Elucidating Olfactory Epithelial Anti-Viral Responses in Persistent Post-Viral Olfactory Dysfunction

项目概要： 持续性病毒感染后嗅觉功能障碍（PVOD）与大范围的病毒相关， 对生活质量产生重大不利影响。嗅上皮细胞分子和细胞的变化 持续性PVOD的病例尚未得到很好的表征。具体而言，我们有兴趣研究 负责外周嗅觉神经发生的嗅觉干细胞的反应和 嗅上皮（OE）损伤后的再生。在嗅觉系统中，组成型TNF α介导的 炎症抑制干细胞分化，而升高的IL-6水平与持续性炎症相关。 嗅觉丧失这些发现提出了延长抗病毒信号传导将抑制嗅干的可能性。 细胞分化，从而损害OE再生并导致嗅觉功能障碍。在我们的初步数据中， 我们发现SARS患者嗅粘膜中I型和II型干扰素信号增加的证据， CoV-2感染和感染小鼠嗅觉干细胞中干扰素信号基因的表达升高 甲型H1N1流感此外，已知Notch信号传导在维持水平基底细胞中是重要的。 细胞（嗅觉干细胞群之一）处于未分化状态。因此，我们假设， PVOD可能与由宿主先天免疫系统驱动的抗病毒嗅觉上皮反应有关， 转化为嗅觉干细胞中炎症信号基因的慢性上调， Notch信号的上调导致干细胞静止和嗅觉再生受损 上皮 在拟议的研究中，我们将调查慢性抗病毒和促炎信号通路 通过细胞因子分析和单细胞免疫组化检测， 转录组测序（Aim 1）。我们将这些细胞因子的表达水平与干扰素 刺激基因与客观措施的嗅觉功能在人类PVOD。此外，我们将 评估细胞因子直接对体外培养的嗅干细胞的影响。在目标2中，我们将评估 使用个体细胞命运图谱研究病毒感染对嗅觉干细胞Notch信号通路的影响 定量免疫染色和单细胞转录组测序。Notch拮抗剂的体内测试 使用病毒感染的小鼠模型将有助于确定病毒影响的可逆性和 Notch途径的治疗靶向。本研究中小鼠和人模型的结合 将允许精确的基因操作和功能测试，以评估嗅觉上皮细胞的命运，以及 允许病毒感染后基因表达的变化直接影响嗅觉的临床结果。在一起， 这些研究将有助于阐明病毒感染后嗅觉持续丧失的细胞和分子机制 这可能会为未来的治疗方法提供潜在的途径。

PROJECT SUMMARY: Persistent post-viral olfactory dysfunction (PVOD) is associated with a large spectrum of viruses with significant adverse impacts on quality of life. The molecular and cellular changes in the olfactory epithelium in cases of persistent PVOD have not been well characterized. Specifically, we are interested in studying the response of the olfactory stem cells which are responsible for peripheral olfactory neurogenesis and the regeneration of olfactory epithelium (OE) following injury. In the olfactory system, constitutive TNF-mediated inflammation suppresses stem cell differentiation while elevated levels of IL-6 are correlated with persistent olfactory loss. These findings raised the possibility that prolonged anti-viral signaling will inhibit olfactory stem cell differentiation and thus impair OE regeneration and result in olfactory dysfunction. In our preliminary data, we show evidence of increased type I and II interferon signaling in the olfactory mucosa of patients with SARS- CoV-2 infection and elevated expression of interferon signaling genes in the olfactory stem cells of mice infected with H1N1 influenza A. Furthermore, Notch signaling is known to be important in maintaining horizontal basal cells (one of the olfactory stem cell populations) in an undifferentiated state. Thus, we hypothesize that persistent PVOD may be related to an anti-viral olfactory epithelial response driven by the host innate immune system that translates to a chronic upregulation of inflammatory signaling genes in the olfactory stem cells and an upregulation of Notch signaling to result in stem cell quiescence and impaired regeneration of the olfactory epithelium. In the proposed studies, we will investigate the chronic anti-viral and pro-inflammatory signaling pathways in the local olfactory epithelial environment and olfactory stem cells through cytokine analysis and single cell transcriptomic sequencing (Aim 1). We will correlate expression levels of these cytokines and interferon stimulated genes with objective measures of olfactory function in humans with PVOD. Furthermore, we will assess the effects of cytokines directly on cultured olfactory stem cells in vitro. In Aim 2, we will assess the impact of viral infection on the Notch signaling pathway in olfactory stem cells using individual cell fate mapping with quantitative immunostaining and single cell transcriptomic sequencing. In vivo testing of Notch antagonists using a mouse model of viral infection will help determine the reversibility of the viral impact and the efficacy of therapeutic targeting of the Notch pathway. The incorporation of both mouse and human models in this study will allow for precise genetic manipulation and functional testing to assess olfactory epithelial cell fate as well as permit direct translation of post-viral changes in gene expression on clinical outcomes of olfaction. Together, these studies will help elucidate the cellular and molecular mechanisms for persistent post-viral olfactory loss that may shed light on potential pathways for future therapeutics.