Role of the type I tumor necrosis factor receptor in inflammation-associated olfactory dysfunction

Abstract

Background: To understand mechanisms of human olfactory dysfunction in chronic rhinosinusitis, an inducible olfactory inflammation (IOI) model has been utilized to chronically express inflammatory cytokines locally, resulting in neuronal loss, diminished odorant responses, and repressed olfactory regeneration. Knockout of the minor tumor necrosis factor α receptor 2 (TNFR2) was previously shown to partially rescue these olfactory changes. The purpose of current study was to investigate the role of the major TNF receptor, TNFR1, in chronic olfactory inflammation.

Methods: Two experimental groups of mice were studied: TNFR1 knockout in IOI background and TNFR1 knockout with allergen-induced inflammation. Olfactory function was assayed by electro-olfactogram (EOG), and olfactory tissue was processed for histology and immunohistochemical staining.

Results: TNF-α was dramatically induced in IOI-TNFR1 knockout mice, but the olfactory epithelium did not show inflammation. EOG responses were normal after either 2 or 8 weeks of TNF-α expression. Ovalbumin-sensitized TNFR1 knockout mice developed markedly diminished eosinophilic inflammatory infiltration.

Conclusion: Genetic deletion of TNFR1 completely blocks TNF-α-induced inflammation and reduces allergen-induced inflammation. Preserved EOG responses suggest a TNFR1-dependent mechanism of TNF-α-induced olfactory neuron dysfunction.

Keywords: TNF-alpha; TNFR1; eosinophils; inflammation; olfactory epithelium; transgenic mice.

FIGURE 1

Histopathological features of the OE in the WT, IOI-TNFR1^−/−, and IOI mice. (Top) Gross appearance of the OE by hematoxylin-eosin staining in the same turbinate of WT and IOI-TNFR1^−/− mice both after 2 and 8 weeks of DOX exposure reveals similarity among the 3 groups. In IOI mice, impairment after 2 weeks is mild, but thickness of OE after 8 weeks is dramatically diminished. Images are at ×20 magnification. (Bottom) An antibody to the CD45 common leukocyte antigen was used to visualize nucleated cells of hematopoietic origin within the olfactory turbinate, to better demarcate the inflammatory cell infiltrate. Number of stained cells is approximately the same in WT and IOI-TNFR1^−/−, either after 2 or 8 weeks, and uniformly distributed throughout the olfactory regions. There is an increase in CD45 staining in IOI after 2 weeks and a very high staining after 8 weeks. Images are at ×40 magnification. Scale bars = 50 μm. Data are representative of at least 6 mice from each group. 2w = 2 weeks; 8w = 8 weeks; DAPI = 4′,6-diamidino-2-phenylindole; DOX = doxycycline; IOI = inducible olfactory inflammation; OE = olfactory epithelium; TNFR1 = tumor necrosis factor α receptor 1; WT = wild-type.

FIGURE 2

Effect of TNFR1 knockout on functional odorant responses in TNF-α–induced olfactory inflammation with DOX for 2 weeks. The bars represent EOG recordings after exposure to vapor phase of 1 × 10⁻⁴ and 1 × 10⁻⁵ AA, AP, and HEP, respectively. The quantitative assessment of responses shows no statistically significant (p < 0.05) difference between WT and IOI-TNFR1^−/− mice for all odorants. There is significant reduction of amplitudes in IOI mice for all odorants when comparing to IOI-TNFR1^−/− mice. Data reflect a minimum of recordings from 4 animals. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001. AA = amyl acetate; AP = acetophenone; DOX = doxycycline; EOG = electro-olfactogram; HEP = heptaldehyde; IOI = inducible olfactory inflammation; SEM = standard error; TNFR1 = tumor necrosis factor α receptor 1; WT = wild-type.

FIGURE 3

Effect of TNFR1 knockout on functional odorant responses in TNF-α–induced olfactory inflammation for 8 weeks. The bars represent EOG recordings after exposure to vapor phase of 1 × 10⁻⁴ and 1 × 10⁻⁵ AA, AP, and HEP, respectively. The quantitative assessment of responses shows no statistically significant (p < 0.05) difference between WT and IOI-TNFR1^−/− mice for all odorants. Reduction of amplitudes in IOI group is substantial. Data reflect a minimum of recordings from 4 animals. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001. AA = amyl acetate; AP = acetophenone; DOX = doxycycline; EOG = electro-olfactogram; HEP = heptaldehyde; IOI = inducible olfactory inflammation; SEM = standard error; TNFR1 = tumor necrosis factor α receptor 1; WT = wild-type.

FIGURE 4

Effect of TNFR1 knockout in allergen-induced olfactory inflammation. (Left column) TNFR1 knockout mice developed markedly diminished eosinophilic inflammatory infiltration in comparison to WT. In general, eosinophils surround axon bundles in subepithelium and do not invade the neuronal layer. (Right column) A trend toward decreased macrophage infiltration is observed in TNFR1 knockout mice. Transition regions to respiratory areas show more robust eosinophil and macrophage infiltration. Images are at ×40 magnification. Scale bars = 50 μm. Data are representative of at least 5 mice from each group. DAPI = 4′,6-diamidino-2-phenylindole; EMBP = eosinophil major basic protein; Krt5 = keratin 5; TNFR1 = tumor necrosis factor α receptor 1; WT = wild-type.

FIGURE 5

Scatter plot for quantification of eosinophil and macrophage staining in WT and TNFR1^−/− mice. Raw values are indicated by circular markers for WT and triangular markers for TNFR1^−/−; blue markers indicate EMBP (eosinophils) and red markers indicate F4/80 (macrophages). Bars represent the mean and the SEM. A reduction in EMBP⁺ cells in TNFR1^−/−mice suggests that TNFR1 plays an important role in eosinophilic allergen-induced inflammation in the olfactory tissue. On the other hand, the number of F4/80⁺ cells in both groups was not significantly different. Data includes measurements of at least 5 mice from each group. ***p < 0.001. EMBP = eosinophil major basic protein; SEM = standard error; TNFR1 = tumor necrosis factor α receptor 1; WT = wild-type.

FIGURE 6

TNFR1 knockout inhibits olfactory neuronal loss in OVA-challenged mice. The mean olfactory epithelium thickness for wild-type (WT) and TNFR1^−/− are 45.89 μm (range, 27.85 to 70.00 μm) and 59.15 μm (range, 49.38 to 76.92 μm) in transitional olfactory epithelium. Data represent a minimum of 5 measurements from each mouse. At least 5 mice from each group were analyzed. Error bars represent SEM. *p < 0.05. SEM = standard error; TNFR1 = tumor necrosis factor α receptor 1; WT = wild-type.