key: cord-0934831-tr0a1ybz authors: Dean, M. J.; Ochoa, J. B.; Sanchez-Pino, M.; Zabaleta, J.; Garai, J.; del Valle, L.; Wyczechowska, D.; Buckner Baiamonte, L.; Philbrook, P.; Majumder, R.; Vander Heide, R.; Dunkenberger, L.; Thylur, R.; Nossaman, R.; Roberts, W. M.; Chapple, A.; Collins, J.; Luke, B.; Johnson, R.; Koul, H.; Rees, C. A.; Morris, C. R.; Garcia-Diaz, J.; Ochoa, A. C. title: Transcriptome and Functions of Granulocytic Myeloid-Derived Suppressor Cells Determine their Association with Disease Severity of COVID-19 date: 2021-03-29 journal: medRxiv : the preprint server for health sciences DOI: 10.1101/2021.03.26.21254441 sha: d496f65b5eba749d57291a3b21ba6eabae048ef8 doc_id: 934831 cord_uid: tr0a1ybz COVID-19 ranges from asymptomatic in 35% of cases to severe in 20% of patients. Differences in the type and degree of inflammation appear to determine the severity of the disease. Recent reports show an increase in circulating monocytic-myeloid-derived suppressor cells (M-MDSC) in severe COVID 19, that deplete arginine but are not associated with respiratory complications. Our data shows that differences in the type, function and transcriptome of Granulocytic-MDSC (G-MDSC) may in part explain the severity COVID-19, in particular the association with pulmonary complications. Large infiltrates by Arginase 1+ G-MDSC (Arg+G-MDSC), expressing NOX-1 and NOX-2 (important for production of reactive oxygen species) were found in the lungs of patients who died from COVID-19 complications. Increased circulating Arg+G-MDSC depleted arginine, which impaired T cell receptor and endothelial cell function. Transcriptomic signatures of G-MDSC from patients with different stages of COVID-19, revealed that asymptomatic patients had increased expression of pathways and genes associated with type I interferon (IFN), while patients with severe COVID-19 had increased expression of genes associated with arginase production, and granulocyte degranulation and function. These results suggest that asymptomatic patients develop a protective type I IFN response, while patients with severe COVID-19 have an increased inflammatory response that depletes arginine, impairs T cell and endothelial cell function, and causes extensive pulmonary damage. Therefore, inhibition of arginase-1 and/or replenishment of arginine may be important in preventing/treating severe COVID-19. COVID-19 disease ranges from asymptomatic (35% of cases) to severe requiring treatment in intensive care units (https://www.cdc.gov/coronavirus/2019-ncov/hcp/clinical-guidance-management-patients.html). Approximately 20% of hospitalized COVID-19 patients develop hypoxemia, respiratory difficulty, hypercoagulation and end organ damage that may result in death. These clinical manifestations are similar to other coronavirus infections including SARS-CoV-1 and MERS-CoV. The pathophysiology is primarily associated with an over-active inflammatory response manifested by a cytokine release syndrome, a surge in granulocytes and decreased lymphocytes 1, 2 . The mechanism(s) that regulate these events are unclear. Genomic studies of peripheral blood suggested in increase genomic signatures associated with neutrophil functions in COVID-19 patients 3 . Early reports also showed that increased neutrophils/lymphocyte ratios were associated with poor outcomes 4, 5 . More recently, Agrati et al 6 inhibit T cell proliferation. MDSC have been best studied in cancer, but also described in chronic infections, autoimmunity, asthma and trauma [9] [10] [11] . MDSC express high arginase 1 (Arg1) that metabolizes arginine to ornithine and urea, effectively depleting this amino acid from the microenvironment. Arginine depletion inhibits T cell receptor signaling resulting in T cell dysfunction 12, 13 . It also impairs nitric oxide production and increases endothelial cell dysfunction promoting intravascular coagulation 14, 15 . Arginine depletion also increases the production of reactive oxygen species (ROS) which can damage infiltrated organs and exacerbate inflammation 16 . We compared the type, function and transcriptomic signature of MDSC in COVID-19 patients to better understand their effect on arginine levels, T cell function and respiratory complications. Our results showed