IL-13 dampens human airway epithelial innate immunity through induction of IL-1 receptor-associated kinase M

Abstract

Background: Impaired airway mucosal immunity can contribute to increased respiratory tract infections in asthmatic patients, but the involved molecular mechanisms have not been fully clarified. Airway epithelial cells serve as the first line of respiratory mucosal defense to eliminate inhaled pathogens through various mechanisms, including Toll-like receptor (TLR) pathways. Our previous studies suggest that impaired TLR2 function in T(H)2 cytokine-exposed airways might decrease immune responses to pathogens and subsequently exacerbate allergic inflammation. IL-1 receptor-associated kinase M (IRAK-M) negatively regulates TLR signaling. However, IRAK-M expression in airway epithelium from asthmatic patients and its functions under a T(H)2 cytokine milieu remain unclear.

Objectives: We sought to evaluate the role of IRAK-M in IL-13-inhibited TLR2 signaling in human airway epithelial cells.

Methods: We examined IRAK-M protein expression in epithelia from asthmatic patients versus that in normal airway epithelia. Moreover, IRAK-M regulation and function in modulating innate immunity (eg, TLR2 signaling) were investigated in cultured human airway epithelial cells with or without IL-13 stimulation.

Results: IRAK-M protein levels were increased in asthmatic airway epithelium. Furthermore, in primary human airway epithelial cells, IL-13 consistently upregulated IRAK-M expression, largely through activation of phosphoinositide 3-kinase pathway. Specifically, phosphoinositide 3-kinase activation led to c-Jun binding to human IRAK-M gene promoter and IRAK-M upregulation. Functionally, IL-13-induced IRAK-M suppressed airway epithelial TLR2 signaling activation (eg, TLR2 and human β-defensin 2), partly through inhibiting activation of nuclear factor κB.

Conclusions: Our data indicate that epithelial IRAK-M overexpression in T(H)2 cytokine-exposed airways inhibits TLR2 signaling, providing a novel mechanism for the increased susceptibility of infections in asthmatic patients.

FIG 1

Increased IRAK-M protein expression in airway epithelial cells from asthmatic patients. Upper panel, Airway epithelial IRAK-M protein quantitative data in endobronchial biopsy specimens of healthy subjects (n = 4) and asthmatic patients (n = 6). Lower panel, Representative IRAK-M immunohistochemistry staining in bronchial epithelial cells and submucosal inflammatory cells (black arrows, original magnification ×200). Data are presented as means (thick horizontal lines) ± SEMs.

FIG 2

IL-13 induces IRAK-M protein expression in cultured human brushed bronchial epithelial cells. Upper panel, IRAK-M protein quantitative data (means ± SEM) in healthy subjects (n = 4) and asthmatic patients (n = 6). Lower panel, Representative IRAK-M and GAPDH Western blots.

FIG 3

PI3K activation is required for IL-13–induced IRAK-M expression. A, Representative phospho-Akt (pAkt) and total Akt Western blots (n = 3 independent experiments). B, Upper level, IRAK-M protein quantitative data (means ± SEMs) are from 4 independent experiments. Lower panel, Representative IRAK-M and GAPDH Western blots. DMSO, Dimethyl sulfoxide; Wort, wortmannin.

FIG 4

c-Jun directly binds to the IRAK-M gene promoter through PI3K activation on IL-13 stimulation. A, Representative phospho–c-Jun and GAPDH Western blots (n = 4 independent experiments). B, Phospho– c-Jun activity data (means ± SEM) are from 3 independent experiments. C, Chromatin immunoprecipitation assay. Left panel, Increased c-Jun binding to IRAK-M gene promoter after IL-13. Data (means ± SEM) are from 3 independent experiments. Right panel, Representative agarose gel electrophoresis. Ab, Antibody; DMSO, dimethyl sulfoxide; Wort, wortmannin.

FIG 5

IL-13 decreases TLR2 protein expression in human airway epithelial cells. A, TLR2 protein quantitative data in cultured brushed bronchial epithelial cells from healthy subjects (n = 4) and asthmatic patients (n = 6). B, PI3K inhibition restored TLR2 protein expression in IL-13–treated normal human tracheobronchial epithelial cells (n = 4). Data are presented as means ± SEMs. DMSO, Dimethyl sulfoxide; Wort, wortmannin.

FIG 6

Role of IRAK-M in IL-13–mediated impairment of epithelial TLR2 signaling. A, IRAK-M shRNA (shIRAK-M) significantly reduced IRAK-M mRNA (left panel) and protein (right panel) expression compared with the control (firefly luciferase shRNA [shLUC]) in normal human brushed bronchial epithelial cells (n = 4). B, NF-κB p65 activity, TLR2 protein, and human hBD2 peptide levels in normal human brushed bronchial epithelial cells that were transduced with shLUC or shIRAK-M, followed by IL-13, Pam2CSK4 (Pam2), or both treatments (n = 4). Short thick horizontal lines represent means.

FIG 7

IRAK-M overexpression decreases TLR2 signaling activation in NCIH292 cells. A, Overexpression of human IRAK-M protein was confirmed by using Western blotting. B, NF-κB p65 activity. C, IL-8 protein levels in cell supernatants. Data are from 3 independent experiments and presented as means ± SEMs. EV, Empty vector; hIRAK-MV, human IRAK-M vector.

FIG 8

IL-4 induces IRAK-M protein expression in cultured normal human tracheobronchial epithelial cells. Representative IRAK-M and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Western blots of 4 replicates are shown.