The Nucleotide-binding Leucine-rich Repeat (NLR) Family Member NLRX1 Mediates Protection against Experimental Autoimmune Encephalomyelitis and Represses Macrophage/Microglia-induced Inflammation

The nucleotide binding domain and leucine-rich repeat-containing (NLR) family of proteins is known to activate innate immunity, and the inflammasome-associated NLRs are prime examples. In contrast, the concept that NLRs can inhibit innate immunity is still debated, and the impact of such inhibitory NLRs in diseases shaped by adaptive immune responses is entirely unexplored. This study demonstrates that, in contrast to other NLRs that activate immunity, NLRX1 plays a protective role in experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. When compared with wild-type controls, Nlrx1^−/− mice have significantly worsened clinical scores and heightened CNS tissue damage during EAE. NLRX1 does not alter the production of encephalitogenic T cells in the peripheral lymphatic tissue, but Nlrx1^−/− mice are more susceptible to adoptively transferred myelin-reactive T cells. Analysis of the macrophage and microglial populations indicates that NLRX1 reduces activation during both active and passive EAE models. This work represents the first case of an NLR that attenuates microglia inflammatory activities and protects against a neurodegenerative disease model caused by autoreactive T cells.     

NLRC5 regulates MHC class I antigen presentation in host defense against intracellular pathogens

NOD-like receptors (NLRs) are a family of intracellular proteins that play critical roles in innate immunity against microbial infection. NLRC5, the largest member of the NLR family, has recently attracted much attention. However, in vitro studies have reported inconsistent results about the roles of NLRC5 in host defense and in regulating immune signaling pathways. The in vivo function of NLRC5 remains unknown. Here, we report that NLRC5 is a critical regulator of host defense against intracellular pathogens in vivo. NLRC5 was specifically required for the expression of genes involved in MHC class I antigen presentation. NLRC5-deficient mice showed a profound defect in the expression of MHC class I genes and a concomitant failure to activate L. monocytogenes-specific CD8⁺ T cell responses, including activation, proliferation and cytotoxicity, and the mutant mice were more susceptible to the pathogen infection. NLRP3-mediated inflammasome activation was also partially impaired in NLRC5-deficient mice. However, NLRC5 was dispensable for pathogen-induced expression of NF-κB-dependent pro-inflammatory genes as well as type I interferon genes. Thus, NLRC5 critically regulates MHC class I antigen presentation to control intracellular pathogen infection.     

Roles of NOD1 (NLRC1) and NOD2 (NLRC2) in innate immunity and inflammatory diseases

NOD1 {nucleotide-binding oligomerization domain 1; NLRC [NOD-LRR (leucine-rich repeat) family with CARD (caspase recruitment domain) 1]} and NOD2 (NLRC2) are among the most prominent members of the NLR (NOD-LRR) family –proteins that contain nucleotide-binding NACHT domains and receptor-like LRR domains. With over 20 members identified in humans, NLRs represent important components of the mammalian innate immune system, serving as intracellular receptors for pathogens and for endogenous molecules elaborated by tissue injury. NOD1 and NOD2 proteins operate as microbial sensors through the recognition of specific PG (peptidoglycan) constituents of bacteria. Upon activation, these NLR family members initiate signal transduction mechanisms that include stimulation of NF-κB (nuclear factor-κB), stress kinases, IRFs (interferon regulatory factors) and autophagy. Hereditary polymorphisms in the genes encoding NOD1 and NOD2 have been associated with an increasing number of chronic inflammatory diseases. In fact, potential roles for NOD1 and NOD2 in inflammatory disorders have been revealed by investigations using a series of animal models. In the present review, we describe recent experimental findings associating NOD1 and NOD2 with various autoimmune and chronic inflammatory disorders, and we discuss prospects for development of novel therapeutics targeting these NLR family proteins.     

