Resetting proteostasis with ISRIB promotes epithelial differentiation to attenuate pulmonary fibrosis

Abstract

Pulmonary fibrosis is a relentlessly progressive and often fatal disease with a paucity of available therapies. Genetic evidence implicates disordered epithelial repair, which is normally achieved by the differentiation of small cuboidal alveolar type 2 (AT2) cells into large, flattened alveolar type 1 (AT1) cells as an initiating event in pulmonary fibrosis pathogenesis. Using models of pulmonary fibrosis in young adult and old mice and a model of adult alveologenesis after pneumonectomy, we show that administration of ISRIB, a small molecule that restores protein translation by EIF2B during activation of the integrated stress response (ISR), accelerated the differentiation of AT2 into AT1 cells. Accelerated epithelial repair reduced the recruitment of profibrotic monocyte-derived alveolar macrophages and ameliorated lung fibrosis. These findings suggest a dysfunctional role for the ISR in regeneration of the alveolar epithelium after injury with implications for therapy.

Keywords: ISRIB; fibrosis; proteostasis.

Fig. 1.

Old mice develop more severe lung fibrosis compared to young adult mice. Young adult mice (3 to 5 mo) and old mice (18 to 24 mo) were administered bleomycin (0.025 unit/50 μL) or crocidolite asbestos (100 μg/50 μL) intratracheally, and the lungs were harvested as indicated. (A) Representative histologic images 28 d after bleomycin (Masson’s trichrome). (Scale bar, 1 mm.) (B) Ashcroft fibrosis score (Mann–Whitney U test with Bonferroni correction for multiple comparisons), lung compliance using Flexivent (one-way ANOVA with Tukey test for multiple comparisons), and soluble collagen in lung homogenates 28 d after bleomycin (one-way ANOVA with Tukey test for multiple comparisons). (C) Soluble collagen 0, 14, 28, and 56 d after the administration of bleomycin. Two-way ANOVA with Dunnett’s multiple comparisons test, comparing data from each day to baseline (day 0). (D) Body weight in young adult and old mice after the administration of bleomycin. Kruskal–Wallis test with Dunn’s multiple comparisons test, comparing data from each day to baseline (day 0). (E) Representative histologic images 28 d after the administration of asbestos (Masson’s trichrome). (Scale bar, 1 mm.) (F) Ashcroft fibrosis score (Mann–Whitney U tests with Bonferroni correction for multiple comparisons), lung compliance using Flexivent (one-way ANOVA with Tukey test for multiple comparisons), and soluble collagen in lung homogenates 28 d after the administration of asbestos (one-way ANOVA with Tukey test for multiple comparisons). (G) Soluble collagen 0, 28, and 56 d after the administration of asbestos. Two-way ANOVA with Dunnett’s multiple comparisons test, comparing data from each day to baseline (day 0). (H) Body weight in young adult and old mice after the administration of asbestos. Kruskal–Wallis test with Dunn’s multiple comparisons test, comparing data from each day to baseline (day 0). All data presented as mean ± SEM, 5 to 10 mice per group. *P < 0.05. Representative data from one of two independent experiments.

Fig. 2.

Lung fibrosis is associated with an increased number of monocyte-derived alveolar macrophages in old mice. Young adult mice (3 to 5 mo) and old mice (18 to 24 mo) were administered bleomycin (0.025 unit/50 μL, intratracheally) or crocidolite asbestos (100 μg/50 μL, intratracheally), and the lungs were harvested 28 d later. Monocyte and macrophage populations in (A) bleomycin-induced lung fibrosis (Bleo) and (B) asbestos-induced lung fibrosis were quantified via flow cytometry. Data presented as mean ± SEM, number of mice shown on the plot, one-way ANOVA with Tukey test for multiple comparisons. *P < 0.05. Representative data from two independent experiments.

Fig. 3.

