Quantification of Inflammatory Markers in Laryngotracheal Stenosis

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Abstract

Objectives

(1) Develop a novel method for serial assessment of gene and protein expression in laryngotracheal stenosis (LTS). (2) Assess cytokine expression and determine an immunophenotype in LTS.

Study Design

A matched comparison of endolaryngeal brush biopsy samples from laryngotracheal scar and normal airway.

Setting

Tertiary care hospital, 2015-2016.

Methods

Brush biopsy specimens of laryngotracheal scar and normal trachea were obtained from 17 patients with LTS at the time of operating room dilation and were used for protein and RNA extraction. Gene expression of the TH1 cytokine interferon γ (INF-γ), TH2 cytokine interleukin 4 (IL-4), transforming growth factor β, and collagen 1 (Coll1) was quantified with quantitative real-time polymerase chain reaction. Cytokine analysis was performed with flow cytometry with a cytometric bead array.

Results

LTS specimens demonstrated a 13.68-fold increase in Coll1 gene expression versus normal (P < .001, N = 17). Additionally, IL-4 gene expression showed a 3.76-fold increase (P < .001, N = 17) in LTS scar. When stratified into iatrogenic LTS and idiopathic subglottic stenosis cohorts, INF-γ gene expression was significantly increased in idiopathic subglottic stenosis (P = .011). Soluble cytokine measurements were below the limit of detection for reliable quantification and thus could not be assessed.

Conclusions

Brush biopsies from LTS samples can be successfully utilized for RNA extraction and demonstrate the expected increase in Coll1 gene expression associated with LTS. Preliminary gene expression suggests that abnormal collagen production may be mediated by the TH2 cytokine IL-4 and that increased INF-γ expression may represent a key difference between iatrogenic LTS and idiopathic subglottic stenosis. Further analysis of soluble cytokines is needed to confirm these findings.

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