NOD2 Activation Research Articles

Inflammatory bowel disease environmental risk factors versus genetics based on migration epidemiological studies.

Inflammatory bowel disease (IBD) experienced a rapid rise in incidence and prevalence in developing countries that parallels rates of accelerated industrialization in these regions.1 Migrant studies can investigate the epidemiological factors driving this change. Migrants form a unique population for investigating the evolving pattern of IBD incidence and prevalence globally. Groups migrating from areas of low IBD incidence to high IBD incidence will experience a shorter time period between risk factor exposure and disease onset. This is relevant for those risk factors related to the lifestyle of westernized countries. Studying the offspring of migrants will also provide new cohort data on generational changes and their relationship with IBD development. Furthermore, such investigations might elucidate information on the critical period of genetic versus environmental influence on IBD pathogenesis.2 Existing migrant studies highlight higher IBD incidence in migrants, suggesting that migrants adopt similar if not higher risks of IBD as their native counterparts in developed countries. The incidence of IBD is higher in first- and second-generation migrants,3 and second-generation migrants have more extensive disease in both adults3 and children.4 Age at time of migration also appears to be important, as those raised from birth in the adoptive country or those who migrated during childhood have the greatest risk of developing IBD compared with non-migrants, as well as those migrating to certain regions such as the Middle East.5 Furthermore, even a period of time living in a more westernized environment and returning to the original milieu might influence IBD risk.6 Proxy measures of the hygiene hypothesis is also more pronounced in migrants. Childhood rural dwelling, farm animal access, bedroom sharing, pet ownership, and pet feeding were associated with decreased development of IBD in migrant Middle Easterners, while not significant for non-migrants in Australia.7 IBD environmental factors might act differentially in different populations. In those who are exposed to an undeveloped environment, hygiene increases IBD development. However, in those who have been in a developed environment for several generations, the hygiene-related risk factors are no longer applicable to disease pathogenesis.8 Thus, the migrant population might be more sensitive for detecting the relationship between IBD and lifestyle of affluent countries. Genetic risk factors also play an important role in IBD pathogenesis as demonstrated by genome wide associations studies. Migrant populations might have a role in studying IBD epigenetics, which has been previously investigated. Epigenetic changes may mediate interactions between genes and environmental factors and thus might explain some of the missing heritability.9 Smoking is an environmental modifier of epigenetic state and NOD2 activation, which can influence the expression of microRNA-29 and indirectly the Th17 pathway.10 Studying key deoxyribonucleic acid methylation site differences between migrant and non-migrant populations while adjusting for lifestyle and dietary factors might provide greater insight into IBD epigenetics. Limitations of migrant studies include possible different health-seeking behavior and health literacy in migrant groups, hence reducing IBD detection. However, this suggests positive findings are likely of importance. Ethnic factors such as diet might be difficult to differentiate from true environmental effects on disease. This might be overcome by recruiting controls with similar characteristics. Overall, migrant studies provide a good opportunity to further delve into the complexities of the environment versus genetics of IBD.

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.

NOD1 Mediates Insulin Resistance in Response to Circulating Free Fatty Acids

Insulin resistance (IR) in type 2 diabetes (T2D) is associated with chronic low-grade inflammation. We have shown that nucleotide binding oligomerization domain (NOD)1, an intracellular pattern recognition receptor for bacterial wall peptidoglycans, is a factor linking the innate immune system to the development of high fat diet-induced IR. Although high fat diet may result in NOD1 activation via increased intestinal absorption of bacterial products, NOD1 has also been shown to be activated by saturated fatty acids in intestinal cells and adipocytes. We hypothesize that NOD1 plays a role in IR caused by chronic high levels of circulating free fatty acids (FFA) as found in obesity and T2D. Wild type (WT) and NOD1-/- mice were infused with ethylpalmitate (resulting in raised circulating palmitate) or ethanol vehicle as a control, for 48 hours to model prolonged elevation of circulating FFA in vivo. Hyperinsulinemic-euglycemic clamp with tracer methodology was then used to assess peripheral and hepatic insulin sensitivity. WT mice treated with ethylpalmitate had decreased peripheral and hepatic insulin sensitivity expressed as stimulation of glucose utilization (Control= 123%±30 and Ethylpalmitate= 25%±25, p<.05) and suppression of glucose production (Control= 51%±10 and Ethylpamitate= 22%±2, p<.05). NOD1-/- mice were protected from palmitate induced IR, showing no difference in stimulation of glucose utilization (Control= 289%±27 and Ethylpalmitate= 352%±64) and suppression of glucose production (Control= 63%±16 and Ethylpalmitate= 95%±25) with or without palmitate. Thus, our data provide compelling evidence that NOD1 activation via elevated plasma FFA contributes to the development of peripheral and hepatic IR. Disclosure S.L. Rivers: None. L. Yeung: None. K.L. Chan: None. Y. Mori: None. D. Philpott: None. A. Klip: None. A. Giacca: None.