a major increase in circulating granulocytic-MDSC expressing high levels of arginase-1 (Arg + G-MDCS). Large accumulations of Arg + G-MDSC expressing NOX1 and NOX2 infiltrated the lungs of patients who died from severe COVID-19 complications. High numbers of Arg + G-MDSC in circulation depleted arginine in plasma, decreased T cell receptor zeta chain (CD3) and increased markers of endothelial cell dysfunction. RNAseq studies demonstrated contrasting transcriptomes, where G-MDSC from asymptomatic COVID-19 patients had increased expression of type I IFN genes and pathways, while G-MDSC from severe COVID-19 patients had instead increased expression of genes associated with granulocyte functions and degranulation. These data support the concept that Arg + G-MDSC may play a significant role in the pathophysiology of COVID-19. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Study participants. The study was conducted under LSU IRB protocol #20-053 and Ochsner Medical Center IRB protocol 21015-101C. All participants were consented prior to inclusion in the study. Peripheral blood from 24 severe, 26 convalescent and 5 asymptomatic COVID-19 patients and 15 healthy (COVID-19 negative) controls were used for these studies (Supplemental Table 1 ). Severity of the disease was determined following WHO classification. All severe COVID-19 patients were being treated in the ICU at the time of sample collection. Treatments included remdesivir, systemic corticosteroids, antibiotics and anticoagulants; with convalescent sera or monoclonal antibodies used in 3 cases. Only 50% of severe COVID-19 patients were on ventilator support. Samples were collected within the first three weeks of admission to ICU (mean= 17.6 days; over ficoll-hypaque, which eliminates high density granulocytes, further revealed a significant increase in G-MDSC in severe COVID-19 patients ranging from 2.3% -76% (mean=29.3%) (1G), which was >10 fold higher than normal controls (mean=2.5%), asymptomatic and convalescent patients (mean=1.2%). In 62% (14/24) of severe COVID-19 patients G-MDSC were >20% of PBMC following ficoll-histopaque separation, i.e. > 4 standard deviations above the mean of normal controls and convalescent patients. G-MDSC levels did not differ between severe COVID-19 patients needing ventilator support, the presence of pneumonia, nor differences in medications (data not shown). Monocytic-MDSC (1H) were also moderately increased, but not as significantly as G-MDSC. Comparison of the flow cytometry results using Log Density Plots (1I) showed significantly higher CD66b/CD3 (p=6.8 x 10 -8 ), G-MDSC/CD3 (4.07 x 10 -12 ) ratios and percent G-MDSC (1.68 x 10 -16 ) in severe COVID-19 patients compared to other patients. These major differences suggest that these ratios and the percent G-MDSC may help identify patients progressing toward a severe form of COVID-19. A frequent complication of severe COVID-19 is hypoxemia and acute respiratory distress. We tested whether G-MDSC might play a role in the pathophysiology of this complication. Lung autopsy samples from ten patients who died from respiratory distress during severe COVID-19 were tested for inflammatory infiltrates ( Figure 2 ). Panel A shows extensive infiltration of broncho-pulmonary parenchyma by inflammatory cells with extensive damage of alveolar spaces, exfoliation of bronchial epithelium and thrombosis of blood vessels. Immunohistochemistry (Panel B) showed a prominent expression All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. in CD66b+ confirming them to be Arg + G-MDSC. Furthermore, staining with anti-NOX-1 and NOX-2 antibodies revealed high expression of these enzymes in clusters of inflammatory and exfoliated epithelial cells (Panel F). NOX-1 and NOX-2 enzymes catalyze the synthesis of reactive oxygen species (ROS). These results suggest that the massive infiltration of the lungs by Arg + G-MDSC simultaneously expressing NOX1/2, may inhibit T cells and caused endothelial cell dysfunction (through depletion of arginine, which is required for nitric oxide production and blood vessel tone), and directly damage alveolar epithelium through the release of ROS. This may in part explain the acute respiratory distress syndrome and coagulopathy frequently seen in