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.