NOD2-mediated downregulation of the TLR pathways contributes to protection against colitis and colorectal tumorigenesis

Abstract NOD-like receptor family member NOD2, which activates the NF-kB and MAPK signaling pathways in response to the bacterial cell wall component MDP, is the first identified inflammatory bowel disease (IBD) susceptibility gene. IBD is a risk factor for colorectal tumorigenesis. However, the role of NOD2 in colorectal tumorigenesis has not been studied adequately and the precise mechanism of NOD2-mediated protection against IBD is elusive. We studied the role of NOD2 in colorectal tumorigenesis using azoxymethane plus dextran sulfate sodium-induced colorectal tumorigenesis in mice. We found that Nod2-deficient mice are highly susceptible to colorectal tumorigenesis with increased tumor burden as compared to wild-type mice. Interestingly, increased tumorigenesis in Nod2−/− mice was independent of gut microbial composition. Increased tumorigenesis in Nod2-deficient mice was associated with higher inflammatory responses and activation of the inflammatory signaling pathways, such as NF-kB, ERK, and STAT3. Consistently, in vitro studies demonstrate that while NOD2 activates NF-kB and ERK in response to MDP, it inhibits TLR-mediated activation of NF-kB and ERK, and production of inflammatory mediators. Notably, NOD2-mediated downregulation of TLR signaling pathways is independent of MDP-mediated activation of NOD2. We further explored the underlying mechanism of NOD2-mediated downregulation of TLR signaling and revealed that NOD2 induces IRF4 which negatively regulates TLR-NF-kB signaling axis. Taken together, this study therefore shed light on a novel function of NOD2 which may be implicated in genetic association of NOD2 mutations with IBD and colorectal cancer.

RIP2 Is a Critical Regulator for NLRs Signaling and MHC Antigen Presentation but Not for MAPK and PI3K/Akt Pathways.

RIP2 is an adaptor protein which is essential for the activation of NF-κB and NOD1- and NOD2-dependent signaling. Although NOD-RIP2 axis conservatively existed in the teleost, the function of RIP2 was only reported in zebrafish, goldfish, and rainbow trout in vitro. Very little is known about the role and mechanisms of piscine NOD-RIP2 axis in vivo. Our previous study showed the protective role of zebrafish NOD1 in larval survival through CD44a-mediated activation of PI3K-Akt signaling. In this study, we examined whether RIP2 was required for larval survival with or without pathogen infection, and determined the signaling pathways modulated by RIP2. Based on our previous report and the present study, our data demonstrated that NOD1-RIP2 axis was important for larval survival in the early ontogenesis. Similar to NOD1, RIP2 deficiency significantly affected immune system processes. The significantly enriched pathways were mainly involved in immune system, such as “Antigen processing and presentation” and “NOD-like receptor signaling pathway” and so on. Furthermore, both transcriptome analysis and qRT-PCR revealed that RIP2 was a critical regulator for expression of NLRs (NOD-like receptors) and those genes involved in MHC antigen presentation. Different from NOD1, the present study showed that NOD1, but not RIP2 deficiency significantly impaired protein levels of MAPK pathways. Although RIP2 deficiency also significantly impaired the expression of CD44a, the downstream signaling of CD44a-Lck-PI3K-Akt pathway remained unchanged. Collectively, our works highlight the similarity and discrepancy of NOD1 and RIP2 in the regulation of immune signaling pathways in the zebrafish early ontogenesis, and confirm the crucial role of RIP2 in NLRs signaling and MHC antigen presentation, but not for MAPK and PI3K/Akt pathways.

