A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury

Abstract

During several months of 2003, a newly identified illness termed severe acute respiratory syndrome (SARS) spread rapidly through the world. A new coronavirus (SARS-CoV) was identified as the SARS pathogen, which triggered severe pneumonia and acute, often lethal, lung failure. Moreover, among infected individuals influenza such as the Spanish flu and the emergence of new respiratory disease viruses have caused high lethality resulting from acute lung failure. In cell lines, angiotensin-converting enzyme 2 (ACE2) has been identified as a potential SARS-CoV receptor. The high lethality of SARS-CoV infections, its enormous economic and social impact, fears of renewed outbreaks as well as the potential misuse of such viruses as biologic weapons make it paramount to understand the pathogenesis of SARS-CoV. Here we provide the first genetic proof that ACE2 is a crucial SARS-CoV receptor in vivo. SARS-CoV infections and the Spike protein of the SARS-CoV reduce ACE2 expression. Notably, injection of SARS-CoV Spike into mice worsens acute lung failure in vivo that can be attenuated by blocking the renin-angiotensin pathway. These results provide a molecular explanation why SARS-CoV infections cause severe and often lethal lung failure and suggest a rational therapy for SARS and possibly other respiratory disease viruses.

Conflict of interest statement

The Institute for Molecular Biotechnology of the Austrian Academy of Sciences has applied for a patent on modulating the renin-angiotension system for treatment of lung edema.

Figures

Figure 1. ACE2 is a crucial receptor for SARS-CoV infections in vivo.

(a,b) SARS-CoV replication (a) and detection of SARS-CoV Spike RNA (b) in wild-type (WT) and Ace2 knockout mice. Viral replication was determined from lung tissue at day 2 of infection. Virus titers (mean log10TCID50 per gram lung tissue) are shown for individual mice. n = 15 per group. SARS-CoV Spike RNA expression was assayed using real-time RT-PCR and normalized to mouse Actb. Data are shown as mean + s.e.m. n = 15 per group. **P < 0.01. (c) Lung histopathology (original magnification ×200) and (d) lung injury scores as defined by leukocyte infiltration of control and SARS-CoV–infected wild-type and Ace2 knockout mice. Lung samples were taken on day 6 after SARS-CoV infection.

Figure 2. Downregulation of ACE2 expression by SARS-CoV infection and SARS-CoV Spike protein.

(a) Schematic diagram of the renin-angiotensin system in acute lung failure and proposed SARS-CoV action. (b) Decreased ACE2 protein, but normal ACE levels, in the lungs of SARS-CoV–infected mice. Lung homogenates were prepared from control and SARS-CoV–infected wild-type or Ace2 knockout (KO) mice on day 2 and analyzed by western blot. (c) Binding of recombinant Spike(S-1190)-Fc protein to human ACE2 (hACE2) and mouse ACE2 (mACE2) in pull-down assays. Spike-Fc but not control-Fc protein pulled down hACE2 and mACE2 from total-cell extracts of A549 human alveolar epithelial cells and IMCD mouse kidney epithelial cells, respectively. Total lysates are shown as controls. (d) Binding of Spike-Fc protein to human and mouse ACE2 in cell culture. 293 cells transfected with hACE2 or mACE2 were incubated with Spike-Fc and the binding was detected by FACS (blue lines). Nontransfected 293 cells incubated with Spike-Fc followed by Fc-specific antibodies are shown as controls (black line). (e) Decreased cell-surface expression of ACE2 after binding to Spike-Fc protein at 37 °C compared to 4 °C in Vero E6 cells. ACE2 surface expression was detected at 3 h of incubation with Spike-Fc using an ACE2-specific monoclonal antibody. Similar data were obtained using Fc-specific antibody to directly detect surface-bound Spike-Fc and to avoid masking of the ACE2 epitope. Representative FACS histograms are shown including a background control with an isotype-matched antibody.

Figure 3. The SARS-CoV Spike protein enhances the severity of acute lung injury.

(a) Lung elastance measurements after saline or acid instillation in Spike-Fc protein–(5.5 nmol/kg) or control-Fc– (5.5 nmol/kg) treated wild-type mice. n = 5–7 per group. *P < 0.05 for the whole time course comparing Spike-Fc–treated and control-Fc–treated wild-type mice after acid injury. (b) Lung histopathology. Representative images are shown. Original magnification, ×200. (c) Lung injury score (Supplementary Table 1 online). **P < 0.01 versus control-Fc–treated wild-type. (d) Wet to dry weight ratios of lungs as readout for pulmonary edema in control and Spike-Fc–treated mice in the presence or absence of acid-induced lung injury. *P < 0.05 between control and Spike-Fc–treated mice with acid challenge. (e) Severe acute lung failure by Spike(S318-510)-Fc (5.5 nmol/kg) treatment in acid-challenged mice. The scheme (upper panel) shows the ACE2-binding domain of Spike (S318-510). Lung elastance measurements (lower panel) showed that Spike(S318-510)-Fc induced severe acute lung failure in acid-challenged wild-type mice, comparable to Spike(S1190)-Fc. n = 5–7 per group. P < 0.05 for the whole time course comparing Spike(S318-510)-Fc or Spike(S1190)-Fc–treated and control-Fc–treated wild-type mice after acid injury. (f) Lung elastance measurements after acid instillation in Spike-Fc protein–(S1190; 5.5 nmol/kg) or control-Fc–(5.5 nmol/kg) treated Ace2 knockout (KO) mice. n = 5–7 for each group.

Figure 4. SARS-CoV Spike mediates lung injury through modulation of the renin-angiotensin system.

(a,b) Localization of intraperitoneally injected Spike(S-1190)-Fc in lung tissue. (a) Spike-Fc was detected by pull-down assay with Protein G Sepharose and western blot with human Fc–specific antibody. Mouse IgG is shown as loading control. (b) Lung immunohistochemistry to detect Spike(S-1190)-Fc or control-Fc protein using a human Fc–specific antibody. Spike(S-1190)-Fc localizes to bronchial epithelial cells (left; original magnification, ×100), inflammatory exudates cells (middle; original magnification, ×200), and alveolar pneumocytes (right; original magnification, ×200). (c) Decreased ACE2 protein expression in the lungs of Spike(S-1190)-Fc– treated mice. Lung homogenates were prepared from control-Fc– and Spike(S-1190)-Fc–treated wild-type mice and analyzed by western blot with ACE2-specific antibody. (d) AngII peptide levels in lungs of Spike(S1190)-Fc protein– or control–Fc-treated wild-type mice after saline or acid aspiration. AngII levels were determined at 3 h by enzyme immunoassay. Bars, mean ± s.e.m. *P < 0.05 comparing Spike(S1190)-Fc– and control-Fc–treated wild-type mice after acid injury. (e) Lung elastance measurements in acid plus Spike(S1190)-Fc–challenged wild-type mice treated with the AT1R inhibitor losartan (15 mg/kg). n = 4–6 per group. P < 0.05 comparing losartan-treated Spike(S1190)-Fc–challenged mice with vehicle-treated Spike(S1190)-Fc–challenged mice. (f) Wet to dry weight ratios of lungs of acid and Spike(S1190)-challenged mice in the presence or absence of losartan (15 mg/kg). n = 4–6 mice per group. *P < 0.05, comparing losartan-treated wild-type with vehicle-treated wild-type mice at 3 h after acid injury.