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

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.