Poster U18 - Antimicrobial Peptides Reduce Bacterial-Induced Subglottic Stenosis in Mice

Introduction: Pediatric subglottic stenosis (SGS) is characterized by the formation of aberrant fibrotic scar tissue from prolonged endotracheal tube (ET) intubation, leading to breathing difficulties and impaired phonation. Most cases require repeated endoscopic interventions to open the airway and severe cases require invasive surgeries like laryngotracheal reconstruction. Independent of severity, most SGS patients experience major complications such as restenosis and laryngeal edema and are left with severe dysphonia, dyspnea, and cognitive delays. We have recently discovered that in SGS-induced mice perturbing the laryngotracheal microbiome with antibiotics has immunomodulatory effects, resulting in decreased SGS severity. During airway injury, the epithelial barrier is damaged permitting bacteria to enter the lamina propria, driving an inflammatory cascade that promotes myofibroblast differentiation and scar tissue formation. Therefore, modulating the airway bacterial communities represents a novel avenue for a preventative SGS treatment. In this work, we demonstrate that the antimicrobial peptide (AMP) Lasioglossin-III can modulate the upper airway microbiome when locally delivered on a coated ET and mitigate inflammation. We then show that decreased airway bacteria abundance leads to reduced fibrosis with negligible scar matrix deposition, and altogether abrogates SGS.

Materials and

Methods: We fabricated mouse-sized ETs coated with a polymeric AMP-eluting layer, quantified AMP release over 10 days, and validated bactericidal activity for both planktonic and biofilm-resident bacteria against S. aureus and P. aeruginosa. Biocompatibility was assessed against airway fibroblasts and epithelial cells. Ex vivo testing: We inserted AMP-ETs and ET controls into excised laryngotracheal complexes of C57BL/6 mice and assessed biofilm formation after 24 hours. In vivo testing: AMP-ETs and ET controls were inserted in sham or SGS-induced LTCs,4 which were then implanted subcutaneously in receptor mice and assessed for immune response and SGS severity after 7 days. H&E and Masson’s trichrome were conducted to visualize lamina propria thickness and collagen deposition, while immunohistochemistry for CD3 and F4/80 revealed infiltrating T cells and macrophages, respectively.

Results, Conclusions, and Discussions: We achieved reproducible, linear AMP release from mouse-sized ETs at 1.16µg/day resulting in strong bacterial inhibition in vitro and ex vivo. This was repeated for 2 additional peptides showcasing the robust platform capable of incorporating many different peptides. Furthermore, Lasioglossin-III was biocompatible with both airway fibroblasts and epithelial cells. In vivo, SGS-induced laryngotracheal complexes exhibited a thickened scar tissue typical of stenosis, while the use of AMP-ET abrogated stenosis (Fig. 1. A-C). Notably, SGS airways exhibited high infiltration of T cells and macrophages, which was reversed with AMP-ET treatment to quantities similar to that of uninjured airways (Fig. 1. D-E). We developed an antimicrobial peptide-eluting endotracheal tube platform that modulates the laryngotracheal microbiome upon insertion into the airway. Upon implantation in an SGS injured airway, our treatment reduces lamina propria thickness or stenosis severity, mitigates macrophage infiltration and activation and antigen specific T cell responses, and prevents myofibroblast differentiation resolving stenosis progression.

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