Activation of the NLRP1 Inflammasome and Its Role in Transmissible Gastroenteritis Coronavirus Infection.

The inflammasome pathway is a critical early response mechanism of the host that detects pathogens, initiates the production of inflammatory cytokines, and recruits effector cells to the infection site. Nonetheless, the mechanism of inflammasome activation in coronavirus infection and its biological functions in host defense remain unclear. Transmissible gastroenteritis virus (TGEV), a member of the genus Alphacoronavirus, is a significant pathogen that mainly infects piglets and causes intestinal inflammation and inflammatory cell infiltration. Here, we investigated the mechanism of inflammasome activation in intestinal epithelial cells (IECs) infected with TGEV. We observed a substantial increase in interleukin 1ß (IL-1ß) and IL-18 levels in both IECs and TGEV-infected porcine intestinal tissues. Furthermore, TGEV infection resulted in increased activation of caspase-1 and the NLRP1 (NOD-like receptor [NLR]-containing pyrin domain [PYD]) inflammasome. Our findings revealed that TGEV infection impeded the interaction between porcine NLRP1 (pNLRP1) and porcine dipeptidyl peptidases 9 (pDPP9), yet it did not reduce the expression of pDPP9. Importantly, the ZU5 domain, not the function-to-find domain (FIIND) reported in human NLRP1, was identified as the minimal domain of pNLRP1 for pDPP9 binding. In addition, the robust type I IFN expression induced by TGEV infection also upregulated pNLRP1 expression and pNLRP1 itself acts as an interferon-stimulated gene to counteract TGEV infection. Our data demonstrate that pNLRP1 has antiviral capabilities against coronavirus infection, which highlights its potential as a novel therapeutic target for coronavirus antiviral therapy. IMPORTANCE Coronavirus primarily targets the epithelial cells of the respiratory and gastrointestinal tracts, leading to damage in both humans and animals. NLRP1 is a direct sensor for RNA virus infection which is highly expressed in epithelial barrier tissues. However, until recently, the precise molecular mechanisms underlying its activation in coronavirus infection and subsequent downstream events remained unclear. In this study, we demonstrate that the alphacoronavirus TGEV induces the production of IL-1ß and IL-18 and upregulates the expression of pNLRP1. Furthermore, we found that pNLRP1 can serve as an interferon-stimulated gene (ISG) to inhibit the infection of enterovirus TGEV. Our research highlights the crucial role of NLRP1 as a regulator of innate immunity in TGEV infection and shows that it may serve as a potential therapeutic target for the treatment of coronavirus infection.

Bat ASC2 suppresses inflammasomes and ameliorates inflammatory diseases.

Bats are special in their ability to live long and host many emerging viruses. Our previous studies showed that bats have altered inflammasomes, which are central players in aging and infection. However, the role of inflammasome signaling in combating inflammatory diseases remains poorly understood. Here, we report bat ASC2 as a potent negative regulator of inflammasomes. Bat ASC2 is highly expressed at both the mRNA and protein levels and is highly potent in inhibiting human and mouse inflammasomes. Transgenic expression of bat ASC2 in mice reduced the severity of peritonitis induced by gout crystals and ASC particles. Bat ASC2 also dampened inflammation induced by multiple viruses and reduced mortality of influenza A virus infection. Importantly, it also suppressed SARS-CoV-2-immune-complex-induced inflammasome activation. Four key residues were identified for the gain of function of bat ASC2. Our results demonstrate that bat ASC2 is an important negative regulator of inflammasomes with therapeutic potential in inflammatory diseases.

