ACE2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia

Abstract

Studies of patients with severe acute respiratory syndrome (SARS) demonstrate that the respiratory tract is a major site of SARS-coronavirus (CoV) infection and disease morbidity. We studied host-pathogen interactions using native lung tissue and a model of well-differentiated cultures of primary human airway epithelia. Angiotensin converting enzyme 2 (ACE2), the receptor for both the SARS-CoV and the related human respiratory coronavirus NL63, was expressed in human airway epithelia as well as lung parenchyma. As assessed by immunofluorescence staining and membrane biotinylation, ACE2 protein was more abundantly expressed on the apical than the basolateral surface of polarized airway epithelia. Interestingly, ACE2 expression positively correlated with the differentiation state of epithelia. Undifferentiated cells expressing little ACE2 were poorly infected with SARS-CoV, while well-differentiated cells expressing more ACE2 were readily infected. Expression of ACE2 in poorly differentiated epithelia facilitated SARS spike (S) protein-pseudotyped virus entry. Consistent with the expression pattern of ACE2, the entry of SARS-CoV or a lentivirus pseudotyped with SARS-CoV S protein in differentiated epithelia was more efficient when applied to the apical surface. Furthermore, SARS-CoV replicated in polarized epithelia and preferentially exited via the apical surface. The results indicate that infection of human airway epithelia by SARS coronavirus correlates with the state of cell differentiation and ACE2 expression and localization. These findings have implications for understanding disease pathogenesis associated with SARS-CoV and NL63 infections.

FIG. 1.

ACE2 is expressed on human airway epithelia. (A) ACE2 protein levels were determined using immunoblot analysis of extracts from human airway and alveolar tissue. The control is recombinant ACE2 released into the supernatant from A549 cells infected with an adenoviral vector expressing human ACE2. The positions of ACE2 and α-tubulin are indicated by arrows. MW, molecular weight in thousands. (B) ACE2 protein location in polarized human airway epithelia was determined using immunofluorescence staining for ACE2 (green), β-tubulin IV (red), and nuclear DAPI (blue). Confocal fluorescence photomicroscopic images are presented en face (top) and from vertical sections (bottom). Colocalization is shown by yellow in the merged images. Bar, 10 μm. (C) ACE2 protein location in polarized human airway epithelia was determined by selective apical or basolateral biotinylation, immunoprecipitation of biotinylated surface proteins, and immunoblot analysis for ACE2 or control basolateral erbB2. MW, molecular weight in thousands. The positions of ACE2 and erbB2 are indicated by arrows.

FIG. 2.

ACE2 expression is associated with airway epithelial cell differentiation. (A) Ciliated epithelial cell differentiation in cultures of primary airway epithelial cells under air-liquid interface or resubmerged conditions was verified by SEM of the apical epithelial surface. Bar, 10 μm. (B) ACE2 mRNA levels were determined using real-time RT-PCR analysis of samples from A549 cells or primary hTBE cells cultured under undifferentiating submerged (Sub), differentiating ALI, or resubmerged (Resub) conditions. Values are expressed as mean mRNA levels relative to control HPRT mRNA levels plus or minus standard deviations (SD) (n = 3), and the asterisk indicates a significant difference in mRNA levels between air-liquid interface and resubmerged conditions. (C) ACE2 protein levels were determined using immunoblot analysis of extracts from A549 or primary hTBE cells. The positions of ACE2, foxj1 (to verify epithelial cell differentiation status), and β-actin are indicated by arrows. MW, molecular weight in thousands. (D) ACE2 protein levels were determined using immunoblot analysis of extracts from hTBE cells under submerged conditions that were infected with a recombinant adenoviral vector that expressed ACE2, control transgene (β-galactosidase), or foxj1. MW, molecular weight in thousands.

FIG. 3.

SARS-CoV S protein-pseudotyped FIV infects differentiated airway epithelia best from the apical surface. (A) β-galactosidase levels were determined by enzyme activity in 293 cells transfected with a plasmid expressing control (Ctl) transgene or human ACE2 and then infected with SARS-S protein-pseudotyped FIV expressing β-galactosidase. ND, not detected. (B) β-galactosidase levels were determined in A549 cells (black bars) or primary hTBE cultured under submerged conditions (white bars) that were first infected with an adenoviral vector expressing ACE2 at the indicated MOI and then infected with SARS-S protein-pseudotyped FIV expressing β-galactosidase. (C) β-Galactosidase levels determined in extracts from A549 cells or primary hTBE cultured under submerged or ALI conditions that were infected from the apical surface with SARS-S protein-pseudotyped FIV expressing β-galactosidase. (D) β-galactosidase levels determined in primary hTBE cultured under ALI conditions that were infected from the apical or basolateral surface with vesicular stomatitis virus-G or SARS-S protein-pseudotyped FIV. In panels A through D, values are expressed as means plus or minus SD (n = 4 to 6) and a significant difference from levels on uninfected cells (A and B), hTBE cells cultured under submerged conditions (C), or cells infected from the apical surface (D) is indicated by an asterisk. RLU, relative light units.

FIG. 4.

Infection of differentiated airway epithelia by SARS-CoV. (A) SARS-CoV N and S gene mRNA levels were determined using real-time RT-PCR analysis of A549 cells or primary hTBE cultured under submerged or ALI conditions and infected with SARS-CoV from the apical surface at an MOI of 0.8 for 24 h. Values are expressed as mean mRNA levels relative to control HPRT mRNA levels plus or minus SD (n = 2). ND, not detected. An asterisk indicates a significant difference in mRNA levels between submerged and ALI conditions. (B) PCR products in panel A for hTBE cells cultured under ALI conditions were visualized by ethidium bromide. bp, base pairs. (C) SARS-CoV nsp1 replicase protein location in polarized human airway epithelia that were left uninfected or infected from the apical or basolateral side with SARS-CoV. Twenty-four hours following infection with SARS-CoV viral replication, complexes were localized using immunofluorescence staining for nsp1 (green) and nuclear To-pro-3 (red). Bar, 50 μm. (D) Colocalization of SARS-CoV nsp1 protein and cilia in polarized human airway epithelia. Airway epithelia were infected as described in the legend for Fig. 4C and then fixed and immunostained for nsp1 (green) or β-tubulin IV (red) as a marker for ciliated cells. The merged image shows colocalization of nsp1 and β-tubulin, indicating that the predominant infected cell types were ciliated epithelia. Bar, 10 μm.