Antagonism of microRNA-126 suppresses the effector function of TH2 cells and the development of allergic airways disease

Abstract

Allergic asthma is an inflammatory disease of the lung characterized by abnormal T helper-2 (T(H)2) lymphocyte responses to inhaled antigens. The molecular mechanisms leading to the generation of T(H)2 responses remain unclear, although toll-like receptors (TLRs) present on innate immune cells play a pivotal role in sensing molecular patterns and in programming adaptive T cell responses. Here we show that in vivo activation of TLR4 by house dust mite antigens leads to the induction of allergic disease, a process that is associated with expression of a unique subset of small, noncoding microRNAs. Selective blockade of microRNA (miR)-126 suppressed the asthmatic phenotype, resulting in diminished T(H)2 responses, inflammation, airways hyperresponsiveness, eosinophil recruitment, and mucus hypersecretion. miR-126 blockade resulted in augmented expression of POU domain class 2 associating factor 1, which activates the transcription factor PU.1 that alters T(H)2 cell function via negative regulation of GATA3 expression. In summary, this study presents a functional connection between miRNA expression and asthma pathogenesis, and our data suggest that targeting miRNA in the airways may lead to anti-inflammatory treatments for allergic asthma.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

HDM-induced miR-126 expression is TLR4- and MyD88-dependent. (A) Fold change in miRNA-126, -16, and -21 expression in the airway wall of mice challenged twice with HDM as compared to baseline (nonallergic, SAL mice). (B) Fold change in miR-126 expression in HDM allergic WT, TLR4−/−, and MyD88−/− mice as compared to baseline (SAL mice). Results are mean ± SEM (n = 3–4 mice/group). *, P < 0.05, HDM versus SAL. Data are representative of two independent experiments.

Fig. 2.

TLR4/MyD88 deficiency abolishes HDM-induced AHR and reduces airway inflammation. (A–C) Total lung resistance is presented as a percentage change over baseline measurement (water) in response to inhaled methacholine in allergic (HDM) versus nonallergic (SAL) wild-type (WT), TLR4−/−, and MyD88−/− mice. Results are mean ± SEM (n = 6–10 mice/group). *, P < 0.05. [D (WT), E, and F] Number of cells in BALF. Results are mean ± SEM (n = 3–4 mice/group). *, P < 0.05. Data are representative of two independent experiments.

Fig. 3.

Silencing miR-126 function by antagomir abolishes HDM-induced airways hyperreactivity and reduces allergic inflammation. (A) HDM-induced miR-126 expression after no treatment (no ant), treatment with a scrambled control sequence (ant-scrambled), or treatment with antagomir specific for the miR-126 sequence (ant-miR-126) in allergic mice. Results are mean ± SEM of the fold change from nonallergic mice treated with saline (SAL) only (baseline) (n = 3–4 mice/group). *, P < 0.05. (B) Total lung resistance as percentage change of baseline measurement (water) in response to inhaled methacholine in HDM challenged allergic mice treated with ant-miR-126 or ant-scrambled versus nonallergic (SAL) mice. Results are mean ± SEM (n = 6–10 mice/group). *, P < 0.05. (C) Number of inflammatory cells in BALF. Results are mean ± SEM (n = 3–4 mice/group). *, P < 0.05. (D) Number of peribronchial eosinophils (×1000) per high-power field (HPF), (E) representative H&E stained histological section showing decreased eosinophil numbers in tissue (arrows), and (F) number of mucus-producing cells (×1000) per HPF. Results are mean ± SEM (n = 3–4 mice/group). **, P < 0.01. Data are representative of two independent experiments.

Fig. 4.

Silencing miR-126 function by antagomir impairs TH2 responses in the lung. (A) IL-5, IL-13, and IFN-γ release from in vitro HDM-stimulated peribronchial lymphnode cells after no treatment (SAL) or treatment with a scrambled control sequence (ant-scrambled) or treatment with antagomir specific for the miR-126 sequence (ant-miR-126) in allergic mice and in nonallergic mice (SAL). Results are mean ± SEM (n = 6–10 mice per group). *, P < 0.05, ant-scrambled versus SAL; **, P < 0.01, ant-miR-126 versus SAL. (B) Percentages of CD4+ cells in lung homogenates and (C) percentages of CD11b+ CD11c+ MHC class II high (myeloid DCs) and pDC+ MHC class II high cells (plasmacytoid DCs) in lung homogenates. Results are mean ± SEM of three experiments (n = 4–6 mice/group per experiment). (D) Fold change in OBF.1/BOB.1 and PU.1 expression in the airway wall and GATA3 expression in the parenchyma of HDM-treated allergic mice that were exposed to ant-miR-126 as compared to those mice treated with ant-scrambled (baseline). **, P < 0.01, ant-miR-126 versus ant-scrambled. Results are mean ± SEM (n = 4 mice/group). Data are representative of two independent experiments.