IL-1b drives SARS-CoV-2 disease in vivo, independently of the inflammasome and pyroptotic signalling

IL-1b drives SARS-CoV-2 disease in vivo, independently of the inflammasome and pyroptotic signalling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article IL-1b drives SARS-CoV-2 disease in vivo, independently of the inflammasome and pyroptotic signalling Marcel Doerflinger, Stefanie M. Bader, Lena Scherer, Jan Schaefer, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4826453/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Feb, 2025 Read the published version in Cell Death & Differentiation → Version 1 posted 12 You are reading this latest preprint version Abstract Excessive inflammation and cytokine release are hallmarks of severe COVID-19. Programmed cell death processes can drive inflammation, however, the relevance in the pathogenesis of severe COVID-19 is unclear. Pyroptosis is a pro-inflammatory form of regulated cell death initiated by inflammasomes and executed by the pore-forming protein gasdermin D (GSDMD). Using an established mouse-adapted SARS-CoV-2 virus and a combination of gene-targeted mice we found that deletion of the inflammasome (NLRP1/3 and the adaptor ASC) and pore forming proteins involved in pyroptosis (GSDMA/C/D/E) did not impact disease outcome or viral loads. Furthermore, we found that SARS-CoV-2 infection did not trigger GSDMD activation in mouse lungs. We did not observe any difference between WT animals and mice with compound deficiencies in upstream caspases C1/11/12 −/− . This indicates that the classical canonical and non-canonical pro-inflammatory caspases known to process and activate IL-1β, IL-18 and GSDMD do not substantially contribute to SARS-CoV-2 pathogenesis. However, the loss of IL-1β, but not the absence of IL-18, ameliorated disease and enhanced survival in older animals compared to wildtype mice. Collectively, these findings indicate that IL-1β is an important factor contributing to severe SARS-CoV-2 disease, but its release was largely independent of inflammasome and pyroptotic pathways. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION SARS-CoV-2 causes a spectrum of symptoms, ranging from mild resolving illness to severe COVID-19 and mortality, but the host factors contributing to the spectrum of SARS-CoV-2 associated disease are yet to be fully characterized. Unravelling the molecular mechanisms that contribute to the dysregulation and aberrant activation of the immune system following SARS-CoV-2 infection is paramount in formulating effective strategies to mitigate the morbidity and mortality caused by this pandemic virus. Emerging evidence suggests that inflammation and certain programmed (regulated) cell death processes play pivotal roles in the pathogenesis of COVID-19 (reviewed in 1 , 2 ). Lytic forms of cell death, such as necroptosis and pyroptosis, have been linked to dysregulation of the immune system associated with COVID-19 through the release of damage associated molecular patterns (DAMPs), cytokines and immune activation and inflammation linked to cell membrane disruption 3 , 4 , 5 , 6 . Pyroptosis is a distinct form of programmed (regulated) cell death characterized by features of necrosis and an inflammatory response. It is triggered by the activation of inflammatory caspases-1 (human and mouse), -4 (human), -5 (human), and/or -11 (mouse) 7 , which proteolytically activate the pyroptotic pore-forming effector protein gasdermin D (GSDMD) 8 . The activation of the pyroptotic caspases is regulated by inflammasomes in response to certain infections or specific host-derived proteins and crystals 9 , 10 . The inflammasome sensors nucleotide oligomerization domain (NOD)-like, leucine-rich repeat (LRR) receptor, absent in melanoma 2 (AIM2), and Pyrin require the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) to form a functional complex to activate caspase-1. Conversely, NLRC4 and NLRP1b inflammasomes can directly bind to and activate caspase-1 without ASC. In addition to proteolytically activating GSDMD, caspase-1 also cleaves the pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18) into their bioactive forms that can be secreted by cells. Upon assembly, the negatively charged GSDMD pore allows the rapid release of these proteolytically processed pro-inflammatory cytokines into the extracellular milieu 7 , 11 . The secreted bioactive form of IL-1β promotes the recruitment of innate immune cells and modulates the activity of adaptive immune cells. Mature IL-18 is involved in the production of interferon-gamma (IFN-γ) and promotes Th1 and Th2 T cell immune responses 12 , 13 . Necroptosis is another lytic form of programmed (regulated) cell death that triggers the release of DAMPs to promote immune activation, cytokine release and inflammation 8 , 14 . Necroptosis can occur downstream of death receptor signaling, such as ligation of TNF receptor 1 (TNFR1) 15 , 16 , through the activation of cytoplasmic nucleic acid sensor Z-DNA binding protein 1 (ZBP1) 15 , 16 and downstream of Toll-like receptors (TLR) through the stimulation of Toll/IL-1 receptor domain-containing adapter-inducing interferon-b (TRIF) 17 , 18 . These signals lead to RIPK3 activation, typically in the absence of proteolytically active caspase-8 19 . Once activated, RIPK3 phosphorylates the pseudokinase mixed lineage kinase domain-like (MLKL) causing its oligomerization and translocation to the plasma membrane, where it causes membrane rupture and a necrotic cell death 20 , 21 , 22 . Lytic forms of programmed (regulated) cell death have been proposed to contribute to COVID-19 pathogenesis, but genetic in vivo evidence is lacking 23 . In our recent study utilizing gene-targeted mouse models of mild and severe COVID-19, we challenged the notion that necroptosis was a significant driver of this disease 24 . This highlighted the importance of further animal studies to dissect complex disease pathogenesis involving several cell types and organ systems. Here, we utilized diverse mouse models to investigate the physiological relevance of lytic programmed cell death pathways and their contribution to inflammation during severe SARS-CoV-2 disease. We found that IL-1b drives SARS-CoV-2 disease severity, independently of pyroptosis and the inflammasome. RESULTS Loss of inflammasome signaling does not prevent pro-inflammatory cytokine release and disease following SARS-CoV-2 infection in vivo. To investigate the role of inflammasome pathways during severe SARS-CoV-2 infection we used a previously characterised mouse adapted strain (P21) 25 . This strain was derived from a clinical isolate that was passaged 21 times in mice to allow adaptations that caused severe disease in WT mice, characterized by weight loss and increased levels of pro-inflammatory cytokines 25 . During SARS-CoV-2 infection, activation of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome pathway is thought to contribute to the release of bioactive IL-1β and IL-18, cytokines associated with severe COVID-19 5, 6, 26 . We infected NLRP3 deficient mice ( Nlrp3 −/− ) with P21 to explore this hypothesis. Interestingly, three days post infection (dpi), Nlrp3 −/− mice displayed similar viral burdens to WT controls and did not show changes in weight loss, an established marker of disease severity in P21-infected mice at this time point ( Fig. 1 A-B ) . NLRP1 is also a potent activator of inflammation 27 , 28 , however, we observed that severe disease and lung viral burdens caused by SARS-CoV-2 infection were not affected by deletion of NLRP1 in mice ( Fig. 1 C-D ) . Many of the inflammasomes, including NLRP1, NLRP3, AIM2 and others, are dependent on the adapter protein ASC 13 , 29 , 30 , 31 . Surprisingly, ASC-deficient mice ( Asc −/− ) exhibited viral burdens and weight loss similar to infected WT animals ( Fig. 1 E-F ) . This demonstrates that ASC-dependent inflammasomes are not essential for SARS-CoV-2 driven pathogenesis. We next determined the abundance of 25 cytokines and chemokines in lung homogenates from infected gene-targeted mice to understand if lytic programmed cell death and canonical inflammasome activation contributed in a more subtle way to SARS-CoV-2 disease pathogenesis. Interestingly, neither of the inflammasome knockout mice we investigated displayed a reduction in IL-18, while only Asc −/− animals had slightly diminished IL-1β levels in their lungs upon SARS-CoV-2 infection (Fig. 1 G). While Nlrp3 −/− , Nlrp1 −/− and Asc −/− inflammasome knockout mice all showed a reduction in the levels of TNF, MIP1α and IL-17a, variable differences in the levels of other cytokines were observed depending on the particular inflammasome deficiency (Fig. 1 G and S1 A). Overall, these results suggest that while NLRP1/3 and ASC do affect cytokine release during SARS-CoV-2 infection, this was not sufficient to impact disease severity (weight loss) or viral burdens in vivo compared to WT mice. To better understand disease in SARS-CoV-2 P21 infected gene-targeted animals, we compared lung histology of knockouts and WT mice at 3 dpi. Lung sections were stained with hematoxylin and eosin (H&E) and analyzed by a board-certified pathologist. At 3 dpi, WT mice displayed multifocal, acute alveolitis (sometimes necrotizing), multifocal pneumonia, as well as moderate to severe acute multifocal perivasculitis (Fig. 1 H). Gene-targeted mice showed similar manifestations of disease with interstitial pneumonia, moderate to severe multifocal perivasculitis and acute alveolitis. Immunohistochemical (IHC) staining for SARS-CoV-2 nucleocapsid showed that the virus localized to the bronchiolar and alveolar epithelium, as well as macrophages in both WT and all gene-targeted mice (Fig. 1 H). Staining for myeloperoxidase (MPO), CD3 and F4/80 revealed a similar number of myeloid cell and T cell infiltrates in WT and all knockout mice (Fig. 1 H). Collectively, these findings show that while canonical ASC-dependent inflammasome pathways do play some role in the cytokine responses associated with severe SARS-CoV-2 pathogenesis, they do not significantly impact disease outcomes. The pyroptosis effector Gasdermin D is not required for pro-inflammatory cytokine release during SARS-CoV-2 infection in vivo. Regardless of the upstream mechanisms responsible for the activation of pyroptosis and cytokine production, the cellular release of cytokines and cell lysis is thought to be primarily dependent on the activation of the pore forming protein GSDMD 32 . We found that GSDMD deficient animals ( Gsdmd −/− ) showed similar viral burdens and weight loss compared to WT mice upon infection with SARS-CoV-2 ( Fig. 2 A-B ) . Analysis of 25 cytokines and chemokines in lung homogenates from infected Gsdmd −/− mice showed that IL-1β levels were slightly reduced compared to WT animals (Fig. 2 C). Interestingly, however, the levels of IL-18 and IL-23 were increased in these animals upon infection (Fig. 2 C and S2 A ) . These data indicate that GSDMD does not exert an essential role in the pathogenic pro-inflammatory cytokine release during SARS-CoV-2 infection. We further compared lung histology of Gsdmd −/− and WT mice at 3 dpi. Lung sections were stained with hematoxylin and eosin (H&E) and analyzed by a board-certified pathologist. Three days post SARS-CoV-2 infection, both Gsdmd −/− and WT mice displayed multifocal, acute alveolitis, multifocal pneumonia, as well as moderate to severe acute multifocal perivasculitis. IHC staining for SARS-CoV-2 nucleocapsid showed that virus localized to the bronchiolar and alveolar epithelium in both WT and Gsdmd −/− mice. MPO, CD3 and F4/80 staining revealed a similar pattern of myeloid cell and T cell infiltrates in WT and Gsdmd −/− animals (Fig. 2 D). We next explored if severe SARS-CoV-2 disease was linked to cleavage (i.e. activation) of GSDMD in WT animals. In line with our previous results, no cleaved GSDMD was detected by IHC in WT animals with severe SARS-CoV-2 disease confirmed with nucleocapsid staining ( Fig. 2 E ) . As a positive control, to ensure that our antibody detects cleaved GSDMD, we stained small intestine tissue from mice infected with the enteric pathogen Cryptosporidium , which is known to trigger pyroptosis and cause Gasdermin D cleavage 33 ( Fig. 2 E bottom panel) . To further investigate GSDMD cleavage during SARS-CoV-2 infection, we performed Western blot analysis of whole lung tissue extracts. Cleavage (i.e. activation) of GSDMD in the lungs of infected animals could not be detected in WT and in Asc −/− mice ( Fig. 2 F, original westerns can be found in the supplemental information ) . Together with our genetic investigations, this indicates that the inflammation associated with severe SARS-CoV-2 disease does not lead to or require the activation of GSDMD in vivo. Regulators of non-canonical pyroptosis are dispensable for SARS-CoV-2 infection induced pathogenesis. We further investigated the role of non-canonical pyroptosis pathways, including those involving GSDME activation. We infected Gsdme −/− mice with SARS-CoV-2 but found no difference in disease phenotype compared to control WT animals. Furthermore, compound loss of GSDMD/E ( Gsdmd/e −/− ) and GSDMA/C/E ( Gsdma/c/e −/− ) did not alter disease phenotypes compared to SARS-CoV-2 infected WT mice ( Fig. 3 A-D ). Functional overlap of programmed cell death pathways, particularly in response to infection, has emerged as a paradigm in recent years 34 , 35 . To examine if necroptosis could be compensating for the loss of pyroptosis, we infected mice lacking essential effectors of both of these lytic programmed (regulated) cell death processes, namely GSDMD and MLKL, with SARS-CoV-2. Compound mutant Gsdmd/Mlkl −/− mice showed similar disease phenotypes compared to infected WT animals ( Fig. 3 E-F ) . We extended our investigation to examine mice that were deficient in NINJ1, a protein that drives plasma membrane rupture and release of high molecular weight DAMP downstream of pyroptosis, apoptosis, ferroptosis and accidental cell lysis, but not necroptosis 36 , 37 . Ninj1 −/− mice showed similar disease phenotypes upon SARS-CoV-2 infection as WT mice ( Fig. 3 G-H ) . Cytokine processing during SARS-CoV-2 infection occurs independently of caspases-1/-11/-12. Inflammasome and pyroptosis signaling have been implicated in the pathogenesis of COVID-19 disease because of their known link to IL-1β and IL-18 release. Both of these cytokines have been associated with increased severity of this disease 4 , 38 , 39 . The prevailing dogma is that these cytokines are released downstream of inflammasome signaling and processed into their bioactive forms by caspases-1, -11 (and possibly − 12) 40 , 41 , 42 . We therefore used gene-targeted mice lacking all these caspases ( C1/11/12 −/− ) to understand their overall contribution to severe SARS-CoV-2 disease in our mouse model. Infected C1/11/12 −/− mice showed similar viral burdens and weight loss compared to infected WT mice ( Fig. 4 A-B ). Analysis of 25 cytokines and chemokines showed that upon SARS-CoV-2 infection, C1/11/12 −/− mice mounted a similar cytokine/chemokine response to infected WT controls, with only GROα being slightly elevated in knockout mice ( Fig. 4 C and S4 A ) . This finding indicates that processing and release of pro-inflammatory IL-1β and IL-18 during SARS-CoV-2 infection can occur independently of the three inflammatory caspases-1/-11/-12. Histological examination confirmed similar lung pathology and immune cell infiltrates in SARS-CoV-2 infected C1/11/12 −/− and WT mice ( Fig. 4 D ). These results show that all components of the pyroptosis machinery, from ASC-dependent inflammasome sensors to catalytic pro-inflammatory caspases and the pore-forming effector proteins, are not essential to drive SARS-CoV-2 viremia or disease manifestations. IL1-b, but not IL-18 contributes to severe SARS-CoV-2-mediated disease. Our results show that SARS-CoV-2 driven inflammation and cytokine release in vivo are independent of key components of the pyroptosis machinery. Several studies reported an association between IL-1β and IL-18 serum levels and severe COVID-19 disease 5 , 6 , 26 . Pyroptosis and caspases-1/-11/-12 are often linked to IL-1β and IL-18 processing. So, if these inflammatory cytokines are critically involved in severe COVID-19 disease, the results from our studies using gene targeted mice indicate a disconnect between the processing and release of IL-1β and IL-18 and pyroptosis. To confirm that one or both of these cytokines do contribute to SARS-CoV-2 driven disease in our animal model, we infected IL-18 deficient ( Il-18 −/− ) mice. Il-18 −/− mice were not significantly protected from SARS-CoV-2 infection displaying similar disease compared to infected WT animals (Fig. S5 A-B) . In contrast, Il-1β −/− mice had significantly lower viral burdens in the lungs, and exhibited less weight loss compared to infected WT animals ( Fig. 5 A-B ) . To better understand the cellular host responses linked to better outcomes in SARS-CoV-2 infected Il-1β −/− mice, we examined lung histology at 3 dpi. Lung sections were stained with hematoxylin and eosin (H&E) and analyzed by a board-certified pathologist. Three days post SARS-CoV-2 infection, WT mice had mild to severe acute multifocal perivasculitis, interstitial pneumonia and necrotizing