NLRP3 inflammasome activation and SARS-CoV-2-mediated hyperinflammation, cytokine storm and neurological syndromes

Abstract

Despite the introduction of vaccines and drugs for SARS-CoV-2, the COVID-19 pandemic continues to spread throughout the world. In severe COVID-19 patients, elevated levels of proinflammatory cytokines have been detected in the blood, lung cells, and bronchoalveolar lavage, which is referred to as a cytokine storm, a consequence of overactivation of the NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome and resultant excessive cytokine production. The hyperinflammatory response and cytokine storm cause multiorgan impairment including the central nervous system, in addition to a detriment to the respiratory system. Hyperactive NLRP3 inflammasome, due to dysregulated immune response, is the primary cause of COVID-19 severity. The severity could be enhanced due to viral evolution leading to the emergence of mutated variants of concern, such as delta and omicron. In this review, we elaborate on the inflammatory responses associated with the NLRP3 inflammasome activation in COVID-19 pathogenesis, the mechanisms for the NLRP3 inflammasome activation and pathway involved, cytokine storm, and neurological complications as long-term consequences of SARS-CoV-2 infection. Also discussed is the therapeutic potential of NLRP3 inflammasome inhibitors for the treatment of COVID-19.

Introduction

Emerging and re-emerging pathogens are a massive threat to the world population and the primary global concern of the current public health [1,2]. The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in Wuhan, China [3]. SARS-CoV-2 is the etiologic agent of coronavirus disease 2019 (COVID-19), the culprit behind the current pandemic [4,5]. This viral pathogen causes flu-like syndrome with mild symptoms in majority of infected individuals [6-8]. However, a subset of infected patients (~15% in the early phase of the COVID-19 pandemic) suffer from severe illnesses that require hospitalization and ventilation support [9,10]. SARS-CoV-2 has astounding infectivity due to global air connectivity, culminating in the worst pandemic ever [11-14]. According to the world health organization (WHO), as of February 23, 2022, there are more than 426 million confirmed cases and over 5 million deaths (https://covid19.who.int/). To treat COVID-19, drug repurposing for expeditious drug development against SARS-CoV-2 is unprecedented [15,16]. Meanwhile, work-speed vaccine pipelines and the application of modern biotechnology have yielded ever quicker vaccines [17,18]. Therefore, vaccination represents one of the most promising counter-pandemic measures to the COVID-19 [19]. However, COVID-19 remains massive and the need for effective therapies urgent, in part due to the emergence of mutated variants of concern (VOCs) [20-22].

Phylogenetically, SARS-CoV-2 is a member of the genus β-coronavirus, which includes 2003 SARS-CoV and 2012 the Middle East Respiratory Syndrome coronavirus (MERS-CoV) [23-25]. SARS-CoV-2 genome sequence is 80% similar to SARS-CoV [5,26,27]. Structurally, SARS-CoV-2 resembles other coronaviruses (CoVs), is spherical with ~100 nm in diameter, and has a single-stranded positive-sense RNA (ssRNA) [28,29]. It encodes 4-structural proteins: membrane glycoprotein (M), spike glycoprotein (S), envelope glycoprotein (E), and nucleocapsid (N) [30]. The N-protein conjugating with genomic RNA forms nucleocapsid, and to enclose nucleocapsid viral envelope assembled by three protein components S, M, and E [29]. The SARS-CoV-2 S-protein binds to the receptors: angiotensin-converting enzyme 2 (ACE2), and the viral entry is facilitated by transmembrane protein serine protease 2 (TMPRSS2) [31-35]. Identification of polybasic cleavage site in the S-protein and its enhanced affinity for ACE2 [26,36,37] may impart in greater transmissibility and increased virulence of SARS-CoV-2 [38,39]. The virus infects cells expressing ACE2, including monocytes, macrophages, alveolar cells, intestinal epithelial cells, endothelial cells, kidney cells, neurons, neuroepithelial cells and glial cells [36,40-42].

In the lungs of SARS-CoV-2 infected patients, an elevated level of cytokine release has been reported. The elevated levels of cytokines trigger an aberrant uncontrolled response known as cytokine storm, also referred to as cytokine release syndrome (CRS) [43-45]. The accumulating cytokines empower SARS-CoV-2 invasion by attracting immune cells, engaging aggressive inflammatory responses, and severe respiratory complications like acute respiratory distress syndrome (ARDS) [46-49]. The ARDS and related acute lung injury (ALI) occur due to the storm of inflammatory cytokines, notably interleukin-1β (IL-1β), IL-6, and tumor necrosis factor (TNF)-α [50]. Although the mechanisms underlying cytokine storm are multifaceted, accumulating evidence indicates that host cells necessitate inflammasome activation to produce inflammatory cytokines leading to the storm [45,51]. Inflammasomes are multiprotein cytosolic platforms with a tendency to aggregate in response to pathogen-induced or host-mediated assaults [52]. The NLR family pyrin domain-containing protein 3 (NLRP3) is a well-characterized inflammasome and initiates an inflammatory response by inducing excessive cytokine production against viral infection [53]. An involvement of NLRP3 inflammasome in SARS-CoV-2-associated intracellular signaling is shown in Figure 1.

