Patient-specific iPSCs carrying an SFTPC mutation reveal the intrinsic alveolar epithelial dysfunction at the inception of interstitial lung disease

Abstract

Alveolar epithelial type 2 cell (AEC2) dysfunction is implicated in the pathogenesis of adult and pediatric interstitial lung disease (ILD), including idiopathic pulmonary fibrosis (IPF); however, identification of disease-initiating mechanisms has been impeded by inability to access primary AEC2s early on. Here, we present a human in vitro model permitting investigation of epithelial-intrinsic events culminating in AEC2 dysfunction, using patient-specific induced pluripotent stem cells (iPSCs) carrying an AEC2-exclusive disease-associated variant (SFTPCI73T). Comparing syngeneic mutant versus gene-corrected iPSCs after differentiation into AEC2s (iAEC2s), we find that mutant iAEC2s accumulate large amounts of misprocessed and mistrafficked pro-SFTPC protein, similar to in vivo changes, resulting in diminished AEC2 progenitor capacity, perturbed proteostasis, altered bioenergetic programs, time-dependent metabolic reprogramming, and nuclear factor κB (NF-κB) pathway activation. Treatment of SFTPCI73T-expressing iAEC2s with hydroxychloroquine, a medication used in pediatric ILD, aggravates the observed perturbations. Thus, iAEC2s provide a patient-specific preclinical platform for modeling the epithelial-intrinsic dysfunction at ILD inception.

Keywords: NF-κB; autophagy; bioenergetics; iPSC-derived alveolar epithelial type 2 cells; idiopathic pulmonary fibrosis; induced pluripotent stem cells; interstitial lung disease; metabolic reprogramming; proteostasis; surfactant protein C.

Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.

Figures

Figure 1.. Generation of patient-specific iPSC lines and their differentiation to alveolar epithelium

(A) Schematic showing the generation of patient-specific iPSCs from dermal fibroblasts or peripheral blood mononuclear cells (PBMCs) from three individuals carrying the most frequent SFTPC pathogenic variant (SFTPCI73T/WT). (B) Chest CT of the SPC2 donor reveals diffuse ground glass opacities and traction bronchiectasis. (C) H&E staining of the SPC2 donor lung explant shows end-stage lung disease with interstitial fibrosis, chronic inflammation, and alveolar remodeling with AEC2 hyperplasia and degenerating macrophages within the residual alveoli. Scale bar, 200 μm. (D) Transcription activator-like effector nucleases (TALEN) targeting strategy and edited SFTPC loci post Cre-mediated antibiotic cassette excision. (E) Schematic of directed differentiation protocol from iPSCs to day 30+ monolayered epithelial iAEC2 spheres (alveolospheres). (F) Schematic of CHIR modulation to achieve iAEC2 maturation and representative flow cytometry dot plots (mean ± SD; n = 6 biological replicates of independent differentiations). (G) Dot plots depicting the normalized expression level of AEC2-marker genes in day 114 and day 131 SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s compared to day 0 iPSCs and week 21 human fetal distal lung (HFL) controls, by bulk RNA sequencing (boxplots represent mean ± SD; n = 3 experimental replicates of independent wells of a differentiation).

Figure 2.. Derivation of parallel self-renewing corrected (SFTPCtdT/WT) and mutant (SFTPCI73T/tdT) iAEC2s

(A) Representative flow cytometry dot plots of day 82 (p2), day 115 (p4), day 149 (p6), day 190 (p8), day 208 (p9), day 243 (p11), and day 285 (p13) SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s. MFI, mean fluorescence intensity. (B) Representative live-cell imaging of SFTPCtdT/WT and SFTPCI73T/tdT alveolospheres (bright-field/tdTomato overlay; day 149). Scale bars, 500 μm. (C) Graph showing yield per input tdT/WT or I73T/tdT SFTPCtdTomato+ cell sorted on day 51 and passaged without further sorting. **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired, two-tailed Student’s t test. (D) Bar graph shows retention of the AEC2 cell fate in SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s maintained in culture for 302 days, measured by flow cytometry as the frequency of cells expressing the SFTPCtdTomato reporter. (E) Histograms show higher proliferation rates in SFTPCtdT/WT compared to SFTPCI73T/tdT iAEC2s, measured by flow cytometry as the frequency of SFTPCtdTomato+ cells that incorporate EdU. (F) Representative confocal immunofluorescence microscopy of SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s stained for activated caspase 3 (green) and DNA (Hoechst, blue) shows absence of significant apoptosis. Scale bars, 50 μm. (A and C–E) Mean ± SD is shown; n = 3 experimental replicates of independent wells of a differentiation.