alveolitis. Infected IL-1β deficient mice also showed perivasculitis, moderate interstitial pneumonia, but no alveolitis ( Fig. 5 C-D ) . IHC staining for SARS-CoV-2 nucleocapsid showed that virus localized to the bronchiolar and alveolar epithelium and macrophages in both WT and Il-1β −/− mice. Histological scoring showed that in contrast to WT controls, Il-1β −/− mice did not present with signs of pleural mesothelial hyperplasia, iBALT or proteinaceous debris in the air space. These differences amounted to an overall significantly reduced histological score of disease ( Fig. 5 C-D ) . These findings in gene-targeted mice corroborate the human correlative data and prove for the first time in an in vivo setting that IL-1β plays a critical pathogenic role during severe SARS-CoV-2 induced disease. Inflammatory IL-1 signaling is activated through binding of IL-1 family cytokines to the membrane bound, type I interleukin-1 receptor (IL-1R) 43 . Interestingly, while Il-1r −/− animals were also protected from severe SARS-CoV-2 induced disease, as measured by a reduction in weight loss, these animals did not have decreased viral burdens but were similar to WT mice ( Fig. 5 E-F ) . To identify the effects of the absence of critical components of the IL-1 pathway on the pro-inflammatory cytokine response, we quantified the levels of 25 cytokines and chemokines in the lungs of WT, Il-1β −/− and Il-1r −/− animals at 3 dpi. The absence of IL-1β led to reductions in GM-CSF, IL17a and IL-5. Il-1r −/− animals tended to express lower levels of a wider range of cytokines and chemokines compared to IL-1β deficient mice (Fig. 5 G and S5 C ). However, the reduction in cytokines was of longer duration, continuing for 6 days post-infection, and was more profound at later time points in IL-1β deficient mice compared to WT animals and Il-1r −/− animals (Fig. 5 H and S5 D ). Age related severity of SARS-CoV-2 disease can be reduced by the absence of IL1-b. Similar to COVID-19 in humans, SARS-CoV-2 P21 driven disease in mice becomes more pronounced with increased age 25 . Upon infection, the majority of infected WT animals > 10 weeks-old lose more than 20% body weight, reaching ethical end-point by 4–6 dpi 25 . To test whether the absence of IL-1β could protect against aged-associated severity of SARS-CoV-2 driven disease, we infected eleven-week-old Il-1β −/− and control WT mice. IL-1β deficient mice were more likely to survive SARS-CoV-2 infection compared to WT controls ( Fig. 6 A ) , however, this survival advantage was attenuated in 6-month-old infected mice ( Fig. 6 B). Despite this, 6-month-old IL-1β deficient mice showed significantly lower viral burdens in the lungs at the peak of infection (3 dpi) compared to WT controls ( Fig. 6 C-D ). Interestingly, in aged animals (> 6 months), while multiple cytokines were decreased at 3 dpi, the only cytokine found to be significantly reduced in lung homogenates from Il-1β −/− mice was IL-1β itself ( Fig. 6 E ). To analyse the potential of anti-IL-1β therapeutics against severe COVID-19 in our mouse model, we administered a single dose of IL-1β neutralizing monoclonal antibodies to 10- to 12-week-old WT mice on the day of infection. Inhibition of IL-1β alone was not sufficient to prevent mortality in P21 infected animals although a trend was observed. Thus, more profound inhibition of IL-1β and concomitant inhibition of other pathogenic cytokines should be explored. DISCUSSION COVID-19 morbidity and mortality are driven by dysregulated host cytokine storms. The pro-inflammatory cytokines IL-18 and IL-1b have been implicated in disease pathogenesis and, based on in vitro experiments, it has been proposed that NLRP3 inflammasome activation and pyroptosis are responsible for the exaggerated production of IL-18 and IL-1b. We confirmed in our mouse model that IL-1 signaling contributes to severe SARS-CoV-2 disease through IL-1b, whereas IL-18 plays only a negligible role. Furthermore, our data challenge the notion that NLRP3, NLRP1 and other ASC-dependent inflammasomes are essential for IL-1b-driven SARS-CoV-2 disease pathogenesis. Although deletion of ASC and GSDMD slightly reduced the levels of IL-1b in the lungs of SARS-CoV-2 infected mice compared to WT controls, this reduction was not sufficient to alter disease outcomes. Collectively, we provide genetic in vivo evidence that inflammasomes and GSDMD are not required for maximal lung IL-1b levels and they are not essential for pathogenesis and viral dissemination. This indicates that inhibitors of inflammasomes are unlikely to offer therapeutic benefit in severe COVID-19. We showed that effectors of pyroptosis, including GSDMD, GSDME and GSDMA/C/E, are not required to induce severe SARS-CoV-2 disease in our model. As we have previously shown, necroptosis was also dispensable for causing severe disease 24 and combined deletion of genes of the essential effectors for pyroptosis and necroptosis ( GsdmD/Mlkl −/− ) did not diminish pathology. We further confirmed that lytic programmed cell death does not play a prominent role in SARS-CoV-2 driven disease using mice lacking NINJ1, which is essential for plasma membrane lysis in pyroptosis, ferroptosis and late-stage apoptosis 36 . Importantly, caspases-1, -11 and − 12 were not required for the release of IL-1b during severe SARS-CoV-2 disease. Collectively, these findings indicate that processes independent of lytic programmed cell death must facilitate IL-1β processing and release during SARS-CoV-2 infection. Several potential mechanisms have been identified (reviewed in 44 ), suggesting that these cytokines can be downstream of pathways other than canonical inflammasome and gasdermin effected pyroptosis (or similar lytic cell death). Recent studies have broadened understanding of the caspases capable of proteolytically activating IL-1b and regulating cytokine transcription 45 , making it temping to speculate that proteolytic processing of IL-1b/a during SARS-CoV-2 infection might be mediated by caspases other than caspases-1/11/12. Although studies have suggested that NLPR3 activation and GSDMD serve as predictive markers for severity of COVID-19 4, 38 , these studies were conducted in vitro only. Published in vivo studies using Nlrp3 −/− animals, utilized adeno-associated virus (AAV) to deliver hACE2 prior to SARS-CoV-2 infection 53 , an additional process that adds complexities, such as triggering inflammatory pathways. To date, clinical trials testing NLRP3 inhibitors in COVID-19 patients have been inconclusive, only showing subtle changes in mortality and not being successful in improving APACHE II scores compared to treatment with standard of care alone 54 . Our data complement this work indicating that inflammasomes and pyroptosis are not promising targets for the treatment of severe COVID-19. Given the critical role of IL-1b in severe disease in our SARS-CoV-2 infection model we compared the effects of IL-1b deficiency to the loss of its cognate receptor IL-1R during SARS-CoV-2 infection. Although there were similarities in disease amelioration compared to infected WT mice, loss of IL-1b caused a more profound drop in inflammatory cytokine levels at later time points during infection compared to mice deficient in its cognate receptor. This indicates that complete ablation of all IL-1R binding cytokines is not more beneficial than IL-1b deletion alone. This conclusion is further supported by the lack of a significant reduction in viral burdens in IL-1R deficient mice compared to WT controls. The IL1R pathway involves signaling through a range of factors, including IL-1a, IL-1Ra, IL-33 and IL-18, some of which may facilitate immune-mediated clearance. Given the importance of IL-1b in promoting severe SARS-CoV-2 induced disease in our model and mounting evidence for a central role of this cytokine in driving severe human COVID-19 disease 26 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , we further investigated IL-1b's contributions in age-driven disease. We have previously shown that genetic deletion of TNF prevented lethality in aged animals infected with P21, but only partially, indicating that TNF is not solely responsible for severe age-related COVID-19 disease. Here, we show that IL-1b is another key cytokine that contributes significantly to severe SARS-CoV-2 induced disease. The deletion of the Il-1b gene prevented mortality in young adult animals and attenuated disease in aged mice (> 6 months). Several clinical trials have examined the therapeutic benefit of inhibiting IL-1 signaling during severe COVID-19. These studies used anakinra, an IL-1R antagonist. Our data, derived from comparing IL-1b knockout with IL-1R knockout mice, revealed that removal of IL-1b alone leads to a more prominent reduction of pro-inflammatory cytokines than loss of IL-1R. This is in line with results from clinical trials using Anakinra that did not show significant efficacy in humans 55 , 56 , 57 . Here, we assessed the impact of IL-1b neutralising monoclonal antibodies. We could not recapitulate the survival advantage conferred by genetic loss of IL-1b using a validated IL-1b neutralizing antibody. Several factors could contribute to this discrepancy, including inadequate neutralization of IL-1b within the relevant tissue(s). Another therapeutic avenue would be to target the upstream mechanisms responsible for IL-1β production. Here, we have ruled out some candidate pathways, including various forms of lytic programmed cell death, such as pyroptosis and necroptosis. In conclusion, we have shown a critical pathogenic role for IL-1β during severe SARS-CoV-2 disease in our mouse model. We provide evidence that pyroptotic cell death is not a major driver responsible of IL-1b production. This underscores the importance of further work to determine what process(es) promote(s) IL-1b release to fuel the cytokine storm driven disease in our SARS-CoV-2 infected mice. Identification of the critical upstream signaling pathways may identify novel therapeutic targets that could underpin novel therapeutic strategies to abrogate IL-1b production and thus overcome potential issues with inadequate blockade. MATERIALS AND METHODS Mice Male or female WT and gene-targeted mice were bred and maintained on a C57BL/6J background in the Specific Pathogen Free (SPF) Physical Containment Level 2 (PC2) Bioresources Facility at The Walter and Eliza Hall Institute of Medical Research (WEHI). All procedures involving animals and live SARS-CoV-2 strains were conducted in an OGTR-approved Physical Containment Level 3 (PC3) facility at WEHI (Cert-3621). Mice were transferred to the PC3 laboratory for all SARS-CoV-2 infection experiments at least 4 days prior to the start of experiments. Animals were age- and sex-matched within experiments (both sexes were used). Experimental mice were housed in individually ventilated microisolator cages under level 3 biological containment conditions with a 12-hour light/dark cycle. Mice were provided with WEHI mouse breeder cubes (Ridley Agri Products) and sterile acidified water ad libitum . SARS-CoV-2 strains The SARS-CoV-2 VIC2089 clinical isolate (hCoV-19/Australia/VIC2089/2020) was obtained from the Victorian Infectious Disease Reference Laboratory (VIDLR). Viral passages were achieved by serial passage of VIC2089 through successive cohorts of young C57BL/6J (WT) mice. Briefly, mice were infected with the SARS-CoV-2 clinical isolate intranasally. At 3 dpi, mice were euthanized and lungs harvested and homogenized in a bullet blender (Next Advance Inc) in 1 mL Dulbecco’s modified Eagle’s medium (DMEM) (Gibco/ThermoFisher) containing steel homogenization beads (Next Advance Inc). Samples were clarified by centrifugation at 10,000 x g for 5 min before intranasal delivery of 30 µL lung homogenate into a new cohort of naïve C57BL/6J mice. This process was repeated a further 20 times to obtain the SARS-CoV-2 VIC2089 P21 isolate. Lung homogenates from all passages were stored at -80°C. Infection of mice with SARS-CoV-2 P21 6–8 week-old, or 6 month-old mice were anesthetized with methoxyflurane and inoculated intranasally with 30 µl SARS-CoV-2. Virus stocks were diluted in serum free DMEM to a final concentration of 10 4 TCID50/mouse. After infection, animals were visually checked and weighed daily for a minimum of 10 days. Mice were euthanized at the indicated times post-infection by CO 2 asphyxiation. For histological analysis, animals were euthanized by cervical dislocation. Lungs were collected and stored at -80°C in serum-free DMEM until further processing. Measurement of viral loads by 50% tissue culture infectious dose (TCID 50 ) TCID 50 was performed as previously described in 58 . Briefly, Vero African green monkey kidney epithelial cells, purchased from ATCC (clone CCL-81), were seeded in flat bottom 96-well plates (1.75x10 4 cells/well) and left to adhere overnight at 37°C/5% CO 2 . Cells were washed twice with PBS and transferred to serum-free DMEM containing TPCK trypsin (0.5 µg/mL working concentration). Infected organs were defrosted, homogenized, clarified by centrifugation at 10,000 x g for 5 min at 4°C and supernatant was added to the first row of cells at a ratio of 1:7, followed by 9 rounds of 1:7 serial dilutions in the other rows. Cells were incubated at 37°C/5% CO 2 for 4 days until virus-induced cytopathic effects (CPE) could be scored. TCID 50 was calculated using the Spearman & Kärber algorithm as described in 58 . Histological analysis and immunohistochemical staining Organs were harvested and fixed in 4% paraformaldehyde (PFA) for 24 h, followed by dehydration in 70% ethanol, paraffin embedding and sectioning. Slides were stained with either hematoxylin and eosin (H&E), or immunohistochemically stained with antibodies against CD3 (1:500, Agilent A045201), MPO (1:1000, Agilent A039829), F4/80 (1:1000, WEHI in-house antibody), or an antibody against SARS-CoV-2 nucleocapsid (1:4000, abcam ab271180) using the automated Omnis EnVision G2 template (Dako, Glostrup, Denmark). De-waxing was performed with Clearify Clearing Agent (Dako) and antigen retrieval was performed using EnVision FLEX TRS, High pH (Dako) at 97°C for 30 min. Primary antibodies were diluted in EnVision Flex Antibody Diluent (Dako) and incubated at 32°C for 60 min. HRP-labeled secondary antibodies (Invitrogen, Waltham, USA) were applied at 32°C for 30 min. Slides were counter-stained with Mayer hematoxylin, dehydrated, cleared, and mounted with MM24 mounting medium (Surgipath-Leica, Buffalo Grove, IL, USA). Slides were scanned with an Aperio ScanScope AT slide scanner (Leica Microsystems, Wetzlar, Germany) and analyzed by an American board-certified pathologist (Smitha Rose Georgy) to describe histological changes. Lung cytokine and chemokine analysis Lungs were thawed, homogenized and clarified by centrifugation at 10,000 x g for 5 min at 4°C. Supernatants were pre-treated for 20 min with 1% Triton-X-100 for viral de-activation and the Cytokine & Chemokine 26-Plex Mouse ProcartaPlex Panel 1 (EPX260-26088-901) was used according to the manufacturer’s instructions. Briefly, 25 µL of clarified lung samples were diluted with 25 µL universal assay buffer, incubated with magnetic capture beads, washed, incubated with detection antibodies and SA-PE. Cytokines were recorded on a Bio-plex 200 system (Bio-Rad) and quantitated by comparison to a standard curve. Analysis was performed using R Studio. Western blot analysis Total cell protein was isolated from whole mouse lungs using cell lysis buffer. Absolute protein content of clarified lysates was determined by BCA Protein Assay (Pierce), and equal quantities (10–30 µg) of total protein were separated under denaturing and reducing conditions (with 5% β-mercapto-ethanol) using 4–12% SDS-PAGE gels (Life Technologies). Proteins were transferred onto a PVDF membrane. These membranes were blocked with 5% skim milk (Devondale, Brunswick, Australia) in TBS with 0.05% Tween-20 (TBST) for 30 min, and proteins of interest were detected using the following primary antibodies: rabbit anti-GSDMD (Abcam, ab209845) and rabbit anti-beta-Actin (Cell Signaling, 13E5; loading control). HRP-conjugated secondary goat anti-rabbit IgG antibodies (Southern Biotech, Birmingham, AL, USA) were then applied to membranes, which were subsequently incubated with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore) and imaged using a ChemiDoc Touch Imaging System (Bio-Rad). A positive control for GSMD activation was prepared by infecting bone marrow derived macrophages with salmonella 34 . Anti-IL-1β treatment of mice For depletion of IL-1β, 10–12 week-old animals were treated intraperitoneally with 200µg of monoclonal antibody (Bio X Cell BE0246) on the day of SARS-CoV-2 infection. Vehicle-treated mice received 200µg of isotype control antibodies (Bio X Cell, BE0091). Animals were monitored daily for weigh change and euthanised upon reaching the humane endpoint (> 20% weight loss). Quantification of data and statistical analyses Statistical analyses were performed using Prism v9.3.1 software (GraphPad Software, Inc.). Unpaired two-tailed t-tests were used for normally distributed data for comparisons between two independent groups. Data that violated the assumption of normality were transformed by generating log 10 prior to statistical analysis. Bars in