Figure 1.

Activation of NLRP3 inflammasome by SARS-CoV-2. SARS-CoV-2 infection occurs by binding of Spike glycoprotein with cell surface receptor ACE2 leading to viral internalization, followed by the release of nucleic acid, viral replication and synthesis of viral proteins. Infection of SARS-CoV-2 upregulates the NFkB pathway leading to increased expression and synthesis of NLRP3 and IL-1β. Viral proteins including S, N, E and viroporins interact with NLRP3 and facilitate inflammasome assembly via oligomerization, the interaction of NLRP3 with ASC and cleavage of caspas1 leading to maturation and release of IL-18 and IL-1β. Ion channels and ion flux are also involved in NLRP3 inflammasome activation. Mitochondrial ROS and lysosomal degradation further impart NLRP3 inflammasome activation.

In addition to its attack on the respiratory system, SARS-CoV-2 was found to induce neurological syndromes including dizziness, hypersomnia, hypogeusia, headaches, myalgia, ataxia, seizures, and impaired consciousness in a high proportion of infected patients [54-56]. Indeed, COVID-19 patients exhibit diverse neurological symptoms that are similar to other respiratory viral infections [57-60]. These neurological symptoms in SARS-CoV-2-infected patients are disabling and quite frequent in their occurrence [61,62]. The neurodegenerative changes, brain edema, and even encephalitis were observed in the severe COVID-19 patients [33,63-65] and some of them were found positive for SARS-CoV-2 in the cerebrospinal fluid (CSF) and brain tissues [66-68]. The presence of SARS-CoV-2 in the CSF and brain tissues in postmortem cases demonstrated that this virus is not only restricted to the respiratory system but can enter the central nervous systems (CNS) inducing neurological manifestations [69]. The consequences of SARS-CoV-2 CNS infection are discussed in section titled NLRP3 inflammasome activation and COVID19-assoociated neurological symptoms and illustrated in Figure 2.

Figure 2.

COVID-19-associated neurological consequences. The graphical representation of impacts of neurological outcomes in COVID-19 patients. These neurological syndromes are diverse in nature including dizziness, headache, loss of sense of taste and smell, brain fog and various inflammatory brain diseases.

Evidently, hyperinflammation and cytokine storm is the key pathophysiological processes leading to COVID-19 severity. This review tentatively covers the current progress on SARS-CoV-2-induced NLRP3 inflammasome activation, hyperinflammation, cytokine storm, and neurological consequences. We scrutinize the COVID-19-mediated neurological symptoms associated with NLRP3 inflammasome activation in microglia, astrocytes, and other CNS cells. Also discussed are the mechanistic pathways, checkpoints of inflammasome activation, cytokine storm, and pyroptosis, which could be the potential targets for intervening/ameliorating the severity of the COVID-19 pandemic.

Inflammasome complexes

The induction of the inflammatory process in cells is often mediated by inflammasomes which are multiprotein cytosolic platforms of the innate immune defense system [52]. Inflammasomes tend to aggregate in response to various microbe-associated and host-generated assaults and orchestrate the development of local and systemic inflammation [52,70]. These harmful threats are detected by components of the host innate immune system, the pattern-recognition receptors (PRRs). To trigger inflammatory pathways for the removal of microbial infection and repairment of tissue damage PRRs recognize pathogen-associated molecular patterns (PAMPs) or endogenous stress generated damage-associated molecular patterns (DAMPs). Inflammasomes are defined by their sensor proteins (PRRs) and get oligomerized in response to PAMPs and DAMPs to activate caspase-1. There are five confirmed members of inflammasomes: nucleotide-binding oligomerization domain (NOD), leucine-rich repeat (LRR)-containing proteins (NLR) family representative NLRP1, NLRP3, NLRC4, absent-in-melanoma 2 (AIM2) and pyrin [71,72]. Additionally, other members of PRRs known for forming inflammasomes are NLRP2, NLRP6, NLRP7, NLRP12 and IFI16 [73-77]. Among various inflammasomes, the NLRP3 inflammasome is a well-characterized and most studied molecular platform that responds to RNA viruses and gets activated against microbial infection (PAMPs) and host cell damage or aggregates (DAMPs). NLRP3 inflammasome is composed of the NLRP3 receptor, the adaptor molecule apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1. NLRP3 is a tripartite protein having an amino-terminal pyrin domain (PYD), a central NOD or NACHT, and an LRR domain [78]. To initiate inflammasome assembly PYD of NLRP3 interacts with PYD of ASC [79]. The NOD of NLRP3 possesses ATPase activity and is crucial for oligomerization of NLRP3 following the activation [80]. A commonly used NLRP3 inhibitor MCC950 targets ATPase activity of NOD [81,82]. Following NLRP3 inflammasome assembly, caspase-1 becomes activated by autoproteolytic cleavage and promotes maturation of proinflammatory cytokines, IL-1β, IL-18, IL-6, TNFα and gasdermin-D (GSDMD), a pore-forming protein that incites pyroptosis (a type of inflammatory programmed cell death) [52,83]. The hallmark of the active inflammasome is the presence of spec (puncta), these puncta or spec are micron-size structures formed by polymerization of ASC as a consequence of the activation by PAMPs, and DAMPs [84,85].