Figure 3.. SFTPCI73T/tdT iAEC2s demonstrate distinct cellular morphology and misprocess and mistraffick pro-SFTPC similarly to in vivo SFTPCI73T-expressing AEC2s

(A) Representative H&E staining of formalin fixed and paraffin embedded sections (i, scale bars, 50 μm) and toluidine blue staining of plastic sections (ii) of SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s. Arrowheads indicate lamellar body-like inclusions; eclipse indicates intraluminal inclusions. (B) Representative confocal immunofluorescence microscopy of SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s for EPCAM (green) and DNA (Hoechst, blue). Scale bars, 10 μm. (C) Representative staining of E18.5 CFlp-SFTPCI73T/I73T and CFlp-SFTPCWT/WT embryos for HA-tagged SFTPC. Scale bars, 70 μm. (D) Representative confocal immunofluorescence microscopy of distal sections of SPC2 donor and healthy donor lung explants stained for EPCAM (red), pro-SFTPC (green), and DNA (Hoechst, blue). Scale bars, 10 μm. (E) Representative TEM images of SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s depicting lamellar bodies. Scale bars, 1 μm. (F) Schematic representing the cellular compartments in which pro-SFTPC processing into mature SFTPC occurs. ER, endoplasmic reticulum; MVB, multi-vesicular body; LB, lamellar body. (G and H) Representative western blots of SFTPCtdT/WT and SFTPCI73T/tdT iAEC2 lysates at the indicated time points were compared to freshly isolated primary human AEC2s lysates for pro-SFTPC (NPRO-SFTPC) (G, left panel), mature SFTPC (G, right panel), and mature SFTPB (H) with β actin as a loading control. (I) Representative confocal immunofluorescence microscopy of SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s for pro-SFTPC (red; with zoom), EPCAM (green; with zoom), SFTPB (red), and DNA (Hoechst, blue). Scale bars, 10 μm. (J) Representative confocal immunofluorescence microscopy of distal sections of SPC2 donor and healthy donor lung explants for pro-SFTPC (green), EPCAM (red), SFTPB (green), and DNA (Hoechst, blue). Scale bars, 10 μm.

Figure 4.. Transcriptomic and proteomic/phosphoproteomic analyses identify candidate disease-associated pathways

(A) Clustering of SFTPCtdT/WT and SFTPCI73T/tdT iAEC2 scRNA-seq transcriptomes at two time points (days (d)30 and 113 of differentiation) using Uniform Manifold Approximation Projection (UMAP). (B) Average expression levels and frequencies (purple dots) for select genes profiled by scRNA-seq in SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s. Comparison is made to a publicly available adult primary distal lung dataset (Habermann et al., 2020), and genes are selected to indicate AEC2, AEC1, airway, endothelial, epithelial, leukocyte, or proliferation programs. (C) Heatmap of top 50 upregulated and top 50 downregulated genes comparing d113 SFTPCtdT/WT versus d113 SFTPCI73T/tdT iAEC2s by scRNA-seq (ranked by average log fold-change, FDR <0.05; row-normalized expression Z scores). A subset of differentially expressed genes is highlighted with large font. (D) Violin plots showing normalized expression for indicated genes or cell cycle phase in d30 and d113 SFTPCtdT/WT versus SFTPCI73T/tdT iAEC2s by scRNA-seq. (E) Heatmap of the 50 upregulated and top 50 downregulated genes in d114 SFTPCtdT/WT versus d114 SFTPCI73T/tdT iAEC2s by bulk RNA seq (ranked by FDR, FDR <0.05; row-normalized expression Z scores). AEC2-marker genes are highlighted with large font. (F) Volcano plots indicating differential protein expression in d113 SFTPCtdT/WT versus d113 SFTPCI73T/tdT iAEC2s by mass spectrometry. (G) Representative confocal immunofluorescence microscopy of distal sections of SPC2 donor and healthy donor lung explants stained for pro-SFTPC (green) and DNA (Hoechst, blue) shows an altered cellular localization pattern and a higher pro-SFTPC fluorescence intensity in the patient’s AEC2s, quantified by relative fluorescence units (RFU). Scale bars, 10 μm (mean ± SD; n = 35 individual SFTPC-positive cells treated as experimental replicates). (H) Top 10 upregulated pathways in d113 SFTPCI73T/tdT versus d113 SFTPCtdT/WT iAEC2s based on global proteomic analysis. (I) Integrated gene set enrichment analyses based on scRNA-seq transcriptomic, proteomic, and phosphoproteomic analyses in d113 SFTPCI73T/tdT versus d113 SFTPCtdT/WT iAEC2s with an FDR <0.1 in at least 2 out of 3 datasets. NES, normalized enrichment score; gene ratio, ratio of enriched genes for a given pathway to the total number of genes in the pathway. The last column represents the number of datasets (out of 3) with an FDR <0.05. **p