Novel identified aluminum hydroxide-induced pathways prove monocyte activation and pro-inflammatory preparedness

Aluminum-based adjuvants are the most widely used adjuvants in human vaccines. A comprehensive understanding of the mechanism of action of aluminum adjuvants at the molecular level, however, is still elusive. Here, we unravel the effects of aluminum hydroxide Al(OH)3 by a systems-wide analysis of the Al(OH)3-induced monocyte response. Cell response analysis by cytokine release was combined with (targeted) transcriptome and full proteome analysis. Results from this comprehensive study revealed two novel pathways to become activated upon monocyte stimulation with Al(OH)3: the first pathway was IFNβ signaling possibly induced by DAMP sensing pathways like TLR or NOD1 activation, and second the HLA class I antigen processing and presentation pathway. Furthermore, known mechanisms of the adjuvant activity of Al(OH)3 were elucidated in more detail such as inflammasome and complement activation, homeostasis and HLA-class II upregulation, possibly related to increased IFNγ gene expression. Altogether, our study revealed which immunological pathways are activated upon stimulation of monocytes with Al(OH)3, refining our knowledge on the adjuvant effect of Al(OH)3 in primary monocytes. SignificanceAluminum salts are the most used adjuvants in human vaccines but a comprehensive understanding of the working mechanism of alum adjuvants at the molecular level is still elusive. Our Systems Vaccinology approach, combining complementary molecular biological, immunological and mass spectrometry-based techniques gave a detailed insight in the molecular mechanisms and pathways induced by Al(OH)3 in primary monocytes. Several novel immunological relevant cellular pathways were identified: type I interferon secretion potentially induced by TLR and/or NOD like signaling, the activation of the inflammasome and the HLA Class-I and Class-II antigen presenting pathways induced by IFNγ. This study highlights the mechanisms of the most commonly used adjuvant in human vaccines by combing proteomics, transcriptomics and cytokine analysis revealing new potential mechanisms of action for Al(OH)3.

Anti-Inflammatory Effect of Columbianetin on Lipopolysaccharide-Stimulated Human Peripheral Blood Mononuclear Cells.

Dysregulated inflammation is increasingly considered as the main cause of many diseases on which NOD1/NF-κB pathway plays an important role. Columbianetin (CBT) is derived from the root of the Chinese herb Radix Angelicae Pubescentis for treating inflammatory diseases. Although the anti-inflammatory effect of CBT has been reported, its anti-inflammatory mechanism was poorly studied. In this study, we explored the anti-inflammatory pathway of CBT in lipopolysaccharide- (LPS-) stimulated human peripheral blood mononuclear cell (PBMC) model. Inflammatory cytokine production in culture supernatant was assessed using ELISA assay, and the possible anti-inflammatory pathway of CBT was screened using qPCR array and enrichment analysis with DAVID6.8. To further confirm the targeted pathway of CBT, we pretreated PBMC with the selective NOD1 inhibitor ML130 and then measured the protein levels of the pathway by Western blotting. The result showed that CBT effectively suppressed the expressions of TNF-α, IL-6, MCP-1, and IL-1β in a dose-dependent manner and significantly downregulated 19 out of 32 differentially expressed genes, most of which were involved in the NOD1/NF-κB pathway, and also showed that CBT remarkably inhibited LPS-induced NOD1, RIP2, and NF-κB activation. Furthermore, the inhibitory effects of CBT on NOD1/NF-κB pathways were blocked by ML130. These findings indicated that CBT inhibits the production of inflammatory cytokines induced by LPS involved in the downregulation of NOD1/NF-κB pathways.

Intracellular NOD2 Activation Promotes Maturation and Antigen-Presenting Functions of Dentritic Cells Exposed to Porphyromonas Gingivalis Lipopolysaccharide.

