Reducing protein oxidation reverses lung fibrosis

Abstract

Idiopathic pulmonary fibrosis is characterized by excessive deposition of collagen in the lung, leading to chronically impaired gas exchange and death^1-3. Oxidative stress is believed to be critical in this disease pathogenesis^4-6, although the exact mechanisms remain enigmatic. Protein S-glutathionylation (PSSG) is a post-translational modification of proteins that can be reversed by glutaredoxin-1 (GLRX)⁷. It remains unknown whether GLRX and PSSG play a role in lung fibrosis. Here, we explored the impact of GLRX and PSSG status on the pathogenesis of pulmonary fibrosis, using lung tissues from subjects with idiopathic pulmonary fibrosis, transgenic mouse models and direct administration of recombinant Glrx to airways of mice with existing fibrosis. We demonstrate that GLRX enzymatic activity was strongly decreased in fibrotic lungs, in accordance with increases in PSSG. Mice lacking Glrx were far more susceptible to bleomycin- or adenovirus encoding active transforming growth factor beta-1 (AdTGFB1)-induced pulmonary fibrosis, whereas transgenic overexpression of Glrx in the lung epithelium attenuated fibrosis. We furthermore show that endogenous GLRX was inactivated through an oxidative mechanism and that direct administration of the Glrx protein into airways augmented Glrx activity and reversed increases in collagen in mice with TGFB1- or bleomycin-induced fibrosis, even when administered to fibrotic, aged animals. Collectively, these findings suggest the therapeutic potential of exogenous GLRX in treating lung fibrosis.

Figure 1

Lower glutaredoxin-1 (GLRX) enzymatic activity and higher protein S-Glutathionylation (PSSG) occur in lung tissues from subjects with IPF and correlate with disease severity. (a) mRNA expression of GLRX in lung tissue from subjects with IPF (n = 160) and without IPF (non-IPF; n = 132). *P < 0.05, Kruskal-Wallis test. Supplementary Table 1 shows subject demographics. (b) GLRX protein in lung tissues from individuals without IPF (Non-IPF; n = 30) and with IPF (n = 29). (c) GLRX enzyme activity following its immunoprecipitation from lung tissues from subjects with IPF (n = 16) and without IPF (non-IPF; n = 15). Results are expressed as nmol/min/mg of input protein. (d) PSSG in lung tissue from cases of IPF (n = 30) and non-IPF (n = 30). (e) In situ PSSG (red) in healthy lungs (n = 4) or lung tissue from subjects with IPF (n = 4). Scale bar, 50μm. White arrowheads, reactive type II pneumocytes. (f-h)Correlations between PSSG and GLRX (f), PSSG and DL_CO (g), and GLRX activity and DL_CO (h), in lungs of subjects with IPF and non-IPF controls. Supplementary Table 2 shows demographics of subject samples used in panels b-h. *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed Student’s t-test.

Figure 2

Bleomycin-induced lung fibrosis and protein S-glutathionylation are elevated in Glrx^−/− mice, and lowered following transgenic overexpression of Glrx in lung epithelial cells. (a) Collagen in the right superior lobe, 21 days post administration of bleomycin (BLM) WT PBS n = 5, BLM n = 7, Glrx^−/− PBS n = 5 & BLM n = 8, pooled from 2 independent experiments. (b) Masson trichrome staining in WT or Glrx^−/− mice described in a. Shown are representative images. Scale bar, 50μm. (c) Overall PSSG in lungs from WT or Glrx^−/− mice exposed to PBS or bleomycin (n = 5 mice/group). (d) Fas-SSG in lung tissues (WLL) from WT or Glrx^−/− mice 21 days post bleomycin. + DTT: negative control, (e) Caspase 3 (Casp3) activity in lungs from WT or Glrx^−/− mice 21 days post-bleomycin (n = 5 mice/group) pooled from 2 independent experiments, (f-j) Attenuation of bleomycin- or AdTGFB1-induced fibrosis, PSSG and Casp3 activation in mice overexpressing Glrx in airway epithelia (Epi-Glrx) compared to WT or Ctr groups. Total PSSG (f; n = 5 mice/group), Fas-SSG (g; Top panels), Casp3 activity (h; n = 5/group) or collagen content (I; Sircol, WT: PBS n = 5, BLM n = 8, Ctr PBS n = 4, BLM n = 5; Epi-Glrx n = 5, BLM n = 7 mice/group) in lung tissues. (g) Bottom panels; Assessment of Fas-SSG in Ctr or Epi-Glrx transgenic mice that received AdTGFB1 or AdCtr. Flag-GLRX confirms transgene expression. Representative blots reflect n = 3-4 mice/group. Ctr: CCSP-rtTA single transgenic mice fed dox. * denotes the Flag-Glrx transgene. j: Hydroxyproline in lungs from CCSP-rtTA/TetOP-Glrx bi-transgenic mice following delayed administration of dox. (ctr; n = 5, TGFB1 n = 8 mice/group). Shown are cropped Western blots. *P < 0.05, **P < 0.01, ***P < 0.001, ANOVA, using Tukey’s post-hoc test.

