Fas, a tumor necrosis factor family receptor, is activated by the membrane protein Fas ligand (FasL) expressed on various immune cells. Fas signaling triggers apoptosis and induces inflammatory cytokine production. Among the Fas induced cytokines, the IL-1β family cytokines require proteolysis to gain biological activity. Inflammasomes, which respond to pathogens and danger signals, cleave IL-1β cytokines via caspase-1. The mechanisms, by which Fas regulates IL-1β activation, however, remain unresolved. Here, we demonstrate that macrophages exposed to TLR ligands upregulate Fas, which renders them responsive to receptor engagement by Fas ligand. Fas signaling activates caspase-8 in macrophages and dendritic cells leading to the maturation of IL-1β and IL-18 independently of inflammasomes or Rip3. Hence, Fas controls a novel non-canonical IL-1β activation pathway in myeloid cells, which could play an essential role in inflammatory processes, tumor surveillance and control of infectious diseases.
Prion diseases are fatal transmissible neurodegenerative diseases, characterized by aggregation of the pathological form of prion protein, spongiform degeneration, neuronal loss and activation of astrocytes and microglia. Microglia can clear prion plaques but on the other hand cause neuronal death via release of neurotoxic species. Elevated expression of the proinflammatory cytokine IL-1β has been observed in brains affected by several prion diseases and IL-1R-deficiency significantly prolonged the onset of the neurodegeneration in mice. We show that microglial cells stimulated by prion protein (PrP) fibrils induced neuronal toxicity. Microglia and macrophages release IL-1β upon stimulation by PrP fibrils, which depends on the NLRP3 inflammasome. Activation of NLRP3 inflammasome by PrP fibrils requires depletion of intracellular K+ and requires phagocytosis of PrP fibrils and consecutive lysosome destabilization. Among the well-defined molecular forms of PrP the strongest NLRP3 activation was observed by fibrils, followed by aggregates, while neither native monomeric nor oligomeric PrP were able to activate the NLRP3 inflammasome. Our results together with previous studies on IL-1R-deficient mice suggest the IL-1 signaling pathway as the perspective target for the therapy of prion disease.
prions; amyloid; inflammasome; NLRP3; IL-1β; neuroinflammation
Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates a number of functions of these organelles that allow them to participate in processes essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3-inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3-inflammasome and caspase-1 in host defense.
Rift Valley fever virus (RVFV) is an emerging RNA virus with devastating economic and social consequences. Clinically, RVFV induces a gamut of symptoms ranging from febrile illness to retinitis, hepatic necrosis, hemorrhagic fever, and death. It is known that type I interferon (IFN) responses can be protective against severe pathology; however, it is unknown which innate immune receptor pathways are crucial for mounting this response. Using both in vitro assays and in vivo mucosal mouse challenge, we demonstrate here that RNA helicases are critical for IFN production by immune cells and that signaling through the helicase adaptor molecule MAVS (mitochondrial antiviral signaling) is protective against mortality and more subtle pathology during RVFV infection. In addition, we demonstrate that Toll-like-receptor-mediated signaling is not involved in IFN production, further emphasizing the importance of the RNA cellular helicases in type I IFN responses to RVFV.
Yersinia pestis, the causative agent of plague, is able to suppress production of inflammatory cytokines IL-18 and IL-1β, which are generated through caspase-1–activating nucleotide-binding domain and leucine-rich repeat (NLR)-containing inflammasomes. Here, we sought to elucidate the role of NLRs and IL-18 during plague. Lack of IL-18 signaling led to increased susceptibility to Y. pestis, producing tetra-acylated lipid A,and an attenuated strain producing a Y. pseudotuberculosis-like hexa-acylated lipid A. We found that the NLRP12 inflammasome was an important regulator controlling IL-18 and IL-1β production after Y. pestis infection, and NLRP12-deficient mice were more susceptible to bacterial challenge. NLRP12 also directed interferon-γ production via induction of IL-18, but had minimal effect on signaling to the transcription factor NF-κB._ These studies reveal a role for NLRP12 in host resistance against pathogens. Minimizing NLRP12 inflammasome activation may have been a central factor in evolution of the high virulence of Y. pestis.
