Phase separation drives RNA virus-induced activation of the NLRP6 inflammasome.

NLRP6 is important in host defense by inducing functional outcomes including inflammasome activation and interferon production. Here, we show that NLRP6 undergoes liquid-liquid phase separation (LLPS) upon interaction with double-stranded RNA (dsRNA) in vitro and in cells, and an intrinsically disordered poly-lysine sequence (K350-354) of NLRP6 is important for multivalent interactions, phase separation, and inflammasome activation. Nlrp6-deficient or Nlrp6K350-354A mutant mice show reduced inflammasome activation upon mouse hepatitis virus or rotavirus infection, and in steady state stimulated by intestinal microbiota, implicating NLRP6 LLPS in anti-microbial immunity. Recruitment of ASC via helical assembly solidifies NLRP6 condensates, and ASC further recruits and activates caspase-1. Lipoteichoic acid, a known NLRP6 ligand, also promotes NLRP6 LLPS, and DHX15, a helicase in NLRP6-induced interferon signaling, co-forms condensates with NLRP6 and dsRNA. Thus, LLPS of NLRP6 is a common response to ligand stimulation, which serves to direct NLRP6 to distinct functional outcomes depending on the cellular context.

FcγR-mediated SARS-CoV-2 infection of monocytes activates inflammation.

SARS-CoV-2 can cause acute respiratory distress and death in some patients1. Although severe COVID-19 is linked to substantial inflammation, how SARS-CoV-2 triggers inflammation is not clear2. Monocytes and macrophages are sentinel cells that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D, leading to inflammatory death (pyroptosis) and the release of potent inflammatory mediators3. Here we show that about 6% of blood monocytes of patients with COVID-19 are infected with SARS-CoV-2. Monocyte infection depends on the uptake of antibody-opsonized virus by Fcγ receptors. The plasma of vaccine recipients does not promote antibody-dependent monocyte infection. SARS-CoV-2 begins to replicate in monocytes, but infection is aborted, and infectious virus is not detected in the supernatants of cultures of infected monocytes. Instead, infected cells undergo pyroptosis mediated by activation of NLRP3 and AIM2 inflammasomes, caspase-1 and gasdermin D. Moreover, tissue-resident macrophages, but not infected epithelial and endothelial cells, from lung autopsies from patients with COVID-19 have activated inflammasomes. Taken together, these findings suggest that antibody-mediated SARS-CoV-2 uptake by monocytes and macrophages triggers inflammatory cell death that aborts the production of infectious virus but causes systemic inflammation that contributes to COVID-19 pathogenesis.

Gasdermin D pore structure reveals preferential release of mature interleukin-1.

As organelles of the innate immune system, inflammasomes activate caspase-1 and other inflammatory caspases that cleave gasdermin D (GSDMD). Caspase-1 also cleaves inactive precursors of the interleukin (IL)-1 family to generate mature cytokines such as IL-1ß and IL-18. Cleaved GSDMD forms transmembrane pores to enable the release of IL-1 and to drive cell lysis through pyroptosis1-9. Here we report cryo-electron microscopy structures of the pore and the prepore of GSDMD. These structures reveal the different conformations of the two states, as well as extensive membrane-binding elements including a hydrophobic anchor and three positively charged patches. The GSDMD pore conduit is predominantly negatively charged. By contrast, IL-1 precursors have an acidic domain that is proteolytically removed by caspase-110. When permeabilized by GSDMD pores, unlysed liposomes release positively charged and neutral cargoes faster than negatively charged cargoes of similar sizes, and the pores favour the passage of IL-1ß and IL-18 over that of their precursors. Consistent with these findings, living-but not pyroptotic-macrophages preferentially release mature IL-1ß upon perforation by GSDMD. Mutation of the acidic residues of GSDMD compromises this preference, hindering intracellular retention of the precursor and secretion of the mature cytokine. The GSDMD pore therefore mediates IL-1 release by electrostatic filtering, which suggests the importance of charge in addition to size in the transport of cargoes across this large channel.

Targeting stem-loop 1 of the SARS-CoV-2 5' UTR to suppress viral translation and Nsp1 evasion.

