Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis (original) (raw)
Features of usual interstitial pneumonia in IPF. Patchy interstitial fibrosis, loss of alveolar structure, and honeycombing, hallmarks of usual interstitial pneumonia (UIP), were present in all IPF explant tissues evaluated after transplant (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/jci.insight.90558DS1). Uniformly thin alveolar septae lined by AT2 and AT1 cells were characteristic of normal lungs. IPF tissues consisted of heterogeneous lesions with dense connective tissue, fibroblastic foci, and cystic lesions, many containing mucus. “Honeycomb” cysts were lined by diverse epithelial cell types, including cuboidal “hyperplastic” AT2 cells, goblet cells, and ciliated cells, the latter two cell types normally primarily restricted to tracheal, bronchial, and bronchiolar epithelium lining cartilaginous airways. Heterogeneous lesions containing disorganized epithelial cells and inflammatory infiltrates were present in all IPF samples.
Gene expression patterns in pulmonary epithelial cells obtained by cell sorting. Lung cells were isolated from peripheral control and IPF lung tissue after protease digestion and viable cells sorted on the basis of their 7AAD–, CD45–, CD31–, CD326+ (EPCAM), HTII-280+ phenotype (herein referred to as HTII-280+ epithelial cells); HTII-280 is a selective surface marker of normal AT2 cells (24). Consistent differences were observed between distal normal donor lung and distal IPF explant lung tissue in the relative abundance of epithelial cell types recognized by anti-CD326 and HTII-280 monoclonal antibodies. Control lung tissue consistently yielded >90% HTII-280 surface reactive epithelial cells, indicative of an abundant AT2 cell fraction; and relatively few NGFR+ or double negative epithelial cells, indicating few airway basal or luminal cell types, respectively. In contrast, distal lung tissue from IPF patients demonstrated a remarkable decline in HTII-280+ cells, decreasing to approximately 5% of total epithelial cells, with a corresponding increase in the abundance of NGFR+ and double negative epithelial cells. Differences in the relative abundance of HTII-280+ cells between control and IPF tissues were associated with disease-dependent changes in their molecular phenotype. RNA sequencing demonstrated clear separation of gene expression between populations of HTII-280+ epithelial cells isolated from IPF and control tissues (Figure 1A). A heatmap illustrates differentially expressed genes based on unsupervised hierarchical clustering (Figure 1B). Transcriptome profiles from normal lung HTII-280+ cells were consistent with expression profiles of AT2 cells that mediate surfactant protein and lipid homeostasis, with this extensive RNA data providing a useful resource for further investigation of human AT2 cell biology. Transcriptome profiles of HTII-280+ epithelial cells from IPF lungs were surprisingly enriched in transcripts normally associated with conducting airway epithelial cells, but also expressed AT2 cell–associated transcripts. Genes related to “cell migration, cell junction/extracellular matrix organization, response to wounding, and epithelial cell proliferation” were induced in IPF (Figure 1, C and D). Ingenuity pathway analysis (IPA) predicted activation of TGF-β–, PI3K/AKT-, HIPPO/YAP-, p53-, and WNT-mediated pathways, whereas those associated with lipid synthesis and metabolism, endosomal protein processing, and unfolded protein responses were suppressed in IPF epithelial cells (Figure 1, C and D). TGFB1, TP53, IGF1, NFKB, and ETS family transcription factors (ETV5, SPDEF, EHF) were predicted to be driving forces in IPF epithelial cells, influencing their transcriptional responses (Supplemental Figure 2). The most upregulated or suppressed RNAs in the CD326+HTII-280+ sorted IPF cells are shown in Supplemental Tables 1 and 2. AT2 cell “signature” genes, including surfactant-associated genes, were highly expressed in sorted cells from both control and IPF tissue samples; however, their expression levels were moderately decreased (P > 0.05) in IPF cells compared with controls. A number of AT1-associated transcripts, on the other hand, were present at relatively high levels in HTII-280+ cells of IPF samples. The finding that the HTII-280+ cells from both control and IPF samples expressed AT2 cell gene signatures suggests that some IPF cells either maintain or acquire some AT2-like identity despite or possibly as a result of extensive tissue remodeling, respectively. AT1 cell–associated transcripts were either absent or were present at low levels in epithelial cells sorted from normal lung. Since the transcriptome of epithelial cells within remodeled lung tissue of IPF patients is likely to be impacted by the altered inflammatory and stromal milieu, we identified transcripts encoding cytokines, chemokines, growth factors, and associated genes that were selectively expressed in IPF epithelial cells. Supplemental Figure 3 demonstrates expression of mediators selectively expressed by IPF cells that are known to influence cell migration, chemotaxis, and epithelial cell growth, supporting their potential roles in the inflammatory and fibrotic processes in IPF.
