key: cord-0767674-9ugpuewj
authors: Plassmeyer, M.; Alpan, O.; Corley, M.; Lillard, K.; Coatney, P.; Vaziri, T.; Michalski, S.; Premeaux, T. A.; Pang, A. P. S.; Bukhari, Z.; Yueng, S. Y.; Evering, T.; Naughton, G.; Latterich, M.; Mudd, P. A.; Spada, A.; Rindone, N.; Loizou, D.; Ndhlovu, L.; Gupta, R.
title: Caspases in COVID-19 Disease and Sequela and the Therapeutic Potential of Caspase Inhibitors
date: 2020-11-04
journal: nan
DOI: 10.1101/2020.11.02.20223636
sha: 82b729a0c7aed98c5681976efcbdb83c267d9c11
doc_id: 767674
cord_uid: 9ugpuewj

At present, there are is no effective vaccine and only one FDA approved early-stage therapy against infection with the SARS-CoV-2 virus to prevent disease progression. The excessive inflammation and tissue damage associated with COVID-19 can lead to immediate (i.e. respiratory failure, sepsis, and ultimately, death) or long-term health problems (i.e. fatigue, dyspnea, cough, joint pain, anosmia) and the risk for these complications are higher in the elderly population, certain ethnic groups, as well as those with various co-morbid conditions. Cellular caspases play a role in the pathophysiology of a number of disorders that overlap with the list of co-morbid conditions seen in severe COVID-19. In this study, we assessed transcriptional states of caspases in immune cells from COVID-19 patients and profiled intra-cellular caspases in immune cells and red blood cells derived from a spectrum of COVID-19 patients hospitalized with acute disease or convalescent. Gene expression levels of select caspases were increased in in vitro SARS-CoV-2 infection models and single cell RNA-Seq data of peripheral blood from COVID-19 patients showed a distinct pattern of caspase expression in T cell, neutrophils, and dendritic cells. Flow cytometric evaluation of CD4 T cells showed up-regulation of caspase-1 in hospitalized COVID-19 patients compared to unexposed controls, with the exception of a subset of patients with asthma and chronic rhinosinusitis (CRS). Convalescent COVID-19 patients with lingering symptoms (long haulers) showed persistent up-regulation of caspase-1 in CD4 T cells that was attenuated ex vivo following co-culture with a select pan-caspase inhibitor. Further, we observed elevated caspase 3 levels in red blood cells from COVID-19 patients compared to controls that were responsive to caspase inhibition. Taken together, our results expose an exuberant caspase response in COVID-19 that may facilitate immune-related pathological processes leading to severe outcomes. Pan-caspase inhibition could emerge as a therapeutic strategy to ameliorate, reduce, or prevent severe COVID-19 outcomes.

to develop a vaccine against SARS-CoV-2, the causal agent of COVID-19, a variety of investigational therapeutic approaches are also being explored (3) . Although the pathology of COVID-19 is now well described, the mechanisms of disease progression is still not clear. In clinical trials dexamethasone reduced severe outcomes in critically ill patients, suggesting of an inflammatory mechanism. However, while the use of specific anti-IL-6 monoclonal antibody therapies attenuated the cytokine storm, and eculizumab reduced soluble inflammatory markers, including c-reactive protein, in moderate-severe COVID-19, the clinical benefit has been marginal (4, 5) . These findings suggest a lack of understanding of the effector molecules responsible for disease progression or that an intervention earlier in the course of the disease is needed, in order to help design effective therapies to ameliorate disease manifestations and its complications (6) (7) (8) .

The scope and severity of COVID-19 varies among those infected, with some patients presenting with 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 and may lead to death (9) . Although the respiratory and the gastrointestinal system are initial targets for SARS-CoV-2, there clearly is a systemic nature to this disease in some individuals that may be driven by micro-emboli and inflammatory processes (10, 11) . Follow up in natural history studies will likely uncover additional post-infection sequelae. Furthermore, the notable impairment in type-I interferon responses and rapid lymphopenia clearly plays a role in disease severity (12) (13) (14) . The scope and severity of COVID-19 is extraordinary, ranging from patients who present with no or minor flu-like symptoms and recover quickly to those who experience sustained fever and have persistent fatigue with a post-viral syndrome, to people who have serious lung involvement that either results in hospitalization or creates intubation needs and intensive care to people who die (14) . This highlights 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.