Activation of the AIM2 Receptor in Circulating Cells of Post-COVID-19 Patients With Signs of Lung Fibrosis Is Associated With the Release of IL-1α, IFN-α and TGF-ß.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), responsible for COVID-19, has caused a global pandemic. Observational studies revealed a condition, herein called as Long-COVID syndrome (PC), that affects both moderately and severely infected patients, reducing quality-of-life. The mechanism/s underlying the onset of fibrotic-like changes in PC are still not well defined. The goal of this study was to understand the involvement of the Absent in melanoma-2 (AIM2) inflammasome in PC-associated lung fibrosis-like changes revealed by chest CT scans. Peripheral blood mononuclear cells (PBMCs) obtained from PC patients who did not develop signs of lung fibrosis were not responsive to AIM2 activation by Poly dA:dT. In sharp contrast, PBMCs from PC patients with signs of lung fibrosis were highly responsive to AIM2 activation, which induced the release of IL-1α, IFN-α and TGF-ß. The recognition of Poly dA:dT was not due to the activation of cyclic GMP-AMP (cGAMP) synthase, a stimulator of interferon response (cGAS-STING) pathways, implying a role for AIM2 in PC conditions. The release of IFN-α was caspase-1- and caspase-4-dependent when AIM2 was triggered. Instead, the release of pro-inflammatory IL-1α and pro-fibrogenic TGF-ß were inflammasome independent because the inhibition of caspase-1 and caspase-4 did not alter the levels of the two cytokines. Moreover, the responsiveness of AIM2 correlated with higher expression of the receptor in circulating CD14+ cells in PBMCs from patients with signs of lung fibrosis.

[Role of inflammasome NLRP3 in the pathophysiology of viral infections: A focus on SARS-CoV-2 infection]. / L'inflammasome NLRP3 dans la physiopathologie des infections virales - Un focus sur la COVID-19.

NLRP3 is one of the best characterized innate immune cytosolic sensor. As part of the innate immune response, the NLRP3 inflammasome detects a wide range of danger signals such as pathogens, tissue damages, cellular stress. The priming and activation of NLRP3 lead to the formation of an oligomeric intracellular complex and to the recruitment and activation of caspase-1. Once activated, not only this inflammasome complex controls the processing and release of pro-inflammatory factors including IL-1ß and IL-18, but also the inflammatory cell death pyroptosis mediated by gasdermin D pores. In this review, we describe the role of the NLRP3 inflammasome activation in viral infections with a particular interest on SARS-CoV-2 infection. In addition, we present therapies evaluated or under evaluation targeting the NLRP3 inflammasome pathway as COVID-19 treatment.

Title: L'inflammasome NLRP3 dans la physiopathologie des infections virales - Un focus sur la COVID-19. Abstract: L'inflammasome NLRP3 est un complexe multiprotéique intracellulaire impliqué dans la réponse immunitaire innée. Après la détection de signaux de dangers, tels que ceux provenant d'agents pathogènes, ce complexe s'assemble afin d'initier la production et la sécrétion de molécules pro-inflammatoires, comme l'IL(interleukine)-1ß et l'IL-18. L'inflammasome NLRP3 régule aussi l'activation de la gasdermine D, une protéine impliquée dans la mort cellulaire inflammatoire, ou pyroptose. Cette revue s'intéresse à l'activation et aux rôles de l'inflammasome NLRP3 dans les infections virales et plus particulièrement dans le cas de l'infection par le SARS-CoV-2. Une attention particulière est portée dans cette revue aux traitements évalués, ou en cours d'évaluation, ciblant la voie de l'inflammasome NLRP3 activée au cours de la COVID-19.

Colchicine for COVID-19: targeting NLRP3 inflammasome to blunt hyperinflammation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is capable of inducing the activation of NACHT, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome, a macromolecular structure sensing the danger and amplifying the inflammatory response. The main product processed by NLRP3 inflammasome is interleukin (IL)-1ß, responsible for the downstream production of IL-6, which has been recognized as an important mediator in coronavirus disease 2019 (COVID-19). Since colchicine is an anti-inflammatory drug with the ability to block NLRP3 inflammasome oligomerization, this may prevent the release of active IL-1ß and block the detrimental effects of downstream cytokines, i.e. IL-6. To date, few randomized clinical trials and many observational studies with colchicine have been conducted, showing interesting signals. As colchicine is a nonspecific inhibitor of the NLRP3 inflammasome, compounds specifically blocking this molecule might provide increased advantages in reducing the inflammatory burden and its related clinical manifestations. This may occur through a selective blockade of different steps preceding NLRP3 inflammasome oligomerization as well as through a reduced release of the main cytokines (IL-1ß and IL-18). Since most evidence is based on observational studies, definitive conclusion cannot be drawn and additional studies are needed to confirm preliminary results and further dissect how colchicine and other NLRP3 inhibitors reduce the inflammatory burden and evaluate the timing and duration of treatment.