figures represent either the mean (± SD) or median of normally or non-normally distributed data sets, respectively, and each symbol represents one mouse. Sample sizes (n), replicate numbers and significance can be found in the figures and figure legends. For all statistical significance indications: *p < 0.05; **p < 0.01; ***p < 0.001; ****p 0.05). Statistical analysis of cytokine data consisted of Wilcoxon rank sum test between group medians, with Bonferroni adjustment for multiple comparisons. Boxplots in figures depict the median and interquartile ranges. Loess smoothing was applied to the infection time course data, with the shaded area indicating 95% confidence intervals. Declarations Acknowledgements We thank Dr Seth Masters for knockout mouse strains, Natalia Mora Torres, Janzelle Marasigan and Thomas Kapitelli for excellent animal husbandry support, Dr Dylan Sheerin for help with the R code to analysis of cytokine profiles. We thank The Walter and Eliza Hall Institute histology and imaging departments for their help. The authors acknowledge the contribution and assistance of Melbourne Health through its Victorian Infectious Diseases Reference Laboratory at the Doherty Institute in providing our laboratory with isolated SARS-CoV-2 material. Conflict of Interest Statement The authors declare no conflicts of interests relating to this work. Author Contribution Statement S.M.B. designed and performed experiments, analysed data and generated figures. J.P.C. and J.S. designed and performed experiments. L.S., L.M. and M.Da. performed experiments. S.R.G. scored histological images. K.C.D, C.C.A. planned and oversaw experiments. M.H. and A.S. contributed critical ideas and gene-targeted mice and were involved in analyzing data. M.P. and M.Doe. supervised the work. S.M.B, M.Doe. and M.P. wrote the manuscript with input from other authors. Ethics Statement All procedures and mouse strains were reviewed and approved by The Walter and Eliza Hall Institute of Medical Research Animal Ethics Committee and all experiments were conducted in accordance with the Prevention of Cruelty to Animals Act (1986) and the Australian National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Scientific Purposes (1997). Funding Statement Australian National Health and Medical Research Council Australia Investigator grant GNT1175011 (MP). Data Availability Statement Viral strains used in this study are available from the authors upon signing of a Materials Transfer Agreement (MTA). All data generated or analysed during this study are included in this published article [and its supplementary information files]. Full histological images reported in this paper will be shared by the lead contact upon request. References Lee S, Channappanavar R, Kanneganti T-D. Coronaviruses: Innate Immunity, Inflammasome Activation, Inflammatory Cell Death, and Cytokines. Trends in Immunology 2020, 41 (12) : 1083-1099. Morais Da Silva M, Lira De Lucena AS, Paiva Júnior SDSL, Florêncio De Carvalho VM, Santana De Oliveira PS, Rosa MM , et al. Cell death mechanisms involved in cell injury caused by SARS‐CoV‐2. Reviews in Medical Virology 2021. Gutierrez KD, Davis MA, Daniels BP, Olsen TM, Ralli-Jain P, Tait SWG , et al. MLKL Activation Triggers NLRP3-Mediated Processing and Release of IL-1β Independently of Gasdermin-D. Journal of immunology (Baltimore, Md : 1950) 2017, 198 (5) : 2156-2164. Rodrigues TS, de Sá KSG, Ishimoto AY, Becerra A, Oliveira S, Almeida L , et al. Inflammasomes are activated in response to SARS-CoV-2 infection and are associated with COVID-19 severity in patients. The Journal of experimental medicine 2021, 218 (3) : e20201707. Courjon J, Dufies O, Robert A, Bailly L, Torre C, Chirio D , et al. Heterogeneous NLRP3 inflammasome signature in circulating myeloid cells as a biomarker of COVID-19 severity. Blood Advances 2021, 5 (5) : 1523-1534. van den Berg DF, Te Velde AA. Severe COVID-19: NLRP3 Inflammasome Dysregulated. Front Immunol 2020, 11: 1580. Vande Walle L, Lamkanfi M. Pyroptosis. Curr Biol 2016, 26 (13) : R568-r572. Lawlor KE, Murphy JM, Vince JE. Gasdermin and MLKL necrotic cell death effectors: Signaling and diseases. Immunity 2024, 57 (3) : 429-445. Guo H, Callaway JB, Ting JPY. Inflammasomes: mechanism of action, role in disease, and therapeutics. Nature Medicine 2015, 21 (7) : 677-687. Swanson KV, Deng M, Ting JPY. The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nature Reviews Immunology 2019, 19 (8) : 477-489. Bergsbaken T, Fink SL, Cookson BT. Pyroptosis: host cell death and inflammation. Nat Rev Microbiol 2009, 7 (2) : 99-109. Dinarello CA. Immunological and Inflammatory Functions of the Interleukin-1 Family. Annu Rev Immunol 2009, 27 (1) : 519-550. He Y, Hara H, Núñez G. Mechanism and regulation of NLRP3 inflammasome activation. Trends in biochemical sciences 2016, 41 (12) : 1012-1021. Pasparakis M, Vandenabeele P. Necroptosis and its role in inflammation. Nature 2015, 517 (7534) : 311-320. Lin J, Kumari S, Kim C, Van T-M, Wachsmuth L, Polykratis A , et al. RIPK1 counteracts ZBP1-mediated necroptosis to inhibit inflammation. Nature 2016, 540 (7631) : 124-128. Jiao H, Wachsmuth L, Kumari S, Schwarzer R, Lin J, Eren RO , et al. Z-nucleic-acid sensing triggers ZBP1-dependent necroptosis and inflammation. Nature 2020, 580 (7803) : 391-395. He S, Liang Y, Shao F, Wang X. Toll-like receptors activate programmed necrosis in macrophages through a receptor-interacting kinase-3-mediated pathway. Proc Natl Acad Sci U S A 2011, 108 (50) : 20054-20059. Kaiser WJ, Sridharan H, Huang C, Mandal P, Upton JW, Gough PJ , et al. Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL. J Biol Chem 2013, 288 (43) : 31268-31279. Tummers B, Green DR. Caspase-8: regulating life and death. Immunol Rev 2017, 277 (1) : 76-89. Murphy JM, Czabotar PE, Hildebrand JM, Lucet IS, Zhang JG, Alvarez-Diaz S , et al. The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity 2013, 39 (3) : 443-453. Hildebrand JM, Tanzer MC, Lucet IS, Young SN, Spall SK, Sharma P , et al. Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death. Proceedings of the National Academy of Sciences 2014, 111 (42) : 15072-15077. Samson AL, Zhang Y, Geoghegan ND, Gavin XJ, Davies KA, Mlodzianoski MJ , et al. MLKL trafficking and accumulation at the plasma membrane control the kinetics and threshold for necroptosis. Nature Communications 2020, 11 (1). Bader SM, Cooney JP, Pellegrini M, Doerflinger M. Programmed cell death: the pathways to severe COVID-19? Biochem J 2022, 479 (5) : 609-628. M. Bader S, Cooney JP, Bhandari R, Mackiewicz L, Dayton M, Sheerin D , et al. Necroptosis does not drive disease pathogenesis in a mouse infective model of SARS-CoV-2 in vivo. Cell Death & Disease 2024, 15 (1) : 100. Bader SM, Cooney JP, Sheerin D, Taiaroa G, Harty L, Davidson KC , et al. SARS-CoV-2 mouse adaptation selects virulence mutations that cause TNF-driven age-dependent severe disease with human correlates. Proceedings of the National Academy of Sciences 2023, 120 (32) : e2301689120. Karki R, Sharma BR, Tuladhar S, Williams EP, Zalduondo L, Samir P , et al. Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes. Cell 2021, 184 (1) : 149-168.e117. Tsu BV, Beierschmitt C, Ryan AP, Agarwal R, Mitchell PS, Daugherty MD. Diverse viral proteases activate the NLRP1 inflammasome. eLife 2021, 10: e60609. Planès R, Pinilla M, Santoni K, Hessel A, Passemar C, Lay K , et al. Human NLRP1 is a sensor of pathogenic coronavirus 3CL proteases in lung epithelial cells. Mol Cell 2022, 82 (13) : 2385-2400.e2389. Faustin B, Lartigue L, Bruey J-M, Luciano F, Sergienko E, Bailly-Maitre B , et al. Reconstituted NALP1 inflammasome reveals two-step mechanism of caspase-1 activation. Molecular cell 2007, 25 (5) : 713-724. Abderrazak A, Syrovets T, Couchie D, El Hadri K, Friguet B, Simmet T , et al. NLRP3 inflammasome: from a danger signal sensor to a regulatory node of oxidative stress and inflammatory diseases. Redox biology 2015, 4: 296-307. Grenier JM, Wang L, Manji GA, Huang W-J, Al-Garawi A, Kelly R , et al. Functional screening of five PYPAF family members identifies PYPAF5 as a novel regulator of NF-κB and caspase-1. FEBS letters 2002, 530 (1-3) : 73-78. Evavold CL, Ruan J, Tan Y, Xia S, Wu H, Kagan JC. The Pore-Forming Protein Gasdermin D Regulates Interleukin-1 Secretion from Living Macrophages. Immunity 2018, 48 (1) : 35-44.e36. Sateriale A, Gullicksrud JA, Engiles JB, McLeod BI, Kugler EM, Henao-Mejia J , et al. The intestinal parasite Cryptosporidium is controlled by an enterocyte intrinsic inflammasome that depends on NLRP6. Proceedings of the National Academy of Sciences 2021, 118 (2) : e2007807118. Doerflinger M, Deng Y, Whitney P, Salvamoser R, Engel S, Kueh AJ , et al. Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection. Immunity 2020, 53 (3) : 533-547.e537. Bedoui S, Herold MJ, Strasser A. Emerging connectivity of programmed cell death pathways and its physiological implications. Nature Reviews Molecular Cell Biology 2020, 21 (11) : 678-695. Kayagaki N, Kornfeld OS, Lee BL, Stowe IB, O'Rourke K, Li Q , et al. NINJ1 mediates plasma membrane rupture during lytic cell death. Nature 2021, 591 (7848) : 131-136. Ramos S, Hartenian E, Santos JC, Walch P, Broz P. NINJ1 induces plasma membrane rupture and release of damage-associated molecular pattern molecules during ferroptosis. Embo j 2024. Junqueira C, Crespo Ã, Ranjbar S, Lewandrowski M, Ingber J, de Lacerda LB , et al. SARS-CoV-2 infects blood monocytes to activate NLRP3 and AIM2 inflammasomes, pyroptosis and cytokine release. Res Sq 2021. Schultze JL, Aschenbrenner AC. COVID-19 and the human innate immune system. Cell 2021, 184 (7) : 1671-1692. Wilson KP, Black J-AF, Thomson JA, Kim EE, Griffith JP, Navia MA , et al. Structure and mechanism of interleukin-lβ converting enzyme. Nature 1994, 370 (6487) : 270-275. Martinon F, Burns K, Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-β. Molecular cell 2002, 10 (2) : 417-426. McIlwain DR, Berger T, Mak TW. Caspase functions in cell death and disease. Cold Spring Harb Perspect Biol 2013, 5 (4) : a008656. Dinarello CA. Overview of the IL-1 family in innate inflammation and acquired immunity. Immunol Rev 2018, 281 (1) : 8-27. Lopez-Castejon G, Brough D. Understanding the mechanism of IL-1β secretion. Cytokine Growth Factor Rev 2011, 22 (4) : 189-195. Pang J, Vince JE. The role of caspase-8 in inflammatory signalling and pyroptotic cell death. Semin Immunol 2023, 70: 101832. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y , et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020, 395 (10223) : 497-506. Peng R, Wang-Kan X, Idorn M, Zhou FY, Orozco SL, McCarthy J , et al. ZBP1 induces inflammatory signaling via RIPK3 and promotes SARS-CoV-2-induced cytokine expression. bioRxiv 2021 : 2021.2010.2001.462460. Li S, Jiang L, Li X, Lin F, Wang Y, Li B , et al. Clinical and pathological investigation of patients with severe COVID-19. JCI Insight 2020, 5 (12). Blanco-Melo D, Nilsson-Payant BE, Liu W-C, Uhl S, Hoagland D, Møller R , et al. Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19. Cell 2020, 181 (5) : 1036-1045.e1039. Mehta P, McAuley DF, Brown M, Sanchez E, Tattersall RS, Manson JJ. COVID-19: consider cytokine storm syndromes and immunosuppression. The Lancet 2020, 395 (10229) : 1033-1034. Zhu L, Yang P, Zhao Y, Zhuang Z, Wang Z, Song R , et al. Single-Cell Sequencing of Peripheral Mononuclear Cells Reveals Distinct Immune Response Landscapes of COVID-19 and Influenza Patients. Immunity 2020, 53 (3) : 685-696.e683. Liao M, Liu Y, Yuan J, Wen Y, Xu G, Zhao J , et al. Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19. Nature Medicine 2020, 26 (6) : 842-844. Zeng J, Xie X, Feng X-L, Xu L, Han J-B, Yu D , et al. Specific inhibition of the NLRP3 inflammasome suppresses immune overactivation and alleviates COVID-19 like pathology in mice. eBioMedicine 2022, 75 . Madurka I, Vishnevsky A, Soriano JB, Gans SJ, Ore DJS, Rendon A , et al. DFV890: a new oral NLRP3 inhibitor-tested in an early phase 2a randomised clinical trial in patients with COVID-19 pneumonia and impaired respiratory function. Infection 2023, 51 (3) : 641-654. Effect of anakinra versus usual care in adults in hospital with COVID-19 and mild-to-moderate pneumonia (CORIMUNO-ANA-1): a randomised controlled trial. Lancet Respir Med 2021, 9 (3) : 295-304. Declercq J, Van Damme KF, De Leeuw E, Maes B, Bosteels C, Tavernier SJ , et al. Effect of anti-interleukin drugs in patients with COVID-19 and signs of cytokine release syndrome (COV-AID): a factorial, randomised, controlled trial. The Lancet Respiratory Medicine 2021, 9 (12) : 1427-1438. Derde LPG. Effectiveness of Tocilizumab, Sarilumab, and Anakinra for critically ill patients with COVID-19 The REMAP-CAP COVID-19 Immune Modulation Therapy Domain Randomized Clinical Trial. medRxiv 2021 : 2021.2006.2018.21259133. Hierholzer JC, Killington RA. Virus isolation and quantitation. Virology Methods Manual 1996 : 25-46. Chiou S, Al-Ani AH, Pan Y, Patel KM, Kong IY, Whitehead LW , et al. An immunohistochemical atlas of necroptotic pathway expression. bioRxiv 2023 : 2023.2010.2031.565039. Additional Declarations There is no duality of interest Supplementary Files LegendsforSupplementalFigures.docx BaderetalCellDeathDiffFigS1.png BaderetalCellDeathDiffFigS2.png BaderetalCellDeathDiffFigS3.png BaderetalCellDeathDiffFigS4.png BaderetalCellDeathDiffFigS5.png OriginalWesterns.png Cite Share Download PDF Status: Published Journal Publication published 28 Feb, 2025 Read the published version in Cell Death & Differentiation → Version 1 posted Editorial decision: Reject after peer review 28 Aug, 2024 Review # 2 received at journal 19 Aug, 2024 Review # 3 received at journal 18 Aug, 2024 Review # 1 received at journal 12 Aug, 2024 Reviewer # 3 agreed at journal 05 Aug, 2024 Reviewer # 2 agreed at journal 02 Aug, 2024 Reviewer # 1 agreed at journal 01 Aug, 2024 Reviewers invited by journal 01 Aug, 2024 Submission checks completed at journal 31 Jul, 2024 First submitted to journal 30 Jul, 2024 Unknown event 30 Jul, 2024 Editor assigned by journal 30 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4826453","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":334743667,"identity":"b1275059-221b-454c-9177-43e019b5149e","order_by":0,"name":"Marcel Doerflinger","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYDCCA0BcUSAB5VUwMBgwA2keQlrOGMC0nCFeC5TD2AbUwkBAC9/x5mMfDhhY5Mm79z5gujnvsJw5OwPjg7dtDPIGB7BrkTxzLHnGAQOJYsMzxw2Yc7cdNrZsZmA2nNvGYLgBhxaDGznGzB8MJBI3zkhjAGq5nbjhMAObNG8bAyNuLfmfGQ6AtMx/BtQy53Y9UAv7b6AWezy2MIO1zJdgA2ppuJ1gALSFGaglEZcWoF+MwVo28KQxHM459t9ww2HGZsk55ySSZ+LQAgyxxwwHKuoS57cfY3ycU5Mmb3D+8MEPb8psbPtwaEG48AAkjoCAsQFISOBWCgPyDYTVjIJRMApGwQgFAAEvYFYY55iwAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-9159-3021","institution":"Walter and Eliza Hall Institute","correspondingAuthor":true,"prefix":"","firstName":"Marcel","middleName":"","lastName":"Doerflinger","suffix":""},{"id":334743668,"identity":"70e68ac1-1166-484a-acef-9910561fc7e5","order_by":1,"name":"Stefanie M. Bader","email":"","orcid":"https://orcid.org/0000-0002-7901-9833","institution":"Walter and Eliza Hall Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Stefanie","middleName":"M.","lastName":"Bader","suffix":""},{"id":334743669,"identity":"7dae9536-6361-479f-8f8f-27dff5f94fa2","order_by":2,"name":"Lena Scherer","email":"","orcid":"","institution":"Walter and Eliza Hall Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Lena","middleName":"","lastName":"Scherer","suffix":""},{"id":334743670,"identity":"22982662-29b3-4dea-81c3-85003bb17bc9","order_by":3,"name":"Jan Schaefer","email":"","orcid":"","institution":"The Walter and Eliza Hall Institute","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Schaefer","suffix":""},{"id":334743671,"identity":"be9c23bf-bea3-458f-8b9f-dbae2d5eaac0","order_by":4,"name":"James Cooney","email":"","orcid":"","institution":"The Walter and Eliza Hall Institute","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"","lastName":"Cooney","suffix":""},{"id":334743672,"identity":"9d65fe9d-6bde-4f96-8ff8-76aca25dad99","order_by":5,"name":"Liana Mackiewicz","email":"","orcid":"","institution":"The Walter and Eliza Hall Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Liana","middleName":"","lastName":"Mackiewicz","suffix":""},{"id":334743673,"identity":"9ccfaca8-4cd9-4eaa-9fd0-3863f7c15abb","order_by":6,"name":"Merle Dayton","email":"","orcid":"","institution":"The Walter and Eliza Hall Institute","correspondingAuthor":false,"prefix":"","firstName":"Merle","middleName":"","lastName":"Dayton","suffix":""},{"id":334743674,"identity":"3ab33c71-dc3f-4e7a-a45a-961f229017ce","order_by":7,"name":"Smitha Georgy","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"Smitha","middleName":"","lastName":"Georgy","suffix":""},{"id":334743675,"identity":"905f88a7-b20c-4292-96a3-5a74f3f07711","order_by":8,"name":"Kathryn Davidson","email":"","orcid":"","institution":"The Walter and Eliza Hall Institute","correspondingAuthor":false,"prefix":"","firstName":"Kathryn","middleName":"","lastName":"Davidson","suffix":""},{"id":334743676,"identity":"9032922f-2268-42bd-81c7-7761460a81b6","order_by":9,"name":"Cody Allison","email":"","orcid":"","institution":"Walter and Eliza