Pattern Recognition Receptor Nucleotide-binding Oligomerization Research Articles

Topical N-phosphonacetyl-l-aspartate is a dual action candidate for treating non-melanoma skin cancer.

Each year, 3.3 million Americans are diagnosed with non-melanoma skin cancers (NMSC) and an additional 40 million individuals undergo treatment of precancerous actinic keratosis lesions. The most effective treatments of NMSC (surgical excision and Mohs surgery) are invasive, expensive and require specialised training. More readily accessible topical therapies currently are 5-fluorouracil (a chemotherapeutic agent) and imiquimod (an immune modulator), but these can have significant side effects which limit their efficacy. Therefore, more effective and accessible treatments are needed for non-melanoma cancers and precancers. Our previous work demonstrated that the small molecule N-phosphonacetyl-L-aspartate (PALA) both inhibits pyrimidine nucleotide synthesis and activates pattern recognition receptor nucleotide-binding oligomerization domain 2. We propose that topical application of PALA would be an effective NMSC therapy, by combining the chemotherapeutic and immune modulatory features of 5-fluorouracil and imiquimod. Daily topical application of PALA to mouse skin was well tolerated and resulted in less irritation, fewer histopathological changes, and less inflammation than caused by either 5-fluorouracil or imiquimod. In an ultraviolet light-induced NMSC mouse model, topical PALA treatment substantially reduced the numbers, areas and grades of tumours, compared to vehicle controls. This anti-neoplastic activity was associated with increased expression of the antimicrobial peptide cathelicidin and increased recruitment of CD8+ T cells and F4/80+ macrophages to the tumours, demonstrating both immunomodulatory and anti-proliferative effects. These findings indicate that topical PALA is an excellent candidate as an effective alternative to current standard-of-care NMSC therapies.

Negative regulator NLRC3: Its potential role and regulatory mechanism in immune response and immune-related diseases.

NLRC3 is a member of the pattern recognition receptors nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) family, and plays a pivotal regulatory role in modulating the activation of immune cells. In macrophages, NLRC3 inhibits the activation of the NF-κB signaling pathway, the STING/TBK1 signaling pathway, and the formation of the inflammasome. In the context of T cells immune response, NLRC3 prevents the activation of T cells by regulating the function of dendritic cells and directly influencing the function of T cells. Different from other pattern recognition receptors, NLRC3 is more closely associated with regulatory activity than pathogens recognition, it influences the fates of cells, for example, prevents proliferation, promotes apoptosis and inhibits pyroptosis. These cellular functions regulated by NLRC3 are involved in the development processes of a variety of diseases, such as infectious disease, sterile inflammatory diseases, and cancer. However, its characteristics, function and regulatory mechanism in immune response and immune-related diseases have not been addressed fully. In this review, we elaborate the potential roles of NLRC3 from several different levels, include molecular mechanism, cellular functions in the immune-related diseases.

Macrophages, smooth muscle cells and NOD1 inhibition in advanced stages of atherosclerosis: A clue to plaque stabilization and atherothrombosis attenuation

Background and Aims: Macrophages and Smooth Muscle Cells (SMCs) are well known to have a preeminent role in plaque necrosis and rupture. These events, added to inflammation and thin layers of collagen unchain atherothrombosis, the main cause of Acute Coronary Syndromes (ACs). Pattern recognition receptor Nucleotide-Binding Oligomerization Domain-1 (NOD1) has previously been linked to inflammation and cardiovascular diseases. The aim of this work was to unveil the function of NOD1 in the plaque stabilization phenomena in the late stages of the disease.

