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 5

Macrophages and fibroblasts promote the accumulation of transitional AECs.

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Macrophages and fibroblasts promote the accumulation of transitional AEC...
(A) Ccr2+/+ or Ccr2–/– mice were treated with bleomycin. Macrophage recruitment was necessary for transitional state accumulation. #P < 0.05 and ##P < 0.01, by 2-way ANOVA for Ccr2+/+ versus Ccr2–/– from days 4–21. n = 5 mice/group. (B) Macrophages and monocytes were a major source of TGF-β and IL-1β in murine and human fibrosis, as determined by scRNA-Seq (54, 58). Bleo, bleomycin. (C) Col1a1CreERT2 Fasfl/fl mice were treated with bleomycin or administered tamoxifen (KO) or corn oil (WT), and euthanized at 9 weeks. Fibroblast-specific Fas knockout induced myofibroblast persistence, which was sufficient for persistence of the AEC transitional state. #P < 0.05, by t test. n = 3 mice/group. (D) Gene expression by AECs cultured on Matrigel or collagen-coated plastic for 3 days. Collagen/stiff substrate promoted transitional state accumulation. Data represent the mean ± SD. #P < 0.05 and ##P < 0.01, by paired t test. (E) Murine AEC2s were cultured in 2D and fixed and immunostained on day 7. Most cells persisted in the transitional state, with some AEC1 differentiation (n = 3). (F and G) Transitional cells were found in small foci of fibrosis in peripheral lung in the PNX and LPS mouse models (*), whereas most of the lung was devoid of fibrosis and transitional cells (**). In the bleomycin model, large areas of lung were characterized by fibrosis and transitional cells (*), whereas some areas were devoid of fibrosis and transitional cells (**). Scale bars: 50 μm. Original magnification, ×20 (enlarged insets in A, C, E, and F). For immunostaining, n = 3/group. PDPN, podoplanin; αSMA, α smooth muscle actin.