Pediatric severe asthma with fungal sensitization is mediated by steroid-resistant IL-33

Abstract

Background: The mechanism underlying severe asthma with fungal sensitization (SAFS) is unknown. IL-33 is important in fungus-induced asthma exacerbations, but its role in fungal sensitization is unexplored.

Objective: We sought to determine whether fungal sensitization in children with severe therapy-resistant asthma is mediated by IL-33.

Methods: Eighty-two children (median age, 11.7 years; 63% male) with severe therapy-resistant asthma were included. SAFS (n = 38) was defined as specific IgE or skin prick test response positivity to Aspergillus fumigatus, Alternaria alternata, or Cladosporium herbarum. Clinical features and airway immunopathology were assessed. Chronic exposure to house dust mite and A alternata were compared in a neonatal mouse model.

Results: Children with SAFS had earlier symptom onset (0.5 vs 1.5 years, P = .006), higher total IgE levels (637 vs 177 IU/mL, P = .002), and nonfungal inhalant allergen-specific IgE. Significantly more children with SAFS were prescribed maintenance oral steroids (42% vs 14%, P = .02). SAFS was associated with higher airway IL-33 levels. In neonatal mice A alternata exposure induced higher serum IgE levels, pulmonary IL-33 levels, and IL-13(+) innate lymphoid cell (ILC) and TH2 cell numbers but similar airway hyperresponsiveness (AHR) compared with those after house dust mite exposure. Lung IL-33 levels, IL-13(+) ILC numbers, TH2 cell numbers, IL-13 levels, and AHR remained increased with inhaled budesonide during A alternata exposure, but all features were significantly reduced in ST2(-/-) mice lacking a functional receptor for IL-33.

Conclusion: Pediatric SAFS was associated with more oral steroid therapy and higher IL-33 levels. A alternata exposure resulted in increased IL-33-mediated ILC2 numbers, TH2 cell numbers, and steroid-resistant AHR. IL-33 might be a novel therapeutic target for SAFS.

Keywords: IL-33; Severe asthma; fungal sensitization; innate immunity; pediatric; steroid resistance.

Fig E1

IL-33 levels were quantified in BAL fluid by means of Western blotting (A), and IL-33⁺ cells stained by means of immunohistochemistry were quantified in the submucosa of endobronchial biopsy specimens (B; n = 14 children). Group differences were assessed by using the Mann-Whitney U test: *P < .05. ns, Not significant.

Fig E2

Endobronchial biopsy specimens from children aged 6 to 16 years with severe asthma receiving high-dose inhaled steroids only were stained by using immunohistochemistry for IL-33 expression. IL-33⁺ cells in the submucosa were counted and expressed per area of submucosa in children with SAFS compared with children with severe asthma without fungal sensitization (no-SAFS; n = 2). Group differences were assessed by using the Mann-Whitney U test: **P < .01.

Fig E3

Neonatal BALB/c mice were exposed to intermittent intranasal A alternata (Alt), HDM, or saline (PBS) for 3 weeks. Total BAL fluid IL-13 levels were measured by means of ELISA (A), and numbers of Lin⁻CD45⁺ICOS⁺ ILCs (B) and CD3⁺CD4⁺ T cells (C) were quantified by means of flow cytometry. *P < .05, **P < .01, and ***P < .001. ns, Not significant.

Fig E4

Neonatal ST2^−/− and wild-type mice were exposed to intermittent intranasal A alternata (Alt; 5 μg for the first 2 weeks and then 10 μg) or saline (PBS) from day 3 of life for 3 weeks. Levels of IL-4 (A), IL-5 (B), IL-33 (C), and MMP-9 (D) were measured in lung homogenates (LH) by means of ELISA. Data are representative of 2 experiments (n = 6-8 per group). *P < .05.

Fig E5

Neonatal BALB/c mice were exposed to intranasal saline (PBS), HDM, or A alternata (Alt) from day 3 of life for 3 weeks, with concomitant intranasal budesonide (0.6 mg/kg) or PBS from day 10 of life for 2 weeks (prevention regimen). These data relate to the same experiment shown in Fig 5. Numbers of eosinophils were quantified by means of flow cytometry in lung digests after A alternata exposure (A) and HDM exposure (B) with and without budesonide (Bud; n = 4-6 per group for PBS and n = 6-8 per group for HDM or A alternata. *P < .05 and **P < .01.