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, along with non-liver transplant controls, suggesting pyroptosis mechanisms may play role in severe COVID-19 (15) .

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 (16) .

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 (17) (18) (19) , and characterized by rapid plasma-membrane rupture and the release of proinflammatory intracellular contents (20, 21) , a marked contrast to the regulated death process of apoptosis (22) . 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 (23) . 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 (24) . Building on our previous findings, we sought to expand our analysis to investigate the expression of not only the inflammatory caspases but also initiator and executionary caspases across the spectrum of COVID-19 disease in multiple immune cell types.

The finding of increases in caspase molecules beyond caspase-1, such as caspase-3 in red blood cells (RBCs), led us to further define the full caspase expression profile of immune system cells.

The impact of unique caspase expression profiles in given cells may impact specific outcomes such as RBCs involvement in coagulopathies in COVID-19 disease and determine the relationship with parameters of disease progression (25, 26) .

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.

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˚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. 

Plasma was separated from whole blood following centrifugation at 960 RCF. Cells (RBC and WBC) were either incubated at 37 °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 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted November 4, 2020. ;  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.

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 (27) . RNA-Seq data from cell lines infected in vitro with SARS-CoV-2 was accessed from GEO: GSE147507 (28) . Expression values for Caspase genes were normalized by DESeq2. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2020. ;  cells were identified on a CD45+ CD3/CD4 plot. Shown are the active caspase-1 MFI and % positive for the three cellular populations indicated.

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.

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<0.05, **p<0.01, ***p<0.001, ****p<0.0001. P-values ≤0.100, but not significant, are noted as statistical trends.

All clinical investigation 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.

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To follow up on our findings of increase caspase-1 expression in T cells of patients with COVID-19, we assayed for multiple caspases in different immune cell types from blood samples of patients with moderate-severe COVID-19. We examined caspase gene expression levels in public transcriptome profiling datasets of single cell RNA-Seq of immune cells from individuals with COVID-19 ( Figure 1 ) and in in vitro SARS-CoV-2 infected cell (Supplemental Figures 1-3) and found transcriptional support of our previous findings showing upregulation of caspase-1 in CD4 T cells. We also found evidence of altered transcriptome levels of caspase genes in natural killer (NK cells), and neutrophils. Interestingly, plasmacytoid dendritic cells were the only immune cell type that showed up-regulation of caspase-9. Neutrophils showed a unique profile with upregulated caspase-5 and 7, an inflammatory and a pro-apoptotic caspase respectively.

IFN-stimulated CD4 T cells show significant upregulation of caspase-7 and -9. We found that RNA-Seq data from several cell lines infected in vitro with SARS-CoV-2 showed an increase in caspase 1 and 4 in select cell lines relative to other viruses, suggesting multiple cellular death mechanisms that may potentially play a role in addition to the caspase-1 pathway.

We designed a laboratory developed test (LDT) to stain intracellular active caspase-1 in CD4 T Table 1 ). Frequency of caspase-1+ CD4 T cells were significantly elevated at baseline in hospitalized (both ICU and All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted November 4, 2020. ; non-ICU) COVID-19 patients compared to healthy participants with and without nigericin stimulation (all p-value<0.0001).

Although we did not see increase in IL-18 and IL-1β in the RNA analysis, serum levels of IL-18 were increased in moderate-severe COVID-19 individuals and showed a positive correlation

with T-helper cell caspase-1 expression (data not shown). 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 2 Table 2 ). Such correlations point out to the complex cellular interactions involved in COVID-19.

