NLRP12-PANoptosome activates PANoptosis and pathology in response to heme and PAMPs.

Cytosolic innate immune sensors are critical for host defense and form complexes, such as inflammasomes and PANoptosomes, that induce inflammatory cell death. The sensor NLRP12 is associated with infectious and inflammatory diseases, but its activating triggers and roles in cell death and inflammation remain unclear. Here, we discovered that NLRP12 drives inflammasome and PANoptosome activation, cell death, and inflammation in response to heme plus PAMPs or TNF. TLR2/4-mediated signaling through IRF1 induced Nlrp12 expression, which led to inflammasome formation to induce maturation of IL-1ß and IL-18. The inflammasome also served as an integral component of a larger NLRP12-PANoptosome that drove inflammatory cell death through caspase-8/RIPK3. Deletion of Nlrp12 protected mice from acute kidney injury and lethality in a hemolytic model. Overall, we identified NLRP12 as an essential cytosolic sensor for heme plus PAMPs-mediated PANoptosis, inflammation, and pathology, suggesting that NLRP12 and molecules in this pathway are potential drug targets for hemolytic and inflammatory diseases.

Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes.

COVID-19 is characterized by excessive production of pro-inflammatory cytokines and acute lung damage associated with patient mortality. While multiple inflammatory cytokines are produced by innate immune cells during SARS-CoV-2 infection, we found that only the combination of TNF-α and IFN-γ induced inflammatory cell death characterized by inflammatory cell death, PANoptosis. Mechanistically, TNF-α and IFN-γ co-treatment activated the JAK/STAT1/IRF1 axis, inducing nitric oxide production and driving caspase-8/FADD-mediated PANoptosis. TNF-α and IFN-γ caused a lethal cytokine shock in mice that mirrors the tissue damage and inflammation of COVID-19, and inhibiting PANoptosis protected mice from this pathology and death. Furthermore, treating with neutralizing antibodies against TNF-α and IFN-γ protected mice from mortality during SARS-CoV-2 infection, sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock. Collectively, our findings suggest that blocking the cytokine-mediated inflammatory cell death signaling pathway identified here may benefit patients with COVID-19 or other infectious and autoinflammatory diseases by limiting tissue damage/inflammation.

Caspase-6 Is a Key Regulator of Innate Immunity, Inflammasome Activation, and Host Defense.

Caspases regulate cell death, immune responses, and homeostasis. Caspase-6 is categorized as an executioner caspase but shows key differences from the other executioners. Overall, little is known about the functions of caspase-6 in biological processes apart from apoptosis. Here, we show that caspase-6 mediates innate immunity and inflammasome activation. Furthermore, we demonstrate that caspase-6 promotes the activation of programmed cell death pathways including pyroptosis, apoptosis, and necroptosis (PANoptosis) and plays an essential role in host defense against influenza A virus (IAV) infection. In addition, caspase-6 promoted the differentiation of alternatively activated macrophages (AAMs). Caspase-6 facilitated the RIP homotypic interaction motif (RHIM)-dependent binding of RIPK3 to ZBP1 via its interaction with RIPK3. Altogether, our findings reveal a vital role for caspase-6 in facilitating ZBP1-mediated inflammasome activation, cell death, and host defense during IAV infection, opening additional avenues for treatment of infectious and autoinflammatory diseases and cancer.

TLR2 senses the SARS-CoV-2 envelope protein to produce inflammatory cytokines.

The innate immune response is critical for recognizing and controlling infections through the release of cytokines and chemokines. However, severe pathology during some infections, including SARS-CoV-2, is driven by hyperactive cytokine release, or a cytokine storm. The innate sensors that activate production of proinflammatory cytokines and chemokines during COVID-19 remain poorly characterized. In the present study, we show that both TLR2 and MYD88 expression were associated with COVID-19 disease severity. Mechanistically, TLR2 and Myd88 were required for ß-coronavirus-induced inflammatory responses, and TLR2-dependent signaling induced the production of proinflammatory cytokines during coronavirus infection independent of viral entry. TLR2 sensed the SARS-CoV-2 envelope protein as its ligand. In addition, blocking TLR2 signaling in vivo provided protection against the pathogenesis of SARS-CoV-2 infection. Overall, our study provides a critical understanding of the molecular mechanism of ß-coronavirus sensing and inflammatory cytokine production, which opens new avenues for therapeutic strategies to counteract the ongoing COVID-19 pandemic.