Objective: This study aimed to study the roles of Muramyl Dipeptide (MDP) which is an agonist of NOD2 in Dendritic Cells (DCs) exposed to Porphyromonas gingivalis lipopolysaccharide ( P. gingivalis - LPS) on maturation and antigen-presenting functions of DCs to provide experimental evidences to explore the possible mechanism of DCs in periodontitis. Methods: Flow cytometry was used to detect CD11c, MHC-II, CD80, CD86, and CD40 expression on DCs and ELISA was used to detect IL-12, IFN-γ, IL-10, and IL-13 secreted by DCs which were stimulated by MDP and P. gingivalis -LPS, respectively or in synergism. RT-PCR analysis was used to detect NOD2, TLR2, and TLR4 mRNA expression in DCs stimulated by MDP and P. gingivalis -LPS, respectively or in synergism. CCK8 was used to assess CD4+ T cells proliferation after co-cultured with DCs stimulated by MDP and P. gingivalis -LPS, respectively or in synergism and ELISA was used to detect IL-2, IFN-γ, IL-10 and IL-13 secreted by these T cells. Results: MDP had weak ability to stimulate DCs maturation but MDP could promote DCs maturation stimulated by P. gingivalis -LPS. MDP was NOD2 agonist to DCs and P. gingivalis - LPS was TLR2 but not TLR4 agonist to DCs. MDP could facilitate TLR2 mRNA expression in DCs exposed to P. gingivalis - LPS. The ability of MDP to promote DCs secreting cytokines was far below P. gingivalis- LPS but MDP could promote the functions of Th2 cell-promoting DCs induced by P. gingivalis -LPS. MDP could promote CD4+T cells proliferation primed by DCs exposed to P. gingivalis -LPS and elevate the ability of DCs exposed to P. gingivalis -LPS to prime Th0 cells to Th2 cells. Conclusion: Intracellular NOD2 in DCs could be activated by MDP and this activation could promote maturation and the ability to prime Th0 cells to Th2 cells of DCs exposed to P. gingivalis -LPS.

NOD2 Modulates Serotonin Transporter and Interacts with TLR2 and TLR4 in Intestinal Epithelial Cells

Background/Aims: Serotonin (5-HT) is a chief modulator of intestinal activity. The effects of 5-HT depend on its extracellular availability, which is mainly controlled by serotonin transporter (SERT), expressed in enterocytes. On the other hand, innate immunity, mediated by Toll-like receptors (TLRs) and nucleotide oligomerization domain (NOD)-like receptors (NLRs), is known to control intestinal microbiota and maintain intestinal homeostasis. The dysregulation of the intestinal serotonergic system and innate immunity has been observed in inflammatory bowel diseases (IBD), the incidence of which has severely increased all over the world. The aim of the present study, therefore, was to analyze the effect of NOD2 on intestinal SERT activity and expression, as well as to study the crosstalk of NOD2 with TLR2 and TLR4. Methods: Intestinal epithelial cell line Caco-2/TC7 was used to analyze SERT activity and SERT, NOD2, TLR2 and TLR4 molecular expression by real-time PCR and western blotting. Moreover, intestinal tract (ileum and colon) from mice deficient in TLR2, TLR4 or TLR2/4 receptors was used to test the interdependence of NOD2 with these TLR receptors. Results: NOD2 activation inhibits SERT activity in Caco-2/TC7 cells, mainly due to the decrement of SERT molecular expression, with RIP2/RICK being the intracellular pathway involved in this effect. This inhibitory effect on SERT would yield an increment of extracellular 5-HT availability. In this sense, 5-HT strongly inhibits NOD2 expression. In addition, NOD2 showed greater interdependence with TLR2 than with TLR4. Indeed, NOD2 expression significantly increased in both cells treated with TLR2 agonists and the intestinal tract of Tlr2-/- mice. Conclusions: It may be inferred from our data that NOD2 could play a role in intestinal pathophysiology not only through its inherent innate immune role but also due to its interaction with other receptors as TLR2 and the modulation of the intestinal serotonergic system decreasing SERT activity and expression.