Hall Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Cody","middleName":"","lastName":"Allison","suffix":""},{"id":334743677,"identity":"b3459a50-c552-404b-b11a-5f8edfebbfee","order_by":10,"name":"Marco Herold","email":"","orcid":"https://orcid.org/0000-0001-7539-7581","institution":"The Walter and Eliza Hall Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Herold","suffix":""},{"id":334743678,"identity":"60c6e12c-f75c-4f2d-8588-fe5bea861bff","order_by":11,"name":"Andreas Strasser","email":"","orcid":"https://orcid.org/0000-0002-5020-4891","institution":"Walter and Eliza Hall Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Strasser","suffix":""},{"id":334743679,"identity":"5b41e570-4914-4a2b-93b3-e4cd89bf36ad","order_by":12,"name":"Marc Pellegrini","email":"","orcid":"","institution":"The Walter and Eliza Hall Institute","correspondingAuthor":false,"prefix":"","firstName":"Marc","middleName":"","lastName":"Pellegrini","suffix":""}],"badges":[],"createdAt":"2024-07-30 07:15:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4826453/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4826453/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41418-025-01459-x","type":"published","date":"2025-02-28T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63491571,"identity":"fcd622ac-1662-4b7d-9e1a-065e2fc9e0af","added_by":"auto","created_at":"2024-08-28 17:56:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4388354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInflammation driven by SARS-CoV-2 infection is independent of inflammasomes. (A-B)\u003c/strong\u003e WT and \u003cem\u003eNlrp3\u003c/em\u003e knockout (\u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e) mice were infected with 10\u003csup\u003e4\u003c/sup\u003e TCID50 of SARS-CoV-2 and monitored at 3 days post-infection (dpi) for \u003cstrong\u003e(A)\u003c/strong\u003e lung viral burden by TCID50 assay and \u003cstrong\u003e(B)\u003c/strong\u003e percent weight change compared to initial weight. Results are pooled from 2 independent experiments (n=13-17 mice per genotype). \u003cstrong\u003e(C-D) \u003c/strong\u003eWT and \u003cem\u003eNlrp1\u003c/em\u003e knockout mice (\u003cem\u003eNlrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e) were challenged intranasally with 10\u003csup\u003e4\u003c/sup\u003e TCID50/mL of SARS-CoV-2 and examined 3 days post-infection (dpi) for \u003cstrong\u003e(C) \u003c/strong\u003elung viral burden using TCID50 assay and \u003cstrong\u003e(D) \u003c/strong\u003epercent weight change compared to initial weight (n=14 mice per genotype). \u003cstrong\u003e(E-F)\u003c/strong\u003e WT and \u003cem\u003eAsc\u003c/em\u003e knockout (\u003cem\u003eAsc\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e) mice were infected with 10\u003csup\u003e4\u003c/sup\u003e TCID50 of SARS-CoV-2 and monitored at 3 dpi for \u003cstrong\u003e(E)\u003c/strong\u003e lung viral burden by TCID50 assay (n=7-8 mice per genotype) and \u003cstrong\u003e(F)\u003c/strong\u003e percent weight change compared to initial weight. Results are pooled from 2 independent experiments (n=12-15 mice per genotype).\u003cstrong\u003e (G)\u003c/strong\u003e Levels of cytokines and chemokines measured by ELISA in lung homogenates of WT and the indicated knockout animals 3 days post SARS-CoV-2 infection (n=5-10 mice per genotype). \u003cstrong\u003e(H)\u003c/strong\u003e Representative images of hematoxylin and eosin (H\u0026amp;E) and immunohistochemistry (IHC) stained lungs testing for SARS-CoV-2 nucleocapsid, F4/80 (marker of macrophages), MPO (marker of neutrophils) and CD3 (marker of T cells). Mice of the indicated genotypes were challenged intranasally with 10\u003csup\u003e4\u003c/sup\u003e TCID50/mL of SARS-CoV-2 and lungs were collected and fixed for histological analysis at 3 dpi. Histological images are representative of at least 3 animals per genotype. Black arrows point to exemplary SARS-CoV-2-positive cells. Scale bar = 250 µm. Unpaired two-tailed Student’s t test after log\u003csub\u003e10 \u003c/sub\u003etransformation (A, C, E), unpaired two-tailed Student’s t test (B, D, F),\u003cem\u003e \u003c/em\u003eWilcoxon rank-sum (G), statistical tests were performed. In (G), significance is shown relative to WT control mice (* \u0026lt; 0.05; **p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"BaderetalCellDeathDiffFig01.png","url":"https://assets-eu.researchsquare.com/files/rs-4826453/v1/91a9de3d2f350f5f0f7a98a9.png"},{"id":63491572,"identity":"6f9adce7-223b-4a2b-a142-c7597fb39806","added_by":"auto","created_at":"2024-08-28 17:56:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3895553,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGSDMD is not essential to drive cytokine release and inflammation during SARS-CoV-2 infection. (A-B)\u003c/strong\u003e WT and \u003cem\u003eGsdmd\u003c/em\u003e knockout (\u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e) mice were infected with 10\u003csup\u003e4\u003c/sup\u003e TCID50 of SARS-CoV-2 and monitored at 3 dpi for \u003cstrong\u003e(A)\u003c/strong\u003e lung viral burden by TCID50 assay and \u003cstrong\u003e(B)\u003c/strong\u003e percent weight change compared to initial weight. Results are pooled from 2 independent experiments (n=14-15 mice per genotype). \u003cstrong\u003e(C)\u003c/strong\u003e Levels of cytokines and chemokines measured by ELISA in lung homogenates of WT and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e animals at 3dpi (n=4 mice per genotype). \u003cstrong\u003e(D)\u003c/strong\u003e WT and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice were challenged intranasally with 10\u003csup\u003e4\u003c/sup\u003e TCID50/mL of SARS-CoV-2 and lungs were collected and fixed for histological analysis at 3 dpi. Representative images of hematoxylin and eosin (H\u0026amp;E) and immunohistochemistry (IHC) stained lungs testing for SARS-CoV-2 nucleocapsid, F4/80 (marker of macrophages), MPO (marker of neutrophils) and CD3 (marker of T cells) are shown. Images are representative of at least 3 animals per genotype. Black arrows point to exemplary SARS-CoV-2-positive cells. Scale bar = 250 µm. \u003cstrong\u003e(E\u003c/strong\u003e) 6-month old WT mice were infected intranasally with 10\u003csup\u003e4\u003c/sup\u003e TCID50/mL of SARS-CoV-2. At 3 dpi mice were euthanized and lungs were collected for histological analysis. Immunohistochemistry (IHC) was performed detecting SARS-CoV-2 nucleocapsid (left panel) or cleaved (i.e. activated) GSDMD (right panel). A positive control was performed for cleaved GSDMD using small intestine sections of mice infected with the enteric pathogen \u003cem\u003eCryptosporidium \u003c/em\u003e(bottom panel). \u003cstrong\u003e(F)\u003c/strong\u003e Western blot analysis of whole lungs from mock (-) or SARS-CoV-2 infected (+) WT, \u003cem\u003eAsc\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice at 3 days after intranasal SARS-CoV-2 infection (10\u003csup\u003e4\u003c/sup\u003e TCID50). Samples were probed for full-length and cleaved (i.e. activated) GSDMD. Probing for Beta-Actin is shown as loading control. Unpaired two-tailed Student’s t test after log\u003csub\u003e10 \u003c/sub\u003etransformation (A), unpaired two-tailed Student’s t test (B),\u003cem\u003e \u003c/em\u003eWilcoxon rank-sum (C), statistical tests were performed (p* \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"BaderetalCellDeathDiffFig02.png","url":"https://assets-eu.researchsquare.com/files/rs-4826453/v1/3ce9f4f0f2735ba8fe50db34.png"},{"id":63491574,"identity":"d3ad2b10-2e70-4fda-81c0-db1391bd4dd0","added_by":"auto","created_at":"2024-08-28 17:57:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":136643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInflammation driven by SARS-CoV-2 infection is independent of the non-canonical lytic forms of programmed cell death. (A-B) \u003c/strong\u003eWT\u003cem\u003e, Gsdme\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e and Gsdmd/e\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice were infected intranasally with 10\u003csup\u003e4\u003c/sup\u003e TCID50 of SARS-CoV-2 and monitored at 3 dpi for \u003cstrong\u003e(A) \u003c/strong\u003elung viral burden using TCID50 assay and \u003cstrong\u003e(B) \u003c/strong\u003epercent weight change compared to initial weight (n=4-7 per genotype). \u003cstrong\u003e(C-D) \u003c/strong\u003eWT and\u003cem\u003e Gsdma/c/e\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice were challenged intranasally with 10\u003csup\u003e4\u003c/sup\u003e TCID50 of SARS-CoV-2 and monitored at 3 dpi for \u003cstrong\u003e(C) \u003c/strong\u003elung viral burden using TCID50 assay and \u003cstrong\u003e(D) \u003c/strong\u003epercent weight change compared to initial weight (n=11-17 mice per genotype). \u003cstrong\u003e(E-F)\u003c/strong\u003e WT and \u003cem\u003eGsdmd/Mlkl\u003c/em\u003e knockout (\u003cem\u003eGsdmd/Mlkl\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e) mice were infected with 10\u003csup\u003e4\u003c/sup\u003e TCID50 of SARS-CoV-2 and monitored at 3 dpi for \u003cstrong\u003e(E)\u003c/strong\u003e lung viral burden by TCID50 assay and \u003cstrong\u003e(F)\u003c/strong\u003e percent weight change compared to initial weight (n=14-19 mice per genotype). \u003cstrong\u003e(G-H)\u003c/strong\u003e WT and \u003cem\u003eNinj1\u003c/em\u003e knockout (\u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e) mice were infected with 10\u003csup\u003e4\u003c/sup\u003e TCID50 of SARS-CoV-2 and monitored at 3 dpi for \u003cstrong\u003e(G)\u003c/strong\u003e lung viral burden by TCID50 assay and \u003cstrong\u003e(H)\u003c/strong\u003e percent weight change compared to initial weight (n=8-11 mice per gentotype). Data are presented as mean ± SD\u003cem\u003e.\u003c/em\u003e Statistical analyses were performed by using one-way ANOVA with multiple comparisons after log\u003csub\u003e10 \u003c/sub\u003etransformation (A), one-way ANOVA with multiple comparisons (B), unpaired two-tailed Student’s t test after log\u003csub\u003e10\u003c/sub\u003e transformation (C, E, G) and unpaired two-tailed Student’s t test (D, F, H).\u0026nbsp;\u003c/p\u003e","description":"","filename":"BaderetalCellDeathDiffFig03.png","url":"https://assets-eu.researchsquare.com/files/rs-4826453/v1/5a8630ac13f7347370208314.png"},{"id":63491817,"identity":"713ed98c-81b7-4b51-8496-a30515882d60","added_by":"auto","created_at":"2024-08-28 18:04:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3234387,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytokine processing during SARS-CoV-2 infection occurs independently of caspases-1/-11/-12. (A-D) \u003c/strong\u003eWT and \u003cem\u003eCaspase-1,-11,-12 \u003c/em\u003etriple knockout (\u003cem\u003eC1/11/12\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e) mice were infected with 10\u003csup\u003e4\u003c/sup\u003e TCID50 of SARS-CoV-2 and monitored at 3 days post-infection (dpi) for \u003cstrong\u003e(A)\u003c/strong\u003e lung viral burden by TCID50 assay and \u003cstrong\u003e(B)\u003c/strong\u003e percent weight change compared to initial weight (n=12-15 mice per genotype). \u003cstrong\u003e(C)\u003c/strong\u003e Levels of cytokines and chemokines were measured by ELISA in lung homogenates of WT and knockout animals at 3 dpi (n=9 mice per genotype). Data are presented as mean ± SD. \u003cstrong\u003e(D) \u003c/strong\u003eHistological analysis of fixed lungs. Representative images of hematoxylin and eosin (H\u0026amp;E) stained and immunohistochemistry (IHC) stained lungs testing for SARS-CoV-2 nucleocapsid, F4/80 (marker of macrophages), MPO (marker of neutrophils) and CD3 (marker of T cells) are shown. Images are representative of at least 3 animals per genotype. Black arrows point to exemplary SARS-CoV-2-positive cells. Scale bar = 250 µm. Statistical analyses were performed by unpaired two-tailed Student’s t test after log10 transformation (A), unpaired two-tailed Student’s t test (B), Wilcoxon rank-sum (C) (***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"BaderetalCellDeathDiffFig04.png","url":"https://assets-eu.researchsquare.com/files/rs-4826453/v1/86debd878bd7d4507dc36a87.png"},{"id":63492208,"identity":"bfb44802-290c-48cb-b093-115642472f1f","added_by":"auto","created_at":"2024-08-28 18:13:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3259542,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-1b, but not IL-18 knockout mice are protected from severe SARS-CoV-2 disease and have reduced viral burdens. (A-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e)\u003c/strong\u003e WT and \u003cem\u003eIl-1b\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice were infected with 10\u003csup\u003e4\u003c/sup\u003e TCID50 of SARS-CoV-2 and monitored at 3 dpi for \u003cstrong\u003e(\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e)\u003c/strong\u003e lung viral burden by TCID50 assay and \u003cstrong\u003e(\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e)\u003c/strong\u003e percent weight change compared to initial weight (n=15-19 mice per genotype). \u003cstrong\u003e(\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e)\u003c/strong\u003e Examination of lung pathology via histological assessment of fixed lungs.\u003cem\u003e \u003c/em\u003eRepresentative images of hematoxylin and eosin (H\u0026amp;E) and immunohistochemistry (IHC) stained lungs testing for SARS-CoV-2 nucleocapsid, F4/80 (marker of macrophages), MPO (marker of neutrophils) and CD3 (marker of T cells). Histological images are representative of at least 3 animals per genotype. Black arrows point to exemplary SARS-CoV-2-positive cells. Scale bar = 250 µm.\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e(D) \u003c/strong\u003eHistological changes based on H\u0026amp;E staining were graded by an American board-certified pathologist. The scores are based on the percentage of lesions: 0 = normal, 1 \u0026lt;10%, 2 = 10-25%, 3 = 25-50%, 4 \u0026gt;50%. Sum of the histological scores of WT and \u003cem\u003eIl-1b\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice are shown on the right. \u003cstrong\u003e(E-F)\u003c/strong\u003e WT and \u003cem\u003eIl-1r\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice were infected with 10\u003csup\u003e4\u003c/sup\u003e TCID50 of SARS-CoV-2 and monitored at 3 dpi for \u003cstrong\u003e(\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e)\u003c/strong\u003e lung viral burden by TCID50 assay and \u003cstrong\u003e(\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e)\u003c/strong\u003e percent weight change compared to initial weight (n=\u003cem\u003e11-16\u003c/em\u003e mice per genotype).\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eG\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) \u003c/strong\u003eLevels of cytokines and chemokines were measured by ELISA in lung homogenates of WT and knockout animals 3 days post SARS-CoV-2 infection (n=8-18 mice per genotype). \u003cstrong\u003e(\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) \u003c/strong\u003eLevels of cytokines and chemokines were measured by ELISA in lung homogenates of WT and knockout animals \u003cem\u003e6\u003c/em\u003e days post SARS-CoV-2 infection (n=8-18 mice per genotype). Data are presented as mean ± SD. Statistical analyses were performed by unpaired two-tailed Student’s t test after log\u003csub\u003e10 \u003c/sub\u003etransformation (A, \u003cem\u003eE\u003c/em\u003e), unpaired two-tailed Student’s t test (B, \u003cem\u003eF\u003c/em\u003e), Wilcoxon rank-sum (\u003cem\u003eG, H\u003c/em\u003e);\u003cem\u003e \u003c/em\u003eIn (G, H), significance is shown relative to WT mice (* \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"BaderetalCellDeathDiffFig05.png","url":"https://assets-eu.researchsquare.com/files/rs-4826453/v1/e0ed3f7e4134e185ba1e4af8.png"},{"id":63491577,"identity":"189038e1-5224-4533-a7cc-003eeabad7fc","added_by":"auto","created_at":"2024-08-28 17:57:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":155866,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAge driven SARS-CoV-2 disease can be reduced by IL1-b\u003c/strong\u003e \u003cem\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edepletion. (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) \u003c/strong\u003eWT and \u003cem\u003eIl-1b\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eadult mice (adult = 11-12 weeks) were intranasally infected with 10\u003csup\u003e4\u003c/sup\u003e TCID50 SARS-CoV-2 and monitored to determine the proportion of mice that became severely ill, reaching predetermined ethical endpoint (n=10-12 mice per genotype). \u003cstrong\u003e(B-E)\u003c/strong\u003e Aged (\u0026gt;6 month-old) WT and Il-1b\u003csup\u003e-/- \u003c/sup\u003emice were infected with 10\u003csup\u003e4\u003c/sup\u003e TCID50 of SARS-CoV-2 and monitored for \u003cstrong\u003e(B)\u003c/strong\u003e proportion of mice that became severely ill, reaching predetermined ethical endpoint (n=9 mice per genotype), \u003cstrong\u003e(C)\u003c/strong\u003e lung viral burden at 3 dpi by TCID50 assay and \u003cstrong\u003e(D)\u003c/strong\u003e percent weight change at 3 dpi compared to initial weight (n=6-10 mice per genotype). \u003cstrong\u003e(E) \u003c/strong\u003eLevels of cytokines and chemokines were measured by ELISA in lung homogenates of WT and knockout animals 6 days post SARS-CoV-2 