Dysfunctional purinergic signaling correlates with disease severity in COVID-19 patients.

Ectonucleotidases modulate inflammatory responses by balancing extracellular ATP and adenosine (ADO) and might be involved in COVID-19 immunopathogenesis. Here, we explored the contribution of extracellular nucleotide metabolism to COVID-19 severity in mild and severe cases of the disease. We verified that the gene expression of ectonucleotidases is reduced in the whole blood of patients with COVID-19 and is negatively correlated to levels of CRP, an inflammatory marker of disease severity. In line with these findings, COVID-19 patients present higher ATP levels in plasma and reduced levels of ADO when compared to healthy controls. Cell type-specific analysis revealed higher frequencies of CD39+ T cells in severely ill patients, while CD4+ and CD8+ expressing CD73 are reduced in this same group. The frequency of B cells CD39+CD73+ is also decreased during acute COVID-19. Interestingly, B cells from COVID-19 patients showed a reduced capacity to hydrolyze ATP into ADP and ADO. Furthermore, impaired expression of ADO receptors and a compromised activation of its signaling pathway is observed in COVID-19 patients. The presence of ADO in vitro, however, suppressed inflammatory responses triggered in patients' cells. In summary, our findings support the idea that alterations in the metabolism of extracellular purines contribute to immune dysregulation during COVID-19, possibly favoring disease severity, and suggest that ADO may be a therapeutic approach for the disease.

SARS-CoV-2 Spike protein alters microglial purinergic signaling.

Despite long-term sequelae of COVID-19 are emerging as a substantial public health concern, the mechanism underlying these processes still unclear. Evidence demonstrates that SARS-CoV-2 Spike protein can reach different brain regions, irrespective of viral brain replication resulting in activation of pattern recognition receptors (PRRs) and neuroinflammation. Considering that microglia dysfunction, which is regulated by a whole array of purinergic receptors, may be a central event in COVID-19 neuropathology, we investigated the impact of SARS-CoV-2 Spike protein on microglial purinergic signaling. Here, we demonstrate that cultured microglial cells (BV2 line) exposed to Spike protein induce ATP secretion and upregulation of P2Y6, P2Y12, NTPDase2 and NTPDase3 transcripts. Also, immunocytochemistry analysis shows that spike protein increases the expression of P2X7, P2Y1, P2Y6, and P2Y12 in BV2 cells. Additional, hippocampal tissue of Spike infused animals (6,5ug/site, i.c.v.) presents increased mRNA levels of P2X7, P2Y1, P2Y6, P2Y12, NTPDase1, and NTPDase2. Immunohistochemistry experiments confirmed high expression of the P2X7 receptor in microglial cells in CA3/DG hippocampal regions after spike infusion. These findings suggest that SARS-CoV-2 Spike protein modulates microglial purinergic signaling and opens new avenues for investigating the potential of purinergic receptors to mitigate COVID-19 consequences.

The Role of Cluster of Differentiation 39 (CD39) and Purinergic Signaling Pathway in Viral Infections.

CD39 is a marker of immune cells such as lymphocytes and monocytes. The CD39/CD73 pathway hydrolyzes ATP into adenosine, which has a potent immunosuppressive effect. CD39 regulates the function of a variety of immunologic cells through the purinergic signaling pathways. CD39+ T cells have been implicated in viral infections, including Human Immunodeficiency Virus (HIV), Cytomegalovirus (CMV), viral hepatitis, and Corona Virus Disease 2019 (COVID-19) infections. The expression of CD39 is an indicator of lymphocyte exhaustion, which develops during chronicity. During RNA viral infections, the CD39 marker can profile the populations of CD4+ T lymphocytes into two populations, T-effector lymphocytes, and T-regulatory lymphocytes, where CD39 is predominantly expressed on the T-regulatory cells. The level of CD39 in T lymphocytes can predict the disease progression, antiviral immune responses, and the response to antiviral drugs. Besides, the percentage of CD39 and CD73 in B lymphocytes and monocytes can affect the status of viral infections. In this review, we investigate the impact of CD39 and CD39-expressing cells on viral infections and how the frequency and percentage of CD39+ immunologic cells determine disease prognosis.

