key: cord-0324585-r923qbb2 authors: Nikitopoulou, I.; Fanidis, D.; Ntatsoulis, K.; Moulos, P.; Mpekoulis, G.; Evangelidou, M.; Dimakopoulou, V.; Vassiliou, A. G.; Jahaj, E.; Tsipilis, S.; Orfanos, S. E.; Dimopoulou, I.; Angelakis, E.; Akinosoglou, K.; Vassilaki, N.; Tzouvelekis, A.; Kotanidou, A.; Aidinis, V. title: Increased Autotaxin levels in severe COVID-19, correlating with IL-6 levels, endothelial dysfunction biomarkers, and impaired functions of dendritic cells date: 2021-07-31 journal: nan DOI: 10.1101/2021.07.30.21261361 sha: c5aad6cf5fa05a38a366a7ef203e3f1384268206 doc_id: 324585 cord_uid: r923qbb2 Autotaxin (ATX; ENPP2) is a secreted lysophospholipase D catalysing the extracellular production of lysophosphatidic acid (LPA), a pleiotropic signalling phospholipid. Genetic and pharmacologic studies have previously established a pathologic role for ATX and LPA signalling in pulmonary injury, inflammation, and fibrosis. Here, increased ENPP2 mRNA levels were detected in immune cells from nasopharyngeal swab samples of COVID-19 patients, and increased ATX serum levels were found in severe COVID-19 patients. ATX serum levels correlated with the corresponding increased serum levels of IL-6 and endothelial damage biomarkers, suggesting an interplay of the ATX/LPA axis with hyperinflammation and the associated vascular dysfunction in COVID-19. Accordingly, dexamethasone (Dex) treatment of mechanically ventilated patients reduced ATX levels, as shown in two independent cohorts, indicating that the therapeutic benefits of Dex include the suppression of ATX. Moreover, large scale analysis of multiple single cell RNAseq datasets revealed the expression landscape of ENPP2 in COVID-19 and further suggested a role for ATX in the homeostasis of dendritic cells, that exhibit both numerical and functional deficits in COVID-19. Therefore, ATX has likely a multifunctional role in COVID-19 pathogenesis, worth of suggesting that its pharmacological targeting might represent an additional therapeutic option. The leading symptom of COVID-19, beyond cough and fever, is hypoxemia, leading to dyspnea in severe cases, attributed to impaired lung mechanics and/or vasoconstriction (1, 2) . Endothelial dysfunction is also a major characteristic of COVID-19 (3), shared with hypertension, diabetes and obesity, the most common comorbidities that are associated with poor prognosis (1, 2) . The respiratory epithelial cell damage that follows viral infection and replication stimulate, depending on the underlying genetic and metabolic context, a hyperinflammatory state denominated "cytokine storm" (4) . The excessive production of proinflammatory cytokines, such as TNF and IL-6, further induces endothelial damage and lung injury, and its more severe form, Acute Respiratory Distress syndrome (ARDS), that can result to respiratory and/or multi-organ failure and death (5) . A subset of surviving COVID-19 ARDS-like patients will develop a fibroproliferative response characterized by fibroblast accumulation and ECM deposition (6) , also evident in postmortem histopathological analysis of the lungs of COVID-19 patients (7) . Moreover, many discharged COVID-19 patients present with abnormally pulmonary architecture and functions (8) (9) (10) (11) (12) , suggesting persisting fibrotic abnormalities, pending long-term follow up studies. Single-cell RNA sequencing (scRNAseq) analysis and transcriptional profiling indicated similarities in expression profiles between Idiopathic pulmonary fibrosis (IPF) and COVID-19 (13, 14) , while CoV-2 infection has been suggested to stimulate the expression of major profibrotic factors including TGFβ (15). Vice versa, patients with interstitial lung diseases (ILD) had an increased risk for severe COVID-19 and poor outcomes (ICU admittance, death) following CoV-2 infection (16-18). Autotaxin (ATX; ENPP2) is a secreted lysophospholipase D that can be found in most biological fluids, including blood and bronchoalveolar lavage fluid (BALF), largely responsible for the extracellular production of lysophosphatidic acid (LPA), a growth factorlike signaling phospholipid. Increased ATX expression and LPA signaling has been reported in cancer as well as in chronic inflammatory diseases (19), including IPF (20, 21). Genetic and pharmacologic studies have further uncovered a therapeutic potential for ATX in IPF (20, 22-24), leading to phase III