and Pyroptosis as Therapeutic Targets for COVID-19 Jeremy K. Y. Yap, Miyu Moriyama and Akiko Iwasaki This information is current as J Immunol published online 3 June 2020 of October 2, 2021. http://www.jimmunol.org/content/early/2020/06/02/jimmun ol.2000513 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 3, 2020, doi:10.4049/jimmunol.2000513

Inflammasomes and Pyroptosis as Therapeutic Targets for COVID-19 x Jeremy K. Y. Yap,* Miyu Moriyama,† and Akiko Iwasaki†,‡, The inflammatory response to severe acute respiratory TLR7, and TLR8 in the endosome as well as retinoic acid– syndrome–related coronavirus 2 infection has a direct inducible gene I (RIG-I)–like receptors in the cytosol (2). impact on the clinical outcomes of coronavirus disease Suggestion of SARS-CoV-2 activating the inflammasomes 2019 patients. Of the many innate immune pathways and pyroptosis being at the core of pathogenesis comes from that are engaged by severe acute respiratory syndrome– the fact that lactate dehydrogenase (LDH) levels are highly related coronavirus 2, we highlight the importance of elevated in patients that go on to develop severe disease the inflammasome pathway. We discuss available phar- (Y. Han, H. Zhang, S. Mu, W. Wei, C. Jin, Y. Xue, C. Tong, Y. Zha, Z. Song, and G. Gu, manuscript posted on medRxiv).

maceutical agents that target a critical component of Downloaded from inflammasome activation, signaling leading to cellular LDH is a cytosolic enzyme that is released to the extracellular pyroptosis, and the downstream as a prom- environment upon membrane rupture. In fact, LDH release is ising target for the treatment of severe coronavirus used to monitor pyroptosis (3). Additionally, re- leased as a result of inflammasome activation, IL-1b, as well as disease 2019–associated diseases. The Journal of Im- its response gene product, IL-1RA, are found to be elevated in munology, 2020, 205: 000–000. the sera of COVID-19 patients (4). The key to overcoming excessive inflammatory activity is to http://www.jimmunol.org/ hile the race for a vaccine against the severe target a crucial regulator of cellular inflammation while leaving acute respiratory syndrome–related coronavirus 2 the antiviral pathways intact. Pathogen- or alarmin-induced W (SARS-CoV-2) is in full swing, current treatments activation of nucleotide-binding oligomerization domain available for coronavirus disease 2019 (COVID-19) are limited (NOD)–like receptors leads to inflammasome assembly into a to supportive care. One of the greatest enigmas surrounding colossal molecular scaffold that generates a platform for the COVID-19 is the diverse disease trajectories of COVID-19 mass recruitment and activation of -1 with the help patients. Some displayed little to no symptoms, whereas others of a “bridge” filament protein, the -associated speck-

develop severe fever and pneumonia, leading to acute respiratory like protein containing a caspase recruitment domain (ASC) by guest on October 2, 2021 distress syndrome and ultimately to . It is becoming in- (Fig. 1). Proteolytic activation of caspase-1 subsequently catalyzes creasingly clear that the is a major player the maturation and secretion of proinflammatory cytokines, in patients’ response to the virus infection. Serum levels of both specifically IL-1b and IL-18 (5). The most well-characterized pro- and anti-inflammatory cytokines are markedly higher in of the inflammasomes is the NOD-like receptor (NLR) family severe cases than in moderate cases of COVID-19, suggesting pyrin domain-containing 3 (NLRP3) inflammasome, which that a cytokine storm, also known as cytokine release syndrome, has been implicated in a plethora of diseases ranging from is associated with increasing disease severity (1). Additionally, autoinflammatory diseases to neurologic disorders. Impor- leukocytosis and lymphocytopenia are hallmark clinical features tantly, the NLRP3 inflammasome is also involved in antiviral of severe cases of COVID-19 (1). These observations allude to responses and virus-associated illnesses. an overdrive in inflammation as a mismanaged antiviral response It is presently unclear if SARS-CoV-2 activates the NLRP3 against SARS-CoV-2 that lead to poor clinical outcomes. inflammasome. However, taking lessons from its predecessor, SARS-CoV-2 is a positive-sense RNA virus. As such, its the severe acute respiratory syndrome–related coronavirus pathogen-associated molecular patterns will be recognized by (SARS-CoV), which caused the severe acute respiratory syn- RNA-sensing pattern recognition receptors, including TLR3, drome global epidemic between 2002 and 2003, was shown

