The inflammasome NLRs in immunity, inflammation, and associated diseases.

Inflammasome activation leads to caspase-1 activation, which causes the maturation and secretion of pro-IL-1ß and pro-IL-18 among other substrates. A subgroup of the NLR (nucleotide-binding domain, leucine-rich repeat containing) proteins are key mediators of the inflammasome. Studies of gene-deficient mice and cells have implicated NLR inflammasomes in a host of responses to a wide range of microbial pathogens, inflammatory diseases, cancer, and metabolic and autoimmune disorders. Determining exactly how the inflammasome is activated in these diseases and disease models remains a challenge. This review presents and integrates recent progress in the field.

The innate immune sensor NLRC3 attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-κB.

Several members of the NLR family of sensors activate innate immunity. In contrast, we found here that NLRC3 inhibited Toll-like receptor (TLR)-dependent activation of the transcription factor NF-κB by interacting with the TLR signaling adaptor TRAF6 to attenuate Lys63 (K63)-linked ubiquitination of TRAF6 and activation of NF-κB. We used bioinformatics to predict interactions between NLR and TRAF proteins, including interactions of TRAF with NLRC3. In vivo, macrophage expression of Nlrc3 mRNA was diminished by the administration of lipopolysaccharide (LPS) but was restored when cellular activation subsided. To assess biologic relevance, we generated Nlrc3(-/-) mice. LPS-treated Nlrc3(-/-) macrophages had more K63-ubiquitinated TRAF6, nuclear NF-κB and proinflammatory cytokines. Finally, LPS-treated Nlrc3(-/-) mice had more signs of inflammation. Thus, signaling via NLRC3 and TLR constitutes a negative feedback loop. Furthermore, prevalent NLR-TRAF interactions suggest the formation of a 'TRAFasome' complex.

NLRP12 suppresses colon inflammation and tumorigenesis through the negative regulation of noncanonical NF-κB signaling.

In vitro data suggest that a subgroup of NLR proteins, including NLRP12, inhibits the transcription factor NF-κB, although physiologic and disease-relevant evidence is largely missing. Dysregulated NF-κB activity is associated with colonic inflammation and cancer, and we found Nlrp12(-/-) mice were highly susceptible to colitis and colitis-associated colon cancer. Polyps isolated from Nlrp12(-/-) mice showed elevated noncanonical NF-κB activation and increased expression of target genes that were associated with cancer, including Cxcl13 and Cxcl12. NLRP12 negatively regulated ERK and AKT signaling pathways in affected tumor tissues. Both hematopoietic- and nonhematopoietic-derived NLRP12 contributed to inflammation, but the latter dominantly contributed to tumorigenesis. The noncanonical NF-κB pathway was regulated upon degradation of TRAF3 and activation of NIK. NLRP12 interacted with both NIK and TRAF3, and Nlrp12(-/-) cells have constitutively elevated NIK, p100 processing to p52 and reduced TRAF3. Thus, NLRP12 is a checkpoint of noncanonical NF-κB, inflammation, and tumorigenesis.

NLRX1 protein attenuates inflammatory responses to infection by interfering with the RIG-I-MAVS and TRAF6-NF-κB signaling pathways.

The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. We showed that Nlrx1(-/-) mice exhibited increased expression of antiviral signaling molecules IFN-ß, STAT2, OAS1, and IL-6 after influenza virus infection. Consistent with increased inflammation, Nlrx1(-/-) mice exhibited marked morbidity and histopathology. Infection of these mice with an influenza strain that carries a mutated NS-1 protein, which normally prevents IFN induction by interaction with RNA and the intracellular RNA sensor RIG-I, further exacerbated IL-6 and type I IFN signaling. NLRX1 also weakened cytokine responses to the 2009 H1N1 pandemic influenza virus in human cells. Mechanistically, Nlrx1 deletion led to constitutive interaction of MAVS and RIG-I. Additionally, an inhibitory function is identified for NLRX1 during LPS activation of macrophages where the MAVS-RIG-I pathway was not involved. NLRX1 interacts with TRAF6 and inhibits NF-κB activation. Thus, NLRX1 functions as a checkpoint of overzealous inflammation.

The Scaffolding Protein IQGAP1 Interacts with NLRC3 and Inhibits Type I IFN Production.