NOD1 downregulates intestinal serotonin transporter and interacts with other pattern recognition receptors.

Serotonin (5-HT) is an essential gastrointestinal modulator whose effects regulate the intestinal physiology. 5-HT effects depend on extracellular 5-HT bioavailability, which is controlled by the serotonin transporter (SERT) expressed in both the apical and basolateral membranes of enterocytes. SERT is a critical target for regulating 5-HT levels and consequently, modulating the intestinal physiology. The deregulation of innate immune receptors has been extensively studied in inflammatory bowel diseases (IBD), where an exacerbated defense response to commensal microbiota is observed. Interestingly, many innate immune receptors seem to affect the serotonergic system, demonstrating a new way in which microbiota could modulate the intestinal physiology. Therefore, our aim was to analyze the effects of NOD1 activation on SERT function, as well as NOD1's interaction with other immune receptors such as TLR2 and TLR4. Our results showed that NOD1 activation inhibits SERT activity and expression in Caco-2/TC7 cells through the extracellular signal-regulated kinase (ERK) signaling pathway. A negative feedback between 5-HT and NOD1 expression was also described. The results showed that TLR2 and TLR4 activation seems to regulate NOD1 expression in Caco-2/TC7 cells. To assess the extend of cross-talk between NOD1 and TLRs, NOD1 expression was measured in the intestinal tract (ileum and colon) of wild type mice and mice with individual knockouts of TLR2, and TLR4 as well as double knockout TLR2/TLR4 mice. Hence, we demonstrate that NOD1 acts on the serotonergic system decreasing SERT activity and molecular expression. Additionally, NOD1 expression seems to be modulated by 5-HT and other immune receptors as TLR2 and TLR4. This study could clarify the relation between both the intestinal serotonergic system and innate immune system, and their implications in intestinal inflammation.

The Absence of NOD1 Enhances Killing of Aspergillus fumigatus Through Modulation of Dectin-1 Expression.

One of the major life-threatening infections for which severely immunocompromised patients are at risk is invasive aspergillosis (IA). Despite the current treatment options, the increasing antifungal resistance and poor outcome highlight the need for novel therapeutic strategies to improve outcome of patients with IA. In the current study, we investigated whether and how the intracellular pattern recognition receptor NOD1 is involved in host defense against Aspergillus fumigatus. When exploring the role of NOD1 in an experimental mouse model, we found that Nod1−/− mice were protected against IA and demonstrated reduced fungal outgrowth in the lungs. We found that macrophages derived from bone marrow of Nod1−/− mice were more efficiently inducing reactive oxygen species and cytokines in response to Aspergillus. Most strikingly, these cells were highly potent in killing A. fumigatus compared with wild-type cells. In line, human macrophages in which NOD1 was silenced demonstrated augmented Aspergillus killing and NOD1 stimulation decreased fungal killing. The differentially altered killing capacity of NOD1 silencing versus NOD1 activation was associated with alterations in dectin-1 expression, with activation of NOD1 reducing dectin-1 expression. Furthermore, we were able to demonstrate that Nod1−/− mice have elevated dectin-1 expression in the lung and bone marrow, and silencing of NOD1 gene expression in human macrophages increases dectin-1 expression. The enhanced dectin-1 expression may be the mechanism of enhanced fungal killing of Nod1−/− cells and human cells in which NOD1 was silenced, since blockade of dectin-1 reversed the augmented killing in these cells. Collectively, our data demonstrate that NOD1 receptor plays an inhibitory role in the host defense against Aspergillus. This provides a rationale to develop novel immunotherapeutic strategies for treatment of aspergillosis that target the NOD1 receptor, to enhance the efficiency of host immune cells to clear the infection by increasing fungal killing and cytokine responses.

A Single Dual-Function Enzyme Controls the Production of Inflammatory NOD Agonist Peptidoglycan Fragments by Neisseria gonorrhoeae.

