Preliminary Evidence for IL-10-Induced ACE2 mRNA Expression in Lung-Derived and Endothelial Cells: Implications for SARS-Cov-2 ARDS Pathogenesis

Abstract

Angiotensin-converting enzyme 2 (ACE2) is a receptor for the spike protein of SARS-COV-2 that allows viral binding and entry and is expressed on the surface of several pulmonary and non-pulmonary cell types, with induction of a "cytokine storm" upon binding. Other cell types present the receptor and can be infected, including cardiac, renal, intestinal, and endothelial cells. High ACE2 levels protect from inflammation. Despite the relevance of ACE2 levels in COVID-19 pathogenesis, experimental studies to comprehensively address the question of ACE2 regulations are still limited. A relevant observation from the clinic is that, besides the pro-inflammatory cytokines, such as IL-6 and IL-1β, the anti-inflammatory cytokine IL-10 is also elevated in worse prognosis patients. This could represent somehow a "danger signal", an alarmin from the host organism, given the immuno-regulatory properties of the cytokine. Here, we investigated whether IL-10 could increase ACE2 expression in the lung-derived Calu-3 cell line. We provided preliminary evidence of ACE2 mRNA increase in cells of lung origin in vitro, following IL-10 treatment. Endothelial cell infection by SARS-COV-2 is associated with vasculitis, thromboembolism, and disseminated intravascular coagulation. We confirmed ACE2 expression enhancement by IL-10 treatment also on endothelial cells. The sartans (olmesartan and losartan) showed non-statistically significant ACE2 modulation in Calu-3 and endothelial cells, as compared to untreated control cells. We observed that the antidiabetic biguanide metformin, a putative anti-inflammatory agent, also upregulates ACE2 expression in Calu-3 and endothelial cells. We hypothesized that IL-10 could be a danger signal, and its elevation could possibly represent a feedback mechanism fighting inflammation. Although further confirmatory studies are required, inducing IL-10 upregulation could be clinically relevant in COVID-19-associated acute respiratory distress syndrome (ARDS) and vasculitis, by reinforcing ACE2 levels.

Keywords: ACE2; ARDS; COVID-19 pro-inflammatory cytokines; IL-10; SARS-COV-2; cytokine storm; metformin; sartans.

Graphical Abstract

ACE2 enzyme is a negative regulator of RAAS providing a crucial link between immunity, inflammation, increased coagulopathy, and cardiovascular disease, thereby serving as a protective mechanism against inflammation, heart failure, myocardial infarction, lung disease, hypertension, vascular permeability, and diabetes. ACE2 function, following SARS-CoV binding, is reduced due to endocytosis and proteolytic cleavage and there are high levels of Ang II in the blood of patients with COVID-19. Consequently, the upregulation of human ACE2 induced by IL-10 in SARS-CoV-2-infected patients could be clinically useful, due to the protection elicited by the increased activity of ACE2 and possibly angiotensin(1–7).

Figure 1

Effects of IL-10 on ACE2 expression in Calu-3 cells by qPCR. (A) The ability of IL-10 (1 to 10 ng/ml) to induce ACE2 expression in Calu-3 cells was determined, following 24 h of stimulation, by qPCR. IL-10 increased ACE2 expression in a dose-dependent manner; N = 5–7. (B) The effects of IL-10 (25 ng/ml) to induce ACE2 expression in Calu-3 cells were compared to the pro-inflammatory cytokines TNFα (50 ng/ml) and IL-1β (25 ng/ml), following 24 h of treatment; N = 5. IL-10 (25 ng/ml) increased ACE2 expression as compared to TNFα (50 ng/ml), IL-1β (25 ng/ml), and control untreated cells. (C) The effects of IL-10 (25 ng/ml) on ACE2 expression in Calu-3 was determined by qPCR, as compared to olmesartan (10 µM) or losartan (10 µM); olmesartan (10 µM) or metformin, (10 mM), by qPCR. N = 5. Non-significant effects was seen with sartans but metformin (10 mM) increased ACE2 expression. Data are shown as mRNA relative expression, normalized to β-actin and control, mean ± SEM, one-way ANOVA, ^** p < 0.01, ^*** p < 0.001, Control: control vehicle cells.

Figure 2

Expression of ACE2 protein in Calu-3 cells. ACE2 expression on Calu-3 cells was evaluated following 24-h exposure to IL-1β (25 ng/ml) and IL-10 (5 and 25 ng/ml) and metformin (10 mM) by Western blot (A). Band intensities were denistometrically quantified (B) and normalized to β-actin and control; N = 4. Data are shown as mean ± SEM. Control, control vehicle cells.

Figure 3

Effects of IL-10, sartans, and metformin on ACE2 expression in HUVECs. The ability of IL-10 (5 and 25 ng/ml) to induce ACE2 expression in HUVECs was determined, following 24 h of stimulation, by qPCR. (A) IL-10 increased ACE2 expression in a dose-dependent manner. (B) The effects of IL-10 (25 ng/ml) on ACE2 expression in HUVECs was determined by qPCR, olmesartan (10 µM), enalapril (10 µM), or metformin (10 mM). Metformin shows a trend in increasing ACE2 expression in HUVECs. Data are shown as mRNA relative expression, normalized to β-actin and control, mean ± SEM, one-way ANOVA, ^* p < 0.05, ^** p < 0.01. Control, control vehicle cells. ns, not significant.

Figure 4

Expression of ACE2 protein in HUVECs. ACE2 expression by HUVECs was evaluated following 24-h exposure to IL-1β (25 ng/ml) or IL-10 (25 ng/ml) (A), IL-10 (5 ng/ml), IL-1β (25 ng/ml) and metformin (10 mM) (B) by Western blot. Eight Western blots were quantified, normalized to β-actin and control, and reported in a histogram (C).