Joseph A. Duncan

NLRX1 is a regulator of mitochondrial antiviral immunity

The RIG-like helicase (RLH) family of intracellular receptors detect viral nucleic acid and signal through the mitochondrial antiviral signalling adaptor MAVS (also known as Cardif, VISA and IPS-1) during a viral infection. MAVS activation leads to the rapid production of antiviral cytokines, including type 1 interferons. Although MAVS is vital to antiviral immunity, its regulation from within the mitochondria remains unknown. Here we describe human NLRX1, a highly conserved nucleotide-binding domain (NBD)- and leucine-rich-repeat (LRR)-containing family member (known as NLR) that localizes to the mitochondrial outer membrane and interacts with MAVS. Expression of NLRX1 results in the potent inhibition of RLH- and MAVS-mediated interferon-β promoter activity and in the disruption of virus-induced RLH–MAVS interactions. Depletion of NLRX1 with small interference RNA promotes virus-induced type I interferon production and decreases viral replication. This work identifies NLRX1 as a check against mitochondrial antiviral responses and represents an intersection of three ancient cellular processes: NLR signalling, intracellular virus detection and the use of mitochondria as a platform for anti-pathogen signalling. This represents a conceptual advance, in that NLRX1 is a modulator of pathogen-associated molecular pattern receptors rather than a receptor, and identifies a key therapeutic target for enhancing antiviral responses.

Akt-dependent regulation of NF-κB is controlled by mTOR and Raptor in association with IKK

While NF-kappaB is considered to play key roles in the development and progression of many cancers, the mechanisms whereby this transcription factor is activated in cancer are poorly understood. A key oncoprotein in a variety of cancers is the serine-threonine kinase Akt, which can be activated by mutations in PI3K, by loss of expression/activity of PTEN, or through signaling induced by growth factors and their receptors. A key effector of Akt-induced signaling is the regulatory protein mTOR (mammalian target of rapamycin). We show here that mTOR downstream from Akt controls NF-kappaB activity in PTEN-null/inactive prostate cancer cells via interaction with and stimulation of IKK. The mTOR-associated protein Raptor is required for the ability of Akt to induce NF-kappaB activity. Correspondingly, the mTOR inhibitor rapamycin is shown to suppress IKK activity in PTEN-deficient prostate cancer cells through a mechanism that may involve dissociation of Raptor from mTOR. The results provide insight into the effects of Akt/mTOR-dependent signaling on gene expression and into the therapeutic action of rapamycin.

Staphylococcus aureus α-Hemolysin Activates the NLRP3-Inflammasome in Human and Mouse Monocytic Cells

Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal α-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus α-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood. Using highly purified recombinant α-hemolysin, we now demonstrate that α-hemolysin activates the Nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 protein (NLRP3)-inflammasome, a host inflammatory signaling complex involved in responses to pathogens and endogenous danger signals. Non-cytolytic mutant α-hemolysin molecules fail to elicit NLRP3-inflammasome signaling, demonstrating that the responses are not due to non-specific activation of this innate immune signaling system by bacterially derived proteins. In monocyte-derived cells from humans and mice, inflammasome assembly in response to α-hemolysin results in activation of the cysteine proteinase, caspase-1. We also show that inflammasome activation by α-hemolysin works in conjunction with signaling by other CA-MRSA-derived Pathogen Associated Molecular Patterns (PAMPs) to induce secretion of pro-inflammatory cytokines IL-1β and IL-18. Additionally, α-hemolysin induces cell death in these cells through an NLRP3-dependent program of cellular necrosis, resulting in the release of endogenous pro-inflammatory molecules, like the chromatin-associated protein, High-mobility group box 1 (HMGB1). These studies link the activity of a major S. aureus virulence factor to a specific host signaling pathway. The cellular events linked to inflammasome activity have clear relevance to the disease processes associated with CA-MRSA including tissue necrosis and inflammation.