*School of Postgraduate Studies, International Medical University, 57000 Kuala Lumpur, Address correspondence and reprint requests to Prof. Akiko Iwasaki, Yale University Malaysia; †Department of Immunobiology, Yale University School of Medicine, New School of Medicine, 300 Cedar Street, TAC S655B, New Haven, CT 06519. E-mail Haven, CT 06520; ‡Department of Molecular, Cellular, and Developmental Biology, Yale address: [email protected] x University, New Haven, CT 06512; and Howard Hughes Medical Institute, Chevy Chase, Abbreviations used in this article: ASC, apoptosis-associated speck-like protein contain- MD 20815 ing a caspase recruitment domain; COVID-19, coronavirus disease 2019; E, envelope; ORCIDs: 0000-0002-5535-0732 (J.K.Y.Y.); 0000-0002-7824-9856 (A.I.). LDH, lactate dehydrogenase; NLR, NOD-like receptor; NLRP3, NOD-like receptor family pyrin domain-containing 3; NOD, nucleotide-binding oligomerization domain; Received for publication May 6, 2020. Accepted for publication May 15, 2020. NSAID, nonsteroidal anti-inflammatory drug; RIG-I, retinoic acid–inducible gene I; This work was supported by National Institutes of Health/National Institute of Allergy ROS, reactive oxygen species; SARS-CoV, severe acute respiratory syndrome–related and Infectious Diseases Grants R01AI127429, 75N93019C00051, 1R01NS111242, coronavirus; SARS-CoV-2, severe acute respiratory syndrome–related coronavirus 2; and U19AI089992 (to A.I.). Laboratory research regarding coronavirus disease 2019 TRAF3, TNFR-associated factor 3. was supported by the Women’s Health Research at Yale Pilot Project Program, a Fast Grant for COVID-19 from Emergent Ventures at the Mercatus Center (George Mason Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 University, Arlington, VA), the Mathers Foundation, and the Ludwig Family Founda- tion. A.I. is an investigator for the Howard Hughes Medical Institute.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000513 2 BRIEF REVIEWS: INFLAMMASOMES AND PYROPTOSIS AS COVID-19 DRUG TARGETS Downloaded from http://www.jimmunol.org/

FIGURE 1. Activation of the NLRP3 inflammasome by SARS-CoV. SARS-CoV E protein induces Ca2+ leakage to the cytosol from Golgi storage, while ORF3a induces K+ efflux at the plasma membrane to the extracellular spaces. This imbalance in the ionic concentration within the cells, and the resultant ROS generated by damaged mitochondria, triggers NLRP3 inflammasome activation. In addition to inducing K+ efflux, ORF3a promotes inflammasome assembly through TRAF3-mediated ubiquitination of ASC. ORF8b interacts directly with leucine-rich repeat of NLRP3 to stimulate its activation independent of ion channel activity. Inflammasome activation induces the formation of gasdermin-D pores on the cell membrane, causing IL-1b and IL-18 secretion, and the influx of water by guest on October 2, 2021 molecules leading to cell swelling and subsequent rupture (pyroptosis). to express at least three proteins that activate the NLRP3 similarity with SARS-CoV and the amino acid sequences of inflammasome: envelope (E), ORF3a, and ORF8b. E protein SARS-CoV-2 and SARS-CoV E protein are 94.7% conserved, localizes at the membrane enfolding the Golgi complex and it is likely that SARS-CoV-2 could similarly activate the the endoplasmic reticulum–Golgi intermediate compartment NLRP3 inflammasome (14, 15). Interestingly, however, a and function as an ion channel (viroporin) that facilitates study on SARS-CoV-2 consensus sequence HKU-SZ-005b Ca2+ leakage to the cytosol (6). In contrast, ORF3a localizes showed a remarkable distinction in ORF8 from that of at the Golgi complex and plasma membrane, acting as a K+ SARS-CoV and a lack of the