CYP19A1 mediates severe SARS-CoV-2 disease outcome in males

Abstract

Male sex represents one of the major risk factors for severe COVID-19 outcome. However, underlying mechanisms that mediate sex-dependent disease outcome are as yet unknown. Here, we identify the CYP19A1 gene encoding for the testosterone-to-estradiol metabolizing enzyme CYP19A1 (also known as aromatase) as a host factor that contributes to worsened disease outcome in SARS-CoV-2-infected males. We analyzed exome sequencing data obtained from a human COVID-19 cohort (n = 2,866) using a machine-learning approach and identify a CYP19A1-activity-increasing mutation to be associated with the development of severe disease in men but not women. We further analyzed human autopsy-derived lungs (n = 86) and detect increased pulmonary CYP19A1 expression at the time point of death in men compared with women. In the golden hamster model, we show that SARS-CoV-2 infection causes increased CYP19A1 expression in the lung that is associated with dysregulated plasma sex hormone levels and reduced long-term pulmonary function in males but not females. Treatment of SARS-CoV-2-infected hamsters with a clinically approved CYP19A1 inhibitor (letrozole) improves impaired lung function and supports recovery of imbalanced sex hormones specifically in males. Our study identifies CYP19A1 as a contributor to sex-specific SARS-CoV-2 disease outcome in males. Furthermore, inhibition of CYP19A1 by the clinically approved drug letrozole may furnish a new therapeutic strategy for individualized patient management and treatment.

Keywords: COVID-19; CYP19A1; estradiol; letrozole; lung health; male sex; testosterone.

Graphical abstract

Figure 1

Association analysis of CYP19A1 gene variation in whole-exome sequencing data of the GEN-COVID cohort (A) Overview of testosterone metabolism. (B) Characteristics and COVID-19 severity grading of the GEN-COVID cohort (n = 2,866 patients). (C and E) Ordered logistic regression (OLR) model in male (C) and female (E) patients, fitted using age to predict the ordinal grading (0, 1, 2, 3, 4, 5) dependent variable. Subjects falling above (severe), below (mild), or matching (intermediate) the expected treatment outcomes according to age are shown as red, blue, and black dots, respectively. (D and F) Pie charts representing the number of male (D) and female (F) patients falling into “milder,” “more severe,” and “predicted by sex and age” categories. (G) Association analysis between low frequency and common variants present in genes involved in testosterone metabolism and COVID-19 severity. (H and I) Graphical presentation of explainability (H) and penetrance (I) under a monogenic model for the Thr201Met CYP19A1 variant. (J and K) CYP19A1 mRNA expression levels (J) and virus titers (K) in Calu-3 cells control-treated (Mock) or infected with H1N1 influenza A virus, SARS-CoV, and SARS-CoV-2 (multiplicity of infection = 0.5) at 24 h post infection (p.i.). A merge of three independent biological replicates, performed in technical triplicates, is shown. Relative CYP19A1 mRNA expression values in mock-treated cells were set to n.d. (not detectable). Values are shown as means; error bars are shown as SD. In (G), (J), and (K) statistical significance was assessed by chi-squared test or one-way ANOVA showing significant differences between mock and infected cells (∗p ≤ 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 2

CYP19A1 expression in the lungs of fatal COVID-19 cases (A) Experimental setup for detecting CYP19A1 protein and mRNA expression levels in deceased COVID-19 patients and controls from three different study sites. (B) Detection of CYP19A1 protein expression (immunohistochemistry) and SARS-CoV-2 RNA (in situ hybridization) in the lungs from deceased male and female COVID-19 patients or non-COVID-19 controls (representative pictures shown from the University Hospital Tübingen cohort). Scale bars, 100 μm. (C–E) CYP19A1 mRNA expression levels in the lungs of deceased male COVID-19 patients, non-COVID-19 controls, and/or deceased patients with other respiratory infections at the University Hospital Hamburg-Eppendorf (C) (non-COVID-19, n = 3; other respiratory infections, n = 11; COVID-19, n = 12), at the University Hospital Tübingen (D) (non-COVID-19, n = 8; COVID-19, n = 9), and at the Erasmus Medical Center Rotterdam (E) (non-COVID-19, n = 4; COVID-19, n = 11). Relative CYP19A1 mRNA expression values in non-COVID-19 males were set to 1. Values are shown as means; error bars are shown as SD. Statistical significance was assessed by Mann-Whitney test (∗∗p < 0.01, ∗∗∗p < 0.001). (F) Visualization of percentage of males who died of COVID-19 with increased pulmonary CYP19A1 expression at the time of death.