Heatmap, principal component analysis, and predicted function in sorted normal and IPF epithelial cells. EPCAM+ (CD326+) and HTII-280+ epithelial cells from control and IPF donors were isolated from peripheral lung tissue by FACS and subjected to RNA sequencing (RNA-seq). (A) Principal component analysis (PCA) RNA-seq data from IPF and control donors (n = 3 per group) shows the primary separation of samples by disease status. (B) Heatmap represents 2D hierarchical clustering of genes and samples and shows differentially expressed genes in IPF versus control samples. (C) Functional enrichment of predicted biological processes and genes induced in IPF is shown. (D) Functional enrichment of predicted biological processes and genes suppressed in IPF is shown. x axis represents the –log10 transformed enrichment P value
Differential gene expression in normal and IPF HTII-280+ cells. Expression of genes involved in early lung morphogenesis (SOX9, CELSR1, SIX1, LGR4) and Wnt signaling PRKX, WNT7B, DKK1, PORCN) were largely induced in IPF epithelial cells, being negligibly expressed in normal (CD326/HTII-280) AT2 cells (Figure 2A). Expression of genes involved in “ion transport” (SLC26A9, SLCO4C1, CA2, SLC6A14, CFTR) were reduced in IPF HTII-280+ cells in comparison with the controls (Figure 2B). Genes associated with “fibrosis, pulmonary fibrosis, and idiopathic pulmonary fibrosis” were compiled from the disease-centered database HuGE Navigator (25) and OMIM (http://www.omim.org/). The overlap among known fibrosis-related genes and genes differentially expressed in CD326/HTII-280 cells from IPF tissues are identified. Relative expression levels of known fibrosis markers in control and IPF CD326/HTII-280 sorted cells were calculated and are shown in Figure 2C.
Representative genes and their relative expression in Control-CD326/HTII-280 versus IPF CD326/HTII-280 cell populations. Genes involved in (A) “branching morphogenesis” and Wnt signaling and (B) “anion transport” were induced and suppressed in IPF epithelial cells, respectively. RNA sequencing data from IPF and control donors (n = 3 per group); data are presented in dot plot with mean ± SEM. (C) Genes associated with fibrosis, pulmonary fibrosis, and idiopathic pulmonary fibrosis were compiled from the disease-centered database HuGE Navigator (ref. 25) and OMIM (http://www.omim.org/). The overlap between the known fibrosis genes and genes induced or suppressed in IPF HTII-280 was identified. Relative expression of known fibrosis-associated transcripts in control and IPF CD326/HTII-280–sorted cells was calculated as shown in the bar graph. The portion of fibrosis related genes in control is noted in blue, and IPF in red.