infection (n=3-4 mice per genotype). \u003cstrong\u003e(F) \u003c/strong\u003eWT and \u003cem\u003eIl-1b\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eadult mice (adult = 11-12 weeks) were treated with anti-Il-1b monoclonal antibodies and intranasally infected with 10\u003csup\u003e4\u003c/sup\u003e TCID50 SARS-CoV-2 and monitored to determine the proportion of mice that became severely ill, reaching predetermined ethical endpoint (n=4-6 mice per genotype and treatment). Data are presented as mean ± SD. Statistical analyses were performed by Log-rank Mantel–Cox test (A, B, F), unpaired two-tailed Student’s t test after log\u003csub\u003e10 \u003c/sub\u003etransformation (C), unpaired two-tailed Student’s t test (D) and Wilcoxon rank-sum (E). In (E), significance is shown relative to WT mice (* \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"BaderetalCellDeathDiffFig06.png","url":"https://assets-eu.researchsquare.com/files/rs-4826453/v1/a5fc01f6c9614de297b14bcb.png"},{"id":77399128,"identity":"293b7370-d8b7-4d6d-90ce-3f8a3053af20","added_by":"auto","created_at":"2025-02-28 08:09:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16432290,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4826453/v1/48e9d77e-0a23-476f-be37-8c5d6e36a4a5.pdf"},{"id":63491569,"identity":"44d4d9cc-e50a-4f2a-b34b-01b80e3a8641","added_by":"auto","created_at":"2024-08-28 17:56:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":84195,"visible":true,"origin":"","legend":"","description":"","filename":"LegendsforSupplementalFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-4826453/v1/f1e86ac53c0b1807f7025951.docx"},{"id":63491570,"identity":"a38b578a-ea3c-4bcf-adfd-b8b6538681d3","added_by":"auto","created_at":"2024-08-28 17:56:59","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":129310,"visible":true,"origin":"","legend":"","description":"","filename":"BaderetalCellDeathDiffFigS1.png","url":"https://assets-eu.researchsquare.com/files/rs-4826453/v1/9fef2b04a01cea18ad382e17.png"},{"id":63491818,"identity":"06cb0b37-a8e5-45ed-bc1a-a67c84c910e7","added_by":"auto","created_at":"2024-08-28 18:05:00","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":96195,"visible":true,"origin":"","legend":"","description":"","filename":"BaderetalCellDeathDiffFigS2.png","url":"https://assets-eu.researchsquare.com/files/rs-4826453/v1/bf98ae5c646b6a4de6f45678.png"},{"id":63492649,"identity":"a205b59a-9653-4930-b497-fe6b8a9730f0","added_by":"auto","created_at":"2024-08-28 18:21:00","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":102679,"visible":true,"origin":"","legend":"","description":"","filename":"BaderetalCellDeathDiffFigS3.png","url":"https://assets-eu.researchsquare.com/files/rs-4826453/v1/3d8354fd0692acb1cbffc9bf.png"},{"id":63491580,"identity":"0143060c-edb9-44e0-887c-0afddbba2763","added_by":"auto","created_at":"2024-08-28 17:57:00","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":234784,"visible":true,"origin":"","legend":"","description":"","filename":"BaderetalCellDeathDiffFigS4.png","url":"https://assets-eu.researchsquare.com/files/rs-4826453/v1/526a2b390797a24e34f63a3d.png"},{"id":63491582,"identity":"5a42e281-7e4e-4bad-90c1-dffd99a8fb9d","added_by":"auto","created_at":"2024-08-28 17:57:01","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":84592,"visible":true,"origin":"","legend":"","description":"","filename":"BaderetalCellDeathDiffFigS5.png","url":"https://assets-eu.researchsquare.com/files/rs-4826453/v1/c7172b68abf6bfccfe104cde.png"},{"id":63491581,"identity":"8cf0aa0b-a61d-4dcb-afaa-054ee23ef160","added_by":"auto","created_at":"2024-08-28 17:57:00","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":792678,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalWesterns.png","url":"https://assets-eu.researchsquare.com/files/rs-4826453/v1/c3dc8717f877f3183d30bcfc.png"}],"financialInterests":"There is no duality of interest","formattedTitle":"IL-1b drives SARS-CoV-2 disease in vivo, independently of the inflammasome and pyroptotic signalling","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSARS-CoV-2 causes a spectrum of symptoms, ranging from mild resolving illness to severe COVID-19 and mortality, but the host factors contributing to the spectrum of SARS-CoV-2 associated disease are yet to be fully characterized. Unravelling the molecular mechanisms that contribute to the dysregulation and aberrant activation of the immune system following SARS-CoV-2 infection is paramount in formulating effective strategies to mitigate the morbidity and mortality caused by this pandemic virus.\u003c/p\u003e \u003cp\u003eEmerging evidence suggests that inflammation and certain programmed (regulated) cell death processes play pivotal roles in the pathogenesis of COVID-19 (reviewed in \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e). Lytic forms of cell death, such as necroptosis and pyroptosis, have been linked to dysregulation of the immune system associated with COVID-19 through the release of damage associated molecular patterns (DAMPs), cytokines and immune activation and inflammation linked to cell membrane disruption \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Pyroptosis is a distinct form of programmed (regulated) cell death characterized by features of necrosis and an inflammatory response. It is triggered by the activation of inflammatory caspases-1 (human and mouse), -4 (human), -5 (human), and/or -11 (mouse) \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, which proteolytically activate the pyroptotic pore-forming effector protein gasdermin D (GSDMD) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The activation of the pyroptotic caspases is regulated by inflammasomes in response to certain infections or specific host-derived proteins and crystals \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The inflammasome sensors nucleotide oligomerization domain (NOD)-like, leucine-rich repeat (LRR) receptor, absent in melanoma 2 (AIM2), and Pyrin require the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) to form a functional complex to activate caspase-1. Conversely, NLRC4 and NLRP1b inflammasomes can directly bind to and activate caspase-1 without ASC. In addition to proteolytically activating GSDMD, caspase-1 also cleaves the pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18) into their bioactive forms that can be secreted by cells. Upon assembly, the negatively charged GSDMD pore allows the rapid release of these proteolytically processed pro-inflammatory cytokines into the extracellular milieu \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The secreted bioactive form of IL-1β promotes the recruitment of innate immune cells and modulates the activity of adaptive immune cells. Mature IL-18 is involved in the production of interferon-gamma (IFN-γ) and promotes Th1 and Th2 T cell immune responses \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNecroptosis is another lytic form of programmed (regulated) cell death that triggers the release of DAMPs to promote immune activation, cytokine release and inflammation \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Necroptosis can occur downstream of death receptor signaling, such as ligation of TNF receptor 1 (TNFR1) \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, through the activation of cytoplasmic nucleic acid sensor Z-DNA binding protein 1 (ZBP1) \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and downstream of Toll-like receptors (TLR) through the stimulation of Toll/IL-1 receptor domain-containing adapter-inducing interferon-b (TRIF) \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. These signals lead to RIPK3 activation, typically in the absence of proteolytically active caspase-8 \u003csup\u003e19\u003c/sup\u003e. Once activated, RIPK3 phosphorylates the pseudokinase mixed lineage kinase domain-like (MLKL) causing its oligomerization and translocation to the plasma membrane, where it causes membrane rupture and a necrotic cell death \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLytic forms of programmed (regulated) cell death have been proposed to contribute to COVID-19 pathogenesis, but genetic in vivo evidence is lacking \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In our recent study utilizing gene-targeted mouse models of mild and severe COVID-19, we challenged the notion that necroptosis was a significant driver of this disease \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. This highlighted the importance of further animal studies to dissect complex disease pathogenesis involving several cell types and organ systems. Here, we utilized diverse mouse models to investigate the physiological relevance of lytic programmed cell death pathways and their contribution to inflammation during severe SARS-CoV-2 disease. We found that IL-1b drives SARS-CoV-2 disease severity, independently of pyroptosis and the inflammasome.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eLoss of inflammasome signaling does not prevent pro-inflammatory cytokine release and disease following SARS-CoV-2 infection in vivo.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the role of inflammasome pathways during severe SARS-CoV-2 infection we used a previously characterised mouse adapted strain (P21) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. This strain was derived from a clinical isolate that was passaged 21 times in mice to allow adaptations that caused severe disease in WT mice, characterized by weight loss and increased levels of pro-inflammatory cytokines \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. During SARS-CoV-2 infection, activation of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome pathway is thought to contribute to the release of bioactive IL-1β and IL-18, cytokines associated with severe COVID-19 \u003csup\u003e5, 6, 26\u003c/sup\u003e. We infected NLRP3 deficient mice (\u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) with P21 to explore this hypothesis. Interestingly, three days post infection (dpi), \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice displayed similar viral burdens to WT controls and did not show changes in weight loss, an established marker of disease severity in P21-infected mice at this time point \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e. NLRP1 is also a potent activator of inflammation \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, however, we observed that severe disease and lung viral burdens caused by SARS-CoV-2 infection were not affected by deletion of NLRP1 in mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D\u003cb\u003e)\u003c/b\u003e. Many of the inflammasomes, including NLRP1, NLRP3, AIM2 and others, are dependent on the adapter protein ASC \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Surprisingly, ASC-deficient mice (\u003cem\u003eAsc\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) exhibited viral burdens and weight loss similar to infected WT animals \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F\u003cb\u003e)\u003c/b\u003e. This demonstrates that ASC-dependent inflammasomes are not essential for SARS-CoV-2 driven pathogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next determined the abundance of 25 cytokines and chemokines in lung homogenates from infected gene-targeted mice to understand if lytic programmed cell death and canonical inflammasome activation contributed in a more subtle way to SARS-CoV-2 disease pathogenesis. Interestingly, neither of the inflammasome knockout mice we investigated displayed a reduction in IL-18, while only \u003cem\u003eAsc\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals had slightly diminished IL-1β levels in their lungs upon SARS-CoV-2 infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). While \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eNlrp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAsc\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e inflammasome knockout mice all showed a reduction in the levels of TNF, MIP1α and IL-17a, variable differences in the levels of other cytokines were observed depending on the particular inflammasome deficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Overall, these results suggest that while NLRP1/3 and ASC do affect cytokine release during SARS-CoV-2 infection, this was not sufficient to impact disease severity (weight loss) or viral burdens in vivo compared to WT mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo better understand disease in SARS-CoV-2 P21 infected gene-targeted animals, we compared lung histology of knockouts and WT mice at 3 dpi. Lung sections were stained with hematoxylin and eosin (H\u0026amp;E) and analyzed by a board-certified pathologist. At 3 dpi, WT mice displayed multifocal, acute alveolitis (sometimes necrotizing), multifocal pneumonia, as well as moderate to severe acute multifocal perivasculitis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Gene-targeted mice showed similar manifestations of disease with interstitial pneumonia, moderate to severe multifocal perivasculitis and acute alveolitis. Immunohistochemical (IHC) staining for SARS-CoV-2 nucleocapsid showed that the virus localized to the bronchiolar and alveolar epithelium, as well as macrophages in both WT and all gene-targeted mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Staining for myeloperoxidase (MPO), CD3 and F4/80 revealed a similar number of myeloid cell and T cell infiltrates in WT and all knockout mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Collectively, these findings show that while canonical ASC-dependent inflammasome pathways do play some role in the cytokine responses associated with severe SARS-CoV-2 pathogenesis, they do not significantly impact disease outcomes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe pyroptosis effector Gasdermin D is not required for pro-inflammatory cytokine release during SARS-CoV-2 infection in vivo.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRegardless of the upstream mechanisms responsible for the activation of pyroptosis and cytokine production, the cellular release of cytokines and cell lysis is thought to be primarily dependent on the activation of the pore forming protein GSDMD \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. We found that GSDMD deficient animals (\u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) showed similar viral burdens and weight loss compared to WT mice upon infection with SARS-CoV-2 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e. Analysis of 25 cytokines and chemokines in lung homogenates from infected \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed that IL-1β levels were slightly reduced compared to WT animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Interestingly, however, the levels of IL-18 and IL-23 were increased in these animals upon infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. These data indicate that GSDMD does not exert an essential role in the pathogenic pro-inflammatory cytokine release during SARS-CoV-2 infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further compared lung histology of \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and WT mice at 3 dpi. Lung sections were stained with hematoxylin and eosin (H\u0026amp;E) and analyzed by a board-certified pathologist. Three days post SARS-CoV-2 infection, both \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and WT mice displayed multifocal, acute alveolitis, multifocal pneumonia, as well as moderate to severe acute multifocal perivasculitis. IHC staining for SARS-CoV-2 nucleocapsid showed that virus localized to the bronchiolar and alveolar epithelium in both WT and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. MPO, CD3 and F4/80 staining revealed a similar pattern of myeloid cell and T cell infiltrates in WT and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eWe next explored if severe SARS-CoV-2 disease was linked to cleavage (i.e. activation) of GSDMD in WT animals. In line with our previous results, no cleaved GSDMD was detected by IHC in WT animals with severe SARS-CoV-2 disease confirmed with nucleocapsid staining \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. As a positive control, to ensure that our antibody detects cleaved GSDMD, we stained small intestine tissue from mice infected with the enteric pathogen \u003cem\u003eCryptosporidium\u003c/em\u003e, which is known to trigger pyroptosis and cause Gasdermin D cleavage \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE \u003cb\u003ebottom panel)\u003c/b\u003e. To further investigate GSDMD cleavage during SARS-CoV-2 infection, we performed Western blot analysis of whole lung tissue extracts. Cleavage (i.e. activation) of GSDMD in the lungs of infected animals could not be detected in WT and in \u003cem\u003eAsc\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, original westerns can be found in the supplemental information\u003cb\u003e)\u003c/b\u003e. Together with our genetic investigations, this indicates that the inflammation associated with severe SARS-CoV-2 disease does not lead to or require the activation of GSDMD in vivo.