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\begin{document}

\title{Caspases in COVID-19 disease and sequela and the therapeutic potential
of caspase inhibitors}



\author[1]{Matthew Plassmeyer}%
\author[2]{Oral Alpan}%
\author[3]{Michael Corley}%
\author[3]{Thomas Premeaux}%
\author[2]{Kimberleigh Lillard}%
\author[2]{Paige Coatney}%
\author[2]{Tina Vaziri}%
\author[2]{Suzan Michalsky}%
\author[3]{Alina Pang}%
\author[4]{Zaheer Bukhari}%
\author[3]{Stephen  Yeung}%
\author[3]{Teresa Evering}%
\author[5]{Gail Naughton}%
\author[5]{Martin Latterich}%
\author[6]{Philip Mudd}%
\author[5]{Alfred Spada}%
\author[2]{Nicole Rindone}%
\author[1]{Denise Loizou}%
\author[1]{Soren Ulrik Sonder}%
\author[7]{Lishomwa Ndhlovu}%
\author[4]{Raavi Gupta}%
\affil[1]{Amerimmune LLC}%
\affil[2]{Amerimmune}%
\affil[3]{Weill Cornell Medicine}%
\affil[4]{SUNY Downstate Medical Center}%
\affil[5]{Histogen}%
\affil[6]{Washington University in St Louis Department of Medicine}%
\affil[7]{Weill Cornell Medical College}%


\vspace{-1em}



  
  \date{May 31, 2024}


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\selectlanguage{english}
\begin{abstract}
COVID-19 can present with lymphopenia and extraordinary complex
multi-organ pathologies that can trigger long-term sequela. Given that
inflammasome products, like caspase-1, play a role in the
pathophysiology of a number of co-morbid conditions, we investigated
caspases across the spectrum of COVID-19 disease. We assessed
transcriptional states of multiple caspases and using flow cytometry,
the expression of active caspase-1 in blood cells from COVID-19 patients
in acute and convalescent stages of disease. Non-COVID-19 subjects
presenting with various co-morbid conditions served as controls.
Single-cell RNA-seq data of immune cells from COVID-19 patients showed a
distinct caspase expression pattern in T cells, neutrophils, dendritic
cells and eosinophils compared to controls. Caspase-1 was upregulated in
CD4+ T-cells from hospitalized COVID-19 patients compared to unexposed
controls. Post-COVID-19 patients with lingering symptoms (long-haulers)
also showed up-regulated caspase-1 activity in CD4+ T-cells that ex vivo
was attenuated with a select pan-caspase inhibitor. We observed elevated
caspase-3/7 levels in red blood cells from COVID-19 patients compared to
controls that was reduced following caspase inhibition. Our preliminary
results suggest an exuberant caspase response in COVID-19 that may
facilitate immune-related pathological processes leading to severe
outcomes. Further clinical correlations of caspase expression in
different stages of COVID-19 will be needed. Pan-caspase inhibition
could emerge as a therapeutic strategy to ameliorate or prevent severe
COVID-19.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Coronavirus Disease 2019 (COVID-19) is the latest global health threat
and, as in two preceding instances of the emergence of coronavirus
respiratory disease, poses critical challenges for the public health,
research, and medical communities (1, 2). Although the pathology of
COVID-19 is now well described, the mechanisms underlying disease
progression remain unclear. While a robust vaccination campaign and the
further development of vaccines against SARS-CoV-2, the causal agent of
COVID-19, are underway, a variety of investigational therapeutic
approaches are also being explored (3). Dexamethasone, plitidepsin, and
monoclonal antibody therapies, such as toclilizumab and eculizumab, have
shown promise in lowering soluble inflammatory markers part of the
cytokine storm and reducing severe outcomes in COVID-19 (4-7). Further
elucidating effector molecules responsible for disease progression to
determine effective interventions earlier in the course of the disease
is needed in order to help design effective therapies to ameliorate
disease manifestations and its complications (8-10).

The scope and severity of COVID-19 varies among those infected. Some
patients exhibit no or minor flu-like symptoms and quick recovery, some
have sustained fever and have persistent fatigue with a post-viral
syndrome, while others experience serious lung involvement that requires
hospitalization that may lead to death (11). Although the respiratory
and the gastrointestinal system are initial targets for SARS-CoV-2,
there clearly is a systemic nature to this disease in severe cases that
may be driven by micro-emboli and inflammatory processes (12, 13). While
follow up in natural history studies will likely uncover additional
post-infection sequelae, the notable impairment in type-I interferon
responses and rapid lymphopenia clearly plays a role in disease severity
(14-16), highlighting the need for novel therapeutics that take into
consideration the mechanism(s) of infection, viral replication, and
effector pathways that lead to COVID-19 associated pathologies.