Innate immunity: the first line of defense against SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus (SARS-CoV)-2, continues to cause substantial morbidity and mortality. While most infections are mild, some patients experience severe and potentially fatal systemic inflammation, tissue damage, cytokine storm and acute respiratory distress syndrome. The innate immune system acts as the first line of defense, sensing the virus through pattern recognition receptors and activating inflammatory pathways that promote viral clearance. Here, we discuss innate immune processes involved in SARS-CoV-2 recognition and the resultant inflammation. Improved understanding of how the innate immune system detects and responds to SARS-CoV-2 will help identify targeted therapeutic modalities that mitigate severe disease and improve patient outcomes.

Inflammasome regulation in driving COVID-19 severity in humans and immune tolerance in bats.

Coronaviruses (CoVs) are RNA viruses that cause human respiratory infections. Zoonotic transmission of the SARS-CoV-2 virus caused the recent COVID-19 pandemic, which led to over 2 million deaths worldwide. Elevated inflammatory responses and cytotoxicity in the lungs are associated with COVID-19 severity in SARS-CoV-2-infected individuals. Bats, which host pathogenic CoVs, operate dampened inflammatory responses and show tolerance to these viruses with mild clinical symptoms. Delineating the mechanisms governing these host-specific inflammatory responses is essential to understand host-virus interactions determining the outcome of pathogenic CoV infections. Here, we describe the essential role of inflammasome activation in determining COVID-19 severity in humans and innate immune tolerance in bats that host several pathogenic CoVs. We further discuss mechanisms leading to inflammasome activation in human SARS-CoV-2 infection and how bats are molecularly adapted to suppress these inflammasome responses. We also report an analysis of functionally important residues of inflammasome components that provide new clues of bat strategies to suppress inflammasome signaling and innate immune responses. As spillover of bat viruses may cause the emergence of new human disease outbreaks, the inflammasome regulation in bats and humans likely provides specific strategies to combat the pathogenic CoV infections.

Innate Immunity as an Executor of the Programmed Death of Individual Organisms for the Benefit of the Entire Population.

In humans, over-activation of innate immunity in response to viral or bacterial infections often causes severe illness and death. Furthermore, similar mechanisms related to innate immunity can cause pathogenesis and death in sepsis, massive trauma (including surgery and burns), ischemia/reperfusion, some toxic lesions, and viral infections including COVID-19. Based on the reviewed observations, we suggest that such severe outcomes may be manifestations of a controlled suicidal strategy protecting the entire population from the spread of pathogens and from dangerous pathologies rather than an aberrant hyperstimulation of defense responses. We argue that innate immunity may be involved in the implementation of an altruistic programmed death of an organism aimed at increasing the well-being of the whole community. We discuss possible ways to suppress this atavistic program by interfering with innate immunity and suggest that combating this program should be a major goal of future medicine.

Inflammasomes and SARS-CoV-2 Infection.

SARS-CoV-2 is a new type of coronavirus that has caused worldwide pandemic. The disease induced by SARS-CoV-2 is called COVID-19. A majority of people with COVID-19 have relatively mild respiratory symptoms. However, a small percentage of COVID-19 patients develop a severe disease where multiple organs are affected. These severe forms of SARS-CoV-2 infections are associated with excessive production of pro-inflammatory cytokines, so called "cytokine storm". Inflammasomes, which are protein complexes of the innate immune system orchestrate development of local and systemic inflammation during virus infection. Recent data suggest involvement of inflammasomes in severe COVID-19. Activation of inflammasome exerts two major effects: it activates caspase-1-mediated processing and secretion of pro-inflammatory cytokines IL-1ß and IL-18, and induces inflammatory cell death, pyroptosis, via protein called gasdermin D. Here, we provide comprehensive review of current understanding of the activation and possible functions of different inflammasome structures during SARS-CoV-2 infection and compare that to response caused by influenza A virus. We also discuss how novel SARS-CoV-2 mRNA vaccines activate innate immune response, which is a prerequisite for the activation of protective adaptive immune response.

Unravelling the Immunomodulatory Effects of Viral Ion Channels, towards the Treatment of Disease.