Characterization of regulatory T cells in SARS-CoV-2 infected hemodialysis patients: relation to clinical and radiological severity.

BACKGROUND: Disordered Treg counts and function have been observed in patients with SARS-Cov-2 and are thought to contribute to disease severity. In hemodialysis patients, scarce data are available on the Treg response to SARS-CoV-2 or its relation to the clinical presentation. METHODS: A cross-sectional study included one hundred patients divided into three groups, thirty SARS-CoV-2-infected hemodialysis patients (COV-HD), and thirty confirmed SARSCoV-2 infected patients (COV), and forty non-infected hemodialysis patients (HD). Flow cytometric analysis of CD4, CD25, FoxP3, and CD39+ Tregs was done for all patients and tested for correlation to in-hospital mortality, clinical, radiological severity indices. RESULTS: COV-HD and COV patients had significantly lower Treg cell count than HD patients (Median value of 0.016 cell/ µl vs 0.28 cell/ µl, respectively- P: 0.001). COV-HD patients had higher CD39+ Tregs (median value of 0.006 cell/ µl vs 0.002 cell/ µl, respectively- P: 0.04). COV-HD patients had significantly lower hospital stay (median value of 3 vs 13 days, P:0.001), ICU admission rates (26.5% vs 46.7%, P:0.005) and in-hospital mortality (20.7% versus 43.3%, P:0.003) than COV patients. Treg and CD39 expressing Treg counts were not correlated to severity indices in both groups. A high neutrophil to lymphocyte ratio is strongly correlated to disease severity in COV-HD patients. CONCLUSIONS: This study provides evidence of T-cell, particularly T-regulatory cell decline in SARS-CoV-2 and suggests that hemodialysis per se does not distinctively impact the T-cell response. COV-HD patients exhibited a higher CD39+ Treg count and a better clinical profile, however, larger studies are needed to extrapolate on these findings.

High and Sustained Ex Vivo Frequency but Altered Phenotype of SARS-CoV-2-Specific CD4+ T-Cells in an Anti-CD20-Treated Patient with Prolonged COVID-19.

Here, we longitudinally assessed the ex vivo frequency and phenotype of SARS-CoV-2 membrane protein (aa145-164) epitope-specific CD4+ T-cells of an anti-CD20-treated patient with prolonged viral positivity in direct comparison to an immunocompetent patient through an MHC class II DRB1*11:01 Tetramer analysis. We detected a high and stable SARS-CoV-2 membrane-specific CD4+ T-cell response in both patients, with higher frequencies of virus-specific CD4+ T-cells in the B-cell-depleted patient. However, we found an altered virus-specific CD4+ T-cell memory phenotype in the B-cell-depleted patient that was skewed towards late differentiated memory T-cells, as well as reduced frequencies of SARS-CoV-2-specific CD4+ T-cells with CD45RA- CXCR5+ PD-1+ circulating T follicular helper cell (cTFH) phenotype. Furthermore, we observed a delayed contraction of CD127- virus-specific effector cells. The expression of the co-inhibitory receptors TIGIT and LAG-3 fluctuated on the virus-specific CD4+ T-cells of the patient, but were associated with the inflammation markers IL-6 and CRP. Our findings indicate that, despite B-cell depletion and a lack of B-cell-T-cell interaction, a robust virus-specific CD4+ T-cell response can be primed that helps to control the viral replication, but which is not sufficient to fully abrogate the infection.

Viral load is associated with mitochondrial dysfunction and altered monocyte phenotype in acute severe SARS-CoV-2 infection.

