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 3

K8 promotes the expression of chemokines but not the accumulation of transitional cells at the expense of AEC1 differentiation.

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K8 promotes the expression of chemokines but not the accumulation of tra...
AEC2s were isolated from Krt8+/+ and Krt8–/– mice and cultured in 2D. RNA-Seq was performed. (A) Average fold change (FC) of composite AEC2, transitional state, or AEC1 marker scores (see also Supplemental Table 2) compared with day 0 for WT AECs. *P < 0.05 compared with day 0. AEC culture recapitulates in vivo stages of alveolar regeneration, as shown by downregulation of AEC2 markers and upregulation of transitional state markers on day 1 of culturing and a gradual upregulation of AEC1 markers by day 7. Far-right panel is a schematic representation of the data. (B) Krt8 deficiency had no effect on transitional cell or AEC1 differentiation. ****P < 0.0001, by unpaired t test on the AUC from days 1–7 for Krt8+/+ versus Krt8–/– cells. (C) Markers of senescence, TGF-β activation, impaired proteostasis, DNA damage, and cell death were upregulated in the transitional state in vitro. *P ≤ 0.05 by t test for day 1 compared with day 0 for all pathway scores. P values for genes in heatmaps are listed in Supplemental Table 3. (D) K8 was not necessary for upregulation of markers of cell-cycle arrest, TGF-β activation, impaired proteostasis, DNA damage, and cell death. For individual genes, the ratio of AUC of expression from days 1–7 in Krt8–/– versus Krt8+/+ cells is shown. (E) K8 was necessary for the expression of SASP chemokines but not proinflammatory cytokines, growth factors, or proteases/antiproteases. #P < 0.05, by t test of the average of the ratio of AUCs of all chemokines; *P < 0.05, by t test for individual genes. n = 3. All data are presented as the mean (A and B) or the mean ± SEM (C–E).