clinical trials (25). Given the associations of COVID-19 with pulmonary fibrosis, the pro-fibrotic properties of ATX, as well the many reported LPA effects on pulmonary cells and especially the vasculature (26), in this study we explored a possible association of ATX with COVID-19. In this context, we quantified ENPP2 mRNA levels in nasopharyngeal swabs, ATX protein levels in the sera of two cohorts of COVID-19 patients, while we performed a large-scale analysis of recently published scRNAseq COVID-19 datasets. All studies were performed in accordance with the Helsinki Declaration principles. All collected data were anonymized in standardized forms and informed consent was obtained from all individuals or patients' next-of-kin for severe cases. All available patient personal, epidemiological, clinical, and experimental data are summarized in the corresponding cohorts' tables (1, 2 and 3) , including the appropriate protocol approvals. ATX and IL-6 protein levels were quantified with dedicated ELISA kits according to the manufacturer's instructions (R&D Systems Inc., Minneapolis, MN, USA, and Invitrogen, ThermoFisher Scientific, CA, USA respectively). Measurements were performed in a blinded fashion in triplicates using the Triturus automated analyser (Grifols, Barcelona, Spain). The presented results on ELISA quantification of soluble E-selectin (sE-sel) and P-selectin (sP-sel), ICAM and ANG2 in the same samples, has been reported previously (27). Total RNA extraction from nasopharyngeal swab samples was performed using the MagNA Pure LC Total Nucleic Acid Isolation Kit using the ΜagNa Pure LC 2.0 automated nucleic acid purifier (Roche), and viral RNA was quantified with the LightMix Modular SARS-CoV-2 RdRP Kit and the LightCycler Multiplex RNA Virus Master kit (Roche). ENPP2 mRNA levels were quantified with Q-RT-PCR using the SYBR Green-based Luna® Universal qPCR Master Mix (New England Biolabs)(ENPP2: forward: 5'-ACT CAT GAA GAT GCA CAC AGC -3'; reverse 5'-CGC TCT CAT ATG TAT GCA GG -3'; product length 131 bp). Normalization was performed with the house-keeping gene 14-3-3-zeta polypeptide (YWHAZ), and the relative quantification method 2 −∆∆Ct was utilised. The available single cell RNA-seq object was mined for each one of the datasets (Table 1) using Seurat package v3 (28). Marker selection and DEA were performed using Wilcoxon Rank Sum test (FC>1.2; Bonferroni adj. p< 0.05). For identifying pDCs in the lung data set of (13), DCs -as initially marked-were isolated, and principal components were calculated post to variable genes identification and data scaling using default parameters. The 30 first principal components were used to construct a SNN graph, while clusters were defined with a resolution of 0.8. pDCs were identified using marker genes reported in the cell atlas of (29). Preprocessed read count matrices of (30) found here were analyzed using metaseqR2 package (31). More specifically, reads where EDASeq normalized, filtered using default parameters and then PANDORA algorithm was used to combine results of DESeq (32), DESeq2 (33), limma-voom (34), edgeR (35) and ABSSeq (36) methods. DEGs were defined using a FC>1.2 and FDR adj. meta p-value < 0.05. Statistical significance was assessed with the Prism (GraphPad) software with the appropriate test according to the distribution of values and their complexity, as detailed in each figure legend. Statistical tests used include the non-parametric Mann-Whitney U test, unpaired t-test, Spearman corelation, 2-way ANOVA followed by Bonferroni post hoc correction, Wilcoxon rank sum test-Bonferroni correction, and Kruskal-Wallis and Dunn post hoc test. As viral infections have been reported to stimulate ENPP2 mRNA expression (37, 38), and to examine if CoV-2 infection has similar effects, we first quantified ENPP2 mRNA levels with Q-RT-PCR in nasopharyngeal swab samples (Table 1) . A significant increase was found in ENPP2 mRNA expression in mild and severe COVID-19 patients, as compared to non-infected subjects (Fig. 1) . Therefore, CoV-2 infection stimulates ENPP2 mRNA expression in the respiratory epithelial or immune cells that compose the nasopharyngeal swab samples. To examine if systemic levels of ATX are possibly increased upon COVID-19, ATX was quantified with an ELISA kit in the serum of COVID-19 patients hospitalised at the Evangelismos University Hospital ( Table 2 ). The cohort