Decidual and placental NOD1 is associated with inflammation in normal and preeclamptic pregnancies

IntroductionInflammation is a normal physiological process that increases to harmful levels in preeclampsia. It affects the interaction between maternal immune cells and fetal trophoblasts at both sites of the maternal-fetal interface; decidua and placenta. The pattern recognition receptor nucleotide-binding oligomerization domain-containing protein (NOD)1 is expressed at both sites. This study aimed to characterize the cellular expression and functionality of NOD1 at the maternal-fetal interface of normal and preeclamptic pregnancies. MethodsWomen with normal or preeclamptic pregnancies delivered by caesarean section were included. Decidual (n = 90) and placental (n = 91) samples were analyzed for NOD1 expression by immunohistochemistry and an automated image-based quantification method. Decidual and placental explants were incubated with or without the NOD1-agonist iE-DAP and cytokine responses measured by ELISA. ResultsNOD1 was markedly expressed by maternal cells in the decidua and by fetal trophoblasts in both decidua and placenta, with trophoblasts showing the highest NOD1 expression. Preeclampsia with normal fetal growth was associated with a trophoblast-dependent increase in decidual NOD1 expression density. Compared to normal pregnancies, preeclampsia demonstrated stronger correlation between decidual and placental NOD1 expression levels. Increased production of interleukin (IL)-6 or IL-8 after in vitro explant stimulation confirmed NOD1 functionality. DiscussionThese findings suggest that NOD1 contributes to inflammation at the maternal-fetal interface in normal pregnancies and preeclampsia and indicate a role in direct maternal-fetal communication. The strong expression of NOD1 by all trophoblast types highlights the importance of combined assessment of decidua and placenta for overall understanding of pathophysiological processes at the maternal-fetal interface.

NOD2 regulates distinct pathways of endogenous thymic regeneration after injury

Abstract Although the thymus has a remarkable capacity for repair following damage, the mechanisms underlying this endogenous regeneration remain poorly understood. Our recent studies have identified two key pathways for thymic regeneration; centered on secretion of BMP4 by endothelial cells (ECs) and IL-22 from innate lymphoid cells (ILCs), which can be utilized individually as therapeutic strategies of immune regeneration. The specific regulatory mechanisms that trigger these regeneration-associated factors after damage remain unclear. Here we detail an unexpected role for the pattern recognition receptor Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in orchestrating these distinct pathways of thymic regeneration. After acute injury, Nod2−/− mice exhibited superior overall thymic regeneration, consistent with increased levels of BMP4, IL-22, and Il-23, a key regulator of IL-22 secreted by dendritic cells (DCs). Furthermore, expression of miR29, a downstream effector of NOD2, was significantly reduced in ECs and DCs after injury, and miR29 mimics suppressed Bmp4 and Il23 expression in ECs or DCs, respectively. Consistent with a non-canonical role for NOD2 in sensing RhoGTPases, we found several RhoGTPase family members were downregulated after TBI; and modulation with small molecule inhibitors targeting Rho GTPases led to robust production of BMP4 and IL-23, with enhanced thymic regeneration after acute injury. These findings provide evidence not only of a novel master regulator of endogenous thymic regeneration, but also offer a superior therapeutic strategy for boosting thymic regeneration and T cell reconstitution after damage such as that caused by infection or cytoreductive therapy.

P131 CHARACTERIZING THE MICROBIOTA-GUT-BRAIN AXIS IN A MURINE MODEL OF PEDIATRIC INFLAMMATORY BOWEL DISEASE

Inflammatory bowel diseases (IBD) are chronic intestinal diseases affecting over 1.4 million Americans. Children and adolescents typically have more severe disease and are more likely to display extra-intestinal effects, including psychosocial deficits, compared to adult patients. Unfortunately, the mechanism(s) through which these extra-intestinal deficits develop is unknown and murine models of pediatric IBD do not exist. Our overall goal was to develop a model of pediatric IBD and assess the microbiota-gut-brain axis in adolescent mice. We hypothesized that induction of colitis at weaning would lead to long-lasting effects on the microbiota-gut-brain axis, including behavior. Colonic inflammation was induced by administration of dextran sodium sulfate (DSS), a chemical colitogen with anticoagulant properties known to cause colitis in rodents. DSS (2% w/v) was given to weanling mice (P21) in drinking water for 5 days and then allowed to recover, modeling acute colitis. Behavior (cognition and anxiety) and inflammation (colon and hippocampus) was assessed at 6-8 weeks of age. In adult mice anxiety (light/dark box) and cognition (novel object recognition [NOR] task) were performed, and tissue samples collected for analysis by qPCR. Compared to control mice, DSS-treated mice showed impaired object recognition and increased anxiety-like behaviors. Gene expression was assessed in the colon and showed increased expression of the pattern recognition receptors nucleotide-binding oligomerization domain Nod1 andNod2 (as well as increased expression of the anti-inflammatory cytokine, interleukin 10 (Il10), and genes involved in NF-κB signaling (IκB) in DSS-treated mice compared to controls. Finally, gene expression in the hippocampus showed increased expression of nod1, nod2, and brain derived neurotropic factor (Bdnf) in DSS-treated mice compared to sham-treated controls. Taken together, these results indicate that acute DSS-induced colitis in early adolescence can have long-lasting effects on the microbiota-gut-brain axis in adulthood.