scRNA-seq analysis identifies distinct epithelial cell types in IPF. We utilized scRNA-seq to test whether the differences in gene expression between sorted IPF and control epithelial cells were related to admixtures of cells from conducting airways and peripheral lung or to fundamental changes in the differentiation state of individual epithelial cells. Viable epithelial cells were FACS enriched following lung tissue dissociation by virtue of their 7AAD–CD31–CD45–CD326+ phenotype and further separated into single cells using the Fluidigm C1 microfluidics system (26). scRNA-seq was obtained from 3 control and 6 IPF patient samples (Figure 3A). The average DNA fragment read per cell was greater than 4 million, average alignment 0.7, average sequencing quality score 34, and average coverage depth 125. In total, 540 cells from control (n = 215) and IPF patients (n = 325) passed quality control and were used for further analysis. We applied our newly developed analytic pipeline Sincera (27) to the dataset and identified 4 distinct cell clusters that we defined as AT2 (C1), “indeterminate” (C2), basal (C3), and goblet/club (C4) cells. The landscape of all cells in 3D and 2D space is shown by PCA and hierarchical clustering (Figure 3, A–C). Expression patterns of a subset of known lung epithelial cell markers are shown in Figure 3B. Epithelial-specific transcripts EPCAM and CDH1 were expressed in virtually all IPF and normal cells, demonstrating successful isolation of single epithelial cells and removal of stromal, vascular, and immune cells. Transcripts associated with AT2 cells, including SFTPC, SLC34A2, and ABCA3 were highly expressed in all AT2 cells from control lungs, consistent with RNA expression profiles in single AT2 cells from the mouse lung (26, 28), as well as those from HTII-280+ cells obtained from normal lung (Figure 1).
Single-cell RNA sequencing analysis from human IPF and normal lung epithelial cells. CD326+ epithelial cells were isolated from peripheral lung tissues by FACS as described in Methods, followed by single-cell isolation using Fluidigm C1 system and RNA sequencing. (A) Hierarchical clustering and principal component analysis (PCA) of 540 single cells from control (n = 3) and IPF patients (n = 6) reveals 4 major cell types (C1–C4), termed as normal AT2 (C1, green), indeterminate (C2, yellow), basal (C3, red), and club/goblet (C4, blue) cells. Single cells are colored by cluster on a 3D space. (B) Heatmap represents the expression of distinct RNAs that identify each of the 4 cell types. (C) Hierarchical clustering of all IPF and control cells using differentially expressed genes involved in epithelial proliferation (GO:0050673), “response to cytokines” (GO:0034097), and “response to growth factors” (GO:0034097) is shown. Minimum expression values were set to 0.01 TPM. Genes (n = 9,154) with specificity >0.7 and with TPM >1 in at least 10 cells in at least 6 samples were selected for hierarchical clustering using Z score–transformed expression.
All 3 IPF predominant cell types expressed increased levels of genes involved in epithelial proliferation (GO:0050673), genes “response to cytokines” (GO:0034097), and “response to growth factors (GO:0034097) (Figure 3C). Heterogeneity and variation among different patients were readily detectable, as shown by distribution of the 4 major epithelial cell types from IPF and from control lungs (Supplemental Figure 4). The relative abundance of the 4 major cell types varied somewhat among IPF patients, e.g., cells from sample 003 selectively expressed RNAs typical of goblet cells and lacked a clear basal cell signature.
Transcripts for signature genes defining normal AT2 cells and IPF-related basal, goblet, and indeterminate cells are shown in Figure 3C. Cluster C1 consisted of 95% of the control cells, sharing gene expression patterns consistent with AT2 cells. AT2 cells were well defined by expression of known AT2 cell-selective transcripts, including those encoding surfactant proteins and related proteins (SFTPB, SFTPC, NAPSA, LPCAT1, SLC34A2, and ABCA3) known to play critical and cell-selective roles in surfactant homeostasis in the alveolus. Potential regulators for cells within the C1 category include FOXA2, NKX2-1, CEBPA, and SREBF1. Functional classes selectively enriched in AT2 cells were absent in IPF, including genes mediating cytoprotection/detoxification and response to oxidative stress (e.g., SRXN1, CAT, FBVLN5, PRDX6, SOD2, SOD3, and GPX4) (Figure 3, B and C, and Table 1). We hypothesize that abnormal responses to oxidative stress may play a role in IPF pathogenesis. Interestingly, only 9 of 325 IPF epithelial cells clustered with normal epithelial cells in the C1 category, a finding that was consistent with dramatic loss of AT2 cells in IPF tissue.