IRF8 Regulates Transcription of Naips for NLRC4 Inflammasome Activation.

Inflammasome activation is critical for host defenses against various microbial infections. Activation of the NLRC4 inflammasome requires detection of flagellin or type III secretion system (T3SS) components by NLR family apoptosis inhibitory proteins (NAIPs); yet how this pathway is regulated is unknown. Here, we found that interferon regulatory factor 8 (IRF8) is required for optimal activation of the NLRC4 inflammasome in bone-marrow-derived macrophages infected with Salmonella Typhimurium, Burkholderia thailandensis, or Pseudomonas aeruginosa but is dispensable for activation of the canonical and non-canonical NLRP3, AIM2, and Pyrin inflammasomes. IRF8 governs the transcription of Naips to allow detection of flagellin or T3SS proteins to mediate NLRC4 inflammasome activation. Furthermore, we found that IRF8 confers protection against bacterial infection in vivo, owing to its role in inflammasome-dependent cytokine production and pyroptosis. Altogether, our findings suggest that IRF8 is a critical regulator of NAIPs and NLRC4 inflammasome activation for defense against bacterial infection.

IRGB10 Liberates Bacterial Ligands for Sensing by the AIM2 and Caspase-11-NLRP3 Inflammasomes.

The inflammasome is an intracellular signaling complex, which on recognition of pathogens and physiological aberration, drives activation of caspase-1, pyroptosis, and the release of the pro-inflammatory cytokines IL-1ß and IL-18. Bacterial ligands must secure entry into the cytoplasm to activate inflammasomes; however, the mechanisms by which concealed ligands are liberated in the cytoplasm have remained unclear. Here, we showed that the interferon-inducible protein IRGB10 is essential for activation of the DNA-sensing AIM2 inflammasome by Francisella novicida and contributed to the activation of the LPS-sensing caspase-11 and NLRP3 inflammasome by Gram-negative bacteria. IRGB10 directly targeted cytoplasmic bacteria through a mechanism requiring guanylate-binding proteins. Localization of IRGB10 to the bacterial cell membrane compromised bacterial structural integrity and mediated cytosolic release of ligands for recognition by inflammasome sensors. Overall, our results reveal IRGB10 as part of a conserved signaling hub at the interface between cell-autonomous immunity and innate immune sensing pathways.

Critical Role for the DNA Sensor AIM2 in Stem Cell Proliferation and Cancer.

Colorectal cancer is a leading cause of cancer-related deaths. Mutations in the innate immune sensor AIM2 are frequently identified in patients with colorectal cancer, but how AIM2 modulates colonic tumorigenesis is unknown. Here, we found that Aim2-deficient mice were hypersusceptible to colonic tumor development. Production of inflammasome-associated cytokines and other inflammatory mediators was largely intact in Aim2-deficient mice; however, intestinal stem cells were prone to uncontrolled proliferation. Aberrant Wnt signaling expanded a population of tumor-initiating stem cells in the absence of AIM2. Susceptibility of Aim2-deficient mice to colorectal tumorigenesis was enhanced by a dysbiotic gut microbiota, which was reduced by reciprocal exchange of gut microbiota with healthy wild-type mice. These findings uncover a synergy between a specific host genetic factor and gut microbiota in determining the susceptibility to colorectal cancer. Therapeutic modulation of AIM2 expression and microbiota has the potential to prevent colorectal cancer.

The transcription factor IRF1 and guanylate-binding proteins target activation of the AIM2 inflammasome by Francisella infection.