NLRX1 is a mitochondrial NOD-like receptor that amplifies NF-kappaB and JNK pathways by inducing reactive oxygen species production

NOD-like receptors (NLRs) are a family of intracellular sensors of microbial- or danger-associated molecular patterns. Here, we report the identification of NLRX1, which is a new member of the NLR family that localizes to the mitochondria. NLRX1 alone failed to trigger most of the common signalling pathways, including nuclear factor-kappaB (NF)-kappaB- and type I interferon-dependent cascades, but could potently trigger the generation of reactive oxygen species (ROS). Importantly, NLRX1 synergistically potentiated ROS production induced by tumour necrosis factor alpha, Shigella infection and double-stranded RNA, resulting in amplified NF-kappaB-dependent and JUN amino-terminal kinases-dependent signalling. Together, these results identify NLRX1 as a NLR that contributes to the link between ROS generation at the mitochondria and innate immune responses.     

Malarial Hemozoin Activates the NLRP3 Inflammasome through Lyn and Syk Kinases

The intraerythrocytic parasite Plasmodium—the causative agent of malaria—produces an inorganic crystal called hemozoin (Hz) during the heme detoxification process, which is released into the circulation during erythrocyte lysis. Hz is rapidly ingested by phagocytes and induces the production of several pro-inflammatory mediators such as interleukin-1β (IL-1β). However, the mechanism regulating Hz recognition and IL-1β maturation has not been identified. Here, we show that Hz induces IL-1β production. Using knockout mice, we showed that Hz-induced IL-1β and inflammation are dependent on NOD-like receptor containing pyrin domain 3 (NLRP3), ASC and caspase-1, but not NLRC4 (NLR containing CARD domain). Furthermore, the absence of NLRP3 or IL-1β augmented survival to malaria caused by P. chabaudi adami DS. Although much has been discovered regarding the NLRP3 inflammasome induction, the mechanism whereby this intracellular multimolecular complex is activated remains unclear. We further demonstrate, using pharmacological and genetic intervention, that the tyrosine kinases Syk and Lyn play a critical role in activation of this inflammasome. These findings not only identify one way by which the immune system is alerted to malarial infection but also are one of the first to suggest a role for tyrosine kinase signaling pathways in regulation of the NLRP3 inflammasome.     

A Role for the Human Nucleotide-binding Domain,Leucine-rich Repeat-containing Family Member NLRC5 in Antiviral Responses

Proteins of the nucleotide-binding domain, leucine-rich repeat (NLR)-containing family recently gained attention as important components of the innate immune system. Although over 20 of these proteins are present in humans, only a few members including the cytosolic pattern recognition receptors NOD1, NOD2, and NLRP3 have been analyzed extensively. These NLRs were shown to be pivotal for mounting innate immune response toward microbial invasion. Here we report on the characterization of human NLRC5 and provide evidence that this NLR has a function in innate immune responses. We found that NLRC5 is a cytosolic protein expressed predominantly in hematopoetic cells. NLRC5 mRNA and protein expression was inducible by the double-stranded RNA analog poly(I·C) and Sendai virus. Overexpression of NLRC5 failed to trigger inflammatory responses such as the NF-κB or interferon pathways in HEK293T cells. However, knockdown of endogenous NLRC5 reduced Sendai virus- and poly(I·C)-mediated type I interferon pathway-dependent responses in THP-1 cells and human primary dermal fibroblasts. Taken together, this defines a function for NLRC5 in anti-viral innate immune responses.     

Crystal structure of the leucine-rich repeat domain of the NOD-like receptor NLRP1: Implications for binding of muramyl dipeptide

The NOD-like receptor NLRP1 (NLR family, pyrin domain containing 1) senses the presence of the bacterial cell wall component l-muramyl dipeptide (MDP) inside the cell. We determined the crystal structure of the LRR domain of human NLRP1 in the absence of MDP to a resolution of 1.65 Å. The fold of the structure can be assigned to the ribonuclease inhibitor-like class of LRR proteins. We compared our structure with X-ray models of the LRR domains of NLRX1 and NLRC4 and a homology model of the LRR domain of NOD2. We conclude that the MDP binding site of NLRP1 is not located in the LRR domain.     

