The cytokine Caspase 4 exacerbates disease severity in SARS-CoV-2 infection


Researchers from Ohio State University have in a new study found that the cytokine Caspase 4 or CASP4 also exacerbates disease severity in SARS-CoV-2 infection by promoting inflammation and thrombosis.

Caspase 4 is an enzyme that proteolytically cleaves other proteins at an aspartic acid residue (LEVD-), and belongs to a family of cysteine proteases called caspases.

The function of caspase 4 is not fully known, but it is believed to be an inflammatory caspase, along with caspase 1, caspase 5 (and the murine homolog caspase 11), with a role in the immune system.

The study findings were published on a preprint server and are currently being peer reviewed.

The COVID-19 pandemic caused by the pathogen SARS-CoV-2 is a worldwide health concern, and new treatment strategies are needed.

Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive.

The study team found that the human caspase-4 (CASP4), and its mouse homologue, caspase-11 (CASP11), are upregulated in SARS-CoV-2 infections, and that CASP4 expression correlates with severity of SARS-CoV-2 infection in humans.

SARS-CoV-2-infected Casp11-/- mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) and gasdermin-D knock out (Gsdmd-/-) mice.

GSDMD is a downstream effector of CASP11 and CASP1. Notably, viral titers were similar in the three genotypes. Global transcriptomics of SARS-CoV-2-infected WT, Casp11-/- and Gsdmd-/- lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11-/- mice.

The study findings confirmed that protein levels of inflammatory mediators IL-1β, IL6, and CXCL1, and neutrophil functions, were reduced in Casp11-/- lungs. Additionally, Casp11-/- lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity.

On the whole, the study findings demonstrate that CASP4/11, promotes detrimental SARS-CoV-2-associated inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.

The COVID-19 Research study findings suggest that targeting the CASP11 homologue, human CASP4, during COVID-19 will prevent severe pneumonia, inflammation, tissue damage as well as thrombosis and accompanying repercussions such as low oxygen, lung failure, need for ventilators and perhaps long-term sequela.

These advantageous effects will be achieved without compromising viral clearance.

It is also plausible that the level of expression of CASP4 could serve as a biomarker to identify patients who will succumb to severe COVID-19.

CASP4/11 expression is elevated in the lungs during SARS-CoV-2 infections of mice and humans and correlates with disease severity in humans
CASP4/11 is weakly expressed or absent in resting cells, but is induced in response to bacterial infections14. The analysis of publicly available RNA sequencing data of nasopharyngeal swab material from subjects with SARS-CoV-2 and healthy donors (GEO accession: GSE163151), revealed that CASP4 is highly expressed in the airway of SARS-CoV-2-infected patients, and that expression levels increase with disease severity (Fig. 1a).

Additionally, we found that human lung sections from COVID-19 patients show higher levels of CASP4 staining compared with healthy lung controls (Fig. 1b), owing to greater numbers of CASP4 positive cells in the infected lung tissue (Fig. 1c). We then performed intranasal infection of C57BL/6 wild-type (WT) mice with pathogenic mouse-adapted SARS-CoV-2 (strain MA10)15, and found that infection strongly induces Casp11 expression throughout murine lung tissue within 4 days of infection as detected by RNAscope in situ hybridization (ISH) (Fig. 1d) and confirmed by qRT-PCR (Fig. 1e).

The level of CASP11 protein, likewise, went from below detection to highly-expressed in response to SARS- CoV-2 infection of murine lungs (Fig. 1f). We further examined infection of K18-hACE2 mice expressing the human ACE2 receptor using human isolate SARS-CoV-2 strain USA-WA1/2020 (WA1). Similar to mouse adapted SARS-CoV-2, the non-adapted human virus strongly induced the lung expression of CASP11 as demonstrated by qRT-PCR (Fig. 1g)16. Overall, CASP4 is highly expressed in the lungs of COVID-19 patients, and CASP11 is similarly induced upon SARS-CoV-2 infection of mice.

Casp11 deficiency reduces disease severity in SARS-CoV-2-infected mice

We next examined whether CASP11 regulates disease severity caused by SARS-CoV-2 infection. Wild-type (WT), Casp11-/- and Gsdmd-/-, and mice were infected with SARS-CoV-2 MA10 for comparison of weight loss, a commonly used indicator of overall infection severity in mice16. We found that WT mice lost a significant percentage of their body weight between days 1 and 4 post-infection, followed by partial recovery of weight up to day 7, at which point we ended our experiments (Fig. 2a). Casp11-/- mice, on the other hand, lost weight only up to day 3, and then rapidly recovered fully to their original weight by day 5 (Fig. 2a).