Inflammasomes are critical for mounting host defense against pathogens. The molecular mechanisms that control activation of the AIM2 inflammasome in response to different cytosolic pathogens remain unclear. Here we found that the transcription factor IRF1 was required for activation of the AIM2 inflammasome during infection with the Francisella tularensis subspecies novicida (F. novicida), whereas engagement of the AIM2 inflammasome by mouse cytomegalovirus (MCMV) or transfected double-stranded DNA did not require IRF1. Infection of F. novicida detected by the DNA sensor cGAS and its adaptor STING induced type I interferon-dependent expression of IRF1, which drove the expression of guanylate-binding proteins (GBPs); this led to intracellular killing of bacteria and DNA release. Our results reveal a specific requirement for IRF1 and GBPs in the liberation of DNA for sensing by AIM2 depending on the pathogen encountered by the cell.

AIM2 forms a complex with pyrin and ZBP1 to drive PANoptosis and host defence.

Inflammasomes are important sentinels of innate immune defence, sensing pathogens and inducing cell death in infected cells1. There are several inflammasome sensors that each detect and respond to a specific pathogen- or damage-associated molecular pattern (PAMP or DAMP, respectively)1. During infection, live pathogens can induce the release of multiple PAMPs and DAMPs, which can simultaneously engage multiple inflammasome sensors2-5. Here we found that AIM2 regulates the innate immune sensors pyrin and ZBP1 to drive inflammatory signalling and a form of inflammatory cell death known as PANoptosis, and provide host protection during infections with herpes simplex virus 1 and Francisella novicida. We also observed that AIM2, pyrin and ZBP1 were members of a large multi-protein complex along with ASC, caspase-1, caspase-8, RIPK3, RIPK1 and FADD, that drove inflammatory cell death (PANoptosis). Collectively, our findings define a previously unknown regulatory and molecular interaction between AIM2, pyrin and ZBP1 that drives assembly of an AIM2-mediated multi-protein complex that we term the AIM2 PANoptosome and comprising multiple inflammasome sensors and cell death regulators. These results advance the understanding of the functions of these molecules in innate immunity and inflammatory cell death, suggesting new therapeutic targets for AIM2-, ZBP1- and pyrin-mediated diseases.

ADAR1 and ZBP1 in innate immunity, cell death, and disease.

ADAR1 and ZBP1 are the only two mammalian proteins that contain Zα domains, which are thought to bind to nucleic acids in the Z-conformation. These two molecules are crucial in regulating diverse biological processes. While ADAR1-mediated RNA editing supports host survival and development, ZBP1-mediated immune responses provide host defense against infection and disease. Recent studies have expanded our understanding of the functions of ADAR1 and ZBP1 beyond their classical roles and established their fundamental regulation of innate immune responses, including NLRP3 inflammasome activation, inflammation, and cell death. Their roles in these processes have physiological impacts across development, infectious and inflammatory diseases, and cancer. In this review, we discuss the functions of ADAR1 and ZBP1 in regulating innate immune responses in development and disease.

Galactosaminogalactan activates the inflammasome to provide host protection.

Inflammasomes are important sentinels of innate immune defence that are activated in response to diverse stimuli, including pathogen-associated molecular patterns (PAMPs)1. Activation of the inflammasome provides host defence against aspergillosis2,3, which is a major health concern for patients who are immunocompromised. However, the Aspergillus fumigatus PAMPs that are responsible for inflammasome activation are not known. Here we show that the polysaccharide galactosaminogalactan (GAG) of A. fumigatus is a PAMP that activates the NLRP3 inflammasome. The binding of GAG to ribosomal proteins inhibited cellular translation machinery, and thus activated the NLRP3 inflammasome. The galactosamine moiety bound to ribosomal proteins and blocked cellular translation, which triggered activation of the NLRP3 inflammasome. In mice, a GAG-deficient Aspergillus mutant (Δgt4c) did not elicit protective activation of the inflammasome, and this strain exhibited enhanced virulence. Moreover, administration of GAG protected mice from colitis induced by dextran sulfate sodium in an inflammasome-dependent manner. Thus, ribosomes connect the sensing of this fungal PAMP to the activation of an innate immune response.

DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome.