Monocytes play a major role in the initial innate immune response to SARS-CoV-2. Although viral load may correlate with several clinical outcomes in COVID-19, much less is known regarding their impact on innate immune phenotype. We evaluated the monocyte phenotype and mitochondrial function in severe COVID-19 patients (n = 22) with different viral burden (determined by the median of viral load of the patients) at hospital admission. Severe COVID-19 patients presented lower frequency of CD14 + CD16- classical monocytes and CD39 expression on CD14 + monocytes, and higher frequency of CD14 + CD16 + intermediate and CD14-CD16 + nonclassical monocytes as compared to healthy controls independently of viral load. COVID-19 patients with high viral load exhibited increased GM-CSF, PGE-2 and lower IFN-α as compared to severe COVID-19 patients with low viral load (p < 0.05). CD14 + monocytes of COVID-19 patients with high viral load presented higher expression of PD-1 but lower HLA-DR on the cell surface than severe COVID-19 patients with low viral load. All COVID-19 patients presented decreased monocyte mitochondria membrane polarization, but high SARS-CoV-2 viral load was associated with increased mitochondrial reactive oxygen species. In this sense, higher viral load induces mitochondrial reactive oxygen species generation associated with exhaustion profile in CD14 + monocytes of severe COVID-19 patients. Altogether, these data shed light on new pathological mechanisms involving SARS-CoV-2 viral load on monocyte activation and mitochondrial function, which were associated with COVID-19 severity.

Assessment of CD39 expression in regulatory T-cell subsets by disease severity in adult and juvenile COVID-19 cases.

COVID-19 is a disease characterized by acute respiratory failure and is a major health problem worldwide. Here, we aimed to investigate the role of CD39 expression in Treg cell subsets in COVID-19 immunopathogenesis and its relationship to disease severity. One hundred and ninety COVID-19 patients (juveniles, adults) and 43 volunteers as healthy controls were enrolled in our study. Flow cytometric analysis was performed using a 10-color monoclonal antibody panel from peripheral blood samples. In adult patients, CD39+ Tregs increased with disease severity. In contrast, CD39+ Tregs were decreased in juvenile patients in an age-dependent manner. Overall, our study reveals an interesting profile of CD39-expressing Tregs in adult and juvenile cases of COVID-19. Our results provide a better understanding of the possible role of Tregs in the mechanism of immune response in COVID-19 cases.

TIM-3 as a potential exhaustion marker in CD4+ T cells of COVID-19 patients.

BACKGROUND: COVID-19 causes a range of clinical symptoms from mild to critical and can be life-threatening. Up to now, it has led to many deaths. We aimed to evaluate exhausted markers on CD4+ T cells of COVID-19 patients. METHODS: In this study, we evaluated 44 patients with confirmed COVID-19 disease and 16 healthy individuals. Patients were divided into moderate/severe and critical groups. Peripheral blood mononuclear cells (PBMCs) were isolated and stained by anti-human CD39, PD-1, TIM-3, and anti-human CD4. The percentage of each CD4+  subpopulation was calculated by flow cytometry. Furthermore, we collected clinical information and laboratory data of both control and patient groups. RESULTS: We detected overexpression of TIM-3 on CD4+  T cells in both critical and moderate/severe patients than in healthy individuals (HIs; p < .01 and p < .0001, respectively). CD4+  TIM-3+ CD39+  lymphocytes were significantly higher in the critical patients than in HI (p < .05). Both Patient groups showed lymphopenia in comparison with HI, but CD4+  lymphocytes did not show any significant difference between study subjects. The increased amount of C-reactive protein, erythrocyte sedimentation rate, creatinine, blood urea nitrogen, and neutrophil count was observed in patients compared to HI. CONCLUSION: T cell exhaustion occurs during COVID-19 disease and TIM-3 is the most important exhausted marker on CD4+ T cells.

Thrombo-Inflammation: A Focus on NTPDase1/CD39.

