ACE2 protein expression in lung tissues of severe COVID-19 infection

Abstract

Angiotensin-converting enzyme 2 (ACE2) is a key host protein by which severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) enters and multiplies within cells. The level of ACE2 expression in the lung is hypothesised to correlate with an increased risk of severe infection and complications in COrona VIrus Disease 2019 (COVID-19). To test this hypothesis, we compared the protein expression status of ACE2 by immunohistochemistry (IHC) in post-mortem lung samples of patients who died of severe COVID-19 and lung samples obtained from non-COVID-19 patients for other indications. IHC for CD61 and CD163 was performed for the assessment of platelet-rich microthrombi and macrophages, respectively. IHC for SARS-CoV-2 viral antigen was also performed. In a total of 55, 44 COVID-19 post-mortem lung samples were tested for ACE2, 36 for CD163, and 26 for CD61, compared to 15 non-covid 19 control lung sections. Quantification of immunostaining, random sampling, and correlation analysis were used to substantiate the morphologic findings. Our results show that ACE2 protein expression was significantly higher in COVID-19 post-mortem lung tissues than in controls, regardless of sample size. Histomorphology in COVID-19 lungs showed diffuse alveolar damage (DAD), acute bronchopneumonia, and acute lung injury with SARS-CoV-2 viral protein detected in a subset of cases. ACE2 expression levels were positively correlated with increased expression levels of CD61 and CD163. In conclusion, our results show significantly higher ACE2 protein expression in severe COVID-19 disease, correlating with increased macrophage infiltration and microthrombi, suggesting a pathobiological role in disease severity.

Figure 1

Haematoxylin and eosin-stained sections from representative areas of lung parenchyma infected with COVID-19. The microphotograph shows (A) diffuse alveolar damage with the hyaline membrane (exudative phase) (× 200). (B) Diffuse alveolar damage with organization (organizing phase) (× 200). (C) Organizing pneumonia with interalveolar capillaries filled with microthrombi (× 200). (D) Alveolar spaces are filled with neutrophilic infiltrate in a case of organizing DAD (not shown here) indicate superimposed acute bronchopneumonia (× 100). (E, F) Hyperplastic pneumocytes (E, arrow) and megakaryocytes (F, arrow) are seen in a case of DAD with acute bronchopneumonia (× 400). (G) Representative image of SARS-CoV2-stained tissue from COVID-19 patient. SARS-COV2 IHC shows cytoplasmic granular positivity in pneumocytes.

Figure 2

Landscape of ACE2. Large patches of ACE2 positive staining in control tissue of (A) testis, (B) kidney, and (C) in adrenal. Representative images of (D) control and (E) COVID-19 infected lung sections showing increased ACE2 staining in COVID-19 cases (brown colour, arrows, and insets, D and E). (F) Quantitative analysis of ACE2 IHC intensity in various control tissues. The difference in the mean intensities was calculated using Wilcox test. Significance denoted by exact P values (n = 13 random images from 15 control lung tissue section, n = 11 images from 2 control testis tissue section, n = 9 images from 2 control kidney tissue section, n = 15 images from 2 control adrenal tissue section). (G, H) Quantitative analysis of ACE2 IHC intensity in control and COVID-19 lung section (G) without sampling analysis (n = 45 images from 15 control lung tissue section and n = 175 images from 44 COVID-19 lung tissue section) and (H) with random sampling analysis (n = 45 images in each group). The difference in the mean intensities was calculated using the Wilcox test, and the exact P value denoted significance.

Figure 3

The landscape of CD163 and CD61. Representative images of (A) control and (B) COVID-19 infected lung sections showing increased CD163 staining in COVID-19 cases, with (C) mean Intensities of CD163 in control and COVID-19 samples. The difference in the mean intensities was calculated using Wilcox test Significance denoted by exact P values (n = 21 images from 6 control lung tissue sections and n = 157 images from 36 COVID-19 lung tissue sections). Detection of CD61 protein in representative sections of (D) control and (E) COVID-19 lung. (F) Mean intensity levels of CD61 staining in control and COVID-19 lungs. (n = 20 images from 6 control lung tissue section and n = 94 images from 26 COVID-19 lung tissue section). (G, H) Intensities of CD163 (n = 21 images/group) and CD61 (n = 20 images/group) protein in control and COVID-19 lung section after random sampling analysis. The difference in the mean intensities was calculated using the Wilcox test. Significance denoted by exact P values. (I, J) Correlation between ACE2-CD163 and ACE2-CD61 has been shown. A regression line was calculated using by fitting a linear model to the data. Label at the top shows correlation value (R) and P value for the linear fit.