External data and methods supplement for:
Ting C, Konopka K, Benedeck RE, Riches DWH, Redente EF, Oldham JM, Zemans RL. Biomarkers Unveil Insights into Pathology of Transitional Epithelial States in Pulmonary Fibrosis. Am J Respir Crit Care Med. 2024 Jun 18. doi: 10.1164/rccm.202310-1839LE. Epub ahead of print. PMID: 38889340
The following links contain zoomable images of larger tissue scans, hosted externally at pathpresenter.net. Loading times may exceed 1 minute for larger images depending on internet connection speed. You are welcome to use these images in your own work - please credit [Ting C, Konopka K, Benedeck RE, Riches DWH, Redente EF, Oldham JM, Zemans RL. Biomarkers Unveil Insights into Pathology of Transitional Epithelial States in Pulmonary Fibrosis. Am J Respir Crit Care Med. 2024 Jun 18. doi: 10.1164/rccm.202310-1839LE. Epub ahead of print. PMID: 38889340] if you decide to do so.
In all images, DAPI: blue, K8: red, and K17: cyan
Idiopathic pulmonary fibrosis
Interstitial lung abnormalities
Repetitive bleomycin model
Single bleomycin model
Evaluation of proteomic and transcriptomic datasets
Significant plasma proteomic biomarkers associated with IPF progression (1), ILA presence, or progression of ILA at followup (2) were determined by taking all proteomic biomarkers with a hazard ratio (1) or odds ratio >1 (2) and adjusted p-value <0.05 for each outcome. UMI count matrices from scRNAseq datasets of IPF lung (3) was obtained from the NCBI Gene Expression Omnibus. In Habermann et al., the cell population "Transitional AT2" is named "KRT8hi/KRT17lo" in our study, and the "KRT5-/KRT17+" cell population is named "KRT8hi/KRT17hi." Z-scores were calculated relative to all cell types. Transcriptomic expression of all plasma proteomic biomarkers associated with the outcome of interest was queried and expressed as an average Z-score.
For the externally hosted figures (Supplemental Figure 1A-C), differentially expressed genes (DEGs) for the KRT8hi/KRT17hi cell state compared to all other epithelial cells were determined. The list of significant plasma proteomic biomarkers was compared to the list of DEGs of the KRT8hi/KRT17hi state to determine overlap. p<0.0001 by Chi Square and Fisher’s exact tests.
Bleomycin Injury Models
Single dose bleomycin and multiple dose bleomycin models were performed as previously described (4, 5). Briefly, 6-8 week old C57BL6 mice were maintained in a pathogen-free environment with free access to food and water. All studies were approved by the University of Michigan Institutional Animal Care and Use Committee. Mice were anesthetized with inhaled isoflurane and given i.t. either a single dose of 1U/kg pharmaceutical grade bleomycin (Hikma NDC 0143-9240-01) in 50 µl endotoxin-free normal saline, or every other week for three total doses for the multiple bleomycin model. Lungs were harvested at day 21 for the single bleomycin model and six weeks after the third dose of bleomycin for the multiple bleomycin model for formalin fixation and paraffin embedding.
Immunostaining
Human pulmonary fibrosis tissue was obtained from the University of Michigan Interstitial Lung Disease Biorepository and from the University of Michigan Department of Pathology. Immunostaining on formalin-fixed and paraffin-embedded tissue was performed as previously described (4). Primary antibodies used were: K8 (University of Iowa Developmental Studies Hybridoma Bank TROMA-1, 1:20), K17 (Abcam ab53707, 1:100). Secondary antibodies used were Cy3-conjugated anti-rat (Jackson ImmunoResearch Laboratories 712-165-153) and Cy5-conjugated anti-rabbit (Jackson ImmunoResearch 711-175-152). Images were acquired using a BX53 microscope (Olympus) or Aperio AT2 DX (Leica) microscope and area measurements performed using cellSens Dimension V4.2 software. K8+K17+ transitional cells were counted manually and represented as number of cells per square millimeter. All images represent n≥3 per group. Scale bars = 200 µm.
1. Oldham JM, Huang Y, Bose S, Ma SF, Kim JS, Schwab A, Ting C, Mou K, Lee CT, Adegunsoye A, Ghodrati S, Vu Pugashetti J, Nazemi N, Strek ME, Linderholm AL, Chen CH, Murray S, Zemans RL, Flaherty KR, Martinez FJ, Noth I. Proteomic Biomarkers of Survival in Idiopathic Pulmonary Fibrosis. Am J Respir Crit Care Med 2023.
2. Axelsson GT, Gudmundsson G, Pratte KA, Aspelund T, Putman RK, Sanders JL, Gudmundsson EF, Hatabu H, Gudmundsdottir V, Gudjonsson A, Hino T, Hida T, Hobbs BD, Cho MH, Silverman EK, Bowler RP, Launer LJ, Jennings LL, Hunninghake GM, Emilsson V, Gudnason V. The Proteomic Profile of Interstitial Lung Abnormalities. Am J Respir Crit Care Med 2022; 206: 337-346.
3. Habermann A, Gutierrez A, Bui L, Yahn S, Winters N, Calvi C, Peter L, Chung M-I, Taylor C, Jetter C, Raju L, Roberson J, Ding G, Wood L, Sucre JMS, Richmond B, Serezani A, McDonnell W, Mallal S, Bacchetta M, Loyd J, Shaver C, Ware L, Bremner R, Walia R, Blackwell T, Banovich N, Kropski J. Single-cell RNA sequencing reveals profibrotic roles of distinct epithelial and mesenchymal lineages in pulmonary fibrosis. Science Advances 2020; 6: eaba1972-eaba1972.
4. Wang F, Ting C, Riemondy KA, Douglas M, Foster K, Patel N, Kaku N, Linsalata A, Nemzek J, Varisco BM, Cohen E, Wilson JA, Riches DW, Redente EF, Toivola DM, Zhou X, Moore BB, Coulombe PA, Omary MB, Zemans RL. Regulation of epithelial transitional states in murine and human pulmonary fibrosis. J Clin Invest 2023; 133.
5. Redente EF, Black BP, Backos DS, Bahadur AN, Humphries SM, Lynch DA, Tuder RM, Zemans RL, Riches DWH. Persistent, Progressive Pulmonary Fibrosis and Epithelial Remodeling in Mice. Am J Respir Cell Mol Biol 2021; 64: 669-676.