TH1 signatures are present in the lower airways of children with severe asthma, regardless of allergic status

Abstract

Background: The pathogenesis of severe asthma in childhood remains poorly understood.

Objective: We sought to construct the immunologic landscape in the airways of children with severe asthma.

Methods: Comprehensive analysis of multiple cell types and mediators was performed by using flow cytometry and a multiplex assay with bronchoalveolar lavage (BAL) specimens (n = 68) from 52 highly characterized allergic and nonallergic children (0.5-17 years) with severe treatment-refractory asthma. Multiple relationships were tested by using linear mixed-effects modeling.

Results: Memory CCR5⁺ T_H1 cells were enriched in BAL fluid versus blood, and pathogenic respiratory viruses and bacteria were readily detected. IFN-γ⁺IL-17⁺ and IFN-γ^-IL-17⁺ subsets constituted secondary T_H types, and BAL fluid CD8⁺ T cells were almost exclusively IFN-γ⁺. The T_H17-associated mediators IL-23 and macrophage inflammatory protein 3α/CCL20 were highly expressed. Despite low T_H2 numbers, T_H2 cytokines were detected, and T_H2 skewing correlated with total IgE levels. Type 2 innate lymphoid cells and basophils were scarce in BAL fluid. Levels of IL-5, IL-33, and IL-28A/IFN-λ2 were increased in multisensitized children and correlated with IgE levels to dust mite, ryegrass, and fungi but not cat, ragweed, or food sources. Additionally, levels of IL-5, but no other cytokine, increased with age and correlated with eosinophil numbers in BAL fluid and blood. Both plasmacytoid and IgE⁺FcεRI⁺ myeloid dendritic cells were present in BAL fluid.

Conclusions: The lower airways of children with severe asthma display a dominant T_H1 signature and atypical cytokine profiles that link to allergic status. Our findings deviate from established paradigms and warrant further assessment of the pathogenicity of T_H1 cells in patients with severe asthma.

Keywords: IFN-γ; IL-23; IL-28A; IL-33; IL-4; IL-5; IgE; Severe asthma; T(H)1 cells; T(H)17 cells; T(H)2 cells; allergic; type 2 innate lymphoid cells.

Figure 1. Serum IgE Increases With Age in Children with Severe Asthma

(A) Total serum IgE levels in children according to age. **p<0.01. (B) Levels of specific IgE antibodies in children according to age. Bars denote geometric means of positive values and dashed lines denote the limit of sensitivity of the assay. *p<0.05 as compared with children ≤6 years of age. D. pter., Dermatophagoides pteronyssinus (dust mite). The prevalence of allergy according to age was as follows: 6 months – 6 years, allergic (n=16) and non-allergic (n=10); >6 years, allergic (n=22) and non-allergic (n=4).

Figure 2. Surface Signature and Intracellular Cytokine Expression in CD4⁺ T Cells in the Peripheral Blood and BAL Fluid of Children with Severe Asthma

(A) Comparison of the percentage of total CD4⁺ T cells expressing CD45RO and CCR5 in blood and BAL fluid specimens obtained within the same subject. Horizontal bars denote geometric mean values. (B) Representative scatter plots showing expression of CD45RO and CCR5 on CD4⁺ T cells. (C) Correlation between serum total IgE and percentages of CCR5⁺CD4⁺ T cells in BAL specimens from asthmatic subjects. (D) The percentage of IL-4⁺, IL-5⁺, IFN-γ⁺ and IL-17⁺ cells within total CD4⁺ T cells in BAL fluid compared with blood, based on intracellular cytokine staining. (E) Comparison of cytokine-positive CD4⁺ and CD8⁺ T cells within the memory subset in BAL fluid. Horizontal bars denote geometric mean values. *p≤0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3. Heterogeneity of Cytokine-Positive CD4⁺ T Cells in BAL Fluid and Relationship to Serum IgE