Systemic infections with Gram-negative bacteria are characterized by high mortality rates due to the “sepsis syndrome,” a widespread and uncontrolled inflammatory response. Though it is well recognized that the immune response during Gram-negative bacterial infection is initiated after the recognition of endotoxin by Toll-like receptor 4, the molecular mechanisms underlying the detrimental inflammatory response during Gram-negative bacteremia remain poorly defined. Here, we identify a TRIF pathway that licenses NLRP3 inflammasome activation by all Gram-negative bacteria. By engaging TRIF, Gram-negative bacteria activate caspase-11. TRIF activates caspase-11 via type I IFN signaling, an event that is both necessary and sufficient for caspase-11 induction and autoactivation. Caspase-11 subsequently synergizes with the assembled NLRP3 inflammasome to regulate caspase-1 activation and leads to caspase-1-independent cell death. These events occur specifically during infection with Gram-negative, but not Gram-positive, bacteria. The identification of TRIF as a regulator of caspase-11 underscores the importance of TLRs as master regulators of inflammasomes during Gram-negative bacterial infection.
Type I interferon (IFN) is an important host defense cytokine against intracellular pathogens, mainly viruses. In assessing IFN production in response to group B streptococcus (GBS), we find that IFN-β was produced by macrophages upon stimulation with both heat-killed and live GBS. Exposure of macrophages to heat-killed GBS activated a Toll-like receptor (TLR)-dependent pathway, whereas live GBS activated a TLR/NOD/RIG-like receptor (RLR)-independent pathway. This latter pathway required bacterial phagocytosis, proteolytic bacterial degradation, and phagolysosomal membrane destruction by GBS pore-forming toxins, leading to the release of bacterial DNA into the cytosol. GBS DNA in the cytosol induced IFN-β production via a pathway dependent on the activation of the serine-threonine kinase TBK1 and phosphorylation of the transcription factor IRF3. Thus, activation of IFN-α/-β production during infection with GBS, commonly considered an extracellular pathogen, appears to result from the interaction of GBS DNA with a putative intracellular DNA sensor or receptor.
Interleukin-1 (IL-1) is an important mediator of innate immunity, but can also promote inflammatory tissue damage. During chronic infections, such as tuberculosis, the beneficial antimicrobial role of IL-1 must be balanced with the need to prevent immunopathology. By exogenously controlling the replication of Mycobacterium tuberculosis in vivo, we obviated the requirement for antimicrobial immunity and discovered that both IL-1 production and infection-induced immunopathology were suppressed by lymphocyte-derived interferon-γ (IFN-γ). This effect was mediated by nitric oxide (NO), which we found to specifically inhibit the assembly of the NLRP3 inflammasome via thiol nitrosylation. These data suggest that the NO produced as a result of adaptive immunity is indispensable in modulating the destructive innate inflammatory responses that are elicited during persistent infections.
Advances in innate immunity over the past decade have revealed distinct classes of pattern recognition receptors (PRRs) which operate to detect pathogens at the cell surface and in intracellular compartments. This has shed light on how herpesviruses, which are large disease-causing DNA viruses that replicate in the nucleus, are initially recognized during cellular infection. Surprisingly, this involves multiple PRRs both on the cell surface, and within endosomes and the cytosol. In this article we describe recent advances in our understanding of innate detection of herpesviruses, how this innate detection translates into anti-herpesvirus host defense, and how the viruses seek to evade this innate detection to establish persistent infections.
Recognition of DNA by the innate immune system is central to anti-viral and anti-bacterial defenses, as well as an important contributor to autoimmune diseases involving self DNA. AIM2 (absent in melanoma 2) and IFI16 (interferon-inducible protein 16) have been identified as DNA receptors that induce inflammasome formation and interferon production, respectively. Here we present the crystal structures of their HIN domains in complex with double-stranded (ds) DNA. Non-sequence specific DNA recognition is accomplished through electrostatic attraction between the positively charged HIN domain residues and the dsDNA sugar-phosphate backbone. An intramolecular complex of the AIM2 Pyrin and HIN domains in an autoinhibited state is liberated by DNA binding, which may facilitate the assembly of inflammasomes along the DNA staircase. These findings provide novel mechanistic insights into dsDNA as the activation trigger and oligomerization platform for the assembly of large innate signaling complexes such as the inflammasomes.
