Inhibition of Macrophage-Specific CHIT1 as an Approach to Treat Airway Remodeling in Severe Asthma

Abstract

Chitotriosidase (CHIT1) is an enzyme produced by macrophages that regulates their differentiation and polarization. Lung macrophages have been implicated in asthma development; therefore, we asked whether pharmacological inhibition of macrophage-specific CHIT1 would have beneficial effects in asthma, as it has been shown previously in other lung disorders. CHIT1 expression was evaluated in the lung tissues of deceased individuals with severe, uncontrolled, steroid-naïve asthma. OATD-01, a chitinase inhibitor, was tested in a 7-week-long house dust mite (HDM) murine model of chronic asthma characterized by accumulation of CHIT1-expressing macrophages. CHIT1 is a dominant chitinase activated in fibrotic areas of the lungs of individuals with fatal asthma. OATD-01 given in a therapeutic treatment regimen inhibited both inflammatory and airway remodeling features of asthma in the HDM model. These changes were accompanied by a significant and dose-dependent decrease in chitinolytic activity in BAL fluid and plasma, confirming in vivo target engagement. Both IL-13 expression and TGFβ1 levels in BAL fluid were decreased and a significant reduction in subepithelial airway fibrosis and airway wall thickness was observed. These results suggest that pharmacological chitinase inhibition offers protection against the development of fibrotic airway remodeling in severe asthma.

Keywords: asthma; chitotriosidase; macrophage.

Figure 1

Increased contribution of CHIT1 in airway remodeling in fatal asthma patients. (A) Representative immunohistochemical staining of CHIT1. (B) Table containing donor and fatal asthmatics characteristics, basement membrane perimeter lengths, and CHIT1-positive area/basement membrane perimeter ratios (µ²/µ). (C) Quantitative representation of CHIT1-positive area/basement membrane perimeter ratios (µ²/µ) in the lungs of donors and fatal asthma individuals. Data presented as mean ± s.e.m. p-values < 0.05 were considered as statistically significant and presented as * for p < 0.05 and*** for p < 0.001.

Figure 2

Activation of chitinases in chronic HDM-induced airway remodeling model in mice. (A) Number of total infiltrated leukocytes (CD45-positive cells) in BAL fluid analyzed by flow cytometry during progression of chronic airway remodeling at 3–7 weeks of HDM administration intranasally 5 times a week. (B) Flow cytometry analysis of alveolar macrophages (Siglec F+, CD11c+) represented as total macrophage numbers in BAL fluid (1 mL) tested after 3, 4, 5, and 7 weeks of HDM administration. (C) A representative dot plot of BALF-isolated CD45-positive cells (Siglec F+, CD11c+) representing alveolar macrophages. (D) Chitinolytic activity in BAL fluid and (E) plasma recovered from naïve mice challenged with PBS or HDM (7 weeks of administration). (F,a–h) Representative histological pictures of samples immunostained for CHIT1 (a–d) and picrosirius red–fast green (e–h) for collagen on mouse lungs; HDM-induced airway remodeling progression was followed at timepoints 3 weeks (b,f), 5 weeks (c,g), and 7 weeks (d,h) of intranasal administration of HDM. Control samples are presented on a and e panels. Data presented as mean ± s.e.m. p-values < 0.05 were considered as statistically significant and presented as * for p < 0.05, ** for p < 0.01, *** for p < 0.001.

Figure 3

OATD-01 reduces goblet cell metaplasia and exhibits anti-inflammatory effects in chronic HDM-induced model. (A) Schematic representation of HDM model and OATD-01 administration regimen. Blue arrows indicated HDM installations while green OATD-01 treatment. (B) Flow cytometry analysis of pulmonary inflammation as represented by total leukocyte numbers (CD45+ cells) and eosinophil population (Siglec F+, CD11c- cells) in particular, in BAL fluid of mice subjected to chronic HDM (7 weeks) and treated with OATD-01 (3 and 30 mg/kg; PO; qd) or vehicle controls. (C) Representative PAS staining of goblet cell metaplasia in chronic HDM-induced airway remodeling model. (D) Semi-quantitative analysis of goblet cell metaplasia by scoring system (0–4) in the lung sections of chronic HDM administration model as compared to vehicle treated controls. Data presented as mean ± s.e.m. p-values < 0.05 were considered as statistically significant and presented as * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001.

Figure 4

OATD-01 decreases chitinolytic activity in BAL fluid and plasma in a dose-dependent manner. (A) Schematic representation of PK/PD study in 3-week-long HDM model with two OATD-01 administration regimens (single dose at 30 mg/kg or 14 day). Blue arrows indicated HDM installations while green OATD-01 treatment. (B) Graphs showing relation between chitinolytic activity (magenta) and OATD-01 concentration (blue) in plasma (a,c) and lung homogenates (b,d) following single dose of OATD-01 at 30 mg/kg on the last day of the study (c,d) or multiple administrations of OATD-01 at 30 mg/kg once a day (a,b).

Figure 5

OATD-01 ameliorates TGFβ1, IL-13, and CHIT1 in chronic HDM model in a dose-dependent fashion. (A) mRNA expression of Il13 in the lungs and (B) active TGFβ1 levels in BAL fluid in mice with chronic HDM (7 week) treated with OATD-01 (3 and 30 mg/kg; PO; single dose) or vehicle control. (C) Number of macrophages in BAL fluid in mice with chronic HDM (7-week-long) treated with OATD-01 (3 and 30 mg/kg; PO; single dose) or vehicle control. The level of CHIT1 protein in BAL fluid of control and HDM-instilled mice treated with vehicle or OATD-01 (30 mg/kg) in chronic airway inflammation model as evaluated by (D) Western blot and (E) corresponding densitometry analysis. Data presented as mean ± s.e.m. p-values < 0.05 were considered as statistically significant and presented as * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001.

Figure 6

OATD-01 reduces features of airway remodeling after chronic HDM administration. (A) Histological evaluation of airway remodeling (bronchiole collagen wall and epithelial thickness) as represented by Masson’s trichrome (a–c) and hematoxylin and eosin (d–f) in a 7-week-long chronic HDM model in C57BL/6 mice treated with OATD-01 (30 mg/kg; PO; qd) (c,f) or vehicle control; Control (a,d) and Chronic HDM (b,e). (B) Quantitative analysis of peribronchial collagen area (collagen wall thickness) stained with Masson’s trichrome in a chronic HDM model with or without OATD-01 treatment (30 mg/kg; PO; qd; n = 8). (C) Quantitative analysis of bronchiole epithelial thickness, measured at the same time as analysis of collagen wall thickness (on Masson’s trichrome-stained samples) in a HDM model with or without OATD-01 treatment (30 mg/kg; PO; qd; n = 8). Data presented as mean ± s.e.m. p-values < 0.05 were considered as statistically significant and presented as **** for p < 0.0001.

Figure 7

Inhibition of macrophage-specific CHIT1 attenuates airway remodeling. Decreased macrophage-specific CHIT1 expression and activity changes phenotype of profibrotic macrophages and blocks TGFβ1 and subsequent Th2-dependent IL-13 production. OATD-01 inhibits CHIT1-dependent macrophage–fibroblast positive feedback loop leading to fibroblast activation and myofibroblast transition. The overall effect of CHIT1 inhibition results in less collagen deposition in extracellular space, decreased goblet cell hyperplasia, and attenuated airway remodeling—features associated with severe asthma.