NOD2 Acts As a Master Regulator of Endogenous Thymic Regeneration

Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence following immunodepletion caused by common cancer therapies such as cytoreductive chemotherapy or radiation; however, the mechanisms governing this regeneration remain poorly understood. Despite this capacity, prolonged T cell deficiency is a major clinical hurdle in recipients of hematopoietic stem cell transplantation (HSCT) and can precipitate high morbidity and mortality from opportunistic infections, and may even facilitate malignant relapse. Our recent studies have revealed that innate lymphoid cells (ILCs) and endothelial cells (ECs), through their production of the regeneration-associated factors (RAFs) IL-22 and BMP4, respectively, have profound reparative effects in the thymus after acute injury; and can be utilized individually as therapeutic strategies of immune regeneration (Dudakov 2012 Science 336:91; Dudakov 2017 Blood 130:933; Wertheimer 2018 Sci Immunol 3:19). These two pathways act by stimulating thymic epithelial cells (TECs), a heterogeneous population of stromal cell in the thymus critical for thymopoiesis. However, the regulation of these endogenous regenerative responses is still poorly understood. Here we reveal an unexpected role for the pattern recognition receptor Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in governing multiple pathways of thymic regeneration.Analysis of thymic recovery following acute injury in mice deficient for NOD2 revealed increased intrathymic BMP4 and IL-23 (Fig. 1a), a key regulator of IL-22 production, and commensurate improved ability to regenerate (Fig. 1b). Although NOD2 is expressed ubiquitously across all populations within the thymus, in regeneration-initiating DCs and ECs, but not thymocytes (damage-targets), we identified a specific reduction in the expression of miR29c after damage, which previous reports suggest could mediate NOD2-induced suppression of IL-23 (Brain 2013 Immunity 39:521). Consistent with these findings, miR29c expression was decreased in the thymus of NOD2-deficient mice (Fig. 1d), and overexpression of miR29c in either ECs or DCs reduced their expression of Bmp4 or Il23, respectively (Fig. 1e). Canonical ligands for NOD2 are peptidoglycans found in the cell wall of bacteria; however, these are unlikely to serve as a NOD2 activator in the thymus since it is typically thought of as a sterile organ. One recently described alternate function of NOD2 is as a cytosolic sensor of activated Rho GTPases. The Rho GTPase family is responsible for a wide range of physiological processes, including the intrathymic regulation of b-selection and positive selection, and inhibition of Rho GTPase signaling is of considerable clinical interest. Consistent with a role in suppressing regeneration, unbiased transcriptome analysis revealed significant downregulation of many members of the RhoGTPase family after damage, corresponding to the increase in production of RAFs. Importantly, suggestive of a potential clinical application, pharmacological suppression of RhoGTPase in vitro significantly induced the expression of Bmp4 in ECs, and Il23 in DCs (Fig. 1f), as well as suppressing the expression of miR29c (Fig. 1g).Although several pathways have been described as contributing to endogenous thymic regeneration, the specific mechanisms regulating their induction has been poorly understood. Here we reveal a common mechanism triggering production of multiple distinct regeneration pathways such as those centered on production of BMP4 and IL-22. Therefore, the mechanistic and pre-clinical studies described not only define an important regulatory mechanism governing endogenous tissue regeneration, but could also offer an innovative therapeutic strategy to boost thymic function and T cell reconstitution in recipients of allo-HSCT, as well as for individuals with T cell deficiencies due to aging, autoimmune diseases, genetic causes, infectious disease, shock, radiation injury (nuclear accident, terrorism) and common cancer treatments such as chemo- and radiation-therapy. DisclosuresNo relevant conflicts of interest to declare.