Enriched functional annotations for cell clusters
Three distinct cell types were identified in the IPF samples by signature gene expression patterns that were consistent with conducting airway epithelial cells, e.g., basal (C3), goblet/club (C4), and indeterminate (C2) cells. Cells belonging to the C3 category harbored transcripts for TP63, KRT5, KRT14, BMP7, LAMB3, LAMC2, and ITGB, known markers of human basal cells (Figure 3B). Signature genes of the C3 cell cluster were involved in “alpha6-beta4 integrin signaling,” “wound healing,” “cell migration,” and “laminin interaction,” consistent with an airway origin for these cells (Table 1). Transcripts for SOX2, TP63, and TGFB1 were predicted as upstream regulators of cells belonging to the C3 cluster. IPF goblet-like cells (C4) selectively expressed SPDEF, a transcription factor regulating goblet cell differentiation (29, 30), MUC5AC, MUC5B, PIGR, AQP3, and SCGB1A1, transcripts that are characteristically expressed by airway secretory cells. Genes associated with “goblet cell morphology” (e.g., SPDEF, TCF7L2, and ELF3) and “O-linked glycosylation of mucins” (e.g., MUC16, MUC20, MUC4, MUC5AC, MUC5B, GALNT6, and GALNT5) were enriched in C4 IPF goblet cells (Table 1 and Figure 3, B and C).
Cells belonging to the C2 cluster express CD326 and CDH1 signature genes, but were not typical of any known epithelial cell type present in the normal lung. C2 cluster cells generally expressed AT2 cell–associated markers at higher levels than IPF basal or goblet/club cells and shared “lipid transport” and “innate immunity” functions with normal AT2 cells; however, C2 cells also expressed markers normally restricted to the proximal airway epithelium. The uniquely enriched functions predicted to be active in the C2 cluster included “activation of myofibroblasts,” “flux of anion,” and “T cell proliferation” (Table 1). The predicted driving forces (key regulators) for the C2 cluster include CTGF, GF11, and FL11) (see below). Although all of the cells expressed CDH1 and EPCAM, the C2 cells did not express clear signature genes associated with any known lung epithelial subtypes, and we termed these cells “indeterminate” IPF cells.
scRNA-seq analysis reveals “bronchiolization” and novel epithelial cell types in IPF. Through the single-cell analysis, we noted that RNAs associated with AT2 cells, including SFTPC, SLC34A2, and ABCA3, were highly expressed in all normal AT2 cells present within the C1 cluster (26, 28). Conducting airway epithelial cell–selective marker transcripts, including SOX2, PAX9, TP63, KRT5, KRT14, MUC5B, and MUC5AC, were generally absent from single cells from normal lung but were present in many IPF cells (Figure 4A). While AT1-associated transcripts (e.g., AQP5, AQP3, AGER, CLIC5) were not enriched in control samples, remarkably, these transcripts were present in relative higher levels in IPF cells. The distinct, squamous morphology and fragility of AT1 cells likely exclude them from the isolation process. Although ciliated cells are readily detected in IPF samples by light and confocal microscopy, there was clear absence of multiciliated cell signature genes, indicating their loss in the cell isolation process. The presence of AT2 and AT1 transcripts and their coexpression with conducting airway and other alveolar markers seen in the single-cell RNA profiles support the hypothesis that the epithelial cells of the remodeled distal lung in IPF acquire atypical mixed differentiation states. While conducting airway-associated genes (SOX2, MUC5B, and PAX9) were rarely or not expressed in single cells from control tissue, these transcripts were frequently expressed in a subset of single IPF cells, even in cells expressing normally AT2-restricted RNAs (Figure 4B). Likewise, single cells expressing goblet cell–associated transcripts — e.g., MUC5AC, SPDEF, LTE, DUSP4, and KRT6A, genes normally selectively expressed in conducting airway epithelial cells — were identified in IPF, but not in control AT2 cells, consistent with the “bronchiolization” typical of IPF. Transcriptomes seen in individual cells demonstrate (a) the loss of normal regional proximal-peripheral cellular identity and (b) coexpression of normally cell type–restricted transcripts in some IPF epithelial cells, indicating loss of normal epithelial cell type identity.