To determine if elevated caspase-1 levels in CD4 T cells is unique to COVID-19 patients, we assessed these levels in non-COVID-19 patients presenting to an Allergy/Immunology Clinic (Supplemental Table 3 ) and assays were performed as a part of patient care during their routine immunological work-up. Data from 102 adult and pediatric subjects are shown in Figure 2 for patients who presented with chronic sinusitis, moderate-severe asthma, chronic idiopathic urticaria and immune deficiencies. Normal ranges for the assay are shown in gray shaded areas ( Figure 3 ). Only patients with asthma showed a baseline elevation of caspase-1 in CD4 T cells ( Figure 3 ). All rights reserved. No reuse allowed without permission.

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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 (29, 30) . Although preliminary reports describe this new feature as "post-COVID syndrome", its mechanisms and natural history remains unknown. We assayed caspase-1 expression on the CD4 T cells of health care workers (HCWs) with persistent symptoms at least 90 days post-SARS-CoV-2 infection (Supplemental Table 4 ). There was 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 ( Figure 4) . 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 were lower in these long haulers. Non-exposed control subjects showed no T cell caspase-1 overexpression.

To assess whether CD4 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) (31) or the selective orally active ICE/caspase-1 inhibitor VX765 (32), followed 24hrs later with or without nigericin stimulation. We found that EMR suppressed CD4 T cell caspase-1 activity in COVID-19 samples or prevented its upregulation in healthy subjects ( Figure 5 ), while VX765 did not show a suppressive effect.

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Recent reports suggest abnormalities in the RBCs in patients with COVID-19 (33) (34) (35) . 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 ( Figure 6A ). This finding was also present in up to 80% of COVID-19 convalescent subjects. Plasma from acutely infected 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. 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 (33) . We found that RBCs from acute COVID-19 subjects showed significant up-regulation of caspase-3 compared to healthy controls ( Figure 6B ). Plasma from these patients also upregulated caspase-3 in healthy subjects' RBCs. We did not see this effect when healthy subjects' RBCs were incubated with plasma from influenza infected patients, although a similar RBC contamination was observed in these samples after Ficoll separation. Furthermore, EMR suppressed the caspase-3 up-regulation in the COVID-19 plasma incubated samples, but did not change the baseline expression levels in influenza-plasma incubated samples.

While COVID-19 disease largely presents with respiratory symptoms, for many patients it is a systemic disease with a wide range of effects on different organ systems and persistent postinfection sequelae. A minority of children present with an autoimmune syndrome suggesting it All rights reserved. No reuse allowed without permission.

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is already a systemic disease for some people at the initial presentation (36) (37) (38) . The blur between acute infections and post-infectious sequelae will only be understood better as detailed follow ups are performed along with immunological testing in natural history studies (39) . In this report, we show preliminary evidence for caspase involvement that may shed further light not only into the systemic nature of the illness, but also the chronicity that is observed in COVID-19 long haulers.

There is accumulating evidence on the role of both apoptotic and pyroptotic cell death in the disease progression seen in COVID-19 (40) . Pyroptosis leads to the production of IL-18 and IL-1β as a result of their cleavage by inflammasome activated caspase-1 (23, 41) . IL-18 induces an IFN-g response, while IL-1β induces neutrophil influx and activation, T and B-cell activation, cytokine and antibody production, and promotes Th17 differentiation (42) (43) (44) (45) . High levels of IL-18, IL-1β, and other proinflammatory cytokines were observed from the lungs and sera of COVID-19 patients (46) . Although the ultimate outcome of activation of the inflammasome in the infected tissue cells (e.g. respiratory epithelial cells) should result in an enhanced immunity against pathogens, an accompanying pyroptosis and apoptosis of immune cells (e.g. T cell and macrophage/dendritic cells) also sending out danger signals may lead to poor outcomes overall.

We hypothesized that T cell lymphopenia, which is a pathognomonic feature for SARS-CoV-2, might be a path in which the virus evades the human host response by creating an adaptive immune defect along with fueling an uncontrolled inflammatory response by the release of cellular contents of both immune and non-immune cells (47) . Since caspase-1 is up-regulated predominantly in the memory CD4 T cell, this could explain why older persons are more susceptible to severe disease compared to the younger population. The end result is likely a self-All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted November 4, 2020. ;  damaging shut down of the immune system that further fuels the inflammation created by the viral infection, resulting in acute virus-induced immune deficiency (AVID). This connection between caspase-1 and COVID-19 has significant therapeutic implications since preventing the pyroptotic lymphocyte death rather than inhibition of the inflammatory response may be a more ideal option. The failure of cytokine targeted therapies could therefore suggest that adaptive immune dysfunction weighs more than an inflammatory response in disease progression (48) .

