(IR)Factor for NAIP Expression.

IRF8 is a master transcription factor for immune cell development. In this issue, Karki et al. reveal that IRF8 governs the constitutive expression of genes encoding for NAIP proteins that are critical for the innate immune sensing of bacteria.

Innate immunity to intracellular LPS.

Monitoring of the cytosolic compartment by the innate immune system for pathogen-encoded products or pathogen activities often enables the activation of a subset of caspases. In most cases, the cytosolic surveillance pathways are coupled to activation of caspase-1 via canonical inflammasome complexes. A related set of caspases, caspase-11 in rodents and caspase-4 and caspase-5 in humans, monitors the cytosol for bacterial lipopolysaccharide (LPS). Direct activation of caspase-11, caspase-4 and caspase-5 by intracellular LPS elicits the lytic cell death called 'pyroptosis', which occurs in multiple cell types. The pyroptosis is executed by the pore-forming protein GSDMD, which is activated by cleavage mediated by caspase-11, caspase-4 or caspase-5. In monocytes, formation of GSDMD pores can induce activation of the NLRP3 inflammasome for maturation of the cytokines IL-1ß and IL-18. Caspase-11-mediated pyroptosis in response to cytosolic LPS is critical for antibacterial defense and septic shock. Here we review the emerging literature on the sensing of cytosolic LPS and its regulation and pathophysiological functions.

Inflammasome Complexes: Emerging Mechanisms and Effector Functions.

Canonical activation of the inflammasome is critical to promote caspase-1-dependent maturation of the proinflammatory cytokines IL-1ß and IL-18, as well as to induce pyroptotic cell death in response to pathogens and endogenous danger signals. Recent discoveries, however, are beginning to unveil new components of the inflammasome machinery as well as the full spectrum of inflammasome functions, extending their influence beyond canonical functions to regulation of eicosanoid storm, autophagy, and metabolism. In addition, the receptor components of the inflammasome can also regulate diverse biological processes, such as cellular proliferation, gene transcription, and tumorigenesis, all of which are independent of their inflammasome complex-forming capabilities. Here, we review these recent advances that are shaping our understanding of the complex biology of the inflammasome and its constituents.

Bacterial Outer Membrane Vesicles Mediate Cytosolic Localization of LPS and Caspase-11 Activation.

Sensing of lipopolysaccharide (LPS) in the cytosol triggers caspase-11 activation and is central to host defense against Gram-negative bacterial infections and to the pathogenesis of sepsis. Most Gram-negative bacteria that activate caspase-11, however, are not cytosolic, and the mechanism by which LPS from these bacteria gains access to caspase-11 in the cytosol remains elusive. Here, we identify outer membrane vesicles (OMVs) produced by Gram-negative bacteria as a vehicle that delivers LPS into the cytosol triggering caspase-11-dependent effector responses in vitro and in vivo. OMVs are internalized via endocytosis, and LPS is released into the cytosol from early endosomes. The use of hypovesiculating bacterial mutants, compromised in their ability to generate OMVs, reveals the importance of OMVs in mediating the cytosolic localization of LPS. Collectively, these findings demonstrate a critical role for OMVs in enabling the cytosolic entry of LPS and, consequently, caspase-11 activation during Gram-negative bacterial infections.

Oligoadenylate-Synthetase-Family Protein OASL Inhibits Activity of the DNA Sensor cGAS during DNA Virus Infection to Limit Interferon Production.

Interferon-inducible human oligoadenylate synthetase-like (OASL) and its mouse ortholog, Oasl2, enhance RNA-sensor RIG-I-mediated type I interferon (IFN) induction and inhibit RNA virus replication. Here, we show that OASL and Oasl2 have the opposite effect in the context of DNA virus infection. In Oasl2-/- mice and OASL-deficient human cells, DNA viruses such as vaccinia, herpes simplex, and adenovirus induced increased IFN production, which resulted in reduced virus replication and pathology. Correspondingly, ectopic expression of OASL in human cells inhibited IFN induction through the cGAS-STING DNA-sensing pathway. cGAS was necessary for the reduced DNA virus replication observed in OASL-deficient cells. OASL directly and specifically bound to cGAS independently of double-stranded DNA, resulting in a non-competitive inhibition of the second messenger cyclic GMP-AMP production. Our findings define distinct mechanisms by which OASL differentially regulates host IFN responses during RNA and DNA virus infection and identify OASL as a negative-feedback regulator of cGAS.

Gasdermin D Restrains Type I Interferon Response to Cytosolic DNA by Disrupting Ionic Homeostasis.