The cellular stress response has a vital role in regulating homeostasis by modulating cell survival and death. Stress granules are cytoplasmic compartments that enable cells to survive various stressors. Defects in the assembly and disassembly of stress granules are linked to neurodegenerative diseases, aberrant antiviral responses and cancer1-5. Inflammasomes are multi-protein heteromeric complexes that sense molecular patterns that are associated with damage or intracellular pathogens, and assemble into cytosolic compartments known as ASC specks to facilitate the activation of caspase-1. Activation of inflammasomes induces the secretion of interleukin (IL)-1ß and IL-18 and drives cell fate towards pyroptosis-a form of programmed inflammatory cell death that has major roles in health and disease6-12. Although both stress granules and inflammasomes can be triggered by the sensing of cellular stress, they drive contrasting cell-fate decisions. The crosstalk between stress granules and inflammasomes and how this informs cell fate has not been well-studied. Here we show that the induction of stress granules specifically inhibits NLRP3 inflammasome activation, ASC speck formation and pyroptosis. The stress granule protein DDX3X interacts with NLRP3 to drive inflammasome activation. Assembly of stress granules leads to the sequestration of DDX3X, and thereby the inhibition of NLRP3 inflammasome activation. Stress granules and the NLRP3 inflammasome compete for DDX3X molecules to coordinate the activation of innate responses and subsequent cell-fate decisions under stress conditions. Induction of stress granules or loss of DDX3X in the myeloid compartment leads to a decrease in the production of inflammasome-dependent cytokines in vivo. Our findings suggest that macrophages use the availability of DDX3X to interpret stress signals and choose between pro-survival stress granules and pyroptotic ASC specks. Together, our data demonstrate the role of DDX3X in driving NLRP3 inflammasome and stress granule assembly, and suggest a rheostat-like mechanistic paradigm for regulating live-or-die cell-fate decisions under stress conditions.

Immune regulator IRF1 contributes to ZBP1-, AIM2-, RIPK1-, and NLRP12-PANoptosome activation and inflammatory cell death (PANoptosis).

The innate immune system provides the first line of defense against pathogens and cellular insults and is activated by pattern recognition receptors sensing pathogen- or damage-associated molecular patterns. This activation can result in inflammation via cytokine release as well as the induction of lytic regulated cell death (RCD). Innate immune signaling can also induce the expression of interferon regulatory factor 1 (IRF1), an important molecule in regulating downstream inflammation and cell death. While IRF1 has been shown to modulate some RCD pathways, a comprehensive evaluation of its role in inflammatory cell death pathways is lacking. Here, we examined the role of IRF1 in cell death during inflammasome and PANoptosome activation using live cell imaging, Western blotting, and ELISA in primary murine macrophages. IRF1 contributed to the induction of ZBP1- (Z-DNA binding protein 1), AIM2- (absent in melanoma-2), RIPK1- (receptor interacting protein kinase 1), and NLRP12 (NOD-like receptor family, pyrin domain-containing 12)-PANoptosome activation and PANoptosis. Furthermore, IRF1 regulated the cell death under conditions where inflammasomes, along with caspase-8 and RIPK3, act as integral components of PANoptosomes to drive PANoptosis. However, it was dispensable for other inflammasomes that form independent of the PANoptosome to drive pyroptosis. Overall, these findings define IRF1 as an upstream regulator of PANoptosis and suggest that modulating the activation of molecules in the IRF1 pathway could be used as a strategy to treat inflammatory and infectious diseases associated with aberrant inflammatory cell death.

The 'cytokine storm': molecular mechanisms and therapeutic prospects.

Cytokine storm syndrome (CSS) has generally been described as a collection of clinical manifestations resulting from an overactivated immune system. Cytokine storms (CSs) are associated with various pathologies, as observed in infectious diseases, certain acquired or inherited immunodeficiencies and autoinflammatory diseases, or following therapeutic interventions. Despite the role of CS in tissue damage and multiorgan failure, a systematic understanding of its underlying molecular mechanisms is lacking. Recent studies demonstrate a positive feedback loop between cytokine release and cell death pathways; certain cytokines, pathogen-associated molecular patterns (PAMPs), and damage-associated molecular patterns (DAMPs), can activate inflammatory cell death, leading to further cytokine secretion. Here, we discuss recent progress in innate immunity and inflammatory cell death, providing insights into the cellular and molecular mechanisms of CSs and therapeutics that might quell ensuing life-threatening effects.