Figure 3

CYP19A1 expression in the lung of SARS-CoV-2-infected hamsters (A) Binding sites for transcription factors in the four promoter regions (P1, P2, P3, and P4) of the CYP19A1 gene. (B) Quantification of CYP19A1-expressing tissue in the lungs of control-treated (PBS) male and female hamsters (n = 5). Data are shown as a box-and-whisker plot. (C) Weight loss of male and female hamsters infected with SARS-CoV-2 (n = 10). (D and G) CYP19A1 mRNA expression levels measured at day 3 p.i. in the lungs of SARS-CoV-2-infected and control-treated (PBS, Poly(I:C)) male (D) and female (G) golden hamsters (n = 10). (E, F, H, and I) IL-6 (E and H) and CYP19A1 (F and I) mRNA expression levels in lung macrophages isolated from uninfected male (E and F) or female (H and I) hamsters upon ex vivo PBS or Poly(I:C) treatment (male, n = 11; female, n = 8). Relative mRNA expression values in PBS-treated hamsters or macrophages were set to 1. Values are shown as means; error bars are shown as SD. Statistical significance was assessed by Mann-Whitney test, unpaired/paired Student’s t test, or one-way ANOVA (∗p ≤ 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 4

Sex hormones and sex hormone receptor expression in SARS-CoV-2-infected lungs as well as lung macrophages of hamsters (A, F, K, and P) Testosterone (A and K) and estradiol (F and P) were measured at the indicated time points in SARS-CoV-2-infected or control-treated (PBS, Poly(I:C)) male (A and F) and female (K and P) hamsters (n = 5; day 3 p.i., n = 5–10). (B, C, G, H, L, M, Q, and R) Androgen receptor (AR) (B and L), zinc transporter 9 (ZIP-9) (C and M), estrogen receptor α (ESR-α) (G and Q), and estrogen receptor β (ESR-β) (H and R) mRNA expression in the lungs of SARS-CoV-2-infected and control-treated (PBS, Poly(I:C)) male (B, C, G, H) and female (L, M, Q, R) hamsters at day 3 p.i. (n = 5; Poly(I:C), n = 4–5). (D, E, I, J, N, O, S, and T) AR (D and N), ZIP-9 (E and O), ESR-α (I and S), and ESR-β (J and T) mRNA expression in lung macrophages isolated from uninfected male (D, E, I, J) and female (N, O, S, T) hamsters (n = 8–11) upon ex vivo PBS or Poly(I:C) treatment. In (B–E), (G–J), (L–O), and (Q–T), relative hormone receptor mRNA expression values in PBS-treated controls were set to 1. Values are shown as means; error bars are shown as SD. Statistical significance was assessed by one-way ANOVA or paired Student’s t test (∗p ≤ 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 5

Lung plethysmography in SARS-CoV-2-infected hamsters Whole-body plethysmography in SARS-CoV-2-infected or control (PBS)-treated male (A, C, E, G, I) and female (B, D, F, H, J) hamsters at the indicated time points (n = 10; day 0 p.i., n = 12): tidal volume (A, B), frequency (C, D), EF₅₀ (expiratory flow rate at the point 50% of tidal volume is expired) (E, F), peak inspiratory flow (G, H), and peak expiratory flow (I, J). Values are shown as means; error bars are shown as SD. Statistical significance was assessed by unpaired Student's t test (∗p ≤ 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 6

Treatment of SARS-CoV-2-infected hamsters with the CYP19A1 inhibitor letrozole (A) Timeline of experimental setup for letrozole treatment. Male and female hamsters were control treated with PBS or infected with SARS-CoV-2 and subjected to daily letrozole treatment from 3 h to 8 days p.i. (B) Docking model of CYP19A1-letrozole complex adapted from Hong et al. (C and D) Letrozole levels in lung (C) and plasma (D) of male and female hamsters treated with vehicle or letrozole. n.d., not detectable. (E–H) Testosterone (E), estradiol (F), as well as lung AR (G) and ESR-β (H) mRNA expression at 3 days p.i. (E, F) or 6 days p.i. (G, H) (n = 11). (G and H) SARS-CoV-2-infected and vehicle-treated hamsters were set as 1. Data are shown as a box-and-whisker plot. (I–L) Whole-body plethysmography in infected male hamsters: Tidal volume (I), frequency (J), EF₅₀ (K), and inspiratory time (L) (n = 5–7; day 0 p.i., n = 12). (M and N) H&E staining (M) or collagen staining with Sirius red (N) of lung sections from infected male hamsters at 21 days p.i. (n = 5–7 per group). Scale bars, 200 μm (M) and 100 μm (N). (O) Weight loss of infected male hamsters (n = 5–7 per group). In (C), (D), (I–L), and (O), values are shown as means; error bars are shown as SD. Statistical significance was assessed by Mann-Whitney test or one-way ANOVA (∗p ≤ 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 7

Genome-wide transcriptome analysis of SARS-CoV-2-infected hamster lungs treated with the CYP19A1 inhibitor letrozole (A–D) Genome-wide lung transcriptomic profiles of PBS-control-treated or SARS-CoV-2-infected male (A and C) and female (B and D) hamsters (n = 5), treated with vehicle or letrozole, at 21days p.i. were compared and are shown as ratio/intensity scatterplots (M/A plot [M-value = log₂ fold change; A-value = log₂ base mean]). (E) Heatmap depicting normalized expression of differentially expressed genes in the lung of male and female hamsters, clustered according to their expression profiles (cutoff log₂ fold change ≥1 or ≤−1, with an adjusted p value of ≤0.05). (F and G) Significant differentially expressed genes in the lung of control (PBS) or SARS-CoV-2-infected male (F) and female (G) hamsters. (H) Gene ontology term enrichment (biological process) of genes that were significantly downregulated in letrozole-treated SARS-CoV-2-infected male hamsters, compared to the vehicle control group.