Interferon-γ Treatment of Human Laryngotracheal Stenosis–Derived Fibroblasts

Abstract

IMPORTANCE Laryngotracheal stenosis (LTS) is a fibroproliferative disorder of the glottis, subglottis, and trachea. In models of fibrosis from other organ systems, the CD4+T-cell response has been shown to regulate extracellular matrix deposition. Specifically, helper T cell 2 (TH2) promotes fibrosis, whereas TH1 and associated cytokines have been shown to be antifibrotic. However, this antifibrotic effect of the TH1 response has not been demonstrated in LTS. OBJECTIVE To determine whether the TH1 cytokine interferon-γ inhibits the function of LTS-derived fibroblasts in vitro. DESIGN, SETTING, AND PARTICIPANTS This in vitro controlled study included 6 patients with iatrogenic LTS undergoing routine surgical subglottic and tracheal dilation at a single institution. Fibroblasts were isolated from biopsy specimens of laryngotracheal scar and normal-appearing trachea. The presence of fibroblasts was confirmed by an immunohistochemical analysis. Laryngotracheal stenosis–derived fibroblasts were treated with interferon-γ and compared with untreated controls (2 sets of untreated, LTS-derived fibroblasts [media did not contain interferon-γ]) and normal airway fibroblasts (fibroblasts isolated from normal trachea). Data were collected from August 2015 through June 2016. INTERVENTIONS Treatment with interferon-γ, 10 ng/mL. MAIN OUTCOMES AND MEASURES Cellular proliferation, fibrosis gene expression (using quantitative reverse transcription polymerase chain reaction analysis), soluble collagen, and cellular histologic features were assessed. RESULTS Among the 6 patients (6 women; mean [SD] age, 38.3 [17.2] years), LTS-derived fibroblast proliferation was reduced in patients who received interferon-γ treatment compared with untreated controls on days 3 (mean difference, −6515 cells; 95% CI, −10 630 to −2600 cells) to 6 (mean difference, −47 521 cells; 95% CI, −81285 to −13 757 cells). Interferon-γ treatment reduced collagen types I and III gene expression by 86% and 68%, respectively, and resulted in lower total collagen production (10.94 vs 14.89 μg/mL). In addition, interferon-γ treatment resulted in a 32% reduction in expression of transforming growth factor β in LTS-derived fibroblasts. CONCLUSIONS AND RELEVANCE Interferon-γ reduced proliferation, soluble collagen production, and collagen expression in LTS-derived fibroblasts while also reducing the expression of the profibrotic cytokine transforming growth factor β. These findings suggest that therapeutics aimed at increasing interferon-γ and the TH1 response could attenuate LTS.

Proteomic and Genomic Methylation Signatures of Idiopathic Subglottic Stenosis

“…Differential methylation patterns can help to elucidate disease mechanisms that occur without (or beyond what is attributable to) genetic mutation, including those underlying cancer, autoimmune, metabolic, and fibrotic diseases; 32,33,56,57 moreover, alterations in methylation pattern can alter the course of monogenic diseases 56 . Whereas other techniques have identified modulation of specific inflammatory and profibrotic pathways in iSGS as well as iatrogenic laryngotracheal stenosis, 2,3,58,59 our data represent the first effort to profile epigenetic regulation in either disease process. We observed hypermethylation—typically associated with transcriptional downregulation—of CpG regions in five genes associated with disparate aspects of cell physiology.…”

Inhibition of glutaminase to reverse fibrosis in iatrogenic laryngotracheal stenosis

“…Aspiration pneumonia secondary to dysphagia 0 (0) Primary fibroblasts were isolated from the biopsy specimens, cultured, and expanded, as previously described. 24 Passage 2 to 4 of the primary fibroblast cell lines were used for the purposes of our experiments.…”

M2 Macrophages Promote Collagen Expression and Synthesis in Laryngotracheal Stenosis Fibroblasts

“…In iLTS, fibroblasts (FBs) are considered the main effector cell responsible for excessive deposition of extracellular matrix in the trachea lamina propria. iLTS FBs have been demonstrated to be hypermetabolic, displaying increased proliferation, collagen expression, and collagen production compared to non‐iLTS or normal airway FBs 9–11 . Whereas FBs represent the end effector cell in iLTS, the specific pathways promoting this pathologic profibrotic phenotype remain to be elucidated.…”

“…iLTS FBs have been demonstrated to be hypermetabolic, displaying increased proliferation, collagen expression, and collagen production compared to non-iLTS or normal airway FBs. [9][10][11] Whereas FBs represent the end effector cell in iLTS, the specific pathways promoting this pathologic profibrotic phenotype remain to be elucidated. Recent investigation has demonstrated that the fibrosis observed in iLTS is associated with an aberrant inflammatory response.…”