Alterations in CD39/CD73 axis of T cells associated with COVID-19 severity.

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 coronavirus disease 2019 (COVID-19) patients. Mild and severe COVID-19 patients had lower extracellular adenosine triphosphate and adenosine levels, and higher cytokines than healthy controls. Mild COVID-19 patients presented lower frequencies of CD4+ CD25+ CD39+ (activated/memory regulatory T cell [mTreg]) and increased frequencies of high-differentiated (CD27- CD28- ) CD8+ T cells compared with healthy controls. Severe COVID-19 patients also showed higher frequencies of CD4+ CD39+ , CD4+ CD25- CD39+ (memory T effector cell), and high-differentiated CD8+ T cells (CD27- CD28- ), and diminished frequencies of CD4+ CD73+ , CD4+ CD25+ CD39+ mTreg cell, 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, indicating increased T-cell apoptosis. Plasma samples collected from severe COVID-19 patients were able to decrease the expression of CD73 on CD4+ and CD8+ T cells of a healthy donor. Interestingly, the in vitro incubation of peripheral blood mononuclear cell from severe COVID-19 patients with adenosine reduced the nuclear factor-κB activation in T cells and monocytes. Together, these data add new knowledge to the COVID-19 immunopathology through purinergic regulation.

Increased LPS levels coexist with systemic inflammation and result in monocyte activation in severe COVID-19 patients.

Mucosal barrier alterations may play a role in the pathogenesis of several diseases, including COVID-19. In this study we evaluate the association between bacterial translocation markers and systemic inflammation at the earliest time-point after hospitalization and at the last 72 h of hospitalization in survivors and non-survivors COVID-19 patients. Sixty-six SARS-CoV-2 RT-PCR positive patients and nine non-COVID-19 pneumonia controls were admitted in this study. Blood samples were collected at hospital admission (T1) (Controls and COVID-19 patients) and 0-72 h before hospital discharge (T2, alive or dead) to analyze systemic cytokines and chemokines, lipopolysaccharide (LPS) concentrations and soluble CD14 (sCD14) levels. THP-1 human monocytic cell line was incubated with plasma from survivors and non-survivors COVID-19 patients and their phenotype, activation status, TLR4, and chemokine receptors were analyzed by flow cytometry. COVID-19 patients presented higher IL-6, IFN-γ, TNF-α, TGF-ß1, CCL2/MCP-1, CCL4/MIP-1ß, and CCL5/RANTES levels than controls. Moreover, LPS and sCD14 were higher at hospital admission in SARS-CoV-2-infected patients. Non-survivors COVID-19 patients had increased LPS levels concomitant with higher IL-6, TNF-α, CCL2/MCP-1, and CCL5/RANTES levels at T2. Increased expression of CD16 and CCR5 were identified in THP-1 cells incubated with the plasma of survivor patients obtained at T2. The incubation of THP-1 with T2 plasma of non-survivors COVID-19 leads to higher TLR4, CCR2, CCR5, CCR7, and CD69 expression. In conclusion, the coexistence of increased microbial translocation and hyperinflammation in patients with severe COVID-19 may lead to higher monocyte activation, which may be associated with worsening outcomes, such as death.

Differential effects of whole blood heat treatment on the ex vivo inflammatory profile of untrained and trained men.