Inflammasome activation in peripheral immune cells and tissues was
recently observed in COVID-19 patients and the level of
inflammasome-derived products, including active caspase-1, associated
with disease severity and poor outcomes (17). We recently reported that
caspase-1 expression in lymphocytes and serum IL-18 levels are increased
in liver transplant patients acutely ill with SARS-CoV-2 infection
suggesting pyroptosis mechanisms may play role in severe COVID-19 (18).
A recent study showed that SARS-CoV-2 infection of rhesus macaques led
to an upregulation of caspase-1 molecular signature in peripheral blood
cells as early as day 2 post-inoculation (19). Pyroptosis, also known as
caspase-1-dependent cell death, is inherently inflammatory, triggered by
various pathological stimuli (i.e. stroke, heart attack, cancer),
crucial for controlling microbial infections (20-22), and characterized
by rapid plasma-membrane rupture and the release of pro-inflammatory
intracellular contents (23, 24), a marked contrast to the regulated
death process of apoptosis (25). Insight into the complex activation and
regulation of the inflammasome complex and the way in which COVID-19
intersects with this pathway is an area of significant investigation
(26). Thus, strategies targeting the inflammasome/pyroptosis pathway
upstream of the production of the effector cytokines may be a novel
approach to reverse COVID-19 induced immune perturbations (27). Building
on our previous findings, we sought to expand our analysis to
investigate caspase-1 activity in SARS-CoV-2 infection, as well as the
role of other caspases, including in red blood cells (RBCs) given the
significance of COVID-19 associated coagulopathies (28, 29).

\textbf{Methods}

\emph{Subjects} . COVID-19 patient blood samples used for
immunophenotyping were obtained during patients' visit or
hospitalizations at SUNY Downstate Medical Center in New York from May
through to July 2020. Patients were defined as 1) non-hospitalized, with
and without presentation of COVID-19 symptoms and 2) hospitalized with
presentation of COVID-19 symptoms. Peripheral blood from venipuncture
was drawn into EDTA and Heparin coated vacutainer tubes for
immunophenotyping and processed within 48h of blood draw. Control blood
samples from healthy volunteers without SARS-CoV-2 infection or
co-morbid conditions were collected after obtaining written informed
consent.

\emph{Flow Cytometry} . Whole blood was stained per the clinical
standard immunophenotyping protocols (Amerimmune LLC, Fairfax, VA). The
samples were stained with the multiple antibody combinations for 30
minutes at 4@\selectlanguage{english}C. RBCs were lysed using BD FACS lysis solution (BD
Bioscience, San Jose, CA) as per manufacture directions. In brief,
freshly obtain peripheral blood mononuclear cells (PBMC) were separated
from 2 mL of whole blood within 24h of collection and diluted 1:1 with
phosphate buffered saline pH 7.2 (PBS) (Thermo Fisher Scientific,
Carlsbad, CA) using Lymphoprep (Stem cell Technologies, Cambridge, MA)
and Accuspin tubes (Sigma-Aldrich, St. Louis, MO) as per manufactures
directions. PBMCs' were washed in PBS and resuspended in 0.5 mL PBS. 100
\selectlanguage{greek}µ\selectlanguage{english}L of the PBMCs were immunostained with a mixture of antibodies at 4
for 1 hour. Cells were washed and resuspended in PBS prior to
acquisition. Antibodies used for the immune phenotyping of patient
samples and gating strategy for active caspase-1 staining are detailed
in the Supplementary information section of the manuscript \textbf{(}
Supplemental Table 1).

Apoptosis (caspase 3/7) and pyroptosis (caspase-1) were measured by flow
cytometry using fluorescent-labeled inhibitors of caspase probe assay
(FLICA; Immunochemistry Technologies, Minneapolis, MN). As a control,
PBMCs were stimulated with nigericin for 2 hours. FAM-FLICA probes
specific for caspase-1 or caspase 3/7 were added to 50 \selectlanguage{greek}µ\selectlanguage{english}l PBMC or whole
blood and incubated for 1h at 37. Cells were subsequently washed and
stained with a cocktail of fluorescently conjugated antibodies against
CD45 PE-CY7 {[}HI30{]}, CD3 AF700 {[}UCHT1{]}, CD4 PE {[}RPA-T4{]},
CD45RO PerCP-EF710 {[}UCHL1{]} and Viability Dye 780 (Thermo Fisher
Scientific, Carlsbad, CA). Red blood cells were identified as
CD235-positive cells in the experiments where caspase3/7 activity was
measured. Samples were acquired on a 3 laser BD FACS Canto 10. CS\&T
beads (BD Bioscience, San Jose, CA) were acquired daily to ensure
consistent performance of the BD FACS Canto 10 Canto10. The BD FACS
Canto 10 utilized for this study has been validated for T, B, NK and
Dendritic cell immunophenotyping clinical diagnostic testing. Denovo FCS
Express v6 clinical edition (De Novo Software, Pasadena, CA) was used
for flow cytometric analyses.