consisted of patients with mild symptoms, hospitalised in the COVID-19 WARD (n=47; no Dex treatment), as well as of patients with severe symptoms, hospitalised in the Intensive Care Unit (ICU); ICU patients were further separated in patients receiving Dexamethazone (Dex) treatment (n=37) or not (NO Dex; n=32). A large proportion of patients suffered from comorbidities, and were receiving a variety of medications prior to admission, while COVID-19-targeted treatment included azithromycin, chloroquine and lopinavir/ritonavir in different combinations per WHO recommendations at that time ( Table 2 ). In comparison with WARD patients, ICU patients were hypoxemic (low ratio of arterial oxygen partial pressure to fractional inspired oxygen; PaO2/FiO2), lymphopenic (low lymphocyte numbers), and had increased LDH levels ( Table 2 ), all three suggested as disease severity markers. Increased ATX serum concentrations were discovered in ICU patients (not receiving Dex) as compared with WARD patients ( Fig. 2A) , suggesting a possible association of ATX with disease severity. However, no substantial, statistically significant corelation was observed independently with the applicable severity markers (data not shown and Table 2 ); no statistically significant differences of ATX levels between the sex or the comorbidities of COVID-19 patients was detected either (Fig. S1 ). However and most importantly, ATX levels corelated significantly with IL-6 levels in the serum of ICU patients (not receiving Dex) (Fig. 2B) , suggesting a possible interplay of ATX/LPA with the cytokine storm in COVID-19. ICU non-survivors in this cohort had higher levels of the endothelial dysfunction markers soluble E-selectin (sE-sel), soluble P-selectin (sP-sel), soluble intercellular adhesion molecule 1 (sICAM-1) and angiopoietin 2 (ANG-2) when compared to survivors, as recently reported using a subset of the current Evangelismos cohort samples (27). Interestingly, the increased ATX protein levels correlated with the increased protein levels of sEsel and sICAM (Fig. S2 ) in ICU patients, suggesting a role for ATX/LPA in COVID-19 induced endothelial dysfunction. The first line of therapy for many inflammatory diseases as well as respiratory infections is Dex, that lowers the expression of pro-inflammatory cytokines including IL-6, and that has been proven effective in COVID-19 patients requiring, invasive or not, oxygenation (39, 40). Therefore, we next examined ATX serum levels in intubated, or not, ICU patients receiving, or not, Dex treatment. Remarkably, Dex treatment was discovered to potently suppress ATX serum levels in ventilated patients (Fig. 3A) , while intubated ICU patients receiving no Dex presented with the highest overall ATX serum levels. Identical results were obtained in another cohort of ICU patients from the University hospital of Patras (Table 3 ) (Fig. 3B) , indicating that the therapeutic benefits of Dex include the suppression of ATX serum levels. Moreover, ATX levels in ICU patients not receiving Dex treatment negatively affected survival, and non-surviving ICU patients receiving no Dex presented with the higher overall ATX serum levels ( Fig. 3 C,D) . To identify possible ATX expressing cells in the nasopharyngeal swab (NS) samples ( Fig. 1) , peripheral blood monocytes (PBMCs) in the circulation (Figs. 2, 3), as well as in BALF and lung tissue cells, we re-analysed and mined several scRNAseq datasets of COVID-19 patients and healthy controls, from recent high impact studies (Table 1) , collectively interrogating the gene expression of more than 10 6 cells; cell clustering and naming followed that of the original analyses, which both varied between studies/datasets. In NS cells, ATX is mainly expressed by natural killer cells (NKs) and monocyte-derived macrophages (MoAM)(Figs. 4A and S3A), as detected in two NS datasets (Table 1 ). In the circulation, and in both PBMCs datasets (Table 1) , ENPP2 expression was mainly detected, surprisingly, in plasmacytoid DCs (pDCs; Figs 4B and S3B). In BALF cells (Table 1) , ENPP2 expression was also mainly detected in pDCs, as well as MoAMs (Figs 4C and S3C). In lung tissue (Table 1) , ENPP2 was found to be primarily expressed in arterial and mesothelial cells, as well as in cells of the monocytic lineage ( Fig. 4D and S3D) . A similar lung tissue profile was also detected ( Fig. S3E ) in an IPF scRNAseq dataset (Table 1) , extending the similarities of pathogenic mechanisms between IPF and COVID-19 and supporting a common role for ATX. Given the ENPP2 expression from monocytic cells and especially pDCs, we next interrogated ENNP2 mRNA levels specifically in pDCs from COVID-19 patients in comparison with control samples, subsets of the datasets analysed in Figure 4 . Confined by the limited numbers of lung pDCs, as well as the detected genes per cell and the relatively low expression levels of ENPP2, the analysis indicated a statistically significant overexpression of ENPP2 in COVID-19 circulating pDCs (Fig. 5B) . Noteworthy, DCs are the highest ENPP2 expressing immune cells during healthy conditions, as identified upon querying a large-scale RNAseq data set interrogating gene expression of 28 immune cell types (79 healthy volunteers and 337 patients from 10 immune-related diseases)(30) (Fig. S4A ). Similar analysis indicated that the main LPA receptor expressed by DCs is LPAR2 (Fig. S4B ), which has been suggested to convey anti-inflammatory LPA signals to DCs (41). Furthermore, increased ENPP2 mRNA expression was detected in pDCs from patients with systemic lupus erythematosous (SLE), adult-onset Still's disease (AOSD), mixed connective tissue disease (MCTD) and idiopathic inflammatory myopathy (IIM) than in DCs from healthy volunteers (Fig. S4C) , suggesting that overexpression of ENPP2 in pDCs may be a common theme in inflammation. Finally, and to gain mechanistic insights into the possible role of ATX in DCs homeostasis upon COVID-19, we first analysed differential gene expression in COVID-19 DCs (as pDCs were too few), from the only COVID-19 lung dataset (13) allowing such analysis, as well as in ENPP2-expressing (ENPP2 + ) DCs (Table S5 ). Increased ENPP2 expression was also detected in all lung DCs (Fig. 5E ), while comparative analysis (Venn diagrams Fig. 5F ) highlighted two genes upregulated in ENPP2 + COVID-19 DCs, transmembrane protein 176B (TMEM176B) and CD1a, that have been both proposed as DC differentiation and/or maturation markers, suggesting that ENPP2 expression may modulate DC homeostasis. Previous studies have shown that HCV, HIV and HBV viral increase Enpp2 mRNA expression in infected cells and/or to raise systemic ATX levels (42-44). As shown here, increased ENPP2 mRNA expression was detected in nasopharyngeal swab samples from COVID-19 patients in comparison to non-infected healthy controls ( Fig. 1) , while scRNAseq re-analysis revealed that the highest ENPP2 expressing cells in swabs are immune cells (Figs. 5A and S5A), suggesting that CoV-2 infection stimulates ENPP2 expression from immune cells in the nasopharynx. LPA, the enzymatic product of ATX and its effector molecule, has been shown to directly affect HCV viral infection and replication (37, 38), suggesting that a similar autocrine mode of action maybe in play in COVID-19, where ATX produced by the infected host cell would stimulate local LPA production, in turn facilitating viral entry and replication. Increased serum ATX protein have been reported in cancer, liver diseases, as well as respiratory diseases including asthma and pulmonary fibrosis (19, 45), while increased levels of serum ATX were recently reported in ARDS (46). Here, increased ATX sera levels were detected in ICU-hospitalised COVID-19 patients (receiving no Dex treatment) compared to patients with less severe disease (Fig. 2) , suggesting increased ATX expression as an additional commonality of ARDS and COVID-19. The origin of serum ATX is not completely deciphered; however, >40% of serum constitutively active ATX has been suggested to originate from the adipose tissue (47), which was shown to be able to modulate the pathophysiology of distant metabolically active organs (48, 49). Moreover, serum ATX has been reported to correlate with insulin resistance in older humans with obesity (50), while mice with heterozygous Enpp2 deficiency were protected from HFD-induced obesity and systemic insulin resistance (49). Several additional reports have incriminated the ATX/LPA axis in the regulation of glucose homeostasis and insulin resistance (reviewed in (51)), among the main comorbidities of COVID-19, suggesting adipose tissuederived ATX as a possible pathologic link between obesity and COVID-19. An additional possible source of serum ATX in disease states, beyond the adipose tissue, is the