Single-cell RNA analysis identifies altered epithelial gene expression and epithelial cell types in IPF. (A) Single cells from human IPF (n = 6) and donor (n = 3) distal lung (CD326+) were prepared using the Fluidigm C1 system. RNA was prepared and analyzed from a total of 325 single cells from IPF and 215 cells from donor lungs. Shown are lung epithelial cell markers: EPCAM and CDH1; alveolar type 1 cell markers: AGER and HOPX; alveolar type 2 cell markers: SFTPC, SLC34A2, and ABCA3; proximal lung epithelial cell markers: SOX2, PAX9, TP63, KRT5, KRT14, MUC5B, and SCGB1A1. Expression values were measured in TPM and square root (sqrt) normalized. Cells are shown in solid colors if the expressions of the markers were greater than 1 (TPM). (B) MUC5B, PAX9, and SOX2 were selectively expressed in subsets of IPF cells (MUC5B: n = 24, PAX9: n = 65; SOX2: n = 24) but not present in C1 control cells. Representative genes clustering with MUC5B, PAX9, and SOX2 in IPF cells are shown in the heatmaps. Equal numbers of control cells were randomly selected. IPF cells expressed a diversity of conducting airway epithelial markers not present in control cells, the latter expressing RNAs characteristic of AT2 cells. (C) Only 9 of 325 IPF cells clustered with control cells, the heatmap indicating “AT2”-like expression patterns; however, these 9 normal IPF cells also coexpressed some of the of IPF-associated disease markers. Expression data (TPM) were log10 transformed.
scRNA-seq analysis identifies potential biomarkers for IPF based on the distinct IPF epithelial cell subtypes. Through the single-cell analysis, we identified signature genes associated with each epithelial cell subtype. Upon validation, these genes may serve as new and cell-selective biomarkers to monitor and predict IPF disease processes that would be useful for diagnosis, prognosis, or therapeutic monitoring. For example, MMP-7, a biomarker indicating IPF prognosis and disease activity in IPF (31, 32), was most highly expressed in the IPF goblet cells (Figure 5). While SOX2 and SOX9 are normally expressed in a mutually exclusive pattern in endoderm of proximal and distal tubules of the developing lung, SOX2 transcripts were enriched in goblet (C4 cluster) and SOX9 transcripts in basal cells (C3 cluster) in IPF. Remarkably, transcripts for SOX2 and SOX9 were frequently coexpressed in indeterminate (C2) cells in IPF, perhaps indicating disruption of proximal-distal patterning (Figure 5). Expression of genes regulating epithelial fluid and electrolyte transport was disrupted in IPF. Chloride transporters, including CLCN2/4/5, SLC26A4, SLC6A14, and CFTR, were significantly decreased in IPF, while the expression of amiloride-sensitive sodium transporter subunits SCNN1G and SCNN1B and bicarbonate transporters CA14, SLC26A8, SLC4A1, and CA5A was induced. Dramatic alternations in expression of associated genes and loss of CFTR are likely to influence mucociliary clearance in a manner similar to that in cystic fibrosis (CF). Expression of ABCA3, a gene essential for AT2 cell lipid transport, was decreased in IPF C2 indeterminate cells and absent in C3 and C4 IPF cells.
Expression of predicted IPF marker genes in 4 epithelial cell types from IPF and control single-cell samples. Violin plots show the expression of the gene markers in all 540 cells from the 4 cell types. Cell types are color coded. Green: AT2 (n = 219); orange: indeterminate (n = 91); red: basal (n = 131); blue: club/goblet (n = 101). One-tailed Welch’s t test was used to identify cell type–specific gene markers. **P < 0.05.
