A genetic model of chronic rhinosinusitis-associated olfactory inflammation reveals reversible functional impairment and dramatic neuroepithelial reorganization

Abstract

Inflammatory sinus and nasal disease is a common cause of human olfactory loss. To explore the mechanisms underlying rhinosinusitis-associated olfactory loss, we have generated a transgenic mouse model of olfactory inflammation, in which tumor necrosis factor alpha (TNF-alpha) expression is induced in a temporally controlled manner specifically within the olfactory epithelium (OE). Like the human disease, TNF-alpha expression leads to a progressive infiltration of inflammatory cells into the OE. Using this model, we have defined specific phases of the pathologic process. An initial loss of sensation without significant disruption is observed, followed by a striking reorganization of the sensory neuroepithelium. An inflamed and disrupted state is sustained chronically by continued induction of cytokine expression. After prolonged maintenance in a deficient state, there is a dramatic recovery of function and a normal histologic appearance when TNF-alpha expression is extinguished. Although obstruction of airflow is also a contributing factor in human rhinosinusitis, this in vivo model demonstrates for the first time that direct effects of inflammation on OE structure and function are important mechanisms of olfactory dysfunction. These features mimic essential aspects of chronic rhinosinusitis-associated olfactory loss, and illuminate underlying cellular and molecular aspects of the disease. This manipulable model also serves as a platform for developing novel therapeutic interventions.

Figure 1.

a, Schematic of cyp2g1-rtTA genetically modified mice. The rtTA DOX regulated transcriptional activator was inserted at the translation initiation codon for the cyp2g1 gene resulting in the deletion of 8 exons. b, In situ hybridization with the cyp2g1 coding region on olfactory cryosections reveals restricted expression in sustentacular cells and the ducts of Bowman's glands. c, The cyp2g1-rtTA mice were crossed to a TRE-lacZ/GFP tandem reporter and whole-mount X-gal stains of the nasal cavity prepared. In the absence of DOX (left), no staining is observed in the tissue. After feeding DOX for 7 d (right), specific, intense staining is observed across the sensory epithelium. RE, Respiratory epithelium; OE, olfactory epithelium; OB, olfactory bulb. d, The reporter induction was visualized by direct imaging of the intrinsic GFP fluorescence in cryosections. At high magnification (left), GFP (green) is restricted to the soma of the apical-located sustentacular cells and their basal processes. Nuclei are identified by DAPI staining (blue). Low-magnification (right) images reveal the broad but not uniform expression in the sustentacular cells. The scale bar in each panel = 25 μm. e, Induction of TNF-α in olfactory epithelium. Expression of TNF-α was assessed by lavage of nasal cavity and quantitative ELISA. The expression of TNF-α is dependent on the cyp2g1-rtTA locus and the DOX inducer (7 d). Error bars represent ±SEM (n = 2 mice, TRE-TNF with dox; n = 3 cyprtTA/TNF no dox, cyprtTA/TNF with dox). Bars indicated with asterisks are at or below the reported sensitivity of the assay. f, Immunofluorescence on olfactory epithelium cryosections with anti-TNF-α antibody reveals DOX-inducible expression of TNF-α in the sustentacular cell soma and enrichment in the endings of their basal processes. The scale bar in each panel = 25 μm.

Figure 2.

Time course of epithelial changes during TNF-α expression. a, Before DOX induction in IOI mice, the epithelium has a normal appearance (left panel) and this tissue maintains a nearly normal appearance after 14 d of DOX exposure (center panel) with some additional cells in the submucosal region and a modest decrease in axon bundle diameter. At 42 d (right panel), the olfactory tissue is markedly abnormal. The epithelium consists primarily of apical sustentacular cells and basal progenitors. The subepithelium is populated by dense infiltration of inflammatory cells and an absence of discernable axon bundles. An OE3-GFP reporter introduced into the IOI mouse documents the initial retention (center) and selective loss of mature and immature ORNs at later times (right). b, Withdrawal of DOX from IOI mice after 42 d of exposure results in essentially complete recovery of olfactory architecture. At 8 d (left), additional cells appear within the epithelial layer and inflammatory cells are sloughing from the apical surface. Inflammation has largely resolved by 14 d after withdrawal although axon bundles retain interstitial cells (center). At 28 d the epithelium is apparently normal (right). The reappearance of neuronal cells and axon bundles is visualized with the OE3-GFP reporter (bottom). The scale bar in each panel = 25 μm.

Figure 3.

Functional loss and recovery in the IOI mouse. a, EOG recordings after exposure to vapor phase of 10⁻³ m amyl acetate and corresponding tissue histology. The responses after 14 d of DOX exposure are reduced despite retention of nearly normal histology. Odor responses are nearly absent at 42 d. The recovery of epithelial architecture is accompanied by an essentially normal EOG. b, Quantitative assessment of EOG responses for three odorants and each time point. Data reflect a minimum of 4 independent recordings. Error bars represent SEM.

Figure 4.

Regenerative state of olfactory epithelium in IOI mice. Proliferating cells are labeled with BrdU and visualized in cryosections. Dividing cells are rare in adult normal olfactory epithelium (left), and the progenitors within the epithelial layer remain quiescent during TNF-α exposure at all time points on DOX. Removal of DOX results in reactivation of proliferation that continues at 28 d. The scale bar in each panel = 25 μm.