\emph{Plasma experiments} . Plasma was separated from whole blood
following centrifugation at 960 RCF. Cells (RBC and WBC) were either
incubated at 37 \selectlanguage{ngerman}°C alone or in the presence of trypsin for 1 hour then
washed with 10 packed cell volumes of RPMI 1640 incomplete medium.
Plasma was either held at room temperature (18 - 25°C) or heat
inactivated at 56°C for 1 hr. Plasma was added back to the RBC/WBC in a
1:1 ratio and incubated overnight, rocking at room temperature. RBC
caspase 3/7 activity was measured as described under flow cytometry.

\emph{Public SARS-CoV-2 and COVID-19 Transcriptome Analyses} . Single
cell RNA-Seq data from three COVID-19 participants that were ventilated
and diagnosed with acute respiratory distress syndrome at 2-16 days
after symptom onset and from 6 healthy controls was accessed from GEO
(30)\textbf{.} RNA-Seq data from cell lines infected \emph{in vitro}with
SARS-CoV-2 was accessed from GEO: GSE147507 (31)\textbf{.}Expression
values for Caspase genes were normalized by DESeq2.

\emph{Ex vivo stimulation studies} . Active caspase-1 in COVID-19
infected patient samples.~ Whole blood from a COVID-19 positive patient
was either (A) untreated or (B) treated with the pan-caspase inhibitor
emricasan (Sigma Aldrich, MO, SML2227-5MG) or selective caspase-1
inhibitor VX765 overnight at 37 degrees in a water bath. Subsequently,
PBMCs were purified (Accuspin System -- Histopaque 1077; Sigma Aldrich,
MO, A6929) and incubated with nigericin (Immunochemistry Technologies,
MN) for 2h.~ A Fam-FLICA probe specific for active caspase-1 was added
to 50 \selectlanguage{greek}µ\selectlanguage{english}l PBMC, incubated for 1h at 37.~ PMBCs were washed with cell
wash buffer (Immunochemistry Technologies, MN) and stained with a
cocktail of fluorescently conjugated antibodies against CD45 PE-CY7
{[}HI30{]}, CD3 AF700 {[}UCHT1{]}, CD4 PE {[}RPA-T4{]}, CD45RO
PerCP-EF710 {[}UCHL1{]} and Viability Dye 780 (Thermo Fisher Scientific,
Carlsbad, CA).~ Lymphocytes were identified using a standard gating
schematic which incorporated gating of lymphocytes on an FSC/SSC plot
and singlets on a FSC-A/FSC-H plot. Lymphocytes were further identified
as CD45+ on a CD45/SSC plot and subsequent CD3-, CD3+ and CD3+CD4+ cells
were identified on a CD45+ CD3/CD4 plot.

\emph{Statistical analysis} . Demographic and HIV-related
characteristics were described using the median, first quartile (Q1),
and third quartile (Q3) for continuous variables and frequency for
categorical variables. Differences among continuous variables were
evaluated by either the Mann-Whitney, student \emph{t} test or
Krustal-Wallis test with Dunn's multiple comparisons. Relationships
among parameters were examined by Pearson correlation for continuous
variables. All statistical tests were performed with GraphPad Prism
version 8.0 (Graphpad Software Inc., CA, USA). Statistical significance
is indicated as *p\textless{}0.05, **p\textless{}0.01,
***p\textless{}0.001, ****p\textless{}0.0001. P-values [?]0.100, but not
significant, are noted as statistical trends.

\emph{Study approval} . All clinical investigations were conducted
according to Declaration of Helsinki principles. All human studies were
approved by institutional review boards (IRB 269846-10 and 1285028
protocols from State University of New York Downstate Medical Center and
Amerimmune respectively). Written informed consent was received from
participants prior to inclusion in the study.