Prediction of active cell signaling pathways in IPF epithelial cells. To identify signaling pathways involved in the pathological changes in IPF, we performed enrichment analysis of the KEGG pathways (http://www.genome.jp/kegg/) using differentially expressed genes in the AT2 cell cluster (C1) and each of the IPF cell clusters (C2, C3, and C4). KEGG pathways enriched or suppressed in IPF epithelium were determined by the following criteria: (a) at least 5 genes in the pathway are expressed (transcripts per kilobase million [TPM] ≥1); (b) at least 30% of expressed genes were differentially expressed; and (c) the ratio between the number of C1 differentially expressed genes and the number of IPF differentially expressed genes in the pathway was ≥1.5 or ≤0.67. Pathways were ranked based on the ratios. The average expression of pathway genes in each individual cell was calculated for each significantly altered KEGG pathway. A heatmap (Figure 6A) represents single-cell gene expression profiles of the top 25 ranked KEGG pathways significantly altered in IPF epithelial cells. HIPPO/YAP, TGF-β, and PI3K/AKT signaling pathways were induced in single cells in IPF, findings consistent with RNA data from FACS-sorted cells (Figure 1). Likewise, processes related to normal AT2 cell functions including lipid synthesis and metabolism were suppressed in IPF. Figure 6B shows the relative expression of representative TGF-β signaling pathway genes (BMP1, BMPR1B, INHBA, INHBB, TGFBR1, TGFB1, TGFB2, and SMAD3) in control (n = 215) and IPF (n = 316) cells, and in the 9 IPF cells that clustered together with control AT2 cells. As shown, even the 9 relatively “normal” IPF AT2-like cells expressed significantly higher levels of TGFBR1 and VIM that may represent less advanced stages of IPF pathology (Figure 6B and Supplemental Table 3). Although all IPF cells maintained epithelial cell identities, vimentin, normally selectively expressed in mesenchymal cells, was increased in basal and indeterminate cells in IPF, with highest expression in the relatively “normal” IPF AT2-like cells, perhaps indicating epithelial-mesenchymal transition (EMT) in the early transitional stage of IPF (Supplemental Table 3).
Expression of altered KEGG pathways in human IPF and control single cells. (A) The heatmap shows the top 25 pathways and differentially expressed genes identified using a 1-tailed Welch’s t test of gene expression between the control AT2 cells (C1) and IPF cell clusters (C2, C3, and C4) using the following criteria: P < 0.01, expressed (TPM ≥1) in at least 80% of cell type with induced gene expression. KEGG pathways enriched or suppressed in IPF epithelium were determined by the following criteria: (a) at least 5 genes in the pathway were expressed (TPM ≥1), (b) at least 30% of expressed genes were differentially expressed, and (c) the ratio between the number of C1 differentially expressed genes and the number of IPF differentially expressed genes in the pathway was ≥1.5 or ≤0.67. 4). Pathways were ranked based on the ratios. The expression of a pathway in a cell was measured by the average expression (TPM + 1, log2 transformed) of differentially expressed genes associated in the pathway. Pathways were clustered using hierarchical clustering analysis with Spearman’s correlation–based distance measure and complete linkage. Cancer- or disease-related pathways were excluded. (B) Representative TGF-β signaling pathway genes (BMP1, BMPR1B, INHBA, INHBB, TGFBR1, TGFB1, TGFB2, and SMAD3) in control (n = 215), IPF (n = 316), and relatively normal IPF cells that clustered with control AT2 cells (n = 9). Data are presented as dot plot with mean ± SEM. P values were determined by Student’s t test. **P < 0.05.
Single-cell transcriptome analysis predicted the activation of EMT from basal to indeterminate cells (Supplemental Figure 5). EMT-related signaling molecules (TGFBR1, Wnt/ β -catenin, EGFR, PI3K/AKT), transcription factors (ZEB1, SNAl2, SMAD2/3), and an mRNA splicing factor involved in EMT (ESRP1) were selectively induced in basal cells. While E-cadherin (CDH1) was suppressed in basal cells, mesenchymal markers acquired in EMT including MMPs, vimentin, N-cadherin (CDH12), and fibronectin (FN1) were selectively induced in either basal or indeterminate cells, indicating that basal cells may be the progenitor of the indeterminate cell type. A number of genes regulating planar polarity or EMTs, e.g., PRICKLE1, VANGL1, SNAI2, and VIM, also were induced in IPF cells, consistent with cell shape alterations, and pan-cytokeratin, E-cadherin, and vimentin staining in IPF epithelial cells (Figure 4, Supplemental Figure 5, Supplemental Figure 6A, and Supplemental Table 3). In spite of the enrichment of transcripts related to epithelial cell proliferation in KEGG cells observed in the pathway analysis of IPF cells (Figure 1 and 3), phospho–histone 3 staining was rarely detected in epithelial cells in either normal or IPF lung tissue (Supplemental Figure 6B).
