Idiopathic subglottic stenosis is associated with activation of the inflammatory IL-17A/IL-23 axis

Abstract

Objectives/hypothesis: Idiopathic subglottic stenosis (iSGS) is a rare and devastating extrathoracic obstruction involving the lower laryngeal and upper tracheal airway. It arises without known antecedent injury or associated disease process. Persistent mucosal inflammation and a localized fibrotic response are hallmarks of the disease. Despite the initial clinical description of iSGS more than 40 year ago, there have been no substantive investigations into the pathogenesis of this enigmatic and progressive airway obstruction. In these studies, we present the initial characterization of the molecular pathogenesis underlying the fibrosing phenotype of iSGS.

Methods: Utilizing 20 human iSGS and healthy control specimens, we applied histologic, immunohistochemical, molecular, and immunologic techniques.

Results: We demonstrate significant activation of the canonical IL-23/IL-17A pathway in the tracheal mucosa of iSGS patients, as well as identify γδ T cells as the primary cellular source of IL-17A.

Conclusion: Our results suggest that aberrant mucosal immune activation is a component in of the pathogenesis of iSGS. Most critically, our work offers new targets for future therapeutic intervention.

Level of evidence: NA Laryngoscope, 126:E356-E361, 2016.

Keywords: IL-17; IL-17A; ISS; iSGS; idiopathic subglottis stenosis; laryngotracheal stenosis; tracheal stenosis.

Figure 1. iSGS Anatomic, Physiologic and Histologic characteristics

Trichome blue staining of iSGS subglottic scar demonstrating extensive blue collagen staining and an intact basement membrane (Magnification 20x, scale bar = 100μM) (A.). Sirius red staining highlights subepithelial collagen deposition in red (Magnification 40x, scale bar = 100μM) (B.). Transmission electron microscopy shows irregular, disordered subepithelial collagen bundles (C.), as well as abundant fibroblasts (D.). qPCR from 10 iSGS patients for extracellular matrix constituents (Types I & III collagen, Fibronectin) from iSGS scar compared with 23 healthy controls (E.), asterisk indicates statistical significance (Two-tailed, Mann Whitney test; p<0.05).

Figure 2. Host Inflammatory Response

qPCR results for panel IL-17A/IL-23 pathway gene products from 10 iSGS patients demonstrating significant upregulation in IL-17A and IL-23 when compared to control healthy trachea, asterisk denotes significance (Two-tailed, Mann Whitney test; p<0.005). IL-1β tended to be higher in iSGS patients but did not reach statistical significance (p=0.06) (A.). Representative images from IHC of IL-17A in surgical specimens from iSGS (n=10), iLTS (n=10), and control (n=10) patients (Magnification 20x, scale bar = 100μM). Accompanying graph summarizing digitally quantified IL-17A levels, depicting significantly more IL-17A expression in iSGS patients, compared with either iLTS, or controls, asterisk denotes significance (Kruskal-Wallis test; p<0.0001) (B.). Flow cytometry from tracheal mucosa in 3 surgical specimens from different iSGS patients, demonstrating that the majority of IL-17A positive cells were γδ TCR positive (C.).