SARS-CoV-2 is a highly pathogenic virus that evades antiviral immunity by interfering with host protein synthesis, mRNA stability, and protein trafficking. The SARS-CoV-2 nonstructural protein 1 (Nsp1) uses its C-terminal domain to block the messenger RNA (mRNA) entry channel of the 40S ribosome to inhibit host protein synthesis. However, how SARS-CoV-2 circumvents Nsp1-mediated suppression for viral protein synthesis and if the mechanism can be targeted therapeutically remain unclear. Here, we show that N- and C-terminal domains of Nsp1 coordinate to drive a tuned ratio of viral to host translation, likely to maintain a certain level of host fitness while maximizing replication. We reveal that the stem-loop 1 (SL1) region of the SARS-CoV-2 5' untranslated region (5' UTR) is necessary and sufficient to evade Nsp1-mediated translational suppression. Targeting SL1 with locked nucleic acid antisense oligonucleotides inhibits viral translation and makes SARS-CoV-2 5' UTR vulnerable to Nsp1 suppression, hindering viral replication in vitro at a nanomolar concentration, as well as providing protection against SARS-CoV-2-induced lethality in transgenic mice expressing human ACE2. Thus, SL1 allows Nsp1 to switch infected cells from host to SARS-CoV-2 translation, presenting a therapeutic target against COVID-19 that is conserved among immune-evasive variants. This unique strategy of unleashing a virus' own virulence mechanism against itself could force a critical trade-off between drug resistance and pathogenicity.

Structures and functions of the inflammasome engine.

Inflammasomes are molecular machines that carry out inflammatory responses on challenges by pathogens and endogenous dangers. Dysregulation of inflammasome assembly and regulation is associated with numerous human diseases from autoimmunity to cancer. In recent years, significant advances have been made in understanding the mechanism of inflammasome signaling using structural approaches. Here, we review inflammasomes formed by the NLRP1, NLRP3, and NLRC4 sensors, which are well characterized structurally, and discuss the structural and functional diversity among them.

Inflammasome Activation and Pyroptosis via a Lipid-regulated SIRT1-p53-ASC Axis in Macrophages From Male Mice and Humans.

Obesity-linked diabetes is associated with accumulation of proinflammatory macrophages into adipose tissue leading to inflammasome activation and pyroptotic secretion of interleukin (IL)-1ß and IL-18. Targeting fatty acid binding protein 4 (FABP4) uncouples obesity from inflammation, attenuates characteristics of type 2 diabetes and is mechanistically linked to the cellular accumulation of monounsaturated fatty acids in macrophages. Herein we show that pharmacologic inhibition or genetic deletion of FABP4 activates silent mating type information regulation 2 homolog 1 (SIRT1) and deacetylates its downstream targets p53 and signal transducer and activator of transcription 3 (STAT3). Pharmacologic inhibition of fatty acid synthase or stearoyl-coenzyme A desaturase inhibits, whereas exogenous addition of C16:1 or C18:1 but not their saturated acyl chain counterparts, activates SIRT1 and p53/STAT3 signaling and IL-1ß/IL-18 release. Expression of the p53 target gene ASC [apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (CARD)] required for assembly of the NLR family pyrin domain containing 3 (NLRP3) inflammasome is downregulated in FABP4 null mice and macrophage cell lines leading to loss of procaspase 1 activation and pyroptosis. Concomitant with loss of ASC expression in FABP4-/- macrophages, inflammasome activation, gasdermin D processing, and functional activation of pyroptosis are all diminished in FABP4 null macrophages but can be rescued by silencing SIRT1 or exogenous expression of ASC. Taken together, these results reveal a novel lipid-regulated pathway linking to SIRT1-p53-ASC signaling and activation of inflammasome action and pyroptosis.

Inflammasome activation at the crux of severe COVID-19.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), results in life-threatening disease in a minority of patients, especially elderly people and those with co-morbidities such as obesity and diabetes. Severe disease is characterized by dysregulated cytokine release, pneumonia and acute lung injury, which can rapidly progress to acute respiratory distress syndrome, disseminated intravascular coagulation, multisystem failure and death. However, a mechanistic understanding of COVID-19 progression remains unclear. Here we review evidence that SARS-CoV-2 directly or indirectly activates inflammasomes, which are large multiprotein assemblies that are broadly responsive to pathogen-associated and stress-associated cellular insults, leading to secretion of the pleiotropic IL-1 family cytokines (IL-1ß and IL-18), and pyroptosis, an inflammatory form of cell death. We further discuss potential mechanisms of inflammasome activation and clinical efforts currently under way to suppress inflammation to prevent or ameliorate severe COVID-19.

SARS-CoV-2 infects blood monocytes to activate NLRP3 and AIM2 inflammasomes, pyroptosis and cytokine release.

SARS-CoV-2 causes acute respiratory distress that can progress to multiorgan failure and death in some patients. Although severe COVID-19 disease is linked to exuberant inflammation, how SARS-CoV-2 triggers inflammation is not understood. Monocytes are sentinel blood cells that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D (GSDMD) pores, leading to inflammatory death (pyroptosis) and processing and release of IL-1 family cytokines, potent inflammatory mediators. Here we show that ~10% of blood monocytes in COVID-19 patients are dying and infected with SARS-CoV-2. Monocyte infection, which depends on antiviral antibodies, activates NLRP3 and AIM2 inflammasomes, caspase-1 and GSDMD cleavage and relocalization. Signs of pyroptosis (IL-1 family cytokines, LDH) in the plasma correlate with development of severe disease. Moreover, expression quantitative trait loci (eQTLs) linked to higher GSDMD expression increase the risk of severe COVID-19 disease (odds ratio, 1.3, p<0.005). These findings taken together suggest that antibody-mediated SARS-CoV-2 infection of monocytes triggers inflammation that contributes to severe COVID-19 disease pathogenesis. ONE SENTENCE SUMMARY: Antibody-mediated SARS-CoV-2 infection of monocytes activates inflammation and cytokine release.

