Interleukin-26 Production in Human Primary Bronchial Epithelial Cells in Response to Viral Stimulation: Modulation by Th17 cytokines

Abstract

Interleukin (IL)-26 is abundant in human airways and this cytokine is involved in the local immune response to a bacterial stimulus in vivo. Specifically, local exposure to the toll-like receptor (TLR) 4 agonist endotoxin does increase IL-26 in human airways and this cytokine potentiates chemotactic responses in human neutrophils. In addition to T-helper (Th) 17 cells, alveolar macrophages can produce IL-26, but it remains unknown whether this cytokine can also be produced in the airway mucosa per se in response to a viral stimulus. Here, we evaluated whether this is the case using primary bronchial epithelial cells from the airway epithelium in vitro, and exploring the signaling mechanisms involved, including the modulatory effects of additional Th17 cytokines. Finally, we assessed IL-26 and its archetype signaling responses in healthy human airways in vivo. We found increased transcription and release of IL-26 protein after stimulation with the viral-related double stranded (ds) RNA polyinosinic-polycytidylic acid (poly-IC) and showed that this IL-26 release involved mitogen-activated protein (MAP) kinases and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The release of IL-26 in response to a viral stimulus was modulated by additional Th17 cytokines. Moreover, there was transcription of IL26 mRNA and expression of the protein in epithelial cells of bronchial brush and tissue biopsies respectively after harvest in vivo. In addition, the extracellular IL-26 protein concentrations in bronchoalveolar lavage (BAL) samples did correlate with increased epithelial cell transcription of an archetype intracellular signaling molecule downstream of the IL-26-receptor complex, STAT1, in the bronchial brush biopsies. Thus, our study suggests that viral stimulation causes the production of IL-26 in lining epithelial cells of human airway structural cells that constitute a critical immune barrier and that this production is modulated by Th17 cytokines.

Keywords: IL-26; MAP kinases; Th17 cytokines; bronchial epithelial cells; signal transduction.

Conflict of interest statement

DISCLOSURE

The authors declare that they have no competing interests as defined by Molecular Medicine or other interests that might be perceived to influence the results and discussion reported in this paper.

Figures

Figure 1.

Primary bronchial epithelial cells produce IL-26 enhanced by viral-related stimuli. Cells were stimulated (24 h) with different viral stimuli (TLR3 agonist poly-IC, TLR7 agonist imiquimod and TLR8 agonist ssRNA). Extracellular concentrations in cell-free conditioned media as well as intracellular expression of IL-26 protein were measured using ELISA and western blot, respectively, and levels of mRNA using real time. (A) IL26 mRNA levels after stimulation with poly-IC (n = 11). (B) Extracellular concentrations of IL-26 in cell-free conditioned media in response to poly-IC at different concentrations (n = 8). (C) Intracellular IL-26 protein (representative western blot). (D) the average protein expression (fold difference) after stimulation with poly-IC (1ug/mL) during 24 h. (E) Extracellular concentrations of IL-26 in cell-free conditioned media in response to imiquimod or ssRNA (n = 8). (F) TLR3, TLR7 and TLR8 mRNA levels (fold) (n = 5). The p values indicated are according to the Student paired t test. p < 0.05 is considered significant.

Figure 2.

The adaptor protein TRIF is involved in poly-IC-induced release of IL-26. Primary bronchial epithelial cells were preincubated (5 h) with TRIF inhibitor and vehicle (25 μmol/L). Extracellular concentrations of IL-26 in cell-free conditioned media as well as intracellular levels were measured using ELISA and western blot respectively and mRNA levels using real time. (A) Extracellular concentrations of IL-26 in cell-free conditioned in response to poly-IC (0.05 μg/mL) in the presence of the TRIF inhibitor (n = 7). (B) Level of IL26 mRNA in response to poly-IC (1μg/mL) in the presence of the TRIF inhibitor (n = 8). (C) Intracellular IL-26 protein (representative western blot) and (D) the average protein expression (fold difference) in response to poly-IC (1 μg/mL) in the presence of the TRIF inhibitor (n = 4).

Figure 3.

