Increased Interleukin-17 and Glucocorticoid Receptor-β Expression in Interstitial Lung Diseases and Corticosteroid Insensitivity

Abstract

Background: Treatment responsiveness to corticosteroids is excellent for cryptogenic organizing pneumonia (COP) and sarcoidosis, but suboptimal for idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia (UIP). We hypothesise that the differential expression of IL-17 contributes to variable corticosteroid sensitivity in different interstitial lung diseases.

Objective: To determine the associations among expression of IL-17, glucocorticoid receptor-β and responsiveness to corticosteroid treatment in interstitial lung diseases.

Methods: Immunohistochemical (IHC) staining was performed on formalin-fixed paraffin-embedded (FFPE) lung tissues obtained by bronchoscopic, CT-guided or surgical biopsies, and quantified by both cell counting (% positive cells) by individuals and by software IHC Profiler plugin of ImageJ (opacity density score). We studied the effect of IL-17 on corticosteroid sensitivity in human fibroblast MRC5 cell line.

Results: Compared with specimens from patients with COP (n =13) and sarcoidosis (n =13), those from IPF patients (n = 21) had greater GR-β and IL-17 expression and neutrophil infiltration. Radiographic progression after oral corticosteroid treatment was positively correlated with the expression in IL-17 and GR-β/GR-α ratio in all patients (COP, sarcoidosis and IPF) and also within the IPF subgroup only. IL-17 expression level was positively associated with GR-β and GR-β/GR-α ratio. In MRC5 cells, exogenous IL-17 increased the production of collagen I and up-regulated GR-β expression and dexamethasone's suppressive effect on collagen I production was impaired by IL-17, and silencing IL-17 receptor A gene attenuated the effect of IL-17.

Conclusion: Up-regulation of GR-β/GR-α ratio by IL-17 could be associated with the relative corticosteroid-insensitivity of IPF.

Keywords: corticosteroid insensitivity; cryptogenic organizing pneumonia (COP); glucocorticoid receptor-beta; idiopathic pulmonary fibrosis; interleukin-17; sarcoidosis.

Figure 1

Increased neutrophil infiltration and more IL-17 expression in IPF lungs. (A) Representative H&E images of lung specimens from subjects with COP, sarcoidosis and IPF. Magnification ×400. (B) IPF lungs exhibited greater number of neutrophils. (C) Representative immunohistochemical image of lung specimens from subjects with COP, sarcoidosis and IPF. Magnification ×200. (D, E) Immunohistochemical staining showed IPF lungs had a greater percentage of IL-17⁺ cells and higher IL-17 OD. (F, G) The expression of IL-17 is positively correlated with neutrophil counts in lung specimens. IL-17, interleukin-17; IPF, idiopathic pulmonary fibrosis; COP, cryptogenic organizing pneumonia; H&E, hematoxylin and eosin; HPF, high power fields (magnification, x400); OD, opacity density score; *p < 0.05; **p < 0.01.

Figure 2

IL-17RA expression in ILD lungs. (A) Representative immunohistochemical image of lung specimens from subjects with COP, sarcoidosis and IPF. Magnification ×200. (B, C) Immunohistochemical staining showed no significant difference in IL-17RA expression amongst lung specimens from patients with COP, sarcoidosis and IPF. (D, E) The correlation between IL-17RA expression and IL-17 expression in ILDs. IL-17, interleukin-17; IL-17RA, interleukin-17 receptor A; IPF, idiopathic pulmonary fibrosis; COP, cryptogenic organizing pneumonia; OD, opacity density score; **p < 0.01.

Figure 3

Greater expression of GR-β in IPF. (A) Representative immunohistochemical stain images of lung specimens from subjects with COP, sarcoidosis and IPF. Magnification X200. Proportion of positive staining cells (B, D, F, H) and OD (C, E, G, I) in lung specimens from subjects with ILDs (COP, sarcoidosis and IPF in combination), determined by both examiners and software-determined opacity density score. IPF patients had greater GR-β and GR-β/GR-α ratio. Horizontal lines represent the means ± SEM values for each group. GR, glucocorticoid receptor; IL-17, interleukin-17; IPF, idiopathic pulmonary fibrosis; COP, cryptogenic organizing pneumonia; OD, opacity density score; *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4

The relationship between IL-17 and GR isoforms in ILDs. (A–D) Immunohistochemical staining showed IL-17 expression was positively associated with GR-β and GR-β/GR-α ratio in ILD lungs (COP, sarcoidosis and IPF in combination). IL-17, interleukin; ILD, interstitial lung disease; IPF, idiopathic pulmonary fibrosis; COP, cryptogenic organizing pneumonia; GR, glucocorticoid receptor; HDAC2, histone deacetylase 2; OD, opacity density score. **p < 0.01, ****p < 0.0001.

Figure 5

GR-β up-regulation, HDAC2 down-regulation and corticosteroid insensitivity induced by IL-17. Human fibroblast MRC5 cells were transfected with Si-IL-17RA or scrambled Si-Ctrl and then incubated with IL-17 (10ng/mL). mRNA was determined by RT-qPCR and protein was determined by western blotting. (A, B) Dexamethasone (10^-7 M) reduces collagen I production at both mRNA level (2h) and protein level (6h), but the suppressive effect was hampered by IL-17. Silencing IL-17RA gene restored dexamethasone’s suppressive effect. The GR-β mRNA expression was up-regulated at 2h (C) and protein was increased at 6h and 12h (D). IL-17 also down-regulated HDAC2 at mRNA and protein level. Silencing IL-17RA gene attenuated IL-17’s effect on GR-β up-regulation and HDAC2 down-regulation. Bars represent means ± SEM of 6 experiments. Si, small interfering RNA; Ctrl, control; IL, interleukin; IL-17RA, interleukin-17 receptor A; Dex, dexamethasone; GR, glucocorticoid receptor; HDAC2, histone deacetylase 2. *p < 0.05.