Local cortisol activation is involved in EGF-induced immunosuppression

Abstract

The major effects of the epidermal growth factor receptor (EGFR) signalling pathway on keratinocytes are cell proliferation, cell differentiation, and wound healing. In addition to these effects, an immunosuppressive effect of EGFR signalling has been reported. However, the precise mechanism of immunosuppression by EGFR signalling is not well understood. In this study, we clarified the involvement of increased local cortisol activation in EGFR signalling-induced immunosuppression in keratinocytes. EGF treatment up-regulated the expression of 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) and supernatant cortisol levels in a dose-dependent manner in keratinocytes. 11β-HSD1 is an enzyme that catalyses the conversion of cellular hormonally inactive cortisone into active cortisol. qRT-PCR and ELISA assays indicated that EGF significantly decreased tumour necrosis factor α (TNF- α)-induced interleukin-6 (IL-6) expression in keratinocytes. Similarly, 11β-HSD1 overexpression significantly decreased TNF-α-induced IL-6 expression. We evaluated the role of 11β-HSD1 in immunosuppression through EGFR signalling. Blockade of 11β-HSD1 via 11β-HSD1 inhibitor reversed both the expression and production of TNF-α-induced IL-6, which was decreased by EGF in keratinocytes. Therefore, increased local cortisol activation by 11β-HSD1 is involved in EGFR signalling-induced immunosuppression in keratinocytes. Finally, we evaluated whether EGFR inhibition by cetuximab affects the expression of 11β-HSD1. We found that 0.1 µg cetuximab decreased 11β-HSD1 transcript levels in keratinocytes. The changes in 11β-HSD1 were more apparent in TNF-α-treated cells. As 11β-HSD1 expression in keratinocytes is associated with inflammation and cell proliferation, this mechanism may be associated with adverse skin reactions observed in patients treated with EGFR inhibitors.

Keywords: 11β-hydroxysteroid dehydrogenase; atopic dermatitis; cortisol; epidermal growth factor receptor; inflammation; keratinocyte;.

Figure 1.

The immunosuppressive effect of EGF and 11β-HSD1 in NHEKs (A and B) qRT-PCR (A) and ELISA (B) analysis of IL-6 in NHEKs. Cells were cultured with or without EGF (10 ng/ml) and treated with TNF-α (10 ng/ml) 24 hours later. Cells lysates were harvested 4 hours after TNF-α treatment for qRT-PCR and supernatant were harvested 24 hours after TNF-α for ELISA. Cortisone (1 μM) was added in all samples 10 minutes before TNF-α addition. GAPDH was used as an internal control [N = 3 (qRT-PCR), N = 4 (ELISA); *P<0.05, **P<0.01, ***P<0.001 as assessed by a one-way ANOVA followed by the Dunnett test for multiple comparisons]. (C) qRT-PCR analysis of HSD11B1 transfected with control or HSD11B1 plasmid (500 or 1000 ng/ml). GAPDH was used as an internal control (N = 3; ***P<0.001 as assessed by a one-way ANOVA followed by the Dunnett test for multiple comparisons). (D and E) qRT-PCR analysis of HSD11B1 (D) and IL-6 (E). HSD11B1 overexpressed cells were treated with TNF-α (10 ng/ml) and harvested 4 hours later. Cortisone (1 μM) was added 10 minutes before TNF-α in all samples. GAPDH was used as an internal control (N = 3; *P<0.05, **P<0.01, ***P<0.001 as assessed by Student's t-test). (F) qRT-PCR analysis of IL-6 expression in NHEKs treated with various doses of cortisol (0, 10⁻¹⁰, 10⁻⁹, 10⁻⁸, 10⁻⁷, or 10⁻⁶ M). Cortisol was added 10 min before TNF-α (10 ng/ml) addition. Cells were harvested 4 h after TNF-α treatment for qRT-PCR. β-actin was used as an internal control. (N = 3; *P<0.05, **P<0.01, ***P<0.001 as assessed by one-way ANOVA followed by Bonferroni tests for multiple comparisons). (G) qRT-PCR analysis of HSD11B1 in NHEKs treated with EGF (0, 1 or 10 ng/ml) for indicated times. GAPDH was used as an internal control (N = 3; **P<0.01, ***P<0.001 as assessed by a one-way ANOVA followed by the Dunnett test for multiple comparisons). A representative western blot of 11β-HSD1 relative to the β-actin loading control is shown. Cells were harvested 48 h after EGF treatment for western blotting (N = 3). (H) ELISA analysis of supernatant cortisol concentration in NHEKs. Cells were cultured with EGF (0, 1 or 10 ng/ml) and cortisone (1 μM) was added 24 hours later. Supernatant was harvested 24 hours after cortisone addition (N = 6; **P<0.01 as assessed by a one-way ANOVA followed by the Dunnett test for multiple comparisons). (I) ELISA analysis of cortisol expression in NHEKs treated with an 11β-HSD1 inhibitor. NHEKs were treated with an 11β-HSD1 inhibitor (10 μM) and/or EGF (10 ng/ml). Cortisone (1 μM) was added 1 d after EGF addition in all samples, and an 11β-HSD1 inhibitor was added 10 min prior to cortisone in the indicated samples. Supernatants were harvested 4 h after cortisone treatment (N = 5; *P<0.05 as assessed by Student's t-test). (J) ELISA analysis of cortisol expression in NHEKs transfected with control siRNA or HSD11B1 siRNA. Cortisone (1 μM) was added 1 d after EGF (10 ng/ml) addition in all samples. Supernatants were harvested 4 h after cortisone treatment (N = 5; **P<0.01 as assessed by Student's t-test).