Therapy with ISRIB attenuates bleomycin-induced lung fibrosis in young adult and old mice. (A) Schematic of the experimental design. Young adult (3 to 5 mo) and old (18 to 24 mo) mice were administered bleomycin (0.025 unit/50 μL, intratracheally) and treated with 2.5 mg/kg of ISRIB or vehicle (intraperitoneally daily) beginning at day 7 and harvested at the indicated time points. (B) Representative images of lung tissue from young adult naïve or bleomycin-treated mice, with or without ISRIB on day 28. Masson’s trichrome staining. (Scale bar, 1 mm.) (C) Ashcroft fibrosis score (Mann–Whitney U tests with Bonferroni multiple test correction). (D) Lung compliance and collagen levels from young adult naïve or bleomycin-treated mice, with or without ISRIB (one-way ANOVA with Tukey test for multiple comparisons). (E) Body weight of young adult mice after administration of bleomycin, with or without ISRIB treatment, five mice per each group. The arrow indicates the start of ISRIB treatment (one-way ANOVA with Dunnet’s multiple comparisons test). (F) Representative images of lung tissue from old naïve or bleomycin-treated mice, with or without ISRIB on day 28. Masson’s trichrome staining. (Scale bar, 1 mm.) (G) Ashcroft fibrosis score (Mann–Whitney U tests with Bonferroni multiple test correction). (H) Lung compliance and collagen levels from old naïve mice or bleomycin-treated mice, with or without ISRIB (one-way ANOVA with Tukey test for multiple comparisons). (I) Body weight of old mice after administration of bleomycin, with or without ISRIB treatment, five mice per each group. The arrow indicates the start of ISRIB treatment (one-way ANOVA with Dunnet’s multiple comparisons test). Data are shown as mean ± SEM, 5 to 7 mice per group. *P < 0.05. Representative data from two independent experiments.

Fig. 4.

ISRIB reduces the number of profibrotic monocyte-derived alveolar macrophages in lung fibrosis. Young adult (3 to 5 mo) and old (18 to 24 mo) mice were administered 0.025 unit of bleomycin (Bleo), treated with 2.5 mg/kg of ISRIB or vehicle (intraperitoneally, every day beginning at day 7), and the lungs were analyzed by flow cytometry on day 21. Alveolar macrophages (AMs) and interstitial macrophages (IMs) and classical monocytes in the lung were quantified in (A) young adult and (B) old mice. Data are shown as mean ± SEM, 4 to 5 mice per group. One-way ANOVA with Tukey test for multiple comparisons. *P < 0.05. Representative data from two independent experiments.

Fig. 5.

ISRIB reduces recruitment of monocyte-derived alveolar macrophages during lung fibrosis. (A) Schematic of the genetic lineage tracing system using Cx₃cr1^ERCre × zsGreen mice in bleomycin-induced lung fibrosis. Tamoxifen treatment permanently labels circulating monocytes, which are largely replaced within 7 d (SI Appendix, Fig. S5B), but tissue-resident alveolar macrophages, which do not express Cx3cr1, are unlabeled. After lung injury, monocytes lineage-tagged with GFP are recruited to the lung where they differentiate into monocyte-derived alveolar macrophages. Filled squares indicate experiments where tamoxifen was given before bleomycin. Open squares indicate experiments where tamoxifen was given 7 d before harvest. (B) The number of total Siglec F^high tissue-resident alveolar macrophages (AMs) and Siglec F^low monocyte-derived AMs in bleomycin model. Cx₃cr1^ERCre × zsGreen mice received a single pulse of tamoxifen 1 d prior to administration of bleomycin and were analyzed at indicated time points. One-way ANOVA with Dunnett’s multiple comparisons test, comparing data from each day to baseline (day 0), 3 to 5 mice per group. (C) The number of GFP⁺ monocyte-derived alveolar macrophages remains stable over the course of bleomycin-induced fibrosis. Cx₃cr1^ERCre × zsGreen mice received a single pulse of tamoxifen 1 d prior to administration of bleomycin (intratracheal) and were analyzed at indicated timepoints. The difference between the days is not significant, one-way ANOVA with Dunnett’s test for multiple comparisons. (D) Recruitment of monocyte-derived alveolar macrophages continues during the first 7 d of bleomycin-induced pulmonary fibrosis but ceases after 21 d. Cx₃cr1^ERCre × zsGreen mice received a pulse of tamoxifen 7 d before analysis. One-way ANOVA with Dunnett’s test for multiple comparisons between day 14 and days 28 and 56. (E and F) ISRIB decreases recruitment of monocyte-derived alveolar macrophages in young adult (E) and old (F) mice. Mice received a single pulse of tamoxifen one day prior to administration of bleomycin (intrathecal) and received either with 2.5 mg/kg of ISRIB or vehicle (intraperitoneally, every day, starting at day 7) and were harvested at indicated timepoints. One-way ANOVA with Tukey test for multiple comparisons. 4 to 6 mice per group. *P < 0.05.