Expression of conducting airway and alveolar cell markers in IPF epithelial cells. Since single-cell transcriptome analysis identified remarkable changes in epithelial cell gene expression patterns, we utilized immunofluorescence confocal microscopy to distinguish epithelial cells in normal and IPF lungs. In normal lung, cuboidal AT2 epithelial cells were identified by coexpression of both HTII-280 (24) and ABCA3. HOPX intensely stained the cytoplasm and nuclei of squamous AT1 cells and was detected at lower levels in normal AT2 cells (Figure 7). Neither ABCA3 nor HTII-280, AT2 cell markers present in normal lungs, was detected in conducting airways. In contrast to normal alveolar and bronchial tissue, lesions in the periphery of IPF lungs contained epithelial cells of atypical shapes and staining characteristics. Abnormally shaped cells intensely costaining for HOPX, ABCA3, and/or HTII-280 were present in all IPF samples, and normal squamous AT1 cells were rarely detected (Figure 7). Ciliated, basal, and goblet cell markers were frequently observed in IPF lesions, found in close proximity to ABCA3-stained cuboidal cells, indicating a loss of regional specification and epithelial cell gene expression (Figure 7A). The epithelial lining of IPF cysts frequently contained p63-positive basal cells located in close proximity to cells expressing ABCA3 or HOPX. Clusters of KRT14+ “basal” cells were also present in these IPF lesions (Figure 7B). Thus, differentiation-specific cell markers that are normally spatially restricted in conducting versus alveolar regions were frequently found in close contiguity within the IPF lesions. Similarly, individual IPF cells coexpressing SFTPB and MUC5B, markers normally restricted to distinct alveolar and goblet epithelial cell types, were readily identified in IPF tissues, findings consistent with the “abnormal” cell differentiation characteristics seen in the single-cell RNA analyses (Supplemental Figure 7). Although α-SMA was not detected in IPF epithelial cells, vimentin, which normally stains only mesenchymal cells, was detected in subsets of pan-cytokeratin– and E-cadherin–stained epithelial cells, perhaps indicating a partial epithelial to mesenchymal transition, findings consistent with Vim and pathway analysis of the single-cell IPF RNA profiles (Supplemental Figure 6A).
Immunofluorescence confocal microscopy identifies atypical epithelial cell differentiation in IPF. (A) Peripheral samples of normal and IPF lung tissue were stained for epithelial cell markers used to identify AT2 (HTII-280 and ABCA3), AT1 (HOPX), ciliated (TUBA4A), goblet (MUC5B) cells. Yellow staining indicates coexpression of the proteins. HTII-280 and ABCA3, normally restricted to peripheral/alveolar epithelial cells in normal lung, were expressed in IPF lesions; cystic lesions were variably lined by hyperplastic AT2 cells that stained for ABCA3 in close proximity to MUC5B (goblet) or TUBA4A (ciliated) stained cells. Abnormally shaped epithelial cells variably staining for HOPX, ABCA3, and HTII-280 were characteristic of IPF tissues that generally lacked normal squamous AT1 cells. (B) Epithelial cells expressing conducting airway and alveolar epithelial cell markers were found in close proximity in the IPF lesions (e.g., TP63, KRT14 and MUC5B) and (ABCA3 and HOPX) respectively are shown. Figures are representative of n = 3–5 control and 9 IPF samples, except for KRT14 (n = 3). Images were obtained at ×10 magnification (scale bars: 200 μm). Insets in yellow boxes are at ×60 magnification