aggregation motif found in channel (7). As NLRP3 is sensitive to high cytosolic Ca2+ but SARS-CoV ORF8b that triggers NLRP3 activation (14). is instead inhibited by high K+ concentration, the viroporin ORF3a share 72% amino acid sequence identity between the activity of SARS-CoV presumably induces inflammasome two viruses, and of note, ORF3b is another region with dis- activation via E protein–mediated Ca2+ leakage from intra- tant sequences with only 32% identity (14). It would be in- cellular storage and ORF3a-mediated cellular K+ efflux at the teresting to determine whether SARS-CoV-2 ORF3a and plasma membrane to the extracellular spaces (7, 8). The re- ORF8 can likewise interact with NLRP3 or at least function sultant disruption of intracellular ionic balance also promotes as ion channels that would indirectly induce inflammasome mitochondrial damage and generation of reactive oxygen activation. Although it is unknown for SARS-CoV2 infec- species (ROS), which coactivates NLRP3 (7). SARS-CoV tion, various innate immune receptor signals are proposed could also activate inflammasomes independent of its viro- as the upstream contributors of RNA virus-induced NLRP3 porin activities. E protein and ORF3a are able to stimulate inflammasome activation. These include Z-DNA binding pro- NF-kB signaling to drive the transcription of inflammatory tein 1 (ZBP1)/receptor interacting protein kinase 1 (RIPK1)/ cytokines and chemokines, including IL-1b, IL-18, and IL-8, RIPK3 signaling (16, 17), and 29,59-oligoadenylate synthetase/ and prime NLRP3 expression to its functional level (9–12). RNaseL pathway (18). Moreover, RIG-I is proposed to in- ORF3a also activates NLRP3 inflammasome by promoting teract with ASC and induce IL-1b secretion after vesicular TNFR-associated factor 3 (TRAF3)–mediated ubiquitination stomatitis virus infection independently of NLRP3 (19). In of ASC (12). Finally, ORF8b activates NLRP3 through parallel with the viral protein-mediated inflammasome activa- direct interaction of the leucine-rich repeat domain of NLRP3 tion, it is possible that the RNA-sensing pathways trigger (13). Given that SARS-CoV-2 shares ∼79% overall genetic inflammasome activation upon SARS-CoV-2 infection. The Journal of Immunology 3

There are various other NLRs beyond NLRP3 and the viral pathogenesis (29). It is predicted that pyroptosis in lung inflammasomes that may be just as consequential in host epithelial cells is likewise detrimental given the severe pneu- immune response against viral infections like SARS-CoV-2. monia experienced by COVID-19 patients. In contrast, These include NLRs that intensify inflammatory processes, pyroptosis in alveolar induces acute lung injury such as NOD1 and NOD2 that similarly form multiprotein and exacerbates lung inflammation by promoting neutrophil complexes known as NODosomes. NOD1 and NOD2 are infiltration into the lungs and augmented alveolar concen- expressed in leukocytes and epithelial cells, and the assembled trations of cytokines IL-6, TNF-a, and IL-1b (30). The NODosomes drive NF-kB signaling and type I IFN pro- combination effects between leukocytosis and pyroptosis duction (20). Conversely, there exists a unique subgroup of may be a major contributor to cytokine storms observed in NLRs that function as negative regulators of inflammation, COVID-19 patients. Another unsettling observation that is including NLRX1, NLRP12, and NLR family CARD do- especially relevant to severe COVID-19 patients is that me- main containing 3 (NLRC3). These NLRs attenuate in- chanical stretch of the lungs further amplifies lung inflam- flammation by modulating NF-kB signaling, type I IFN mation via NLRP3 activation in alveolar response, and ROS production, among other processes (20). and MAPK kinase 6–mediated high-mobility group box 1 Interestingly, it was reported that SARS-CoV-2 ORF9c protein (HMGB1) protein expression in alveolar epithelial cells (31, can activate negative regulators of host inflammatory responses, 32). Therefore, the use of NLRP3 suppressors in patients including NLRX1, to block mitochondrial antiviral-signaling requiring the use of ventilators might be helpful in