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRegulators of non-canonical pyroptosis are dispensable for SARS-CoV-2 infection induced pathogenesis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe further investigated the role of non-canonical pyroptosis pathways, including those involving GSDME activation. We infected \u003cem\u003eGsdme\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice with SARS-CoV-2 but found no difference in disease phenotype compared to control WT animals. Furthermore, compound loss of GSDMD/E (\u003cem\u003eGsdmd/e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) and GSDMA/C/E (\u003cem\u003eGsdma/c/e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) did not alter disease phenotypes compared to SARS-CoV-2 infected WT mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFunctional overlap of programmed cell death pathways, particularly in response to infection, has emerged as a paradigm in recent years \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. To examine if necroptosis could be compensating for the loss of pyroptosis, we infected mice lacking essential effectors of both of these lytic programmed (regulated) cell death processes, namely GSDMD and MLKL, with SARS-CoV-2. Compound mutant \u003cem\u003eGsdmd/Mlkl\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed similar disease phenotypes compared to infected WT animals \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F\u003cb\u003e)\u003c/b\u003e. We extended our investigation to examine mice that were deficient in NINJ1, a protein that drives plasma membrane rupture and release of high molecular weight DAMP downstream of pyroptosis, apoptosis, ferroptosis and accidental cell lysis, but not necroptosis \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eNinj1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed similar disease phenotypes upon SARS-CoV-2 infection as WT mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCytokine processing during SARS-CoV-2 infection occurs independently of caspases-1/-11/-12.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eInflammasome and pyroptosis signaling have been implicated in the pathogenesis of COVID-19 disease because of their known link to IL-1β and IL-18 release. Both of these cytokines have been associated with increased severity of this disease \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The prevailing dogma is that these cytokines are released downstream of inflammasome signaling and processed into their bioactive forms by caspases-1, -11 (and possibly \u0026minus;\u0026thinsp;12) \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. We therefore used gene-targeted mice lacking all these caspases (\u003cem\u003eC1/11/12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e to understand their overall contribution to severe SARS-CoV-2 disease in our mouse model. Infected \u003cem\u003eC1/11/12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice showed similar viral burdens and weight loss compared to infected WT mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B\u003cb\u003e).\u003c/b\u003e Analysis of 25 cytokines and chemokines showed that upon SARS-CoV-2 infection, \u003cem\u003eC1/11/12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice mounted a similar cytokine/chemokine response to infected WT controls, with only GROα being slightly elevated in knockout mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. This finding indicates that processing and release of pro-inflammatory IL-1β and IL-18 during SARS-CoV-2 infection can occur independently of the three inflammatory caspases-1/-11/-12. Histological examination confirmed similar lung pathology and immune cell infiltrates in SARS-CoV-2 infected \u003cem\u003eC1/11/12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and WT mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e).\u003c/b\u003e These results show that all components of the pyroptosis machinery, from ASC-dependent inflammasome sensors to catalytic pro-inflammatory caspases and the pore-forming effector proteins, are not essential to drive SARS-CoV-2 viremia or disease manifestations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIL1-b, but not IL-18 contributes to severe SARS-CoV-2-mediated disease.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur results show that SARS-CoV-2 driven inflammation and cytokine release in vivo are independent of key components of the pyroptosis machinery. Several studies reported an association between IL-1β and IL-18 serum levels and severe COVID-19 disease \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Pyroptosis and caspases-1/-11/-12 are often linked to IL-1β and IL-18 processing. So, if these inflammatory cytokines are critically involved in severe COVID-19 disease, the results from our studies using gene targeted mice indicate a disconnect between the processing and release of IL-1β and IL-18 and pyroptosis. To confirm that one or both of these cytokines do contribute to SARS-CoV-2 driven disease in our animal model, we infected IL-18 deficient (\u003cem\u003eIl-18\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice. \u003cem\u003eIl-18\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were not significantly protected from SARS-CoV-2 infection displaying similar disease compared to infected WT animals \u003cb\u003e(Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eA-B)\u003c/b\u003e. In contrast, \u003cem\u003eIl-1β\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice had significantly lower viral burdens in the lungs, and exhibited less weight loss compared to infected WT animals \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo better understand the cellular host responses linked to better outcomes in SARS-CoV-2 infected \u003cem\u003eIl-1β\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, we examined lung histology at 3 dpi. Lung sections were stained with hematoxylin and eosin (H\u0026amp;E) and analyzed by a board-certified pathologist. Three days post SARS-CoV-2 infection, WT mice had mild to severe acute multifocal perivasculitis, interstitial pneumonia and necrotizing alveolitis. Infected IL-1β deficient mice also showed perivasculitis, moderate interstitial pneumonia, but no alveolitis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D\u003cb\u003e)\u003c/b\u003e. IHC staining for SARS-CoV-2 nucleocapsid showed that virus localized to the bronchiolar and alveolar epithelium and macrophages in both WT and \u003cem\u003eIl-1β\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. Histological scoring showed that in contrast to WT controls, \u003cem\u003eIl-1β\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice did not present with signs of pleural mesothelial hyperplasia, iBALT or proteinaceous debris in the air space. These differences amounted to an overall significantly reduced histological score of disease \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D\u003cb\u003e)\u003c/b\u003e. These findings in gene-targeted mice corroborate the human correlative data and prove for the first time in an in vivo setting that IL-1β plays a critical pathogenic role during severe SARS-CoV-2 induced disease.\u003c/p\u003e \u003cp\u003eInflammatory IL-1 signaling is activated through binding of IL-1 family cytokines to the membrane bound, type I interleukin-1 receptor (IL-1R) \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Interestingly, while \u003cem\u003eIl-1r\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals were also protected from severe SARS-CoV-2 induced disease, as measured by a reduction in weight loss, these animals did not have decreased viral burdens but were similar to WT mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F\u003cb\u003e)\u003c/b\u003e. To identify the effects of the absence of critical components of the IL-1 pathway on the pro-inflammatory cytokine response, we quantified the levels of 25 cytokines and chemokines in the lungs of WT, \u003cem\u003eIl-1β\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eIl-1r\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals at 3 dpi. The absence of IL-1β led to reductions in GM-CSF, IL17a and IL-5. \u003cem\u003eIl-1r\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals tended to express lower levels of a wider range of cytokines and chemokines compared to IL-1β deficient mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003eS5\u003c/span\u003eC\u003cb\u003e).\u003c/b\u003e However, the reduction in cytokines was of longer duration, continuing for 6 days post-infection, and was more profound at later time points in IL-1β deficient mice compared to WT animals and \u003cem\u003eIl-1r\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eH and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003eS5\u003c/span\u003eD\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eAge related severity of SARS-CoV-2 disease can be reduced by the absence of IL1-b.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSimilar to COVID-19 in humans, SARS-CoV-2 P21 driven disease in mice becomes more pronounced with increased age \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Upon infection, the majority of infected WT animals\u0026thinsp;\u0026gt;\u0026thinsp;10 weeks-old lose more than 20% body weight, reaching ethical end-point by 4\u0026ndash;6 dpi \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. To test whether the absence of IL-1β could protect against aged-associated severity of SARS-CoV-2 driven disease, we infected eleven-week-old \u003cem\u003eIl-1β\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and control WT mice. IL-1β deficient mice were more likely to survive SARS-CoV-2 infection compared to WT controls \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, however, this survival advantage was attenuated in 6-month-old infected mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Despite this, 6-month-old IL-1β deficient mice showed significantly lower viral burdens in the lungs at the peak of infection (3 dpi) compared to WT controls \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D\u003cb\u003e).\u003c/b\u003e Interestingly, in aged animals (\u0026gt;\u0026thinsp;6 months), while multiple cytokines were decreased at 3 dpi, the only cytokine found to be significantly reduced in lung homogenates from \u003cem\u003eIl-1β\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice was IL-1β itself \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eE\u003cb\u003e).\u003c/b\u003e To analyse the potential of anti-IL-1β therapeutics against severe COVID-19 in our mouse model, we administered a single dose of IL-1β neutralizing monoclonal antibodies to 10- to 12-week-old WT mice on the day of infection. Inhibition of IL-1β alone was not sufficient to prevent mortality in P21 infected animals although a trend was observed. Thus, more profound inhibition of IL-1β and concomitant inhibition of other pathogenic cytokines should be explored.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCOVID-19 morbidity and mortality are driven by dysregulated host cytokine storms. The pro-inflammatory cytokines IL-18 and IL-1b have been implicated in disease pathogenesis and, based on in vitro experiments, it has been proposed that NLRP3 inflammasome activation and pyroptosis are responsible for the exaggerated production of IL-18 and IL-1b. We confirmed in our mouse model that IL-1 signaling contributes to severe SARS-CoV-2 disease through IL-1b, whereas IL-18 plays only a negligible role. Furthermore, our data challenge the notion that NLRP3, NLRP1 and other ASC-dependent inflammasomes are essential for IL-1b-driven SARS-CoV-2 disease pathogenesis. Although deletion of ASC and GSDMD slightly reduced the levels of IL-1b in the lungs of SARS-CoV-2 infected mice compared to WT controls, this reduction was not sufficient to alter disease outcomes. Collectively, we provide genetic in vivo evidence that inflammasomes and GSDMD are not required for maximal lung IL-1b levels and they are not essential for pathogenesis and viral dissemination. This indicates that inhibitors of inflammasomes are unlikely to offer therapeutic benefit in severe COVID-19.\u003c/p\u003e \u003cp\u003eWe showed that effectors of pyroptosis, including GSDMD, GSDME and GSDMA/C/E, are not required to induce severe SARS-CoV-2 disease in our model. As we have previously shown, necroptosis was also dispensable for causing severe disease \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and combined deletion of genes of the essential effectors for pyroptosis and necroptosis (\u003cem\u003eGsdmD/Mlkl\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) did not diminish pathology. We further confirmed that lytic programmed cell death does not play a prominent role in SARS-CoV-2 driven disease using mice lacking NINJ1, which is essential for plasma membrane lysis in pyroptosis, ferroptosis and late-stage apoptosis \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Importantly, caspases-1, -11 and \u0026minus;\u0026thinsp;12 were not required for the release of IL-1b during severe SARS-CoV-2 disease. Collectively, these findings indicate that processes independent of lytic programmed cell death must facilitate IL-1β processing and release during SARS-CoV-2 infection. Several potential mechanisms have been identified (reviewed in \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e), suggesting that these cytokines can be downstream of pathways other than canonical inflammasome and gasdermin effected pyroptosis (or similar lytic cell death). Recent studies have broadened understanding of the caspases capable of proteolytically activating IL-1b and regulating cytokine transcription \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, making it temping to speculate that proteolytic processing of IL-1b/a during SARS-CoV-2 infection might be mediated by caspases other than caspases-1/11/12.\u003c/p\u003e \u003cp\u003eAlthough studies have suggested that NLPR3 activation and GSDMD serve as predictive markers for severity of COVID-19 \u003csup\u003e4, 38\u003c/sup\u003e, these studies were conducted in vitro only. Published in vivo studies using \u003cem\u003eNlrp3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e animals, utilized adeno-associated virus (AAV) to deliver hACE2 prior to SARS-CoV-2 infection \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, an additional process that adds complexities, such as triggering inflammatory pathways. To date, clinical trials testing NLRP3 inhibitors in COVID-19 patients have been inconclusive, only showing subtle changes in mortality and not being successful in improving APACHE II scores compared to treatment with standard of care alone \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Our data complement this work indicating that inflammasomes and pyroptosis are not promising targets for the treatment of severe COVID-19.\u003c/p\u003e \u003cp\u003eGiven the critical role of IL-1b in severe disease in our SARS-CoV-2 infection model we compared the effects of IL-1b deficiency to the loss of its cognate receptor IL-1R during SARS-CoV-2 infection. Although there were similarities in disease amelioration compared to infected WT mice, loss of IL-1b caused a more profound drop in inflammatory cytokine levels at later time points during infection compared to mice deficient in its cognate receptor. This indicates that complete ablation of all IL-1R binding cytokines is not more beneficial than IL-1b deletion alone. This conclusion is further supported by the lack of a significant reduction in viral burdens in IL-1R deficient mice compared to WT controls. The IL1R pathway involves signaling through a range of factors, including IL-1a, IL-1Ra, IL-33 and IL-18, some of which may facilitate immune-mediated clearance. Given the importance of IL-1b in promoting severe SARS-CoV-2 induced disease in our model and mounting evidence for a central role of this cytokine in driving severe human COVID-19 disease \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, we further investigated IL-1b's contributions in age-driven disease. We have previously shown that genetic deletion of TNF prevented lethality in aged animals infected with P21, but only partially, indicating that TNF is not solely responsible for severe age-related COVID-19 disease. Here, we show that IL-1b is another key cytokine that contributes significantly to severe SARS-CoV-2 induced disease. The deletion of the \u003cem\u003eIl-1b\u003c/em\u003e gene prevented mortality in young adult animals and attenuated disease in aged mice (\u0026gt;\u0026thinsp;6 months).\u003c/p\u003e \u003cp\u003eSeveral clinical trials have examined the therapeutic benefit of inhibiting IL-1 signaling during severe COVID-19. These studies used anakinra, an IL-1R antagonist. Our data, derived from comparing IL-1b knockout with IL-1R knockout mice, revealed that removal of IL-1b alone leads to a more prominent reduction of pro-inflammatory cytokines than loss of IL-1R. This is in line with results from clinical trials using Anakinra that did not show significant efficacy in humans \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Here, we assessed the impact of IL-1b neutralising monoclonal antibodies. We could not recapitulate the survival advantage conferred by genetic loss of IL-1b using a validated IL-1b neutralizing antibody. Several factors could contribute to this discrepancy, including inadequate neutralization of IL-1b within the relevant tissue(s). Another therapeutic avenue would be to target the upstream mechanisms responsible for IL-1β production. Here, we have ruled out some candidate pathways, including various forms of lytic programmed cell death, such as pyroptosis and necroptosis.