Fig E6

Endobronchial biopsy specimens from children aged 6 to 16 years with severe asthma were stained by means of immunohistochemistry for IL-13 expression. A, IL-13⁺ cells in the submucosa were counted and expressed per area of submucosa in children with SAFS compared with children with severe asthma without fungal sensitization (no SAFS). B-E, IL-13 (Fig E6, B), IL-5 (Fig E6, C), IL-6 (Fig E6, D), and IL-8 (Fig E6, E) levels in BAL fluid from children with SAFS and non-SAFS were quantified by using the Luminex multiplex assay (Bio-Rad). ns, Not significant.

Fig 1

Increased IL-33 levels (A) and MMP-9 activity (B) in BAL fluid and increased submucosal IL-33⁺ cell numbers in endobronchial biopsy specimens (C) from children with STRA and SAFS compared with those without fungal sensitization (no SAFS). There were 18 specimens for BAL data, and 33 specimens for biopsy data. *P < .05 and ***P < .001.

Fig 2

Fungal exposure in neonatal mice resulted in more severe atopy and inflammation than HDM exposure, but AHR was similar with both allergens. Neonatal BALB/c mice were challenged with intranasal HDM (20 μg for the first 2 weeks and then 25 μg) or A alternata (Alt; 5 μg for the first 2 weeks and then 10 μg) for 3 weeks (A), and assessments of AHR (B), total serum IgE levels (C), allergen-specific IgE levels (D), total pulmonary inflammation (E), and eosinophilic inflammation (F) were made. Data are representative of 2 experiments (n = 4-8 per group). *P < .05 and **P < .01. ns, Not significant.

Fig 3

A alternata exposure resulted in significantly increased lung IL-33 levels, but lung IL-13 levels were similar to those after HDM exposure. Pulmonary IL-4 (A), IL-5 (B), IL-13 (C), and IL-33 (D) levels; BAL fluid MMP-9 levels (E); IL-13⁺ Lin⁻CD45⁺ICOS⁺ ILC numbers (F); and IL-13⁺CD3⁺CD4⁺ T-cell numbers (G) in neonatal BALB/c mice exposed to intranasal saline (PBS), HDM, or A alternata (Alt) for 3 weeks are shown. Data are representative of 2 experiments (n = 4-8 per group). *P < .05, **P < .01, and ***P < .001.

Fig 4

Neonatal ST2^−/− mice have significantly reduced allergic airways disease after A alternata exposure compared with wild-type mice. Neonatal mice were exposed to intermittent intranasal A alternata (Alt; 5 μg for the first 2 weeks and then 10 μg) or saline (PBS) from day 3 of life for 3 weeks. AHR (A and B), total serum IgE levels (C), pulmonary IL-13 levels (D), and IL-13⁺Lin⁻CD45⁺ICOS⁺ ILCs (E) and IL-13⁺CD3⁺CD4⁺ T cells (F) were assessed. Data are representative of 2 experiments (n = 6-8 per group). *P < .05 and **P < .01. MCh, Methacholine; ns, not significant.

Fig 5

AHR is unaffected by steroids after A alternata exposure, whereas inflammation and serum IgE levels are reduced. A, Neonatal BALB/c mice were exposed to intranasal (I-N) saline (PBS), HDM, or A alternata (Alt) from day 3 of life for 3 weeks, with concomitant intranasal budesonide (0.6 mg/kg) or PBS from day 10 of life for 2 weeks (prevention regimen). B-G, AHR (Fig 5, B and C), total lung inflammation (Fig 5, D), BAL inflammation (Fig 5, E), and serum immunoglobulin levels (Fig 5, F and G) were assessed (n = 4-6 per group [PBS] or n = 6-8 per group [HDM or Alt]. *P < .05 and **P < .01. MCh, Methacholine; ns, not significant.

Fig 6

Steroids did not reduce IL-33 levels. Lung IL-13 levels and IL-13⁺ ILC and IL-13⁺ T-cell numbers only remained increased after steroids with A alternata exposure. Neonatal BALB/c mice were exposed to intranasal saline (PBS), HDM, or A alternata (Alt) for 3 weeks from day 3 of life, with either concomitant intranasal budesonide or saline (PBS). Lung IL-13⁺Lin⁻CD45⁺ICOS⁺ ILCs (A) and IL-13⁺CD3⁺CD4⁺ T cells (B) were quantified by means of flow cytometry. Levels of pulmonary IL-13 (C) and IL-33 (D) were measured by means of ELISA. There were 4 to 6 per group for the PBS group and 6 to 8 per group for the HDM or A alternata groups. *P < .05, **P < .01, and ***P < .001. ns, Not significant.