Selective hACE2 expression in lung epithelial cells confers nonlethal pneumonia after SARS-CoV-2 infection.

(A) Generation of LSL-hACE2 mice using gene targeting of the mouse Rosa26 locus. WPRE, SV40, PGK-NeoR. (B) Immunoblotting of whole lung lysates from WT, LSL-hACE2+/0, and ShhCre/+;LSL-hACE2+/0 mice was performed using anti-hACE2 and anti-β-actin antibodies. Each lane represents a single animal. Representative of n = 3 per genotype and two independent experiments. (C) Immunohistochemistry of lung from LSL-hACE2+/0 and ShhCre/+;LSL-hACE2+/0 mice was performed using anti-panACE2 and anti-hACE2 antibodies. Representative of N = 3 per genotype and two independent experiments. (D) Immunohistochemistry of lung from LSL-hACE2+/0 and ShhCre/+;LSL-hACE2+/0 mice was performed using antibodies to detect SARS-CoV-2 nucleocapsid, AT1 cell PDPN, and AT2 cell DC-LAMP 2 days after infection with 104 PFU of SARS-CoV-2 virus. Representative of n = 4 per genotype. (E) Immunohistochemistry of lung from LSL-hACE2+/0 and ShhCre/+;LSL-hACE2+/0 mice was performed using antibodies to detect SARS-CoV-2 nucleocapsid, AT1 cell PDPN, and AT2 cell DC-LAMP 6 days after infection with 104 PFU of SARS-CoV-2 virus. Representative of n = 4 per genotype. (F) Immunohistochemistry of cerebral cortex from LSL-hACE2+/0 and ShhCre/+;LSL-hACE2+/0 mice was performed using antibodies to detect SARS-CoV-2 nucleocapsid, neuronal NeuN, and glial cell GFAP 6 days after infection with 104 PFU of SARS-CoV-2 virus. Representative of n = 4 per genotype. (G) HE staining of LSL-hACE2+/0 and ShhCre/+;LSL-hACE2+/0 lung tissue 2 days after exposure to 104 PFU of SARS-CoV-2 virus. Arrows, sites of inflammatory cell infiltrate. Hashtag, acute emphysematous changes. Representative of n = 4 animals per genotype. (H) HE staining of LSL-hACE2+/0 and ShhCre/+;LSL-hACE2+/0 lung tissue 6 days after exposure to 104 PFU of SARS-CoV-2 virus. Arrows, sites of alveolar inflammatory infiltrate and hyalinosis. Representative of n = 4 animals per genotype. (I) Pulse oximetry measured in LSL-hACE2+/0 and ShhCre/+;LSL-hACE2+/0 mice 6, 10, and 14 days after exposure to 105 PFU of SARS-CoV-2 virus. (J, K) Weight loss and survival of LSL-hACE2+/0 and ShhCre/+;LSL-hACE2+/0 mice after infection with 105 PFU of SARS-CoV-2. N = 6 (LSL-hACE2+/0) and 7 (ShhCre/+;LSL-hACE2+/0 mice), two independent experiments. Scale bars in all images 50 μm. Note: Images in each panel were taken at lower and/or higher magnification from the same tissue section respective to genotype and highlight different pathology. ns, not significant, p > 0.05 **, p t test, one-way ANOVA with Holm–Sidak correction for multiple comparisons, or log-rank Mantel Cox test. Numerical data in corresponding S1 Metadata tab. AT1, alveolar type 1; AT2, alveolar type 2; hACE2, human ACE2; HE, hematoxylin–eosin; PDPN, Podoplanin; PFU, plaque-forming unit; PGK-NeoR, Phosphoglycerate kinase promoter-driven Neomycin resistance cassette; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; SV40, simian virus 40; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; WT, wild-type.