Beckley K. Davis

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-1β 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 NLR gene family: a standard nomenclature.

Iimmune regulatory proteins such as CIITA, NAIP, IPAF, NOD1, NOD2, NALP1, cryopyrin/NALP3 are members of a family characterized by the presence of a nucleotide-binding domain (NBD) and leucine-rich repeats (LRR). Members of this gene family encode a protein structure similar to the NB-LRR subgroup of disease-resistance genes in plants and are involved in the sensing of pathogenic products and the regulation of cell signaling and apoptosis. Several members of this family have been associated with immunologic disorders. NOD2 for instance is associated with both Crohns disease and Blau syndrome. A variety of different names are currently used to describe this gene family, its subfamilies and individual genes, including CATERPILLER (CLR), NOD-LRR, NACHT-LRR, CARD, NALP, NOD, PAN and PYPAF, and this lack of consistency has led to a pressing need to unify the nomenclature. Consequently, we collectively propose the family designation NLR (nucleotide-binding domain and leucine-rich repeat containing) and provide unique and standardized gene designations for all family members.

CorrespondenceThe NLR Gene Family: A Standard Nomenclature

Iimmune regulatory proteins such as CIITA, NAIP, IPAF, NOD1, NOD2, NALP1, cryopyrin/NALP3 are members of a family characterized by the presence of a nucleotide-binding domain (NBD) and leucine-rich repeats (LRR). Members of this gene family encode a protein structure similar to the NB-LRR subgroup of disease-resistance genes in plants and are involved in the sensing of pathogenic products and the regulation of cell signaling and apoptosis. Several members of this family have been associated with immunologic disorders. NOD2 for instance is associated with both Crohns disease and Blau syndrome. A variety of different names are currently used to describe this gene family, its subfamilies and individual genes, including CATERPILLER (CLR), NOD-LRR, NACHT-LRR, CARD, NALP, NOD, PAN and PYPAF, and this lack of consistency has led to a pressing need to unify the nomenclature. Consequently, we collectively propose the family designation NLR (nucleotide-binding domain and leucine-rich repeat containing) and provide unique and standardized gene designations for all family members.

Cutting Edge: Monarch-1 Suppresses Non-Canonical NF-κB Activation and p52-Dependent Chemokine Expression in Monocytes

CATERPILLER (NOD, NBD-LRR) proteins are rapidly emerging as important mediators of innate and adaptive immunity. Among these, Monarch-1 operates as a novel attenuating factor of inflammation by suppressing inflammatory responses in activated monocytes. However, the molecular mechanisms by which Monarch-1 performs this important function are not well understood. In this report, we show that Monarch-1 inhibits CD40-mediated activation of NF-κB via the non-canonical pathway in human monocytes. This inhibition stems from the ability of Monarch-1 to associate with and induce proteasome-mediated degradation of NF-κB inducing kinase. Congruently, silencing Monarch-1 with shRNA enhances the expression of p52-dependent chemokines.

Discovery of a viral NLR homolog that inhibits the inflammasome.

A viral homolog of a host microbial sensor inhibits viral detection by the host and subsequent antiviral immunity. The NLR (nucleotide binding and oligomerization, leucine-rich repeat) family of proteins senses microbial infections and activates the inflammasome, a multiprotein complex that promotes microbial clearance. Kaposi’s sarcoma–associated herpesvirus (KSHV) is linked to several human malignancies. We found that KSHV Orf63 is a viral homolog of human NLRP1. Orf63 blocked NLRP1-dependent innate immune responses, including caspase-1 activation and processing of interleukins IL-1β and IL-18. KSHV Orf63 interacted with NLRP1, NLRP3, and NOD2. Inhibition of Orf63 expression resulted in increased expression of IL-1β during the KSHV life cycle. Furthermore, inhibition of NLRP1 was necessary for efficient reactivation and generation of progeny virus. The viral homolog subverts the function of cellular NLRs, which suggests that modulation of NLR-mediated innate immunity is important for the lifelong persistence of herpesviruses.

Monocytic microparticles activate endothelial cells in an IL-1β–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-1β 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-1β 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-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.

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

Background: Nucleotide binding and oligomerization domain-containing protein 2 (NOD2) is a protein involved in the recognition of bacterial pathogens through detection of muramyl dipeptide. Results: Purified recombinant NOD2 was found to bind ATP and muramyl dipeptide. Conclusion: NOD2 is an intracellular signaling receptor for muramyl dipeptide. Significance: These results help to define the molecular events involved in NOD2 signaling. 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.

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-1β response is mediated by innate receptors in macrophages and lung epithelial cells. NLRP3 is crucial in macrophages; however, which sensors elicit IL-1β 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-1β response to IAV in primary lung epithelial cells. To activate IL-1β 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-1β 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-1β 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.

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

The inflammasome activates caspase-1 and the release of interleukin-1β (IL-1β) 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−/−/ApcMin/+ than in APCMin/+ mice. The effects of AIM2 on CAC were independent of inflammasome activation and IL-1β 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.