Sensing of cytosolic nucleotides is a critical initial step in the elaboration of type I IFN. One of several upstream receptors, cyclic GMP-AMP synthase, binds to cytosolic DNA and generates dicyclic nucleotides that act as secondary messengers. These secondary messengers bind directly to stimulator of IFN genes (STING). STING recruits TNFR-associated NF-κB kinase-binding kinase 1 which acts as a critical node that allows for efficient activation of IFN regulatory factors to drive the antiviral transcriptome. NLRC3 is a recently characterized nucleotide-binding domain, leucine-rich repeat containing protein (NLR) that negatively regulates the type I IFN pathway by inhibiting subcellular redistribution and effective signaling of STING, thus blunting the transcription of type I IFNs. NLRC3 is predominantly expressed in lymphoid and myeloid cells. IQGAP1 was identified as a putative interacting partner of NLRC3 through yeast two-hybrid screening. In this article, we show that IQGAP1 associates with NLRC3 and can disrupt the NLRC3-STING interaction in the cytosol of human epithelial cells. Furthermore, knockdown of IQGAP1 in THP1 and HeLa cells causes significantly more IFN-ß production in response to cytosolic nucleic acids. This result phenocopies NLRC3-deficient macrophages and fibroblasts and short hairpin RNA knockdown of NLRC3 in THP1 cells. Our findings suggest that IQGAP1 is a novel regulator of type I IFN production, possibly via interacting with NLRC3 in human monocytic and epithelial cells.

The Poly(ADP-ribose) Polymerase Enzyme Tankyrase Antagonizes Activity of the ß-Catenin Destruction Complex through ADP-ribosylation of Axin and APC2.

Most colon cancer cases are initiated by truncating mutations in the tumor suppressor, adenomatous polyposis coli (APC). APC is a critical negative regulator of the Wnt signaling pathway that participates in a multi-protein "destruction complex" to target the key effector protein ß-catenin for ubiquitin-mediated proteolysis. Prior work has established that the poly(ADP-ribose) polymerase (PARP) enzyme Tankyrase (TNKS) antagonizes destruction complex activity by promoting degradation of the scaffold protein Axin, and recent work suggests that TNKS inhibition is a promising cancer therapy. We performed a yeast two-hybrid (Y2H) screen and uncovered TNKS as a putative binding partner of Drosophila APC2, suggesting that TNKS may play multiple roles in destruction complex regulation. We find that TNKS binds a C-terminal RPQPSG motif in Drosophila APC2, and that this motif is conserved in human APC2, but not human APC1. In addition, we find that APC2 can recruit TNKS into the ß-catenin destruction complex, placing the APC2/TNKS interaction at the correct intracellular location to regulate ß-catenin proteolysis. We further show that TNKS directly PARylates both Drosophila Axin and APC2, but that PARylation does not globally regulate APC2 protein levels as it does for Axin. Moreover, TNKS inhibition in colon cancer cells decreases ß-catenin signaling, which we find cannot be explained solely through Axin stabilization. Instead, our findings suggest that TNKS regulates destruction complex activity at the level of both Axin and APC2, providing further mechanistic insight into TNKS inhibition as a potential Wnt pathway cancer therapy.

Type I IFN triggers RIG-I/TLR3/NLRP3-dependent inflammasome activation in influenza A virus infected cells.

Influenza A virus (IAV) triggers a contagious and potentially lethal respiratory disease. A protective IL-1ß response is mediated by innate receptors in macrophages and lung epithelial cells. NLRP3 is crucial in macrophages; however, which sensors elicit IL-1ß secretion in lung epithelial cells remains undetermined. Here, we describe for the first time the relative roles of the host innate receptors RIG-I (DDX58), TLR3, and NLRP3 in the IL-1ß response to IAV in primary lung epithelial cells. To activate IL-1ß secretion, these cells employ partially redundant recognition mechanisms that differ from those described in macrophages. RIG-I had the strongest effect through a MAVS/TRIM25/Riplet-dependent type I IFN signaling pathway upstream of TLR3 and NLRP3. Notably, RIG-I also activated the inflammasome through interaction with caspase 1 and ASC in primary lung epithelial cells. Thus, NS1, an influenza virulence factor that inhibits the RIG-I/type I IFN pathway, strongly modulated the IL-1ß response in lung epithelial cells and in ferrets. The NS1 protein derived from a highly pathogenic strain resulted in increased interaction with RIG-I and inhibited type I IFN and IL-1ß responses compared to the least pathogenic virus strains. These findings demonstrate that in IAV-infected lung epithelial cells RIG-I activates the inflammasome both directly and through a type I IFN positive feedback loop.

Pathogen sensing by nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is mediated by direct binding to muramyl dipeptide and ATP.