Inflammasome-activated caspase-1 cleaves gasdermin D to unmask its pore-forming activity, the predominant consequence of which is pyroptosis. Here, we report an additional biological role for gasdermin D in limiting cytosolic DNA surveillance. Cytosolic DNA is sensed by Aim2 and cyclic GMP-AMP synthase (cGAS) leading to inflammasome and type I interferon responses, respectively. We found that gasdermin D activated by the Aim2 inflammasome suppressed cGAS-driven type I interferon response to cytosolic DNA and Francisella novicida in macrophages. Similarly, interferon-ß (IFN-ß) response to F. novicida infection was elevated in gasdermin D-deficient mice. Gasdermin D-mediated negative regulation of IFN-ß occurred in a pyroptosis-, interleukin-1 (IL-1)-, and IL-18-independent manner. Mechanistically, gasdermin D depleted intracellular potassium (K+) via membrane pores, and this K+ efflux was necessary and sufficient to inhibit cGAS-dependent IFN-ß response. Thus, our findings have uncovered an additional interferon regulatory module involving gasdermin D and K+ efflux.

TRIF licenses caspase-11-dependent NLRP3 inflammasome activation by gram-negative bacteria.

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.

Nitric oxide controls the immunopathology of tuberculosis by inhibiting NLRP3 inflammasome-dependent processing of IL-1ß.

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 specifically inhibited assembly of the NLRP3 inflammasome via thiol nitrosylation. Our data indicate that the NO produced as a result of adaptive immunity is indispensable in modulating the destructive innate inflammatory responses elicited during persistent infections.

Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function.

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 many functions of these organelles that allow phagosomes to participate in processes that are 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.

Regulation of inflammasome signaling.

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 1ß (IL-1ß), 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.

Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome.

Autophagy, a cellular process for organelle and protein turnover, regulates innate immune responses. Here we demonstrate that depletion of the autophagic proteins LC3B and beclin 1 enhanced the activation of caspase-1 and secretion of interleukin 1ß (IL-1ß) and IL-18. Depletion of autophagic proteins promoted the accumulation of dysfunctional mitochondria and cytosolic translocation of mitochondrial DNA (mtDNA) in response to lipopolysaccharide (LPS) and ATP in macrophages. Release of mtDNA into the cytosol depended on the NALP3 inflammasome and mitochondrial reactive oxygen species (ROS). Cytosolic mtDNA contributed to the secretion of IL-1ß and IL-18 in response to LPS and ATP. LC3B-deficient mice produced more caspase-1-dependent cytokines in two sepsis models and were susceptible to LPS-induced mortality. Our study suggests that autophagic proteins regulate NALP3-dependent inflammation by preserving mitochondrial integrity.

The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses.

Inflammasomes regulate the activity of caspase-1 and the maturation of interleukin 1beta (IL-1beta) and IL-18. AIM2 has been shown to bind DNA and engage the caspase-1-activating adaptor protein ASC to form a caspase-1-activating inflammasome. Using Aim2-deficient mice, we identify a central role for AIM2 in regulating caspase-1-dependent maturation of IL-1beta and IL-18, as well as pyroptosis, in response to synthetic double-stranded DNA. AIM2 was essential for inflammasome activation in response to Francisella tularensis, vaccinia virus and mouse cytomegalovirus and had a partial role in the sensing of Listeria monocytogenes. Moreover, production of IL-18 and natural killer cell-dependent production of interferon-gamma, events critical in the early control of virus replication, were dependent on AIM2 during mouse cytomegalovirus infection in vivo. Collectively, our observations demonstrate the importance of AIM2 in the sensing of both bacterial and viral pathogens and in triggering innate immunity.

The NLRP12 inflammasome recognizes Yersinia pestis.

Yersinia pestis, the causative agent of plague, is able to suppress production of inflammatory cytokines IL-18 and IL-1ß, 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-1ß 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.

Bacterial RNA:DNA hybrids are activators of the NLRP3 inflammasome.

Enterohemorrhagic Escherichia coli (EHEC) is an extracellular pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. The proinflammatory cytokine, interleukin-1ß, has been linked to hemolytic uremic syndrome. Here we identify the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome as an essential mediator of EHEC-induced IL-1ß. Whereas EHEC-specific virulence factors were dispensable for NLRP3 activation, bacterial nucleic acids such as RNA:DNA hybrids and RNA gained cytosolic access and mediated inflammasome-dependent responses. Consistent with a direct role for RNA:DNA hybrids in inflammasome activation, delivery of synthetic EHEC RNA:DNA hybrids into the cytosol triggered NLRP3-dependent responses, and introduction of RNase H, which degrades such hybrids, into infected cells specifically inhibited inflammasome activation. Notably, an E. coli rnhA mutant, which is incapable of producing RNase H and thus harbors increased levels of RNA:DNA hybrid, induced elevated levels of NLRP3-dependent caspase-1 activation and IL-1ß maturation. Collectively, these findings identify RNA:DNA hybrids of bacterial origin as a unique microbial trigger of the NLRP3 inflammasome.