Figure 2.

11β-HSD1 inhibition or knockdown reversed EGF-induced immunosuppression in NHEK (A) NHEKs treated with an 11β-HSD1 selective inhibitor (0, 1, 5, or 10 μM) for 48 h were assessed by a MTS assay. DMSO was used as a control (N = 7 per group). (B and C) qRT-PCR (B) and ELISA (C) analysis of IL-6 in NHEKs treated with an 11β-HSD1 inhibitor. NHEKs were treated with an 11β-HSD1 inhibitor (10 μM) and/or EGF (10 ng/ml) with or without TNF-α (10 ng/ml). TNF-α was added 1 day after EGF addition. Cortisone (1 μM) was added 10 minutes before TNF-α addition in all samples and an 11β-HSD1 inhibitor was added 10 minutes prior to cortisone in indicated samples. Cells were harvested 4 hours after TNF-α treatment for qRT-PCR and supernatant were harvested 24 hours after TNF-α treatment for ELISA. GAPDH was used as an internal control [N = 3 (qRT-PCR), N = 4 (ELISA); **P<0.01, ***P<0.001 as assessed by Student's t-test]. (D) qRT-PCR analysis of HSD11B1 in NHEKs transfected with control siRNA or HSD11B1 siRNA for 48 hours. GAPDH was used as an internal control. HSD11B1 expression 48 h after transfection of control siRNA or HSD11B1 siRNA. A representative western blot of 11β-HSD1 relative to the β-actin loading control is shown (N = 3). (E, F and G) qRT-PCR (E and F) and ELISA (G) analysis of IL-6 and HSD11B1 in NHEKs transfected with control siRNA or HSD11B1 siRNA. TNF-α was added 1 day after EGF addition. Cortisone (1 μM) was added 10 minutes before TNF-α addition in all samples and cells were harvested 4 hours after TNF-α treatment for qRT-PCR and supernatant were harvested 24 hours after TNF-α treatment for ELISA. GAPDH was used as an internal control [N = 3 (qRT-PCR), N = 4 (ELISA); **P<0.01, ***P<0.001 as assessed by Student's t-test].

Figure 3.

EGFR inhibitor, cetuximab, decreased the expression of HSD11B1 in NHEK (A, C) qRT-PCR analysis of IL-6 (A) and HSD11B1 (C) expression in NHEKs treated with cetuximab (0, 0.01, or 0.1 μg/ml). TNF-α (10 ng/ml) was added 1 d after cetuximab addition. Cortisone (1 μM) and cortisol (1 μM) were added 10 min before TNF-α addition. Cells were harvested 4 h after TNF-α treatment. HPRT was used as an internal control (N = 4; *P<0.05, **P<0.01, ***P<0.001 as assessed by one-way ANOVA followed by Dunnett tests for multiple comparisons). (B, D) ELISA analysis of IL-6 (B) and cortisol (D) expression in NHEKs treated with cetuximab (0, 0.01, or 0.1 μg/ml). TNF-α (10 ng/ml) was added 1 d after cetuximab addition. Cortisone (1 μM) was added 10 min before TNF-α addition. Supernatants were harvested 24 h after TNF-α treatment for ELISA analysis (N = 4 per group, *P<0.05, **P<0.01, ***P<0.001 as assessed by one-way ANOVA followed by Dunnett tests for multiple comparisons).