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

doi:10.21203/rs.3.rs-4826453/v1

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

https://doi.org/10.21203/rs.3.rs-4826453/v1

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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.

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.

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. 1A-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. 1C-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. 1E-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. 1G). 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. 1G and S1A). 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. 1H). 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. 1H). 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. 1H). 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. 2A-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. 2C). Interestingly, however, the levels of IL-18 and IL-23 were increased in these animals upon infection (Fig. 2C and S2A). 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. 2D).

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. 2E). 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. 2E 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. 2F, 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. 3A-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. 3E-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. 3G-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. 4A-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. 4C and S4A). 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. 4D). 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. S5A-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. 5A-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. 5C-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. 5C-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. 5E-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. 5G and S5C). 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. 5H and S5D).

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. 6A), however, this survival advantage was attenuated in 6-month-old infected mice (Fig. 6B). 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. 6C-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. 6E). 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.

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.

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 10⁴ 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 CO₂ 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 (TCID₅₀)

TCID₅₀ 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 (1.75x10⁴ cells/well) and left to adhere overnight at 37°C/5% CO₂. 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% CO₂ for 4 days until virus-induced cytopathic effects (CPE) could be scored. TCID₅₀ 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 log₁₀ 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 < 0.0001; ns, not statistically significant (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.

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.

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

Status:

Version 1

Abstract

Figures

INTRODUCTION

RESULTS

DISCUSSION

MATERIALS AND METHODS

Mice

SARS-CoV-2 strains

Infection of mice with SARS-CoV-2 P21

Measurement of viral loads by 50% tissue culture infectious dose (TCID₅₀)

Histological analysis and immunohistochemical staining

Lung cytokine and chemokine analysis

Western blot analysis

Anti-IL-1β treatment of mice

Quantification of data and statistical analyses

Declarations

Data Availability Statement

References

Additional Declarations

Supplementary Files

Status:

Version 1

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

Status:

Version 1

Abstract

Figures

INTRODUCTION

RESULTS

DISCUSSION

MATERIALS AND METHODS

Mice

SARS-CoV-2 strains

Infection of mice with SARS-CoV-2 P21

Measurement of viral loads by 50% tissue culture infectious dose (TCID50)

Histological analysis and immunohistochemical staining

Lung cytokine and chemokine analysis

Western blot analysis

Anti-IL-1β treatment of mice

Quantification of data and statistical analyses

Declarations

Data Availability Statement

References

Additional Declarations

Supplementary Files

Status:

Version 1

Measurement of viral loads by 50% tissue culture infectious dose (TCID₅₀)