SARS-CoV-2 Infection of Pluripotent Stem Cell-Derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response

Abstract

A hallmark of severe COVID-19 pneumonia is SARS-CoV-2 infection of the facultative progenitors of lung alveoli, the alveolar epithelial type 2 cells (AT2s). However, inability to access these cells from patients, particularly at early stages of disease, limits an understanding of disease inception. Here, we present an in vitro human model that simulates the initial apical infection of alveolar epithelium with SARS-CoV-2 by using induced pluripotent stem cell-derived AT2s that have been adapted to air-liquid interface culture. We find a rapid transcriptomic change in infected cells, characterized by a shift to an inflammatory phenotype with upregulation of NF-κB signaling and loss of the mature alveolar program. Drug testing confirms the efficacy of remdesivir as well as TMPRSS2 protease inhibition, validating a putative mechanism used for viral entry in alveolar cells. Our model system reveals cell-intrinsic responses of a key lung target cell to SARS-CoV-2 infection and should facilitate drug development.

Keywords: COVID-19; SARS-CoV-2; alveolar epithelial cell; alveolar type 2 cell; human induced pluripotent stem cells; iPSCs; inflammation; lung.

Graphical abstract

Figure 1

iPSC-Derived Alveolar Epithelial Type 2 Cells (iAT2s) Express Functional SARS-CoV-2 Entry Factors ACE2 and TMPRSS2 (A and B) iAT2s, carrying a tdTomato reporter targeted to the endogenous SFTPC locus by gene editing (SPC2 line), can be serially passaged while maintaining >90% SFTPC^tdTomato+ expression in 3D sphere cultures (day 160 of differentiation, passage 8 shown; scRNA-seq profile provided in Figure S1 and compared to Habermann et al., 2020). (C and D) scRNA-seq data of iAT2s (SPC2 line at day 114 of differentiation) visualized in SPRING plots (Weinreb et al., 2018) based on reanalysis of a dataset previously published in (Hurley et al., 2020) showing expression of (C) SFTPC as well as (D) an eight-gene benchmark of AT2 cell differentiation (SFTPC, CLDN18, LAMP3, SFTPB, SFTPD, NAPSA, SLC34A2, and CXCL8 as characterized in [Hurley et al., 2020]). (E) iAT2s (RUES2 line) express ACE2 and TMPRSS2 transcripts at comparable levels to purified primary adult human lung AT2s (day 15 PLP, primordial lung progenitors derived from pluripotent stem cells at day 15 of differentiation; early HFL, primary early human fetal lung alveolar epithelium at 16–17.5 weeks gestation; late HFL, alveolar epithelium at 20–21 weeks gestation; and adult AT2s, adult alveolar epithelial type 2 cells from 3 different individuals freshly sorted using the antibody against HTII-280. Primary adult and fetal sample procurement described in detail in Hurley et al., [2020]). (F–H) iAT2s (SPC2 line) cultured at air-liquid interface (ALI) (F) express ACE2 protein, as observed by flow cytometry, n = 9 (G; additional scRNA-seq profiling in Figure S1), which is apically localized, as observed by immunofluorescence staining (scale bar, 10 μm) (H). (I and J) iAT2s infected with a GFP-expressing lentivirus pseudotyped with either VSVG or SARS-CoV-2 Spike envelopes, n = 3. ^∗p < 0.05, one-way ANOVA. All bars represent mean ± standard deviation. See also Figure S1.