Nucleotide binding and oligomerization domain-containing protein 2 (NOD2/Card15) is an intracellular protein that is involved in the recognition of bacterial cell wall-derived muramyl dipeptide. Mutations in the gene encoding NOD2 are associated with inherited inflammatory disorders, including Crohn disease and Blau syndrome. NOD2 is a member of the nucleotide-binding domain and leucine-rich repeat-containing protein gene (NLR) family. Nucleotide binding is thought to play a critical role in signaling by NLR family members. However, the molecular mechanisms underlying signal transduction by these proteins remain largely unknown. Mutations in the nucleotide-binding domain of NOD2 have been shown to alter its signal transduction properties in response to muramyl dipeptide in cellular assays. Using purified recombinant protein, we now demonstrate that NOD2 binds and hydrolyzes ATP. Additionally, we have found that the purified recombinant protein is able to bind directly to muramyl dipeptide and can associate with known NOD2-interacting proteins in vitro. Binding of NOD2 to muramyl dipeptide and homo-oligomerization of NOD2 are enhanced by ATP binding, suggesting a model of the molecular mechanism for signal transduction that involves binding of nucleotide followed by binding of muramyl dipeptide and oligomerization of NOD2 into a signaling complex. These findings set the stage for further studies into the molecular mechanisms that underlie detection of muramyl dipeptide and assembly of NOD2-containing signaling complexes.

Regulation of class I major histocompatibility complex (MHC) by nucleotide-binding domain, leucine-rich repeat-containing (NLR) proteins.

Most of the nucleotide-binding domain, leucine-rich repeat (NLR) proteins regulate responses to microbial and damage-associated products. Class II transactivator (CIITA) has a distinct function as the master regulator of class II major histocompatibility complex (MHC-II) transcription. Recently, human NLRC5 was found to regulate MHC-I in cell lines; however, a host of conflicting positive and negative functions has been attributed to this protein. To address the function of NLRC5 in a physiologic setting, we generated an Nlrc5(-/-) strain that contains a deletion in the exon that encodes the nucleotide-binding domain. We have not detected a role for this protein in cytokine induction by pathogen-associated molecular patterns and viruses. However, Nlrc5(-/-) cells showed a dramatic decrease of classical (H-2K) and nonclassical (Tla) MHC-I expression by T/B lymphocytes, natural killer (NK) cells, and myeloid-monocytic lineages. As a comparison, CIITA did not affect mouse MHC-I expression. Nlrc5(-/-) splenocytes and bone marrow-derived macrophages were able to up-regulate MHC-I in response to IFN-γ; however, the absolute levels of MHC-I expression were significantly lower than WT controls. Chromatin immunoprecipitation of IFN-γ-treated cells indicates that Nlrc5 reduced the silencing H3K27me3 histone modification, but did not affect the activating AcH3 modification on a MHC-I promoter. In summary, we conclude that Nlrc5 is important in the regulation of MHC-I expression by reducing H3K27me3 on MHC-I promoter and joins CIITA as an NLR subfamily that controls MHC gene transcription.

Monocytic microparticles activate endothelial cells in an IL-1ß-dependent manner.

Microparticles (MPs) are shed from activated and dying cells. They can transmit signals from cell to cell, locally or at a distance through the circulation. Monocytic MPs are elevated in different diseases, including bacterial infections. Here, we investigated how monocytic MPs activate endothelial cells. We found that MPs from lipopolysaccharide (LPS)-treated THP-1 monocytic cells bind to and are internalized by human endothelial cells. MPs from LPS-treated THP-1 cells, but not untreated cells, induced phosphorylation of ERK1/2, activation of the nuclear factor-κB pathway and expression of cell adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. Similar results were observed using MPs from LPS-treated peripheral blood mononuclear cells. We next investigated the mechanism by which monocytic MPs activated endothelial cells and found that they contain IL-1ß and components of the inflammasome, including apoptosis-associated speck-like protein containing a CARD, caspase-1, and NLRP3. Importantly, knockdown of NLRP3 in THP-1 cells reduced the activity of the MPs and blockade of the IL-1 receptor on endothelial cells decreased MP-dependent induction of cell adhesion molecules. Therefore, monocytic MPs contain IL-1ß and may amplify inflammation by enhancing the activation of the endothelium.

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-1ß 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-1ß, 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-1ß, 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.

DJ-1 enhances cell survival through the binding of Cezanne, a negative regulator of NF-kappaB.

