Expression of SARS-CoV-2 Entry Factors in Human Alveolar Type II Cells in Aging and Emphysema

Abstract

Alveolar type II (ATII) cells proliferate and restore the injured epithelium. It has been described that SARS-CoV-2 infection causes diffuse alveolar damage in the lungs. However, host factors facilitating virus infection in ATII cells are not well known. We determined the SARS-CoV-2-related genes and protein expression using RT-PCR and Western blotting, respectively, in ATII cells isolated from young and elderly non-smokers, smokers, and ex-smokers. Cells were also obtained from lung transplants of emphysema patients. ACE2 has been identified as the receptor for SARS-CoV-2, and we found significantly increased levels in young and elderly smokers and emphysema patients. The viral entry depends on TMPRSS2 protease activity, and a higher expression was detected in elderly smokers and ex-smokers and emphysema patients. Both ACE2 and TMPRSS2 mRNA levels were higher in this disease in comparison with non-smokers. CD209L serves as a receptor for SARS-CoV-2, and we found increased levels in ATII cells obtained from smokers and in emphysema patients. Also, our data suggest CD209L regulation by miR142. Endoplasmic reticulum stress was detected in ATII cells in this disease. Our results suggest that upregulation of SARS-CoV-2 entry factors in ATII cells in aging, smokers, and emphysema patients may facilitate infection.

Keywords: aging; alveolar type II cells; emphysema; smoking.

Figure 1

ACE2 and TMRPSS2 expression in ATII cells in young and elderly non-smokers, smokers, and ex-smokers. Freshly isolated ATII cells from lungs obtained from young non-smokers (yNS), young smokers (ySM), elderly non-smokers (eNS), elderly smokers (eSM), and elderly ex-smokers (e-exSM) were used to determine protein and mRNA expression. (A) Representative cytospins of freshly isolated ATII cells using SP-C and DAPI by immunofluorescence (n = 3), scale bar, 50 µm. (B) Western blot images of ACE2 and TMRPSS2 expression (n = 5–14 per group). (C) Quantification of protein expression normalized to GAPDH. (D) ACE2 and TMPRSS2 mRNA levels (n = 5–14 per group). * p < 0.05. Data are shown as means ± SEM.

Figure 2

High ACE2 and TMRPSS2 expression in ATII cells in emphysema. ATII cells were isolated from non-smokers (NS), smokers (SM), and emphysema patients (EM). (A) Representative images of ACE2 and TMRPSS2 expression using Western blotting (n = 8–20 per group). (B) Densitometric quantification of protein expression normalized to GAPDH. (C) ACE2 and TMPRSS2 mRNA levels (n = 8–23). * p < 0.05. Data are shown as means ± SEM.

Figure 3

The regulation of CD209L expression by miR142 in A549 cells. (A) Predicted homology between CD209L 3′UTR (top) and miR142 (bottom). (B) The miR142 gene was overexpressed in A549 cells, and its levels were confirmed by RT-PCR and normalized to the U6 levels. (C) CD209L mRNA expression in A549 cells with miR142 overexpression by RT-PCR. (D) CD209L protein levels in A549 cells with miR142 overexpression by Western blotting. Quantification is also shown (n = 3). * p < 0.05, ** p < 0.01. Data are shown as means ± SEM.

Figure 4

A relationship between miR142 and CD209L in ATII cells. Panel I—ATII cells were isolated from young non-smokers (yNS) and young smokers (ySM). (A) CD209L levels determined by RT-PCR (n = 5-6 per group). (B) miR142 expression (n = 5–7 per group). (C) CD209L protein expression by Western blotting (n = 6–8 per group). (D) Quantification of CD209L protein expression normalized to GAPDH. Panel II—ATII cells were obtained from elderly non-smokers (eNS), elderly smokers (eSM), and elderly ex-smokers (e-exSM). (A) CD209L mRNA expression (n = 5–8 per group). (B) miR142 levels were analyzed by RT-PCR (n = 5–9 per group). (C) CD209L protein levels were determined by Western blotting (n = 5–8 per group). (D) Densitometric quantification of CD209L protein expression normalized to GAPDH. Panel III—ATII cells were isolated from non-smokers (NS), smokers (SM), and emphysema patients (EM). (A) CD209L expression was analyzed by RT-PCR (n = 6–14 per group). (B) miR142 levels (n = 6–15 per group). (C) Representative images of CD209L expression using Western blotting. (D) Quantification of protein expression normalized to GAPDH. * p < 0.05, ** p < 0.001, *** p < 0.0001. Data are shown as means ± SEM.

Figure 5

GRP78 and HERPUD1 levels in ATII cells in young and elderly non-smokers, smokers, and ex-smokers. ATII cells were obtained from young non-smokers (yNS), young smokers (ySM), elderly non-smokers (eNS), elderly smokers (eSM), and elderly ex-smokers (e-exSM). (A) GRP78 and HERPUD1 expression by Western blotting (n = 5–12 per group). (B) Densitometric quantification of protein levels normalized to GAPDH. (C) GRP78 and HERPUD1 mRNA levels were determined by RT-PCR (n = 5–14 per group). *p < 0.05. Data are shown as means ± SEM.

Figure 6

Expression of GRP78 and HERPUD1 in ATII cells isolated from non-smokers, smokers, and emphysema patients. Lungs from non-smokers (NS), smokers (SM), and emphysema patients (EM) were used to isolate ATII cells. (A) GRP78 and HERPUD1 protein expression (n = 8–23 per group). (B) Densitometric quantification of GRP78 and HERPUD1 levels normalized to GAPDH. (C) GRP78 and HERPUD1 mRNA levels (n = 6-14 per group). * p < 0.05, ** p < 0.001. Data are shown as means ± SEM.

Figure 7

Expression of CD147 in ATII cells isolated from young and elderly non-smokers, smokers, and ex-smokers. ATII cells were isolated from young non-smokers (yNS), young smokers (ySM), elderly non-smokers (eNS), elderly smokers (eSM), and elderly ex-smokers (e-exSM). (A) Representative CD147 expression determined by Western blotting (n = 4–6 per group). (B) Quantification of CD147 expression normalized to GAPDH. (C) CD147 mRNA levels by RT-PCR (n = 6–8 per group). * p < 0.05. Data are shown as means ± SEM.