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

项目摘要： 喉气管狭窄（LTS）是喉、声门下和气管的病理性狭窄 继发于长时间插管造成的粘膜损伤。这种狭窄会导致呼吸困难、发音困难， 迅速发展为气道损伤。用于管理LTS的治疗干预措施仅限于 连续扩张、气管切除或永久性气管造口术，进一步损害沟通。医疗 LTS的治疗受到我们对LTS发病机制的认识不足的限制。更好地了解 需要促进LTS的机制来改善这种使人衰弱的疾病的治疗。 先前的研究表明，完整的免疫反应对LTS的发展至关重要。 LTS中免疫应答的表征已经证明了增加的CD 4 + T细胞群体， 巨噬细胞在小鼠LTS模型中的初步研究表明， 减弱LTS纤维化，暗示其病理作用。然而，局部免疫介质和细胞信号传导 在LTS中促进病理性巨噬细胞的途径是未知的。 巨噬细胞活化通过刺激Toll样受体（TLR）来调节。TLRs高度 识别病原体或损伤相关分子模式的保守受体（PAMP/DAMP）和 导致控制表型调节蛋白的下游活化。使用单细胞RNA测序 PI显示LTS巨噬细胞中TLR 4-MyD 88信号通路的表达增加。 此外，我们已经鉴定了LTS组织中DAMP S100 A8/A9的表达增加。S100 A8/A9是一款 已知的TLR 4-MyD 88信号传导激活剂，和我们的鼠LTS模型中的纤维化。这些发现 表明巨噬细胞中TLR 4-MyD 88信号通路可能对LTS发病机制至关重要。然而，在这方面， S100 A8/A9、TLR 4-MyD 88信号传导和病理性巨噬细胞之间的关系尚未被阐明， 在LTS或其他纤维化疾病中探索，并且可能代表驱动病理性纤维化的关键信号传导轴。 在这项研究中，我们将阐明促进喉气管病理性巨噬细胞的信号网络， 狭窄在目标1中，我们将评估S100 A8/A9对小鼠LTS中巨噬细胞表型和功能的影响 模型，建立了S100 A8/A9 s促纤维化作用是由巨噬细胞介导的，并确定了关键来源 人LTS和小鼠模型中病理性S100 A8/A9的表达。在目标2中，我们将展示 TLR 4-MyD 88信号在LTS中促进病理性巨噬细胞的作用，并阐明IL 1 β在LTS中促进病理性巨噬细胞的作用。 LTS纤维化。最后，在目标3中，我们将评估S100 A8/A9作为开发 长期插管患者的喉气管狭窄。总的来说，这一应用将导致 深入了解促进巨噬细胞介导的失调的细胞信号网络 炎症和随后的纤维化。确定促进病理性的关键调节途径 LTS中的巨噬细胞将作为减轻纤维化的靶向治疗策略的基础。

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.