NOD2 acts as a master regulator of regeneration pathways in the thymus

Abstract Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence following stress, infection and cytotoxic cancer treatments. Although there is continuous thymic involution and regeneration in response to stress and infection in the healthy state, prolonged T cell deficiency is common after profound thymic damage, such as that caused by the conditioning required for hematopoietic stem cell transplant. Our previous studies have revealed two pathways important for thymic repair, centered around the production of IL-22 and BMP4 by innate lymphoid cells and endothelial cells (ECs), respectively. However, the specific mechanisms that trigger these pathways are poorly understood. Here we reveal an unexpected role for the pattern recognition receptor Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in governing multiple pathways of thymic regeneration. Mice deficient in NOD2 showed increased intrathymic levels of L-22, IL-23 and BMP4 and increased thymus cellularity after damage caused by total body irradiation (TBI). Although NOD2 is classically defined as a bacterial sensor, members of the Rho GTPase family have been implicated to promote NOD signaling, even in the absence of pattern recognition. Consistent with this, inhibition of RhoA or Rac1 can induce the production of BMP4 by ECs in vitro, and after TBI there is a significant reduction in these two Rho GTPases. These studies not only enhance our understanding of endogenous tissue regeneration, but in identifying a master regulator of multiple pathways, offers a superior therapeutic strategy to boost thymic function in patients whose immune system has been decimated due to age, infection or cancer therapies.

NOD2 promotes cell proliferation and inflammatory response by mediating expression of TSLP in human airway smooth muscle cells

The newly discovered intracytosolic pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) has been studied as an important indicator of T helper 2 (Th2) inflammation, and its effect on regulatory T (Treg) cells is likely to modulate the immune response. In this study, we attempted to study the expression of NOD2 and its impact in human airway smooth muscle cells (HASMC). Quantitative real-time PCR (qRT-PCR) was used to measure the expression level of NOD2 in HASMC and comparisons were made between those from asthmatic and non-asthmatic donors; we found that NOD2 was significantly upregulated in asthma patient tissues and cell lines. In addition, overexpression of NOD2 apparently promotes cell proliferation and migration in HASMC. Gain-of-function in vitro experiments further showed that NOD2 overexpression significantly promotes pro-inflammatory cytokine release in HASMC. Subsequent experimental analysis indicated that thymic stromal lymphopoietin (TSLP) is involved in NOD2-mediated cellular effects in HASMC. Therefore, our results indicate that NOD2 is an asthma-related factor that can promote cell proliferation and inflammatory response by mediated expression of TSLP in HASMC. Taken together, our results indicate that NOD2 could serve as a potential diagnostic biomarker and therapeutic option for human asthma in the near future.

Nucleotide-binding oligomerization domain 2 receptor is expressed in platelets and enhances platelet activation and thrombosis.

Pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) is well investigated in immunity, but its expression and function in platelets has never been explored. Using reverse transcription polymerase chain reaction and Western blot, we show that both human and mouse platelets express NOD2, and its agonist muramyl dipeptide induced NOD2 activation as evidenced by receptor dimerization. NOD2 activation potentiates platelet aggregation and secretion induced by low concentrations of thrombin or collagen, and clot retraction, as well. These potentiating effects of muramyl dipeptide were not seen in platelets from NOD2-deficient mice. Plasma from septic patients also potentiates platelet aggregation induced by thrombin or collagen NOD2 dependently. Using intravital microscopy, we found that muramyl dipeptide administration accelerated in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model. Platelet depletion and transfusion experiments confirmed that NOD2 from platelets contributes to the in vivo thrombosis in mice. NOD2 activation also accelerates platelet-dependent hemostasis. We further found that platelets express receptor-interacting protein 2, and provided evidence suggesting that mitogen activated-protein kinase and nitric oxide/soluble guanylyl cyclase/cGMP/protein kinase G pathways downstream of receptor-interacting protein mediate the role of NOD2 in platelets. Finally, muramyl dipeptide stimulates proinflammatory cytokine interleukin-1β maturation and accumulation in human and mouse platelets NOD2 dependently. NOD2 is expressed in platelets and functions in platelet activation and arterial thrombosis, possibly during infection. To our knowledge, this is the first study on NOD-like receptors in platelets that link thrombotic events to inflammation.