This study evaluated the effects of heat stress on the ex vivo inflammatory profile in untrained and trained men. Whole blood samples from untrained (UT) and trained (TR) individuals were incubated for 2 h at 37 °C or 40 °C. The whole blood of a subsample of the participants (n = 5 in both TR and UT groups) were stimulated with lipopolysaccharide (LPS, 10 ng/mL) concomitant to heat treatment (37 °C versus 40 °C). Flow cytometry was used to assess the intracellular NF-κB activation in CD4+ T cells and CD14+ monocytes, the expression of Toll-Like Receptor-4 (TLR-4), the frequencies of CD4+CD25-CD39+ and CD4+CD25+CD39+ T cells and monocyte subsets (CD14+CD16-; CD14+CD16+; CD14-CD16+), the mitochondrial membrane potential (MMP) and the reactive oxygen species (ROS) production by lymphocytes and monocytes. The production of interleukin (IL)-6, IL-10, and tumor necrosis factor-alpha (TNF-α) by LPS-stimulated whole blood were also evaluated. Heat treatment (40 °C) increased the proportions of CD14+CD16- and CD14+CD16+ monocytes and the lymphocyte MMP in the UT group. The frequencies of CD14-CD16+ monocytes and the activation of NF-κB in CD14+ monocytes decreased in UT and TR groups after heat treatment, while a reduction in CD4+CD25-CD39+ T-cells was observed only in the UT group. Higher TLR-4 and NF-κB activation were found in LPS-stimulated monocytes of UT men concomitant with higher TNF-α production and diminished IL-10 production after heat treatment. TR individuals presented lower NF- κB activation in LPS-stimulated monocytes after heat treatment. Our data suggest that the training status of individuals may impact on the anti-inflammatory response of heat treatment.

DNA damage in mononuclear cells following maximal exercise in sedentary and physically active lean and obese men.

We evaluated the impact of maximal exercise on oxidative stress and DNA damage in peripheral blood mononuclear cells (PBMC) from sedentary and exercised lean and obese men. PBMC were collected before, immediately and 1-h after exercise and exposed to hydrogen peroxide (H2O2; 25 and 50 µM, 4 h). A leukocytosis was induced by maximal exercise immediately and 1-h after exercise in all groups. However, a lymphopenia was observed 1-h after exercise in the Sedentary obese group. In the control condition, low DNA damage index concomitant to increases in intracellular glutathione content (GSH) was identified immediately after exercise in all groups. However, higher DNA damage index and lipid peroxidation occurred 1-h after the bout in Sedentary and Exercised Obese groups. PBMC exposed to both H2O2 25 and 50 µM experienced higher DNA damage and lipid peroxidation index immediately after exercise in all groups. Both lipid peroxidation and DNA damage index remained higher in PBMC of Sedentary Lean, Sedentary Obese, and Exercised obese groups obtained 1-h after exercise in both H2O2 25 and 50 µM, with the highest values identified in PBMC from Sedentary Obese group. However, increases in GSH content were identified in treated PBMC from sedentary and exercised lean groups as well as exercised obese group 1-h after exercise. Habitual exercise confers increased resistance of PBMC to DNA damage induced by oxidative stress, reducing the detrimental effects of obesity.Keywords: Exercise, physical activity, DNA damage, obesity, mutagenesis, oxidative stress.

Immunoregulation induced by autologous serum collected after acute exercise in obese men: a randomized cross-over trial.

In this study, we evaluated the effects of autologous serum collected after two types of exercise on the in vitro inflammatory profile and T cell phenotype of resting peripheral blood mononuclear cells (PBMCs) in obese men. Serum samples and PBMCs were obtained from eight obese men who performed two exercise bouts-high intensity interval exercise (HIIE) and exhaustive exercise session to voluntary fatigue-in a randomized cross-over trial. Pre-exercise PBMCs were incubated with 50% autologous serum (collected before and after each exercise bout) for 4 h. In vitro experiments revealed that post-HIIE serum reduced the histone H4 acetylation status and NF-κB content of PBMCs and suppressed the production of both TNF-α and IL-6 by PBMCs, while increasing IL-10 production. Post-exhaustive exercise serum induced histone H4 hyperacetylation and mitochondrial depolarization in lymphocytes and increased TNF-α production. In vitro post-HIIE serum incubation resulted in an increase in the frequencies of CD4 + CTLA-4 + and CD4 + CD25+ T cells expressing CD39 and CD73. Post-exhaustive exercise serum decreased the frequency of CD4 + CD25 + CD73+ T cells but increased CD4 + CD25-CD39 + T cell frequency. Both post-exercise serums increased the proportions of CD4 + PD-1 + and CD8 + PD-1+ T cells. Blood serum factors released during exercise altered the immune response and T cell phenotype. The type of exercise impacted the immunomodulatory activity of the post-exercise serum on PBMCs.