mitigating protein (MAVS) to hinder NF-kB–mediated cytokine pro- excessive lung tissue damage. duction (21). Widespread pyroptosis could lead to excessive tissue in- Downloaded from As a mechanism to drastically intensify disease pathogenesis, flammation, organ failure, and death within minutes (33). inflammasome activation can trigger cellular pyroptosis, a Uncontrolled pyroptosis is especially detrimental in the elderly type of programmed characterized by gasdermin who are already experiencing an age-related chronic inflamma- D–mediated membrane rupture, and spontaneous release tory condition known as “inflammaging” (34). Moreover, ageing of cytosolic contents into the extracellular spaces. Upon individuals have impaired capacity to produce type I and type III inflammasome activation, caspase-1 and other noncanonical IFNs because of TRAF3 degradation (35). These IFNs are not http://www.jimmunol.org/ inflammasome such as caspase-4, caspase-5 and only key antiviral resistance factors but are also potent regu- caspase-11 activate gasdermin-D, which subsequently form lators of the inflammasomes (36). When tested in a mouse pores on the cell membrane (22). These gasdermin-D pores model of influenza A virus disease, the absence of innate re- facilitate the secretion of IL-1b and IL-18, and importantly, sistance (due to deficiencies in TLR7 and RIG-I–like receptor they also enable simultaneous influx of Na+ and water signaling) led to a lethal disease only in the presence of molecules, causing excessive cell swelling to the point of caspase-1/caspase-11 activation (37). In this setting, recruit- membrane rupture (22, 23). Pyroptosis of macrophages that ment of neutrophils to the lung and activation of NETosis led have phagocytosed viruses rapidly releases a myriad of alar- to the pathological and lethal disease. Treatment with DNase by guest on October 2, 2021 mins,includingviralparticles,cytokines,chemokines,LDH, (to break up the DNA released by the NETs) as well as IL-1R ATP, and ROS, prompting an immediate reaction from antagonist (Anakinra) was able to reduce the severity of the surrounding immune cells and thus inducing a pyroptotic disease. Thus, the impairment in antiviral resistance and un- chain reaction. Moreover, pyroptosis would allow viral Ags regulated inflammasome activation may underlie the perfect and RNA to be disseminated in the circulation and possibly storm for the severe disease we observe in the COVID-19 generate immune complex and deposition in target organs, patients. such as kidney, to initiate severe inflammatory cascade. Although there is indeed ample evidence advocating for SARS-CoV-2–induced inflammasome activation and pyrop- inflammasome inhibition as a viable solution to treatment of tosis in alveolar macrophages and recruited monocyte-derived hyperinflammatory responses in virus infections, there have macrophages could drastically aggravate symptoms of pneumo- been conflicting results in the roles of immune receptors in host nia, including acute respiratory distress syndrome and fever. It immune defense against virus infections. For example, it was was established that the route of SARS-CoV-2 entry into cells shown that mice lacking NLRP3 and caspase-1 exhibit much is through the angiotensin-converting enzyme 2 (ACE2) re- greater mortality to influenza A virus infection due to com- ceptor, and these are indeed expressed by cells in the lungs, promised immune response, including a reduction in neu- including alveolar type 2 cells, respiratory epithelial cells, and trophil and monocyte migration, as well as decreased secretion macrophages, making them suitable targets for viral infection of cytokines and chemokines (38, 39). This implies the sig- and potential inflammasome induction leading to pyroptosis nificant temporal, cell type, and disease-specific functions that (24, 25). The epithelial cells lining the airways are particularly would alter their therapeutic potential in a particular con- vulnerable to pathogenic insults, owing to its large area of text, and it is imperative that these factors should be taken exposure to external environment. Following influenza A virus