Angiotensin-converting enzyme 2 (ACE2) mediates influenza H7N9 virus-induced acute lung injury

Abstract

Since March 2013, the emergence of an avian-origin influenza A (H7N9) virus has raised concern in China. Although most infections resulted in respiratory illness, some severe cases resulted in acute respiratory distress syndrome (ARDS), which is a severe form of acute lung injury (ALI) that further contributes to morbidity. To date, no effective drugs that improve the clinical outcome of influenza A (H7N9) virus-infected patients have been identified. Angiotensin-converting enzyme (ACE) and ACE2 are involved in several pathologies such as cardiovascular functions, renal disease, and acute lung injury. In the current study, we report that ACE2 could mediate the severe acute lung injury induced by influenza A (H7N9) virus infection in an experimental mouse model. Moreover, ACE2 deficiency worsened the disease pathogenesis markedly, mainly by targeting the angiotensin II type 1 receptor (AT1). The current findings demonstrate that ACE2 plays a critical role in influenza A (H7N9) virus-induced acute lung injury, and suggest that might be a useful potential therapeutic target for future influenza A (H7N9) outbreaks.

Figure 1. ACE2 plays a critical role in 2013 influenza H7N9 virus-induced ALI.

(A) Downregulated ACE2 expression in the lungs of Hb01/H7N9 virus-infected mice. Lung tissue homogenates prepared from control and Hb01/H7N9 virus-infected WT mice on day 3 were analyzed by western blotting. (B) Plasma levels of Ang II in control and Hb01/H7N9 virus-infected ACE2 KO or WT mice on day 3 (n = 8). (C) Ang II levels in the lungs of control and Hb01/H7N9 virus-infected WT mice on day 3 were measured using enzyme immunoassays (n = 8). (D) Plasma levels of Ang II in healthy and H7N9 virus-infected patients were measured using enzyme immunoassays. *, p < 0.05; ** p < 0.01 between groups.

Figure 2. Loss of ACE2 expression worsens H7N9-induced ALI.

(A) The survival rates of WT and ACE2 KO mice (n = 10). (B) H&E staining and infiltrating cell counts (n = 200 fields) in the lung tissues of WT B6 and ACE2 KO mice (n = 5) at day 5 post-infection. (C) The wet-to-dry ratio of lungs from WT B6 and ACE2 KO mice (n = 8) at day 5 post-infection. (D) Detection of Hb01/H7N9 virus NP RNA from WT B6 and ACE2 KO mice (n = 8) at day 5 post-infection. (E) Lung viral titers in Hb01/H7N9 virus-infected WT B6 and ACE2 KO mice (n = 8) at day 5 post-infection.

Figure 3. The Ang II receptor AT1 regulates H7N9-induced lung injury.

(A) The wet-to-dry ratio of the lungs of WT mice treated with control or AT1 inhibitor (losartan, 15 mg/kg) 30 min before Hb01/H7N9 virus infection (n = 8). (B) H&E staining and infiltrating cell counts (n = 200 fields) in the lung tissue of Hb01/H7N9 influenza virus-infected B6 mice treated with PBS control or AT1 inhibitor (losartan, 15 mg/kg) at day 5 post-infection. (C) Detection of Hb01/H7N9 virus NP RNA in WT mice treated with PBS control or AT1 inhibitor (losartan, 15 mg/kg) 30 min before Hb01/H7N9 virus infection (n = 8) at day 5 post-infection. NP mRNA expression was quantified using real-time PCR, and was normalized to GAPDH (n = 10). (D) Lung viral titers in WT mice treated with PBS control or AT1 inhibitor (losartan, 15 mg/kg) before Hb01/H7N9 virus infection (n = 8) at day 5 post-infection. **p < 0.01(two-tailed t-test).

Figure 4. Inhibiting AT1 attenuates H7N9-induced lung injury in ACE2 KO mice.

(A) The wet-to-dry ratio of the lungs of ACE2 KO mice treated with PBS control or AT1 inhibitor (losartan, 15 mg/kg) 30 min before Hb01/H7N9 virus infection (n = 8). (B) H&E staining and infiltrating cell counts (n = 200 fields) in the lung tissue of Hb01/H7N9 virus-infected ACE2 KO mice treated with PBS control or AT1 inhibitor (losartan, 15 mg/kg) at day 5 post-infection. **p < 0.01 (two-tailed t-test). (C) Detection of Hb01/H7N9 virus NP RNA in ACE2 KO mice treated with PBS control or AT1 inhibitors at day 5 post-infection. NP mRNA expression was assessed using real-time PCR, and was normalized to GAPDH (n = 10). (D) Lung viral titers in ACE2 KO mice treated with PBS control or AT1 inhibitor at day 5 post-infection. **p < 0.01 (two-tailed t-test). (E) Schematic diagram of the role of the renin-angiotensin system in ALI and influenza A H7N9 virus infection.