Aberrant expression of regulatory cytokine IL-35 and pattern recognition receptor NOD2 in patients with allergic asthma.

We investigated the plasma concentration of the novel regulatory cytokine IL-35 and intracytosolic pattern recognition receptors nucleotide-binding oligomerization domain (NOD)-like receptors in granulocytes and explored their potential implication in disease severity monitoring of allergic asthma. The expression of circulating IL-35 and other pro-inflammatory mediators in asthmatic patients or control subjects were evaluated using enzyme-linked immunosorbent assay (ELISA). The intracellular expressions of NOD1 and NOD2 in CCR3+ granulocytes were assessed using flow cytometry. Plasma concentrations of IL-35, IL-17A, basophil activation marker basogranulin, and eosinophilic airway inflammation biomarker periostin were significantly elevated in allergic asthmatic patients compared to non-atopic control subjects (all probability (p) <0.05). Both granulocyte markers exhibited significant and positive correlation with plasma IL-35 concentration in asthmatic patients (all p < 0.05). Significant positive correlation was also identified between plasma concentrations of IL-35 and periostin with disease severity score in asthmatic patients (both p < 0.05). The basophil activation allergenicity test was positive in allergic asthmatic patients but not in control subjects. Despite significantly elevated eosinophil count in allergic asthmatic patients, downregulation of NOD2 in CCR3+ granulocytes was observed in these patients (both p < 0.05). A negative correlation between plasma concentrations of tumor necrosis factor family member LIGHT and soluble herpesvirus entry mediator was observed in patients with elevated plasma concentration of IL-35 (p < 0.05). Aberrant expression of NOD2 in granulocytes may be contributed to the impaired innate immunity predisposing allergic asthma. IL-35 may serve as a potential surrogate biomarker for disease severity of allergic asthma.

Engagement of Nucleotide-binding Oligomerization Domain-containing Protein 1 (NOD1) by Receptor-interacting Protein 2 (RIP2) Is Insufficient for Signal Transduction

Following activation, the cytoplasmic pattern recognition receptor nucleotide-binding oligomerization domain-containing protein 1 (NOD1) interacts with its adaptor protein receptor-interacting protein 2 (RIP2) to propagate immune signaling and initiate a proinflammatory immune response. This interaction is mediated by the caspase recruitment domain (CARD) of both proteins. Polymorphisms in immune proteins can affect receptor function and predispose individuals to specific autoinflammatory disorders. In this report, we show that mutations in helix 2 of the CARD of NOD1 disrupted receptor function but did not interfere with RIP2 interaction. In particular, N43S, a rare polymorphism, resulted in receptor dysfunction despite retaining normal cellular localization, protein folding, and an ability to interact with RIP2. Mutation of Asn-43 resulted in an increased tendency to form dimers, which we propose is the source of this dysfunction. We also demonstrate that mutation of Lys-443 and Tyr-474 in RIP2 disrupted the interaction with NOD1. Mapping the key residues involved in the interaction between NOD1 and RIP2 to the known structures of CARD complexes revealed the likely involvement of both type I and type III interfaces in the NOD1·RIP2 complex. Overall we demonstrate that the NOD1-RIP2 signaling axis is more complex than previously assumed, that simple engagement of RIP2 is insufficient to mediate signaling, and that the interaction between NOD1 and RIP2 constitutes multiple CARD-CARD interfaces.