Heightened DJ-1 (Park7) expression is associated with a reduction in chemotherapeutic-induced cell death and poor prognosis in several cancers, whereas the loss of DJ-1 function is found in a subgroup of Parkinson disease associated with neuronal death. This study describes a novel pathway by which DJ-1 modulates cell survival. Mass spectrometry shows that DJ-1 interacts with BBS1, CLCF1, MTREF, and Cezanne/OTUD7B/Za20d1. Among these, Cezanne is a known deubiquitination enzyme that inhibits NF-κB activity. DJ-1/Cezanne interaction is confirmed by co-immunoprecipitation of overexpressed and endogenous proteins, maps to the amino-terminal 70 residues of DJ-1, and leads to the inhibition of the deubiquitinating activity of Cezanne. Microarray profiling of shRNA-transduced cells shows that DJ-1 and Cezanne regulate IL-8 and ICAM-1 expression in opposing directions. Similarly, DJ-1 enhances NF-κB nuclear translocation and cell survival, whereas Cezanne reduces these outcomes. Analysis of mouse Park7(-/-) primary cells confirms the regulation of ICAM-1 by DJ-1 and Cezanne. As NF-κB is important in cellular survival and transformation, IL-8 functions as an angiogenic factor and pro-survival signal, and ICAM-1 has been implicated in tumor progression, invasion, and metastasis; these data provide an additional modality by which DJ-1 controls cell survival and possibly tumor progression via interaction with Cezanne.

Deletion of ripA alleviates suppression of the inflammasome and MAPK by Francisella tularensis.

Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F. tularensis ripA deletion mutant (LVSΔripA) provoked significant release of IL-1ß, IL-18, and TNF-α by resting macrophages. IL-1ß and IL-18 secretion was dependent on inflammasome components pyrin-caspase recruitment domain/apoptotic speck-containing protein with a caspase recruitment domain and caspase-1, and the TLR/IL-1R signaling molecule MyD88 was required for inflammatory cytokine synthesis. Complementation of LVSΔripA with a plasmid encoding ripA restored immune evasion. Similar findings were observed in a human monocytic line. The presence of ripA nearly eliminated activation of MAPKs including ERK1/2, JNK, and p38, and pharmacologic inhibitors of these three MAPKs reduced cytokine induction by LVSΔripA. Animals infected with LVSΔripA mounted a stronger IL-1ß and TNF-α response than that of mice infected with wild-type live vaccine strain. This analysis revealed novel immune evasive mechanisms of F. tularensis.

TRAF3IP3 negatively regulates cytosolic RNA induced anti-viral signaling by promoting TBK1 K48 ubiquitination.

Innate immunity to nucleic acids forms the backbone for anti-viral immunity and several inflammatory diseases. Upon sensing cytosolic viral RNA, retinoic acid-inducible gene-I-like receptors (RLRs) interact with the mitochondrial antiviral signaling protein (MAVS) and activate TANK-binding kinase 1 (TBK1) to induce type I interferon (IFN-I). TRAF3-interacting protein 3 (TRAF3IP3, T3JAM) is essential for T and B cell development. It is also well-expressed by myeloid cells, where its role is unknown. Here we report that TRAF3IP3 suppresses cytosolic poly(I:C), 5'ppp-dsRNA, and vesicular stomatitis virus (VSV) triggers IFN-I expression in overexpression systems and Traf3ip3-/- primary myeloid cells. The mechanism of action is through the interaction of TRAF3IP3 with endogenous TRAF3 and TBK1. This leads to the degradative K48 ubiquitination of TBK1 via its K372 residue in a DTX4-dependent fashion. Mice with myeloid-specific gene deletion of Traf3ip3 have increased RNA virus-triggered IFN-I production and reduced susceptibility to virus. These results identify a function of TRAF3IP3 in the regulation of the host response to cytosolic viral RNA in myeloid cells.

C-C chemokine receptor 5 on pulmonary fibrocytes facilitates migration and promotes metastasis via matrix metalloproteinase 9.