\textbf{Results}

\emph{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\textsuperscript{+} T cells was observed
using a published single cell RNA-Seq immune profiling dataset of
patients with moderate-severe COVID-19 (\textbf{Supplementary Figure 1}
) (30, 31). CD4\textsuperscript{+} 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 of\emph{in vitro} SARS-CoV-2 infection models
(\textbf{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\textsuperscript{+} T cells of non-ICU and ICU patients with COVID-19
and healthy individuals for comparison (\textbf{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\textsuperscript{+} T cells were
significantly elevated at baseline in hospitalized (both ICU and
non-ICU) COVID-19 patients compared to healthy participants
(\textbf{Figure 1A-D} ; all p-value\textless{}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\textsuperscript{+} T cells compared to
controls (all p-value\textless{}0.0001; \textbf{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-1\selectlanguage{ngerman}ß
processing and release, were also elevated in COVID-19
patients\textbf{(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\textsuperscript{+} 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 (\textbf{Supplemental
Table 1} ). Furthermore, CD4\textsuperscript{+} T cell active caspase-1
levels in patients with COVID-19 correlated with CRTH2+ T-cells, \selectlanguage{greek}γ/δ
\selectlanguage{english}T-cells, CD3-CD16+/CD56+ lymphocytes, and plasmacytoid dendritic cells
(\textbf{Figure 1 E-G} and \textbf{Supplemental Table 1} ).
CD4\textsuperscript{+} T cell active caspase-1 expression directly
correlated with elevated serum levels of IL-18 in hospitalized COVID-19
individuals \textbf{(Supplemental Figure 6)} .

Next, we wanted to determine if elevated active caspase-1 activity in
CD4\textsuperscript{+} 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 (\textbf{Figure 2)} . Among adults, there
were statistically significant elevation of baseline T-helper cell
caspase-1 in only asthmatics (p\textless{}0.001), nigericin stimulated
T-helper cell caspase in asthmatics (p\textless{} 0.0001) and chronic
rhinosinusitis (p\textless{}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\textless{} 0.001), further providing preliminary evidence
on the role active caspase-1 in this high-risk population.

\emph{Caspase-1 up-regulation is not limited to the acute stage of
COVID-19 disease}

Up to 87\% of inpatients and 35\% of outpatients who recover from
COVID-19 report persistence of at least 1 symptom, particularly fatigue
and dyspnea (32, 33). Although preliminary reports describe this new
feature as ``post-COVID-19 syndrome'', its mechanisms and natural
history remains unknown. We assayed caspase-1 activity in
CD4\textsuperscript{+} T cells of health care workers (HCWs) with
persistent respiratory and/or neurological (fatigue) symptoms at least
90 days post-SARS-CoV-2 infection (\textbf{Table 2} ). There was
statistically significant up-regulation of baseline as well as nigericin
stimulated T-helper cell caspase-1 levels only in symptomatic
``post-COVID-19'' HCWs, also known as long haulers (\textbf{Figure 3} ).
An asymptomatic group of patients with history of PCR+ virus infection
no flu like illness preceding the PCR test and absent seroconversion
showed elevated T-helper cell caspase-1 expression. Interestingly,
PCR-negative symptomatic HCWs with history of flu-like illness in early
2020 as well as those with positive IgG to SARS-CoV-2 also had increase
caspase-1 expression. The level of expression of nigericin stimulated
caspase-1 was comparable to those with active infection as seen in
Figure 2, although the baseline caspase-1 levels are lower in long
haulers. Non-exposed control subjects showed no T cell caspase-1
overexpression. The non-exposed subjects were identified from a
different geographical area (Fairfax, Virginia, USA), at a time when the
pandemic was at its lowest numbers (August-September 2020). These
subjects were also exercising strict self-isolation.

\emph{Pan-caspase inhibitor suppresses elevated caspase-1 activity in
CD4} \textsuperscript{+} \emph{T cells derived from moderate-severe
COVID-19 patients}

To assess whether CD4\textsuperscript{+} T cell caspase-1 activity can
be suppressed by small molecule caspase inhibitors, we incubated whole
blood samples with either the oral pan-caspase inhibitor emricasan (EMR)
(34) or the selective orally active ICE/caspase-1~inhibitor VX765 (35),
followed 24hrs later with or without nigericin stimulation. We found
that EMR suppressed CD4\textsuperscript{+} T cell caspase-1 activity in
COVID-19 samples or prevented its upregulation in healthy subjects
(\textbf{Figure 4} ), while VX765 showed only minimal suppressive
effect.