Inflammasome activation and regulation

Inflammasomes are key signaling platforms responsible for detecting pathogenic microorganisms and sterile stressors resulting in the activation and secretion of proinflammatory cytokines. The mechanisms of activation and regulation of inflammasomes are complex and ensure an effective but balanced inflammasome-mediated immune response. Owing to the well-characterized and most studied inflammasome NLRP3, we will further contemplate activation and regulation mechanism using this. Despite the controversy, a two-step signaling model for the activation of NLRP3 inflammasome is widely accepted, consisting of primming (first signal) and activation steps (second signal) [86-88]. Primming signals are generally ligands for toll-like receptors (TLRs), NLRs or cytokine receptors which in turn activate the NF-kB. Primming signals regulate the NLRP3 inflammasome by upregulating transcription of key components (NLRP3 and IL-1β), as a basal expression of these components is insufficient for activation of inflammasome in resting cells [78,89]. In response to TLR ligands, NF-kB induces signaling components MyD88 and TRIF, which regulate the induction of NLRP3 as well as pro-IL-1β [89]. However, the primming signal does not alter the expression profile of ACS, pro-caspase-1, and pro-IL-18 [78]. Further, it was reported that apoptotic signaling molecules caspase-8 and FADD prime NLRP3 induction [90,91]. Interaction of IKK complex with caspase-8 promotes NFkB transcription and translocation for downstream signaling [92]. In contrast, the transcription-independent role of priming was reported using rapid priming with lipopolysaccharide (LPS), where NLRP3 activation occurs in the absence of its induction [93]. This rapid transcription-independent priming is believed to be mediated by a signaling molecule downstream of TLRs and MyD88 known as IL-1 receptor-associated kinase 1 (IRAK-1), [94]. Phosphorylation of IRAK-1 induced by LPS primes activation of inflammasome in IKK complex independent manner [95]. Nevertheless, beyond the transcriptional regulation, the primming step does more to license NLRP3 inflammasome activation.

Following the primming step, activation of NLRP3 inflammasome can be induced by a plethora of stimuli which includes, ATP, K⁺ ionophores [96], particulate matter [97,98], heme [99], pathogen-associated RNA [100,101], microbial toxins and integrant [102-104]. These biochemically diverse agonists induce multiple cellular and molecular signals including ionic flux, dysfunction of mitochondria, generation of reactive oxygen species (ROS), and lysosomal damage for the NLRP3 inflammasome activation. Activated inflammasome plays a critical role in host defense in anticipation of infectious agents mounting immune responses. Stringent inflammasome activation regulation is required as dysregulated NLRP3 inflammasome has been involved in the pathogenesis of numerous inflammatory diseases. Precise regulation of activation of NLRP3 inflammasome is critical for adequate immune protection to the host by subverting the tissue damage. The mechanisms for the NLRP3 inflammasome regulation include post-translational alterations of NLRP3 as well as its interacting partners.

NLRP3 inflammasome in COVID-19

The involvement of excessive inflammation and resultant cytokine storm owing to uncontrolled release of cytokines is accountable for the unfavorable clinical outcomes of the COVID-19 [105,106]. The mechanism of inflammasome activation in SARS-CoV-2 is poorly explored, despite confirmed participation of NLRP3 inflammasome in SARS-Co-V and MERS-CoV [107,108]. However, the engagement of pyroptosis and cytokine storm, where inflammasome-associated products such as IL-1β, IL-18, and LDH were detected in COVID-19 patient sera, suggests the association of inflammasome in the COVID-19 [105,106,109-111]. Further, as an inflammasome-independent pathway can also produce inflammatory substances, it demands a definitive confirmation of the involvement of inflammasome in the SARS-CoV-2 infection [112-114]. In an elegant article by Rodrigue and coworkers [85] on COVID-19, the involvement of NLRP3 inflammasome was confirmed in patients with moderate to severe infection of SARS-CoV-2. Other contemporary studies have shown the involvement of SARS-CoV-2-induced activation of NLRP3 inflammasome in the COVID-19 [45,115-119].

