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. 2020 Dec 8;6(1):e141618.
doi: 10.1172/jci.insight.141618.

Loss of Fas signaling in fibroblasts impairs homeostatic fibrosis resolution and promotes persistent pulmonary fibrosis

Elizabeth F Redente  1   2   3 , Sangeeta Chakraborty  1 , Satria Sajuthi  4 , Bart P Black  1 , Ben L Edelman  1 , Max A Seibold  1   2   4 , David Wh Riches  1   2   3   5
Affiliations

Loss of Fas signaling in fibroblasts impairs homeostatic fibrosis resolution and promotes persistent pulmonary fibrosis

Elizabeth F Redente et al. JCI Insight. .

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible fibrotic disease of the distal lung alveoli that culminates in respiratory failure and reduced lifespan. Unlike normal lung repair in response to injury, IPF is associated with the accumulation and persistence of fibroblasts and myofibroblasts, as well as continued production of collagen and other extracellular matrix (ECM) components. Prior in vitro studies have led to the hypothesis that the development of resistance to Fas-induced apoptosis by lung fibroblasts and myofibroblasts contributes to their accumulation in the distal lung tissues of IPF patients. Here, we test this hypothesis in vivo in the resolving model of bleomycin-induced pulmonary fibrosis in mice. Using genetic loss-of-function approaches to inhibit Fas signaling in fibroblasts, potentially novel flow cytometry strategies to quantify lung fibroblast subsets, and transcriptional profiling of lung fibroblasts by bulk and single cell RNA sequencing, we show that Fas is necessary for lung fibroblast apoptosis during homeostatic resolution of bleomycin-induced pulmonary fibrosis in vivo. Furthermore, we show that loss of Fas signaling leads to the persistence and continued profibrotic functions of lung fibroblasts. Our studies provide insights into the mechanisms that contribute to fibroblast survival, persistence, and continued ECM deposition in the context of IPF and how failure to undergo Fas-induced apoptosis impairs fibrosis resolution.