NOD-like receptors and the innate immune system: coping with danger, damage and death

Members of the family of NOD-like receptors (NLRs) play essential roles in innate immunity by detecting intracellular 'pathogen-associated molecular patterns' (PAMPs) and 'danger-associated molecular patterns' (DAMPs). These molecules reveal the presence of pathogenic infection, abiotic stress, environmental insults, cellular damage, and cell death. NLR family members can be divided in two functional groups. One group consists of intracellular receptors, such as NLRP1, NLRP3, NLRP6 and NLRC4, which mediate the assembly of inflammasome complexes leading to the activation of procaspase-1. The second group includes members such as NOD1 and NOD2, and mediates the assembly of complexes that activate MAPK and NF-κB signaling pathways. We review the roles of NLR family members in health and disease, with emphasis on the signaling mechanisms in cell death and inflammation.     

Inflammasome-dependent pyroptosis and IL-18 protect against Burkholderia pseudomallei lung infection while IL-1β is deleterious

Burkholderia pseudomallei is a Gram-negative bacterium that infects macrophages and other cell types and causes melioidosis. The interaction of B. pseudomallei with the inflammasome and the role of pyroptosis, IL-1β, and IL-18 during melioidosis have not been investigated in detail. Here we show that the Nod-like receptors (NLR) NLRP3 and NLRC4 differentially regulate pyroptosis and production of IL-1β and IL-18 and are critical for inflammasome-mediated resistance to melioidosis. In vitro production of IL-1β by macrophages or dendritic cells infected with B. pseudomallei was dependent on NLRC4 and NLRP3 while pyroptosis required only NLRC4. Mice deficient in the inflammasome components ASC, caspase-1, NLRC4, and NLRP3, were dramatically more susceptible to lung infection with B. pseudomallei than WT mice. The heightened susceptibility of Nlrp3^-/- mice was due to decreased production of IL-18 and IL-1β. In contrast, Nlrc4^-/- mice produced IL-1β and IL-18 in higher amount than WT mice and their high susceptibility was due to decreased pyroptosis and consequently higher bacterial burdens. Analyses of IL-18-deficient mice revealed that IL-18 is essential for survival primarily because of its ability to induce IFNγ production. In contrast, studies using IL-1RI-deficient mice or WT mice treated with either IL-1β or IL-1 receptor agonist revealed that IL-1β has deleterious effects during melioidosis. The detrimental role of IL-1β appeared to be due, in part, to excessive recruitment of neutrophils to the lung. Because neutrophils do not express NLRC4 and therefore fail to undergo pyroptosis, they may be permissive to B. pseudomallei intracellular growth. Administration of neutrophil-recruitment inhibitors IL-1ra or the CXCR2 neutrophil chemokine receptor antagonist antileukinate protected Nlrc4^-/- mice from lethal doses of B. pseudomallei and decreased systemic dissemination of bacteria. Thus, the NLRP3 and NLRC4 inflammasomes have non-redundant protective roles in melioidosis: NLRC4 regulates pyroptosis while NLRP3 regulates production of protective IL-18 and deleterious IL-1β.     

Direct Proteolytic Cleavage of NLRP1B Is Necessary and Sufficient for Inflammasome Activation by Anthrax Lethal Factor

