Regulation of epithelial transitional states in murine and human pulmonary fibrosis
Fa Wang, Christopher Ting, Kent A. Riemondy, Michael Douglas, Kendall Foster, Nisha Patel, Norihito Kaku, Alexander Linsalata, Jean Nemzek, Brian M. Varisco, Erez Cohen, Jasmine A. Wilson, David W.H. Riches, Elizabeth F. Redente, Diana M. Toivola, Xiaofeng Zhou, Bethany B. Moore, Pierre A. Coulombe, M. Bishr Omary, Rachel L. Zemans
Fa Wang, Christopher Ting, Kent A. Riemondy, Michael Douglas, Kendall Foster, Nisha Patel, Norihito Kaku, Alexander Linsalata, Jean Nemzek, Brian M. Varisco, Erez Cohen, Jasmine A. Wilson, David W.H. Riches, Elizabeth F. Redente, Diana M. Toivola, Xiaofeng Zhou, Bethany B. Moore, Pierre A. Coulombe, M. Bishr Omary, Rachel L. Zemans
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Research Article Pulmonology

Regulation of epithelial transitional states in murine and human pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease arising from impaired regeneration of the alveolar epithelium after injury. During regeneration, type 2 alveolar epithelial cells (AEC2s) assume a transitional state that upregulates multiple keratins and ultimately differentiate into AEC1s. In IPF, transitional AECs accumulate with ineffectual AEC1 differentiation. However, whether and how transitional cells cause fibrosis, whether keratins regulate transitional cell accumulation and fibrosis, and why transitional AECs and fibrosis resolve in mouse models but accumulate in IPF are unclear. Here, we show that human keratin 8 (KRT8) genetic variants were associated with IPF. Krt8–/– mice were protected from fibrosis and accumulation of the transitional state. Keratin 8 (K8) regulated the expression of macrophage chemokines and macrophage recruitment. Profibrotic macrophages and myofibroblasts promoted the accumulation of transitional AECs, establishing a K8-dependent positive feedback loop driving fibrogenesis. Finally, rare murine transitional AECs were highly senescent and basaloid and may not differentiate into AEC1s, recapitulating the aberrant basaloid state in human IPF. We conclude that transitional AECs induced and were maintained by fibrosis in a K8-dependent manner; in mice, most transitional cells and fibrosis resolved, whereas in human IPF, transitional AECs evolved into an aberrant basaloid state that persisted with progressive fibrosis.

Authors

Fa Wang, Christopher Ting, Kent A. Riemondy, Michael Douglas, Kendall Foster, Nisha Patel, Norihito Kaku, Alexander Linsalata, Jean Nemzek, Brian M. Varisco, Erez Cohen, Jasmine A. Wilson, David W.H. Riches, Elizabeth F. Redente, Diana M. Toivola, Xiaofeng Zhou, Bethany B. Moore, Pierre A. Coulombe, M. Bishr Omary, Rachel L. Zemans

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Figure 2

K8 promotes fibrosis and accumulation of transitional AECs.

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K8 promotes fibrosis and accumulation of transitional AECs. (A) Using a ...
(A) Using a genome-wide association meta-analysis of IPF (75), a nested candidate gene study for the keratins expressed in the transitional state was performed. Regional association plot showing all SNPs that overlap with KRT8. Gray dotted line indicates genome-wide significance; red dotted line indicates statistical significance of the nested candidate gene study for the keratin genes; blue curve indicates the estimated recombination rate. Seven KRT8 SNPs were associated with IPF (P < 1.4 × 10–4). The most significant variant, rs4531558 (P = 5.2 × 10–5), shown as a purple diamond, is in linkage disequilibrium (LD) (R2 > 0.8) with all other statistically significant variants. (B–G) Krt8+/+ and Krt8–/– mice were treated with bleomycin. Krt8–/– mice were protected from fibrosis, as determined by hydroxyproline assay (B), trichrome staining (C), and myofibroblast accumulation (D). Arrowhead in C indicates a small area of fibrosis. Krt8–/– mice were not protected from lung injury at day 4, as determined by inflammation (E) and permeability (F). (G) Compared with Krt8–/– mice, transitional cells accumulated with incomplete AEC1 regeneration in Krt8+/+ mice. (B, E, and F) Data are represented as box-and-whisker plots, with box (25th to 75th percentiles), median (line), and whiskers (minimum to maximum). *P < 0.05 and **P < 0.01, by 1-way ANOVA with post hoc Bonferroni’s test. (G) Data indicate the mean ± SD. *P < 0.05, by 2-way ANOVA with post hoc Šidák’s multiple-comparison test. Scale bars: 200 μm. Original magnification, ×20 (enlarged insets in D). n = 3 mice/group except bleomycin-treated mice in B, n = 14 mice/group and E, n = 6 mice/group.