COVID-19 induced NLRP3 inflammasome activation and cytokine storm

The immune system defends our body against invaders such as SARS-CoV-2. This defense system includes 2-arms, the innate and adaptive immune systems. The innate-immunity-mediated antiviral responses are triggered by recognizing PAMPs, leading to the induction of adaptive immunity which is critical to protecting the host [120,121]. The host cells in response to viral infection release cytokines, chemokines, leukotrienes, proteases, and ROS to enable viral clearance [122]. A stringent equilibrium between antagonistic signals and cellular response maintains the immune response evoked by pathogens and is responsible for preventing host tissue damage, by preventing continuous activation of the immune system [122]. Usually, acute viral infections evoke systemic inflammatory responses leading to excessive synthesis and release of proinflammatory cytokines as a defense measure [122]. This systemic inflammatory response triggered by infections and drugs causes cytokine storm or CRS. Previously, influenza viral infection causing respiratory illness has been characterized as a stimulus for CRS [123]. Influenza patients undergo robust cytokine-mediated responses which are associated with fever, hypoxemia and hypotension [10,123]. The syndrome may be mild or can develop into persistent high-grade fevers, vasodilatory shock with hemodynamic instability demanding mechanical ventilation [10,123]. Similarly, the existence of elevated inflammatory cytokine levels in COVID-19 patients leading to CRS or cytokine storm culminate in lung collapse, respiratory failure and may lead to multiorgan failures [105]. Enhanced induction in IL-1β, TNFα, IL-6, IL-18, IL-10, IL-1RA, and C-X-C motif chemokine ligand 10 (CXCL10) was reported in severe COVID-19 patients exhibiting cytokine storm phenotype [106,124]. These excessively released cytokines may produce eosinopenia (low count of eosinophil) and lymphocytopenia (low count of CD4⁺T, CD8⁺T, and NK cells) and can induce naïve B-cell activation, Th17 differentiation, neutrophil recruitment and monocyte stimulation [125-128]. Recently, it was demonstrated that SARS-CoV-2 infection could lead to NLRP3 overactivation resulting in cytokine storms in the hematopoietic stem cells [116]. The NLRP3 is one of the most critical innate immune components that accelerate inflammation by releasing IL-1β, IL-18 and provokes pyroptosis. There have been reports of a positive correlation of IL-18 and caspase-1 with other inflammatory markers, including C-reactive protein (CRP), LDH, and IL-6 in the activation of inflammasome in COVID-19 patients [85]. Further investigation to find out the cytokine storm in other organs and tissues causing the disease severity in COVID-19 is imperative. Thus, to control cytokine storm in SARS-CoV-2 infected patient NLRP3 inflammasome inhibitors can be harnessed alongside other inflammatory inhibitors.

NLRP3 inflammasome activation and COVID 19-associated neurological symptoms

The activation of NLRP3 inflammasome and associated pathophysiology of various neurological disorders have been reported in numerous neurological disorders, such as in Alzheimer’s disease (AD) [129-133], Parkinson’s disease (PD) [134-136], multiple sclerosis (MS) [137,138], and traumatic brain injury [139-141]. Viral infection in the brain (neuroinvasion) leads to inflammatory response resulting in neuroinflammation [142]. At the beginning of the COVID-19 pandemic, a general belief was that SARS-CoV-2 infection solely affects the respiratory system of humans [143], but the appearance of newer symptoms such as olfactory and taste indicates an involvement of the CNS [144]. There is evidence that SARS-CoV-2 can infect neuronal cells, the BBB endothelial cells, microglia, and astrocyte via ACE2 and another receptor known as cluster differentiation 147 (CD147) [145,146] and cause a neurological deficit in a substantial proportion of COVID-19 patients [147,148]. Figure 2 is the graphical representation of COVID-19 induced neurological syndromes.

In the CNS, the critical player of neuroinflammation is microglial cells, a major source of proinflammatory cytokines [149]. As resident macrophages in the brain, microglia are accountable for phagocytosis and removal of pathogens and neurotoxic agents [150]. They are also the target cells in SARS-CoV-2 CNS infection. Studies using postmortem samples of COVID-19 patients have demonstrated that SARS-CoV-2 was present in the brain compartments [151,152]. SARS-CoV-2 infection to the CNS was further confirmed by the observation that this virus can infect neural progenitors and brain organoids [153]. The entry of SARS-CoV-2 into the brain may take a similar approach as other viruses to invade brain tissues, the hematogenous route, the retrograde route [154], or the direct access to the brain via the olfactory canal [105,155]. Following entry, the virus and viral proteins can cause direct neural cell injury or promote microglial pro-inflammatory responses causing an indirect neural injury [156]. Inflammasome activation through SARS-CoV-2 infection or entrance of viral proteins into the brain and other CNS compartments was reported. [157,158]. It has been shown that incubation of spike glycoprotein S1 of SARS-CoV-2 with BV2 microglial cells elevated levels of NLRP3, IL-1β, TNFα, IL-6, nitric oxide (NO) and enhanced activity of NFkB and caspase-1 [157]. The S1 protein also triggered the production of interferon-beta (IFNβ), TNFα, and NFkB in the human microglia [159].

Extensive activation of cascades of neuroinflammation is provoked by excessive release of cytokines and chemokines, which drives neuronal hyperexcitability through activation of glutamate receptors accompanied by induction of seizures [160,161]. COVID-19 patients may give rise to inflammatory injury and brain edema owing to the immune system over-exuberance response may lead to defective consciousness [162]. Such immunologic response resulting in hyperinflammation may further amplify the cytokine storm [163]. It was presumed that intracranial cytokine storm further potentiates the breakdown of BBB and the consequent leukocytes migration. COVID-19-induced defects in gustation and olfaction could be a result of olfactory bulb injury due to, nasal cavity inflammation. Thus, the binding of odorants to the olfactory receptors is blocked due to nasal inflammation resulting in dysfunction in the olfactory response [164]. Additionally, loss of taste (ageusia) occurs due to dysfunction in the taste buds [165]. In COVID-19 patients, it usually takes a longer time for the regeneration and recovery of damaged neurons in the olfactory lobes [164]. Depending on the extent of viral insult, the loss of smell and taste may persist for months to years [164]. It is widely recognized that senior individuals with compromised health conditions are more vulnerable to COVID-19-associated fatal and long-term consequences. The profile of NLRP3 inflammasome involvement in neurological consequences induced by COVID-19 is an unmet need for research. Considering age-related neurological deficits, COVID-19 brain infection could further exacerbate neurological symptoms in senior patients.

