Airway surface mycosis in chronic TH2-associated airway disease

Abstract

Background: Environmental fungi have been linked to TH2 cell-related airway inflammation and the TH2-associated chronic airway diseases asthma, chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP), and allergic fungal rhinosinusitis (AFRS), but whether these organisms participate directly or indirectly in disease pathology remains unknown.

Objective: To determine the frequency of fungus isolation and fungus-specific immunity in patients with TH2-associated and non-TH2-associated airway disease.

Methods: Sinus lavage fluid and blood were collected from sinus surgery patients (n = 118) including patients with CRSwNP, patients with CRS without nasal polyps, patients with AFRS, and non-CRS/nonasthmatic control patients. Asthma status was determined from medical history. Sinus lavage fluids were cultured and directly examined for evidence of viable fungi. PBMCs were restimulated with fungal antigens in an enzyme-linked immunocell spot assay to determine total memory fungus-specific IL-4-secreting cells. These data were compared with fungus-specific IgE levels measured from plasma by ELISA.

Results: Filamentous fungi were significantly more commonly cultured in patients with TH2-associated airway disease (asthma, CRSwNP, or AFRS: n = 68) than in control patients with non-TH2-associated disease (n = 31): 74% vs 16%, respectively (P < .001). Both fungus-specific IL-4 enzyme-linked immunocell spot (n = 48) and specific IgE (n = 70) data correlated with TH2-associated diseases (sensitivity 73% and specificity 100% vs 50% and 77%, respectively).

Conclusions: The frequent isolation of fungi growing directly within the airways accompanied by specific immunity to these organisms only in patients with TH2-associated chronic airway diseases suggests that fungi participate directly in the pathogenesis of these conditions. Efforts to eradicate airway fungi from the airways should be considered in selected patients.

Keywords: Allergic; T(H)2-associated airway disease; airway; asthma; chronic rhinosinusitis; fungal; mycosis.

Figure 1

Fungal recovery from sinus lavage is more likely in the context of Th2-associated airway disease. (A-C) Percent of patients yielding positive fungal cultures from sinus lavage comparing (A) control patients to CRS without nasal polyps, CRS with nasal polyps, and AFRS patients; B) control patients and non-asthmatics to asthmatics; and C) Non-Th2-associated to Th2-associated patients. * p<0.05, ** p<0.01, *** p<0.001. (D, E) Representative Sabouraud’s plate fungal cultures of sinus lavage fluid from two subjects. (F) Photomicrograph of unstained sinus lavage mucus from a representative AFRS patient showing extensive hyphal network (400×). §Control is a subset of all non-asthmatics.

Figure 2

Fungus-specific IL-4 and IgE responses predict Th2-associated disease. Subjects with and without Th2-associated disease (Th2 and no Th2, respectively) were assessed for (A) specific serum IgE and (B) IL-4 memory recall responses from PBMC against whole cell antigens derived from the fungi A. alternata, A. fumigatus, and A. niger, both individually and combined. For the combined analysis, the largest positive response obtained with any of the three fungal antigens was plotted. Values above dotted lines represent positive responses. (C) Relative Operating Characteristic (ROC) curve analyses for both IgE and IL-4 reactivity using optimal IgE & IL-4 cutoff values of 6 kIU/L and 50 cells/10⁶ PBMCs, respectively. AUC: area under the curve.* p<0.05, ** p<0.01, *** p<0.001.

Figure 3

Fungus-specific PBMC IL-4 responses occur in the majority of Th2-associated disease patients. (A-C) PBMC isolated from the indicated subject populations were cultured in media alone or supplemented with 5 mg of antigen derived from nine fungi and IL-4 secretion by ELISpot analysis was performed 24 hours later. A positive reaction to any antigen was >50 IL-4-secreting cells/10⁶ PBMC above media control. Th2-associated airway disease patients have either CRS with nasal polyps or asthma or both D) Maximal fungus specific IL-4 ELISpot responses comparing Th2-associated and non-associated disease subjects. E) Distribution of the number of distinct fungi inducing positive IL-4 ELISpot responses in subjects with allergic airway disease. F) Percentage of Th2-associated disease patients reacting to the indicated fungal allergens. * p<0.05, ** p<0.01, *** p<0.001. §Control is a subset of all non-asthmatics.

Figure 4

Fungal-specific IL-4 responses are highly concordant with the species isolated. (A-C) IL-4 ELISpot data were determined using antigens matched to fungi derived from the same patient and were otherwise collected as in Figure 3 * p<0.05, ** p<0.01, *** p<0.001. §Control is a subset of all non-asthmatics.