Figure 5.. Autophagy perturbations in SFTPCI73T iAEC2s

(A) Schematic illustrating the autophagy pathway. Key proteins involved in the pathway (p62/SQSTM1 and LC3) and the mechanisms of action of bafilomycin A1 and torin are depicted. (B) Representative western blot of SFTPCtdT/WT and SFTPCI73T/tdT iAEC2 lysates for p62 and LC3 with GAPDH and β actin as loading controls with densitometric quantification (mean ± SD; n = 11 for LC3 and n = 7 for p62 independent experiments). (C) Western blots of cell lysates for LC3 and β actin from autophagic flux studies using SFTPCtdT/WT or SFTPCI73T/tdT iAEC2s treated with either torin (5 μM) for 4, 8, or 18 h or bafilomycin A1 (BafA1) (50 or 100 nM) for 18 h or vehicle (DMSO) for 4 or 18 h (blots shown are representative of n = 2 independent experiments). (D) A repeat autophagic flux study, as in (C) but using BafA1 (50 nM) for 1, 2, 4, and 18 h or vehicle (DMSO) for 18 h (representative of n = 2 independent experiments). (E) SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s transduced with a lentiviral LC3:GFP fusion protein and exposed to BafA1 (50 nM) to quantify autophagosomes and autophagolysosomes. Scale bar, 10 μm (representative confocal fluorescence microscopy images of n = 3 experimental replicates of independent wells of a differentiation). (F) Representative TEM image of SFTPCI73T/tdT iAEC2s shows a double-membrane autophagosome (inset, arrowheads). Scale bar, 1 μm. (G) Real-time qPCR showing fold change in gene expression compared to WT in primary mouse SftpcI73T AEC2s (mean ± SEM; n = 6 for SftpcI73T and n = 5 for WT mice). (H) Representative western blots of primary mouse WT and SftpcI73T AEC2 lysates for pro-SFTPC (NPRO), LC3, and p62 with β actin as a loading control (see also Figure 3G) (n = 3 WT and SftpcI73T mice). (I) Representative live-cell confocal fluorescence microscopy of SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s stained with LysoTracker green. Scale bars, 10 μm. (J) Quantification of acidic organelles expressed as percentage of covered cell area (mean ± SD; n = 11–14 independent alveolospheres treated as experimental replicates). (K) Quantification of lysosomal pH based on Lysosensor yellow/blue-dextran ratio (mean ± SD; n = 2 independent experiments with 20–30 cells analyzed per experiment; see also Figure S4). *p

