Th17 cytokines induce pro-fibrotic cytokines release from human eosinophils

Abstract

Background: Subepithelial fibrosis is one of the most critical structural changes affecting bronchial airway function during asthma. Eosinophils have been shown to contribute to the production of pro-fibrotic cytokines, TGF-β and IL-11, however, the mechanism regulating this process is not fully understood.

Objective: In this report, we investigated whether cytokines associated with inflammation during asthma may induce eosinophils to produce pro-fibrotic cytokines.

Methods: Eosinophils were isolated from peripheral blood of 10 asthmatics and 10 normal control subjects. Eosinophils were stimulated with Th1, Th2 and Th17 cytokines and the production of TGF-β and IL-11 was determined using real time PCR and ELISA assays.

Results: The basal expression levels of eosinophil derived TGF-β and IL-11 cytokines were comparable between asthmatic and healthy individuals. Stimulating eosinophils with Th1 and Th2 cytokines did not induce expression of pro-fibrotic cytokines. However, stimulating eosinophils with Th17 cytokines resulted in the enhancement of TGF-β and IL-11 expression in asthmatic but not healthy individuals. This effect of IL-17 on eosinophils was dependent on p38 MAPK activation as inhibiting the phosphorylation of p38 MAPK, but not other kinases, inhibited IL-17 induced pro-fibrotic cytokine release.

Conclusions: Th17 cytokines might contribute to airway fibrosis during asthma by enhancing production of eosinophil derived pro-fibrotic cytokines. Preventing the release of pro-fibrotic cytokines by blocking the effect of Th17 cytokines on eosinophils may prove to be beneficial in controlling fibrosis for disorders with IL-17 driven inflammation such as allergic and autoimmune diseases.

Figure 1

Basal expression of pro-fibrotic cytokines by human eosinophils isolated from asthmatic and controls subjects. Eosinophils were isolated from 10 asthmatic and 10 controls subjects and total RNA was extracted from 2×10⁶ million cells and quantified using real-time PCR. A: Level of expression of TGF-β1 and IL-11 mRNA in eosinophils of asthmatic versus control subjects (n = 10). B: Levels of TGF-β1 and IL-11 cytokines within the supernatant of un-stimulated eosinophils (n = 10) as determined by ELISA assay. (C-D) Effect of Th1 and Th2 cytokines on asthmatic eosinophil TGF-β1 and IL-11 transcripts levels. Level of expression of TGF-β1 (C) and IL-11 (D) mRNA as quantified by real-time PCR following 4 hours exposure to mediators. Data is presented as percentage of basal expression (n = 10).

Figure 2

IL-17 and IL-23 enhance eosinophil expression of pro-fibrotic cytokines. (A) Surface expression of IL-17R on eosinophils (1×10⁶ cells) isolated from healthy and asthmatics was determined by flow cytometry. Blots are representative data for eosinophils isolated from one healthy control and one asthmatic patient. The graph shows arithmetic mean ± SD of IL-17R positive eosinophils as percentage of total eosinophils (n = 5). 2×10⁶ peripheral blood eosinophils isolated from 10 asthmatic and 10 controls subjects were stimulated with IL-17A, F, and IL-23 (50 ng/ml or 25 ng/ml) alone or in combination for 4 hrs. Total RNA was extracted and mRNA levels of TGF-β and IL-11 were then quantified using real-time PCR. mRNA expression levels of TGF-β (B) and IL-11 (C) were normalized with GAPDH for asthmatic versus healthy individuals.

Figure 3

Th17 cytokines enhance eosinophil production and release of pro-fibrotic cytokines. Levels of TGF-β (A) and IL-11 (B) in the supernatant of stimulated eosinophils (1×10⁶ cells/0.5 ml) were determined 24 hrs following Th17 cytokine stimulation (0-100 ng/ml) using ELISA assay. Results are expressed as the arithmetic mean ± SD from 5 independent experiments. * = p < 0.05.

Figure 4

P38 MAP Kinase activation is required for IL-17 enhancement of eosinophil derived pro-fibrotic cytokines. Eosinophils were isolated from peripheral blood of 10 asthmatic patients and 2×10⁶/ml cells were treated, or not, with p38 MAPK or PI3K inhibitors (SB2035802 and PI103, respectively) 2 hours prior to stimulation with IL-17 (50 ng/ml). Levels of TGF-β (A) and IL-11 (B) in the supernatant of stimulated eosinophils were then determined 24 hrs following Th17 cytokine stimulation using ELISA assay (n = 10). (C) Induction of p38 MAPK phosphorylation by a combination of IL-17A and IL-17 F (50 ng/ml each) is detected by western analysis. The western data shown represent one of similar results from 4 independent experiments. * = p < 0.05 compared to non-stimulated (NS). ** = p < 0.05 compared to stimulated not inhibited.