There is increasing evidence for a link between inflammation and thrombosis. Following tissue injury, vascular endothelium becomes activated, losing its antithrombotic properties whereas inflammatory mediators build up a prothrombotic environment. Platelets are the first elements to be activated following endothelial damage; they participate in physiological haemostasis, but also in inflammatory and thrombotic events occurring in an injured tissue. While physiological haemostasis develops rapidly to prevent excessive blood loss in the endothelium activated by inflammation, hypoxia or by altered blood flow, thrombosis develops slowly. Activated platelets release the content of their granules, including ATP and ADP released from their dense granules. Ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1)/CD39 dephosphorylates ATP to ADP and to AMP, which in turn, is hydrolysed to adenosine by ecto-5'-nucleotidase (CD73). NTPDase1/CD39 has emerged has an important molecule in the vasculature and on platelet surfaces; it limits thrombotic events and contributes to maintain the antithrombotic properties of endothelium. The aim of the present review is to provide an overview of platelets as cellular elements interfacing haemostasis and inflammation, with a particular focus on the emerging role of NTPDase1/CD39 in controlling both processes.

B cell analysis in SARS-CoV-2 versus malaria: Increased frequencies of plasmablasts and atypical memory B cells in COVID-19.

B cells play a central role in antiviral and antiparasitic immunity, not only as producers of antibodies, but also as APCs and mediators of inflammation. In this study, we used 16-color flow cytometry analysis to investigate the frequency, differentiation, and activation status of peripheral B cells of patients with SARS-CoV-2 infection or acute Plasmodium falciparum malaria compared with the healthy individuals. As a main result, we observed an increase of the frequency of (CD27-, CD21-) atypical memory B cells and (CD19+, CD27+, CD38+) plasmablasts in malaria and COVID-19 patients. Additionally, CD86, PD-1, CXCR3, and CD39 expression was up-regulated, whereas CD73 was down-regulated on plasmablasts of COVID-19 and malaria patients compared with the bulk B cell population. In particular, there was a more pronounced loss of CD73+ B cells in malaria. The frequency of plasmablasts positively correlated with serum levels of CRP, IL-6, and LDH of COVID-19 patients. In the longitudinal course of COVID-19, a rapid normalization of the frequency of atypical memory B cells was observed. The role and function of plasmablasts and atypical memory B cells in COVID-19 and other acute infections remain to be further investigated. The role of B cells as either "driver or passenger" of hyperinflammation during COVID-19 needs to be clarified.

Pronounce expression of Tim-3 and CD39 but not PD1 defines CD8 T cells in critical Covid-19 patients.

BACKGROUND: During viral infection, inhibitory receptors play a key role in regulating CD8 T-cell activity. The objective of this research was to investigate programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin domain-containing protein-3 (TIM-3), and CD39 exhaustion markers in CD8 T cells of new coronavirus disease-2019 (COVID-19) patients. METHODS: A total of 44 patients with COVID-19 (17 subjects in a critical group and 27 patients in a non-critical group) and 14 healthy controls, who were admitted to Hospitals in Babol, were recruited to the study. In subjects' peripheral blood mononuclear cells (PBMCs), we compared the phenotype of CD8 T lymphocytes, expressing PD-1, TIM-3, or CD39, both alone and in various combinations. RESULTS: The findings showed that the percentage of CD8+ cells was significantly lower in patients. Critical and non-critical patients were more likely than healthy controls to have an escalated frequency of CD8+ TIM-3+, CD8+ CD39+, and CD8+ TIM-3+ CD39+ cells. No significant differences were observed between all groups in the CD8+ PD-1+ cell counts. There was also no difference between three groups regarding the counts of CD8+ TIM-3+ PD-1+, CD8+ PD-1+ CD39+, and CD8+ TIM-3+ PD-1+ CD39+ cells. The counts of non-exhausted cells were significantly lower in critical and non-critical individuals compared to the healthy individuals' value. CONCLUSION: Patients, infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), altered exhausted CD8 T lymphocytes with CD39 and TIM-3 exhaustion markers, which may account the dysregulated immune response found in COVID-19.

Targeting adenosinergic pathway and adenosine A2A receptor signaling for the treatment of COVID-19: A hypothesis.

