Angiotensin-Converting Enzyme Inhibition and/or Angiotensin Receptor Blockade Modulate Cytokine Profiles and Improve Clinical Outcomes in Experimental COVID-19 Infection

Abstract

The regulation of angiotensin-converting enzyme 2 (ACE2) expression by medications such as ACE inhibitors (ACEis) and angiotensin receptor blockers (ARBs) has raised critical questions regarding their potential benefits and risks during COVID-19. ACE2, a regulator of blood pressure through the renin-angiotensin system (RAS), is the primary receptor for SARS-CoV-2. ACEis and ARBs can modulate ACE2 expression, potentially exacerbating viral load. However, the risks of higher viral load could be mitigated by favorable anti-inflammatory responses associated with ACEi and ARB use, highlighting the complexity of their impact on viral replication and disease outcomes. This study investigates the effects of sustained Losartan monotherapy (ARB) and combination Losartan + Lisinopril (ARB + ACEi) on viral replication, inflammation, lung function, and clinical measures of disease severity in a murine model of severe COVID-19 involving humanized ACE2 transgenic mice infected with SARS-CoV-2 Wuhan strain. Both ARB and ARB + ACEi treatments led to increased ACE2 expression in the lungs and higher viral load post-infection. Despite this, the ARB + ACEi combination improved clinical scores, reduced weight loss and inflammatory cytokine levels, and preserved lung function, though it did not improve survival. Overall, the results of these controlled experiments provide insight into the complex dynamics of ACEi and ARB use in COVID-19; while these drugs induce expression of the ACE2 receptor and increase viral load, they provide compensatory modulation of the inflammatory response that appears to diminish severity of the infection.

Keywords: COVID-19; K18-hACE2 mice; Losartan; SARS-CoV-2; lisinopril.

Figure 1

(A) Analysis of weight loss (%) and (B) clinical score (daily sum) of uninfected control and SARS-CoV-2-infected K18-hACE2 mice treated with Losartan or Losartan + Lisinopril combination. UU = Untreated Uninfected, CTI = Combination-treated Infected, LTI = Losartan-treated Infected, and UI = Untreated Infected. Signs indicate significant differences: (#) all groups versus UU group; (&) UI versus UU group; (*) UI versus CTI or LTI: * p < 0.05, ** p < 0.005, *** p < 0.0005 and **** p < 0.0001. Mice in the UU group showed clinical scores of zero at all times. Data presented as mean ± standard error of the mean (SEM). Analysis performed by mixed-effects model with Geisser–Greenhouse correction, followed by Tukey’s post hoc test. n = 30 per group.

Figure 2

Assessment of lung capacity through the parameters of respiratory frequency (A), Rpef (B), minute volume (Mv) (C), and Pause (D) in uninfected control or SARS-CoV-2 infected K18-hACE2 mice treated or not with Losartan or Losartan + Lisinopril combination at 6/7 days post-infection. UU = Untreated Uninfected, CTI = Combination-treated Infected, LTI = Losartan-treated Infected, and UI = Untreated Infected. The normality of the data was assessed using the Shapiro–Wilk test. Data of (A–C) showed normal distribution and were analyzed using a parametric test, one-way ANOVA followed by the Bonferroni post-test. Data of (D) did not show normal distribution, and a non-parametric analysis was performed, with the Kruskal–Wallis test followed by the Dunn post-test. (#) Significant difference, all groups versus the UU group. (*) Significant difference between specific groups as indicated by the lines: * p < 0.05, ** p < 0.005, *** p < 0.0005 and **** p < 0.0001. n = 10 per group.

Figure 3

(A) Quantification of ACE2, on lungs, by ELISA in uninfected mice treated or not with Losartan or Losartan + Lisinopril combination over 21 days (black and white columns; UU: uninfected untreated; CTU: combination-treated, uninfected; LTU: Losartan-treated, uninfected) and in the same animals after infection with SARS-CoV-2 (colored columns; CTI [gray]: combination-treated, infected; LTI [green]: Losartan-treated, infected; UI [red]: untreated, infected). (B) Viral load in SARS-CoV-2-infected K18-hACE2 mice treated or not with Losartan or Losartan-Lisinopril combination on days 3, 5, or 6/7 post-infection. (*) Significant difference between groups: * p < 0.05, ** p < 0.005, **** p < 0.0001. The normality of the data was assessed using the Shapiro–Wilk test. A one-way ANOVA was performed, followed by the Bonferroni post-test (A-Treated non-infected), and mixed-effects analyses were conducted, followed by the Tukey post-test (A-Treated infected/B). (*) Significant difference between specific groups as indicated by the lines: * p < 0.05, ** p < 0.005, **** p < 0.0001. Data are presented as mean ± SEM. n = 10 per time point.

Figure 4

Analysis of IL-6 and TNF-α in serum (A,B) and lung (C,D) of SARS-CoV-2-infected K18-hACE2 mice treated or not with Losartan or Losartan + Lisinopril combination at 6/7 dpi. CTI = Combination-treated Infected, LTI = Losartan-treated Infected, and UI = Untreated Infected. (*) Significant difference: * p < 0.05, ** p < 0.005 and *** p < 0.0005. Analysis was performed using ANOVA, followed by Bonferroni’s multiple comparisons test. Data presented as mean ± SEM. n = 10 mice per group.

Figure 5

Histopathological analysis of lungs at days 6/7 post-infection of SARS-CoV-2-infected K18-hACE2 mice treated of not with Losartan or Losartan-Lisinopril combination. CTI = Combination-treated Infected, LTI = Losartan-treated Infected, and UI = Untreated Infected. (A) Results obtained from the sum of the degrees of intensity (maximum score of 44), *** p < 0.0005. Data are presented as individual scores and mean ± SD. The normality was assessed using the Shapiro–Wilk test. The Kruskal–Wallis test was used, followed by the Dunn post-test. (B–D) Representative histopathological images, emphasizing inflammatory infiltrate and thickness: (B) CTI-mice (score 1), (C) LTI-mice (score 1), and (D) UI-mice (score 4). (E) Histopathological findings, data presented as mean. n = 10 animals per group. Bar = 50 µm.

Figure 6

(A) Survival curve of uninfected control (black line, n = 10) and SARS-CoV-2-infected K18-hACE2 mice untreated (red line, n = 20) or treated with Losartan (green line, n = 18) or Losartan-Lisinopril combination (gray line, n = 18). Untreated Uninfected did not show mortality. No significant differences in survival were observed between infected groups. The survival log-rank (Mantel–Cox) test was applied. (B) Analysis of weight loss of uninfected controls (black columns) or survivor SARS-CoV-2-infected K18-hACE2 mice untreated (red columns) or treated with Losartan (green columns) or Losartan-lisinopril (gray columns) at day 0 (pre-infection) and 15 days post-infection (dpi). (C) clinical scores of the experimental groups at 15 dpi. Normality was assessed using the Shapiro–Wilk test. Analysis was performed using ANOVA, followed by Bonferroni’s multiple comparisons test. (*) Significant differences: ** p < 0.005, *** p < 0.0005 and **** p < 0.0001.