Angiotensin-converting enzyme 2 inhibits lung injury induced by respiratory syncytial virus

Abstract

Respiratory syncytial virus (RSV) infection is a major cause of severe lower respiratory illness in infants and young children, but the underlying mechanisms responsible for viral pathogenesis have not been fully elucidated. To date, no drugs or vaccines have been employed to improve clinical outcomes for RSV-infected patients. In this paper, we report that angiotensin-converting enzyme-2 (ACE2) protected against severe lung injury induced by RSV infection in an experimental mouse model and in pediatric patients. Moreover, ACE2 deficiency aggravated RSV-associated disease pathogenesis, mainly by its action on the angiotensin II type 1 receptor (AT1R). Furthermore, administration of a recombinant ACE2 protein alleviated the severity of RSV-induced lung injury. These findings demonstrate that ACE2 plays a critical role in preventing RSV-induced lung injury, and suggest that ACE2 is a promising potential therapeutic target in the management of RSV-induced lung disease.

Figure 1. Angiotensin-converting enzyme-2 (ACE2) plays a critical role in respiratory syncytial virus (RSV)-induced lung injury.

(a) Plasma angiotensin II (Ang II) levels in healthy children and RSV-infected patients were measured using enzyme immunoassays. (b) Plasma Ang II levels in control, wild-type (WT) RSV virus (RSV BJ016) and A2 virus-infected mice on day 3 (n = 8 per group). (c) Ang II levels in the lungs of control, RSV BJ016 and A2 virus-infected mice on day 3 were examined using enzyme immunoassays (n = 8 per group). (d) ACE2 expression in lung homogenates of control, RSV BJ016 and A2 virus-infected mice were detected by Western blotting. Mice were sacrificed on day 3 post infection.*p < 0.05; **p < 0.01 (two-tailed t-test).

Figure 2. Loss of ACE2 increases severity of RSV BJ016 virus-induced lung injury.

(a) Survival rates of WT and ACE2 knockout (KO) mice (n = 10 per group). (b) Body weight changes of WT and ACE2 KO mice (n = 10 per group). (c) Hematoxylin and eosin (H&E) staining and infiltrating cell counts (n = 200 fields) in lung sections of WT C57BL/6 (B6) and ACE2 KO mice (n = 5) at 5 days post injection (DPI). (d) Lung wet-to-dry weight ratios from WT B6 and ACE2 KO mice (n = 8) at 5 DPI. (e) Lung viral titers in RSV BJ016 virus-infected WT B6 and ACE2 KO mice (n = 8) at 5 DPI. (f) Detection of plasma Ang II in WT B6 and ACE2 KO mice (n = 8) at 5 DPI.

Figure 3. Recombinant hACE2 protects against lung injury induced by RSV BJ016 viral infection in mouse models.

(a) Body weight changes of RSV BJ016 virus-infected WT mice. Mice were treated intravenously with 0.1 mg/kg soluble recombinant hACE2 protein or control, 1 day before infection, 1 DPI and 3 DPI (n = 10/treatment group). (b) H&E staining and infiltrating cell counts (n = 200 fields) in lung sections of RSV BJ016 virus-infected mice at 5 DPI (n = 3). (c) Lung wet-to-dry weight ratios in mice infected with RSV BJ016 virus at 5 DPI (n = 5). (d) Lung viral titers in mice infected with RSV BJ016 virus at 5 DPI (n = 5). (e) Plasma levels of Ang II in RSV BJ016 virus-infected mice treated with hACE2 proteins (n = 6). All experiments were performed in triplicate at least.

Figure 4. Ang II receptor angiotensin II type 1 receptor (AT1R) regulates RSV BJ016 virus-induced lung injury.

(a) H&E staining and infiltrating cell counts (n = 200 fields) in lung sections of RSV BJ016 virus-infected B6 mice treated with PBS control or AT1R inhibitor (Losartan, 15 mg/kg) at 5DPI. (b) Lung wet-to-dry weight ratio of WT mice treated with vehicle control or AT1R inhibitor (losartan, 15 mg/kg) 30 min before RSV BJ016 viral infection (n = 8). (c) Lung viral titers in WT mice treated with PBS control or AT1R inhibitor (losartan, 15 mg/kg) before RSV BJ016 viral infection (n = 8) at 5 DPI. (d) Detection of plasma levels of Ang II in WT mice treated with PBS control or AT1R inhibitor (Losartan, 15 mg/kg) 30 min before RSV BJ016 viral infection (n = 8), and at 5 DPI (n = 8). *p < 0.05; **p < 0.01 (two-tailed t-test).

Figure 5. Inhibition of AT1R attenuates RSV BJ016 virus-induced lung injury in ACE2 knockout mice.

(a) H&E staining and infiltrating cell counts (n = 200 fields) in lung sections of RSV BJ016 virus-infected ACE2 KO mice treated with PBS control or AT1R inhibitor (losartan, 15 mg/kg) at 5 DPI. (b) Lung wet-to-dry weight ratios of ACE2 KO mice treated with PBS control or AT1R inhibitor (Losartan, 15 mg/kg) 30 min before RSV BJ016 viral infection (n = 8). (c) Lung viral titers in ACE2 KO mice treated with PBS control or AT1R inhibitor at 5 DPI. *p < 0.05; **p < 0.01 (two-tailed t-test). (d) Schematic diagram of the role of the renin-angiotensin system in lung injury and RSV infection.