Age-determined expression of priming protease TMPRSS2 and localization of SARS-CoV-2 in lung epithelium

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) novel coronavirus 2019 (COVID-19) global pandemic has led to millions of cases and hundreds of thousands of deaths. While older adults appear at high risk for severe disease, hospitalizations and deaths due to SARS-CoV-2 among children have been relatively rare. Integrating single-cell RNA sequencing (scRNA-seq) of developing mouse lung with temporally resolved immunofluorescence in mouse and human lung tissue, we found that expression of SARS-CoV-2 Spike protein primer TMPRSS2 was highest in ciliated cells and type I alveolar epithelial cells (AT1), and TMPRSS2 expression increased with aging in mice and humans. Analysis of autopsy tissue from fatal COVID-19 cases detected SARS-CoV-2 RNA most frequently in ciliated and secretory cells in airway epithelium and AT1 cells in peripheral lung. SARS-CoV-2 RNA was highly colocalized in cells expressing TMPRSS2. Together, these data demonstrate the cellular spectrum infected by SARS-CoV-2 in lung epithelium and suggest that developmental regulation of TMPRSS2 may underlie the relative protection of infants and children from severe respiratory illness.

Keywords: Expression profiling; Pulmonology.

Conflict of interest statement

Conflict of interest: JAK has received advisory board fees from Boehringer Ingelheim Inc. and has research contracts with Genentech. TSB has received advisory board fees from Boehringer Ingelheim Inc., Orinove, GRI Bio, Morphic, and Novelstar, and has research contracts with Genentech and Celgene.

Figures

Figure 1. Time-series scRNA-seq of developing mouse lung.

(A) Overview of murine developmental stages and time points for scRNA-seq time series. (B) Workflow of scRNA-seq time series. Single-cell suspensions were generated from at least 4 mice at each time point. Viable, Cd45–, Ter119– cells were selected for scRNA-seq library preparation using the 10X Genomics Chromium 5′ platform. Uniform Manifold Approximation and Projection (UMAP) embedding of 67,629 cells annotated by (C) developmental time point and (D) cell-type. (E) Violin plot depicting expression of key SARS-CoV2 receptor Ace2 and coreceptor Tmprss2 across cell types. (F) UMAP embedding of 4,369 epithelial cells after subsetting and reclustering. (G, H) UMAP plots depicting relative expression of Ace2 and Tmprss2. (I) Relative expression of putative SARS-CoV-2 priming proteases during development, shown as violin plots to indicate relative expression, with individual cells shown as dots. *P < 3.125 × 10–3 by 1-way ANOVA across developmental time points.

Figure 2. Spatial and temporal localization of Tmprss2 expression across lung development.

(A) RNA-ISH of Tmprss2 expression (white) with epithelial markers Scgb1a1 (secretory cells, blue), Foxj1 (ciliated cells, magenta), Sftpc (surfactant protein C, AT2 cells, green), Hopx (type 1 alveolar epithelial cells, red). FFPE tissue from lungs at E18, P0, P7, P14, 2 months, 12 months, and 24 months was used, with representative image data from P0, P7, 2 months, 12 months, and 24 months shown in the figure. Lungs from 3 mice at each time point were used, with ten ×40 images obtained per slide. Scale bars: 100 μm. (B, C) Quantification of Tmprss2 expression in each epithelial subtype across development by automated image analysis (HALO, Indica Labs) measured as (B) percentage of cellular area covered by Tmprss2 probe in each cell expressing both Tmprss2 and the epithelial marker (all data points are shown with mean ± SD) and (C) percentage of cells expressing the epithelial marker that also express Tmprss2, with positive Tmprss2 expression defined as having 5 or more copies of Tmprss2 probe (box and whisker plots are shown with mean and error bars reflecting minimum and maximum values for each time point). More than 1000 cells were counted at each time point. ****P < 0.0001 by 1-way ANOVA. (D) RNA-ISH of Foxj1 expression (red) or Hopx expression (red) with protein immunofluorescence for TMPRSS2 protein (white). Five ×40 images per slide were obtained. Scale bars: 100 μm.

Figure 3. Spatial and temporal localization of TMPRSS2 expression in lung across the human lifespan.

(A) RNA-ISH of TMPRSS2 expression (white) with epithelial markers SCGB1A1 (secretory cells, blue), FOXJ1 (ciliated cells, magenta), SFTPC (surfactant protein C, AT2 cells, green), AGER (AT1 cells, red). FFPE tissue from 20 human lungs between the ages of birth and 69 years was analyzed. Ten ×40 images were obtained per slide for analysis. Scale bars: 100 μm. (B, C) Quantification of TMPRSS2 expression in each epithelial subtype across development by automated image analysis (HALO, Indica Labs), measured as (B) percentage of cellular area covered by TMPRSS2 probe for each cell expressing both TMPRSS2 and the epithelial marker (all data points are shown with mean ± SD and (C) percentage of cells expressing the epithelial marker that also express TMPRSS2, with positive TMPRSS2 expression defined has having 5 or more copies of TMPRSS2 probe (box and whisker plots are shown with mean and error bars reflecting minimum and maximum values for each time point). More than 1000 cells were counted at each time point. ****P < 0.0001, **P < 0.01 by 1-way ANOVA. (D) RNA-ISH of FOXJ1 expression (red) or AGER expression (red) with protein immunofluorescence for TMPRSS2 protein (white). Data are shown with mean ± SD. Five ×40 images per slide were obtained. Scale bars: 100 μm.

Figure 4. Spatial localization of SARS-CoV-2 RNA in lung autopsy tissue from fatal COVID-19.

(A) RNA-ISH of SARS-CoV-2 RNA (red) with epithelial markers SCGB1A1 (secretory cells, cyan), FOXJ1 (ciliated cells, white), SFTPC (AT2 cells, green), AGER (AT1 cells, white). Scale bars: 100 μm. (B) Quantification of cells containing SARS-CoV-2 RNA by epithelial subtype as determined by percentage of cellular area covered by SARS-CoV-2 probe in all cells positive for the epithelial marker; more than 150 cells were counted for each subtype. All data points are shown with mean ± SD. (C) RNA-ISH of large airway from the same patient demonstrating RNA transcripts for TMPRSS2 (white) in the same cells containing SARS-CoV-2 RNA (red), with secretory cells labeled in green (SCGB1A1) for context. Scale bars: 100 μm. (D) Quantification of TMPRSS2 expression in SARS-CoV-2+ cells; more than 1000 cells were counted from the 3 subjects, P < 0.05. All percentages are shown in graphs with mean ± SD. (E) RNA-ISH of SARS-CoV-2 (red) with protein immunofluorescence for TMPRSS2 protein (white). Scale bars: 100 μm.