Cytokines from Macrophages Activated by Spike S1 of SARS-CoV-2 Cause eNOS/Arginase Imbalance in Endothelial Cells

Abstract

Multiple lines of evidence suggest that endothelial dysfunction is a key player in the pathogenesis of COVID-19, with cytokine storm as one of the main primary causes. Among the mechanisms underlying endothelial damage, clinical findings identify alterations in arginine metabolism, as patients with severe COVID-19 exhibit lower levels of nitric oxide synthase (eNOS) and upregulated arginase. In this study, we investigated, in human endothelial cells (HUVECs), the effect of conditioned medium from macrophages activated with SARS-CoV-2 Spike protein (CM_S1) on arginine metabolism. The results indicate that CM_S1 causes a marked decrease in eNOS and an increase in arginase, along with a greater intracellular arginine content and the induction of the CAT2 transporter. These effects are ascribable to the inflammatory mediators released by macrophages in CM_S1, mainly TNFα and IL-1β. Since infliximab, an antibody targeting TNFα, and baricitinib, an inhibitor of the JAK/STAT pathway, correct the observed imbalance between eNOS and arginase, our findings suggest the potential efficacy of a combined therapy to counteract endothelial dysfunction in COVID-19.

Keywords: TNFα; arginase; arginine; cytokines; eNOS; endothelial dysfunction; macrophages.

Figure 1

HUVECs were incubated in conditioned medium (CM) obtained from monocyte-derived macrophages untreated (CM_cont) or treated for 24 h with 5 nM S1 (CM_S1); for control, cells were maintained in RPMI1640 medium. After 4 and 24 h, the mRNA levels of NOS3 (A) and ARG2 (D) were assessed with RT-qPCR and shown as a fold change in control (=1). Bars are means ± SEM of four experiments performed in duplicate. * p < 0.05, *** p < 0.001 vs. control with Student’s t test. eNOS (B) and arginase (E) protein expression was determined with Western Blot analysis, as detailed in the Methods section. Representative blots are shown, along with the mean ± SEM of the densitometry analysis in three different experiments. ** p < 0.01, *** p < 0.001 vs. control cells with One-way ANOVA. (C). After 48 h of incubation in the presence of CM_S1, the amount of nitrites in the culture medium was assessed (see Methods). Bars are means ± SEM of three independent experiments.

Figure 2

(A) The amount of the indicated cytokines in CM_S1 was determined as described in the Methods section. (B–E) HUVECs were incubated in RPMI1640 medium, in the absence (control) or in the presence of the indicated cytokines, employed alone or combined (cytomix). Where indicated, 5 nM of Spike S1 was added. The mRNA expression of NOS3 (B) and ARG2 (C) was measured after 24 h of incubation using RT-qPCR. Data are expressed as a fold change in the expression in control cells (=1). Bars are means ± SEM of four experiments, each performed in duplicate. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control, untreated cells with One-way ANOVA. After 48 h, the expression of eNOS (D) and arginase (E) proteins was assessed with Western Blot (see Methods). Representative blots are shown, along with mean ± SEM of the densitometry analysis in three different experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control cells with One-way ANOVA. (F). After 48 h of treatment in the presence of cytomix, the amount of nitrites in the culture medium was assessed (see Methods). Bars are means ± SEM of three independent experiments.

Figure 3

HUVECs were incubated in CM_S1 (A) or in the presence of cytomix (B). After the indicated times, the expression of the indicated proteins was assessed with Western Blot (see Methods). Representative blots are shown, along with mean ± SEM of the densitometry analysis in three different experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control cells with One-way ANOVA.

Figure 4

(A–C) HUVECs were treated as in Figure 1. (A) After 48 h, the intracellular content of arginine was determined as described in the Methods section. Bars are the means ± SEM of four experiments. * p < 0.05 vs. CM_cont with Student’s t test. (B) At the times indicated, arginine uptake was determined by measuring the activity of systems y+ and y+L, as described in the Methods section. Data are the mean ± SEM of three experiments, each performed in quadruplicate. * p < 0.05 vs. CM_cont with Student’s t test. (C) After 24 h, the expression of arginine transporters was measured using RT-qPCR and expressed as a fold change in cells maintained in RPMI1640 medium (control = 1). Bars are means ± SEM of three experiments, each performed in duplicate. *** p < 0.001 vs. control with Student’s t test. (D,E) HUVECs were incubated in RPMI1640 medium, either in the absence (control) or in the presence of the indicated cytokines, employed alone or combined (cytomix). (D) After 48 h, the arginine intracellular content was determined as described in the Methods section. Bars are means ± SEM of four experiments. *** p < 0.001 vs. control cells with One-way ANOVA. (E) SLC7A2/CAT2 mRNA expression was measured after 24 h of incubation by means of RT-qPCR. Data are expressed as a fold change in gene levels in control (=1). Bars are the means ± SEM of four experiments, each performed in duplicate. *** p < 0.001 vs. control, untreated cells with One-way ANOVA.

Figure 5

HUVECs were incubated with CM_cont or CM_S1; the drugs infliximab (IFX) or baricitinib and the antibodies against the indicated cytokines were added to CM_S1, as indicated. After 24 h, the mRNA levels of SLC7A2 (A), NOS3 (B), and ARG2 (C) were measured by means of RT-qPCR and expressed as a fold change in CM_cont (=1). Bars are means ± SEM of three experiments, each performed in duplicate. * p < 0.05 vs. none with One-way ANOVA. After 48 h, the expression of eNOS (D) and arginase (E) proteins were assessed through Western Blot analysis (see Methods). Representative blots are shown, along with the mean ± SEM of the densitometry analysis of three different experiments. ** p < 0.01, *** p < 0.001 vs. none with One-way ANOVA.