key: cord-0726876-v9fyjouw
authors: Mathi, Kavita; Rosenberg-Hasson, Yael; Maecker, Holden; Carlo, Dennis J.; Moss, Ronald B.
title: Brief report: Tempol, a novel antioxidant, inhibits both activated T cell and antigen presenting cell derived cytokines in-vitro from COVID-19 patients
date: 2021-08-21
journal: Clin Immunol
DOI: 10.1016/j.clim.2021.108828
sha: 686338f6734b40e22d99171caa4c5501c2364cc7
doc_id: 726876
cord_uid: v9fyjouw

COVID-19 is characterized by a dysregulation of inflammatory cytokines ultimately resulting a cytokine storm that can result in significant morbidity and mortality. We developed an in-vitro assay using activated peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharide (LPS) or CD3 + CD28 to examine secretion of cytokines from antigen presenting cells (APCs) and T cells, respectively, in donor patients with a history of COVID-19 (convalescent) and uninfected negative controls. We hypothesized that a novel antioxidant called Tempol may decrease cytokines from activated peripheral blood cells from both COVID-19 patients and normal donors. Preincubation of immune cells with Tempol resulted in a significant (P < 0.05) decrease in multiple T cell and APC-derived cytokines from both cells of COVID-19 (n = 7) and uninfected donors (n = 7). These preliminary results suggest that Tempol has strong in-vitro anti-cytokine activity and supports additional studies examining the use of Tempol for the treatment of COVID-19.

The immunopathogenesis of SARS-CoV-2, the causative agent for COVID-19, remains incompletely understood [1] . The early phases of SARS-CoV-2 infection appear to be characterized by viral replication along with an upregulation of inflammatory cytokines [2] . Indeed, severe inflammation accompanied by systemic markers of inflammation such as C-reactive protein (CRP) along with an upregulation of inflammatory cytokines are poor prognostic indicators of outcome for COVID-19 patients [3, 4] . In addition, for those who survive the initial infection, long-term clinical sequalae to COVID-19 have been observed secondary to damage caused by an initial dysfunctional inflammatory response [5, 6] .

The National Institute of Health has released Coronavirus Disease 2019 Treatment Guidelines, which include information on therapeutic options for COVID-19 [7] . Per the guidelines, the use of the antiviral Remdesivir has been recommended in severe hospitalized cases. Furthermore, dexamethasone, a corticosteroid, is also recommended in patients requiring supplemental or mechanical ventilation but has not been shown to be effective in earlier diseased patients. This recommendation is consistent with the pathogenesis of COVID-19 infection, which involves multi-system inflammation. However, because of its pan-immunosuppressive effects, corticosteroids would not likely be used in early COVID-19 infection.

Oxidative stress is thought to play a critical role in SARS-CoV infection [8] as well as other respiratory infections, including influenza [9] [10] [11] . As with other respiratory pathogens, it has been hypothesized that damage from free radicals due to oxidative stress may result in host systemic injury perpetuated by an aberrant immune response in patients with COVID-19 [12] [13] [14] . An antioxidant and anti-inflammatory that protects healthy cells could be hypothesized to prevent both the progression of the disease in high-risk individuals and long-term complications of

Tempol is a redox cycling nitroxide that promotes the metabolism of many harmful reactive oxygen species (ROS) and improves nitric oxide bioavailability [15] . In-vitro and preclinical studies of Tempol have demonstrated an attenuation of LPS induced lung damage in both acute and chronic lung injury in-vivo models in small animals [16] [17] [18] . In addition, in these animal models Tempol decreases inflammatory cytokines from a variety of cell types [19] . Table 1 . Donors were roughly matched for age and sex, and COVID-19 convalescent donors ranged from mild to severe disease.

The effect of increasing in-vitro doses of Tempol on T cell secreted cytokines (CD3+CD28) is shown in Figure 1a from both convalescent COVID donors (red) and normal donors (blue) for each donor. Statistical analysis revealed To ensure that decreases in cytokine production observed with Tempol were not simply due to toxicity, we checked cell viability at the end of the incubation period. Differences in cell viability with increasing doses of Tempol are shown in Figure 3 . While some decrease in viability was seen at the highest Tempol dose, the viability changes did not appear sufficient to account for the level of cytokine effect. A summary of the effects of Tempol on activated Table 2 . There is a trend for COVID-19 convalescent subjects to have lower cytokine production in the presence and absence of Tempol. This was statistically significant Table 1 ). This difference was independent of the suppressive effect of Tempol, which was seen in both control and COVID-19 convalescent subjects.