Figure 2

SARS-CoV-2 Infects iAT2s in a Dose- and Time-Dependent Manner (A) Schematic of the iAT2 directed differentiation protocol in which robustly self-renewing iAT2s can be plated at ALI for SARS-CoV-2 infections. “CK+DCI,” distal lung medium components detailed in the STAR★Methods. (B and C) Immunofluorescence images of viral nucleoprotein (N, green) of iAT2s infected with SARS-CoV-2 (MOI = 5) at 1 and 4 days post infection (dpi) (B) or with increasing MOIs (0.5, 2.5, 5) shown at 1 dpi (C) (20×, scale bar, 50 μm). (D) Efficiency of iAT2 infections scored by representative FACS plots of SARS-CoV-2 N at 1 and 4 dpi (MOI 5) compared to mock; mean gated percentages ± standard deviation for n = 3 replicates are shown; results representative of three independent experiments. (E) RT-qPCR of viral N gene expression at 1 and 4 dpi via a range of low MOIs of an unpurified SARS-CoV-2 virus stock, n = 3. (F and G) RT-qPCR of N gene expression at 1 and 4 dpi via a purified virus stock to infect with an MOI of 0.4 (F) or an MOI of 5 (G), n = 3. Fold change expression compared to Mock [2^-ΔΔCt] after 18S normalization is shown. (H) RT-qPCR of N gene expression of BU3 and SPC2 iAT2s at MOIs 0.04 and 0.4 at 1 dpi. (I) RT-qPCR of N gene expression at an MOI of 0.4 in iAT2s in alveolospheres and iAT2s at ALI at 2 dpi. (J) Viral titers were determined in apical washes and basolateral media at 1 and 4 dpi (n = 5). (K) Mean percent fragmented nuclei in immunofluorescence images of infected iAT2s at 1 and 4 dpi (MOI 5; n = 3). (L–N) Electron micrograph of infected iAT2s showing virions (arrow heads) intracellularly, including in a lamellar body and (M) extracellular virions around tubular myelin (N, arrow). Tubular myelin meshwork (inset from M) that forms upon secretion of pulmonary surfactant (scale bar, 200 nm). (O) Higher magnification of a coronavirus virion at 1 dpi (MOI 5) (scale bar, 100 nm). All bars represent mean ± standard deviation with biological replicates indicated for each panel. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001, unpaired, two-tailed Student’s t test (I and K) or one-way ANOVA with multiple comparisons (E, F, G, H, and J) were performed.

Figure 3

SARS-CoV-2 Elicits Transcriptomic Changes in iAT2s That Highlight Epithelial-Intrinsic Inflammatory Responses to Infection (A) Schematic of the iAT2 ALI samples (starting with day 208 iAT2s) infected with SARS-CoV-2 (MOI 5) and collected at 1 and 4 dpi (mock collected 1 dpi) for bulk RNA sequencing (RNA-seq). (B) Principal-component analysis (PCA) of iAT2 samples (n = 3 biological replicates per condition) showing global transcriptomic variance (%) of PC1 and PC2 components. (C) Local regression (LOESS) plots of viral, AT2, NF-κB, and interferon (IFN) gene expression levels quantified by RNA-seq normalized expression (counts per million reads). (D) Gene set enrichment analysis (GSEA, Camera using Hallmark gene sets) of the top ten upregulated gene sets in 1 dpi versus mock or 4 dpi versus 1 dpi conditions (black color indicates statistical significance; FDR < 0.05). (E and F) Unsupervised hierarchical clustered heatmaps of differentially expressed genes (DEGs; FDR < 0.05) in the Hallmark gene sets “TNFA signaling in NFkB” (E) and “interferon gamma response” (F) as plotted with row normalized Z score; a selected subset of these DEGs are highlighted with large font. (G and H) Volcano plots of differentially expressed genes in 1 dpi versus mock (G) and 4 dpi versus 1 dpi (H).

Figure 4

Infection of iAT2s with SARS-CoV-2 Prompts the Loss of the Lung AT2 Program, Activation of the NF-κB Pathway, and Delayed Activation of IFN Signaling (A) Immunofluorescence staining of pro-surfactant protein C (pro-SFTPC) in tissue sections of non-COVID-19 and COVID-19 lungs with zoomed insets showing the typical cytoplasmic punctate appearance of lamellar body-localized pro-SFTPC. The arrow indicates AT2 hyperplasia (right panel), a typical and non-specific response to injury, juxtaposed with regions that have a paucity of pro-SFTPC (middle panel). (B) COVID-19 decedents’ autopsied lung tissue sections, stained with H&E. (C) Non-COVID-19 and COVID-19 decedents’ sections stained with cytokeratin AE1/AE3 (brown) showing early acute phase of diffuse alveolar damage with sloughed alveolar epithelium (200× magnification). (D and E) RT-qPCR of AT2 (D) and NF-κB-related (E) transcripts in iAT2s infected with SARS-CoV-2 (MOI 5; n = 3; Fold change expression over “Mock” = 2^-ΔΔCt) at 1 and 4 dpi. (F) Luminex analysis of apical washes and basolateral media collected from iAT2 ALI cultures (n = 5). (G) RT-qPCR of interferon-stimulated genes (ISGs) in infected (MOI 5) iAT2s at 1 and 4 dpi (n = 3). (H and I) RT-qPCR of N gene expression at 2 dpi (MOI 0.04) with (H) vehicle control, camostat (TMPRSS2 inhibitor), E-64d (cathepsin B/L inhibitor), or (I) remdesivir treatment, n = 3. All bars represent mean ± standard deviation.^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001, one-way ANOVA with multiple comparisons were performed.