Mucosal Type 2 Innate Lymphoid Cells Are a Key Component of the Allergic Response to Aeroallergens

Abstract

Rationale: Newly characterized type 2 innate lymphoid cells (ILC2s) display potent type 2 effector functionality; however, their contribution to allergic airways inflammation and asthma is poorly understood. Mucosal biopsy used to characterize the airway mucosa is invasive, poorly tolerated, and does not allow for sequential sampling.

Objectives: To assess the role of ILC2s during nasal allergen challenge in subjects with allergic rhinitis using novel noninvasive methodology.

Methods: We used a human experimental allergen challenge model, with flow cytometric analysis of nasal curettage samples, to assess the recruitment of ILC2s and granulocytes to the upper airways of subjects with atopy and healthy subjects after allergen provocation. Soluble mediators in the nasal lining fluid were measured using nasosorption.

Measurements and main results: After an allergen challenge, subjects with atopy displayed rapid induction of upper airway symptoms, an enrichment of ILC2s, eosinophils, and neutrophils, along with increased production of IL-5, prostaglandin D2, and eosinophil and T-helper type 2 cell chemokines compared with healthy subjects. The most pronounced ILC2 recruitment was observed in subjects with elevated serum IgE and airway eosinophilia.

Conclusions: The rapid recruitment of ILC2s to the upper airways of allergic patients with rhinitis, and their association with key type 2 mediators, highlights their likely important role in the early allergic response to aeroallergens in the airways. The novel methodology described herein enables the analysis of rare cell populations from noninvasive serial tissue sampling.

Keywords: ILC2; Th2; allergic airway inflammation; asthma.

Figures

Figure 1.

Increased nasal symptom scores in subjects with atopy after nasal allergen challenge (NAC). Nasal symptoms were assessed at baseline (30 min before NAC) and at the time points shown after NAC (0 h) in subjects with atopy (n = 9) and healthy controls (n = 8) using a nasal symptom questionnaire. Subjects scored the severity of their nasal running, blockage, itching, and sneezing from 0 to 3; the total nasal symptom score represents the cumulative score. Statistical analysis was performed using a Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney U tests; data are displayed as median (25th, 75th percentiles). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2.

Increased recruitment of granulocytes to the upper airways of subjects with atopy after an allergen challenge. Subjects with atopy (n = 9) and healthy control subjects (n = 8) were challenged intranasally with timothy grass pollen. Nasal curettage samples were collected from the inferior turbinate, and granulocytes were enumerated via flow cytometric analysis. (A) Eosinophils (CD9+ CD16−) and neutrophils (CD9− CD16+) were determined within the single, live, HLA-DR− gate. (B) Representative flow cytometric plots display eosinophils and neutrophils from two healthy subjects and two subjects with atopy at baseline and 6 hours after nasal allergen challenge (NAC). (C) Eosinophil and (D) neutrophil numbers for healthy subjects and subjects with atopy at baseline at 6 hours after NAC are shown. Bars represent median values. Kruskal-Wallis tests were used to assess differences between groups; further statistical analysis for paired data was calculated using Wilcoxon matched-pairs test, and unpaired data were calculated using the Mann-Whitney U test. *P < 0.05; **P < 0.01; ***P < 0.001; P < 0.1 displayed. FSC-A/H = forward scatter area/height; IR = infrared.

Figure 3.

Induction of type 2 mediators in the nasal lining fluid of subjects with atopy after nasal allergen challenge (NAC). Nasal lining fluid samples were collected using nasosorption, at baseline (30 min before NAC) and at 2-hour intervals up to 8 hours after NAC. Levels of (A) IL-5, (B) prostaglandin D2 (PGD2), (C) CCL17/TARC, (D) CCL11/eotaxin-1, (E) CCL26/eotaxin-3, (F) IFN-γ, and (G) IL-6 were determined in subjects with atopy and healthy control using Meso-Scale Discovery and a PGD2-methoxime enzyme immunoassay. Levels 6 hours after NAC are displayed. Bars represent median values, and dashed lines represent the limit of detection. Statistical analysis for paired data was calculated using Wilcoxon matched-pairs test, and unpaired data were calculated using the Mann-Whitney U test. *P < 0.05; **P < 0.01; P < 0.1 displayed. CCL = chemokine ligand.

Figure 4.

Individuals with atopy have increased recruitment of type 2 innate lymphoid cells (ILC2s) to the upper airways after an allergen challenge. Subjects with atopy (n = 9) and healthy control subjects (n = 8) were challenged intranasally with timothy grass pollen. Nasal curettage samples were collected from the inferior turbinate. (A) ILC2s were enumerated via flow cytometry analysis using the following gating strategy: live, single, lineage− (CD2, CD3, CD14, CD16, CD19, CD56, CD235a) CD123− FcεR1α−, and CD127+ CRTh2+ cells. (B) Representative flow cytometric plots of CD127+ CRTh2+ ILC2s from two healthy subjects and two subjects with atopy are shown at baseline and 6 hours after nasal allergen challenge (NAC). (C) ILC2 numbers from healthy subjects and subjects with atopy before and after NAC are displayed; bars represent median values. Kruskal-Wallis tests were used to assess differences between groups, and further statistical analysis for paired data was calculated using Wilcoxon matched-pairs test, and unpaired data were calculated using the Mann-Whitney U test. *P < 0.05; ***P < 0.001; P < 0.1 displayed. FSC-A/H = forward scatter area/height; IR = infrared.

Figure 5.

Markers of type 2 responses correlate with type 2 innate lymphoid cell (ILC2) recruitment. Subjects with atopy (n = 9) and healthy control subjects (n = 8) were challenged intranasally with timothy grass pollen. The number of ILC2s in the upper airways 6 hours after nasal allergen challenge (NAC) correlated with (A) eosinophil number 6 hours after NAC, (B) baseline serum IgE levels (n = 7 healthy control subjects), and (C) IL-5 levels 6 hours after NAC, using Spearman’s correlation coefficient.