Targeting pathologic macrophage activation through inhibition of MyD88 to attenuate laryngotracheal stenosis

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Project Summary: Laryngotracheal Stenosis (LTS) is the pathologic narrowing of the larynx, subglottis, and trachea secondary to mucosal injury from prolonged intubation. This narrowing leads to dyspnea, dysphonia, and can rapidly progress to airway compromise. Therapeutic interventions for the management of LTS are limited to serial dilation, tracheal resection, or permanent tracheostomy which further impairs communication. Medical therapies for LTS are limited by our poor understanding of LTS pathogenesis. Improved understanding of the mechanisms promoting LTS is needed to improve treatment of this debilitating disease. Previous investigation has revealed that an intact immune response is critical to the development of LTS. Characterization of the immune response in LTS has demonstrated increased populations of CD4+ T-cells and macrophages. Preliminary studies in a murine LTS model reveal that depletion of the macrophage population attenuates LTS fibrosis, implicating their pathologic role. However, the local immune mediators and cell signaling pathways promoting pathologic macrophages in LTS are unknown. Macrophage activation is regulated through stimulation of Toll-like receptors (TLRs). TLRs are highly conserved receptors recognizing Pathogen or Damage Associated Molecular Patterns (PAMPs/DAMPs) and lead to downstream activation of regulatory proteins controlling phenotype. Using single cell RNA sequencing the PI has demonstrates increased expression of the TLR4-MyD88 signaling pathway in LTS macrophages. Furthermore, we have identified increased expression of the DAMP S100A8/A9 in LTS tissue. S100A8/A9 is a known activator of TLR4-MyD88 signaling, and worsens fibrosis in our murine LTS model. These findings indicate that TLR4-MyD88 signaling pathways in macrophages may be critical to LTS pathogenesis. However, the relationship between S100A8/A9, TLR4-MyD88 signaling, and pathologic macrophages has not been explored in LTS or other fibrotic diseases, and may represent a critical signaling axis driving pathologic fibrosis. For this study, we will elucidate the signaling networks promoting pathologic macrophages in laryngotracheal stenosis. In Aim 1 we will assess effect of S100A8/A9 on macrophage phenotype and function in a murine LTS model, establish that S100A8/A9s profibrotic effect is mediated by macrophages, and identify the key sources of pathologic S100A8/A9 in human LTS and a murine model. In Aim 2, we will demonstrate the critical role of TLR4-MyD88 signaling in promoting pathologic macrophages in LTS, and elucidate the role of IL1β in promoting LTS fibrosis. Finally, in Aim 3 we will assess S100A8/A9 as a candidate biomarker for the development of laryngotracheal stenosis in patients who have had prolonged intubation. Collectively, this application will lead to an in-depth understanding of the cell signaling networks promoting dysregulated macrophage mediated inflammation and subsequent fibrosis in LTS. The identification of key regulatory pathways promoting pathologic macrophages in LTS will serve as the foundation for targeted treatment strategies that attenuate fibrosis.