Stimulation with poly-IC induces phosphorylation of MAP kinases and NF-κB and is inhibited by TRIF. Cells were preincubated with the TRIF inhibitor (ihh-TRIF) or vehicle (V-TRIF) (25 μmol/L for 5 h) and/or stimulated with poly-IC (0.05 μg/mL or 0.5 μg/mL) for another 1.5 h. Adherent cells were then lysed and the lysate used to measure phosphorylated levels of p38, JNK1–3, ERK1/2 and NF-κB by phosphorTracer ELISA. Panels (n = 8) show relative fluorescence units (RFU) of (A) Phosphorylated (p) p38, (B) pJNK1–3, (C) pERK1–3, (D) pNF-κB and (E) comparative levels for the different molecules. The p values indicated are according to Student paired t test and a p < 0.05 is considered significant.

Figure 4.

Inhibition of p38, JNK1–3, ERK1/2 and NF-κB attenuates poly-IC induced release of IL-26. Primary bronchial epithelial cells were preincubated (1 h) with MAP kinase inhibitors for p38 (SB203580), JNK1–3 (SP600125), ERK1/2 (AZD6244) as well as combined (p38 plus ERK1/2, p38 plus JNK1–3 and JNK1–3 plus ERK1/2). Cells were preincubated for 5 h with the NF-κB inhibitor (SC17741). Cells were then stimulated with poly-IC (0.05 μg/mL) for another 24 h. Figure panels show the different concentrations of each inhibitor used during poly-IC stimulation. Extracellular concentrations of IL-26 in cell-free conditioned media as well as intracellular levels were measured using ELISA and western blot respectively and mRNA expression by real time. Panels show IL-26 concentrations after the inhibition of (A) p38, (B) JNK1–3, (C) ERK1/2, (D) p38 plus JNK1–3, (E) p38 plus ERK1/2 and (F) JNK1–3 plus ERK1/2 and (G) NF-κB (n = 7 for all panels). (C) Intracellular IL-26 (representative western blot) and (D) the average protein levels (fold difference) in response to poly-IC (1 μg/mL) in the presence of the MAP kinase or NF-κB inhibitors (n = 5). The p values indicated are according to Student paired t test and p < 0.05 is considered significant.

Figure 5.

Th17 cytokines modulate poly-IC induced release of IL-26. Primary bronchial epithelial cells were stimulated with poly-IC (0.05 μg/mL) in the presence or absence of rhIL-17A (100 ng/mL) or rhIL-22 (100 ng/mL) or rhIL-17A (100 ng/mL) plus rhIL-22 (100 ng/mL) (24 h). Extracellular concentrations of IL-26 in cell-free conditioned media as well as intracellular levels were measured using ELISA and western blot respectively and mRNA level by real time PCR. (A) Extracellular concentrations of IL-26 in cell-free conditioned media in response to rhIL-17A and/or rhIL-22 (n = 14). (B) IL26 mRNA level in response to rhIL-17A (n = 9). (C) Extracellular concentrations of IL-26 in cell-free conditioned media in response to poly-IC plus rhIL-17A (n = 6). (D) Extracellular concentrations of IL-26 in cell-free conditioned media in response to poly-IC plus IL-22 (n = 6). (E) Extracellular concentrations of IL-26 in cell-free conditioned media in response to poly-IC plus rhIL-17A plus rhIL-22. (n = 6). (F) Intracellular IL-26 protein (representative western blot) and (G) the average protein level (fold difference) in response to IL-17A during 24 h (n = 10). The p values indicated are cording to the Student paired t test. p < 0.05 is considered significant

Figure 6.

IL-26 in bronchial brush biopsies and bronchial tissue biopsies. The gene expression profile of epithelial cells in bronchial brush biopsies was determined by mRNA analyses. (A) IL26 and TLR3, TLR7 and TLR8 mRNA levels (n = 10). (B) The mRNA level for the IL-26 associated genes IL26, IL10R2, IL20R1, STAT1 and STAT3 (n = 10). (C–F) Correlation between BAL IL-26 protein (67pg/mL [7–238pg/mL]) and mRNA levels for STAT1, STAT3, IL10R2 and IL20R1 respectively (n = 10). Cellular IL-26 protein expression in bronchial tissue biopsies was determined using IHC. (G) Monoclonal IgG2b isotype control antibody and (H) positive staining (brown) with monoclonal specific IL-26 antibody. Arrows show granular and cytoplasmic IL-26 expression. Each panel show a representative staining out of 8 subjects at a magnification of 400×. The p values indicated are according to the Mann–Whitney test (A and B) and Spearman correlation test (C–F) and p < 0.05 is considered significant.