Caspase-8 modulates dectin-1 and complement receptor 3-driven IL-1ß production in response to ß-glucans and the fungal pathogen, Candida albicans.

Inflammasomes are central mediators of host defense to a wide range of microbial pathogens. The nucleotide-binding domain and leucine-rich repeat containing family (NLR), pyrin domain-containing 3 (NLRP3) inflammasome plays a key role in triggering caspase-1-dependent IL-1ß maturation and resistance to fungal dissemination in Candida albicans infection. ß-Glucans are major components of fungal cell walls that trigger IL-1ß secretion in both murine and human immune cells. In this study, we sought to determine the contribution of ß-glucans to C. albicans-induced inflammasome responses in mouse dendritic cells. We show that the NLRP3-apoptosis-associated speck-like protein containing caspase recruitment domain protein-caspase-1 inflammasome is absolutely critical for IL-1ß production in response to ß-glucans. Interestingly, we also found that both complement receptor 3 (CR3) and dectin-1 play a crucial role in coordinating ß-glucan-induced IL-1ß processing as well as a cell death response. In addition to the essential role of caspase-1, we identify an important role for the proapoptotic protease caspase-8 in promoting ß-glucan-induced cell death and NLRP3 inflammasome-dependent IL-1ß maturation. A strong requirement for CR3 and caspase-8 also was found for NLRP3-dependent IL-1ß production in response to heat-killed C. albicans. Taken together, these results define the importance of dectin-1, CR3, and caspase-8, in addition to the canonical NLRP3 inflammasome, in mediating ß-glucan- and C. albicans-induced innate responses in dendritic cells. Collectively, these findings establish a novel link between ß-glucan recognition receptors and the inflammatory proteases caspase-8 and caspase-1 in coordinating cytokine secretion and cell death in response to immunostimulatory fungal components.

TRIL is involved in cytokine production in the brain following Escherichia coli infection.

TLR4 interactor with leucine-rich repeats (TRIL) is a brain-enriched accessory protein that is important in TLR3 and TLR4 signaling. In this study, we generated Tril(-/-) mice and examined TLR responses in vitro and in vivo. We found a role for TRIL in both TLR4 and TLR3 signaling in mixed glial cells, consistent with the high level of expression of TRIL in these cells. We also found that TRIL is a modulator of the innate immune response to LPS challenge and Escherichia coli infection in vivo. Tril(-/-) mice produce lower levels of multiple proinflammatory cytokines and chemokines specifically within the brain after E. coli and LPS challenge. Collectively, these data uncover TRIL as a mediator of innate immune responses within the brain, where it enhances neuronal cytokine responses to infection.

Cutting edge: Mycobacterium tuberculosis but not nonvirulent mycobacteria inhibits IFN-ß and AIM2 inflammasome-dependent IL-1ß production via its ESX-1 secretion system.

Mycobacterium tuberculosis extracellular DNA gains access to the host cell cytosol via the ESX-1 secretion system. It is puzzling that this extracellular DNA of M. tuberculosis does not induce activation of the AIM2 inflammasome because AIM2 recognizes cytosolic DNA. In this study, we show that nonvirulent mycobacteria such as Mycobacterium smegmatis induce AIM2 inflammasome activation, which is dependent on their strong induction of IFN-ß production. In contrast, M. tuberculosis, but not an ESX-1-deficient mutant, inhibits the AIM2 inflammasome activation induced by either M. smegmatis or transfected dsDNA. The inhibition does not involve changes in host cell AIM2 mRNA or protein levels but led to decreased activation of caspase-1. We furthermore demonstrate that M. tuberculosis inhibits IFN-ß production and signaling, which was partially responsible for the inhibition of AIM2 activation. In conclusion, we report a novel immune evasion mechanism of M. tuberculosis that involves the ESX-1-dependent, direct or indirect, suppression of the host cell AIM2 inflammasome activation during infection.

Mouse, but not human STING, binds and signals in response to the vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid.

Vascular disrupting agents such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA) represent a novel approach for cancer treatment. DMXAA has potent antitumor activity in mice and, despite significant preclinical promise, failed human clinical trials. The antitumor activity of DMXAA has been linked to its ability to induce type I IFNs in macrophages, although the molecular mechanisms involved are poorly understood. In this study, we identify stimulator of IFN gene (STING) as a direct receptor for DMXAA leading to TANK-binding kinase 1 and IFN regulatory factor 3 signaling. Remarkably, the ability to sense DMXAA was restricted to murine STING. Human STING failed to bind to or signal in response to DMXAA. Human STING also failed to signal in response to cyclic dinucleotides, conserved bacterial second messengers known to bind and activate murine STING signaling. Collectively, these findings detail an unexpected species-specific role for STING as a receptor for an anticancer drug and uncover important insights that may explain the failure of DMXAA in clinical trials for human cancer.