liver. Increased ATX expression has been reported in chronic liver diseases of different aetiologies, associated with shorter overall survival (37), while the genetic deletion of ATX from hepatocytes (37), or as discussed above adipocytes (48), attenuated liver steatosis and fibrosis. Therefore, increased levels of serum ATX are expected upon liver damage, whereas aberrant liver functions have been reported in COVID-19, irrespectively of pre-existing liver disease (52). On the other hand, cirrhotic patients have high rates of liver failure and death from respiratory failure upon CoV-2 infection, attributed to increased systemic inflammation, immune dysfunction, and vasculopathy (52). Therefore, ATX could be also a pathologic link between liver damage and COVID-19. Plasma ATX levels have been recently reported to corelate with IL-6 levels in severe ARDS patients (46), as well as acute-on-chronic liver failure (ACLF) patients (53), as shown here in the serum of ICU COVID-19 patients (Fig. 2) . Increased serum IL-6 levels have been reported in COVID-19 patients corelating with the severity of COVID-19 pneumonia and mortality risk (54), or respiratory failure and the need for mechanical ventilation (55). Metaanalyses of published studies on COVID-19 laboratory findings indicated that serum levels of IL-6 were among the most predictive biomarkers (56, 57). Interestingly, components of the COVID-19 cytokine storm (IL-6, TNF and IL-1β) have been suggested to stimulate ATX expression and/or activity in different cell types, while, vice versa, LPA has been reported to stimulate TNF and IL-6 expression in different contexts (22), suggesting a possible interplay of the COVID-19 cytokine storm and the ATX/LPA axis. Dex treatment, a potent suppressor of systemic inflammation including IL-6, has been shown to reduce mortality in hospitalised COVID-19 patients under oxygen supplementation treatment or mechanical ventilation (39, 40). Dex treatment has been shown to decrease ATX (as well as IL-6) levels in the mouse adipose tissue and plasma (58), as well as in irradiated mammary fat pad (59). As shown here (Fig. 3) , Dex treatment of mechanically ventilated patients drastically reduced their ATX serum levels, indicating that the therapeutic effects of Dex in COVID-19 include the suppression of ATX serum levels. An essential role for ATX/LPA in embryonic vasculature has been well established through genetic studies in both mice (60-62) and zebrafish (63). In adult mice, Enpp2 has been discovered as a high priority candidate gene for pulmonary hemorrhage upon SARS/MERS-CoV infection (64, 65), while vascular leak has been suggested to be among the main pathological effects of ATX/LPA in pulmonary pathophysiology and fibrosis in mice (21, 22). As shown here, ENPP2 mRNA expression in the COVID-19 lung tissue was detected mainly in artery cells (Fig. 4D, S3D) , while high ATX expression from ECs in HEVs in lymph nodes has been previously reported (66) . Moreover, and in the same context, a plethora of LPA in vitro effects on endothelial cells have been suggested, with some controversy, including endothelial permeability, leukocyte adhesion, and cytokine expression, as previously reported in detail (26). Among them, LPA has been suggested to stimulate the expression of E-Sel from human aortic endothelial cells (67-69), a cell surface adhesion molecule regulating interaction with leukocytes. As shown here, ATX serum levels corelated with the corresponding sE-sel and sICAM serum levels (Fig. S2) , which has been independently associated, in the same samples, with mortality of COVID-19 ICU patients (27), suggesting that ATX/LPA effects in COVID-19 may also include vasculopathy. IPF macrophages have been previously shown to stain for ATX, and conditional genetic deletion of ATX from macrophages in mice, reduced BALF ATX levels and disease severity in modeled pulmonary fibrosis (20). scRNAseq analysis of BALF cells from COVID-19 patients, where macrophages predominate, indicated that ENPP2 mRNA expression was detected in different macrophage subpopulations (Fig. 4C /UMAP, S3C/UMAP), where it could modulate their maturation in an autocrine mode via LPA (70) (71) (72) . LPA has been also suggested to stimulate, in vitro, the conversion of monocytes to DCs (41, 73, 74) . Interestingly, ENPP2 mRNA expression was mainly detected in pDCs among all PBMCs and BALF cells in