\emph{Red Blood cells show increased caspase-3/7 activity in COVID-19
disease which is suppressed by a pan-caspase inhibitor}

Recent reports suggest abnormalities in the RBCs in patients with
COVID-19 (36-38). In the process of Ficoll separation, we observed a
layer of RBCs contaminating the PBMC layer that was universally present
in all samples from COVID-19 individuals (\textbf{Figure 5A} ). This
finding was also present in up to 80\% of COVID-19 convalescent
subjects. Plasma from acutely infected COVID-19 subjects induced a
similar finding when incubated overnight with plasma depleted whole
blood of healthy patients. Treatment of the plasma samples with trypsin,
DNAse, or heat inactivation did not abolish this effect (data not
shown), suggesting a cell intrinsic process rather than due to cell
surface changes. Cellular caspases are not limited to immune cells. RBCs
do not express caspase-1, but have been shown to have detectable
caspase-3 that increases with various disorders (36). We found that RBCs
from acute COVID-19 subjects showed up-regulation of caspase-3/7
activity compared to healthy controls (\textbf{Figure 5B} ). Plasma from
these patients also upregulated caspase-3 in healthy subjects' RBCs.
When healthy subjects' RBCs were incubated with plasma from influenza
infected patients this effect was not observed, although a similar RBC
contamination was observed in these samples after Ficoll separation.
Furthermore, EMR suppressed the caspase-3 up-regulation in samples
incubated with COVID-19 patient-derived plasma, but did not change the
baseline expression levels in influenza-plasma incubated samples.

\textbf{Discussion}

While COVID-19 disease presents primarily with respiratory symptoms, for
many patients including children, it is a systemic disease with a wide
range of effects on many organs (39-41). In this report, we show
preliminary evidence for association of caspase molecules that play role
in cell death and immunity, not only in the acute phase but also late
stages of COVID19 (42). The changes are seen in multiple caspase
molecules, and a number of different circulating blood cells, a finding
that will further lead to exploration of the systemic nature of this
disease.

Caspase-1 has been proposed to play role in the pathophysiology of
COVID-19 (43). In addition to leading to a lytic form of cell death
called pyroptosis, caspase-1 induces the formation of biologically
active IL-18 and IL-1b (26, 44). IL-18 induces an IFN-\selectlanguage{greek}γ \selectlanguage{english}response, while
IL-1\selectlanguage{greek}β \selectlanguage{english}induces neutrophil influx and activation, T and B-cell activation,
cytokine and antibody production, and promotes Th17 differentiation
(45-48). Elevated levels of IL-18, IL-1\selectlanguage{greek}β, \selectlanguage{english}and other proinflammatory
cytokines were observed from the lungs and sera of COVID-19 patients
(49). Although activation of the inflammasome enhances immunity against
pathogens, the accompanying danger and inflammatory signals originating
from pyroptosing immune system cells (e.g., T cell and
macrophage/dendritic cells) can be damaging to the host in several ways.
First, it will result in immune cell lymphopenia, such as that observed
with T cells, a pathognomonic feature for SARS-CoV-2, creating an
adaptive immune defect. Second, the host will have difficulty
controlling the inflammation created in the setting of this immune
deficiency as the ``danger signals'' would also be originating from
dying immune system cells (50-52). The end result is likely a
self-damaging shut down of the immune system, resulting in acute
virus-induced immune deficiency (AVID). Preventing the pyroptotic
lymphocyte death by using caspase inhibitors may lead to better success
rather than inhibition of the inflammatory response from the cell death
itself. The failure of cytokine targeted therapies could be due to that
adaptive immune dysfunction due to AVID weighing more heavily than an
inflammatory response in disease progression (18).

In our active T-cell caspase-1 assay, we analyzed the sensitivity to
nigericin stimulation. This provided further information on the cell
surface pannexin-1 expression, which is upregulated by cellular caspases
and can play role in disease pathogenesis. Also, cell surface expression
level of pannexin-1 can vary between healthy controls, which can explain
the differences in response to nigericin. We also found that EMR is
effective in reducing active caspase-1 T cells from COVID-19 patients,
while VX765 failed to significantly do so. VX765 is a prodrug that needs
hydrolyzation to form into its active form and is a reversible inhibitor
of caspase-1, as opposed to EMR which is an irreversible inhibitor.
Furthermore, EMR is transported into cells via active transport with
cell membrane channels, whereas VX765 is internalized by passive
diffusion. All these factors may explain the differences we see between
these two caspase inhibitors. Further studies are needed to explain the
differences in inhibition between the two molecules, particularly in the
context of SARS-CoV-2 infection.