The current COVID-19 pandemic has highlighted the need for the research community to develop a better understanding of viruses, in particular their modes of infection and replicative lifecycles, to aid in the development of novel vaccines and much needed anti-viral therapeutics. Several viruses express proteins capable of forming pores in host cellular membranes, termed "Viroporins". They are a family of small hydrophobic proteins, with at least one amphipathic domain, which characteristically form oligomeric structures with central hydrophilic domains. Consequently, they can facilitate the transport of ions through the hydrophilic core. Viroporins localise to host membranes such as the endoplasmic reticulum and regulate ion homeostasis creating a favourable environment for viral infection. Viroporins also contribute to viral immune evasion via several mechanisms. Given that viroporins are often essential for virion assembly and egress, and as their structural features tend to be evolutionarily conserved, they are attractive targets for anti-viral therapeutics. This review discusses the current knowledge of several viroporins, namely Influenza A virus (IAV) M2, Human Immunodeficiency Virus (HIV)-1 Viral protein U (Vpu), Hepatitis C Virus (HCV) p7, Human Papillomavirus (HPV)-16 E5, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Open Reading Frame (ORF)3a and Polyomavirus agnoprotein. We highlight the intricate but broad immunomodulatory effects of these viroporins and discuss the current antiviral therapies that target them; continually highlighting the need for future investigations to focus on novel therapeutics in the treatment of existing and future emergent viruses.

Instructions for Flow Cytometric Detection of ASC Specks as a Readout of Inflammasome Activation in Human Blood.

Inflammasome activation is linked to the aggregation of the adaptor protein ASC into a multiprotein complex, known as the ASC speck. Redistribution of cytosolic ASC to this complex has been widely used as a readout for inflammasome activation and precedes the downstream proteolytic release of the proinflammatory cytokines, IL-1ß and IL-18. Although inflammasomes are important for many diseases such as periodic fever syndromes, COVID-19, gout, sepsis, atherosclerosis and Alzheimer's disease, only a little knowledge exists on the precise and cell type specific occurrence of inflammasome activation in patient samples ex vivo. In this report, we provide detailed information about the optimal conditions to reliably identify inflammasome activated monocytes by ASC speck formation using a modified flow cytometric method introduced by Sester et al. in 2015. Since no protocol for optimal sample processing exists, we tested human blood samples for various conditions including anticoagulant, time and temperature, the effect of one freeze-thaw cycle for PBMC storage, and the fast generation of a positive control. We believe that this flow cytometric protocol will help researchers to perform high quality translational research in multicenter studies, and therefore provide a basis for investigating the role of the inflammasome in the pathogenesis of various diseases.

Viral Glycoproteins Induce NLRP3 Inflammasome Activation and Pyroptosis in Macrophages.

Infections with viral pathogens are widespread and can cause a variety of different diseases. In-depth knowledge about viral triggers initiating an immune response is necessary to decipher viral pathogenesis. Inflammasomes, as part of the innate immune system, can be activated by viral pathogens. However, viral structural components responsible for inflammasome activation remain largely unknown. Here we analyzed glycoproteins derived from SARS-CoV-1/2, HCMV and HCV, required for viral entry and fusion, as potential triggers of NLRP3 inflammasome activation and pyroptosis in THP-1 macrophages. All tested glycoproteins were able to potently induce NLRP3 inflammasome activation, indicated by ASC-SPECK formation and secretion of cleaved IL-1ß. Lytic cell death via gasdermin D (GSDMD), pore formation, and pyroptosis are required for IL-1ß release. As a hallmark of pyroptosis, we were able to detect cleavage of GSDMD and, correspondingly, cell death in THP-1 macrophages. CRISPR-Cas9 knockout of NLRP3 and GSDMD in THP-1 macrophages confirmed and strongly support the evidence that viral glycoproteins can act as innate immunity triggers. With our study, we decipher key mechanisms of viral pathogenesis by showing that viral glycoproteins potently induce innate immune responses. These insights could be beneficial in vaccine development and provide new impulses for the investigation of vaccine-induced innate immunity.

miRNA-223 as a regulator of inflammation and NLRP3 inflammasome, the main fragments in the puzzle of immunopathogenesis of different inflammatory diseases and COVID-19.