Figure 3

Direct administration of recombinant Glrx to the lung protects against the progression of, and reverses existing pulmonary fibrosis. (a) Schematic depicting experimental design. Glrx 1X-5X; number of administrations of Glrx (Ctr; HBSS vehicle). (b) Detection of Alexa 647 or Alexa labeled-Glrx (red) in lungs from control animals 18 hours post oropharyngeal administration. Optical slice = 0.1μm, Scale bar = 50μm. n = 3 mice/group. Arrows; labeled epithelial cells, Arrowheads; cells resembling macrophages. (c-f) Impact of sequential administrations of Glrx, or HBSS (ctr), on Glrx activity (c), PSSG (d), hydroxyproline (e) in mice exposed to bleomycin. (f) Pyridinoline (PYD) in lungs following administration of HBSS or Glrx. c-f n = 3/4 mice/group/time period. (g) Assessment of dose-dependence of Glrx administered starting on day 14 on bleomycin-induced elevations of hydroxyproline. Mice were euthanized on day 24. PBS n = 5, BLM n = 9, + Ctr n = 5, +1 / 3 / 6 and 12μg n = 6 mice/group. Shown are data pooled from two independent experiments. (h) Representative images showing total lung collagen via picrosirius red staining. Left: bright field images, Right: corresponding polarized light images. Scale bar =100 μm. (i) Quantification of total collagen in h. PBS/HBSS n = 7, PBS/Glrx n = 7, 14 day BLM n = 3, BLM/HBSS n = 5, BLM/Glrx n = 7. (j) Schematic depicting administration of Glrx into lungs of mice with existing AdTGFB1-induced fibrosis, (k) Hydroxyproline in the right superior lung lobe of mice of mice with AdTGFB1-induced fibrosis treated with WT or C23S Glrx. (21 days Ad-Ctr/TGFB1 n = 5, 42 days Ad-Ctr n = 5, 42 days AdTGFB1 HBSS n = 11, Mut Glrx n = 9 and WT Glrx n = 11 mice/group). Shown are data pooled from two independent experiments. * P < 0.05, ** p < 0.01, ***p < 0.001, ANOVA, using Tukey’s post-hoc test, ns: not significant.

Figure 4

The aging-associated enhanced susceptibility to pulmonary fibrosis induced by bleomycin is associated with low Glrx activity and can be rescued following oropharyngeal administration of Glrx. Glrx activity (a) and PSSG (b) in lung tissues of naïve mice of different ages.(3 month n = 8, 18 month n = 6, 24 month n = 3). Glrx activity (c; 3 month PBS, n = 5, BLM n = 4, 18 month PBS n = 3, BLM n = 6) and hydroxyproline (d; n = 6) in 3 month or 18 month old mice exposed to PBS or bleomycin, (e-j) Impact of Glrx administration into airways of 18 month old mice with existing bleomycin-induced fibrosis. 21 days post-bleomycin, mice received HBSS (Ctr), WT Glrx (12 μg) or C23S Glrx (Mut, 12 μg) oropharyngeally, every 3^rd day. Mice were euthanized on day 42.(e) Glrx activity n = 6 mice/group, (f) PSSG, via non-reducing Western blot analysis using an anti-GSH antibody, β-actin: loading control n = 3 mice/group, (g) Hydroxyproline (21 days PBS/BLM n = 6, 42 days HBSS n= 11, WT Glrx n = 14, and Mut Glrx n = 10). Shown is pooled data from 2 independent experiments, (h) Gene expression of Vim, Acta2 and Cdh1 lung tissue (HBSS n = 5, WT n = 8, Mut n = 7 mice/group). Data are expressed relative to mice exposed to PBS at 21 days. (i) Casp3 activity (n = 6 mice/group). RLU: Relative luminescence units. (j) Collagen degrading activity in lung lysates (n = 3 mice/group form a single analysis). Additional information is found in Supplementary Fig. 8a-b. M: marker, PC: positive control using collagen plus recombinant collagenase, and NC: negative control (rat tail collagen). α, β, and γ: the subunits of collagen. Bulky arrows indicate degradation products. *P < 0.05, **P < 0.01, ***P < 0.001, ANOVA, using Tukey’s post-hoc test, ns: not significant.