How the Noninflammasome NLRs Function in the Innate Immune System

NLR (nucleotide-binding domain, leucine-rich repeat–containing) proteins have rapidly emerged as central regulators of immunity and inflammation with demonstrated relevance to human diseases. Much attention has focused on the ability of several NLRs to activate the inflammasome complex and drive proteolytic processing of inflammatory cytokines; however, NLRs also regulate important inflammasome-independent functions in the immune system. We discuss several of these functions, including the regulation of canonical and noncanonical NF-κB activation, mitogen-activated protein kinase activation, cytokine and chemokine production, antimicrobial reactive oxygen species production, type I interferon production, and ribonuclease L activity. We also explore the mechanistic basis of these functions and describe current challenges in the field.

Cryopyrin/NALP3 binds ATP/dATP, is an ATPase, and requires ATP binding to mediate inflammatory signaling

The CATERPILLER (CLR/NLR) gene family encodes a family of putative nucleotide-binding proteins important for host defense. Although nucleotide binding is thought to be central to this family, this aspect is largely unstudied. The CATERPILLER protein cryopyrin/NALP3 regulates IL-1β processing by assembling the multimeric inflammasome complex. Mutations within the exon encoding the nucleotide-binding domain are associated with hereditary periodic fevers characterized by constitutive IL-1β production. We demonstrate that purified cryopyrin binds ATP, dATP, and ATP-agarose, but not CTP, GTP, or UTP, and exhibits ATPase activity. Mutation of the nucleotide-binding domain reduces ATP binding, caspase-1 activation, IL-1β production, cell death, macromolecular complex formation, self-association, and association with the inflammasome component ASC. Disruption of nucleotide binding abolishes the constitutive activation of disease-associated mutants, identifying nucleotide binding by cryopyrin as a potential target for antiinflammatory pharmacologic intervention.

NLRP3 (NALP3, Cryopyrin) Facilitates In Vivo Caspase-1 Activation, Necrosis, and HMGB1 Release via Inflammasome-Dependent and -Independent Pathways

Bacterial infection elicits a range of beneficial as well as detrimental host inflammatory responses. Key among these responses are macrophage/monocyte necrosis, release of the proinflammatory factor high-mobility group box 1 protein (HMGB1), and induction of the cytokine IL-1. Although the control of IL-1β has been well studied, processes that control macrophage cell death and HMGB1 release in animals are poorly understood. This study uses Klebsiella pneumonia as a model organism because it elicits all three responses in vivo. The regulation of these responses is studied in the context of the inflammasome components NLRP3 and ASC, which are important for caspase-1 activation and IL-1β release. Using a pulmonary infection model that reflects human infection, we show that K. pneumonia-induced mouse macrophage necrosis, HMGB1, and IL-1β release are dependent on NLRP3 and ASC. K. pneumoniae infection of mice lacking Nlrp3 results in decreased lung inflammation and reduced survival relative to control, indicating the overall protective role of this gene. Macrophage/monocyte necrosis and HMGB1 release are controlled independently of caspase-1, suggesting that the former two responses are separable from inflammasome-associated functions. These results provide critical in vivo validation that the physiologic role of NLRP3 and ASC is not limited to inflammasome formation.

Neisseria gonorrhoeae Activates the Proteinase Cathepsin B to Mediate the Signaling Activities of the NLRP3 and ASC-Containing Inflammasome

Neisseria gonorrhoeae is a common sexually transmitted pathogen that significantly impacts female fertility, neonatal health, and transmission of HIV worldwide. N. gonorrhoeae usually causes localized inflammation of the urethra and cervix by inducing production of IL-1β and other inflammatory cytokines. Several NLR (nucleotide-binding domain, leucine-rich repeat) proteins are implicated in the formation of pro-IL-1β-processing complexes called inflammasomes in response to pathogens. We demonstrate that NLRP3 (cryopyrin, NALP3) is the primary NLR required for IL-1β/IL-18 secretion in response to N. gonorrhoeae in monocytes. We also show that N. gonorrhoeae infection promotes NLRP3-dependent monocytic cell death via pyronecrosis, a recently described pathway with morphological features of necrosis, including release of the strong inflammatory mediator HMBG1. Additionally, N. gonorrhoeae activates the cysteine protease cathepsin B as measured by the breakdown of a cathepsin B substrate. Inhibition of cathepsin B shows that this protease is an apical controlling step in the downstream activities of NLRP3 including IL-1β production, pyronecrosis, and HMGB1 release. Nonpathogenic Neisseria strains (Neisseria cinerea and Neisseria flavescens) do not activate NLRP3 as robustly as N. gonorrhoeae. Conditioned medium from N. gonorrhoeae contains factors capable of initiating the NLRP3-mediated signaling events. Isolated N. gonorrhoeae lipooligosaccharide, a known virulence factor from this bacterium that is elaborated from the bacterium in the form of outer membrane blebs, activates both NLRP3-induced IL-1β secretion and pyronecrosis. Our findings indicate that activation of NLRP3-mediated inflammatory response pathways is an important venue associated with host response and pathogenesis of N. gonorrhoeae.