The Pattern Recognition Receptor NOD2 Mediates Staphylococcus aureus–Induced IL-17C Expression in Keratinocytes

IL-17C is an important epithelial cell-derived cytokine activating innate immunity by the induction of antimicrobial peptides and cytokines. Here, we investigated the role of the cytosolic pattern recognition receptor nucleotide-binding oligomerization domain-containing protein 2 (NOD2) for the Staphylococcus aureus-mediated induction of IL-17C. Activation of NOD2 in HEK293 cells overexpressing NOD2 induced the IL-17C promoter, an activity that was significantly reduced in cells overexpressing the Crohn's disease-associated NOD2 mutation 3020insC (1007fs) or the Crohn's disease- and atopic dermatitis-associated NOD2-R702W variant. The first NF-κB-binding site in the IL-17C promoter was critical for NOD2-mediated IL-17C induction. Infection of human primary keratinocytes with S. aureus induced NOD2 and IL-17C gene expression. Overexpression of NOD2 in keratinocytes augmented S. aureus-mediated IL-17C gene expression as compared with NOD2-R702W overexpression. S. aureus-induced IL-17C expression was diminished in NOD2 small interfering RNA (siRNA)-treated keratinocytes. Moreover, significantly less S. aureus bacteria survived in keratinocytes overexpressing NOD2 but not in cells overexpressing the NOD2-R702W variant. Finally, S. aureus showed an increased survival in keratinocytes treated with NOD2 or IL-17C siRNA. In summary, our study provides evidence that S. aureus activates NOD2 in keratinocytes, resulting in an increased expression of IL-17C, a mechanism that may be dysregulated in atopic dermatitis.

Calcineurin/NFAT signaling and innate host defence: a role for NOD1-mediated phagocytic functions

The calcineurin/nuclear factor of activated T cells (NFATs) signaling pathway plays a central role in T cell mediated adaptive immune responses, but a number of recent studies demonstrated that calcineurin/NFAT signaling also plays a key role in the control of the innate immune response by myeloid cells. Calcineurin inhibitors, such as cyclosporine A (CsA) and tacrolimus (FK506), are commonly used in organ transplantation to prevent graft rejection and in a variety of immune diseases. These immunosuppressive drugs have adverse effects and significantly increase host’s susceptibility towards bacterial or fungal infections. Recent studies highlighted the role of NFAT signaling in fungal infection and in the control of the pattern recognition receptor nucleotide-binding oligomerization domain-containing protein 1 (NOD1), which predominantly senses invasive Gram-negative bacteria and mediates neutrophil phagocytic functions. This review summarises some of the current knowledge concerning the role of NFAT signaling in the innate immune response and the recent advances on NFAT-dependent inhibition of NOD1-mediated innate immune response caused by CsA, which may contribute to sensitizing transplant recipients to bacterial infection.

NOD2 Activation Induces Muscle Cell-Autonomous Innate Immune Responses and Insulin Resistance

Insulin resistance is associated with chronic low-grade inflammation in vivo, largely mediated by activated innate immune cells. Cytokines and pathogen-derived ligands of surface toll-like receptors can directly cause insulin resistance in muscle cells. However, it is not known if intracellular pathogen sensors can, on their own, provoke insulin resistance. Here, we show that the cytosolic pattern recognition receptors nucleotide-binding oligomerization domain-containing protein (NOD)1 and NOD2 are expressed in immune and metabolic tissues and hypothesize that their activation in muscle cells would result in cell-autonomous responses leading to insulin resistance. Bacterial peptidoglycan motifs that selectively activate NOD2 were directly administered to L6- GLUT4myc myotubes in culture. Within 3 h, insulin resistance arose, characterized by reductions in each insulin-stimulated glucose uptake, GLUT4 translocation, Akt Ser(473) phosphorylation, and insulin receptor substrate 1 tyrosine phosphorylation. Muscle cell-autonomous responses to NOD2 ligand included activation of the stress/inflammation markers c-Jun N-terminal kinase, ERK1/2, p38 MAPK, degradation of inhibitor of κBα, and production of proinflammatory cytokines. These results show that NOD2 alone is capable of acutely inducing insulin resistance within muscle cells, possibly by activating endogenous inflammatory signals and/or through cytokine production, curbing upstream insulin signals. NOD2 is hence a new inflammation target connected to insulin resistance, and this link occurs without the need of additional contributing cell types. This study provides supporting evidence for the integration of innate immune and metabolic responses through the involvement of NOD proteins and suggests the possible participation of cell autonomous immune responses in the development of insulin resistance in skeletal muscle, the major depot for postprandial glucose utilization.