Keywords: Apoptosis; Fas signaling; Fibrosis; Pulmonology.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Fas expression in mesenchymal cells is essential for their spontaneous apoptosis during fibrosis resolution.
(A) Cell surface expression on cultured fibroblasts from WT and Fas-deficient mice. (B) Caspase 3 activity in WT and Fas-deficient fibroblasts after Fas ligation. (C) Hydroxyproline levels in the lungs over time after bleomycin in Dermo1-Cre;Fas+/+ and Dermo1-Cre;Fas–/– mice. (D and E) Representative H&E-stained (D) and Picrosirius red–stained (E) lung sections over time in Dermo1-Cre;Fas+/+ and Dermo1-Cre;Fas–/– mice. Box-and-whisker plots show median, minimum, and maximum values. Time course is mean ± SEM, n = 6–8. *P < 0.05, **P < 0.01, 2-tailed t test with Welch’s correction. Total magnification, 200× (upper panels) and 400× (lower panels).
Figure 2
Figure 2. Fas expression in collagen producing cells is essential for their spontaneous apoptosis during fibrosis resolution.
(A) Sketch illustrating instillation time, tamoxifen dosing, and harvest time points in Col1-CreERT2;Fasfl/fl mice. (B) Hydroxyproline levels in the lungs over time after bleomycin in Col1-CreERT2;Fas+/+ and Col1-CreERT2;Fas–/– mice. Representative H&E-stained (C) and Picrosirius red–stained (D) lung sections over time in Col1-CreERT2;Fas+/+ and Col1-CreERT2;Fas–/– mice. Box-and-whisker plots show median, minimum and maximum values. Time course is mean ± SEM, n = 6-8. **P < 0.01, 2-tailed t test with Welch’s correction. Total original magnification, 200× (upper panels) and 400× (lower panels).
Figure 3
Figure 3. Fibroblasts persist in fibrotic lungs in the absence of Fas.
(A) α-SMA (green) and S100A4 (red) immunofluorescence staining of lungs over time after bleomycin in Dermo1-Cre;Fas+/+ and Dermo1-Cre;Fas–/– mice. (B–D) Quantification of Lin–, PDGFRα+, and PDGFRα– fibroblasts populations. (E–G) Quantification of CD90+CD26–, CD26+CD90–, and CD90–CD26– fibroblast subsets. Time course is mean ± SEM, n = 6–8. *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed t test with Welch’s correction. Total original magnification, 200× (upper panels) and 400× (lower panels).
Figure 4
Figure 4. Deletion of Fas in Col-1 fibroblasts permits their persistence during fibrosis.
(A) α-SMA (green) and S100A4 (red) immunofluorescence staining of lungs over time after bleomycin in Col1-CreERT2;Fas+/+ and Col1-CreERT2;Fas–/– mice. (B–D) Quantification of Lin–, PDGFRα+ and PDGFRα– fibroblast populations. (E–G) Quantification of CD90+CD26–, CD26+CD90–, and CD90–CD26– fibroblast subsets. Time course is mean ± SEM, n = 6–9. *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed t test with Welch’s correction. Total original magnification, 200× (upper panels) and 400× (lower panels).
Figure 5
Figure 5. Loss of Fas reduces apoptosis of fibroblasts during fibrosis resolution in vivo.
(A and B) Immunofluorescence staining and quantification of fibroblasts using antibodies for α-SMA (green), S100A4 (red), and TUNEL (white) in Dermo1-Cre;Fas+/+ and Dermo1-Cre;Fas–/– mice over time after bleomycin. (C and D) Immunofluorescence staining and quantification of fibroblasts using antibodies for SMA (green), S100A4 (red), and TUNEL (white) in Col1-CreERT2;Fas+/+ and Col1-CreERT2;Fas–/– mice over time after bleomycin. Time course is mean ± SEM, n = 10 images per animal/time point. *P < 0.05, 2-tailed t test with Welch’s correction. Total original magnification, 200× (upper panels) and 400× (lower panels).
Figure 6
Figure 6. Fibroblast expression of Col1 and α-SMA during fibrosis development and resolution.
(A) GFP expression of Col1a1 in fibroblasts over time after bleomycin in Col1-GFP mice. (B–D) Quantification and representative flow cytometry plots of GFP+ CD90+CD26, CD26+CD90–, and CD90–CD26– fibroblast subsets over time. (E) RFP expression of α-SMA in fibroblasts over time after bleomycin in α-SMA–RFP mice. (F–H) Quantification and representative flow cytometry plots of RPF+ CD90+CD26–, CD26+CD90–, and CD90–CD26– fibroblast subsets over time. Time course is mean ± SEM, n = 5–12. Total original magnification, 200× (upper panels) and 400× (lower panels).
Figure 7
Figure 7. Fibroblast expression of Col1 and α-SMA during fibrosis persistence.
(A) GFP expression of Col1a1 in fibroblasts 6 weeks after bleomycin in Col1-CreERT2;Fas+/+;Col1-GFP and Col1-CreERT2;Fas–/–;Col1-GFP mice. (B–D) Quantification of GPF+ CD90+CD26–, CD26+CD90–, and CD90–CD26– fibroblast subsets. (E) RFP expression of α-SMA in fibroblasts 6 weeks after bleomycin in Col1-CreERT2;Fas+/+;α-SMA–RFP and Col1-CreERT2;Fas–/–;α-SMA–RFP mice. (F–H) Quantification of RPF+ CD90+CD26–, CD26+CD90–, and CD90–CD26– fibroblast subsets. Box-and-whisker plots show median, minimum, and maximum values. n = 5. *P < 0.05, 2-tailed t test with Welch’s correction.
Figure 8
Figure 8. Bulk sequencing reveals a sustained profibrotic signature in fibroblasts in the absence of Fas.
(A) PCA plot of global transcription patterns in Col1-CreERT2;Fas+/+ and Col1-CreERT2;Fas–/– Lin– fibroblasts from control and bleomycin-treated mice after 3 and 6 weeks. (B) Representation of enrichment for GO pathway categories 6 weeks after bleomycin from Col1-CreERT2;Fas+/+ and Col1-CreERT2;Fas–/– Lin– fibroblasts. (C) Box-and-whisker plots of genes expressed during fibrosis and in nonresolving Col1-CreERT2;Fas–/– fibroblasts. Box-and-whisker plots show median, minimum, and maximum values. n = 4. *P < 0.05, **P < 0.01, 2-tailed t test with Welch’s correction.
Figure 9
Figure 9. scRNA-seq reveals distinct fibroblast populations in naive and fibrotic lungs.
(A) UMAP plot of all samples and the identified population clusters and from Fas-sufficient naive mice 3 and 6 weeks after bleomycin. (B) Violin plots showing genes highly associated with Wnt2+ Lipofibroblasts (clusters 1 and 2). (C) Violin plots showing genes highly associated with Col14a1+ mesenchymal alveolar niche cells (MANCs) (clusters 3 and 4). (D) Violin plots showing genes highly associated with profibrotic fibroblasts (clusters 5 and 6). O, combined expression of other clusters (clusters 3, 6, 8, 9, 11, 13, 15). (E) Representation of enrichment for GO pathway categories for clusters 1, 2, 3, 4, 5, and 6, a total of 6 weeks after bleomycin. (F) Pseudotime trajectory analysis for 2 scenarios. Violin plots show median. n = 4 pooled mice/group.
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