ABSTRACTNeisseria gonorrhoeae gonococcus (GC) is a Gram-negative betaproteobacterium and causative agent of the sexually transmitted infection gonorrhea. During growth, GC releases lipooligosaccharide (LOS) and peptidoglycan (PG) fragments, which contribute significantly to the inflammatory damage observed during human infection. In ascending infection of human Fallopian tubes, inflammation leads to increased risk of ectopic pregnancy, pelvic inflammatory disease, and sterility. Of the PG fragments released by GC, most are disaccharide peptide monomers, and of those, 80% have tripeptide stems despite the observation that tetrapeptide stems make up 80% of the assembled cell wall. We identified a serine-protease l,d-carboxypeptidase, NGO1274 (LdcA), as the enzyme responsible for converting cell wall tetrapeptide-stem PG to released tripeptide-stem PG. Unlike characterized cytoplasmic LdcA homologs in gammaproteobacteria, LdcA in GC is exported to the periplasm, and its localization is critical for its activity in modifying PG fragments for release. Distinct among other characterized l,d-carboxypeptidases, LdcA from GC is also capable of catalyzing the cleavage of specific peptide cross-bridges (endopeptidase activity). To define the role of ldcA in pathogenesis, we demonstrate that ldcA disruption results in both loss of NOD1-dependent NF-κB activation and decreased NOD2-dependent NF-κB activation while not affecting Toll-like receptor (TLR) agonist release. Since the human intracellular peptidoglycan receptor NOD1 (hNOD1) specifically recognizes PG fragments with a terminal meso-DAP rather than d-alanine, we conclude that LdcA is required for GC to provoke NOD1-dependent responses in cells of the human host.

TIFA Signaling in Gastric Epithelial Cells Initiates the cag Type 4 Secretion System-Dependent Innate Immune Response to Helicobacter pylori Infection.

ABSTRACTHelicobacter pylori is a bacterial pathogen that colonizes the human stomach, causing inflammation which, in some cases, leads to gastric ulcers and cancer. The clinical outcome of infection depends on a complex interplay of bacterial, host genetic, and environmental factors. Although H. pylori is recognized by both the innate and adaptive immune systems, this rarely results in bacterial clearance. Gastric epithelial cells are the first line of defense against H. pylori and alert the immune system to bacterial presence. Cytosolic delivery of proinflammatory bacterial factors through the cag type 4 secretion system (cag-T4SS) has long been appreciated as the major mechanism by which gastric epithelial cells detect H. pylori. Classically attributed to the peptidoglycan sensor NOD1, recent work has highlighted the role of NOD1-independent pathways in detecting H. pylori; however, the bacterial and host factors involved have remained unknown. Here, we show that bacterially derived heptose-1,7-bisphosphate (HBP), a metabolic precursor in lipopolysaccharide (LPS) biosynthesis, is delivered to the host cytosol through the cag-T4SS, where it activates the host tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with forkhead-associated domain (TIFA)-dependent cytosolic surveillance pathway. This response, which is independent of NOD1, drives robust NF-κB-dependent inflammation within hours of infection and precedes NOD1 activation. We also found that the CagA toxin contributes to the NF-κB-driven response subsequent to TIFA and NOD1 activation. Taken together, our results indicate that the sequential activation of TIFA, NOD1, and CagA delivery drives the initial inflammatory response in gastric epithelial cells, orchestrating the subsequent recruitment of immune cells and leading to chronic gastritis.

The Role of Nucleotide-Binding Oligomerization Domain-Like Receptors in Pulmonary Infection.

Pneumonia is caused by both viral and bacterial pathogens and is responsible for a significant health burden in the Unites States. The innate immune system is the human body's first line of defense against these pathogens. The recognition of invading pathogens via pattern recognition receptors leads to proinflammatory cytokine and chemokine production, followed by recruitment and activation of effector immune cells. The nonspecific inflammatory nature of the innate immune response can result in immunopathology that is detrimental to the host. In this review, we focus on one class of pattern recognition receptors, the nucleotide-binding oligomerization domain (NOD)-like receptors, specifically NOD1 and NOD2, and their role in host defense against viral and bacterial pathogens of the lung, including influenza, respiratory syncytial virus, Streptococcus pneumoniae, Chlamydophila pneumoniae, and Staphylococcus aureus. It is hoped that improved understanding of NOD1 and NOD2 activity in pneumonia will facilitate the development of novel therapies and promote improved patient outcomes.