Figure 6.. SFTPCI73T-expressing AEC2s demonstrate metabolic reprogramming

(A) Representative live-cell confocal fluorescence microscopy of SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s stained with MitoTracker green. Scale bars, 10 μm. (B–D) Quantitative analyses of morphometric data from fluorescence images (mean ± SD; n = 24 SFTPCtdT/WT and n = 18 SFTPCI73T/tdT independent alveolospheres treated as experimental replicates). (B) Increased mitochondrial mass in SFTPCI73T/tdT iAEC2s, quantified as the percentage of cell area covered by mitochondria. (C) Increased mitochondrial size in SFTPCI73T/tdT iAEC2s. (D) SFTPCI73T/tdT iAEC2 mitochondria are more fragmented, assessed by the aspect ratio. (E) Representative western blot of SFTPCtdT/WT and SFTPCI73T/tdT iAEC2 lysates for TOM20 with β actin as loading control with densitometric quantification (mean ± SD; n = 4 [days 128, 176, 223, 223] SFTPCtdT/WT and n = 3 [days 128, 223, 223] SFTPCI73T/tdT independent experiments). (F) Early time point OCR was measured under basal conditions followed by addition of oligomycin, FCCP, and antimycin A (Ant A), as indicated. (G) SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s demonstrate no significant differences in proton leak, maximal respiration, and spare capacity measured as percent change over basal respiration (mean ± SEM; n = 2 independent experiments). (H) Early time point ECAR under basal conditions and following addition of oligomycin. (I) Late time point OCR. (J) SFTPCI73T/tdT iAEC2s demonstrate significantly lower maximal respiration and spare respiratory capacity (mean ± SEM; n = 3 independent experiments) measured as percent change over basal respiration. (K) Late time point reveals a significantly higher ECAR in SFTPCI73T/tdT iAEC2s (mean ± SD; n = 3 independent experiments). (L) Representative live-cell confocal fluorescence microscopy of SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s stained with BODIPY 493/503 dye with quantitative analyses. Scale bars, 10 μm (mean ± SD; n = 10 SFTPCtdT/WT and n = 7 SFTPCI73T/tdT independent alveolospheres treated as experimental replicates). (M) OCR measured under basal conditions and following addition of oligomycin, FCCP, and Ant A, as indicated (mean ± SD; n = 7 SftpcI73T and n = 5 WT mice). (N) ECAR measured under basal conditions in WT and SftpcI73T mouse AEC2s (mean ± SEM; n = 5 WT mice and n = 7 SftpcI73T). OCR, oxygen consumption rate; ECAR, extracellular acidification rate. (G and J) Depict super plots: small shapes represent replicate values within each experiment and large shapes represent the average of each independent experiment, tdT/WT and I73T/tdT are color-matched between experiments. *p

Figure 7.. SFTPCI73T/tdT iAEC2s display activation of the NF-κB pathway and are more susceptible to hydroxychloroquine treatment

(A) Unsupervised hierarchical clustered heatmap of differentially expressed proteins (FDR in vivo inactivation of the viral LTR promoter; Ψ, Psi lentiviral packaging sequence; GFP, green fluorescence protein. (C) Representative live-cell imaging of lentivirally transduced SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s (bright-field/tdTomato/GFP overlay). Scale bars, 500 μm. (D) Sort gates used to purify transduced (GFP+) SFTPCtdT/WT and SFTPCI73T/tdT iAEC2s. (E) Bioluminescence quantification shows increased luciferase activity in SFTPCI73T/tdT versus SFTPCtdT/WT iAEC2s. *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired, two-tailed Student’s t test for all panels. (F) Luminex analysis of supernatants collected from SFTPCI73T/tdT and SFTPCtdT/WT iAEC2s for the NF-κB target proteins GM-CSF, CXCL5, and MMP-1. *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired, two-tailed Student’s t test for all panels. (G) SFTPCtdT/WT or SFTPCI73T/tdT iAEC2s were treated with hydroxychloroquine (HCQ; 10 μM) or vehicle (ddH2O) for 18 h or 7 days. Lysates were subjected to western blotting for LC3 with β actin as loading control. Densitometric quantification is shown. See also Figure S5. *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired, two-tailed Student’s t test for all panels. (H) Graph showing yield in cell number per input SFTPCtdT/WT or SFTPCI73T/tdT iAEC2 with or without treatment with HCQ (10 μM). ****p < 0.0001 across all groups by one-way ANOVA; p < 0.001 for d10, p < 0.01 for d20, p < 0.0001 for d31, and p < 0.05 for d44 for corrected versus mutant iAEC2s by unpaired, two-tailed Student’s t test. Mean ± SD is shown; n = 3 (E and F) or n = 2 (G) experimental replicates of independent wells of a differentiation or n = 3 (H) biological replicates of independent differentiations.