Contribution of IL-17 in Steroid Hyporesponsiveness in Obese Asthmatics Through Dysregulation of Glucocorticoid Receptors α and β

Abstract

Obesity is on the rise worldwide and is one of the most common comorbidities of asthma. The chronic inflammation seen in obesity is believed to contribute to this process. Asthma and obesity are associated with a poorer prognosis, more frequent exacerbations, and poor asthma control to standard controller medication. Difficult-to-treat asthma is associated with increased levels of Th17 cytokines which have been shown to play a central role in the upregulation of glucocorticoid receptor-beta (GR-β), a dominant-negative inhibitor of the classical GR-α. In this study, we studied the role of IL-17 cytokines in steroid hyporesponsiveness in obese asthmatics. We stimulated lean and obese adipocytes with IL-17A and IL-17F. Adipocytes obtained from obese patients cultured in vitro in the presence of IL-17A for 48 h showed a decrease in GRα/GRβ ratio as compared to adipocytes from lean subjects where GR-α/GR-β ratio was increased following IL-17A and IL-17F stimulation. At protein level, GR-β was increased in obese adipocytes with IL-17A and IL-17F stimulation. IL-8 and IL-6 expression was increased in IL-17-stimulated obese adipocytes. Pre-incubation with Dexamethasone (Dexa) led to a decrease in GR-α/GR-β ratio in obese adipocytes which was further affected by IL-17A whereas Dexa led to an increase in GR-α/GR-β ratio in lean adipocytes which was decreased in response to IL-17A. TGF-β mRNA expression was decreased in obese adipocytes in response to Th17 cytokines. We next sought to validate these findings in obese asthmatic patients. Serum obtained from obese asthmatic subjects showed a decrease in GRα/GRβ protein expression with an increase in IL-17F and IL-13 as compared to serum obtained from non-obese asthmatics. In conclusion, steroid hyporesponsiveness in obese asthmatic patients can be attributed to Th17 cytokines which are responsible for the dysregulation of the GRα/GRβ ratio and the inflammatory response.

Keywords: IL-17; asthma; glucocorticoid receptors; inflammation; obesity; steroid hyporesponsiveness.

Figure 1

Stimulation with IL-17A & F and IL-17A alone induces changes in GR-α/GR-β mRNA ratio. Adipocytes from lean and obese subjects were stimulated with 100 ng/mL of IL-17A and IL-17F in combination or IL-17A alone for 48 h. Cells were collected and qRT-PCR analysis was performed in duplicate using TaqMan probes to assess GR-α and GR-β mRNA expression. One independent experiment performed per subject. n = 4 lean subjects, n = 3 obese subjects, One-Way ANOVA, Mean ± SE; ^*P < 0.05, ^**P < 0.01.

Figure 2

IL-17A & F and IL-17A alone induces changes in protein levels of GR-β. GR-α (A) and GR- β (B) protein expression in adipocytes from lean and obese subjects following 48 h stimulation with IL-17A&F combination or IL-17A alone (C) Ratio of GR-α/GR-β. n = 4 lean subjects, n = 3 obese subjects, One-way ANOVA, Mean ± SE; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. Data is representative of three experiments.

Figure 3

Pre-treatment with Dexamethasone followed by IL-17A stimulation induces changes in GR-α/GR-β mRNA ratio. Adipocytes from lean and obese subjects were pre-treated with 500 ng/ml dexamethasone followed by a 48 h stimulation with 100 ng/ml of IL-17A. mRNA expression of GR-α and GR-β were measured by qRT-PCR in duplicates using TaqMan probes. One independent experiment preformed per subject: n = 4 lean subjects, n = 3 obese subjects, One-Way ANOVA, Mean ± SE; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 4

Stimulation with IL-17A&F and IL-17A alone induces changes in mRNA expression of inflammatory mediators in adipocytes from lean and obese subjects. qRT-PCR analysis of detected mRNA expression of inflammatory markers: IL-8 (A), IL-6 (B), TGF-β (C), IL-1β (D) in mature adipocytes after 48 h stimulation with combination of IL-17A&F or IL-17A alone. n = 4 lean subjects, n = 3 obese subjects, One-way ANOVA, Mean ± SE; ^*P < 0.05, ^***P < 0.001.

Figure 5

Stimulation with IL-17A&F and IL-17A alone induces changes in inflammatory cytokine profiles in adipocytes from lean and obese subjects. A multiplex assay was used to measure the levels of inflammatory cytokines: IL-8 (A), IL-6 (B), IL-5 (C), IL-4 (D), and IFN-γ (E) secreted by mature adipocytes after 48 h stimulation with IL-17A & F combination or IL-17A alone. One independent experiment preformed per subject. n = 4 lean subjects, n = 3 obese subjects, One-way ANOVA, Mean ± SE; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 6

GR-α/GR-β ratio is decreased in serum of obese asthmatic subjects. Serum was obtained from lean (BMI < 25 kg/m²), overweight (BMI < 30 kg/m²), obese (BMI < 35 kg/m²), and morbidly obese (BMI > 35 kg/m²) asthmatic subjects. (A,C) ELISA was used to assess protein levels of GR-α and GR-β. (B) Pearson correlation between BMI and GR-α/GR-β ratio. n = 43 non-obese subjects (n = 18 lean, n = 26 overweight), n = 57 obese subjects (n = 44 obese, n = 13 morbidly obese), Mean ± SE; ^*P < 0.05.

Figure 7

IL-17F production is increased in serum of obese asthmatics. Serum was obtained from lean (BMI < 25 kg/m²), overweight (BMI < 30 kg/m²), obese (BMI < 35 kg/m²), and morbidly obese (BMI > 35 kg/m²) asthmatic subjects. (A–C) ELISA was used to assess protein levels of IL-17A, IL-17F, IL-13, respectively. (D) GR-α/GR-β ratio in subjects with high IL-17F and low IL-17F. n = 43 non-obese subjects, n = 57 obese subjects, Mean ± SE; ^*P < 0.05.