Fig. 6.

Transcriptomic profiling does not reveal evidence of activation of the ISR in alveolar macrophage populations during lung fibrosis. (A) Transcriptomes of sorted monocyte-derived alveolar macrophages in young adult and old mice exposed to bleomycin (day 21) with and without ISRIB treatment. k-means clustering of all differentially expressed genes (ANOVA-like test on negative binomial generalized log-linear model, FDR q-value < 0.05). (Right) The characteristic Gene Ontology biological processes and genes associated with each cluster. (B) Representative flow cytometry plots gated on CD45⁺CD64⁺ macrophages in old Cx₃cr1^ERCre × zsGreen mice treated with tamoxifen 1 d prior to administration of bleomycin (see Fig. 5A for design). Median fluorescence intensity (MFI) of Siglec F levels on GFP-positive alveolar macrophages (AM) in young and old mice 21 d after bleomycin, with and without ISRIB treatment. Data are shown as mean ± SEM, 3 to 5 mice per group. Unpaired t test. *P < 0.05.

Fig. 7.

ISRIB promotes alveolar epithelial differentiation after bleomycin. (A) Schematic of the experiment design. Young adult Sftpc^ERCre × zsGreen mice (3 to 5 mo) received 10 mg of tamoxifen via oral gavage on 2 sequential days 22 d prior to administration of bleomycin (0.025 units, intrathecal). Mice were treated with ISRIB 25 mg/kg intraperitoneally or vehicle 7 d after administration of bleomycin and analyzed 3 d later. (B) Representative flow cytometry data gated on GFP⁺ epithelial cells. (C) The number of GFP⁺PDPN⁺ epithelial cells in vehicle- and ISRIB-treated mice was determined by flow cytometry. Unpaired t test, *P < 0.05. (D) Representative lung sections stained with antibodies against KRT8 and PDPN from mice treated with bleomycin and a single dose of ISRIB or vehicle according to the timeline in A. The arrows indicate GFP⁺KRT8⁺ epithelial cells, and the arrow heads indicate GFP⁺PDPN⁺ AT1 cells. (Scale bar, 100 μm.) (E) Quantification of GFP⁺KRT8⁺ and GFP⁺PDPN⁺ epithelial cells by immunofluorescence. Average cell counts were calculated from at least five nonoverlapping areas per section. Data are shown as mean ± SEM, 4 to 5 mice per group. Mann–Whitney U test. *P < 0.05. (F) Quantification of double positive TUNEL and pro-SPC cells on histological sections after bleomycin. Data are shown as mean ± SEM, 4 to 5 mice per group. Mann–Whitney U test. *P < 0.05.

Fig. 8.

ISRIB promotes alveolar epithelial differentiation after pneumonectomy. (A) Schematic of the experiment design. Young adult Sftpc^ERCre × zsGreen mice (3 to 5 mo) received 10 mg of tamoxifen via oral gavage on 2 sequential days 14 d prior to pneumonectomy (PNX). Mice were treated with ISRIB 25 mg/kg intraperitoneally or vehicle 1 d after pneumonectomy, analysis performed on day 7 after pneumonectomy on postcaval lobe. (B) Representative flow cytometry plots, from Sftpc^ERCre × zsGreen mice, gated on CD45^–EpCAM⁺MHC II⁺GFP⁺ cells. (C) Quantification of GFP⁺PDPN^– AT cells and GFP⁺PDPN⁺ AT1 cells in vehicle- and ISRIB-treated mice. Data are shown as mean ± SEM, 3 to 8 mice per group. One-way ANOVA with Tukey–Kramer test for multiple comparison, *P < 0.05. (D) Representative images of lung tissue (postcaval lobe) from young adult mice 7 d after pneumonectomy and a single dose of ISRIB or vehicle at day 1 postpneumonectomy. The arrows indicate GFP⁺PDPN⁺ AT1 cells derived from GFP+ AT2 cells. (Scale bar, 100 μm.)