Dysregulated Macrophages Are Present in Bleomycin-Induced Murine Laryngotracheal Stenosis

Abstract

Objective: To define the inflammatory cell infiltrate preceding fibrosis in a laryngotracheal stenosis (LTS) murine model.

Study design: Prospective controlled murine study.

Setting: Laboratory.

Subjects and methods: Chemomechanical injury mice (n = 44) sustained bleomycin-coated wire-brush injury to the laryngotracheal complex while mechanical injury controls (n = 42) underwent phosphate-buffered saline (PBS)-coated wire-brush injury. Mock surgery controls (n = 34) underwent anterior transcervical tracheal exposure only. Inflammatory and fibrosis protein and gene expression were assessed in each condition. Immunohistochemistry served as a secondary outcome.

Results: In chemomechanical injury mice, there was an upregulation of collagen I (P < .0001, P < .0001), Tgf-β (P = .0023, P = .0008), and elastin (P < .0001, P < .0001) on day 7; acute inflammatory gene Il1β (P = .0027, P = .0008) on day 1; and macrophage gene CD11b (P = .0026, P = .0033) on day 1 vs mechanical and mock controls, respectively. M1 marker inducible nitric oxide synthase (iNOS) expression decreased (P = .0014) while M2 marker Arg1 (P = .0002) increased on day 7 compared with mechanical controls. Flow cytometry demonstrated increased macrophages (P = .0058, day 4) and M1 macrophages (P = .0148, day 4; P = .0343, day 7; P = .0229, day 10) compared to mock controls. There were similarities between chemomechanical and mechanical injury mice with an increase in M2 macrophages at day 10 (P = .0196).

Conclusions: The bleomycin-induced LTS mouse model demonstrated increased macrophages involved with the development of fibrosis. Macrophage immunophenotype suggested that dysregulated M2 macrophages have a role in abnormal laryngotracheal wound healing. These data delineate inflammatory cells and signaling pathways in LTS that may potentially be modulated to lessen fibroblast proliferation and collagen deposition.

Keywords: airway epithelial injury; laryngotracheal stenosis; mouse model; subglottic stenosis; trachea.

Figure 1

Mouse LTS (red columns) fibrosis-related gene expression peaked at 7 days (red) compared to that in the mechanical injury (blue) and mock (green) controls.

Figure 2

Chemomechanical injury demonstrates an increase in acute inflammation markers Tnfa and IL1b, macrophages marker Cd11b, and M2 marker Arg1.

Figure 3

CD11b+/CD11c+ macrophages increase at day 4 in the experimental group versus mock. CD11b+/CD86+ (M1) expression increases versus mock. CD11b+/CD206+ (M2) expression significantly increases on day 10, differentiating from the mechanical injury control. Dotted line=baseline expression.

Figure 4

H&E stain shows a thickened lamina propria (A&C) with F4/80 immunohistochemical staining showing high density macrophages (red) at day 7 (B). Day 14 shows a fibrotic appearance (C) with reduced macrophage presence (D). 20x magnification.

Figure 5

Schematic representation of macrophage immunophenotype extrapolated from bleomycin-induced laryngotracheal stenosis. Chemomechanical injury mice demonstrate a similar M1 response versus mechanical injury with a subsequent prolonged M2 response.