Cutting Edge: Caspase-8 Is a Linchpin in Caspase-3 and Gasdermin D Activation to Control Cell Death, Cytokine Release, and Host Defense during Influenza A Virus Infection.

Programmed cell death (PCD) is essential for the innate immune response, which serves as the first line of defense against pathogens. Caspases regulate PCD, immune responses, and homeostasis. Caspase-8 specifically plays multifaceted roles in PCD pathways including pyroptosis, apoptosis, and necroptosis. However, because caspase-8-deficient mice are embryonically lethal, little is known about how caspase-8 coordinates different PCD pathways under physiological conditions. Here, we report an anti-inflammatory role of caspase-8 during influenza A virus infection. We generated viable mice carrying an uncleavable version of caspase-8 (Casp8 DA/DA). We demonstrated that caspase-8 autoprocessing was responsible for activating caspase-3, thereby suppressing gasdermin D-mediated pyroptosis and inflammatory cytokine release. We also found that apoptotic and pyroptotic pathways were activated at the same time during influenza A virus infection, which enabled the cell-intrinsic anti-inflammatory function of the caspase-8-caspase-3 axis. Our findings provide new insight into the immunological consequences of caspase-8-coordinated PCD cross-talk under physiological conditions.

Single cell analysis of PANoptosome cell death complexes through an expansion microscopy method.

In response to infection or sterile insults, inflammatory programmed cell death is an essential component of the innate immune response to remove infected or damaged cells. PANoptosis is a unique innate immune inflammatory cell death pathway regulated by multifaceted macromolecular complexes called PANoptosomes, which integrate components from other cell death pathways. Growing evidence shows that PANoptosis can be triggered in many physiological conditions, including viral and bacterial infections, cytokine storms, and cancers. However, PANoptosomes at the single cell level have not yet been fully characterized. Initial investigations have suggested that key pyroptotic, apoptotic, and necroptotic molecules including the inflammasome adaptor protein ASC, apoptotic caspase-8 (CASP8), and necroptotic RIPK3 are conserved components of PANoptosomes. Here, we optimized an immunofluorescence procedure to probe the highly dynamic multiprotein PANoptosome complexes across various innate immune cell death-inducing conditions. We first identified and validated antibodies to stain endogenous mouse ASC, CASP8, and RIPK3, without residual staining in the respective knockout cells. We then assessed the formation of PANoptosomes across innate immune cell death-inducing conditions by monitoring the colocalization of ASC with CASP8 and/or RIPK3. Finally, we established an expansion microscopy procedure using these validated antibodies to image the organization of ASC, CASP8, and RIPK3 within the PANoptosome. This optimized protocol, which can be easily adapted to study other multiprotein complexes and other cell death triggers, provides confirmation of PANoptosome assembly in individual cells and forms the foundation for a deeper molecular understanding of the PANoptosome complex and PANoptosis to facilitate therapeutic targeting.

Caspase-6 promotes activation of the caspase-11-NLRP3 inflammasome during gram-negative bacterial infections.

The innate immune system acts as the first line of defense against infection. One key component of the innate immune response to gram-negative bacterial infections is inflammasome activation. The caspase-11 (CASP11)-nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is activated by cytosolic lipopolysaccharide, a gram-negative bacterial cell wall component, to trigger pyroptosis and host defense during infection. Although several cellular signaling pathways have been shown to regulate CASP11-NLRP3 inflammasome activation in response to lipopolysaccharide, the upstream molecules regulating CASP11 activation during infection with live pathogens remain unclear. Here, we report that the understudied caspase-6 (CASP6) contributes to the activation of the CASP11-NLRP3 inflammasome in response to infections with gram-negative bacteria. Using in vitro cellular systems with bone marrow-derived macrophages and 293T cells, we found that CASP6 can directly process CASP11 by cleaving at Asp59 and Asp285, the CASP11 auto-cleavage sites, which could contribute to the activation of CASP11 during gram-negative bacterial infection. Thus, the loss of CASP6 led to impaired CASP11-NLRP3 inflammasome activation in response to gram-negative bacteria. These results demonstrate that CASP6 potentiates activation of the CASP11-NLRP3 inflammasome to produce inflammatory cytokines during gram-negative bacterial infections.