SARS-CoV-2 infects blood monocytes to activate NLRP3 and AIM2 inflammasomes, pyroptosis and cytokine release.

SARS-CoV-2 causes acute respiratory distress that can progress to multiorgan failure and death in a minority of patients. Although severe COVID-19 disease is linked to exuberant inflammation, how SARS-CoV-2 triggers inflammation is not understood. Monocytes and macrophages are sentinel immune cells in the blood and tissue, respectively, that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D (GSDMD) pores, leading to inflammatory death (pyroptosis) and processing and release of IL-1 family cytokines, potent inflammatory mediators. Here we show that expression quantitative trait loci (eQTLs) linked to higher GSDMD expression increase the risk of severe COVID-19 disease (odds ratio, 1.3, p<0.005). We find that about 10% of blood monocytes in COVID-19 patients are infected with SARS-CoV-2. Monocyte infection depends on viral antibody opsonization and uptake of opsonized virus by the Fc receptor CD16. After uptake, SARS-CoV-2 begins to replicate in monocytes, as evidenced by detection of double-stranded RNA and subgenomic RNA and expression of a fluorescent reporter gene. However, infection is aborted, and infectious virus is not detected in infected monocyte supernatants or patient plasma. Instead, infected cells undergo inflammatory cell death (pyroptosis) mediated by activation of the NLRP3 and AIM2 inflammasomes, caspase-1 and GSDMD. Moreover, tissue-resident macrophages, but not infected epithelial cells, from COVID-19 lung autopsy specimens showed evidence of inflammasome activation. These findings taken together suggest that antibody-mediated SARS-CoV-2 infection of monocytes/macrophages triggers inflammatory cell death that aborts production of infectious virus but causes systemic inflammation that contributes to severe COVID-19 disease pathogenesis.

Higher-order assemblies in innate immune and inflammatory signaling: A general principle in cell biology.

Higher-order supramolecular complexes-dubbed signalosomes carry out key signaling and effector functions in innate immunity and inflammation. In this review, we present several recently discovered signalosomes that are formed either by stable protein-protein interactions or by dynamic liquid-liquid phase separation. Structural features of these signalosomes are highlighted to elucidate their functions and biological insights.

Centrosomes are required for proper ß-catenin processing and Wnt response.

The Wnt/ß-catenin signaling pathway is central to metazoan development and routinely dysregulated in cancer. Wnt/ß-catenin signaling initiates transcriptional reprogramming upon stabilization of the transcription factor ß-catenin, which is otherwise posttranslationally processed by a destruction complex and degraded by the proteasome. Since various Wnt signaling components are enriched at centrosomes, we examined the functional contribution of centrosomes to Wnt signaling, ß-catenin regulation, and posttranslational modifications. In HEK293 cells depleted of centrosomes we find that ß-catenin synthesis and degradation rates are unaffected but that the normal accumulation of ß-catenin in response to Wnt signaling is attenuated. This is due to accumulation of a novel high-molecular-weight form of phosphorylated ß-catenin that is constitutively degraded in the absence of Wnt. Wnt signaling operates by inhibiting the destruction complex and thereby reducing destruction complex-phosphorylated ß-catenin, but high-molecular-weight ß-catenin is unexpectedly increased by Wnt signaling. Therefore these studies have identified a pool of ß-catenin effectively shielded from regulation by Wnt. We present a model whereby centrosomes prevent inappropriate ß-catenin modifications that antagonize normal stabilization by Wnt signals.

The benefits of local depletion: The centrosome as a scaffold for ubiquitin-proteasome-mediated degradation.

The centrosome is the major microtubule-organizing center in animal cells but is dispensable for proper microtubule spindle formation in many biological contexts and is thus thought to fulfill additional functions. Recent observations suggest that the centrosome acts as a scaffold for proteasomal degradation in the cell to regulate a variety of biological processes including cell fate acquisition, cell cycle control, stress response, and cell morphogenesis. Here, we review the body of studies indicating a role for the centrosome in promoting proteasomal degradation of ubiquitin-proteasome substrates and explore the functional relevance of this system in different biological contexts. We discuss a potential role for the centrosome in coordinating local degradation of proteasomal substrates, allowing cells to achieve stringent spatiotemporal control over various signaling processes.