Mechanism of TH2/TH17-predominant and neutrophilic TH2/TH17-low subtypes of asthma

Abstract

Background: The mechanism of T_H2/T_H17-predominant and T_H2/T_H17-low asthma is unknown.

Objective: We sought to study the immune mechanism of T_H2/T_H17-predominant and T_H2/T_H17-low asthma.

Methods: In a previously reported cohort of 60 asthmatic patients, 16 patients were immunophenotyped with T_H2/T_H17-predominant asthma and 22 patients with T_H2/T_H17-low asthma. We examined bronchoalveolar lavage (BAL) fluid leukocytes, cytokines, mediators, and epithelial cell function for these asthma subgroups.

Results: Patients with T_H2/T_H17-predominant asthma had increased IL-1β, IL-6, IL-23, C3a, and serum amyloid A levels in BAL fluid, and these correlated with IL-1β and C3a levels. T_H2/T_H17 cells expressed higher levels of the IL-1 receptor and phospho-p38 mitogen-activated protein kinase. Anakinra, an IL-1 receptor antagonist protein, inhibited BAL T_H2/T_H17 cell counts. T_H2/T_H17-low asthma had 2 distinct subgroups: neutrophilic asthma (45%) and pauci-inflammatory asthma (55%). This contrasted with patients with T_H2/T_H17-predominant and T_H2-predominant asthma, which included neutrophilic asthma in 6% and 0% of patients, respectively. BAL fluid neutrophils strongly correlated with BAL fluid myeloperoxidase, IL-8, IL-1α, IL-6, granulocyte colony-stimulating factor, and GM-CSF levels. Sixty percent of the patients with neutrophilic asthma had a pathogenic microorganism in BAL culture, which suggested a subclinical infection.

Conclusion: We uncovered a critical role for the IL-1β pathway in patients with T_H2/T_H17-predminant asthma. A subgroup of patients with T_H2/T_H17-low asthma had neutrophilic asthma and increased BAL fluid IL-1α, IL-6, IL-8, granulocyte colony-stimulating factor, and GM-CSF levels. IL-1α was directly involved in IL-8 production and likely contributed to neutrophilic asthma. Sixty percent of neutrophilic patients had a subclinical infection.

Keywords: Asthma endotype; C3a; IL-1; T(H)2/T(H)17-predominant asthma; infection; neutrophilic asthma.

Figure 1

Expression of Th2/Th17-inducing cytokines in BAL from three immunophenotypes of asthma. BAL fluid from Th2-predominant (N=22), Th2/Th17-predominant (N=16) and Th2/Th17-low (N=22) asthmatic patients were assayed for IL1α (A), IL1β (B), IL6 (C) and IL23 (D) by ELISA. Each symbol represents a single patient. P values are shown above the graphs.

Figure 2

DAMPs in three immunophenotypes of asthma. The levels of C3a (A), serum amyloid A (B) and uric acid (C) in BAL were measured in Th2-predominant (N=22), Th2/Th17-predominant (N=16) and Th2/Th17-low (N=22) asthma. Each symbol represents a single patient. P values are shown above the graphs.

Figure 3

A: Correlation of cytokines and DAMPs with Th2/Th17 cells. B: Correlation of IL1β with Th2/Th17. C: Correlation of IL1β with C3a. D: Expression of IL1 receptor (IL1R) and phosphor-p38 MAPK in IL4+ (Th2), IL17+ (Th17), IL4/IL17+ (Th2/Th17) and total cells. BAL cells from asthmatic patients were analyzed by flow cytometry for expression of CD4, and intracellular IL4, IL17, IL1R and p-p38 MAPK. *P<0.04, N=6. E: Inhibition of Th2/Th17 cells by anakinra. BAL cells were cultured with increasing concentrations of anakinra for 3 days and then analyzed for dual IL4/IL17+ CD4 T cells by flow cytometry. Each symbol represents an asthmatic patient. P value (on the top of the graph) was calculated by Kruskal Wallis test. F: Inhibition of p-p38 MAPK in BAL T cells by anakinra. BAL cells were cultured with anakinra for 24 hours and then examined for expression of p-p38 MAPK in CD4 T cells by flow cytometry. Each symbol represents an asthmatic patient. P value was calculated by Kruskal Wallis test.

Figure 4

BAL neutrophils in various immunophenotypes of asthma. A: BAL neutrophils in Th2-predominant (N=22), Th2/Th17-predominant (N=16) and Th2/Th17-low (N=22) asthma. Flow cytometric detection of IL4+ and IL4/IL17 dual positive BAL CD4 T cells was performed and the immunophenotype was assigned as reported in ref. 11. B & C: BAL myeloperoxidase (B) and IL-8 (C) in Th2-predominant, Th2/Th17-predominant and Th2/Th17 -low asthma. D: Correlation of BAL neutrophils and IL8 in Th2/Th17-low asthmatic patients. E: Effect of type 2 cytokines on IL8 production. Airway epithelial cells BEAS-2B were cultured with increasing concentration of IL4 and IL13 for 3 days and the culture supernatant was assayed for IL8 by ELISA (**P<0.05, N=4). F: Correlation between IL4 and IL8 in BAL from all 3 study groups (N=42).

Figure 5

A-C: Comparison of the levels of IL17A (A), GM-CSF (B) and G-CSF (C) in BAL from Th2-predominant, Th2/Th17-predominant and Th2/Th17-low asthmatic patients. D: Effect of cytokines/mediators on epithelial IL8 production. Freshly isolated human bronchial epithelial cells (HBE) and BEAS-2B cells were cultured with various cytokines/mediators (10 ng/ml) singly or in combination for 3 days. The BEAS-2B cell cultures were primed with a subthreshold dose of LPS (0.25 μg/ml). Culture supernatant was assayed for IL8 by ELISA. *P<0.05 (N=4). E: Freshly isolated human bronchial epithelial cells were cultured with DMSO or SB212190 (10 μM) in the presence of IL1α (10 ng/ml) for 3 days. The culture supernatant was assayed for IL8 by ELISA. *P<0.05 (N=3).