Cytokines play an important role in the immunopathogenesis of COVID-19 and the level of inflammatory cytokines in the plasma have been shown to be independent predictors of mortality and morbidity (2) . Indeed, the measurement of cytokines in-vitro may have a role as important prognostic biomarkers. The cytokine release syndrome also known as cytokine storm observed with COVID-19 has also been observed with immune therapies for cancer including chimeric antigen receptor (CAR) therapy and after infusion of muromonab-CD3 therapy (OKT3) [22] . Thus, the use of the in-vitro immune assay developed in this study may be useful biomarkers to examine cytokine release for cancer immunotherapy as well as other anti-cytokines therapies for COVID-19.

In this cross-sectional study, Tempol, a unique antioxidant, was examined in-vitro with activated immune cells from both COVID positive and COVID negative donors. The results suggest that pre-incubation with Tempol with activated T cell or APCs inhibited the release of multiple cytokines in both donor populations. The broad immune suppressive activity of Tempol appeared to target mostly activated rather than un-activated cells.

A number of single anti-cytokine agents are currently in clinical development for the treatment of COVID-19 including anti-IL6 and anti-TNF [23, 24] . Of note, we observed that pre-incubation with Tempol decreased the secretion of multiple cytokines secreted from activated immune cells. The inhibition of secreted cytokines from preincubation with Tempol was broad, dose-dependent, and included cytokines such as IL-6, IL-1beta, IFN-, IL-2, beta chemokines, IL 17, IL-13, IL-10, IL-4, and GM-CSF. In addition, it appears that Tempol in-vitro inhibited release of cytokines from both APCs and T cells, both of which play a role in the immunopathogenesis of COVID19. Thus, the ability to inhibit broader cytokines may have additional therapeutic utility compared to treatment with a single anti-cytokine in COVID-19 patients. Interestingly, except for four cytokines (IL2, IL-10, IL-13, IFN- and GM-CSF), Tempol did not significantly suppress cytokines from unstimulated cells.

This is the first published study that we know of that has examined the effects of Tempol in-vitro on immune cells from COVID patients. Although the patient's cells examined herein were convalescing from SARS-CoV-2 infection, long-term effects of COVID-19 have now been described [25] . Other studies are warranted to examine the in-vitro effects of Tempol in patients with active COVID-19 infection. A recent study examined the effects of Tempol on iron-cofactors required for replication of SARS-COV-2 [25] . Tempol was shown to disassemble iron cofactors sites, inhibiting the RNA dependent RNA polymerase and blocked virus replication in cell culture.

Therefore, it may be speculated that Tempol may have both antiviral and anti-inflammatory activity against COVID- 

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Biomarkers associated with COVID-19 disease progression

Elevated levels of IL-6 and CRP predict the need for mechanical ventilation in COVID-19

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Devilishly radical NETwork in COVID-19: Oxidative stress, neutrophil extracellular traps (NETs), and T cell suppression

Targeting COVID-19 Inflammation and Oxidative Stress

Effects of tempol and redox-cycling nitroxides in models of oxidative stress

Tempol, a membrane-permeable radical scavenger, ameliorates lipopolysaccharide-induced acute lung injury in mice: a key role for superoxide anion

Effects of tempol, a membrane-permeable radical scavenger, in a rodent model of carrageenan-induced pleurisy

Inflammation induced by increased frequency of intermittent hypoxia is attenuated by tempol administration., Braz

Tempol, an intracellular antioxidant, inhibits tissue factor expression, attenuates dendritic cell function, and is partially protective in a murine model of cerebral malaria

Cryopreservation of Peripheral Blood Mononuclear Cells from Whole Blood

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We thank all patients who have donated samples for this investigation and Dr Kari Nadeau and the Stanford Blood Center for providing the PBMC samples for this study.

 Tables:   Table 1 . Demographic