\u003c/p\u003e \u003cp\u003eIn conclusion, we have shown a critical pathogenic role for IL-1β during severe SARS-CoV-2 disease in our mouse model. We provide evidence that pyroptotic cell death is not a major driver responsible of IL-1b production. This underscores the importance of further work to determine what process(es) promote(s) IL-1b release to fuel the cytokine storm driven disease in our SARS-CoV-2 infected mice. Identification of the critical upstream signaling pathways may identify novel therapeutic targets that could underpin novel therapeutic strategies to abrogate IL-1b production and thus overcome potential issues with inadequate blockade.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eMale or female WT and gene-targeted mice were bred and maintained on a C57BL/6J background in the Specific Pathogen Free (SPF) Physical Containment Level 2 (PC2) Bioresources Facility at The Walter and Eliza Hall Institute of Medical Research (WEHI).\u003c/p\u003e \u003cp\u003e All procedures involving animals and live SARS-CoV-2 strains were conducted in an OGTR-approved Physical Containment Level 3 (PC3) facility at WEHI (Cert-3621). Mice were transferred to the PC3 laboratory for all SARS-CoV-2 infection experiments at least 4 days prior to the start of experiments. Animals were age- and sex-matched within experiments (both sexes were used). Experimental mice were housed in individually ventilated microisolator cages under level 3 biological containment conditions with a 12-hour light/dark cycle. Mice were provided with WEHI mouse breeder cubes (Ridley Agri Products) and sterile acidified water \u003cem\u003ead libitum\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSARS-CoV-2 strains\u003c/h2\u003e \u003cp\u003eThe SARS-CoV-2 VIC2089 clinical isolate (hCoV-19/Australia/VIC2089/2020) was obtained from the Victorian Infectious Disease Reference Laboratory (VIDLR). Viral passages were achieved by serial passage of VIC2089 through successive cohorts of young C57BL/6J (WT) mice. Briefly, mice were infected with the SARS-CoV-2 clinical isolate intranasally. At 3 dpi, mice were euthanized and lungs harvested and homogenized in a bullet blender (Next Advance Inc) in 1 mL Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (Gibco/ThermoFisher) containing steel homogenization beads (Next Advance Inc). Samples were clarified by centrifugation at 10,000 x g for 5 min before intranasal delivery of 30 \u0026micro;L lung homogenate into a new cohort of na\u0026iuml;ve C57BL/6J mice. This process was repeated a further 20 times to obtain the SARS-CoV-2 VIC2089 P21 isolate. Lung homogenates from all passages were stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eInfection of mice with SARS-CoV-2 P21\u003c/h2\u003e \u003cp\u003e6\u0026ndash;8 week-old, or 6 month-old mice were anesthetized with methoxyflurane and inoculated intranasally with 30 \u0026micro;l SARS-CoV-2. Virus stocks were diluted in serum free DMEM to a final concentration of 10\u003csup\u003e4\u003c/sup\u003e TCID50/mouse. After infection, animals were visually checked and weighed daily for a minimum of 10 days. Mice were euthanized at the indicated times post-infection by CO\u003csub\u003e2\u003c/sub\u003e asphyxiation. For histological analysis, animals were euthanized by cervical dislocation. Lungs were collected and stored at -80\u0026deg;C in serum-free DMEM until further processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of viral loads by 50% tissue culture infectious dose (TCID\u003csub\u003e50\u003c/sub\u003e)\u003c/h2\u003e \u003cp\u003eTCID\u003csub\u003e50\u003c/sub\u003e was performed as previously described in \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Briefly, Vero African green monkey kidney epithelial cells, purchased from ATCC (clone CCL-81), were seeded in flat bottom 96-well plates (1.75x10\u003csup\u003e4\u003c/sup\u003e cells/well) and left to adhere overnight at 37\u0026deg;C/5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were washed twice with PBS and transferred to serum-free DMEM containing TPCK trypsin (0.5 \u0026micro;g/mL working concentration). Infected organs were defrosted, homogenized, clarified by centrifugation at 10,000 x g for 5 min at 4\u0026deg;C and supernatant was added to the first row of cells at a ratio of 1:7, followed by 9 rounds of 1:7 serial dilutions in the other rows. Cells were incubated at 37\u0026deg;C/5% CO\u003csub\u003e2\u003c/sub\u003e for 4 days until virus-induced cytopathic effects (CPE) could be scored. TCID\u003csub\u003e50\u003c/sub\u003e was calculated using the Spearman \u0026amp; K\u0026auml;rber algorithm as described in \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eHistological analysis and immunohistochemical staining\u003c/h2\u003e \u003cp\u003eOrgans were harvested and fixed in 4% paraformaldehyde (PFA) for 24 h, followed by dehydration in 70% ethanol, paraffin embedding and sectioning. Slides were stained with either hematoxylin and eosin (H\u0026amp;E), or immunohistochemically stained with antibodies against CD3 (1:500, Agilent A045201), MPO (1:1000, Agilent A039829), F4/80 (1:1000, WEHI in-house antibody), or an antibody against SARS-CoV-2 nucleocapsid (1:4000, abcam ab271180) using the automated Omnis EnVision G2 template (Dako, Glostrup, Denmark). De-waxing was performed with Clearify Clearing Agent (Dako) and antigen retrieval was performed using EnVision FLEX TRS, High pH (Dako) at 97\u0026deg;C for 30 min. Primary antibodies were diluted in EnVision Flex Antibody Diluent (Dako) and incubated at 32\u0026deg;C for 60 min. HRP-labeled secondary antibodies (Invitrogen, Waltham, USA) were applied at 32\u0026deg;C for 30 min. Slides were counter-stained with Mayer hematoxylin, dehydrated, cleared, and mounted with MM24 mounting medium (Surgipath-Leica, Buffalo Grove, IL, USA). Slides were scanned with an Aperio ScanScope AT slide scanner (Leica Microsystems, Wetzlar, Germany) and analyzed by an American board-certified pathologist (Smitha Rose Georgy) to describe histological changes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eLung cytokine and chemokine analysis\u003c/h2\u003e \u003cp\u003eLungs were thawed, homogenized and clarified by centrifugation at 10,000 x g for 5 min at 4\u0026deg;C. Supernatants were pre-treated for 20 min with 1% Triton-X-100 for viral de-activation and the Cytokine \u0026amp; Chemokine 26-Plex Mouse ProcartaPlex Panel 1 (EPX260-26088-901) was used according to the manufacturer\u0026rsquo;s instructions. Briefly, 25 \u0026micro;L of clarified lung samples were diluted with 25 \u0026micro;L universal assay buffer, incubated with magnetic capture beads, washed, incubated with detection antibodies and SA-PE. Cytokines were recorded on a Bio-plex 200 system (Bio-Rad) and quantitated by comparison to a standard curve. Analysis was performed using R Studio.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eTotal cell protein was isolated from whole mouse lungs using cell lysis buffer. Absolute protein content of clarified lysates was determined by BCA Protein Assay (Pierce), and equal quantities (10\u0026ndash;30 \u0026micro;g) of total protein were separated under denaturing and reducing conditions (with 5% β-mercapto-ethanol) using 4\u0026ndash;12% SDS-PAGE gels (Life Technologies). Proteins were transferred onto a PVDF membrane. These membranes were blocked with 5% skim milk (Devondale, Brunswick, Australia) in TBS with 0.05% Tween-20 (TBST) for 30 min, and proteins of interest were detected using the following primary antibodies: rabbit anti-GSDMD (Abcam, ab209845) and rabbit anti-beta-Actin (Cell Signaling, 13E5; loading control). HRP-conjugated secondary goat anti-rabbit IgG antibodies (Southern Biotech, Birmingham, AL, USA) were then applied to membranes, which were subsequently incubated with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore) and imaged using a ChemiDoc Touch Imaging System (Bio-Rad).\u003c/p\u003e \u003cp\u003eA positive control for GSMD activation was prepared by infecting bone marrow derived macrophages with salmonella \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnti-IL-1β treatment of mice\u003c/h2\u003e \u003cp\u003eFor depletion of IL-1β, 10\u0026ndash;12 week-old animals were treated intraperitoneally with 200\u0026micro;g of monoclonal antibody (Bio X Cell BE0246) on the day of SARS-CoV-2 infection. Vehicle-treated mice received 200\u0026micro;g of isotype control antibodies (Bio X Cell, BE0091). Animals were monitored daily for weigh change and euthanised upon reaching the humane endpoint (\u0026gt;\u0026thinsp;20% weight loss).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of data and statistical analyses\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using Prism v9.3.1 software (GraphPad Software, Inc.). Unpaired two-tailed t-tests were used for normally distributed data for comparisons between two independent groups. Data that violated the assumption of normality were transformed by generating log\u003csub\u003e10\u003c/sub\u003e prior to statistical analysis. Bars in figures represent either the mean (\u0026plusmn;\u0026thinsp;SD) or median of normally or non-normally distributed data sets, respectively, and each symbol represents one mouse. Sample sizes (n), replicate numbers and significance can be found in the figures and figure legends. For all statistical significance indications: *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; ns, not statistically significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eStatistical analysis of cytokine data consisted of Wilcoxon rank sum test between group medians, with Bonferroni adjustment for multiple comparisons. Boxplots in figures depict the median and interquartile ranges. Loess smoothing was applied to the infection time course data, with the shaded area indicating 95% confidence intervals.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr Seth Masters for knockout mouse strains,\u0026nbsp;Natalia Mora Torres, Janzelle Marasigan and Thomas Kapitelli\u0026nbsp;for excellent animal husbandry support, Dr Dylan Sheerin for help with the R code to analysis of cytokine profiles. We thank The Walter and Eliza Hall Institute histology and imaging departments for their help. The authors acknowledge the contribution and assistance of Melbourne Health through its Victorian Infectious Diseases Reference Laboratory at the Doherty Institute in providing our laboratory with isolated SARS-CoV-2 material.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interests relating to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.M.B. designed and performed experiments, analysed data and generated figures. J.P.C. and J.S. designed and performed experiments. L.S., L.M. and M.Da. performed experiments. S.R.G. scored histological images. K.C.D, C.C.A. planned and oversaw experiments. M.H. and A.S. contributed critical ideas and gene-targeted mice and were involved in analyzing data. M.P. and M.Doe. supervised the work. S.M.B, M.Doe. and M.P. wrote the manuscript with input from other authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures and mouse strains were reviewed and approved by The Walter and Eliza Hall Institute of Medical Research Animal Ethics Committee and all experiments were conducted in accordance with the Prevention of Cruelty to Animals Act (1986) and the Australian National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Scientific Purposes (1997).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAustralian\u0026nbsp;National Health and Medical Research Council Australia Investigator grant GNT1175011 (MP).\u003c/p\u003e\n\u003ch2\u003eData Availability Statement\u003c/h2\u003e\n\u003cul\u003e\n \u003cli\u003eViral strains used in this study are available from the authors upon signing of a Materials Transfer Agreement (MTA).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/li\u003e\n \u003cli\u003eFull histological images reported in this paper will be shared by the lead contact upon request.\u0026nbsp;\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLee S, Channappanavar R, Kanneganti T-D. Coronaviruses: Innate Immunity, Inflammasome Activation, Inflammatory Cell Death, and Cytokines. \u003cem\u003eTrends in Immunology\u003c/em\u003e 2020, \u003cstrong\u003e41\u003c/strong\u003e(12)\u003cstrong\u003e:\u003c/strong\u003e 1083-1099.\u003c/li\u003e\n \u003cli\u003eMorais Da Silva M, Lira De Lucena AS, Paiva J\u0026uacute;nior SDSL, Flor\u0026ecirc;ncio De Carvalho VM, Santana De Oliveira PS, Rosa MM\u003cem\u003e, et al.\u003c/em\u003e Cell death mechanisms involved in cell injury caused by SARS‐CoV‐2. \u003cem\u003eReviews in Medical Virology\u003c/em\u003e 2021.\u003c/li\u003e\n \u003cli\u003eGutierrez KD, Davis MA, Daniels BP, Olsen TM, Ralli-Jain P, Tait SWG\u003cem\u003e, et al.\u003c/em\u003e MLKL Activation Triggers NLRP3-Mediated Processing and Release of IL-1\u0026beta; Independently of Gasdermin-D. \u003cem\u003eJournal of immunology (Baltimore, Md : 1950)\u003c/em\u003e 2017, \u003cstrong\u003e198\u003c/strong\u003e(5)\u003cstrong\u003e:\u003c/strong\u003e 2156-2164.\u003c/li\u003e\n \u003cli\u003eRodrigues TS, de S\u0026aacute; KSG, Ishimoto AY, Becerra A, Oliveira S, Almeida L\u003cem\u003e, et al.\u003c/em\u003e Inflammasomes are activated in response to SARS-CoV-2 infection and are associated with COVID-19 severity in patients. \u003cem\u003eThe Journal of experimental medicine\u003c/em\u003e 2021, \u003cstrong\u003e218\u003c/strong\u003e(3)\u003cstrong\u003e:\u003c/strong\u003e e20201707.\u003c/li\u003e\n \u003cli\u003eCourjon J, Dufies O, Robert A, Bailly L, Torre C, Chirio D\u003cem\u003e, et al.\u003c/em\u003e Heterogeneous NLRP3 inflammasome signature in circulating myeloid cells as a biomarker of COVID-19 severity. \u003cem\u003eBlood Advances\u003c/em\u003e 2021, \u003cstrong\u003e5\u003c/strong\u003e(5)\u003cstrong\u003e:\u003c/strong\u003e 1523-1534.\u003c/li\u003e\n \u003cli\u003evan den Berg DF, Te Velde AA. Severe COVID-19: NLRP3 Inflammasome Dysregulated. \u003cem\u003eFront Immunol\u003c/em\u003e 2020, \u003cstrong\u003e11:\u003c/strong\u003e 1580.\u003c/li\u003e\n \u003cli\u003eVande Walle L, Lamkanfi M. Pyroptosis. \u003cem\u003eCurr Biol\u003c/em\u003e 2016, \u003cstrong\u003e26\u003c/strong\u003e(13)\u003cstrong\u003e:\u003c/strong\u003e R568-r572.\u003c/li\u003e\n \u003cli\u003eLawlor KE, Murphy JM, Vince JE. Gasdermin and MLKL necrotic cell death effectors: Signaling and diseases. \u003cem\u003eImmunity\u003c/em\u003e 2024, \u003cstrong\u003e57\u003c/strong\u003e(3)\u003cstrong\u003e:\u003c/strong\u003e 429-445.\u003c/li\u003e\n \u003cli\u003eGuo H, Callaway JB, Ting JPY. Inflammasomes: mechanism of action, role in disease, and therapeutics. \u003cem\u003eNature Medicine\u003c/em\u003e 2015, \u003cstrong\u003e21\u003c/strong\u003e(7)\u003cstrong\u003e:\u003c/strong\u003e 677-687.\u003c/li\u003e\n \u003cli\u003eSwanson KV, Deng M, Ting JPY. The NLRP3 inflammasome: molecular activation and regulation to therapeutics. \u003cem\u003eNature Reviews Immunology\u003c/em\u003e 2019, \u003cstrong\u003e19\u003c/strong\u003e(8)\u003cstrong\u003e:\u003c/strong\u003e 477-489.\u003c/li\u003e\n \u003cli\u003eBergsbaken T, Fink SL, Cookson BT. Pyroptosis: host cell death and inflammation. \u003cem\u003eNat Rev Microbiol\u003c/em\u003e 2009, \u003cstrong\u003e7\u003c/strong\u003e(2)\u003cstrong\u003e:\u003c/strong\u003e 99-109.\u003c/li\u003e\n \u003cli\u003eDinarello CA. Immunological and Inflammatory Functions of the Interleukin-1 Family. \u003cem\u003eAnnu Rev Immunol\u003c/em\u003e 2009, \u003cstrong\u003e27\u003c/strong\u003e(1)\u003cstrong\u003e:\u003c/strong\u003e 519-550.\u003c/li\u003e\n \u003cli\u003eHe Y, Hara H, N\u0026uacute;\u0026ntilde;ez G. Mechanism and regulation of NLRP3 inflammasome activation. \u003cem\u003eTrends in biochemical sciences\u003c/em\u003e 2016, \u003cstrong\u003e41\u003c/strong\u003e(12)\u003cstrong\u003e:\u003c/strong\u003e 1012-1021.\u003c/li\u003e\n \u003cli\u003ePasparakis M, Vandenabeele P. Necroptosis and its role in inflammation. \u003cem\u003eNature\u003c/em\u003e 2015, \u003cstrong\u003e517\u003c/strong\u003e(7534)\u003cstrong\u003e:\u003c/strong\u003e 311-320.\u003c/li\u003e\n \u003cli\u003eLin J, Kumari S, Kim C, Van T-M, Wachsmuth L, Polykratis A\u003cem\u003e, et al.\u003c/em\u003e RIPK1 counteracts ZBP1-mediated necroptosis to inhibit inflammation. \u003cem\u003eNature\u003c/em\u003e 2016, \u003cstrong\u003e540\u003c/strong\u003e(7631)\u003cstrong\u003e:\u003c/strong\u003e 124-128.\u003c/li\u003e\n \u003cli\u003eJiao H, Wachsmuth L, Kumari S, Schwarzer R, Lin J, Eren RO\u003cem\u003e, et al.\u003c/em\u003e Z-nucleic-acid sensing triggers ZBP1-dependent necroptosis and inflammation. \u003cem\u003eNature\u003c/em\u003e 2020, \u003cstrong\u003e580\u003c/strong\u003e(7803)\u003cstrong\u003e:\u003c/strong\u003e 391-395.