The changes in caspase expression are not only limited to T cells, as we
show changes in caspase-3 in RBCs and caspase-5 in neutrophils. Caspase
activation has been shown to induce changes in the RBC morphology
(53-55), which can explain the contamination of the PBMC layer during
cell separation as a result of a reduction in their density.
Furthermore, their overexpression of caspase 3/7 can subsequently
contribute to the formation or advancement of inflammatory microvascular
thrombi, which is prominently found in the lung, kidney, and heart of
patients with COVID-19 (56, 57). Although viral illnesses typically will
impact the function or the life-cycle of lymphocytes, presenting with
either lymphocytosis, such as in with CMV, influenza, varicella, or more
rarely, lymphopenia, as in H5N1, H1N1, HIV, the finding of neutrophilia
in the setting of moderate to severe COVID-19 has been a common, but
intriguing finding (58). In the absence of significant overexpression of
apoptotic caspases, the increase in the inflammatory caspase-5 in
neutrophils may play a part in the neutrophilia observed with COVID-19.
Furthermore, the production of IL-10 by neutrophils with increase
caspase activity, can further suppress the proliferation of T
lymphocytes, hence contributing to the adaptive immune deficiency.

Caspase molecules have been studied extensively in many forms of
inflammatory conditions, such as obesity, diabetes and nonalcoholic
steatohepatisis (NASH) (59-61). Caspase-1-dependent inflammasome
activation has been shown to have a crucial function in the
establishment of diabetic nephropathy (62). In an animal model of
hypertension apoptosis of myocardial cells were demonstrated, and the
apoptosis becomes more serious with the constantly elevated level and
prolonged duration of hypertension. The activity of caspase-3 was shown
to have a close correlation with cardiomyocyte apoptosis (63). Our data
showing increased expression of active caspase-1 in T-helper cells of
patients with asthma and immune deficiencies correlates with their
high-risk classification for severe COVID-19 as provided by the centers
for disease control (CDC). Perhaps, the changes in cellular caspases
seen in COVID-19 may not only explain the multisystem involvement in
this disease but may allow for identification for those at risk for
complications, including long haulers, based on caspase expression in
blood cells.

Our findings suggest a novel alternate therapeutic approach against
COVID-19 through the use of caspase inhibition early on in the course of
infection to alleviate or prevent disease progression. As an oral
formulation, EMR has been shown to reduce serum markers of apoptosis
(caspase-3/7), liver enzymes, function (e.g., reducing ALT, MELD \&
Child-Pugh scores, INR and total bilirubin) and inflammatory biomarkers
(CK-18) in patients w/ hepatitis C virus and NASH (64). Although there
was no improvement in liver histology, it is possible that the pathology
of this disease has mechanisms that are caspase-independent or with the
timing of therapy (65, 66). Although SARS-CoV-2 does not seem to infect
immune system cells (with the possible exception of macrophage or
dendritic cells), the outcome of T cell depletion in severe forms of the
disease seems to be through caspase-1 activation, a mechanism also
proposed in HIV (67). A better understanding of the impact of different
co-morbid conditions on T cell caspase expression at baseline, before
exposure to SARS-CoV-2, may identify those that are at highest risk for
developing severe disease. There is a large body of evidence pointing
out to an activated inflammasome in a wide variety of disorders that
overlap with high-risk conditions for severe COVID-19 (15, 51, 52, 68).
Ultimately \emph{in vivo} clinical data is necessary to test the
hypothesis of whether pan-caspase inhibition can prevent inflammasome
activation in early onset SARS-CoV-2 patients and subsequent lymphopenia
and sequelae development. Furthermore, the pan-caspase inhibitor, EMR
has been shown in a bioinformatics computational screen to bind to the
COVID-19 receptor ACE2, suggesting a potential block to cell entry (69).
In a separate unrelated study, a screen of \textasciitilde{}6,070 drugs
with a known 28 previous history of use in humans was conducted to
identify compounds that inhibit the activity of SARS-CoV-2 main protease
Mpro \emph{in vitro} (70). EMR was shown to be among 50 compounds with
activity against Mpro with an overall hit rate \textless{}0.75\%.
Preliminary evidence on this multimodal therapeutic effect of EMR raise
a relevant key question that will need to be answered through a
randomized clinical trial in the setting of COVID-19 (Figure 6).