The influence of fetal sex on the fetal–placental adaptation to stressors

The newly discovered intracytosolic pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) has been studied as an important indicator of T helper 2 (Th2) inflammation, and its effect on regulatory T (Treg) cells is likely to modulate the immune response. In this study, we attempted to study the expression of NOD2 and its impact in human airway smooth muscle cells (HASMC). Quantitative real-time PCR (qRT-PCR) was used to measure the expression level of NOD2 in HASMC and comparisons were made between those from asthmatic and non-asthmatic donors; we found that NOD2 was significantly upregulated in asthma patient tissues and cell lines. In addition, overexpression of NOD2 apparently promotes cell proliferation and migration in HASMC. Gain-of-function in vitro experiments further showed that NOD2 overexpression significantly promotes pro-inflammatory cytokine release in HASMC. Subsequent experimental analysis indicated that thymic stromal lymphopoietin (TSLP) is involved in NOD2-mediated cellular effects in HASMC. Therefore, our results indicate that NOD2 is an asthma-related factor that can promote cell proliferation and inflammatory response by mediated expression of TSLP in HASMC. Taken together, our results indicate that NOD2 could serve as a potential diagnostic biomarker and therapeutic option for human asthma in the near future.

Toll-like receptor-induced granulocyte-macrophage colony-stimulating factor secretion is impaired in Crohn's disease by nucleotide oligomerization domain 2-dependent and -independent pathways

Pattern recognition receptors (PRRs) are an integral part of the innate immune system and govern the early control of foreign microorganisms. Single nucleotide polymorphisms (SNPs) in the intracellular pattern recognition receptor nucleotide-binding oligomerization domain-containing protein (NOD2, nucleotide oligomerization domain 2) are associated with Crohn's disease (CD). We investigated the impact of NOD2 polymorphisms on cytokine secretion and proliferation of peripheral blood mononuclear cells (PBMCs) in response to Toll-like receptor (TLR) and NOD2 ligands. Based on NOD2 SNP analyses, 41 CD patients and 12 healthy controls were studied. PBMCs were stimulated with NOD2 and TLR ligands. After 18 h culture supernatants were measured using multiplex assays for the presence of human cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-1 beta and tumour necrosis factor (TNF)-alpha. In CD patients, TLR-induced GM-CSF secretion was impaired by both NOD2-dependent and -independent mechanisms. Moreover, TNF-alpha production was induced by a TLR-2 ligand, but a down-regulatory function by the NOD2 ligand, muramyl dipeptide, was impaired significantly in CD patients. Intracellular TLR ligands had minimal effect on GM-CSF, TNF-alpha and IL-1beta secretion. CD patients with NOD2 mutations were able to secrete TNF-alpha, but not GM-CSF, upon stimulation with NOD2 and TLR-7 ligands. CD patients have impaired GM-CSF secretion via NOD2-dependent and -independent pathways and display an impaired NOD2-dependent down-regulation of TNF-alpha secretion. The defect in GM-CSF secretion suggests a hitherto unknown role of NOD2 in the pathogenesis of CD and is consistent with the hypothesis that impaired GM-CSF secretion in part constitutes a NOD2-dependent disease risk factor.

Susceptibility to asthma and eczema from mucosal and epidermal expression of distinctive genes

The past several years have seen an increase in the rate at which genes that are associated with allergic asthma and eczema are discovered. This review -examines genetic association, gene expression, and functional studies that have identified genes that are expressed in the epithelial cells of the skin and lung and are involved in the pathogenesis of allergic asthma and eczema. This includes the genes encoding thymic stromal lympho-poietin (TSLP) and suppressor of cytokine signaling (SOCS3) that are involved in the activation of T-helper 2 cells, the microbial pattern recognition receptors nucleotide-binding oligomerization domain (NOD) genes (CARD4 and CARD15), Toll-like receptors (TLR2 and TLR4), and filaggrin, a protein required for effective barrier defense of the skin. Therefore, the development of allergic disease involves both the adaptive and innate immune systems, and the expression of these genes in the skin and lungs suggests a link to environmental -triggers at body surfaces.