Hypothetical protein Cpn0423 triggers NOD2 activation and contributes to Chlamydia pneumoniae-mediated inflammation

BackgroundChlamydia pneumoniae (C. pneumoniae) is pathogenic to humans, by causing pulmonary inflammation or bronchitis in both adolescents and young adults. However, the molecular signals linking C. pneumoniae components to inflammation remain elusive. This study was to investigate the effect of Chlamydia-specific Cpn0423 of C. pneumoniae on C. pneumoniae-mediated inflammation.ResultsCpn0423 was detected outside of C. pneumoniae inclusions, which induced production of several cytokines including macrophage inflammatory protein-2 (MIP-2) and interleukins (ILs). Production of the Cpn0423-induced cytokines was markedly reduced in cells pretreated with NOD2-siRNA, but not with negative control oligonucleotides. Mice treated with Cpn0423 through intranasal administration exhibited pulmonary inflammation as evidenced by infiltration of inflammatory cells, increased inflammatory scores in the lung histology, recruitment of neutrophils and increased cytokines levels in the BALF.ConclusionCpn0423 could be sensed by NOD2, which was identified as an essential element in a pathway contributing to the development of C. pneumoniae -mediated inflammation.

SLC46 Family Transporters Facilitate Cytosolic Innate Immune Recognition of Monomeric Peptidoglycans.

Tracheal cytotoxin (TCT), a monomer of DAP-type peptidoglycan from Bordetella pertussis, causes cytopathology in the respiratory epithelia of mammals and robustly triggers the Drosophila Imd pathway. PGRP-LE, a cytosolic innate immune sensor in Drosophila, directly recognizes TCT and triggers the Imd pathway, yet the mechanisms by which TCT accesses the cytosol are poorly understood. In this study, we report that CG8046, a Drosophila SLC46 family transporter, is a novel transporter facilitating cytosolic recognition of TCT, and plays a crucial role in protecting flies against systemic Escherichia coli infection. In addition, mammalian SLC46A2s promote TCT-triggered NOD1 activation in human epithelial cell lines, indicating that SLC46As is a conserved group of peptidoglycan transporter contributing to cytosolic immune recognition.

Innate And Adaptive Immunity are Progressively Activated in Parallel with Renal Injury in the 5/6 Renal Ablation Model

The mechanisms triggering renal inflammation in chronic kidney disease (CKD) are unclear. We performed a detailed analysis of the time course of innate and adaptive immunity activation in the 5/6 renal ablation (Nx) model. Munich-Wistar rats undergoing Nx were studied 15, 60 and 120 days after ablation. Hypertension, albuminuria, creatinine retention, interstitial expansion and infiltration by macrophages and T-lymphocytes were already evident 15 days after Nx. PCR-array was used to screen for altered gene expression, whereas gene and protein expressions of TLR4, CASP1, IL-1β and NLRP3 were individually assessed. Tlr4, Tlr5, Lbp, Nlrp3, Casp1, Irf7 and Il1b were already upregulated 15 days after Nx, while activation of Tlr2, Tlr7, Tlr9, Nod2, Tnf and Il6 was seen after 60 days post-ablation. The number of genes related to innate or adaptive immunity grew steadily with time. These observations indicate that parallel activation of innate and adaptive immunity antecedes glomerular injury and involves a growing number of intricate signaling pathways, helping to explain the difficulty in detaining renal injury in Nx as CKD advances, and, stressing the need for early treatment. Additionally, these findings may contribute to the search of therapeutic targets specific for advanced phases of CKD.

The effect of PRR ligands on the membrane potential of intestinal epithelial cells.