Mechanisms of NLRP3 inflammasome activation in COVID-19

Inflammasomes are large cytosolic multiprotein oligomers of the innate immune system which assemble in response to PAMPs and DAMPs leading to proinflammatory cytokine release and lytic cell death termed pyroptosis [52,53]. The NLRP3 is a well-characterized sensor of the NLR family, detects a broad range of microbial motifs in addition to environmental irritants and endogenous danger signals. The exact molecular mechanism for activation of NLRP3 inflammasome is largely unknown. It occurs in a two-step process: primming, the first signal and subsequent activation is the second signal. For SARS-CoV-2 primming triggers are not well established, however, a recent study has shown that spike glycoprotein can initiate inflammasome activation [166]. Generally, NLRP3 inflammasome responds to an agglomeration of affront to the cells, which causes Ca²⁺ influx, K⁺ efflux, ROS production by mitochondria, mitochondrial dysfunction, and lysosomal rupture [52,167-169]. In addition, the NLRP3 inflammasome can be activated by pore-forming toxins, extracellular ATP and large extracellular aggregates including cholesterol and uric acid crystals, and amyloid [53]. Viral infection evokes the NLRP3 inflammasome activation, which is also admissible with coronaviruses [170,171].

Previous studies using SARS-CoV have demonstrated that activation of the inflammasome occurs by E and ORF3a proteins via altering the permeability of K⁺ ions across the plasma membrane and ROS production by mitochondria [50,172]. In a recent study, inflammasome-derived products including Casp1p20, IL-1β, IL-6 and IL-18 were also detected at higher levels in SARS-CoV-2 infected patients and were correlated with the severity of disease [85,117]. It is conventionally agreed that activation of NLRP3 occurs in viral infection by assembling NLRP3 with ASC and recruiting caspase-1, resulting in proinflammatory cytokine production and GSDMD-mediated pyroptosis [45,85,170,173]. Further, SARS-CoV-2 N-protein was shown to directly interact with the NLRP3 and promote the ASC assembly. This sequel of N-protein and NLRP3 interaction enhances proteolytic activity of caspase-1 and enhanced release of IL-1β and IL-6, resulting in hyperinflammation and cytokine storm in the lungs [118]. Using the NLRP3 double knockout mice, Pan and colleagues have demonstrated that the lung NLRP3 inflammasomes activation was the cause of ARDS and resulting fetal death [118].

Other contemporary studies have revealed that SARS-CoV-2 S, E, ORF3a and ORF8 can induce inflammasome activation and hyperinflammation, leading to severe clinical outcomes [45,119,174-176]. Nevertheless, investigations on the mechanisms of inflammasome activation induced by SARS-CoV-2 could be of immense interest in the battle against the pandemic of COVID-19. In the following section, we report current advances in NLRP3 inflammasome activation induced by proteins of SARS-CoV-2.

SARS-CoV-2 proteins-mediated NLRP3 activation

In addition to SARS-CoV-2 infection-associated activation of the NLRP3 inflammasome, SARS-CoV-2 proteins were also found to induce NLRP3 activation. Among them are S, N, E, Viroprin (ORF3a), and ORF8 proteins [45,118,119,157,174-176]. How these proteins cause NLRP3 inflammasome activation and resultant biological consequences are discussed tentatively in the following subsections.

SARS-CoV-2 Spike (S)

The SARS-CoV-2 S-protein is a glycoprotein and a crucial player in infectivity as it binds to host cell receptors ACE2, which facilitates virus entry into the cells [177,178]. It has been consistently, demonstrated that the SARS-CoV-2 S-protein interacts with ACE2 and causes the release of various cytokines including IL-1β, IL-6, IL-8, and IL-18 via the NLRP3 inflammasome-mediated activation of caspase-1 [171,174,179]. The recombinant nucleic acid-based and subunit vaccines are against this antigenic protein [17,18,180]. The S-protein can trigger NLRP3 inflammasome activation, resulting in hyperinflammation and related cytokine storms [157,166,174]. It has been reported that incubation of recombinant spike glycoprotein S1 of SARS-CoV-2 with peripheral blood mononuclear cells (PBMCs) of humans evoked excessive cytokine production, which was abolished by dexamethasone [174]. The spike glycoprotein S1 evoked cytokine release through mechanisms involving activation of NFkB, p38MAPK, and NLRP3 inflammasome as demonstrated by harnessing different specific inhibitors [174]. Further studies on BV2 microglial cells demonstrated that spike glycoprotein S1 activated the NLRP3 inflammasome resulting in enhancement of pro-inflammatory cytokine production and nitric oxide [157]. These results indicate that viral spike glycoprotein S1, perhaps other viral proteins as well, plays a crucial role in the activation of the NLRP3 inflammasome. This exemplifies the involvement of NLRP3 inflammasome in alveolar cells in COVID-19. Using primary rat microglial cells and spike glycoprotein S1 of SARS-CoV-2, we observed activation of the NLRP3 inflammasome by measuring enhanced production of IL-1β and other cytokines, as well as the expression of NO, and iNOS (Dutta and Xiong, unpublished data). Our results further demonstrated the neuroinflammatory properties of spike glycoprotein S1 via activation of the NLRP3 inflammasome. The spike glycoprotein S1-mediated inflammasome activation in rat microglial cultures was blocked by an NLRP3 specific inhibitor MCC950 (Dutta and Xiong, unpublished data). In addition, the S-protein of SARS-CoV-2 was found to damage hematopoietic stem/progenitor cells through pyroptosis mediated by NLRP3 inflammasome-dependent mechanism [115]. Nevertheless, these experimental results, including ours, demonstrated that SARS-CoV-2 spike glycoprotein induces NLRP3 inflammasome activation.