The CATERPILLER Protein Monarch-1 Is an Antagonist of Toll-like Receptor-, Tumor Necrosis Factor α-, and Mycobacterium tuberculosis-induced Pro-inflammatory Signals

The CATERPILLER (CLR, also NOD and NLR) proteins share structural similarities with the nucleotide binding domain (NBD)-leucine-rich repeat (LRR) superfamily of plant disease-resistance (R) proteins and are emerging as important immune regulators in animals. CLR proteins contain NBD-LRR motifs and are linked to a limited number of distinct N-terminal domains including transactivation, CARD (caspase activation and recruitment), and pyrin domains (PyD). The CLR gene, Monarch-1/Pypaf7, is expressed by resting primary myeloid/monocytic cells, and its expression in these cells is reduced by Toll-like receptor (TLR) agonists tumor necrosis factor (TNF) α and Mycobacterium tuberculosis. Monarch-1 reduces NFκB activation by TLR-signaling molecules MyD88, IRAK-1 (type I interleukin-1 receptor-associated protein kinase), and TRAF6 (TNF receptor (TNFR)-associated factor) as well as TNFR signaling molecules TRAF2 and RIP1 but not the downstream NFκB subunit p65. This indicates that Monarch-1 is a negative regulator of both TLR and TNFR pathways. Reducing Monarch-1 expression with small interference RNA in myeloid/monocytic cells caused a dramatic increase in NFκB activation and cytokine expression in response to TLR2/TLR4 agonists, TNFα, or M. tuberculosis infection, suggesting that Monarch-1 is a negative regulator of inflammation. Because Monarch-1 is the first CLR protein that interferes with both TLR2 and TLR4 activation, the mechanism of this interference is significant. We find that Monarch-1 associates with IRAK-1 but not MyD88, resulting in the blockage of IRAK-1 hyperphosphorylation. Mutants containing the NBD-LRR or PyD-NBD also blocked IRAK-1 activation. This is the first example of a CLR protein that antagonizes inflammatory responses initiated by TLR agonists via interference with IRAK-1 activation.

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.

Staphylococcus aureus α-Hemolysin Mediates Virulence in a Murine Model of Severe Pneumonia Through Activation of the NLRP3 Inflammasome

Staphylococcus aureus is a dangerous pathogen that can cause necrotizing infections characterized by massive inflammatory responses and tissue destruction. Staphylococcal α-hemolysin is an essential virulence factor in severe S. aureus pneumonia. It activates the nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 (NLRP3) inflammasome to induce production of interleukin-1β and programmed necrotic cell death. We sought to determine the role of α-hemolysin-mediated activation of NLRP3 in the pathogenesis of S. aureus pneumonia. We show that α-hemolysin activates the NLRP3 inflammasome during S. aureus pneumonia, inducing necrotic pulmonary injury. Moreover, Nlrp3(-/-) mice have less-severe pneumonia. Pulmonary injury induced by isolated α-hemolysin or live S. aureus is independent of interleukin-1β signaling, implicating NLRP3-induced necrosis in the pathogenesis of severe infection. This work demonstrates the exploitation of host inflammatory signaling by S. aureus and suggests the NLRP3 inflammasome as a potential target for pharmacologic interventions in severe S. aureus infections.