\u003c/li\u003e\n \u003cli\u003eHe S, Liang Y, Shao F, Wang X. Toll-like receptors activate programmed necrosis in macrophages through a receptor-interacting kinase-3-mediated pathway. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e 2011, \u003cstrong\u003e108\u003c/strong\u003e(50)\u003cstrong\u003e:\u003c/strong\u003e 20054-20059.\u003c/li\u003e\n \u003cli\u003eKaiser WJ, Sridharan H, Huang C, Mandal P, Upton JW, Gough PJ\u003cem\u003e, et al.\u003c/em\u003e Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL. \u003cem\u003eJ Biol Chem\u003c/em\u003e 2013, \u003cstrong\u003e288\u003c/strong\u003e(43)\u003cstrong\u003e:\u003c/strong\u003e 31268-31279.\u003c/li\u003e\n \u003cli\u003eTummers B, Green DR. Caspase-8: regulating life and death. \u003cem\u003eImmunol Rev\u003c/em\u003e 2017, \u003cstrong\u003e277\u003c/strong\u003e(1)\u003cstrong\u003e:\u003c/strong\u003e 76-89.\u003c/li\u003e\n \u003cli\u003eMurphy JM, Czabotar PE, Hildebrand JM, Lucet IS, Zhang JG, Alvarez-Diaz S\u003cem\u003e, et al.\u003c/em\u003e The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. \u003cem\u003eImmunity\u003c/em\u003e 2013, \u003cstrong\u003e39\u003c/strong\u003e(3)\u003cstrong\u003e:\u003c/strong\u003e 443-453.\u003c/li\u003e\n \u003cli\u003eHildebrand JM, Tanzer MC, Lucet IS, Young SN, Spall SK, Sharma P\u003cem\u003e, et al.\u003c/em\u003e Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e 2014, \u003cstrong\u003e111\u003c/strong\u003e(42)\u003cstrong\u003e:\u003c/strong\u003e 15072-15077.\u003c/li\u003e\n \u003cli\u003eSamson AL, Zhang Y, Geoghegan ND, Gavin XJ, Davies KA, Mlodzianoski MJ\u003cem\u003e, et al.\u003c/em\u003e MLKL trafficking and accumulation at the plasma membrane control the kinetics and threshold for necroptosis. \u003cem\u003eNature Communications\u003c/em\u003e 2020, \u003cstrong\u003e11\u003c/strong\u003e(1).\u003c/li\u003e\n \u003cli\u003eBader SM, Cooney JP, Pellegrini M, Doerflinger M. Programmed cell death: the pathways to severe COVID-19? \u003cem\u003eBiochem J\u003c/em\u003e 2022, \u003cstrong\u003e479\u003c/strong\u003e(5)\u003cstrong\u003e:\u003c/strong\u003e 609-628.\u003c/li\u003e\n \u003cli\u003eM. Bader S, Cooney JP, Bhandari R, Mackiewicz L, Dayton M, Sheerin D\u003cem\u003e, et al.\u003c/em\u003e Necroptosis does not drive disease pathogenesis in a mouse infective model of SARS-CoV-2 in vivo. \u003cem\u003eCell Death \u0026amp; Disease\u003c/em\u003e 2024, \u003cstrong\u003e15\u003c/strong\u003e(1)\u003cstrong\u003e:\u003c/strong\u003e 100.\u003c/li\u003e\n \u003cli\u003eBader SM, Cooney JP, Sheerin D, Taiaroa G, Harty L, Davidson KC\u003cem\u003e, et al.\u003c/em\u003e SARS-CoV-2 mouse adaptation selects virulence mutations that cause TNF-driven age-dependent severe disease with human correlates. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e 2023, \u003cstrong\u003e120\u003c/strong\u003e(32)\u003cstrong\u003e:\u003c/strong\u003e e2301689120.\u003c/li\u003e\n \u003cli\u003eKarki R, Sharma BR, Tuladhar S, Williams EP, Zalduondo L, Samir P\u003cem\u003e, et al.\u003c/em\u003e Synergism of TNF-\u0026alpha; and IFN-\u0026gamma; Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes. \u003cem\u003eCell\u003c/em\u003e 2021, \u003cstrong\u003e184\u003c/strong\u003e(1)\u003cstrong\u003e:\u003c/strong\u003e 149-168.e117.\u003c/li\u003e\n \u003cli\u003eTsu BV, Beierschmitt C, Ryan AP, Agarwal R, Mitchell PS, Daugherty MD. Diverse viral proteases activate the NLRP1 inflammasome. \u003cem\u003eeLife\u003c/em\u003e 2021, \u003cstrong\u003e10:\u003c/strong\u003e e60609.\u003c/li\u003e\n \u003cli\u003ePlan\u0026egrave;s R, Pinilla M, Santoni K, Hessel A, Passemar C, Lay K\u003cem\u003e, et al.\u003c/em\u003e Human NLRP1 is a sensor of pathogenic coronavirus 3CL proteases in lung epithelial cells. \u003cem\u003eMol Cell\u003c/em\u003e 2022, \u003cstrong\u003e82\u003c/strong\u003e(13)\u003cstrong\u003e:\u003c/strong\u003e 2385-2400.e2389.\u003c/li\u003e\n \u003cli\u003eFaustin B, Lartigue L, Bruey J-M, Luciano F, Sergienko E, Bailly-Maitre B\u003cem\u003e, et al.\u003c/em\u003e Reconstituted NALP1 inflammasome reveals two-step mechanism of caspase-1 activation. \u003cem\u003eMolecular cell\u003c/em\u003e 2007, \u003cstrong\u003e25\u003c/strong\u003e(5)\u003cstrong\u003e:\u003c/strong\u003e 713-724.\u003c/li\u003e\n \u003cli\u003eAbderrazak A, Syrovets T, Couchie D, El Hadri K, Friguet B, Simmet T\u003cem\u003e, et al.\u003c/em\u003e NLRP3 inflammasome: from a danger signal sensor to a regulatory node of oxidative stress and inflammatory diseases. \u003cem\u003eRedox biology\u003c/em\u003e 2015, \u003cstrong\u003e4:\u003c/strong\u003e 296-307.\u003c/li\u003e\n \u003cli\u003eGrenier JM, Wang L, Manji GA, Huang W-J, Al-Garawi A, Kelly R\u003cem\u003e, et al.\u003c/em\u003e Functional screening of five PYPAF family members identifies PYPAF5 as a novel regulator of NF-\u0026kappa;B and caspase-1. \u003cem\u003eFEBS letters\u003c/em\u003e 2002, \u003cstrong\u003e530\u003c/strong\u003e(1-3)\u003cstrong\u003e:\u003c/strong\u003e 73-78.\u003c/li\u003e\n \u003cli\u003eEvavold CL, Ruan J, Tan Y, Xia S, Wu H, Kagan JC. The Pore-Forming Protein Gasdermin D Regulates Interleukin-1 Secretion from Living Macrophages. \u003cem\u003eImmunity\u003c/em\u003e 2018, \u003cstrong\u003e48\u003c/strong\u003e(1)\u003cstrong\u003e:\u003c/strong\u003e 35-44.e36.\u003c/li\u003e\n \u003cli\u003eSateriale A, Gullicksrud JA, Engiles JB, McLeod BI, Kugler EM, Henao-Mejia J\u003cem\u003e, et al.\u003c/em\u003e The intestinal parasite \u0026lt;i\u0026gt;Cryptosporidium\u0026lt;/i\u0026gt; is controlled by an enterocyte intrinsic inflammasome that depends on NLRP6. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e 2021, \u003cstrong\u003e118\u003c/strong\u003e(2)\u003cstrong\u003e:\u003c/strong\u003e e2007807118.\u003c/li\u003e\n \u003cli\u003eDoerflinger M, Deng Y, Whitney P, Salvamoser R, Engel S, Kueh AJ\u003cem\u003e, et al.\u003c/em\u003e Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection. \u003cem\u003eImmunity\u003c/em\u003e 2020, \u003cstrong\u003e53\u003c/strong\u003e(3)\u003cstrong\u003e:\u003c/strong\u003e 533-547.e537.\u003c/li\u003e\n \u003cli\u003eBedoui S, Herold MJ, Strasser A. Emerging connectivity of programmed cell death pathways and its physiological implications. \u003cem\u003eNature Reviews Molecular Cell Biology\u003c/em\u003e 2020, \u003cstrong\u003e21\u003c/strong\u003e(11)\u003cstrong\u003e:\u003c/strong\u003e 678-695.\u003c/li\u003e\n \u003cli\u003eKayagaki N, Kornfeld OS, Lee BL, Stowe IB, O\u0026apos;Rourke K, Li Q\u003cem\u003e, et al.\u003c/em\u003e NINJ1 mediates plasma membrane rupture during lytic cell death. \u003cem\u003eNature\u003c/em\u003e 2021, \u003cstrong\u003e591\u003c/strong\u003e(7848)\u003cstrong\u003e:\u003c/strong\u003e 131-136.\u003c/li\u003e\n \u003cli\u003eRamos S, Hartenian E, Santos JC, Walch P, Broz P. NINJ1 induces plasma membrane rupture and release of damage-associated molecular pattern molecules during ferroptosis. \u003cem\u003eEmbo j\u003c/em\u003e 2024.\u003c/li\u003e\n \u003cli\u003eJunqueira C, Crespo \u0026Atilde;, Ranjbar S, Lewandrowski M, Ingber J, de Lacerda LB\u003cem\u003e, et al.\u003c/em\u003e SARS-CoV-2 infects blood monocytes to activate NLRP3 and AIM2 inflammasomes, pyroptosis and cytokine release. \u003cem\u003eRes Sq\u003c/em\u003e 2021.\u003c/li\u003e\n \u003cli\u003eSchultze JL, Aschenbrenner AC. COVID-19 and the human innate immune system. \u003cem\u003eCell\u003c/em\u003e 2021, \u003cstrong\u003e184\u003c/strong\u003e(7)\u003cstrong\u003e:\u003c/strong\u003e 1671-1692.\u003c/li\u003e\n \u003cli\u003eWilson KP, Black J-AF, Thomson JA, Kim EE, Griffith JP, Navia MA\u003cem\u003e, et al.\u003c/em\u003e Structure and mechanism of interleukin-l\u0026beta; converting enzyme. \u003cem\u003eNature\u003c/em\u003e 1994, \u003cstrong\u003e370\u003c/strong\u003e(6487)\u003cstrong\u003e:\u003c/strong\u003e 270-275.\u003c/li\u003e\n \u003cli\u003eMartinon F, Burns K, Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-\u0026beta;. \u003cem\u003eMolecular cell\u003c/em\u003e 2002, \u003cstrong\u003e10\u003c/strong\u003e(2)\u003cstrong\u003e:\u003c/strong\u003e 417-426.\u003c/li\u003e\n \u003cli\u003eMcIlwain DR, Berger T, Mak TW. Caspase functions in cell death and disease. \u003cem\u003eCold Spring Harb Perspect Biol\u003c/em\u003e 2013, \u003cstrong\u003e5\u003c/strong\u003e(4)\u003cstrong\u003e:\u003c/strong\u003e a008656.\u003c/li\u003e\n \u003cli\u003eDinarello CA. Overview of the IL-1 family in innate inflammation and acquired immunity. \u003cem\u003eImmunol Rev\u003c/em\u003e 2018, \u003cstrong\u003e281\u003c/strong\u003e(1)\u003cstrong\u003e:\u003c/strong\u003e 8-27.\u003c/li\u003e\n \u003cli\u003eLopez-Castejon G, Brough D. Understanding the mechanism of IL-1\u0026beta; secretion. \u003cem\u003eCytokine Growth Factor Rev\u003c/em\u003e 2011, \u003cstrong\u003e22\u003c/strong\u003e(4)\u003cstrong\u003e:\u003c/strong\u003e 189-195.\u003c/li\u003e\n \u003cli\u003ePang J, Vince JE. The role of caspase-8 in inflammatory signalling and pyroptotic cell death. \u003cem\u003eSemin Immunol\u003c/em\u003e 2023, \u003cstrong\u003e70:\u003c/strong\u003e 101832.\u003c/li\u003e\n \u003cli\u003eHuang C, Wang Y, Li X, Ren L, Zhao J, Hu Y\u003cem\u003e, et al.\u003c/em\u003e Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. \u003cem\u003eLancet\u003c/em\u003e 2020, \u003cstrong\u003e395\u003c/strong\u003e(10223)\u003cstrong\u003e:\u003c/strong\u003e 497-506.\u003c/li\u003e\n \u003cli\u003ePeng R, Wang-Kan X, Idorn M, Zhou FY, Orozco SL, McCarthy J\u003cem\u003e, et al.\u003c/em\u003e ZBP1 induces inflammatory signaling via RIPK3 and promotes SARS-CoV-2-induced cytokine expression. \u003cem\u003ebioRxiv\u003c/em\u003e 2021\u003cstrong\u003e:\u003c/strong\u003e 2021.2010.2001.462460.\u003c/li\u003e\n \u003cli\u003eLi S, Jiang L, Li X, Lin F, Wang Y, Li B\u003cem\u003e, et al.\u003c/em\u003e Clinical and pathological investigation of patients with severe COVID-19. \u003cem\u003eJCI Insight\u003c/em\u003e 2020, \u003cstrong\u003e5\u003c/strong\u003e(12).\u003c/li\u003e\n \u003cli\u003eBlanco-Melo D, Nilsson-Payant BE, Liu W-C, Uhl S, Hoagland D, M\u0026oslash;ller R\u003cem\u003e, et al.\u003c/em\u003e Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19. \u003cem\u003eCell\u003c/em\u003e 2020, \u003cstrong\u003e181\u003c/strong\u003e(5)\u003cstrong\u003e:\u003c/strong\u003e 1036-1045.e1039.\u003c/li\u003e\n \u003cli\u003eMehta P, McAuley DF, Brown M, Sanchez E, Tattersall RS, Manson JJ. COVID-19: consider cytokine storm syndromes and immunosuppression. \u003cem\u003eThe Lancet\u003c/em\u003e 2020, \u003cstrong\u003e395\u003c/strong\u003e(10229)\u003cstrong\u003e:\u003c/strong\u003e 1033-1034.\u003c/li\u003e\n \u003cli\u003eZhu L, Yang P, Zhao Y, Zhuang Z, Wang Z, Song R\u003cem\u003e, et al.\u003c/em\u003e Single-Cell Sequencing of Peripheral Mononuclear Cells Reveals Distinct Immune Response Landscapes of COVID-19 and Influenza Patients. \u003cem\u003eImmunity\u003c/em\u003e 2020, \u003cstrong\u003e53\u003c/strong\u003e(3)\u003cstrong\u003e:\u003c/strong\u003e 685-696.e683.\u003c/li\u003e\n \u003cli\u003eLiao M, Liu Y, Yuan J, Wen Y, Xu G, Zhao J\u003cem\u003e, et al.\u003c/em\u003e Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19. \u003cem\u003eNature Medicine\u003c/em\u003e 2020, \u003cstrong\u003e26\u003c/strong\u003e(6)\u003cstrong\u003e:\u003c/strong\u003e 842-844.\u003c/li\u003e\n \u003cli\u003eZeng J, Xie X, Feng X-L, Xu L, Han J-B, Yu D\u003cem\u003e, et al.\u003c/em\u003e Specific inhibition of the NLRP3 inflammasome suppresses immune overactivation and alleviates COVID-19 like pathology in mice. \u003cem\u003eeBioMedicine\u003c/em\u003e 2022, \u003cstrong\u003e75\u003c/strong\u003e.\u003c/li\u003e\n \u003cli\u003eMadurka I, Vishnevsky A, Soriano JB, Gans SJ, Ore DJS, Rendon A\u003cem\u003e, et al.\u003c/em\u003e DFV890: a new oral NLRP3 inhibitor-tested in an early phase 2a randomised clinical trial in patients with COVID-19 pneumonia and impaired respiratory function. \u003cem\u003eInfection\u003c/em\u003e 2023, \u003cstrong\u003e51\u003c/strong\u003e(3)\u003cstrong\u003e:\u003c/strong\u003e 641-654.\u003c/li\u003e\n \u003cli\u003eEffect of anakinra versus usual care in adults in hospital with COVID-19 and mild-to-moderate pneumonia (CORIMUNO-ANA-1): a randomised controlled trial. \u003cem\u003eLancet Respir Med\u003c/em\u003e 2021, \u003cstrong\u003e9\u003c/strong\u003e(3)\u003cstrong\u003e:\u003c/strong\u003e 295-304.\u003c/li\u003e\n \u003cli\u003eDeclercq J, Van Damme KF, De Leeuw E, Maes B, Bosteels C, Tavernier SJ\u003cem\u003e, et al.\u003c/em\u003e Effect of anti-interleukin drugs in patients with COVID-19 and signs of cytokine release syndrome (COV-AID): a factorial, randomised, controlled trial. \u003cem\u003eThe Lancet Respiratory Medicine\u003c/em\u003e 2021, \u003cstrong\u003e9\u003c/strong\u003e(12)\u003cstrong\u003e:\u003c/strong\u003e 1427-1438.\u003c/li\u003e\n \u003cli\u003eDerde LPG. Effectiveness of Tocilizumab, Sarilumab, and Anakinra for critically ill patients with COVID-19 The REMAP-CAP COVID-19 Immune Modulation Therapy Domain Randomized Clinical Trial. \u003cem\u003emedRxiv\u003c/em\u003e 2021\u003cstrong\u003e:\u003c/strong\u003e 2021.2006.2018.21259133.\u003c/li\u003e\n \u003cli\u003eHierholzer JC, Killington RA. Virus isolation and quantitation. \u003cem\u003eVirology Methods Manual\u003c/em\u003e 1996\u003cstrong\u003e:\u003c/strong\u003e 25-46.\u003c/li\u003e\n \u003cli\u003eChiou S, Al-Ani AH, Pan Y, Patel KM, Kong IY, Whitehead LW\u003cem\u003e, et al.\u003c/em\u003e An immunohistochemical atlas of necroptotic pathway expression. \u003cem\u003ebioRxiv\u003c/em\u003e 2023\u003cstrong\u003e:\u003c/strong\u003e 2023.2010.2031.565039.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4826453/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4826453/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExcessive inflammation and cytokine release are hallmarks of severe COVID-19. Programmed cell death processes can drive inflammation, however, the relevance in the pathogenesis of severe COVID-19 is unclear. Pyroptosis is a pro-inflammatory form of regulated cell death initiated by inflammasomes and executed by the pore-forming protein gasdermin D (GSDMD). Using an established mouse-adapted SARS-CoV-2 virus and a combination of gene-targeted mice we found that deletion of the inflammasome (NLRP1/3 and the adaptor ASC) and pore forming proteins involved in pyroptosis (GSDMA/C/D/E) did not impact disease outcome or viral loads. Furthermore, we found that SARS-CoV-2 infection did not trigger GSDMD activation in mouse lungs. We did not observe any difference between WT animals and mice with compound deficiencies in upstream caspases \u003cem\u003eC1/11/12\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e. This indicates that the classical canonical and non-canonical pro-inflammatory caspases known to process and activate IL-1β, IL-18 and GSDMD do not substantially contribute to SARS-CoV-2 pathogenesis. However, the loss of IL-1β, but not the absence of IL-18, ameliorated disease and enhanced survival in older animals compared to wildtype mice. Collectively, these findings indicate that IL-1β is an important factor contributing to severe SARS-CoV-2 disease, but its release was largely independent of inflammasome and pyroptotic pathways.\u003c/p\u003e","manuscriptTitle":"IL-1b drives SARS-CoV-2 disease in vivo, independently of the inflammasome and pyroptotic signalling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-28 17:56:54","doi":"10.21203/rs.3.rs-4826453/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Reject after peer review","date":"2024-08-28T15:46:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-08-19T13:21:39+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-08-18T17:09:22+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-08-12T18:08:22+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-08-05T07:28:02+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-08-02T14:28:27+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-08-01T15:34:17+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-08-01T12:22:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-31T09:09:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Differentiation","date":"2024-07-30T23:01:02+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2024-07-30T09:23:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-30T07:06:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b2ad2cc1-222b-44cb-9afc-37be9ffaf882","owner":[],"postedDate":"August 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-28T08:09:09+00:00","versionOfRecord":{"articleIdentity":"rs-4826453","link":"https://doi.org/10.1038/s41418-025-01459-x","journal":{"identity":"cell-death-and-differentiation","isVorOnly":false,"title":"Cell Death \u0026 Differentiation"},"publishedOn":"2025-02-28 05:00:00","publishedOnDateReadable":"February 28th, 2025"},"versionCreatedAt":"2024-08-28 17:56:54","video":"","vorDoi":"10.1038/s41418-025-01459-x","vorDoiUrl":"https://doi.org/10.1038/s41418-025-01459-x","workflowStages":[]},"version":"v1","identity":"rs-4826453","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4826453","identity":"rs-4826453","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}