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.