Expansion of Myeloid Derived Suppressor Cells Contributes to Platelet Activation by L-Arginine Deprivation during SARS-CoV-2 Infection

Massive platelet activation and thrombotic events characterize severe COVID-19, highlighting their critical role in SARS-CoV-2-induced immunopathology. Since there is a well-described expansion of myeloid-derived suppressor cells (MDSC) in severe COVID-19, we evaluated their possible role in platelet activation during SARS-CoV-2 infection. During COVID-19, a lower plasmatic L-arginine level was observed compared to healthy donors, which correlated with MDSC frequency. Additionally, activated GPIIb/IIIa complex (PAC-1) expression was higher on platelets from severe COVID-19 patients compared to healthy controls and inversely correlated with L-arginine plasmatic concentration. Notably, MDSC were able to induce PAC-1 expression in vitro by reducing L-arginine concentration, indicating a direct role of PMN-MDSC in platelet activation. Accordingly, we found a positive correlation between ex vivo platelet PAC-1 expression and PMN-MDSC frequency. Overall, our data demonstrate the involvement of PMN-MDSC in triggering platelet activation during COVID-19, highlighting a novel role of MDSC in driving COVID-19 pathogenesis.


Introduction
The ongoing COVID-19 pandemic due to the coronavirus SARS-CoV-2 remains a global health emergency. The clinical features of COVID-19 range from asymptomatic to severe pneumonia and fulminant disease [1], but the mechanisms responsible for this wide clinical presentation are not completely clear. In severe COVID-19, coagulation abnormalities appear, inducing a hypercoagulable state and an increased rate of thrombotic and thromboembolic events [2].
The high inflammatory response may contribute to the thrombotic complications by impairing procoagulant-anticoagulant balance, thus facilitating the development of microthrombosis and disseminated intravascular coagulation [3]. Further, the expression of SARS-CoV-2 receptor (angiotensin converting enzyme 2, ACE-2) on platelet membranes suggests a possible direct role of SARS-CoV-2 in platelet activation [4].
In this study, we assessed the capability of PMN-MDSC to activate platelets during SARS-CoV-2 infection. Our results showed a novel role of PMN-MDSC from COVID-19 patients, being able to increase platelet activation by reducing L-arginine concentration, thus contributing to the platelet hyperactivity observed in severe COVID-19.
The study was approved by the institutional review board (approval number: 9/2020) and signed written informed consent was obtained from patients.

Plasma Samples Preparation
Heparin anti-coagulated whole blood samples were centrifuged at 100× g for 15 min and platelet rich plasma (PRP) was collected for further use.

Platelets-PMN-MDSC Culture
Purified PMN-MDSCs (2 × 10 5 ) were seeded in 96-well plate (Corning-Incorporated, New York, NJ, USA) in the above described medium without FBS. Twenty microliters of PRP from HD were added and cultured at 37 • C. After 4 h, platelet activation was evaluated by flowcytometry, and supernatants were collected for L-Arginine quantification.

Flow Cytometry
Platelet activation was analyzed by using anti-human REAfinity CD41 APC and anti-human REAfinity activated GPIIb/IIIa complex (PAC-1 PE mAb, Bergisch Gladbach, Miltenyi Biotec) on ice in the dark. After 15 min, 1% paraformaldehyde was added and samples were acquired by Cytoflex Flow Cytometer (Beckman-Coulter, Brea, CA, USA).

L-arginine Quantification
Plasma samples and co-culture supernatants were centrifuged at 2000 rpm for 10 min to eliminate platelets and debris. L-arginine level was evaluated by UPLC-MS/MS by using Kairos Amino Acid Kit (Waters, Milford, MA, USA) according to the manufacturer's instruction. Chromatographic separation was performed using an ACQUITY-UPLC system, followed by detection on a Xevo-TQD (Waters, Milford, MA, USA).

Statistical Analysis
GraphPad Prism version 8.00 (GraphPad Software) was used to perform statistical analyses. The non-parametric Kruskal-Wallis with Dunn's post hoc test or the Wilcoxon matched-pairs signed rank test were used. Correlations were evaluated with the nonparametric Spearman test. The p < 0.05 was considered significant.

Plasmatic L-Arginine in COVID-19 Patients was Correlated to PMN-MDSC Frequency
We evaluated the plasmatic concentration of L-arginine in patients with moderate (no ICU) and severe (ICU) COVID-19 and HD. A lower plasmatic L-arginine level was observed in both patient groups compared to HD ( Figure 1a). Moreover, L-arginine was lower in ICU compared to no ICU patients, suggesting possible association with disease severity.

PMN-MDSC Induced Platelet Activation by Reducing L-Arginine
Next, we evaluated whether PMN-MDSCs were directly involved in platelet activation. We found a positive correlation between PAC-1 platelet expression and PMN-MDSC frequency (Figure 2A), suggesting that PMN-MDSC may be involved in arginine deprivation. To confirm that PMN-MDSC may induce platelet activation, we cultured platelet rich plasma (PRP) from HD with PMN-MDSCs isolated from COVID-19 patients and, after 4 h, the expression of PAC-1 on platelets was evaluated by flow cytometry. We found that PMN-MDSCs from COVID-19 were able to activate resting healthy platelets by increasing the expression of PAC-1 ( Figure 2B,C). As a control, PBMC depleted from MDSCs were used and no effect was observed. Accordingly, a reduction of L-arginine in the culture supernatants ( Figure 2D) was found, indicating that PMN-MDSCs may activate platelets by depriving L-arginine from the microenvironment.