severe COVID-19 patients. The high numbers of Arg + G-MDSC in circulation and lungs of COVID-19 patients, contrasts with the findings in cancer where G-MDSC concentrate around tumors with increases in peripheral blood found mostly in patients with advanced disease [17] [18] [19] . T cell and endothelial cell function 16 20,21 . Figure 3A shows representative Western blot data from 7 severe and 7 convalescent COVID-19 patients and 3 healthy controls. All severe COVID-19 patients had high Arg1 protein expression, compared to only 2/7 convalescent patients tested and none of the healthy controls. Quantification of these Arg1 expression levels reveals a roughly 5-fold increase in Arg1 in severe COVID-19 patients compared to controls and convalescent individuals. SYBR Green qRT-PCR using an MDSC primer panel confirmed a 7.5 fold higher expression of Arg1 in the PBMC of severe COVID-19 patients compared to convalescent and healthy controls ( Figure 3B ). These results also showed increased expression of genes associated with MDSC including MMP9, S100A9, CEBP and genes associated with granulocyte functions such as myeloperoxidase (MPO) and neutrophil degranulation proteins PRTN3. These findings were further confirmed by RNAseq from purified G-MDSC (shown in Figure 4) The metabolic consequence of high numbers of Arg + G-MDSC was a significant decrease in plasma arginine in severe COVID-19 patients (mean=45M; range 18-120M), compared to healthy controls (mean=75M; range 50-130M) and convalescent patients (mean=55M; range 26-92) ( Figure 3C ). As expected, there was an inverse correlation between Arg1 expression (by W. blot) and arginine plasma levels ( Figure 3D ), but nitrite levels were unchanged ( Figure 3E ). These observations are significant in that T cells cultured in arginine <50M lose the T cell receptor zeta chain (CD3) expression, impairing proliferation and IFN production 13, 20 . This effect was also observed in purified T cells from severe COVID-19 patients tested ( Figure 3F ). Arginine depletion also causes endothelial cell dysfunction through interfering with nitric oxide production 16 . This was All rights reserved. No reuse allowed without permission. (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 March 29, 2021. ; https://doi.org/10.1101/2021.03.26.21254441 doi: medRxiv preprint confirmed here by decreased levels of Protein S and increased plasminogen activator inhibitor-1 (PAI-1) (Figure G) , which can increase the risk for intravascular coagulation. To better understand the differences in inflammatory mechanisms in patients with different stages and severity of COVID-19, we compared the transcriptome of purified G-MDSC using RNAseq. Unsupervised hierarchical cluster analysis of the differentially expressed genes (DEGs) in G-MDSC from severe COVID-19 patients vs healthy controls ( Figure 4A ), convalescent ( Figure 4B ) and asymptomatic patients ( Figure 4C ) demonstrated clear differences between these groups. A Venn diagram (Supplemental Figure 1 ) illustrating shared and unique genes revealed the most significant differences in RNA transcripts (FDR <0.05; fold change ≥ 2) was between severe and asymptomatic patients (3675 transcripts), followed by severe and healthy controls (2452 RNA transcripts) and severe and convalescent (863 genes). An initial analysis using MetaCore software identified gene ontology processes that were significantly different among the groups. Specifically, G-MDSC from severe vs convalescent patients showed major differences in the expression of genes associated granulocyte functions and degranulation ( Figure 4E ). In contrast, differences between severe COVID-19 patients and healthy controls or asymptomatic patients were primarily associated with normal cell activation, signaling and regulation (Figure 4 D, E) . Additional analysis using Key Pathway Advisor software identified the top 25 Pathways with predictive positive or negative functional consequences. Comparison of severe vs asymptomatic patients showed that G-MDSC from severe patients had significantly decreased expression of genes from pathways associated with type I IFN responses (Immune response_IFN-alpha/beta signaling via JAK/STAT) and its down-stream signaling mechanisms (Immune response_IFN