOD2 mediates resistance to mycobacterial infection via both innate and adaptive immunity.     

Nod1-mediated lipolysis promotes diacylglycerol accumulation and successive inflammation via PKCδ-IRAK axis in adipocytes

Chronic inflammation contributes to obesity mediated metabolic disturbances, including insulin resistance. Obesity is associated with altered microbial load in metabolic tissues that can contribute to metabolic inflammation. Different bacterial components such as, LPS, peptidoglycans have been shown to underpin metabolic disturbances through interaction with host innate immune receptors. Activation of Nucleotide-binding oligomerization domain-containing protein 1 (Nod1) with specific peptidoglycan moieties promotes insulin resistance, inflammation and lipolysis in adipocytes. However, it was not clear how Nod1-mediated lipolysis and inflammation is linked. Here, we tested if Nod1-mediated lipolysis caused accumulation of lipid intermediates and promoted cell autonomous inflammation in adipocytes. We showed that Nod1-mediated lipolysis caused accumulation of diacylglycerol (DAG) and activation of PKCδ in 3T3-L1 adipocytes, which was prevented with a Nod1 inhibitor. Nod1-activated PKCδ caused downstream stimulation of IRAK1/4 and was associated with increased expression of proinflammatory cytokines such as, IL-1β, IL-18, IL-6, TNFα and MCP-1. Pharmacological inhibition or siRNA mediated knockdown of IRAK1/4 attenuated Nod1-mediated activation of NF-κB, JNK, and the expression of proinflammatory cytokines. These results reveal that Nod1-mediated lipolysis promoted accumulation of DAG, which engaged PKCδ and IRAK1/4 to augment inflammation in 3T3-L1 adipocytes.     

Effective T-cell immune responses in the absence of the serine/threonine kinase RIP2

The serine/threonine kinase RIP2 has been reported to be essential for Nod1 and Nod2 mediated cell activation, and has been suggested to play a role in the signaling cascade downstream of the T-cell receptor. We sought to ascertain the exact role of RIP2 in T-helper cell differentiation and CD8+ T-cell effector function in vivo and in vitro. In contrast to previous reports, we found that RIP2-deficient T cells did not exhibit impaired proliferation upon TCR engagement in vitro, and differentiation to cytokine producing Th1 or Th2 cells was normal in the absence of RIP2. These results were confirmed in vivo, as wild-type and RIP2-deficient virus-specific CD8+ T cells expanded comparably in mice after LCMV infection. Wild-type and RIP2-deficient CD4+ and CD8+ T cells from infected mice also showed similar proliferation and cytokine production when restimulated with full or partial agonist peptides ex vivo. Furthermore, no significant difference in adaptive T-cell responses could be observed between wild-type and RIP2-deficient mice after Listeria monocytogenes infection. Thus contrary to early reports, our data show that RIP2 is not an essential component of the TCR signaling machinery.     

A role for Erbin in the regulation of Nod2-dependent NF-kappaB signaling

Nod2 is an intracellular sensor of a specific bacterial cell wall component, muramyl dipeptide, and activation of Nod2 stimulates an inflammatory response. Specific mutations of Nod2 have been associated with two inflammatory diseases, Crohn disease and Blau syndrome, and are thought to contribute to disease susceptibility through altering Nod2 signaling. Association of disease with inappropriate activation of Nod2 highlights the importance of proper regulation of Nod2 activity. However, little is known about specific regulation of the Nod2 pathway. We performed a biochemical screen to discover potential regulators of Nod2 and identified Erbin, a protein involved in cell polarity, receptor localization, and regulation of the mitogen-activated protein kinase pathway, as a novel Nod2-interacting protein. In our studies, we demonstrate specific interaction of Erbin and Nod2 both in vitro and in vivo and characterize the regions required for interaction in both proteins. We found that Nod2-dependent activation of NF-kappaB and cytokine secretion is inhibited by Erbin overexpression, whereas Erbin-/- mouse embryo fibroblasts show an increased sensitivity to muramyl dipeptide. These studies identify Erbin as a regulator of Nod2 signaling and demonstrate a novel role for Erbin in inflammatory responses.     

Genetic variation and activity of mouse Nod2, a susceptibility gene for Crohn's disease

Genetic variation in human Nod2 has been associated with susceptibility to Crohn's disease. The mouse Nod2 locus is located at chromosome 8 and composed of 12 exons, 11 of which encode the Nod2 protein. Sequence analysis of Nod2 from 45 different strains of Mus musculus and Mus spretus revealed extensive polymorphism involving all exons of Nod2. Of the 140 polymorphic sites identified, 68 were located in the coding region, of which 28 created amino acid substitutions in Nod2. Expression of mouse Nod2 activated NF-kappaB and conferred responsiveness to bacterial components, an activity that was deficient in mutants corresponding to those associated with susceptibility to Crohn's disease. These studies demonstrate a conserved role for Nod2 in the response to bacterial components and suggest that selective evolutionary pressure exerted by pathogens may have contributed to the high level of variability of Nod2 sequences in both humans and mice.