A dysregulated caspase expression profile of immune cells can impact outcomes of an immune response. Caspases not only play an essential role during apoptotic cell death, but a subfamily of them, the inflammatory caspases, are associated with immune responses to microbial pathogens (49) . Caspases can be subdivided into initiator and effector caspases. The subset of caspases that cleave selected substrates to produce the changes associated with apoptosis are known as effector caspases, which in mammals are caspase-3, -6, and -7. In most instances, these executioner caspases are activated by the initiator caspases, i.e., caspase-8, caspase-10, caspase-2, and caspase-9. Some of the "apoptotic" caspases, in particular caspase-8, have been shown to have additional roles in proliferation and differentiation. For example, caspase-8 activity appears to be necessary for lymphocyte proliferation (50) . The clinical consequences of this dysregulated caspase activation can correlate with the hematological and immunological findings in COVID-19 patients (51) . Although viral illnesses typically will impact the function or the life-cycle of lymphocytes, presenting with either lymphocytosis (for example CMV, influenza, varicella) or more rarely, lymphopenia (for example in H5N1, H1N1, HIV), the finding of neutrophilia in the setting of COVID-19 has been a common, but intriguing finding (52) . The unique combination of inflammatory and apoptotic caspase upregulation All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted November 4, 2020. ;  coupled to the timing of the activation in the neutrophil response to SARS-CoV-2 may result in this unique laboratory finding observed in COVID-19.

An extensive body of evidence from the past three decades has implicated dysregulated caspase activation as a causal disease mechanism in tumorigenesis, autoimmunity, autoinflammation and infectious pathologies. As research into signaling pathways of inflammatory caspases progressed, an unexpected amount of crosstalk with apoptotic caspases has emerged over the past years. Pathogens use specific caspase pathways to evade the host, such as caspase-8 and (53) . Although caspases are most often associated with apoptosis, there has been persistent evidence that some of these enzymes can also influence proliferation (54) . One of the earliest observations was that can be one mechanism through which the virus keeps the host immune responses in check.

However, caspase-3 may have the opposite effect, as B cells lacking caspase-3 showed increased proliferation in vivo and hyperproliferation after mitogenic stimulation in vitro (56) . Although the impact on caspase-3 is not seen in the immune cells circulating in the blood, tissue macrophages in postmortem analysis and circulating red blood cells do show significant increases. Caspase activity can be a double-edged sword and SARS CoV2 may be using this strategy to evade the host in way that's beneficial for its own survival to move on to the next host to infect. All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted November 4, 2020. ;  Little is known about the natural stimuli that lead to the assembly of complexes activating inflammatory caspases. The only physiological stimulus that is reported to activate caspase-1 in phagocytes is LPS (57) . Very few studies extended this model of caspase-1 activation, mainly because it only induces moderate caspase-1 activity. The best-studied model of caspase-1 activation relates to the exposure of cells to extracellular ATP acting on P2X7 receptors.

P2X7 receptors belong to a family of ion channel receptors gated by extracellular ATP and widely distributed in non-neuronal cells (57) . Hence, at least two mechanisms are able to trigger activation of caspase-1, one as a consequence of a bacterial product and the other following changes in the intracellular ionic environment. It is of interest to see SARS-CoV-2 added to this list and further elucidating mechanisms on how this virus activates caspase-1 will be of importance in understanding disease mechanisms in COVID-19.

Taken together, our findings implicate that an induced acute immunodeficiency resulting from the necrotic cell death of lymphocytes may play an important factor in COVID-19 progression.