Type I interferons (IFNs), predominantly IFN-α and -β, play critical roles in both innate and adaptive immune responses against viral infections. Interferon regulatory factor 7 (IRF7), a key innate immune molecule in the type I IFN signaling pathway, is essential for the type I IFN response to many viruses, including lymphocytic choriomeningitis virus (LCMV). Here, we show that although IRF7 knockout (KO) mice failed to control the replication of LCMV in the early stages of infection, they were capable of clearing LCMV infection. Despite the lack of type I IFN production, IRF7 KO mice generated normal CD4+ T cell responses, and the expansion of naïve CD8+ T cells into primary CD8+ T cells specific for LCMV GP33–41 was relatively normal. In contrast, the expansion of the LCMV NP396-specific CD8+ T cells was severely impaired in IRF7 KO mice. We demonstrated that this defective CD8+ T cell response is due neither to an impaired antigen-presenting system nor to any intrinsic role of IRF7 in CD8+ T cells. The lack of a type I IFN response in IRF7 KO mice did not affect the formation of memory CD8+ T cells. Thus, the present study provides new insight into the impact of the innate immune system on viral pathogenesis and demonstrates the critical contribution of innate immunity in controlling virus replication in the early stages of infection, which may shape the quality of CD8+ T cell responses.
Innate immune surveillance mechanisms lie at the heart of the antiviral response. A growing number of germ-line encoded pattern recognition receptors have been identified which protect the host from infection by sensing the presence of viral molecules and inducing antiviral defenses. Most compartments that viruses gain access to are under active surveillance by one or more pattern recognition receptors. Members of the Toll-like receptor family guard the extracellular milieu and endosomal compartment where they are activated by viral glycoproteins or nucleic acids, respectively. More recently, the cytosolic compartment has emerged as the frontline in the arsenal of the host’s antiviral defenses. Families of receptors in the cytosol recognize viral RNA or DNA or perturbations of cellular homeostasis and orchestrate effector responses to eliminate the invader. Here, we review this expanding area of innate immunity by focusing on the molecular mechanisms of cytosolic host-defenses.
We report that in the presence of signal 1 (NF-κB), the NLRP3 inflammasome was activated by mitochondrial apoptotic signaling that licensed production of interleukin-1β (IL-1β). NLRP3 secondary signal activators such as ATP induced mitochondrial dysfunction and apoptosis, resulting in release of oxidized mitochondrial DNA (mtDNA) into the cytosol, where it bound to and activated the NLRP3 inflammasome. The anti-apoptotic protein Bcl-2 inversely regulated mitochondrial dysfunction and NLRP3 inflammasome activation. Mitochondrial DNA directly induced NLRP3 inflammasome activation, because macrophages lacking mtDNA had severely attenuated IL-1β production, yet still underwent apoptosis. Both binding of oxidized mtDNA to the NLRP3 inflammasome and IL-1β secretion could be competitively inhibited by the oxidized nucleoside, 8-OH-dG. Thus, our data reveal that oxidized mtDNA released during programmed cell death causes activation of the NLRP3 inflammasome. These results provide a missing link between apoptosis and inflammasome activation, via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome.
The innate immune system senses infection by detecting evolutionarily conserved molecules essential for microbial survival or abnormal location of molecules. Here we demonstrate the existence of a novel innate detection mechanism, which is induced by fusion between viral envelopes and target cells. Virus-cell fusion specifically stimulated a type I interferon (IFN) response with expression of IFN-stimulated genes (ISGs), in vivo recruitment of leukocytes, and potentiation of Toll-like receptor 7 and 9 signaling. The fusion dependent response was dependent on stimulator of interferon genes (STING) but independent of DNA, RNA and viral capsid. We suggest that membrane fusion is sensed as a danger signal with potential implications for defense against enveloped viruses and various conditions of giant cell formation.