COVID-19 (Figs. 4B,C, S3B,C) . pDCs are the principal interferon (IFN) type I producing cells in the human blood and can be rapidly recruited to inflamed sites (75) . Circulating and lung pDCs have been shown to be diminished in COVID-19 (76, 77) , while IFN type I responses were highly impaired (78, 79) . ENPP2 mRNA expression was found upregulated in circulating pDCs (Fig. 5B) , and lung DCs (Fig. 5E ) from COVID-19 patients in comparison to cells from healthy controls. pDC development and homeostasis are regulated by the transcription TCF4 (80) , which has been reported to get modulated by LPA in colon cancer cells (81) , suggesting that ENPP2 expression from pDCs and the local production of LPA modulates, in an autocrine way, pDC development and homeostasis. The hypothesis is further supported from the genes that have been discovered, to be increased in COVID-19 DCs, possibly regulated by ENPP2 (Fig. 5E ). CD1a binds and presents to T-cells lipid metabolites and PLA2-synthesised fatty acid neoantigens and has been found to be expressed in immature DCs in mucosal surfaces, including the bronchus (82) (83) (84) . Tmem176B has been also associated with an immature state of dendritic cells (85, 86) , suggesting that ENPP2 expression from COVID-19 pDCs, via LPA, delays their maturation. Although LPA signals in most cell types are considered proinflammatory and pro-surviving, an anti-inflammatory role of LPA, via LPAR2 -the main subtype expressed in DCs (Fig. S4B) , has been proposed previously for DCs (41), further supporting a possible role for ATX/LPA in supressing DC responses. Taken together, a role for ATX/LPA in COVID-19 pathogenesis seems likely, possibly as a component of the cytokine storm perpetuating hyperinflammation and stimulating endothelial damage, as well as a regulator of the mononuclear phagocyte system and a suppressor of (p)DCs responses, non-withstanding its established role in fibrosis. Dex treatment in mechanically ventilated patients decreased ATX levels, indicating that the therapeutic effects of Dex in COVID-19 include the suppression of the ATX/LPA axis and that ATX levels can be druggable. Additional large-scale studies of serum ATX levels, e.g. retrospectively from recent clinical trials (a-IL-6R, Dex, antiviral), will be required to possibly establish ATX as a diagnostic/prognostic marker. Moreover, perspective FACS analysis of BALF DCs from COVID-19 patients will be necessary to validate the scRNAseq -based hypotheses data on DC homeostasis. More importantly and given that COVID-19 and IPF share risk factors for disease severity, such as age/sex and comorbidities, existing and developing anti-fibrotic therapies have been suggested as additional therapeutic opportunities in COVID-19 (87) (88) (89) . One of the novel candidates target ATX, is currently in clinical trials phase III in IPF (90). Given the multiple possible roles of ATX in COVID-19, ATX inhibition could offer additional therapeutic options in COVID-19 management, both during and after hospitalization. This work has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call Research -Create -Innovate (project code: T1EDK-0049; recipient VA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Statistical significance was assessed with one-way ANOVA followed by Bonferroni post hoc correction; ****denotes p<0.0001. ATX values are presented at Figure 1 . (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 July 31, 2021. ; (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. The copyright holder for this preprint this version posted July 31, 2021. ; https://doi.org/10.1101/2021.07.30.21261361 doi: medRxiv preprint Table S2 . The corresponding spreadsheets can be found at: https://www.dropbox.com/s/zdq0z0ikxrraj37/Table%20S2.xlsx?dl=0 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 July 31, 2021. (Table 1) . UMAP plots (on the left) depict the cellular composition of these sites, while dot plots (on the right) the expression pattern of ENPP2 in the detected cell types. Marker genes, denoted by stars, were detected using a Wilcoxon rank sum test; FC>1.2, Bonferroni corrected p<0.05; *** denotes p< 0.01) (PMIDs: A. 32591762; B. 32810438; C. 32398875; D. 33257409). 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 July 31, 2021. All rights reserved. No reuse allowed without permission. 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