The origins and roles of macrophages in postnatal tympanic membrane development, homeostasis, and repair

PROJECT SUMMARY (Abstract) The integrity of the tympanic membrane (TM, eardrum) is essential for sound transduction and hearing. TM also serves as a protective barrier between the external auditory canal and the middle ear against invading pathogens. TM perforation, damage-induced otitis media, and conductive hearing loss are common clinical problems affecting millions of people worldwide. Macrophages, the first line of innate immune defense, are distributed in all organs and tissues of the body, including the eardrum. They are essential in tissue development, homeostasis, inflammation, and repair. However, the specific role of macrophages in TM biology under steady- state and injured conditions has been largely overlooked and remains poorly studied. When studying macrophages in the inner ear, we captured the large population of tissue-resident macrophages (TRMs) embedded in the TM. The TRMs in the neonatal stage largely colonized `nascent' blood vessels and `underdeveloped' peripheral nerve bundles. Their population dropped, and morphology notably changed from neonate to young adult. Postnatal development of the TRMs intriguingly paralleled vascular maturation and neuronal development. When the TM is acutely perforated in adults, monocyte-derived macrophages rapidly congregate in the wound area and angiogenesis is initiated. Our pilot data also reveal the TM is remarkably rich in TRPV1+ sensory nerve fibers. Our bulk RNA seq data shows high upregulation of Tac1 (Tachykinin Precursor 1, encoding for tachykinin neuropeptides such as substance P and neurokinin A) in the perforated TM at an early stage of injury. Neuropeptides are primarily secreted by neurons following injury. These initial findings raise fundamental questions: What is the origin of the neonatal TRMs? Are they critical for the postnatal development of the TM and required for vascular and neuronal maturation? What molecular signal elicits monocyte recruitment and angiogenesis? Does TM injury activate TRPV1+ neurons to release Tac1-encoded neuropeptides, stimulating wound healing by promoting macrophage recruitment and angiogenesis? To address these questions, we will use a comprehensive set of cutting-edge research methodologies, including genetic fate mapping, chimeric bone marrow transplantation, high-resolution OCT, and genetic mutation, to thoroughly investigate the embryonic source of TRMs, their core function in postnatal TM development, and the molecular signaling involved in adult TM wound healing. Characterization of the origin, diversity, and postnatal dynamics of macrophages in the TM will reveal the previously unknown complexity of macrophage biology. In particular, it will reveal how the innate immune system in the TM is constructed, how it is maintained, and how it functions. Identification of the molecular signaling in TM wound healing in this project will also spur the development of new therapeutics to facilitate the repair of damaged TM.