Virus; Fusion; type I IFN; STING
Innate immune responses have the ability to both combat infectious microbes and drive pathological inflammation. Inflammasome complexes are a central component of these processes through their regulation of interleukin 1β (IL-1β), IL-18 and pyroptosis. Inflammasomes recognize microbial products or endogenous molecules released from damaged or dying cells both through direct binding of ligands and indirect mechanisms. The potential of the IL-1 family of cytokines to cause tissue damage and chronic inflammation emphasizes the importance of regulating inflammasomes. Many regulatory mechanisms have been identified that act as checkpoints for attenuating inflammasome signaling at multiple steps. Here we discuss the various regulatory mechanisms that have evolved to keep inflammasome signaling in check to maintain immunological balance.
Cytokines regulated by the inflammasome pathway have been extensively implicated in various age-related immune pathologies. We set out to elucidate the contribution of the nod-like receptor protein 3 (NLRP3) inflammasome pathway to the previously described deficiencies in IL-1β production by macrophages from aged mice. We examined the production of pro-IL-1β and its conversion into IL-1β as two separate steps and compared these cytokine responses in bone marrow derived macrophages from young (6–8 weeks) and aged (18–24 months) C57BL/6 mice.
Relative to macrophages from young mice, macrophages from aged mice produced less pro-IL-1β after TLR4 stimulation with LPS. However upon activation of the NLRP3 inflammasome with ATP, macrophages from young and aged mice were able to efficiently convert and secrete intracellular pro-cytokines as functional cytokines.
Lower levels of IL-1β production are a result of slower and lower overall production of pro-IL-1β in macrophages from aged mice.
Ageing; Macrophages; Aged mice; NLRP3; Inflammasome; IL-1β
Toll-like receptors (TLRs) are innate sentinels required for clearance of bacterial and fungal infections of the cornea, but their role in viral immunity is currently unknown. We report TLR signaling is expendable in HSV-1 containment as depicted by plaque assays of knockout mice (MyD88−/−, Trif−/− and MyD88−/− Trif−/− DKO) resembling wild type controls. To identify the key sentinel in viral recognition of the cornea, in vivo knockdown of the DNA sensor IFI-16/p204 in corneal epithelium was performed and resulted in a loss of IRF-3 nuclear translocation, interferon-α production, and viral containment. The sensor appears to have a similar function in other HSV clinically-relevant sites such as the vaginal mucosa in which a loss of p204/IFI-16 results in significantly more HSV-2 shedding. Thus, we have identified an IRF-3 dependent, IRF-7 and TLR - independent innate sensor responsible for HSV containment at the site of acute infection.
Although Toll-like receptor 9 (TLR9) has been implicated in regulating cytokine and type I interferon (IFN) production during malaria in humans and mice, the high AT content of the Plasmodium falciparum genome prompted us to examine the possibility that malarial DNA triggered TLR9-independent DNA sensing pathways. Over 6000 ATTTTTAC (“AT-rich”) motifs are present in the genome of P. falciparum, which we show here potently induce type I IFNs. Parasite DNA, parasitized erythrocytes and oligonucleotides containing the AT-r motif induce type I IFNs via a pathway that did not involve previously described sensors including TLR9, DAI, RNA polymerase-III or IFI16/p204. Rather, AT-rich DNA sensing involved an unknown receptor that coupled to STING, TBK1 and IRF3-IRF7 signaling pathway. Mice lacking both IRF3 and IRF7, the kinase TBK1 or the type I IFN receptor were resistant to otherwise lethal cerebral malaria. Collectively, these observations implicate AT-rich DNA sensing via STING, TBK1 and IRF3-IRF7 in P. falciparum malaria.
Lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)-α requires the recruitment of two pairs of adaptors to the Toll-like receptor 4 cytoplasmic domain. The contribution of one pair – Toll-interleukin-1 receptor domain-containing adaptor inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM) – to TNF-α expression is not well understood. To clarify this issue, we studied TRAM knockout bone marrow-derived macrophages (BMDM). LPS-stimulated TRAM-deficient BMDM had decreased TNF-α protein expression even at times when TNF-α mRNA levels were normal, suggesting impaired translation. Consistent with this idea, knockdown of TRAM in RAW264.7 macrophages decreased translation of a reporter controlled by the TNF-α 3′ untranslated region, while transfection of TRAM in HEK293T cells increased translation of this reporter. Also consistent with a role for TRAM in TNF-α translation, LPS-induced activation of MK2, a kinase involved in this process, was impaired in TRAM-deficient BMDM. TRIF did not increase translation of the TNF-α 3′ untranslated region reporter when expressed in HEK293T cells. However, BMDM that lacked functional TRIF produced reduced levels of TNF-α protein in response to LPS despite normal amounts of the mRNA. Unlike BMDM, LPS-stimulated TRAM-deficient peritoneal macrophages displayed equivalent reductions in TNF-α protein and mRNA. Our results indicate that TRAM- and TRIF-dependent signals have a previously unappreciated, cell type-specific role in regulating TNF-α translation.
Copyright © 2011 S. Karger AG, Basel
Macrophage; Lipopolysaccharide; Toll-like receptor; Inflammation; Signal transduction
Murine Aim2 and Ifi202 genes (encoding for the Aim2 and p202 proteins) are members of the interferon (IFN)-inducible Ifi200-gene family. The Aim2-deficiency in mice activates IFN-signaling and stimulates the expression of lupus susceptibility gene, the Ifi202, located within the Nba2 interval. Given that the deficiency in the expression of the Fcgr2b gene (encoding for the inhibitory FcγRIIB receptor) is associated with increased lupus susceptibility in mice, we investigated whether the Aim2 protein could regulate the expression of Fcgr2b gene. Here we report that Aim2-deficiency in mice suppresses the expression of the FcγRIIB receptor. Interestingly, the Fcgr2b-deficient cells expressed increased levels of the IFN-β, activated IFN-signaling, and expressed reduced levels of the Aim2 protein. Treatment of splenic cells with IFN-α or γ reduced levels of the FcγRIIB mRNA and protein, and also decreased the activity of the FcγRIIB p(−729/+ 585) Luc reporter. Moreover, levels of the FcγRIIB receptor were significantly higher in the Stat1-deficient splenic cells than the wild type cells. Accordingly, increased expression of IFN-β in lupus-prone B6.Nba2-ABC mice, as compared with non lupus-prone B6 or B6.Nba2-C mice, was associated with reduced expression of the FcγRIIB receptor. Notably, overexpression of the p202 protein in cells decreased the expression of the Aim2 gene, activated the IFN-response, and suppressed the expression of the Fcgr2b gene. These observations demonstrate that the expression of Aim2 protein is required to maintain the expression of the Fcgr2b gene and also predict epistatic interactions between the Ifi200-genes and the Fcgr2b gene within the Nba2 interval.
