Chitinase 1 is a biomarker for and therapeutic target in scleroderma-associated interstitial lung disease that augments TGF-β1 signaling

Abstract

Interstitial lung disease (ILD) with pulmonary fibrosis is an important manifestation in systemic sclerosis (SSc, scleroderma) where it portends a poor prognosis. However, biomarkers that predict the development and or severity of SSc-ILD have not been validated, and the pathogenetic mechanisms that engender this pulmonary response are poorly understood. In this study, we demonstrate in two different patient cohorts that the levels of chitotriosidase (Chit1) bioactivity and protein are significantly increased in the circulation and lungs of SSc patients compared with demographically matched controls. We also demonstrate that, compared with patients without lung involvement, patients with ILD show high levels of circulating Chit1 activity that correlate with disease severity. Murine modeling shows that in comparison with wild-type mice, bleomycin-induced pulmonary fibrosis was significantly reduced in Chit1⁻/⁻ mice and significantly enhanced in lungs from Chit1 overexpressing transgenic animals. In vitro studies also demonstrated that Chit1 interacts with TGF-β1 to augment fibroblast TGF-β receptors 1 and 2 expression and TGF-β-induced Smad and MAPK/ERK activation. These studies indicate that Chit1 is potential biomarker for ILD in SSc and a therapeutic target in SSc-associated lung fibrosis and demonstrate that Chit1 augments TGF-β1 effects by increasing receptor expression and canonical and noncanonical TGF-β1 signaling.

FIGURE 1

Chit1 in the circulation and tissues from patients with SSc-ILD. (A) In the Yale cohort, comparison of normal controls (left, n = 27) versus SSc-ILD subjects (right, n = 17) reveals significantly increased Chit1 activity in SSc-ILD. (B–F) Chit1 expression is enhanced in SSc-ILD lungs. (B) Compared to nonfibrotic control (left), the lungs from patients with SSc-ILD (right) contain more Chit1⁺ cells per high-powered field. (C) Chit1 IHC staining of normal lung reveals minimal staining in macrophages and alveolar epithelial cells. (D) Chit1 staining of SSc-ILD lung in the absence of primary Ab reveals no signal. (E) Chit1 staining of SSc-ILD lung reveals increased staining in hyperplastic epithelial cells (open arrow) and (F) alveolar macrophages (solid arrow). (G and H) Chit1 staining with lower magnification for control (G) and SSc-ILD (H) lungs. Scale bars, 30 μm. ***p < 0.001.

FIGURE 2

Chit1 expression is increased in SSc patients with ILD and correlates inversely with lung function. (A) In the Chicago cohort, comparison of SSc patients without (left) and with ILD (right) reveals a significant increase in circulating Chit1 activity in SSc-ILD patients. (B and C) Relationship between Chit1 activity and pulmonary function (FVC) in SSc patients in Yale (B) and Chicago (C). (D and E) Negative correlations between Chit1 activity and disease severity (represented by percentage predicted FVC) in SSc-ILD patients in cohorts from Yale (D) (Spearman r, −0.62, p = 0.0074, n = 17) and Chicago (E) (Spearman r, −0.43, p = 0.01, n = 33). *p < 0.05.

FIGURE 3

Bleomycin and IL-13 stimulate Chit1 in the lung. (A–C) WT mice were exposed to i.p. bleomycin (Bleo) for 28 d and the levels of Chit1 protein, mRNA, and activity were evaluated (n = 4–5 mice/group, *p < 0.05). (D and E) BAL Chit1 protein and whole-lung mRNA expression in CC10/IL-13 Tg mice. (F and G) IHC and confocal analysis demonstrating the colocalization of Chit1 and the cell-specific markers pro-SPC (alveolar type 2 cells) and F4/80 (macrophages) after bleomycin administration. The values in the panels (A)–(E) and are the means ± SEM of evaluations in a minimum of four animals. Panels in (F) and (G) are representative of a minimum of five similar experiments. Arrows highlight Chit1. Scale bars, 10 μm.

FIGURE 4

Role of Chit1 in bleomycin- and IL-13–induced pulmonary fibrosis and inflammation. (A–C) Two-month-old WT (Chit1^+/+) and Chit1^−/− were exposed to i.p. bleomycin (Bleo) and pulmonary fibrosis (A), lung collagen content (B), and BAL inflammation (C) were evaluated by Mallory’s trichrome stains, Sircol collagen assays, and BAL cell counts, respectively. (D–F) Evaluation of pulmonary fibrosis (D), collagen content (E), and BAL inflammation (F) in lungs from IL-13 Tg mice with and without Chit1-null mutations. The values in (B), (C), (E), and (F) are the means ± SEM of evaluations in a minimum of four animals. Panels in (A) and (C) are representative of a minimum of five mice per group. Scale bars, 200 μm. *p < 0.05.

FIGURE 5

Transgenic Chit1 enhances bleomycin-induced pulmonary fibrosis. (A–C) Two-month-old WT and Chit1 Tg mice were treated with doxycycline and exposed to intratracheal bleomycin (Bleo; 1.25 U/kg). Pulmonary fibrosis and BAL inflammation were quantitated using Mallory’s trichrome stains (A), Sircol collagen assays (B), and BAL cell counts (C). The values in (B) and (C) are the means ± SEM of evaluations in a minimum of four animals. Panels in (A) are representative of a minimum of five mice per group. Scale bars, 200 μm. *p < 0.05.

FIGURE 6

Chit1 increases TGF-β receptor expression. (A) mRNA expression of TGFR1 and TGFR2 assessed by real-time RT-PCR. (B) The expression of TGFR1 and TGFR2 evaluated by Western blot in MRC5 human lung fibroblast cells after stimulation with rChit1 (250 ng/ml) alone or in combination with rTGF-β1 (10 ng/ml) for 24 h. The values in the (A) are the means ± SEM of triplicate evaluations; (B) is representative of a minimum of three separate experiments. **p < 0.01.

FIGURE 7

Chit1 increases TGF-β signaling. (A) Mv1Lu cells were stimulated with rChit1 and rTGF-β1, alone and in combination, and luciferase activity was assessed. (B) Smad 2 activation (phosphorylation) was assessed by Western blot in the MRC5 cells stimulated by rChit1 and rTGF-β1, alone and in combination. (C) Smad activation was evaluated by dual luciferase reporter assay in HEK293 cells after stimulation with rChit1 and rTGF-β1, alone and in combination. (D) MAPK/ERK activation in MRC5 cells detected by Western blot after stimulation with rChit1 and rTGF-β1, alone and in combination, was evaluated by Western blot analysis after stimulations with rChit1 and rTGF-β1, alone and in combination. The values in (A) and (C) are the means ± SEM of triplicate evaluations. (B) and (D) are representative of a minimum of three separate experiments. **p < 0.01.