Fungal extracts stimulate solitary chemosensory cell expansion in noninvasive fungal rhinosinusitis

Abstract

Background: Solitary chemosensory cells (SCCs) are rare epithelial cells enriched in nasal polyps and are the primary source of interleukin-25 (IL-25), an innate cytokine eliciting T-helper 2 (Th2) immune response. Although it is proposed that SCCs are stimulated by antigens released by upper airway pathogens, the exogenous triggers of human SCCs remain elusive. We studied patients with noninvasive fungal rhinosinusitis to determine whether extracts of Aspergillus fumigatus and Alternaria alternata stimulate SCC proliferation as an early event in type 2 inflammation.

Methods: Multicolor flow cytometry, immunofluorescence, and enzyme-linked immunoassay were used to interrogate mucosa from patients with mycetomas and allergic fungal rhinosinusitis (AFRS) for SCCs and IL-25. Primary sinonasal epithelial cells from AFRS patients and noninflamed inferior turbinates were stimulated with fungal extracts for 72 hours, and SCC population frequency as well as mitotic activity were quantified using flow cytometry.

Results: SCCs producing IL-25 are enriched in inflamed mucosa compared with intrapatient noninflamed control tissue (38.6% vs 6.5%, p = 0.029). In cultured sinonasal epithelial cells from AFRS nasal polyps, Aspergillus fumigatus and Alternaria alternata stimulated higher SCC frequency compared with controls (27.4% vs 10.6%, p = 0.002; 18.1% vs 10.6%, p = 0.046), which led to increased IL-25 secretion in culture media (75.5 vs 3.3 pg/mL, p < 0.001; 32.3 vs 3.3 pg/mL, p = 0.007). Ki-67 expression was higher in SCCs grown in fungal stimulation conditions compared with controls.

Conclusion: Although fungal antigens are known to potentiate immune response through innate cytokines, including IL-25, the early expansion of SCCs in the presence of fungus has not been described. This early event in the pathogenesis of noninvasive fungal rhinosinusitis may represent a target for intervention.

Keywords: IL-25; allergic fungal rhinosinusitis; fungal antigens; mycetoma; solitary chemosensory cells; type 2 inflammation.

Figure 1:

(A) Computed tomographic scan of a patient with mycetoma of the right maxillary sinus. Immunofluorescent staining was performed on a tissue sample from the right maxillary sinus (top) and the inferior turbinate (bottom). (B-C) Representative staining images of inflamed mucosa from patients with mycetoma (n = 3 patients). DAPI blue, IL-25 green, T2R8 green, GNAT3 red. Arrow heads demonstrate solitary chemosensory cell.

Figure 2:

(A) Representative gating strategy to identify SCCs in primary epithelial cells grown in culture (See Supplementary Figure 1 for gating strategy in tissue samples). (B) Frequency of SCCs in tissue samples among all epithelial cells quantified by flow cytometry (n = 3 AFRS patients; n = 1 mycetoma patients) compared to matched inferior turbinates. (C) Frequency of SCCs in primary sinonasal epithelial cultures stimulated for 72 hours with fungal antigens (n = 6 AFRS patients’ epithelium from nasal polyps; n = 4 patients’ epithelium from non-inflamed turbinates). Error bars represent standard error of mean. [***p <0.0005; **p<0.005; *p<0.05]

Figure 3:

(A) Representative mean fluorescent intensity measured by flow cytometry of Ki-67 expression among SCCs in epithelial cultures grown under three conditions. (B) Frequency of Ki-67 positive SCCs and non-SCC epithelial cells in primary sinonasal cell cultures stimulated for 72 hours with fungal antigens (n = 6 AFRS patients’ epithelium from nasal polyps; n = 4 patients’ epithelium from non-inflamed turbinates). Error bars represent standard error of mean. [***p <0.0005; **p<0.005; *p<0.05]

Figure 4:

IL-25 secreted by epithelial cells into culture media detected by ELISA. (n = 6 AFRS patients’ epithelium from nasal polyps; n = 4 patients’ epithelium from non-inflamed turbinates). Error bars represent standard error of mean. [***p <0.0005; **p<0.005; *p<0.05]