We propose that the inflammatory response is secondary to the "danger signals" from necrotic cell death of immune system cells, resulting in a heightened inflammation compared to the one induced by dying tissue cells (58) (59) (60) . The end result is likely a self-damaging cell death and subsequent cytokine storm that further fuels the inflammation created by the viral infection, resulting in AVID. The failure of cytokine targeted therapies could be due to that adaptive immune dysfunction weighs more heavily than an inflammatory response in disease progression (15) . This connection between caspase-1 and COVID-19 has significant therapeutic implications since preventing the pyroptotic lymphocyte death rather than inhibition of the inflammatory All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted November 4, 2020. ;  response may be a more efficacious therapeutic option. Furthermore, an optimal therapeutic approach would be to also prevent progression into this end-stage disease by the use of therapeutics targeting the virus, pyroptosis or both.

The caspase mediated disease process in COVID-19 is most likely not limited to caspase-1 and T cells, as we demonstrate changes in RBCs that may additionally involve the caspase-3 pathway.

Inflammatory microvascular thrombi are present in the lung, kidney, and heart, containing neutrophil extracellular traps associated with platelets and fibrin along with changes in RBC morphology (61, 62) , which raise the possibility of RBCs playing a role in the clotting disorder observed in COVID-19 patients. Although the RBC layer contaminating the PBMCs in COVID-19 blood samples is also observed in other infections, such as influenza, the caspase-3 upregulation may lead to changes in the RBC that may mechanistically link them to complications of the disease (63) (64) (65) .

Both metabolic inflammation and apoptosis play a central role in the pathogenesis of metabolic disease such as obesity, diabetes and the progression of nonalcoholic steatohepatisis (NASH) to more severe liver disease (66) (67) (68) . Caspase-1-dependent inflammasome activation has been shown to have a crucial function in the establishment of diabetic nephropathy (69) . 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 (70). The pan-caspase inhibitor, EMR has been shown in a bioinformatics computational screen to binding to the COVID-19 receptor ACE2, suggesting the potential to block cell entry (71) . In All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted November 4, 2020. ; a separate unrelated study, a screen of ~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 in vitro (72) . EMR was shown to be among 50 compounds with activity against Mpro with an overall hit rate <0.75%. As an oral formulation, EMR has been shown to be welltolerated, reduced 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. There was no impact on histology of the disease characterized by liver fibrosis. (73) . Lack of improvement in liver histology with EMR, despite such a dramatic and universal reduction in serum biomarkers of disease activity suggest a mechanisms that lead to liver injury, resulting in increase in liver enzymes that EMR has a positive impact on is different than those causing fibrosis in NASH (74, 75) . One can also consider an overwhelming cellular injury that EMR is not able to reverse, or lack of efficacy due to therapy being initiated late in the course of the liver disease.

Our findings support a novel alternate therapeutic approach against COVID-19 through the use of a caspase inhibitor early on in the course of infection to alleviate or prevent disease progression. 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 a similar mechanism to that seen in HIV; caspase-1 activation (76) . Perhaps it will be important to better understand the impact of different comorbid conditions on T cell caspase expression at baseline, before exposure to SARS-CoV-2, which may be the determining factor 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 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted November 4, 2020. ;  with high-risk conditions for severe COVID-19 (13, 59, 60, 77) . Ultimately 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. Preliminary evidence on the therapeutic effect of EMR on SARS-CoV-2 viral protease and ACE2 receptor inhibition raise a relevant key question that will need to be answered through a randomized clinical trial on the multi-modal actions of this pan-caspase inhibitor in the setting of COVID-19.

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The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.11.02.20223636 doi: medRxiv preprint Figure 1 . Human caspase single cell gene expression level. Data was compiled from three COVID-19 participants and from 6 healthy controls. Expression values for the caspase genes were normalized by DESeq2. All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted November 4, 2020. ; Patients with no exposure history and negative PCR to SARS-CoV-2 were used as controls. Solid circles represent nigericin stimulated cells. Red circles represent symptomatic, black represent non-symptomatic patients. Exposure indicates being in close proximity to SARS-CoV-2 infected patients in the absence of personal protection equipment.