SARS-CoV-2 nucleocapsid

It was shown that SARS-CoV-2 nucleocapsid (N-protein) interacts with NLRP3 and resulted in NLRP3 inflammasome activation in both cell cultures in vitro and a mouse model of viral infection in vivo [118]. The study demonstrated that N-protein interacted in a dose-dependent manner with NLRP3 and promoted the IL-1β maturation and release. Application of a specific NLRP3 inflammasome inhibitor MCC950 and an inhibitor for Caspase-1 Ac-YVAD-cmk blocked the NLRP3 inflammasome activation [118]. This is a classic finding with an elucidation of a mechanistic view of NLRP3 inflammasome activation by N-protein, where N-protein not only interacted with NLRP3 but also facilitated its assembly with ASC, a process of polymerization and activation. This was detected by increased spec or puncta formation after N-protein treatment. This study further identified the exact domain of N-protein (CTD, 260-340aa), which interacts with the NLRP3 inflammasome using deletion mutation constructs and Co-IP. Deletion of this particular domain of N-protein resulted in a significant reduction in p17, p20, and ASC oligomerization, demonstrating that the physical interaction between NLRP3 and N-protein was indispensable for the activation of the NLRP3 inflammasome [118]. Hence, treatments targeting N-protein might suppress cytokine storms and reduce lung injury and complications in other organs mediated by SARS-CoV-2-associated NLRP3 overactivation.

The N-protein of SARS-CoV-2 possesses dual actions on innate immune responses. At lower doses, it suppresses type I interferon (IFN-I) signaling as well as inflammatory cytokine expression. In contrast, it promotes IFN-I signaling and expression of inflammatory cytokine at higher doses [181]. Such dual functions were also observed in regulating the phosphorylation status of IRF3, STAT1, and STAT2 and their nuclear translocation [181]. N-protein combined with TRIM25-protein was found to suppress the ubiquitination as well as activation of the retinoic acid-inducible gene (RIG-I) [181]. In addition, N-protein bounds to GSDMD linker region and hindered GSDMD cleavage by caspase-1 [182]. These findings indicate that the N-protein of SARS-CoV-2 plays a pivotal role in triggering inflammatory responses in COVID-19.

SARS-CoV-2 envelope

The envelope protein (E-protein) of SARS-CoV-2 is a small structural protein and plays a crucial role in the activation of the NLRP3 inflammasome [176]. The E-protein was shown to have dual roles in the modulation of NLRP3 inflammasome in human and murine macrophages. It suppressed activation of NLRP3 inflammasome in the stage of early infection but activated NLRP3 inflammasome at the advanced stage of the infection [176]. The mechanisms underlying its dual effects on NLRP3 inflammasome are not well characterized at present and further investigations are definitely desirable.

SARS-CoV-2 ORF3a (viroporin)

Open reading frame 3a (ORF3a) is an accessory protein conserved in both SARS-CoV and SARS-CoV-2 [183,184]. It is a viroporin, a transmembrane protein, that works as an ion channel and helps in the viral release [185,186]. In SARS-CoV, ORF3a protein activates NLRP3 inflammasomes leading to dysregulated immune response and elevated levels of proinflammatory cytokines [48,187]. It was shown that SARS-CoV ORF3a protein can activate the NLRP3 inflammasome in lipopolysaccharide primed macrophages [172]. Owing to conservation in ORF3a protein in SARS-CoV and SARS-CoV-2 [183,184], the role of ORF3a in NLRP3 inflammasome activation is apparent. A recent study has shown that ectopically expressed SARS-CoV-2 ORF3a activated the NLRP3 inflammasome by triggering IL-1β expression via the NFkB pathway [175]. Furthermore, the study revealed that this viroporin primed and activated inflammasome through both ASC-dependent and independent pathways. It was also shown that ORF3a-mediated activation of inflammasome was mediated via potassium ion efflux and by NEK7 and NLRP3 oligomerization. Application of MCC950 blocked ORF3a-mediated inflammasome activation.