Inflammasomes are multimeric protein complexes that respond to infection by recruitment and activation of the Caspase-1 (CASP1) protease. Activated CASP1 initiates immune defense by processing inflammatory cytokines and by causing a rapid and lytic cell death called pyroptosis. Inflammasome formation is orchestrated by members of the nucleotide-binding domain and leucine-rich repeat (NLR) or AIM2-like receptor (ALR) protein families. Certain NLRs and ALRs have been shown to function as direct receptors for specific microbial ligands, such as flagellin or DNA, but the molecular mechanism responsible for activation of most NLRs is still poorly understood. Here we determine the mechanism of activation of the NLRP1B inflammasome in mice. NLRP1B, and its ortholog in rats, is activated by the lethal factor (LF) protease that is a key virulence factor secreted by Bacillus anthracis, the causative agent of anthrax. LF was recently shown to cleave mouse and rat NLRP1 directly. However, it is unclear if cleavage is sufficient for NLRP1 activation. Indeed, other LF-induced cellular events have been suggested to play a role in NLRP1B activation. Surprisingly, we show that direct cleavage of NLRP1B is sufficient to induce inflammasome activation in the absence of LF. Our results therefore rule out the need for other LF-dependent cellular effects in activation of NLRP1B. We therefore propose that NLRP1 functions primarily as a sensor of protease activity and thus could conceivably detect a broader spectrum of pathogens than just B. anthracis. By adding proteolytic cleavage to the previously established ligand-receptor mechanism of NLR activation, our results illustrate the remarkable flexibility with which the NLR architecture can be deployed for the purpose of pathogen-detection and host defense.     

Bone marrow‐derived mesenchymal stem cells promote Helicobacter pylori‐associated gastric cancer progression by secreting thrombospondin‐2

ObjectivesBone marrow‐derived cells (BMDCs), especially mesenchymal stem cells (MSCs), may be involved in the development of Helicobacter pylori‐associated gastric cancer (GC) in mice, but the specific mechanism remains unclear, and evidence from human studies is lacking.Materials and MethodsTo verify the role of BM‐MSCs in H pylori‐associated GC, green fluorescent protein (GFP)‐labelled BM‐MSCs were transplanted into the subserosal layers of the stomach in a mouse model of chronic H pylori infection. Three months post‐transplantation, the mice were sacrificed, and the gastric tissues were subjected to histopathological and immunofluorescence analyses. In addition, we performed fluorescence in situ hybridization (FISH) and immunofluorescence analyses of gastric tissue from a female patient with H pylori infection and a history of acute myeloid leukaemia who received a BM transplant from a male donor.ResultsIn mice with chronic H pylori infection, GFP‐labelled BM‐MSCs migrated from the serous layer to the mucosal layer and promoted GC progression. The BM‐MSCs differentiated into pan‐cytokeratin‐positive epithelial cells and α‐smooth muscle actin‐positive cancer‐associated fibroblasts (CAFs) by secreting the protein thrombospondin‐2. FISH analysis of gastric tissue from the female patient revealed Y‐chromosome‐positive cells. Immunofluorescence analyses further confirmed that Y‐chromosome‐positive cells showed positive BM‐MSCs marker. These results suggested that allogeneic BMDCs, including BM‐MSCs, can migrate to the stomach under chronic H pylori infection.ConclusionsTaken together, these findings imply that BM‐MSCs participate in the development of chronic H pylori‐associated GC by differentiating into both gastric epithelial cells and CAFs.     

NLRX1/FUNDC1/NIPSNAP1‐2 axis regulates mitophagy and alleviates intestinal ischaemia/reperfusion injury