alpha/beta signaling via PI3K and NF-pathways) ( Figure 4I ). To further illustrate these findings we plotted the expression of representative genes associated with granulocyte functions and genes associated with type I IFN pathways. These data confirmed increased expression of genes associated with anti-viral responses such as IFI27, IFIT2 and IFIT3 [22] [23] [24] [25] in G-MDSC from asymptomatic patients( Figure 4J) . In contrast, G-MDSC from severe COVID-19 patients had increased expression of genes associated with granulocyte functions/degranulation and chronic inflammation including Arg1, MMP8, MMP9, S100A8 and A9, MPO, IL18RA, elastase, PRTN3 (azurophilic granule protein 7) ( Figure 4K ). These results demonstrate contrasting inflammatory and immune responses between asymptomatic and severe COVID-19 patients. The increased expression of type I IFN associated pathways and down-stream signaling mechanisms in G-MDSC from asymptomatic patients suggested the development of a protective anti-viral immune response. It is unclear whether G-MDSC can themselves have anti-viral activity or serve as effective antigen presenting cells. In contrast patients with severe COVID-19 develop a granulocyte inflammatory response that exacerbates the disease. What genomic characteristics regulate the degree and type of inflammatory response is still unclear, but these findings may help identify individuals that are more likely to develop severe disease. The complete lists of differentially expressed genes are included in Supplemental Table 2 . All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Arginine depletion can also be a mechanism for endothelial cell dysfunction and increased intravascular coagulation. Inhibition of arginase-1 restores endothelial function and production of nitric oxide 14, 15, 29, 30 . Our data shows that severe COVID-19 patients had decreased plasma levels of Protein S, suggesting it is rapidly being consumed, and increased PAI-1, which is a direct manifestation of endothelial cell dysfunction. Arginase can also decrease NO production by endothelial cells because of its faster kinetics and higher avidity for arginine compared to nitric oxide synthase 2 (NOS2). Thus, arginine deficiency can cause vasoconstriction, increase platelet adherence, and further promote hypercoagulation 30 . These observations also suggest novel therapeutic approaches including the use of arginase inhibitors or the replenishment of arginine. Arg1 inhibitors are currently in early phase clinical trials in cancer (Calithera/Incyte; 31, 32 ), while the replenishment of arginine or arginine precursors has been tested in Sickle cell disease resulting in a significant reduction in vaso-occlusive complications 33, 34 Arginine replenishment has also been tested in surgery where it successfully blunted the surge of G-MDSC, prevented T cell dysfunction and decreased infectious complications [35] [36] [37] . Therefore arginase 1 inhibition and/or arginine replenishment should be considered as an adjuvant to the prevention/treatment of COVID-19. Methods: Detailed methods can be found in the supplemental data file All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Plasma protein S and Plasminogen activator inhibitor-1 (PAI-1) in plasma. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Figure 4. Differential Gene Expression in G-MDSC from Patients and Healthy Controls. Comparison of the transcriptome of purified G-MDSC using RNAseq from severe (n=4), asymptomatic (n=4), and convalescent (n=4) COVID-19 patients and healthy controls (n=4) G-I) Analysis using Key Pathway Advisor software identified the Top 25 differentially expressed pathways. J,K) Dot plots comparing differentially expressed genes of "Immune response IFNalpha/beta signaling via JAK/STAT All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder This project was supported by funding from LSU Health and Ochsner Medical Center. Core facilities, personnel and services partially supported by P20-GM103501, P20-CA233374 and P20-GM121288. We would like to acknowledge the dedication of doctors, nurses and researchers at LSU Health and Ochsner Medical Center who provide care and research for COVID-19 patients, and to patients who participated in this study.