into consideration in the design and use of inflammasome infection, the RIG-I receptor is essential in regulating NLRP3 inhibitors. inflammasome activation in response to elevated type I Inflammasome activation and pyroptosis could be under- IFN production to induce pyroptosis of lung epithelial cells appreciated events that are central to COVID-19 pathogenesis. (26, 27). Pyroptosis of lung epithelial cells may confer pro- Abnormalities in blood coagulation leading to thrombotic tection against pathogens, as demonstrated in mice models complications, including pulmonary embolism, are associated of (28). However, inflammasome signaling in with poor prognosis in COVID-19 patients (40, 41). The lung epithelial cells is significantly enhanced in asthmatic suppression of inflammasome-mediated pyroptosis in macro- patients, which aggravates tissue inflammation and worsens phages might mitigate anomalous blood clotting by preventing 4 BRIEF REVIEWS: INFLAMMASOMES AND PYROPTOSIS AS COVID-19 DRUG TARGETS the release of tissue factor, which is an initiator of blood co- of inflammasomes, including IL-1b,usingAnakinra,which agulation cascades (42). Recently, another complication of are currently being tested in phase 3 clinical trials for COVID-19 described among children takes the form of pe- COVID-19 (ClinicalTrials.gov identifier: NCT04330638 diatric inflammatory multisystem syndrome, otherwise referred and NCT04324021). Treatment with Anakinra was previ- to as Kawasaki disease, characterized by acute inflammation of ously shown to be effective in alleviating vasculitis, coronary the medium-sized arteries, including the coronary arteries, lesions, and myocarditis in a mouse model of Kawasaki resulting in artery aneurysms that, if left untreated, would cause disease (57) and was followed by successful use as clinical myocardial ischaemia, infarction, and death (43, 44). Although intervention for cases of severe and resistant Kawasaki dis- the precise etiology of Kawasaki disease is yet obscure, it is ease in newborn and very young children (58, 59). suspected that pathogenic infections play a role in disease Various dedicated inhibitors of NLRP3 have existed mostly pathogenesis because of the seasonality of this disease, and in the form of clinically unproven experimental compounds, SARS-CoV-2 is likely contributing to the present emergence of both of natural and synthetic origins (60). These compounds symptoms among pediatric COVID-19 patients (45). A could either inhibit NLRP3 indirectly or directly target the comparison of 146 Kawasaki disease patients found an over- NLRP3 core protein or its constituents, such as ASC and whelming signature for an acute disease, involving IL-1 signaling caspase-1. Pharmacological compounds that disrupt the sig- pathways (46), further substantiating the roles of aberrant naling pathways upstream of inflammasome activation also inflammasome activity and justifying the use of inflammasome hold promise. For example, the natural polyphenolic flavo- inhibitors in COVID-19 treatments. In another study, inhi- noid silymarin (61), the plant sesquiterpene lactone parthe- bition of complement-induced pyroptosis was able to reduce nolide, as well as the synthetic IkB kinase-b inhibitor Bay Downloaded from local inflammation at the lungs and spleen of mice infected 11-7082, inhibits the NF-kB pathway, thereby preventing the with the Middle East respiratory syndrome coronavirus (47). priming step of NLRP3 activation and the transcription of Yet another potential benefit of NLRP3 inhibition is the pos- inflammatory cytokines (62). Inhibition of NF-kB–mediated sibility of ameliorating comorbidities associated with COVID- inflammation was shown to improve survivability of SARS- 19, including hypertension, chronic obstructive pulmonary CoV–infected mice (9). Furthermore, quercetin, a natural disease, , and cardiovascular disease, as NLRP3 antioxidative flavonoid found in many plants, exhibited po- http://www.jimmunol.org/ inflammasome activation is implicated in these diseases, as well tent suppressive ability against inflammasomes, including (48–51). These comorbidities strongly influence