Aim2 inflammasome; interferon; FcγRIIB; SLE
Poxviruses are large DNA viruses that replicate in the cytoplasm of infected cells. Myxoma virus is a rabbit poxvirus that belongs to the Leporipoxvirus genus. It causes a lethal disease called myxomatosis in European rabbits but cannot sustain any detectable infection in nonlagomorphs. Vaccinia virus is a prototypal orthopoxvirus that was used as a vaccine to eradicate smallpox. Myxoma virus is nonpathogenic in mice, whereas systemic infection with vaccinia virus can be lethal even in immunocompetent mice. Plasmacytoid dendritic cells (pDCs) are potent type I interferon (IFN)-producing cells that play important roles in antiviral innate immunity. How poxviruses are sensed by pDCs to induce type I IFN production is not well understood. Here we report that infection of primary murine pDCs with myxoma virus, but not with vaccinia virus, induces IFN-α, IFN-β, tumor necrosis factor (TNF), and interleukin-12p70 (IL-12p70) production. Using pDCs derived from genetic knockout mice, we show that the myxoma virus-induced innate immune response requires the endosomal DNA sensor TLR9 and its adaptor MyD88, transcription factors IRF5 and IRF7, and the type I IFN positive-feedback loop mediated by IFNAR1. It is independent of the cytoplasmic RNA sensing pathway mediated by the mitochondrial adaptor molecule MAVS, the TLR3 adaptor TRIF, or the transcription factor IRF3. Using pharmacological inhibitors, we demonstrate that myxoma virus-induced type I IFN and IL-12p70 production in murine pDCs is also dependent on phosphatidylinositol 3-kinase (PI3K) and Akt. Furthermore, our results reveal that the N-terminal Z-DNA/RNA binding domain of vaccinia virulence factor E3, which is missing in the orthologous M029 protein expressed by myxoma virus, plays an inhibitory role in poxvirus sensing and innate cytokine production by murine pDCs.
DNA viruses are a significant contributor to human morbidity and mortality. The immune system protects against viral infections through coordinated innate and adaptive immune responses. While the antigen-specific adaptive mechanisms have been extensively studied, the critical contributions of innate immunity to anti-viral defenses have only been revealed in the very recent past. Central to these anti-viral defenses is the recognition of viral pathogens by a diverse set of germ-line encoded receptors that survey nearly all cellular compartments for the presence of pathogens. In this review, we discuss the recent advances in the innate immune sensing of DNA viruses and focus on the recognition mechanisms involved.
The type I interferons (IFNs), IFN-α and -β, are key effector molecules of the immune response to viruses. The anti-viral action of IFNs on virus-infected cells and surrounding tissues is mediated by expression of hundreds of IFN-stimulated genes. Viperin (virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible) is an Interferon stimulated gene (ISG), which is induced by type I, II, and III IFNs or after infection with a broad range of DNA and RNA viruses. Recent evidence indicates that Viperin disrupts lipid rafts to block influenza virus budding and release and interferes with replication of hepatitis C virus by binding to lipid droplets, small organelles involved in lipid homeostasis that are essential for hepatitis C virus replication. Viperin is also induced by nonviral microbial products such as lipopolysaccharide (LPS) and by a wide range of bacteria, suggesting a broader role in innate antimicrobial defenses.
Interleukin (IL)-1β is a cytokine critical to several inflammatory diseases in which pathogenic TH17 responses are implicated. Activation of the NLRP3 inflammasome by microbial and environmental stimuli can enable the caspase-1 dependent processing and secretion of IL-1β. The acute phase protein serum amyloid A (SAA) is highly induced during inflammatory responses, wherein it participates in systemic modulation of innate and adaptive immune responses. Elevated levels of IL-1β, SAA, and IL-17 are present in subjects with severe allergic asthma, yet the mechanistic relationship between these mediators has yet to be identified. Herein, we demonstrate that Saa3 is expressed in the lung of mice exposed to several mixed Th2/Th17-polarizing allergic sensitization regimens. SAA instillation into the lungs elicits robust TLR2-, MyD88-, and IL-1-dependent pulmonary neutrophilic inflammation. Furthermore, SAA drives production of IL-1α, IL-1β, IL-6, IL-23, and PGE2, causes dendritic cell maturation, and requires TLR2, MyD88, and the NLRP3 inflammasome for secretion of IL-1β by dendritic cells and macrophages. CD4+ T cells polyclonally stimulated in the presence of conditioned media from SAA-exposed dendritic cells produced IL-17 and the capacity of polyclonally-stimulated splenocytes to secrete IL-17 is dependent upon IL-1, TLR2, and the NLRP3 inflammasome. Additionally, in a model of allergic airway inflammation, administration of SAA to the lungs functions as an adjuvant to sensitize mice to inhaled ovalbumin, resulting in leukocyte influx after antigen challenge and a predominance of IL-17 production from restimulated splenocytes that is dependent upon IL-1 receptor signaling.