The most serious health issue today is the rapid outbreak of Coronavirus Disease 2019 (COVID-19). More than 6,973,427 confirmed cases were diagnosed in nearly 213 countries and territories around the world and two international conveyances, causing globally over 400,000 deaths. Epidemiology, risk factors, and clinical characteristics of COVID-19 patients have been identified, but the factors influencing the immune system against COVID-19 have not been well established. Upon infection or cell damage, high amounts of adenosine triphosphate (ATP) are released from damaged cells, which serve as mediators of inflammation through purinergic cell surface receptor signaling. As a protective mechanism to prevent excessive damage to host tissue, adenosine counteracts ATP's effects by adenosine receptor stimulation to suppress the pro-inflammatory response. Adenosine is seen as a major obstacle to the efficacy of immune therapies, and the adenosinergic axis components are critical therapeutic targets for cancer and microbial infections. Pharmacologic inhibitors or antibodies specific to adenosinergic pathway components or adenosine receptors in microbial and tumor therapy have shown efficacy in pre-clinical studies and are entering the clinical arena. In this review, we provide a novel hypothesis explaining the potential for improving the efficiency of innate and adaptive immune systems by targeting adenosinergic pathway components and adenosine A2A receptor signaling for the treatment of COVID-19.

Purinergic signaling in infectious diseases of the central nervous system.

The incidence of infectious diseases affecting the central nervous system (CNS) has been increasing over the last several years. Among the reasons for the expansion of these diseases and the appearance of new neuropathogens are globalization, global warming, and the increased proximity between humans and wild animals due to human activities such as deforestation. Neurotropism affecting normal brain function is shared by organisms such as viruses, bacteria, fungi, and parasites. Neuroinfections caused by these agents activate immune responses, inducing neuroinflammation, excitotoxicity, and neurodegeneration. Purinergic signaling is an evolutionarily conserved signaling pathway associated with these neuropathologies. During neuroinfections, host cells release ATP as an extracellular danger signal with pro-inflammatory activities. ATP is metabolized to its derivatives by ectonucleotidases such as CD39 and CD73; ATP and its metabolites modulate neuronal and immune mechanisms through P1 and P2 purinergic receptors that are involved in pathophysiological mechanisms of neuroinfections. In this review we discuss the beneficial or deleterious effects of various components of the purinergic signaling pathway in infectious diseases that affect the CNS, including human immunodeficiency virus (HIV-1) infection, herpes simplex virus type 1 (HSV-1) infection, bacterial meningitis, sepsis, cryptococcosis, toxoplasmosis, and malaria. We also provide a description of this signaling pathway in emerging viral infections with neurological implications such as Zika and SARS-CoV-2.

Defining the CD39/CD73 Axis in SARS-CoV-2 Infection: The CD73- Phenotype Identifies Polyfunctional Cytotoxic Lymphocytes.

The ectonucleotidases CD39 and CD73 regulate immune responses by balancing extracellular ATP and adenosine in inflammation and are likely to be involved in the pathophysiology of COVID-19. Here, we analyzed CD39 and CD73 on different lymphocyte populations in a small cohort of COVID-19 patients and in healthy individuals. We describe a significantly lower level of expression of CD73 on cytotoxic lymphocyte populations, including CD8+ T, natural killer T (NKT), and natural killer (NK) cells, during COVID-19. Interestingly, the decrease of CD73 on CD8+ T cells and NKT cells correlated with serum ferritin levels. Furthermore, we observed distinct functional differences between the CD73+ and CD73- subsets of CD8+ T cells and NKT cells with regard to cytokine/toxin secretion. In COVID-19 patients, the majority of the CD73-CD8+ T cells were capable of secreting granzyme B, perforin, tumor necrosis factor (TNF-α) or interferon-gamma (IFN-γ). To conclude, in this first study of CD39 and CD73 expression of lymphocytes in COVID-19, we show that CD8+ T cells and NKT cells lacking CD73 possess a significantly higher cytotoxic effector functionality compared to their CD73+ counterparts. Future studies should investigate differences of cellular CD39 and CD73 expression in patients at different disease stages and their potential as prognostic markers or targets for immunomodulatory therapies.