An important aspect of our study is the demonstration of caspase-1
expression well past the acute stage of COVID-19, suggesting a role in
the convalescent phase or disease sequelae. Such persistent changes can
not only be limited to immune system cells but can be seen in tissues
such as endothelial cells, which could be a causal impact on multiple
organ systems (19, 39, 71). Assessing the sequelae, such as fatigue,
dyspnea, cough, joint pain, anosmia, among others (32, 72), in
correlation with the changes in caspase molecules in natural history
studies are warranted. Sequelae targeting populations where caspase
elevations are more common, such as the elderly, and those with other
co-morbid conditions, such as heart disease, diabetes, hypertension,
provides further evidence for the association of caspases with poor
outcomes from COVID-19 (1). Dampening the inflammatory response early in
the disease process may be a strategy to prevent sequelae, such as in
rheumatic fever, where treating streptococcus early on in the disease
through the co-treatment of penicillin and anti-inflammatories can
prevent severe disease sequelae.

\textbf{Author contributions}

MP, SUS, TV, KL and PC conducted experiments and acquired data. MP, OA,
TAP, ZB analyzed the data. MP, OA, MJC, KL, PC, TV, SM, TAP, APSP, ZB,
STY, THE, GN, ML, PM, AS, NR, DL, LCN, RG wrote the manuscript. OA, MP,
LCN and RG contributed to the study design and concept. All authors
reviewed the manuscript.

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Table 1. Demographics and co-morbidities of COVID-19 patients and
healthy controls\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& \textbf{Hospitalized} & \textbf{SARS-CoV-2 PCR +} &
\textbf{Healthy}\tabularnewline
& \textbf{Critical/ICU} & \textbf{Non-Critical/Non-ICU} &
\textbf{SARS-CoV-2 Ab Neg}\tabularnewline
\textbf{n} & 18 & 11 & 28\tabularnewline
\textbf{Age (Median)} & 66 & 71.5 & 52\tabularnewline
80+ & 5 & 1 & 5\tabularnewline
71-80 & 4 & 3 & 6\tabularnewline
61-70 & 2 & 2 & 5\tabularnewline
51-60 & 3 & 3 & 4\tabularnewline
41-50 & 2 & 1 & 5\tabularnewline
0-40 & 2 & 1 & 3\tabularnewline
\textbf{Ethnicity} & & &\tabularnewline
African/American & 11 & 4 & 10\tabularnewline
Other & 7 & 7 & 18\tabularnewline
\textbf{Gender} & & &\tabularnewline
Male & 9 & 5 & 13\tabularnewline
Female & 9 & 6 & 14\tabularnewline
\textbf{Mean BMI} & 31 & 29 & 26\tabularnewline
\textbf{Comorbid Conditions} & & &\tabularnewline
Asthma & 3 & 3 & 0\tabularnewline
Autoimmune disease & 3 & 2 & 0\tabularnewline
Cancer & 1 & 3 & 0\tabularnewline
COPD & 8 & 3 & 0\tabularnewline
Coronary Artery Disease & 7 & 6 & 0\tabularnewline
Congestive Heart Failure & 6 & 5 & 0\tabularnewline
CKD without dialysis & 2 & 4 & 0\tabularnewline
CKD/ESRD with dialysis & 7 & 2 & 0\tabularnewline
Diabetes Mellitus & 6 & 4 & 0\tabularnewline
Hyperlipidemia & 4 & 5 & 0\tabularnewline
Hypertension & 11 & 5 & 0\tabularnewline
Immune suppression & 3 & 2 & 0\tabularnewline
\bottomrule
\end{longtable}

Table 2. Demographics of post-COVID-19 health care workers (HCWs)\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
& \textbf{HCW} & \textbf{Co-morbidities}\tabularnewline
\textbf{n} & 36 &\tabularnewline
\textbf{Age (Median)} & 64 &\tabularnewline
71-80 & 3 (8) & 2 (67)\tabularnewline
61-70 & 4 (11) & 2 (50)\tabularnewline
51-60 & 16 (44) & 8 (50)\tabularnewline
41-50 & 4 (11) & 1 (25)\tabularnewline
18-40 & 9 (25) & 2 (22)\tabularnewline
\textbf{Ethnicity} & &\tabularnewline
African/American & 12 (33) & 7 (58)\tabularnewline
Other & 24 (67) & 8 (33)\tabularnewline
\textbf{Gender} & &\tabularnewline
Male & 15 (42) & 9 (60)\tabularnewline
Female & 21 (68) & 6 (40)\tabularnewline
\bottomrule
\end{longtable}