ObjectivesMitophagy is considered to be a key mechanism in the pathogenesis of intestinal ischaemic reperfusion (IR) injury. NOD‐like receptor X1 (NLRX1) is located in the mitochondria and is highly expressed in the intestine, and is known to modulate ROS production, mitochondrial damage, autophagy and apoptosis. However, the function of NLRX1 in intestinal IR injury is unclear.Materials and methodsNLRX1 in rats with IR injury or in IEC‐6 cells with hypoxia reoxygenation (HR) injury were measured by Western blotting, real‐time PCR and immunohistochemistry. The function of NLRX1‐FUNDC1‐NIPSNAP1/NIPSNAP2 axis in mitochondrial homeostasis and cell apoptosis were assessed in vitro.ResultsNLRX1 is significantly downregulated following intestinal IR injury. In vivo studies showed that rats overexpressing NLRX1 exhibited resistance against intestinal IR injury and mitochondrial dysfunction. These beneficial effects of NLRX1 overexpression were dependent on mitophagy activation. Functional studies showed that HR injury reduced NLRX1 expression, which promoted phosphorylation of FUN14 domain‐containing 1 (FUNDC1). Based on immunoprecipitation studies, it was evident that phosphorylated FUNDC1 could not interact with the mitophagy signalling proteins NIPSNAP1 and NIPSNAP2 on the outer membrane of damaged mitochondria, which failed to launch the mitophagy process, resulting in the accumulation of damaged mitochondria and epithelial apoptosis.ConclusionsNLRX1 regulates mitophagy via FUNDC1‐NIPSNAP1/NIPSNAP2 signalling pathway. Thus, this study provides a potential target for the development of a therapeutic strategy for intestinal IR injury.     

Polymorphisms in Inflammasome Genes and Risk of Coal Workers' Pneumoconiosis in a Chinese Population

Background

Coal workers'' pneumoconiosis (CWP), resulting from the inhalation of silica-containing coal mine dust, is characterized by fibrosing nodular lesions that eventually develop into progressive pulmonary fibrosis. Recently, it has been hypothesized that inflammasomes could have a crucial role in the host response to silica and recent studies show that the inflammasome contributes to inflammation and pulmonary fibrosis. NLRP3, CARD8 are components of the NLRP3 inflammasome, which triggers caspase 1-mediated IL-1β and IL-18 release. In the present study, we investigated whether common single nucleotide polymorphisms (SNPs) in inflammasome genes are associated with CWP.

Methods

We performed an association study analyzing 3 NLRP3, 1 CARD8, 1 IL-1β, 2 IL-18 SNPs in a case-control study of 697 CWP and 694 controls. Genotyping was carried out by the TaqMan method.

Results

The NLRP3 rs1539019 TT genotype was associated with a significantly increased risk of CWP (adjusted odds ratio (OR) = 1.39, 95% confidence interval (CI) = 1.07–1.81), compared with the GG/GT genotype, in particular among smokers (adjusted OR = 1.67, 95%CI = 1.15–2.42). In addition, the polymorphism was significantly associated with risk of CWP patients with stage I.

Conclusions

This is the first report showing an association between the NLRP3 rs1539019 polymorphism and CWP, and suggests that this polymorphism may confer increased risk for the development of the disease. Further studies are warranted to confirm our findings.     

Cutting edge: NLRC5-dependent activation of the inflammasome

The nucleotide-binding domain leucine-rich repeat-containing proteins, NLRs, are intracellular sensors of pathogen-associated molecular patterns and damage-associated molecular patterns. A subgroup of NLRs can form inflammasome complexes, which facilitate the maturation of procaspase 1 to caspase 1, leading to IL-1β and IL-18 cleavage and secretion. NLRC5 is predominantly expressed in hematopoietic cells and has not been studied for inflammasome function. RNA interference-mediated knockdown of NLRC5 nearly eliminated caspase 1, IL-1β, and IL-18 processing in response to bacterial infection, pathogen-associated molecular patterns, and damage-associated molecular patterns. This was confirmed in primary human monocytic cells. NLRC5, together with procaspase 1, pro-IL-1β, and the inflammasome adaptor ASC, reconstituted inflammasome activity that showed cooperativity with NLRP3. The range of pathogens that activate NLRC5 inflammasome overlaps with those that activate NLRP3. Furthermore, NLRC5 biochemically associates with NLRP3 in a nucleotide-binding domain-dependent but leucine-rich repeat-inhibitory fashion. These results invoke a model in which NLRC5 interacts with NLRP3 to cooperatively activate the inflammasome.     