Incubation of healthy and COVID-19 subjects samples with caspase inhibitors: EMR or VX765. Caspase-1 expression was measured by flow cytometry.

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 expression 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. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Table 2 . Lymphocyte immune phenotyping results of healthy and COVID-19 subjects and correlation with CD3+CD4+ , CD3+ and CD3-caspase+ T cells.

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; https://doi.org/10.1101/2020.11.02.20223636

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The copyright holder for this preprint this version posted November 4, 2020. z Nigericin stim / no comorbidities Nigericin stim/ with comorbidities Unstim. / no comorbidities Unstim./ with comorbidities

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Multisystem Inflammatory Syndrome Related to COVID-19 in Previously Healthy Children and Adolescents

COVID-19 and multisystem inflammatory syndrome in children and adolescents

Update on the COVID-19-associated inflammatory syndrome in children and adolescents; paediatric inflammatory multisystem syndrome-temporally associated with SARS-CoV-2

Presenting Characteristics, Comorbidities, and Outcomes Among 5700 Patients Hospitalized With COVID-19 in the New York City Area

Inflammasomes and Pyroptosis as Therapeutic Targets for COVID-19

Mechanism of NLRP3 inflammasome activation

Interleukins (from IL-1 to IL-38), interferons, transforming growth factor beta, and TNF-alpha: Receptors, functions, and roles in diseases

IL-1, IL-18, and IL-33 families of cytokines

Mechanism and Regulation of NLRP3 Inflammasome Activation

Activation and regulation of the inflammasomes

Heightened Innate Immune Responses in the Respiratory Tract of COVID-19 Patients

Mitochondrial induced T cell apoptosis and aberrant myeloid metabolic programs define distinct immune cell subsets during acute and recovered SARS-CoV-2 infection. medRxiv

The dynamic changes in cytokine responses in COVID-19: a snapshot of the current state of knowledge

Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases

Caspase-8-dependent control of NK-and T cell responses during cytomegalovirus infection

Hematological findings and complications of COVID-19

Neutrophils and Neutrophil Extracellular Traps Drive Necroinflammation in COVID-19

The induction and consequences of Influenza A virus-induced cell death

Caspase functions in cell death and disease

Caspase-8 is essential for maintaining chromosomal stability and suppressing B-cell lymphomagenesis

Caspase-3 regulates cell cycle in B cells: a consequence of substrate specificity

The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis

Interferons and inflammasomes: Cooperation and counterregulation in disease

Tolerance, danger, and the extended family

The danger model: a renewed sense of self

Morphological anomalies of circulating blood cells in COVID-19

A Controllable Inflammatory Response and Temporary Abnormal Coagulation in Moderate Disease of COVID-19 in Wuhan

Protein kinase C mediates caspase 3 activation: A role for erythrocyte morphology changes

Increased caspase-3 immunoreactivity of erythrocytes in STZ diabetic rats

Oxidative stress and caspasemediated fragmentation of cytoplasmic domain of erythrocyte band 3 during blood storage

The effects of obesity on CD47 expression in erythrocytes

Management of COVID-19 Respiratory Distress

Potential Treatment Options for COVID-19: A Comprehensive Review of Global Pharmacological Development Efforts

Functional consequences of caspase activation in cardiac myocytes

Advanced bioinformatics rapidly identifies existing therapeutics for patients with coronavirus disease-2019 (COVID-19)

A drug repurposing screen identifies hepatitis C antivirals as inhibitors of the SARS-CoV-2 main 1 protease

Emricasan Improves Liver Function in Patients With Cirrhosis and High Model for End-Stage Liver Disease Scores Compared With Placebo

The pan-caspase inhibitor Emricasan (IDN-6556) decreases liver injury and fibrosis in a murine model of non-alcoholic steatohepatitis

A randomized, placebo-controlled trial of emricasan in patients with NASH and F1-F3 fibrosis

Cell death by pyroptosis drives CD4 T-cell depletion in HIV-1 infection

Viral presence and immunopathology in patients with lethal COVID-19: a prospective autopsy cohort study