Disproportionate signaling through intestinal epithelial pattern recognition receptors (PRR) plays a role in IBD (inflammatory bowel disease) pathophysiology. Diarrhea is a clinical trademark of IBD and altered activity of K+ channels (KC) may contribute to the low sodium absorption state. Here we sought to study the impact of PRR activation on the membrane potential of human intestinal epithelial cells and the role of KC in it. All assays were performed in cultured HT-29 cells. KC activity was assessed by spectrofluorometry, measuring changes in cell membrane potential (MP) with the anionic fluorophore DiBAC4(3). PRRs were activated by specific ligands (MDP, LTA, MPLA, flagellin, loxoribine and ODN2216). KC modulators employed were BaCl2, pinacidil, noxiustoxin and AMP-PNP. Activation of NOD2, TLR5, TLR7 and TLR9 hyperpolarized the membrane (at 103ng/ml, the normalized AUC of the fluorescence intensity variation from the control were respectively (mean±SEM): -725.3±111.5; -1517.4±95.0; -857.8±61.1 and -995.6±53.6), while TLR2 and TLR4 stimulation induced membrane depolarization (1110.4±73.1 and 3890.3±342.7 at 103ng/ml, respectively). MPD effect on MP was abolished by BaCl2, partially reversed by AMP-PNP (aKATP channel inhibitor) and insensitive to noxiustoxin (a voltage-gated KC inhibitor). It was shown for the first time that PRR activation affects MP in human intestinal epithelial cells. KC appear to be important mediators in this phenomenon; in particular, KATP channels may partake in NOD2-derived effects.

NOD2 promotes endothelial-to-mesenchymal transition of glomerular endothelial cells via MEK/ERK signaling pathway in diabetic nephropathy

Endothelial-to-mesenchymal transition (EndMT) of glomerular vascular endothelial cells (GEnCs) is now considered to play a critical role in diabetic nephropathy (DN). NOD2 is newly discovered to be closely related to DN renal injury. However, the relationship between NOD2 and EndMT of GEnCs has never been reported. In the present study, we found that NOD2 over-expression was positively correlated with the severity of DN injury in human renal biopsy samples. Immunohistochemical staining of DN renal slices showed gradual absence of endothelial character and gain of mesenchymal character, both of which were associated with NOD2 over-expression. In high glucose stimulated GEnCs, NOD2 was increased. What's more, over-expression and activation of NOD2 could both promote EndMT of GEnCs. On the other hand, silencing of NOD2 markedly attenuated EndMT induced by high glucose. Mechanically, we further found that MEK/ERK signaling pathway was involved in NOD2-regulated EndMT. Collectively, our results indicate that NOD2 has a regulatory role in EndMT via activation of MEK/ERK in high glucose-treated GEnCs. Targeting this pathway is a promising strategy for intervention of DN endothelial dysfunction.

Enhancement of Muramyl Dipeptide‐Dependent NOD2 Activity by a Self‐Derived Peptide

Nucleotide-binding and oligomerization domain like receptors (NLR) are pattern recognition receptors used to provide rapid immune response by detecting intracellular pathogen-associated molecules. Loss of NLR activity is implicated in genetic disorders, disruption of adaptive immunity, and chronic inflammation. One NLR protein, NOD2, is frequently mutated in Crohn's disease (CD), which is an inflammatory disease of the gastrointestinal tract. Three commonly occurring CD-associated NOD2 mutations, R702W, G908R, and L1007fs, are clustered near the regulatory domain, leucine rich region (LRR), and lowers the activity of NOD2 in response to muramyl dipeptide (MDP). As LRR is also the ligand binding domain, this suggests that the mutations either affect the binding of MDP or how the molecule responds to ligand binding. To model the role of R702 in ligand-dependent activation of NOD2, we used homology modeling to map the residue R702 to the interface between the oligomerization domain and LRR. We show that a peptide derived from NOD2(697-718) binds LRR in vitro, and upon co-expressing or importing the peptide into HEK293 expressing NOD2, there is an increase in the MDP-dependent NOD2 activity. The study thus suggests that the R702W mutation interferes with the conformational changes needed for MDP binding and activation. J. Cell. Biochem. 118: 1227-1238, 2017. © 2016 Wiley Periodicals, Inc.