Lung-Targeted Delivery of Dimethyl Fumarate Promotes the Reversal of Age-Dependent Established Lung Fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF), a severe and deadly form of lung fibrosis, is widely regarded as a disease of aging. We previously demonstrated that aged mice with persistent lung fibrosis and IPF lung myofibroblasts exhibit deficient Nrf2-mediated antioxidant responses. Tecfidera is an orally administered FDA-approved drug for the treatment of multiple sclerosis, where the active pharmaceutical ingredient is dimethyl fumarate (DMF), an active Nrf2 activator. However, no studies have evaluated the efficacy of DMF for age-associated persistent lung fibrosis. Here, we demonstrate that in IPF lung fibroblasts, DMF treatment inhibited both TGF-β-mediated pro-fibrotic phenotypes and led to a reversal of established pro-fibrotic phenotypes. We also evaluated the pre-clinical efficacy of lung-targeted (inhaled) vs. systemic (oral) delivery of DMF in an aging murine model of bleomycin-induced persistent lung fibrosis. DMF or vehicle was administered daily to aged mice by oral gavage or intranasal delivery from 3-6 weeks post-injury when mice exhibited non-resolving lung fibrosis. In contrast to systemic (oral) delivery, only lung-targeted (inhaled) delivery of DMF restored lung Nrf2 expression levels, reduced lung oxidative stress, and promoted the resolution of age-dependent established fibrosis. This is the first study to demonstrate the efficacy of lung-targeted DMF delivery to promote the resolution of age-dependent established lung fibrosis.

Keywords: Nrf2; aging; antioxidant; dimethyl fumarate; idiopathic pulmonary fibrosis; reactive oxygen species.

Figure 1

DMF treatment inhibits pro-fibrotic phenotypes in IPF lung fibroblasts. Primary fibroblasts were isolated from the lung of a patient with biopsy-proven IPF. (A,B) IPF lung fibroblasts were treated with DMF (1 µM) or vehicle (DMSO) followed by treatment ± TGF-β (2 ng/mL). H₂O₂ levels were evaluated at 24 h by Amplex Red assay (A), and whole-cell lysates were assessed for collagen-1α expression at 48 h by Western blot; densitometric analyses are shown (B). (C,D) IPF lung fibroblasts were treated with DMF (1 µM) or vehicle (DMSO). Culture supernatant was evaluated for H₂O₂ levels at 24 h by Amplex Red assay (C), and whole-cell lysates were assessed for collagen-1α expression at 24 h by Western blot; densitometric analyses are shown (D). All values represent means  ±  SEM; technical replicates (n = 3); * p < 0.05; ** p < 0.01; *** p < 0.01 using Student’s two-tailed t-test to compare the means between two groups and one-way ANOVA multiple comparisons with a Tukey’s post-test to compare the means among three groups.

Figure 2

Aged-injured mice exhibit Nrf2 deficiency associated with oxidative stress and lung fibrosis. (A,B) Lung protein was assessed for Nrf2 expression by Western blot (A) and densitometric analysis (B). (C) The level of lung oxidized glutathione was analyzed by quantitative oxidized glutathione assay. (D–G) Lung protein was assessed for fibronectin (D,E) and collagen-1α (Col-1α) (F,G) expression by Western blot and densitometric analysis. Representative Western blot images are shown. All values represent means of biological replicates ± SEM; n = 4 mice per group; * p < 0.05; ** p < 0.01 using Student’s two-tailed t-test.

Figure 3

Schematic diagram illustrating age-dependent persistent lung fibrosis and treatment protocol. C57BL/6J aged (18 months) female mice received an intratracheal instillation of bleomycin (0.02875 U/mouse). DMF was administered daily by oral gavage (240 µg/150 µL in sterile PBS/mouse) or intranasal instillation (80 µg/50 µL in sterile PBS/mouse) from 3–6 weeks post-injury.

Figure 4

Intranasal (but not oral gavage) administration of DMF promotes reversal of age-associated established lung fibrosis. (A) Lung tissue was assessed by H&E staining for histopathology (top panels) and Masson’s trichrome blue staining for collagen (bottom panels). (B–E) Lung protein extract was assessed for fibronectin (B,C) and collagen-1α (D,E) expression by Western blot and densitometric analysis. Representative Western blot images are shown. Oral-vehicle (n = 6); oral-DMF (n = 5); intranasal-vehicle (n = 4); intranasal-DMF (n = 6). (F) The resolution of fibrosis was analyzed by quantitative hydroxyproline assay. Oral-vehicle (n = 5); oral-DMF (n = 5); intranasal-vehicle (n = 5); intranasal-DMF (n = 6). All values represent means of biological replicates ± SEM; ns: not significant; * p < 0.05 using Student’s two-tailed t-test.

Figure 5

Intranasal (but not oral gavage) administration of DMF restores lung Nrf2 levels in aged-injured mice. (A) Blood ROS levels were analyzed by Amplex Red assay. Oral-DMF (n = 7); intranasal-DMF (n = 6). (B,C) Lung protein extract was assessed for Nrf2 expression by Western blot (B) and densitometric analysis (C). Representative Western blot images are shown. Oral-vehicle (n = 7); oral-DMF (n = 7); intranasal-vehicle (n = 4); intranasal-DMF (n = 4). (D) The level of lung oxidized glutathione was analyzed by a quantitative oxidized glutathione assay. Oral-vehicle (n = 5); oral-DMF (n = 5); intranasal-vehicle (n = 3); intranasal-DMF (n = 4). (E) The level of lung lipid peroxidation was analyzed by quantitative lipid peroxidation assay. Oral-vehicle (n = 5); oral-DMF (n = 5); intranasal-vehicle (n = 4); intranasal-DMF (n = 3). All values represent means of biological replicates ± SEM; ns: not significant; * p < 0.05; ** p < 0.01 using Student’s two-tailed t-test.