Previously, our group has used a B16-F10 melanoma model to show that C-C chemokine receptor 5 (CCR5) knockout (CCR5(-/-)) mice form fewer pulmonary metastases than wild-type mice. This advantage can be eliminated by injecting CCR5(-/-) mice with wild-type pulmonary mesenchymal cells before tumor injection. In this article, we present the mechanisms underlying this finding. First, we demonstrate that wild-type mesenchymal cells migrate to CCL4 more efficiently in vitro than CCR5(-/-) cells. Wild-type mesenchymal cells were also 3.6 (1.85 to 5.85) times more efficient than CCR5(-/-) cells at migrating into the lung after intravenous injection (P < 0.01). The injection of wild-type but not CCR5(-/-) mesenchymal cells led to a 7.0 +/- 1.6 (P < 0.05)-fold induction of matrix metalloproteinase 9 (MMP9) in the host lung. Neither wild-type nor CCR5(-/-) cells caused significant increases in MMP2, MMP3, or MMP8. Inhibition of the gelatinase activity of MMP9 decreased the number of metastases and restored the advantage that CCR5(-/-) mice have over wild-type mice. Further analysis showed that the CCR5(+) mesenchymal cells expressed CD45(+) and CD13(+) but did not express alpha-smooth muscle actin. This phenotype is characteristic of a subset of mesenchymal cells called fibrocytes. Together, these data suggest a novel role for CCR5 in the migration of pulmonary fibrocytes and the promotion of metastasis.

Derivation of Macrophages from Mouse Bone Marrow.

Macrophages are cellular components of the immune system that are essential for responding to pathogens, initiating inflammation and maintaining tissue homeostasis. Isolation, culture, and functional characterization of bone marrow-derived macrophages from mice are exceptionally powerful in vitro techniques used to examine aspects of macrophage biology, including effector functions, such as phagocytosis, cytokine secretion, oxidative burst, migration, and antigen processing and presentation. These studies can be carried out using wild-type, gene-ablated, and/or transgenic mice. The quantity, purity, and ease of culture of these cells enhance their utility for primary cell cultures to understand macrophage biology. Mouse macrophages have become a cognate animal model for the study of human macrophage biology and disease. This chapter outlines protocols used to generate, polarize, quantitate, and functionally evaluate macrophages derived from bone marrow precursor cells.

Inflammasome-independent role of AIM2 in suppressing colon tumorigenesis via DNA-PK and Akt.

The inflammasome activates caspase-1 and the release of interleukin-1ß (IL-1ß) and IL-18, and several inflammasomes protect against intestinal inflammation and colitis-associated colon cancer (CAC) in animal models. The absent in melanoma 2 (AIM2) inflammasome is activated by double-stranded DNA, and AIM2 expression is reduced in several types of cancer, but the mechanism by which AIM2 restricts tumor growth remains unclear. We found that Aim2-deficient mice had greater tumor load than Asc-deficient mice in the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colorectal cancer. Tumor burden was also higher in Aim2(-/-)/Apc(Min/+) than in APC(Min/+) mice. The effects of AIM2 on CAC were independent of inflammasome activation and IL-1ß and were primarily mediated by a non-bone marrow source of AIM2. In resting cells, AIM2 physically interacted with and limited activation of DNA-dependent protein kinase (DNA-PK), a PI3K-related family member that promotes Akt phosphorylation, whereas loss of AIM2 promoted DNA-PK-mediated Akt activation. AIM2 reduced Akt activation and tumor burden in colorectal cancer models, while an Akt inhibitor reduced tumor load in Aim2(-/-) mice. These findings suggest that Akt inhibitors could be used to treat AIM2-deficient human cancers.

Emerging significance of NLRs in inflammatory bowel disease.

Pattern recognition receptors are essential mediators of host defense and inflammation in the gastrointestinal system. Recent data have revealed that toll-like receptors and nucleotide-binding domain and leucine-rich repeat-containing proteins (NLRs) function to maintain homeostasis between the host microbiome and mucosal immunity. The NLR proteins are a diverse class of cytoplasmic pattern recognition receptors. In humans, only about half of the identified NLRs have been adequately characterized. The majority of well-characterized NLRs participate in the formation of a multiprotein complex, termed the inflammasome, which is responsible for the maturation of interleukin-1ß and interleukin-18. However, recent observations have also uncovered the presence of a novel subgroup of NLRs that function as positive or negative regulators of inflammation through modulating critical signaling pathways, including NF-κB. Dysregulation of specific NLRs from both proinflammatory and inhibitory subgroups have been associated with the development of inflammatory bowel disease (IBD) in genetically susceptible human populations. Our own preliminary retrospective data mining efforts have identified a diverse range of NLRs that are significantly altered at the messenger RNA level in colons from patients with IBD. Likewise, studies using genetically modified mouse strains have revealed that multiple NLR family members have the potential to dramatically modulate the immune response during IBD. Targeting NLR signaling represents a promising and novel therapeutic strategy. However, significant effort is necessary to translate the current understanding of NLR biology into effective therapies.