key: cord-0281005-alatd73x authors: Dorneles, G. P.; Teixeira, P. C.; da Silva, I.; Schipper, L. d. L.; Santana Filho, P. C.; Rodrigues Junior, L. C.; Bonorino, C.; Peres, A.; Fonseca, S.; Monteiro, M.; Boeck, C.; Eller, S.; Oliveira, T. F.; Wendland, E.; Romao, P. R. title: Alterations in CD39/CD73 Axis of T cells associated with COVID-19 severity date: 2021-09-22 journal: nan DOI: 10.1101/2021.09.18.21263782 sha: e5bc93c49ddd657f4c6c84f97cbf051c78f0f6d0 doc_id: 281005 cord_uid: alatd73x Purinergic signaling modulates immune function and is involved in the immunopathogenesis of several viral infections. This study aimed to investigate alterations in purinergic pathways in COVID-19 patients. We evaluated the systemic levels of adenine-based purines, the frequencies of CD4+ and CD8+ T cells expressing CD39/CD73 ectonucleotidases, CD8+CD27-/+CD28-/+ T cells expressing PD-1 and the rates of lymphocyte apoptosis in the peripheral blood of patients with mild or severe COVID-19. Lower plasma ATP and adenosine levels were identified in mild and severe COVID-19 patients associated with higher systemic levels of IL-6, IL-10 and IL-17A compared to health controls. Mild COVID-19 patients presented lower frequencies of CD4+CD25+CD39+ (activated/memory Treg) and CD4+CD25+CD39+CD73+ T cells, and increased frequencies of high differentiated (CD27-CD28-) CD8+T cells compared to health controls. Severe COVID-19 patients also showed higher frequencies of CD4+CD39+, CD4+CD25-CD39+ (memory T effector cell), high differentiated CD8+ T cells (CD27-CD28-) and diminished frequencies of CD4+CD73+, CD4+CD25+CD39+ mTreg, CD4+CD25+CD39+CD73+, CD8+CD73+ and low-differentiated CD8+ T cells (CD27+CD28+) in the blood in relation to mild COVID-19 patients and controls. Moreover, severe COVID-19 patients presented higher expression of PD-1 on low-differentiated CD8+ T cells. Both severe and mild COVID-19 patients presented higher frequencies of CD4+Annexin-V+ and CD8+Annexin-V+ T cells, showing increased T cell apoptosis. Together, these data add new knowledge regarding the immunopathology of COVID-19 through purinergic regulation. The disease caused by SARS-CoV-2 (COVID- 19) is an hyperinflammatory disease, that can be asymptomatic or manifested as a broad spectrum of disease ranging from few symptoms (mild COVID-19 cases) to severe pneumonia that may evolve to SARS and death (severe COVID-19 cases) (1) . Some typical clinical symptoms of patients with COVID-19 are fatigue, fever, dry cough, dyspnea, and shortness of breath. Although a higher number of SARS-CoV-2 infections generate asymptomatic or mild COVID-19, while a proportion of the remaining cases show severe and critical pneumonia requiring oxygen support and mechanical ventilation (2) . Host factors and the physiological environment determine the type and the strength of the immune response during the viral infection, as well as the disease outcomes. Thus, the biology of several immune cells, including lymphocytes, can be influenced by blood extracellular adenine nucleotides -adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) and nucleoside (adenosine) (3) . The purinergic signaling regulates a number or immune cell functions, such as cellto-cell interactions, cell death, cytokine and chemokine secretion, surface antigen shedding, and cell proliferation (4) . During infections, once released by damaged cells, ATP acts as a damage-associated molecular pattern through P2 receptors (P2X and P2Y receptors) activating immune cells and inducing strong pro-inflammatory effects (5) . Interestingly, severe COVID-19 patients had higher ATP content in the bronchoalveolar lavage supernatant associated with P2RX7-inflammasome activation in macrophage compared to non-COVID-19 patients (6) . Thus, it is possible that alterations in adeninebased purine molecules may contribute to the hyperinflammation and immune dysfunction observed in COVID-19 pathophysiology. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 22, 2021. ; https://doi.org/10.1101/2021.09. 18.21263782 doi: medRxiv preprint The ectoenzyme CD39 (ecto-nucleoside triphosphate diphosphohydrolase 1, E-NTPDase1) converts extracellular ATP into AMP, and then CD73 (ecto-5'-nucleotidase, E-5'NTase) dephosphorylates AMP into adenosine (4, 7) . Adenosine signaling is mediated by G-protein-coupled adenosine receptors (AR) which acts as a mechanism for regulating intracellular cyclic AMP (cAMP) levels to induce immunosuppressive events (8). CD39 and CD73 are present in several immune cells, such as CD4+ T cells (including regulatory T cells and effector memory T cells), CD8+ T cells, CD19+ B cells, and Natural Killer cells, and participate in the regulation of the duration and magnitude of the immune response (4) . The expression and activity of both CD39 and CD73 changes under the pathological context of acute and chronic viral infections (9-11). In addition, CD39 expression in CD8+ T cell has also been described as a marker of exhaustion in virus infection (12). To date, Ahmadi and colleagues (13) found lower proportions of CD8+CD73-T cells in the peripheral blood of COVID-19 patients and demonstrated that this cells possess a significantly higher cytotoxic effector phenotype (13). In addition, CD4+ and CD8+ T cells from severe COVID-19 patients present a dysregulated status of activation, characterized by higher expression of human leukocyte antigen-DR (HLA-DR) and CD38 activation marker, associated with phenotypical and functional T cell exhaustion (14, 15) . To better understand the role of CD39/CD73 pathway in the immunopathology of SARS-CoV-2 infection, this study evaluated the systemic levels of ATP, ADP, AMP and adenosine and the frequencies of CD4+ and CD8+ T cells expressing CD39 and CD73 ectonucleotidases, the proportions of CD8+CD27 -/+ CD28 -/+ expressing PD-1, as well as the rates of lymphocyte apoptosis in the peripheral blood from mild and severe COVID-19 patients. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint We prospectively evaluated a convenience sample of mild and severe positive patients admitted at the Hospital Moinhos de Vento between July/2020 and November/2020 and volunteering uninfected healthy individuals. Infection with SARS-CoV-2 was confirmed by reverse transcription polymerase chain reaction (RT-PCR) with nasopharyngeal and oropharyngeal swab samples. This study was approved by the Moinhos de Vento Ethics Committee N 3977144 (Porto Alegre/Brazil) and informed consent was obtained from all participants. Blood samples (5 mL) were obtained from patients with mild or severe COVID-19 into K2EDTA tubes (Becton & Dickinson, USA) within 6 h from hospital admission. Flow cytometry experiments (described below) were conducted with fresh blood. The remaining blood was centrifuged (1500 g, 10 min), plasma was aliquoted and kept at -80 ºC until plasma analysis. Disease severity was classified according to the World Health Organization classification after completing the follow-up questionnaire (16) . Clinical and sociodemographic data were collected from the patient's electronic medical records upon admission to the unit. Body mass index was calculated from weight and height data. An aliquot of 150 μ L of acetonitrile was added to the plasma samples (50 μ L), and the mixture was shaken for 60 seconds. After centrifugation for 6 min at 9000 g, the supernatant was collected and a 25 μ L aliquot was directly injected into the LC MS/MS system. The analytical system consisted of a Nexera UFLC system . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The column oven was kept at 30 °C. The data were processed using LabSolutions software (Shimadzu, Kyoto, Japan). In addition, LPS concentration was determined by quantitation of 3-hydroxytetradecanoic acid as described by Teixeira and coworkers (17). The plasma concentrations of IL 17A (from PeproTech, USA), and IL 6, IL 10 and TGF-β (all from eBioscience, ThermoFisher, USA), were quantified by enzyme linked immunosorbent assay (ELISA) in microplate reader (EzReader, EUA). The detection limits of each cytokine were IL 6, 2 200 pg/mL; IL 10, 2 300 pg/mL; IL 17A, 2 1000 pg/ mL; TGF-β, 20 -500 pg/mL. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The mitochondrial membrane potential (ΔΨm) was quantified according to a method previously described (19) , using the fluorescent dye rhodamine 123 (Rh 123, Sigma-Aldrich). The detection of apoptosis was performed by using additional labeling with Annexin V-FITC following manufacturer's guidelines (Invitrogen, ThermoFisher, USA). Analyses were performed by using CELLQuest Pro Software (BD Bioscience) on a FACSCalibur flow cytometer (BD Bioscience). Normality of data was checked by Kolmogorov Smirnov, and the values were presented as mean ± standard deviation (SD). Categorical variables were presented as relative frequency and analyzed by Qui-Square test. To identify associations between adenine-based purine levels and symptoms we used Mann-Whitney U-test to compare SARS-CoV-2 infected patients with and without specific disease symptoms. A one way ANOVA followed by Bonferroni's post hoc for multiple comparisons was used to verify between-groups differences. Pearson's Coefficient Test was performed to check correlations between the variables. P value ≤ 0.05 were considered statistically significant. The SPSS 20.0 (IBM Inc, EUA) software was used in all analysis. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 22, 2021. ; https://doi.org/10.1101/2021.09.18.21263782 doi: medRxiv preprint The characteristics of severe (n=22), mild (n=24) COVID-19 patients, and healthy control (n=13) participants are presented in Table 1 . All patients had COVID-19 confirmed by RT-PCR. Severe COVID-19 patients were older (p<0.001), presented more days from symptom onset (p<0.001) and required oxygen use during hospitalization (p<0.001) compared to mild COVID-19 patients. Furthermore, several symptoms were reported by severe COVID-19 patients, including cough (p=0.007), dysgeusia (p=0.04), anosmia (p=0.001), vomiting (p=0.007), diarrhea (p=0.004), skin rash (p=0.01), fatigue (p=0.01) and stuffy nose (p=0.005). Regarding associated medical condition, severe COVID-19 patients reported hypertension (p=0.002), diabetes mellitus (p=0.001), cardiovascular diseases (p=0.001), heart failure (p=0.001), chronic obstructive pulmonary disease (p=0.001), asthma (p=0.001) and dyslipidemia (p=0.001). (Table 1) . . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint B M I , b o d y m a s s i n d e x ; C O P D , c h r o n i c o b s t r u c t i v e p u l m o n a r y d i s e a s e ; H I V h u m a n i m m u n o d e f i c i e n c y v i r u s . Q u a l i t a t i v e d a t a p r e s e n t e d a s r e l a t i v e f r e q u e n c y . B e t w e e n g r o u p s c o m p a r i s o n t h r o u g h Q u i -s q u a r e t e s t ( p < 0 . 0 5 ) . The . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The T cell phenotype was evaluated in healthy controls (n=8), mild COVID-19 (n=12) and severe COVID-19 patients (n=13). The peripheral frequencies of CD4+, CD4+CD25-and CD4+CD25+ T cells expressing CD39+ and CD73+ ectonucleotidases were presented in Figure 3 . In total CD4+ T cells, severe COVID-19 group had higher frequencies of CD4+CD39+CD73-(p=0.01 vs. healthy controls; p=0.03 vs. mild COVID-19; Fig.3A ), but lower CD4+CD39-CD73+ (p=0.04 vs. healthy controls; . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Fig.4E ). The expression of PD-1 was higher in low-differentiated CD8+ T cells of severe COVID-19 patients (p=0.008 vs. healthy controls, Fig.4G ). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint Next, we evaluated the apoptosis and mitochondrial membrane polarization in healthy controls (n=6), mild COVID-19 (n=6) and severe COVID-19 (n=6) individuals ( Figure 5 COVID-19 subjects (p=0.03). (Fig.5B) . Here, for the first time we showed that COVID-19 patients present lower adenosine levels in the blood, which may contribute to the uncontrolled inflammation, and severity of disease. Adenosine binds the P1 receptors, mainly the AR2A subtype, of immune cells (i.e. lymphocytes, monocytes and macrophages) to induce several events to suppress the inflammatory response, such as the downregulation of the master inflammatory transcription factor nuclear factor kappaB (NF-kB) (8,23,24). Furthermore, our data revealed higher levels of IL-6, IL-10 and IL-17A plasma concentrations in severe COVID-19 group, indicating a Th1/Th17 inflammatory polarization. Acute hypercytokinemia is a common feature of severe COVID-19 that may contribute to the multiorgan impairment and coagulopathy disorders (17, 25, 26) . Reinforcing the regulatory effect of adenosine in the inflammation and cytokine storm, the use of inhaled adenosine in COVID-19 patients has successfully decreased the length of hospitalization and improved the prognosis of patients (30, 31) . Collectively, our data indicate alterations in adenine-based purinergic molecules, mainly ATP and adenosine levels, during the initial phase of SARS-CoV-2 infection. Here, we also described a negative correlation between plasma adenosine levels and the CD4+ T cell apoptosis rate. Previous studies have suggested that adenosine and its analog are related to anti-apoptotic events through A2A receptor activation in CD4+ T cells (27). On the other hand, mitochondrial dysfunction is involved in the induction of apoptosis, thus increasing the depolarization of transmembrane potential (28) . Here, lymphocytes of COVID-19 patients presented a state of mitochondrial membrane depolarization and higher rates of apoptosis. Interesting, mitochondria have a central . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 22, 2021. ; https://doi.org/10.1101/2021.09.18.21263782 doi: medRxiv preprint role in T cell activation by producing ATP, and higher mitochondrial dysfunction may leads to failure in the purinergic regulation and cell homeostasis (29) . The increased expression of CD39 and CD73 in lymphocytes rapidly converts extracellular ATP to adenosine to induce anti-inflammatory effects (4). Here, we In summary, we describe for the first time an imbalance in the levels of extracellular adenine-based purine molecules and alterations in CD39/CD73 ectonucleotidases axis in CD4+ and CD8+ T cells of COVID-19 patients with different degrees of disease severity. The reduced adenosine extracellular levels and alterations in T cells phenotype may impact on COVID-19 severity. Collectively, these data add new knowledge regarding the immunopathology of COVID-19 through purinergic regulation. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted September 22, 2021. ; https://doi.org/10.1101/2021.09.18.21263782 doi: medRxiv preprint Clinical and immunologic features in severe and moderate forms of Coronavirus Disease COVID-19 diagnosis and management: a comprehensive review Purinergic regulation of the immune system CD39 and CD73 in immunity and inflammation Nucleotide signalling during inflammation Discriminating mild from critical COVID-19 by innate and adaptive immune single-cell profiling of bronchoalveolar lavages Available from: . 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Front Pharmacol CD39/NTPDase-1 activity and expression in normal leukocytes Deep immune profiling of COVID-19 patients reveals patient heterogeneity and distinct immunotypes with implications for therapeutic interventions It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted September 22, 2021. ; https://doi.org/10.1101/2021.09.18.21263782 doi: medRxiv preprint It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted September 22, 2021. ; https://doi.org/10.1101/2021.09.18.21263782 doi: medRxiv preprint