Differential expression patterns of purinergic ectoenzymes and the antioxidative role of IL-6 in hospitalized COVID-19 patient recovery

Abstract

Introduction: We have acquired significant knowledge regarding the pathogenesis of severe acute respiratory syndrome caused by coronavirus 2 (SARS-CoV-2). However, the underlying mechanisms responsible for disease recovery still need to be fully understood.

Methods: To gain insights into critical immune markers involved in COVID-19 etiopathogenesis, we studied the evolution of the immune profile of peripheral blood samples from patients who had recovered from COVID-19 and compared them to subjects with severe acute respiratory illness but negative for SARS-CoV-2 detection (controls). In addition, linear and clustered correlations between different parameters were determined.

Results: The data obtained revealed a significant reduction in the frequency of inflammatory monocytes (CD14+CD16+) at hospital discharge vs. admission. Remarkably, nitric oxide (NO) production by the monocyte compartment was significantly reduced at discharge. Furthermore, interleukin (IL)-6 plasma levels were negatively correlated with the frequency of NO+CD14+CD16+ monocytes at hospital admission. However, at the time of hospital release, circulating IL-6 directly correlated with the NO production rate by monocytes. In line with these observations, we found that concomitant with NO diminution, the level of nitrotyrosine (NT) on CD8 T-cells significantly diminished at the time of hospital release. Considering that purinergic signaling constitutes another regulatory system, we analyzed the kinetics of CD39 and CD73 ectoenzyme expression in CD8 T-cells. We found that the frequency of CD39+CD8+ T-cells significantly diminished while the percentage of CD73+ cells increased at hospital discharge. In vitro, IL-6 stimulation of PBMCs from COVID-19 patients diminished the NT levels on CD8 T-cells. A clear differential expression pattern of CD39 and CD73 was observed in the NT+ vs. NT-CD8+ T-cell populations.

Discussion: The results suggest that early after infection, IL-6 controls the production of NO, which regulates the levels of NT on CD8 T-cells modifying their effector functions. Intriguingly, in this cytotoxic cell population, the expression of purinergic ectoenzymes is tightly associated with the presence of nitrated surface molecules. Overall, the data obtained contribute to a better understanding of pathogenic mechanisms associated with COVID-19 outcomes.

Keywords: CD39; CD73; SARS-CoV-2; cytokines; inflammatory monocytes; nitric oxide; nitrotyrosine.

Figure 1

Cytokine plasma levels. Comparison of cytokine concentrations of COVID-19 patients at admission vs. discharge (n=27) (Wilcoxon signed-rank test). The results are expressed as mean ± SD (ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 2

Frequency of circulating monocyte profiles at admission vs. discharge (A) Gate strategy analysis employed to determinate nitric oxide (NO) produced by monocytes. (B) Frequency of circulating CD14+CD16- and (C) CD14+CD16+ cells (n=26). (D, E) Mean Fluorescence Intensity (MFI) of NO-producing monocyte subsets (n=21). (B–E) Paired comparisons (admission vs. discharge) were analyzed using the Wilcoxon test. (F, G) MFI of NO-producing monocyte subsets in COVID-19 patients at hospital admission and discharge (n=21) vs. non-COVID-19 subjects (n=10). Unpaired comparisons (i.e., admission vs. non-COVID-19 or release vs. non-COVID-19) were analyzed using the Mann-Whitney test. The results are expressed as mean ± SD (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 3

Correlation analysis between IL-6 plasma levels and subpopulations of NO+ monocytes. (A) Frequency of CD14+CD16+ monocytes producing NO at admission (n=18) and (B, C) NO production in different monocyte subsets at hospital discharge (n=20) vs. IL-6 plasma concentrations. Dotted lines represent the confidence interval for the correlation coefficient.

Figure 4

Levels of NT and purinergic ectoenzymes in CD8 T cells. (A) Gate strategy employed to determine the MFI of nitrotyrosine (NT) in CD8 T-cells from COVID-19 patients. (B) Levels of NT in circulating CD8 T-cells in COVID-19 patients at admission and discharge (n=26). Wilcoxon test. (C) Clustered correlation matrix between NO production by monocytes, NT+CD8+ T-cell, and cytokine plasma concentration at hospital admission. (D) Principal component analysis (PCA), based on NO production in monocytes and NT in CD8 T-cells in COVID-19 patients at hospital admission and discharge, and non-COVID-19 patients. (E) Frequency and expression levels of CD39 and CD73 on CD8 T- cells at hospital admission vs. discharge (n=26). The results are expressed as mean ± SD (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 5

In vitro stimulation of PBMCs from COVID-19 patients with recombinant IL-6. Frequency and expression levels of nitrotyrosine (NT) in CD8+ T-cells stimulated with (A) 20 ng/mL or (B) 20 pg/mL of IL-6 for 48 h (n = 7) (ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 6

Analysis of the expression of purinergic ectoenzymes in CD8 T-cells stimulated in vitro with recombinant IL-6. Frequency of CD39+CD73-CD8+ T-cells (A) unstimulated (medium) or stimulated with (B) 20 ng/mL or (C) 20 pg/mL of IL-6. Frequency of CD39-CD73+ CD8+ T-cells (D) unstimulated (medium) or stimulated with (E) 20 ng/mL or (F) 20 pg/mL of IL-6. Frequency of CD39+CD73+CD8+ T-cells (G) unstimulated (medium) or stimulated with (H) 20 ng/mL and (I) 20 pg/mL of IL-6 for 48 h (n = 7) (* p < 0.05; ** p < 0.01; *** p < 0.001).