Olfactory epithelium organoid models identify Ddit3 as a potential therapeutic target against inflammation-related olfactory sensory neuronal loss and functional deficit

Background: Inflammatory activation is a major cause to nasal diseases, such as chronic rhinosinusitis and allergic rhinitis. However, in vitro research model to mimic the process of olfactory inflammation and to screen new therapeutic target is still lacking. Methods: We established three inflammatory models based on olfactory epithelium (OE) organoids, using lipopolysaccharide (LPS), TNFα treatment and doxycycline induction. The efficacy of these models was evaluated by immunostaining, RNA sequencing, qPCR, and functional assays. Results: These inflammatory organoid models mimicked impairment in cell proliferation and neuronal genesis, and showed upregulation of inflammation-related signaling pathway and downregulation of cell cycle-related pathway. We identified that DNA damage inducible transcript 3 (Ddit3) was upregulated in all inflammatory organoid models. Ddit3 downregulation counteracted apoptosis, alleviated cell proliferation and neuronal differentiation, and recovered the functional response to odor stimulation in all three inflammatory organoid models. Ddit3 deficiency counteracted effect of LPS instillation by promoting cell proliferation, recovering neurogenesis, attenuating inflammation, and improving electrophysiological response to odor mixes in the OE. Single-cell RNA sequencing analysis showed that Ddit3 upregulation in mature olfactory sensory neurons of inducible inflammation model and patients with aging-related olfactory dysfunction correlated with endoplasmic reticulum stress and neuron apoptotic process. Conclusions: We established olfactory inflammation organoid models, and made use of these models to identify Ddit3 as a potential therapeutic target against inflammation-related olfactory neuronal loss and functional deficit.