NLRP3 inflammasome as a therapeutic target for COVID-19

Because of poor immune fitness, dysregulated NLRP3 inflammasome and resultant hyperinflammation contribute to the COVID-19 severity [10,188]. Inhibitors targeting the pathways of NLRP3 inflammasome activation can be contemplated as a prospective therapy. The specific inhibitor against NLRP3, MCC950, may have therapeutic potential for patients at the early stage of the disease to prevent cytokine storms, ameliorate complications, and reduce fatal outcomes [118]. Other inhibitors, like Ac-YVAD-cmk which is a specific inhibitor for caspase-1, can also be considered for the treatment, either alone or in combination with MCC950. The therapeutic efficacies of these inhibitors need to be evaluated by in vitro and in vivo models of disease and ultimately in clinical trials. Indeed, in the treatment of cardiovascular disease, the use of NLRP3 inhibitors has been reviewed in the literature [189], paving the path for using NLRP3 inhibitors in COVID-19. It is worth noting that inflammasome and pyroptosis are proposed as potential therapeutic targets for the COVID-19 treatment [190]. Thus, inhibitors targeting critical components of inflammasome activation, cellular pyroptosis, and downstream cytokine can be implicated in the treatment of COVID-19 [43]. Indeed, it has been shown that targeting NLRP3 inflammasome could be a promising immune intervention against the severe COVID-19 [191]. Thus, we tentatively discuss a few NLRP3 inhibitors, both specific (selectively blocks the NLRP3 inflammasome) and non-specific (indirect inhibition of NLRP3-mediated signaling), on their therapeutic potential for COVID-19. Treatment with these inhibitors can block caspase-1 activity, resulting in an inhibition of IL-1β and other cytokine production, and consequent reduction in inflammation (Figure 3).

Figure 3.

Inhibition of NLRP3 inflammasome may have therapeutic potential for COVID-19 and related neurological syndromes. Graphical representation of inflammasome inhibition by various inhibitors. The NLRP3 inflammasome is a prospective target for therapeutic approaches in the treatment of COVID-19. Inhibitors against NLRP3 inflammasome may ameliorate SARS-CoV-2-induced hyperinflammation and related cytokine storms. These inhibitors, in addition to their beneficial effects in lung and other organs, could also have therapeutic potential for COVID-19 neurological syndromes associated with microglial NLRP3 inflammasome activation.

MCC950 (CRID3 or CP-456,773)

MCC950 is a specific inhibitor for NLRP3 inflammasome with a small molecular weight. MCC950 blocks the ATPase activity by binding non-covalently near the Walker B motif of NLRP3 resulting in inhibition of NLRP3 [81,192-197]. Pigs and mice treated with MCC950 exhibited a decreased infiltration of neutrophils, reduced expression of myocardial IL-1β and diminished infarct size and cardiac dysfunction [198-200]. In lung ischemia-reperfusion (IR), the NLRP3 inflammasome was found overactive in the murine lung IR model [201]. Animals pretreated with MCC950 significantly alleviated IR-induced lung injury by restricting the proinflammatory cytokine release and inhibiting infiltration of neutrophils [201]. As neutrophil infiltration has been reported in severe COVID-19 patients along with cytokine storms [202], the use of MCC950 may attenuate neutrophil infiltrations and cytokine storms. In a recent study using monocytes from healthy donors, it was shown that MCC950 can inhibit SARS-CoV-2 infection-induced caspase-1 activation leading to IL-1β production [85]. Also, SARS-CoV-2 N-protein- and S-protein-induced NLRP3 inflammasome induction in lung cells, PBMCs, BV2 neural cells, and mice were inhibited when treated with MCC950 [118,157,174]. In addition to its treatment in lung injury and cardiovascular complications associated with NLRP3 inflammasome activation, MCC950 was used to treat neuroinflammation-related injury as well. A study on spinal cord injury mice model showed that MCC950 restricted the inflammatory response and improved neurological sequel [203]. Such therapeutic effects were achieved by blocking the assembly of the NLRP3 inflammasome, including NLRP3-ACS and NLRP3-caspase-1 complex formation, and inhibiting the release of cytokines TNFα, IL-18 and IL-1β [203]. Nevertheless, NLRP3 inflammasome inhibition by MCC950 has been applied for the treatment of multiple sclerosis (MS)-associated central neuropathic pain in patients with RR-MS [204]. Inspired by this study, MCC950 could be considered for treatment of neuroinflammatory disease conditions due to SARS-CoV-2 infection.

Glyburide

Glyburide is the first NLRP3 inhibitor that works in vitro but at a high dose [205]. It was shown that glyburide can inhibit NLRP3 inflammasome and lung tumorigenesis in mice [206], which implicates for the treatment of lung hyperinflammation in COVID-19 patients.

BAY11-7082

As a synthetic NFkB inhibitor, BAY11-7082 acts by alkylating the cysteine residue of the ATPase region of NLRP3 resulting in inhibition of the NF-kB pathway [207]. To explore its anti-inflammatory properties, BAY11-7-82 was tested in PBMCs treated with spike glycoprotein S1 of SARS-CoV-2. Treatment with S1 increased phosphorylation of NFkB p65, IkBα, and IkBα degradation, resulting in NFkB activation [174]. Such effects were blocked by either dexamethasone or BAY11-7082, leading to an inhibition of inflammasome activation [174]. In another study aiming to recapitulate neuroinflammation by SARS-CoV-2 in BV2 cells, it was shown that BAY11-7082 inhibited the SARS-CoV-2 S1-induced activation of NLRP3 inflammasome as assayed by a reduction in IL-1β, TNFα and IL-6 production [157]. These observations suggest that BAY11-7082 might have therapeutic potential for COVID-19 cytokine storm and neurological consequences associated with neuroinflammation.