In comparison, weight loss of Gsdmd-/- mice was not significantly different from that of WT mice (Fig. 2a). These data indicate that CASP11 promotes disease severity during SARS-CoV-2 infection, and that this function is not mediated by GSDMD.
To determine whether differences in disease severity could be explained by differences in viral replication, we quantified live virus titers in WT, Casp11-/- and Gsdmd-/- mouse lungs at 2 and 4 days post-infection.

We found that viral loads were similar with no statistical difference between the mice genotypes at either time point (Fig. 2b). We also observed that, in agreement with previous reports17, viral titers were decreased at day 4 compared with day 2 in all groups, demonstrating that neither CASP11 nor GSDMD are required for viral clearance mechanisms

(Fig. 2b). To corroborate these findings, lung sections from WT and Casp11-/- mice were stained for SARS-CoV-2 nucleocapsid protein and similar staining patterns were observed with prominent infection of cells lining the airways and neighboring alveoli (Fig. 2c). Overall, these results demonstrate that loss of CASP11, but not GSDMD, prevents severe disease in SARS-CoV-2 infection without affecting virus replication or clearance.

Figure 1: CASP4 is upregulated in humans and mice infected with SARS-CoV-2. a, CASP4 expression levels from RNA sequencing of nasopharyngeal swab samples from patients with no disease, mild SARS-CoV- 2, or severe SARS-CoV-2 [GSE145926], one way ANOVA with Tukey’s multiple comparisons test. b, Human lung samples from 3 donors with healthy lungs or from 3 donors who died of SARS-CoV-2 were stained for CASP4 (brown). Black boxes (i, ii) outline zoomed regions. c, Quantification of CASP4 positive cells from lungs in b, unpaired t test. d-f, Mice were infected for 4 days with mouse adapted SARS-CoV-2 (MA10, 105 pfu). d, Casp11 RNA (green, RNAscope in situ hybridization) and DAPI (blue) were visualized (3D Intensity projection image) in lung sections using 20x objective. e, Casp11 RNA levels were quantfied in lung samples (N=3) by qRT-PCR, unpaired t test. f, CASP11 protein levels in lungs described in d (N=3) were examined by Western blot. g, K18-hACE2 mice were infected for 4 days with human SARS-CoV-2 (WA1, 105 pfu) and Casp11 RNA levels were quantitated in lung samples (N=4) by qRT-PCR, unpaired t test. *p<0.05, **p<0.005, ****p<0.0001.

Figure 2: Casp11-/- mice show decreased SARS-CoV-2 infection severity without affecting viral titers but by modulating specific inflammatory programs. a-c, WT, Casp11-/- and Gsdmd-/- mice were infected with SARS-CoV-2 (MA10, 105 pfu). Weight loss was tracked for 7 d, *p<0.05, **p<0.005, ****p<0.0001, ANOVA with Bonferonni’s multiple comparisons test (a), Day 0-4 WT (N=7), Casp11-/- (N=10), Gsdmd-/- (N=9); Day 5-7 WT (N=4), Casp11-/- (N=7), Gsdmd-/- (N=6). b, TCID50 viral titers were quantified in lung tissue homogenates. c, Sections from non-infected control lungs or lungs collected at 4 days post-infection were stained for SARS-CoV- 2 nucleocapsid protein (brown staining, images representative of at least 3 mice per group). d-f, WT, Casp11-/-,and Gsdmd-/- mice (N=3) were infected with SARS-CoV-2 (MA10, 105 pfu) for 2 days. RNA was extracted from lungs and subjected to RNA sequencing. d, Principal component analysis (PCA) of SARS-CoV2-infected lung gene expression with points representing individual WT (grey), Casp11-/-(blue), and Gsdmd-/- (green) mice. e, Top 30 significant Gene Ontology Biological Pathways are depicted. Node size indicates the number of transcripts within each functional category. Edges connect overlapping gene sets. Numbers represent individual replicates and color indicates relative upregulation (red) or downregulation (blue) in gene expression. f, Heatmap of significantly changed cytokine and chemokine genes when comparing Casp11-/- infected lungs versus WT. Expression scaling is relative to WT and Gsdmd-/- mice for comparisons (N=3) (p<0.05)..

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