DDX3X coordinates host defense against influenza virus by activating the NLRP3 inflammasome and type I interferon response.

Viruses and hosts have coevolved for millions of years, leading to the development of complex host-pathogen interactions. Influenza A virus (IAV) causes severe pulmonary pathology and is a recurrent threat to human health. Innate immune sensing of IAV triggers a complex chain of host responses. IAV has adapted to evade host defense mechanisms, and the host has coevolved to counteract these evasion strategies. However, the molecular mechanisms governing the balance between host defense and viral immune evasion is poorly understood. Here, we show that the host protein DEAD-box helicase 3 X-linked (DDX3X) is critical to orchestrate a multifaceted antiviral innate response during IAV infection, coordinating the activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, assembly of stress granules, and type I interferon (IFN) responses. DDX3X activated the NLRP3 inflammasome in response to WT IAV, which carries the immune evasive nonstructural protein 1 (NS1). However, in the absence of NS1, DDX3X promoted the formation of stress granules that facilitated efficient activation of type I IFN signaling. Moreover, induction of DDX3X-containing stress granules by external stimuli after IAV infection led to increased type I IFN signaling, suggesting that NS1 actively inhibits stress granule-mediated host responses and DDX3X-mediated NLRP3 activation counteracts this action. Furthermore, the loss of DDX3X expression in myeloid cells caused severe pulmonary pathogenesis and morbidity in IAV-infected mice. Together, our findings show that DDX3X orchestrates alternate modes of innate host defense which are critical to fight against NS1-mediated immune evasion strategies during IAV infection.

Innate immunity, cytokine storm, and inflammatory cell death in COVID-19.

The innate immune system serves as the first line of defense against invading pathogens; however, dysregulated innate immune responses can induce aberrant inflammation that is detrimental to the host. Therefore, careful innate immune regulation is critical during infections. The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has resulted in global morbidity and mortality as well as socio-economic stresses. Innate immune sensing of SARS-CoV-2 by multiple host cell pattern recognition receptors leads to the production of various pro-inflammatory cytokines and the induction of inflammatory cell death. These processes can contribute to cytokine storm, tissue damage, and acute respiratory distress syndrome. Here, we discuss the sensing of SARS-CoV-2 to induce innate immune activation and the contribution of this innate immune signaling in the development and severity of COVID-19. In addition, we provide a conceptual framework for innate immunity driving cytokine storm and organ damage in patients with severe COVID-19. A better understanding of the molecular mechanisms regulated by innate immunity is needed for the development of targeted modalities that can improve patient outcomes by mitigating severe disease.

Interferon inducible GBPs restrict Burkholderia thailandensis motility induced cell-cell fusion.

Innate immunity responds to pathogens by producing alarm signals and activating pathways that make host cells inhospitable for pathogen replication. The intracellular bacterium Burkholderia thailandensis invades the cytosol, hijacks host actin, and induces cell fusion to spread to adjacent cells, forming multinucleated giant cells (MNGCs) which promote bacterial replication. We show that type I interferon (IFN) restricts macrophage MNGC formation during B. thailandensis infection. Guanylate-binding proteins (GBPs) expressed downstream of type I IFN were required to restrict MNGC formation through inhibition of bacterial Arp2/3-dependent actin motility during infection. GTPase activity and the CAAX prenylation domain were required for GBP2 recruitment to B. thailandensis, which restricted bacterial actin polymerization required for MNGC formation. Consistent with the effects in in vitro macrophages, Gbp2-/-, Gbp5-/-, GbpChr3-KO mice were more susceptible to intranasal infection with B. thailandensis than wildtype mice. Our findings reveal that IFN and GBPs play a critical role in restricting cell-cell fusion and bacteria-induced pathology during infection.