Exercise-induced BDNF production by mononuclear cells of sedentary and physically active obese men.

BACKGROUND: Obesity and low physical activity changes the redox state and neurotrophin secretion by leukocytes. However, the role of exercise on brain derived neurotrophic factor (BDNF) production and oxidative stress markers of peripheral blood mononuclear cells (PBMC) remains unknown. This study aimed to verify the impact of acute maximal exercise on oxidative stress markers and the BDNF production by stimulated PBMC from sedentary and physically active obese men. METHODS: PBMC from twelve sedentary obese (SED group) and twelve regular exercisers (EXE group) obese men were collected before, immediately and 1-h after maximal exercise. PBMC were stimulated with lipopolysaccharide (LPS) to evaluate the BDNF and nitrite production, lipid peroxidation, and antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) activities. RESULTS: PBMC from EXE group presented higher BDNF production (P=0.03) and lower TBARS levels than SED group at baseline. Maximal exercise increased BDNF and nitrite production, and lipid peroxidation immediately and 1-h after the bout in both groups. The EXE group presented higher superoxide dismutase activity immediately after bout and higher catalase activity 1-h after bout in PBMC. On the other hand, PBMC from SED group had lower superoxide dismutase activity immediately after exercise. Furthermore, PBMC from EXE group presented higher BDNF production and SOD activity and lower TBARS concentrations than SED group immediately after maximal exercise. CONCLUSIONS: Maximal exercise changes the redox state and enhances BDNF production by LPS-stimulated PBMC in obese individuals.

Physical fitness modulates the expression of CD39 and CD73 on CD4+ CD25- and CD4+ CD25+ T cells following high intensity interval exercise.

AIM: To investigate the impact of physical fitness on the mobilization of CD4+ CD25 - CD39 + and CD4 + CD25 + CD39 + T cells in response to acute exercise. METHODS: Fifteen high physical fitness (25.3 ± 1.4 years) and 15 low physical fitness (26.1 ± 1.9 years) men performed a single bout of high-intensity interval exercise (HIIE, 10 bouts of 60 seconds at 85% HRmax intercepted by 75 seconds of recovery at 50% HRmax). Blood lymphocytes were isolated before, immediately after and 1 hour after exercise for assessment of cell surface expression of CD25, CD39, and CD73 on CD4+ T cells. Effector memory T cells (mTeff) were identified by CD4 + CD25 - CD39 + coexpression, and memory regulatory T cells (mTReg) were defined as CD4 + CD25 + CD39 + T cells. RESULTS: Exercise increased CD4+ and CD4 + CD25 + T cell frequencies immediately after followed by a decrease bellow to baseline values at 1 hour after the bout in both low and high physical fitness groups. At baseline, the proportions of mTeff were higher, while mTreg were lower in low physical fitness individuals. The frequency of mTreg increased immediately after HIIE in both groups, and remained higher 1 hour after the bout. However, high physical fitness individuals presented higher mTreg frequency in all periods evaluated. A significantly mobilization of mTeff cells was identified in both groups immediately after HIIE. High physical fitness individuals displayed a decrease in mTeff cells bellow to baseline, while the frequency of mTeff remained higher in low physical fitness group 1 hour after the bout. The peripheral frequency of CD4 + CD25 + CD73 + T cells increased in a similar way immediately after the bout in both groups, returning to the baseline values 1 hour after exercise. No differences in CD4 + CD25 - CD73 + T cells were observed after HIIE in both groups. CONCLUSION: Our results highlight the impact of physical activity status in the redistribution of CD4+ T cells expressing ectonucleotidases in response to HIIE.