(A) Representative scatter plots showing IL-4⁺, IL-5⁺, IL-13⁺, IL-17A⁺ and IFN-γ⁺ cells within the memory CD4⁺ T-cell subset in BAL fluid. (B) Pie charts comparing the distribution of cytokine positive subpopulations expressing various combinations of IL-4, IL-5, IL-13, IL-17A and IFN-γ within memory CD4⁺ T cells present in BAL. Pie charts show data from 6 subjects. Each slice of the pie denotes a single T-cell subpopulation within the memory T-cell subset, and different arc colors denote each cytokine expressed within a given subpopulation. Bacterial culture negative (Bac-) or positive (Bac+). Specific IgE to Cat (C) Aspergillus (Asp), Alternaria (Alt), cow's milk (CM), egg (E), and peanut (PN) is indicated. (C) Summary data for 8 allergic and 6 non-allergic subjects. (D) Correlations between serum total IgE and the percentage of CD4⁺ T cells expressing Th1 or Th2 cytokines, and Th1/Th2 ratio in BAL fluid. Correlation values in parentheses were adjusted for age and inhaled corticosteroid dose. A, allergic; NA, non-allergic. (E) Representative scatter plots from subjects with high and low IgE showing expression of IL-4 and IFN-γ in CD4⁺ T cells in BAL T cells.

Figure 4. Signatures of Secreted Cytokines in BAL Fluid of Asthmatics

(A) Heat map of cytokines in BAL fluid from 68 specimens (includes specimens from 2 different lung regions in 20 subjects). LLL, left lower lobe; RLL, right lower lobe; LUL, left upper lobe; RUL, right upper lobe; RML, right middle lobe; LING, lingular lobe. Samples with undetectable levels are shown in grey. Samples with detectable signals below the standard curve are colored blue to white, while samples within standard curve ranges (>0.02 pg/ml) are scaled red. Green bars (top of heat map) denote samples from allergic subjects. (B) Violin plots of cytokine levels for BAL specimens and relationship to the standard curve (black open dots). Each dot corresponds to the “average” concentration for a given subject derived from all observed MFI values run in at least duplicates and interpolated from the standard curve. Red, green and blue dots correspond to BAL specimens from multi-sensitized (IgE to >3 allergens; 18 subjects), allergic (IgE to 1–3 allergens; 16 subjects) and non-allergic (14 subjects) asthmatics respectively. Horizontal line denotes the lowest value of all standard curves (0.02 pg/ml).

Figure 5. Levels of IL-33, IL-28A/IFN-λ2 and IL-5 are Increased in Asthmatics with Specific IgE to Inhalant Allergens

(A) Box plots of MFI values for cytokines in BAL fluid from asthmatics without and with specific IgE to dust mite, ryegrass, and fungi (Aspergillus and Alternaria). Benjamin Hochberg adjusted P values are denoted. Samples were run in at least duplicates and all MFI values were plotted. (B) Absolute concentration of cytokines in allergic asthmatics with specific IgE to Alternaria. Values are shown for 68 BAL specimens from 48 subjects. Each dot corresponds to the “average” concentration for a given subject derived from all observed MFI values (≥ duplicate runs) and interpolated from the standard curve.

Figure 6. Plasmacytoid and Myeloid DCs are Present in BAL Fluid

(A) Representative density plots of basophils and DC types within lineage-negative cells in BAL fluid and blood. (B) Percentages of basophils and dendritic cell types within lineage-negative cells of blood and BAL. Horizontal bars denote geometric means. (C & D) Collective data and representative histograms comparing expression of FcεRI on each cell type in blood and BAL. (E) Percentage of FcεRI⁺ mDC that expressed IgE. Insufficient cell events were available to reliably phenotype pDC. (F) Representative data showing co-expression of IgE with IgE receptor on BAL mDC. Sample sizes varied based on sufficient cell events for analysis. *p≤0.05. **p=0.002. ND, not done owing to insufficient basophils in BAL. FMO, fluorescence minus one control.