Antifungal immune reactivity in nasal polyposis

Abstract

As a fungal etiology has been proposed to underlie severe nasal polyposis, the present study was undertaken to assess local antifungal immune reactivity in nasal polyposis. For this purpose, microbial colonization, along with the pattern of T helper 1 (Th1)/Th2 cytokine production and Toll-like receptor (TLR) expression, was evaluated in patients with nasal symptoms and with and without polyposis and in healthy subjects. The results show that Th2 reactivity was a common finding for patients with nasal polyposis regardless of the presence of microbes. The production of interleukin-10 was elevated in patients with bacterial and, particularly, fungal colonization, while both TLR2 expression and TLR4 expression were locally impaired in microbe-colonized patients. Eosinophils and neutrophils, highly recruited in nasal polyposis, were found to exert potent antifungal effector activities toward conidia and hyphae of the fungus and to be positively regulated by TLR2 or TLR4 stimulation. Therefore, a local imbalance between activating and deactivating signals to effector cells may likely contribute to fungal pathogenicity and the expression of local immune reactivity in nasal polyposis.

FIG. 1.

Microbial colonization in nasal lavage specimens of NP⁺ and NP⁻ patients or healthy controls. The nasal lavages and microbial growth were carried out as described in Materials and Methods.

FIG. 2.

Local cytokine production in NP⁺ patients. Cytokine production was detected by ELISA (A) or real time RT-PCR (B) in the nasal lavage specimens (black bars) or nasal polyps (white bars) of NP⁺ patients with or without (None) microbial colonization. Levels of cytokines were below the detection limit of the assays in the nasal lavage specimens from healthy donors. In the RT-PCR assay, the results are expressed as relative levels of cytokine mRNA (change in cycle threshold [ΔΔCt]) in polyps from NP⁺ patients compared to those in nasal brush specimens from healthy subjects. *, P < 0.05 for results for patients with bacterial or fungal colonization versus those for noncolonized patients.

FIG. 3.

TLR expression in NP⁺ patients. TLR expression in nasal polyps that were positive for bacteria, fungi, or neither (None) as determined by RT-PCR is shown. TLR expression was inconsistently detected in the nasal brush specimens from healthy subjects. cDNA levels were normalized against levels of the hypoxanthine phosphoribosyltransferase gene. Shown are the results from one experiment representative of five done.

FIG. 4.

Antifungal and immunomodulatory activities of PMNs and eosinophils. Purified PMNs and eosinophils from peripheral blood were incubated with resting conidia and assessed for phagocytosis, fungicidal activity, and oxidant and cytokine production as detailed in Materials and Methods. The hyphal damage activity was assessed as detailed in Materials and Methods. The numbers inside the micrographs are percentages (means ± SEs) of phagocytosis. Photographs were taken with a high-resolution-microscopy camera (AxioCam color). Experiments were done in triplicate. Values are means ± SEs of results for samples taken from five independent experiments. *, P < 0.05 for results for conidium-stimulated versus unstimulated (None) cells. ROIs, reactive oxygen intermediates.

FIG. 5.

Effects of TLR agonists on the antifungal functions of eosinophils and PMNs. Peripheral PMNs and eosinophils were pretreated with zymosan (ZYM; 10 μg/ml) or lipopolysaccharide (LPS; 10 μg/ml) for 120 min before the assessment of effector functions, after a subsequent incubation with resting conidia (120 min). For phagocytosis and fungicidal activity, see Materials and Methods. Values are percentages (means ± SEs) for samples taken from five independent experiments. *, P < 0.05 for functions with and without (None) TLR ligands.