Changes in intracellular active Caspase 1 levels in immune cells in hospitalized patients with COVID-19 disease
Transcriptional support of our previous findings showing increased gene expression of caspase-1 in CD4+ T cells was observed using a published single cell RNA-Seq immune profiling dataset of patients with moderate-severe COVID-19 (Supplementary Figure 1 ) (30, 31). CD4+ T cells also showed an upregulation of caspase-7 and -9 upon IFN stimulation. Interestingly, there were altered caspase gene expression in other cellular subsets, including neutrophils (all inflammatory and apoptotic caspases), plasmacytoid dendritic cells (caspases 7 and 9) and eosinophils (caspase 6). Examination of caspase gene expression levels in public transcriptome profiling datasets ofin vitro SARS-CoV-2 infection models (Supplemental Figures 2-4 ) show further evidence of caspase gene expression upregulation upon SARS-CoV-2 infection in target cells.
To follow up on our previous findings of increased T cell caspase-1 expression in COVID-19, we analyzed intracellular active caspase-1 in CD4+ T cells of non-ICU and ICU patients with COVID-19 and healthy individuals for comparison (Table 1 ) using our laboratory developed test (LDT) that has been analytically validated it in a CLIA certified and CAP accredited flow cytometry laboratory. Frequency of caspase-1+ CD4+ T cells were significantly elevated at baseline in hospitalized (both ICU and non-ICU) COVID-19 patients compared to healthy participants (Figure 1A-D ; all p-value<0.0001). Nigericin was used as a positive control as it is crucial for oligomerization of the NLRP3 inflammasome and activation of caspase-1, and found with nigericin stimulation hospitalized COVID-19 patients still had a higher frequency of active caspase-1 in CD4+ T cells compared to controls (all p-value<0.0001; Figure 1A-D ). However, we also found that the levels of pannexin-1, an intermediatory protein involved in nigericin signaling induced caspase-1 activation and IL-1ß processing and release, were also elevated in COVID-19 patients(Supplemental Figure 5) . Variation in the expression levels of pannexin-1 between individual healthy and COVID-19 subjects may explain the differences in the nigericin response to upregulate T-cell caspase-1.
We next correlated active caspase-1 with cellular subsets and cytokines associated with its activation. We observed active caspase-1 expression is predominantly in the CD45RO+ memory population and showed a weak but statistically significant correlation with older age, a finding that might potentially explain advanced age as one of the biggest risk factor for poor outcomes in COVID-19 (Supplemental Table 1 ). Furthermore, CD4+ T cell active caspase-1 levels in patients with COVID-19 correlated with CRTH2+ T-cells, γ/δ T-cells, CD3-CD16+/CD56+ lymphocytes, and plasmacytoid dendritic cells (Figure 1 E-G and Supplemental Table 1 ). CD4+ T cell active caspase-1 expression directly correlated with elevated serum levels of IL-18 in hospitalized COVID-19 individuals (Supplemental Figure 6) .
Next, we wanted to determine if elevated active caspase-1 activity in CD4+ T cells is unique to COVID-19 patients. We assessed caspase-1 activity in pediatric or adult non-COVID-19 patients (n=104), including those that presented chronic sinusitis, asthma, common variable immune deficiency, or chronic idiopathic urticaria based on ICD-10 diagnosis codes indicated in the patient’s chart, in which T cell caspase-1 measurement was performed as a part of patient care during immunological work-up (Figure 2) . Among adults, there were statistically significant elevation of baseline T-helper cell caspase-1 in only asthmatics (p<0.001), nigericin stimulated T-helper cell caspase in asthmatics (p< 0.0001) and chronic rhinosinusitis (p<0.05). There was no elevation of baseline T-helper cell caspase-1 in any of the disease categories in the pediatric population, however, nigericin stimulated T-helper cell caspase was elevated in pediatric asthma and common variable immune deficiency (p< 0.001), further providing preliminary evidence on the role active caspase-1 in this high-risk population.