Neutrophil elastase promotes myofibroblast differentiation in lung fibrosis

Abstract

IPF is a progressive lung disorder characterized by fibroblast proliferation and myofibroblast differentiation. Although neutrophil accumulation within IPF lungs has been negatively correlated with outcomes, the role played by neutrophils in lung fibrosis remains poorly understood. We have demonstrated previously that NE promotes lung cancer cell proliferation and hypothesized that it may have a similar effect on fibroblasts. In the current study, we show that NE(-/-) mice are protected from asbestos-induced lung fibrosis. NE(-/-) mice displayed reduced fibroblast and myofibroblast content when compared with controls. NE directly both lung fibroblast proliferation and myofibroblast differentiation in vitro, as evidenced by proliferation assays, collagen gel contractility assays, and αSMA induction. Furthermore, αSMA induction occurs in a TGF-β-independent fashion. Treatment of asbestos-recipient mice with ONO-5046, a synthetic NE antagonist, reduced hydroxyproline content. Thus, the current study points to a key role for neutrophils and NE in the progression of lung fibrosis. Lastly, the study lends rationale to use of NE-inhibitory approaches as a novel therapeutic strategy for patients with lung fibrosis.

Keywords: IRS-1; asbestos; fibroblasts.

Figure 1.. NE^−/− mice are protected from asbestos-induced lung fibrosis.

WT and NE^−/− mice were treated with 0.1 mg crocidolite asbestos (Asb) or TiO₂ control. (A) Representative images from H&E (10×)- and Masson's trichrome (20×)-stained lung sections, 14 days after asbestos treatment. (B) Hydroxyproline content was measured at days 7 (left) and 14 (right); n = 6–8/group. (C) Fibrosis index was generated according to the criteria described in Materials and Methods; n = 6–8/group. (D) BAL neutrophil (PMN), macrophage (Mac), and lymphocyte (Lymph) counts for 14 days post-TiO₂ treatment (left; n = 6), day 7 postasbestos treatment (middle; n = 8), and day 14 postasbestos treatment (right; n = 8). Installations were performed at least 2 times, and the data were pooled. All data presented as mean value ± sem. Two-tailed Student’s t-test was used to compare differences between groups. *P < 0.05; **P < 0.01.

Figure 2.. Decreased fibroblast and myofibroblast content in NE^−/− mice.

WT and NE^−/− mice were treated with 0.1 mg crocidolite asbestos or TiO₂ control and studied 14 days later. (A) Representative images and quantification of IHC staining for FSP-1⁺ cells. Results expressed as positive cells/hpf from 10 fields/mouse (n = 8 each group). (B) Representative confocal images from αSMA (green)-, DAPI (blue)-, and FSP-1 (red)-stained frozen lung sections. Laser voltages were set such that the secondary antibody-only controls exhibited no fluorescence. (C) Representative flow cytometric dot plots of single-cell suspensions obtained from day 14 asbestos-treated whole lungs denoting percentages of fibroblasts (CD45⁻FSP-1⁺) and myofibroblasts (CD45⁻FSP-1⁺αSMA⁺). Gates were set according to isotype controls as illustrated. Total cell numbers of (D) fibroblasts (CD45⁻FSP-1⁺), (E) myofibroblasts (CD45⁻FSP-1⁺αSMA⁺), (F) pericytes/smooth muscle cells (CD45⁻FSP-1⁻αSMA⁺), and (G) fibrocytes (CD45⁺FSP-1⁺CD11c⁻F4/80⁻) were obtained by multiplying cell percentages by the absolute whole lung cell count obtained by hemacytometer counts (n = 7 mice/group). (H) Representative dot plots used in obtaining CD45⁺FSP-1⁺CD11c⁻F4/80- fibrocyte counts in (G). These plots have been previously gated on CD45⁺FSP-1⁺ cells. All data presented as the mean value ± sem. FACS experiments were performed twice with 1 representative experiment shown. Two-tailed Student’s t-test was used to compare differences between the 2 groups. *P < 0.05.

Figure 3.. NE induces fibroblast proliferation.

MTS proliferation assay for LL47 cells treated with (A) NE, (B) heat-inactivated NE, and (C) PMSF-inactivated NE and (D) PMF fibroblasts treated with NE at the indicated concentrations (n = 12 wells/treatment). Western blots performed on (E) LL47 and (F) PMF fibroblast cell lysates for IRS-1, pAkt, Akt, p38MAPK, p-p38MAPK, and GAPDH following NE treatment for 1 h at the indicated concentrations. (G) NE-Alexa 488 (4nM, green) and Trypsin (Tryp)-Alexa 488 (4nM, green) were incubated with LL47 fibroblasts for 60 min and washed with PBS. Cells were stained with anti-EEA-1/anti-rabbit-Alexa 594 secondary (red), counterstained with DAPI (blue), and imaged by use of confocal microscopy. Original magnification, 1200×. The percentage of cells in each field that contain colocalized NE and EEA-1 (yellow) was quantified (H) by counting ten confocal fields/condition (containing at least 75 cells in total). (I) MTS proliferation assay for WT and IRS-1^fl/fl fibroblasts, conducted at 24 (left) and 48 (right) h after an overnight treatment with AdCre (MOI 100:1; n = 4 for each condition, representative of 2 independent experiments). (J) Western blot depicting knockdown of IRS-1 in primary fibroblasts described in I. Lysates were prepared at t = 0 before conducting the 24 and 48 h proliferation experiments. All data presented as the mean value ± sem. Each study was performed twice with 1 representative experiment shown. Two-tailed Student’s t-test was used to compare differences between groups. *P < 0.05.

Figure 4.. NE promotes myofibroblast differentiation.

(A and B) Wound healing assay for LL47 fibroblasts plated on scratched tissue-culture plastic and treated with NE (0, 4, or 8 nM) for 1 h. Data are mean value of percent wound closure ± sem from a representative experiment in triplicate. (C and D) LL47 fibroblasts were embedded in a type I collagen matrix in 24-well plates. Gels were treated with NE (0–40 nM) or TGF-β for 1 h. Con, Control. Data are mean value of gel area ± sem (n = 8 wells/condition), pooled from 2 independent experiments. All NE and TGF-β treatment groups are significantly different than untreated wells at each time-point, as indicated by the 3 asterisks in C. (E) LL47 fibroblasts were incubated with 4 nM NE for 1 h. Cells were stained for αSMA (green) and counterstained with phalloidin (red) and DAPI (blue). Original magnification, 600×. (F) Representative Western blots for αSMA, pAkt, pSMAD3, and GAPDH for NE-treated fibroblasts in the presence and absence of the TGF-β inhibitor (TGF-βRi) SB431542 (10 µM; top) and the PI3K inhibitor (PI3Ki), LY294002 (50 µM; bottom). TGF-β-treated fibroblasts in the presence and absence of the TGF-β inhibitor SB431542 are also depicted (middle). One-way ANOVA was performed to determine statistical significance between groups. *P < 0.05.

Figure 5.. ONO-5046 reduces asbestos-induced lung injury.

(A) Hydroxyproline assay for asbestos-recipient C57BL/6 mice treated with ONO-5046 (100 mg/kg) or vehicle control, i.p. daily for 14 days (n = 8 mice/group). This study was performed twice with 1 representative experiment shown. Data are mean value ± sem. (B) A fibrosis index was generated, according to the criteria described in Materials and Methods (n = 14 mice/group, pooled from 2 independent experiments). (C) Representative H&E images (10×) of the lungs from ONO-5046-treated and vehicle control mice. Two-tailed Student’s t-test was used to compare differences between groups. *P < 0.001; **P < 0.01.