COVID-19 NLRP3, by diminishing ASC speck formation and oligo- severity, and mitigating them might improve COVID-19 merization (63). The same study demonstrated that quercetin prognosis and significantly decrease the risk of death. likewise relieves vasculitis in a mouse model of Kawasaki Several repurposed compounds with regulatory effects on disease, thus offering a plausible alternative to the use of inflammasome activity are currently being appraised in clinical Anakinra, although its clinical relevance remained to be de- trials as treatment for COVID-19. An example is tranilast, a termined. Another plant-derived dehydrocostus lactone found tryptophan analogue that has a direct inhibitory action against in traditional Chinese medicinal herbs, such as the roots of by guest on October 2, 2021 NLRP3 (52), which is currently undergoing a randomized Sassuria lappa, similarly suppressed NLRP3 inflammasome control trial in COVID-19 patients (registration number: activity by blocking ASC oligomerization, in addition to re- ChiCTR2000030002 on the Chinese Clinical Trial Registry). ducing neutrophil recruitment (64). These promising results Tranilast is initially approved for the treatment of allergic and illustrated a couple of noteworthy ideas: first, the effectiveness inflammatory diseases such as bronchial asthma, atypical in a multipronged approach in inflammation inhibition, and dermatitis, allergic conjunctivitis, keloids, and hypertrophic second, the prospective benefits in screening for additional scars (53). Now, its effectiveness has also been recognized in naturally occurring substances with anti-inflammatory prop- the treatment of fibrosis, proliferative disorders, , car- erties and assessing their clinical potential in the treatment of diovascular problems, autoimmune disorders, ocular diseases, COVID-19. diabetes, and renal diseases (53). Tranilast might be an at- Clinically approved drugs such as nonsteroidal anti- tractive intervention for COVID-19 patients with comor- inflammatory drugs (NSAIDs) can also be repurposed to bidities, given its wide array of therapeutic effects with selectively inhibit NLRP3. NSAIDs of the fenemate type such minimal side effects. However, tranilast should not be used as flufenamic acid and mefenamic acid were shown to inhibit together with the anticoagulant drug warfarin, in the event NLRP3 inflammasome by reversibly blocking volume-regulated 2 that the latter is used clinically to control blood clotting in anion channels, which regulate Cl transport across plasma COVID-19 patients, as the two drugs are known to interact membrane (65). Additionally, it was suggested that NSAIDs with each other synergistically to produce serious side effects also contribute to limiting the secretion of proinflammatory (54). In an exciting recent development, a study shows that cytokines through their cyclooxygenase-1 (COX-1)–independent disulfiram, a US Food and Drug Administration–approved activity. At present, there is no evidence for or against the drug used to treat alcohol addiction, is a potent inhibitor of use of NSAIDs as COVID-19 treatment. Nevertheless, it is pyroptosis and gasdermin D–dependent cytokine release (55) recommended that NSAIDs should be prescribed cautiously and holds promise for COVID-19 therapy. Preventing to COVID-19 patients, including when used as analgesic gasdermin-D pore formation without disrupting inflamma- (66). some activation represents a promising approach, as one can Finally, as discussed above, type I and type III IFNs can be restrict viral replication within cells by eliciting inflammasome- used to suppress transcription of both IL-1b as well as mediated apoptotic cell death instead of pyroptosis and cyto- inflammasome components. Type I IFNs have demonstrated kine release, thus limiting widespread tissue inflammation (56). efficacy against SARS-CoV infection in in vitro experiments Efforts are also on the way to block the cytokines downstream but have generally failed in human trials (67). However, the The Journal of Immunology 5 useoftypeIIFNinCOVID-19mightstillbeeffectivefor 8. 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