Millions of people around the world are involved with COVID-19 due to infection with SARS-CoV-2. Virological features of SARS-CoV-2, including its genomic sequence, have been identified but the mechanisms governing COVID-19 immunopathogenesis have remained uncertain. miR-223 is a hematopoietic cell-derived miRNA that is implicated in regulating monocyte-macrophage differentiation, neutrophil recruitment, and pro-inflammatory responses. The miR-223 controls inflammation by targeting a variety of factors, including TRAF6, IKKα, HSP-70, FOXO1, TLR4, PI3K/AKT, PARP-1, HDAC2, ITGB3, CXCL2, CCL3, IL-6, IFN-I, STMN1, IL-1ß, IL-18, Caspase-1, NF-κB, and NLRP3. The key role of miR-223 in regulating the inflammatory process and its antioxidant and antiviral role can suggest this miRNA as a potential regulatory factor in the process of COVID-19 immunopathogenesis.

The microglial NLRP3 inflammasome is involved in human SARS-CoV-2 cerebral pathogenicity: A report of three post-mortem cases.

We herein report, by using confocal immunofluorescence, the colocalization of the SARS-CoV-2 nucleocapsid within neurons, astrocytes, oligodendrocytes and microglia in three deceased COVID-19 cases, of between 78 and 85 years of age at death. The viral nucleocapsid was detected together with its ACE2 cell entry receptor, as well as the NLRP3 inflammasome in cerebral cortical tissues. It is noteworthy that NLRP3 was colocalized with CD68 + macrophages in the brain and lung of the deceased, suggesting the critical role of this type of inflammasome in SARS-CoV-2 lesions of the nervous system/lungs and supporting its potential role as a therapeutic target.

Inflammasome Activation in Children With Kawasaki Disease and Multisystem Inflammatory Syndrome.

[Figure: see text].

Scientific premise for the involvement of neutrophil extracellular traps (NETs) in vaccine-induced thrombotic thrombocytopenia (VITT).

Following on from the devastating spread of COVID-19, a major global priority has been the production, procurement, and distribution of effective vaccines to ensure that the global pandemic reaches an end. However, concerns were raised about worrying side effects, particularly the occurrence of thrombosis and thrombocytopenia after administration of the Oxford/AstraZeneca and Johnson & Johnson's Janssen COVID-19 vaccine, in a phenomenon being termed vaccine-induced thrombotic thrombocytopenia (VITT). Similar to heparin-induced thrombocytopenia (HIT), this condition has been associated with the development of anti-platelet factor 4 antibodies, purportedly leading to neutrophil-platelet aggregate formation. Although thrombosis has also been a common association with COVID-19, the precise molecular mechanisms governing its occurrence are yet to be established. Recently, increasing evidence highlights the NLRP3 (NOD-like, leucine-rich repeat domains, and pyrin domain-containing protein) inflammasome complex along with IL-1ß and effete neutrophils producing neutrophil extracellular traps (NETs) through NETosis. Herein, we propose and discuss that perhaps the incidence of VITT may be due to inflammatory reactions mediated via IL-1ß/NLRP3 inflammasome activation and consequent overproduction of NETs, where similar autoimmune mechanisms are observed in HIT. We also discuss avenues by which such modalities could be treated to prevent the occurrence of adverse events and ensure vaccine rollouts remain safe and on target to end the current pandemic.

Comparative Computational Modeling of the Bat and Human Immune Response to Viral Infection with the Comparative Biology Immune Agent Based Model.

Given the impact of pandemics due to viruses of bat origin, there is increasing interest in comparative investigation into the differences between bat and human immune responses. The practice of comparative biology can be enhanced by computational methods used for dynamic knowledge representation to visualize and interrogate the putative differences between the two systems. We present an agent based model that encompasses and bridges differences between bat and human responses to viral infection: the comparative biology immune agent based model, or CBIABM. The CBIABM examines differences in innate immune mechanisms between bats and humans, specifically regarding inflammasome activity and type 1 interferon dynamics, in terms of tolerance to viral infection. Simulation experiments with the CBIABM demonstrate the efficacy of bat-related features in conferring viral tolerance and also suggest a crucial role for endothelial inflammasome activity as a mechanism for bat systemic viral tolerance and affecting the severity of disease in human viral infections. We hope that this initial study will inspire additional comparative modeling projects to link, compare, and contrast immunological functions shared across different species, and in so doing, provide insight and aid in preparation for future viral pandemics of zoonotic origin.

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.