OLT1177

OLT1177 is a specific small-molecule inhibitor for the NLRP3 functioning by blocking the ATPase activity [133,208,209]. It acts against mutants of NLRP3 in the cryopyrin-associated periodic syndrome patients [210] and may have therapeutic potential for COVID-19. In a phase 2A clinical trial, OLT1177 was found safe and effective in reducing targeted joint pain in gout patients [211]. As the compound is already in a clinical trial, testing its efficacy in COVID-19 patients may open another avenue for the treatment of COVID-19.

Colchicine

This is a tricyclic alkaloid compound that is currently in use to treat familial Mediterranean fever, gout and acute as well as chronic pericarditis [212,213]. Colchicine impedes NLRP3 and ASC interaction via disruption of microtubule and inhibits the inflammasome activation [214]. In a recent review, colchicine was suggested to repurpose for COVID-19 treatment owing to its anti-inflammatory and immunomodulatory nature [215]. Based on its current use in the CNS disease treatment, neuro-behcet’s syndrome, a severe chronic inflammatory vascular disease [216], colchicine may have the potential in treating COVID-19-induced neuroinflammation.

Ac-YVAD-cmk

This is an efficacious inhibitor of caspase-1 [217]. Caspase-1 plays a terminal effector role in inflammasome by converting proinflammatory pro-IL-18 and pro-IL-1β into respective mature forms by cleavage [218]. Inhibition of caspase 1 blocks inflammatory processes associated with inflammasome activation. Ac-YVAD-cmk possesses activity to effectively block activation of inflammasomes and display anti-inflammatory and anti-pyroptotic effects [219,220]. Recently, it was shown that SARS-CoV-2 N-protein mediated activation of NLRP3 inflammasome was inhibited using Ac-YVAD-cmk [118]. In a mouse model of depression, its inhibitory effect on caspase-1 was also demonstrated where immune activation and NLRP3-mediated depression were ameliorated by Ac-YVAD-cmk [220]. In view of its ability to selectively block IL-1β converting enzyme, Ac-YVAD-cmk could be considered as a potential treatment option for neuroinflammation and multiorgan damage in COVID-19 patients.

Anakinra

As an interleukin-1 type I receptor (IL-1RI) antagonist, anakinra is in use for the treatment against multiple autoinflammatory diseases including familial Mediterranean fever, MS, and rheumatoid arthritis [221-223]. These clinical disorders, which benefited from anakinra, share the same pathophysiological hallmarks of COVID-19, including macrophage activation syndrome (MAS), hemophagocytic lymphohystiocytosis, and septic shock [224-226], raising the hope for its application for COVID-19 patients. The use of anakinra was indeed found to have clinical benefits in saving the lives of hospitalized COVID-19 patients with moderate to severe manifestations [227].

Nevertheless, suitable NLRP3 inflammasome inhibitors with high efficacy and safety in clinical trials may be considered for immediate COVID-19 treatment emergency use authorization. While dexamethasone and other immunosuppressive drugs block type I IFN signaling, this may leave viral replication unchecked. The advantage of NLRP3 inflammasome inhibitors in COVID-19 leaves type I IFN signaling intact, which increases virus clearance by limiting the viral replication [228]. Also, it was shown that the NLRP3 inflammasome was repressed by type I IFN via reducing the pro-IL-1β level and its cleavage into mature IL-1β [229]. Thus, treatment of COVID-19 patients with inflammasome inhibitors will encounter dual effects: leaving type I IFN levels high promoting antiviral state and restraining the hyperinflammation induced by the NLRP3 inflammasome.

Summary

As SARS-CoV-2 is the etiologic agent for global COVID-19 pandemic fatalities, research into the underlying mechanisms is an unmet need. While specific molecular mechanisms influencing disease severity remain to be determined, a number of studies have demonstrated that activation of inflammasomes and inflammatory mediators including IL-1β, IL-18, IL-6 and LDH are intimately associated with the severity of COVID-19. While it is not restricted to infect the respiratory system, SARS-CoV-2 can attack other organ systems including the brain. COVID-19 gives rise to a group of disease manifestations, and one of the dreadful consequences is the massive inflammatory response mediated by the NLRP3 inflammasome. Inflammasome signaling on one hand provides defense against microbial invasion, and on the other hand, as a countermeasure, produces hyperinflammatory responses. The NLRP3 is one of the well-characterized inflammasomes which plays a salient role in hyperinflammatory responses in SARS-CoV and MERS-CoV as well as in SARS-CoV-2 infection. NLRP3 inflammasome activation induced by infection of SARS-CoV-2 not only causes severe respiratory complications but provokes neurological syndromes. This review has assembled current advances on the SARS-CoV-2-mediated activation of NLRP3 inflammasome that may help us better understand the disease in a broader way and find ways to contain the COVID-19 severity and death.

Disclosure of conflict of interest

None.