Figure 1. Caspase-1 expression in immune cells. Caspase-1 expression is
shown for (A-C) total (CD3\textsuperscript{+}) T-cells,
CD4\textsuperscript{+} T cells, non-CD3\textsuperscript{+} T cell
lymphocytes, and myeloid cells. (D-F) Caspase 1 expression in
CD4\textsuperscript{+} T cells is correlated with \selectlanguage{greek}γ/δ \selectlanguage{english}T cells, NK cells
(CD3-CD56/16+) and CRTH2+ CD4\textsuperscript{+} T cells. Individual
patient data are shown and the frequency of caspase-1+
CD4\textsuperscript{+} T cells are significantly elevated at baseline in
COVID-19 patients (\emph{n} =29) compared to healthy (\emph{n} =28)
participants with and without nigericin stimulation. All p-values are by
unpaired and 2-tailed Student's~\emph{t} ~test or linear regression
analysis. *p\textless{}0.05, ***p\textless{}0.001,
****p\textless{}0.0001

Figure 2. Caspase-1 expression in CD4\textsuperscript{+} T cells of
non-COVID-19 patients (unexposed and uninfected adult and pediatric
patients with allergic/immunological disorders). Open symbols are
resting non-stimulated CD4\textsuperscript{+} T cells. Closed symbols
represent caspase-1 expression in nigericin stimulated
CD4\textsuperscript{+} T cells. Different symbols represent different
disease states. Adults (\textgreater{}18 years) are represented with an
A (\emph{n} =65) and pediatric subjects (\textless{}18 years)
represented with a P (\emph{n} =39) in the bottom of the graph. CRS;
chronic rhinosinusitis, CVID; common variable immune deficiency, and
CIU; chronic idiopathic urticaria. The diagnosis and T-cell caspase-1
data are retrospective data from medical records of patients presenting
to an Allergy Immunology Clinic for an immunological evaluation. Control
patient data was generated during clinical assay validation of T-helper
cell caspase-1 assay (\emph{n} =45 for adults and \emph{n} =39 for
pediatrics). All p-values are by unpaired and 2-tailed
Student's~\emph{t} ~test. *p\textless{}0.05, ***p\textless{}0.001,
****p\textless{}0.0001.

Figure 3. Caspase-1 expression on CD4\textsuperscript{+} T cells in
post-COVID-19 Health Care Workers. Blood samples were analyzed at least
90 days after SARS-CoV-2 exposure in healthcare workers. Patients with
no exposure history and negative PCR to SARS-CoV-2 were used as
controls. Solid black circles represent symptomatic, green circles
represent non-symptomatic patients. Exposure indicates being in close
proximity to SARS-CoV-2 infected patients in the absence of personal
protection equipment. Persistent post-COVID19 symptoms correlated with
elevated caspase-1 expression in T-helper cells (p\textless{} 0.05).

Figure 4. Effect of caspase inhibition on CD4\textsuperscript{+} T cells
in COVID-19 patients. Samples from healthy and COVID-19 subjects
incubated with caspase inhibitors: EMR or VX765. Activated caspase-1 was
measured by flow cytometry using a Fam-FLICA probe. Emricasan at 1\selectlanguage{greek}μ\selectlanguage{english}M
concentration induces the strongest suppression of
CD4\textsuperscript{+} T cell caspase-1 in unstimulated cells
(p\textless{} 0.01), whereas the selective caspase-1 inhibitor VX-765
does not induce a similar effect. Krustal-Wallis ANOVA test with Tukey
multiple comparisons for \textgreater{}2 group comparisons were used.
For P values are as follows:~*p\textless{}0.05,~**p\textless{}0.01.
Experiments represent \emph{n} =3.

Figure 5. Caspase 3/7 activity in red blood cells (RBC) derived from
COVID-19 patients. Blood samples were analyzed from hospitalized
patients with SARS-CoV-2 infection. A) RBC contamination of the PBMC
layer after Ficoll separation. B) Analysis of caspase 3/7 activity in
COVID-19 patients and healthy controls. Some experiments were done using
plasma from COVID-19 or subjects with influenza with incubated with RBCs
from health uninfected donors as indicated. COVID-19 patients RBCs show
elevated caspase 3/7 (p\textless{} 0.01) and EMR has a significant
suppressive activity on this expression (p\textless{} 0.05). Plasma from
hospitalized COVID-19 patients induces caspase 3/7 in health RBCs on
overnight incubation (p\textless{} 0.01). Krustal-Wallis ANOVA test with
Tukey multiple comparisons for \textgreater{}2 group comparisons were
used. For p values are as
follows:~*p\textless{}0.05,~**p\textless{}0.01. Experiments
represent\emph{n} =3.

Figure 6. Emricasan mechanism of action.

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