The Role of TLR2, TLR4 and CD14 Genetic Polymorphisms in Gastric Carcinogenesis: A Case-Control Study and Meta-Analysis

Background

In addition to Helicobacter pylori infection, host genetic factors contribute to gastric cancer (GC). Recognition of H. pylori is known to involve Toll-like receptors (TLR), which subsequently leads to activation of NF-κB. Thus, the overall aim of this study was to estimate for the first time the pooled effect size of polymorphisms in TLR2, TLR4 and CD14 on GC development through a meta-analysis.

Methods

A case-control study comprising 284 ethnic Chinese individuals (70 non-cardia GC cases and 214 functional dyspepsia controls) was conducted for the genotyping of TLR2 -196 to -174del, CD14 -260 C/T and TLR4 rs11536889 using PCR, RT-PCR and mass spectrometry. Case-control studies of TLR2, TLR4 and CD14 polymorphisms and GC were searched up to June 2012. Pooled odds ratios and 95% confidence intervals were obtained by means of the random effects model.

Results

In our ethnic Chinese case-control study, the TLR4 rs11536889 C allele increased the risk of GC (OR: 1.89, 95%CI: 1.23–2.92) while the CD14 -260 T allele was protective (OR: 0.62, 95%CI: 0.42–0.91). TLR2 -196 to -174 increased the risk of GC only in H. pylori-infected individuals (OR: 3.10, 95%CI: 1.27–7.60). In the meta-analysis, TLR4 Asp299Gly showed borderline results in the general analysis (pooled OR: 1.58, 95%CI: 0.98–2.60), nevertheless, stratified analysis by ethnicity showed that the mutant allele was a definitive risk factor for GC in Western populations (pooled OR: 1.87, 95%CI: 1.31–2.65). There was a potential association between the TLR2 -196 to -174 deletion allele and GC in Japanese (pooled OR: 1.18, 95%CI: 0.96–1.45). TLR4 Thr399Ile did not provide significant results.

Conclusions

TLR4 rs11536889 and CD14 -260 C/T are associated with non-cardia GC in Chinese. Based on our meta-analysis, the TLR signalling pathway is involved in gastric carcinogenesis, TLR4 Asp299Gly and TLR2 -196 to -174del showing associations with GC in an ethnic-specific manner.     

NLRC5 potentiates anti-tumor CD8+ T cells responses by activating interferon-β in endometrial cancer

ObjectivesNLR family CARD domain containing 5 (NLRC5) could promote major histocompatibility complex class I (MHC-I)-dependent CD8⁺ T cell-mediated anticancer immunity. In this study, the immunosurveillance role and underlying mechanisms of NLRC5 in endometrial cancer (EC) were characterized.MethodsCD8⁺ T cells were separated from healthy women's peripheral blood by using magnetic beads. The effect of NLRC5 and interferon-β (IFN-β) on immunosurveillance of EC were examined through a mouse tumor model and a CD8⁺ T cell-EC cell coculture system after NLRC5 overexpression and IFN-β overexpression or depletion. The effect of NLRC5 on IFN-β expression was examined with gain- and loss-of-function experiments.ResultsNLRC5 overexpression in the EC cell and CD8⁺ T cell coculture system inhibited EC cell proliferation and migration and promoted EC cell apoptosis and CD8⁺ T cell proliferation. In vivo, NLRC5 overexpression increased the proportion of CD8⁺ T cells and inhibited EC progression. Furthermore, IFN-β overexpression in the EC cell and CD8⁺ T cell coculture system activated CD8⁺ T cell proliferation; however, genetic depletion of IFN-β exerted the opposite effects. In addition, NLRC5 could negatively regulate IFN-β expression in EC cells. Mechanistically, NLRC5 potentiated the antitumor responses of CD8⁺ T cells to EC by activating IFN-β.ConclusionsTaken together, our findings demonstrated that NLRC5 potentiates anti-tumor CD8⁺ T cells responses by activating interferon-β in EC, suggesting that genetically escalated NLRC5 and IFN-β may act as potential candidates for the clinical translation of adjuvant immunotherapies to patients with EC.