PMN-MDSC Induced Platelet Activation by Reducing L-Arginine
Next, we evaluated whether PMN-MDSCs were directly involved in platelet activation. We found a positive correlation between PAC-1 platelet expression and PMN-MDSC frequency (Figure 2a), suggesting that PMN-MDSC may be involved in arginine deprivation. To confirm that PMN-MDSC may induce platelet activation, we cultured platelet rich plasma (PRP) from HD with PMN-MDSCs isolated from COVID-19 patients and, after 4 h, the expression of PAC-1 on platelets was evaluated by flow cytometry. We found that PMN-MDSCs from COVID-19 were able to activate resting healthy platelets by increasing the expression of PAC-1 (Figure 2b,c). As a control, PBMC depleted from MDSCs were used and no effect was observed. Accordingly, a reduction of L-arginine in the culture supernatants (Figure 2d) was found, indicating that PMN-MDSCs may activate platelets by depriving L-arginine from the microenvironment.

Discussion
Patients with severe COVID-19 commonly present thrombotic disorders, and these conditions have been associated with a higher mortality rate [10]. Moreover, severe COVID-19 is characterized by a strong neutrophilia that persists overtime. Among neutrophils, a strong inflammatory-driven expansion of PMN-MDSCs was observed in severe patients, which significantly reduces the adaptive immune response to SARS-CoV-2 and predicts a fatal clinical outcome [7,8].
In this paper, we analyzed an MDSC function never explored before, to the best of our knowledge, and showed that PMN-MDSCs from COVID-19 patients may be involved in platelet activation by reducing L-arginine availability, highlighting a new interplay between immune regulatory cells and platelet function.
According to previous published data [11], a decrease of L-arginine in the plasma from COVID-19 patients was found. In the present work, we also observed that L-arginine level inversely correlated with PMN-MDSC frequency and with platelet activation. Moreover, PMN-MDSC frequency directly correlated with platelet activation, suggesting a role of PMN-MDSCs in platelet activation during COVID-19. This hypothesis is corroborated by the high level of the enzymes involved in L-arginine catabolism, Arg I, and iNOS, expressed by PMN-MDSCs in COVID-19 patients [7]. Our in vitro experiments further provide a formal proof of the direct role of PMN-MDSC in inducing platelet activation through L-arginine consumption. Platelet activation has been described during SARS-CoV-2 infection, and it is known to contribute to thromboembolic complications. Besides hyper-inflammation, factors such as a direct SARSCoV-2 infection and antibody-mediated mechanisms have been proposed to contribute to platelet hyperactivity [4]. In the present study, we demonstrated the PMN-MDSC as a new player in platelet homeostasis, highlighting an unprecedented function of the PMN-MDSC during COVID-19. Correlations between PMN-MDSC frequency with L-arginine concentration and with platelet activation have been shown during other infections such as severe fever caused by a bunyavirus [12]. Herein, we demonstrated that PMN-MDSC, by decreasing L-arginine, might directly contribute to platelet activation, shedding light on a novel role of PMN-MDSCs besides immune suppression.

Discussion
Patients with severe COVID-19 commonly present thrombotic disorders, and these conditions have been associated with a higher mortality rate [10]. Moreover, severe COVID-19 is characterized by a strong neutrophilia that persists overtime. Among neutrophils, a strong inflammatory-driven expansion of PMN-MDSCs was observed in severe patients, which significantly reduces the adaptive immune response to SARS-CoV-2 and predicts a fatal clinical outcome [7,8].
In this paper, we analyzed an MDSC function never explored before, to the best of our knowledge, and showed that PMN-MDSCs from COVID-19 patients may be involved in platelet activation by reducing L-arginine availability, highlighting a new interplay between immune regulatory cells and platelet function.
According to previous published data [11], a decrease of L-arginine in the plasma from COVID-19 patients was found. In the present work, we also observed that L-arginine level inversely correlated with PMN-MDSC frequency and with platelet activation. Moreover, PMN-MDSC frequency directly correlated with platelet activation, suggesting a role of PMN-MDSCs in platelet activation during COVID-19. This hypothesis is corroborated by the high level of the enzymes involved in L-arginine catabolism, Arg I, and iNOS, expressed by PMN-MDSCs in COVID-19 patients [7]. Our in vitro experiments further provide a formal proof of the direct role of PMN-MDSC in inducing platelet activation through L-arginine consumption. Platelet activation has been described during SARS-CoV-2 infection, and it is known to contribute to thromboembolic complications. Besides hyper-inflammation, factors such as a direct SARSCoV-2 infection and antibody-mediated mechanisms have been proposed to contribute to platelet hyperactivity [4]. In the present study, we demonstrated the PMN-MDSC as a new player in platelet homeostasis, highlighting an unprecedented function of the PMN-MDSC during COVID-19. Correlations between PMN-MDSC frequency with L-arginine concentration and with platelet activation have been shown during other infections such as severe fever caused by a bunyavirus [12]. Herein, we demonstrated that PMN-MDSC, by decreasing L-arginine, might directly contribute to platelet activation, shedding light on a novel role of PMN-MDSCs besides immune suppression.

Conclusions
Our findings demonstrate the direct involvement of PMN-MDSCs in platelet activation during COVID-19, confirming the MDSC expansion as one of the main events driving COVID-19 pathogenesis. These results also reveal new therapeutic perspectives targeting MDSC number and function, a promising strategy already under evaluation in cancer patients [13,14].  Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of National Institute for Infectious Diseases "Lazzaro Spallanzani" (approval number: 9/2020).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
Publicly available datasets were analyzed in this study. This data can be found here: [https://rawdata.inmi.it/].