EGFR signaling blunts allergen-induced IL-6 production and Th17 responses in the skin and attenuates development and relapse of atopic dermatitis

Abstract

Despite the important role for epidermal growth factor (EGF) in epithelial homeostasis and wound healing, it has not been investigated in atopic dermatitis (AD). We used AD animal models to explore the role of EGF in AD. In an acute AD model, skin transepidermal water loss was significantly attenuated in EGF-treated mice. Blockade of EGFR signaling genetically or pharmacologically confirms a protective role for EGFR signaling in AD. In a chronic/relapsing AD model, EGF treatment of mice with established AD resulted in an attenuation of AD exacerbation (skin epithelial thickness, cutaneous inflammation, and total and allergen specific IgE) following cutaneous allergen rechallenge. EGF treatment did not alter expression of skin barrier junction proteins or antimicrobial peptides in the AD model. However, EGF treatment attenuated allergen-induced expression of IL-17A, CXCL1, and CXCL2 and neutrophil accumulation in AD skin following cutaneous allergen exposure. IL-17A production was decreased in the in vitro restimulated skin-draining lymph node cells from the EGF-treated mice. Similarly, IL-17A was increased in waved-2 mice skin following allergen exposure. Whereas IL-6 and IL-1β expression was attenuated in the skin of EGF-treated mice, EGF treatment also suppressed allergen-induced IL-6 production by keratinocytes. Given the central role of IL-6 in priming Th17 differentiation in the skin, this effect of EGF on keratinocytes may contribute to the protective roles for EGFR in AD pathogenesis. In conclusion, our study provides evidence for a previously unrecognized protective role for EGF in AD and a new role for EGF in modulating IL-17 responses in the skin.

Figure 1

EGFR signaling protects from skin barrier function impairment following cutaneous allergen exposure. A. Overview of the acute AD sensitization protocol. Mice were exposed to A. fumigatus (ASP) or saline (SAL) patch 3 times as indicated and TEWL was assessed 24 hours after the last patch was removed in B. BALB/c mice treated with EGF as indicated; C. Waved-2 (WD2) mice and C57Bl/6 wild type mice; D. BALB/c mice treated with erlotinib as indicated. n=4–8 mice/group *P<0.05, ***P<0.001. Each experiment was performed a minimum of 3 times.

Figure 2

EGF treatment attenuates AD exacerbation following cutaneous allergen re-challenge. A. Overview of the chronic AD sensitization and re-challenge protocol. Twenty four hours after the last patch was removed, the following were assessed: B. TEWL; C. H&E staining of patched skin; D. Epidermal thickness of patched skin; Values are expressed as mean±SEM (n=6–10 mice/group). *P<0.05, **P<0.01, and ***P<0.001. Each experiment was performed a minimum of 3 times. Scale bars, 25μm.

Figure 3

EGF treatment attenuates skin inflammation and serum total and allergen-specific IgE levels in allergen-exposed mice. A. CD3 staining and B. CD3+ cells counts of the patched skin samples from mice subjected to protocol in Figure 2A; C. CD4+ and CD8+ cells counts of the patched skin samples as above. D. Total and A. fumigatus-specific IgE levels were determined in serum from mice subjected to protocol in Figure 2A. Values are expressed as mean±SEM (n=6–10 mice/group). *P<0.05, **P<0.01, and ***P<0.001. Each experiment was performed a minimum of 3 times. Scale bars, 50μm.

Figure 4

EGF treatment results in attenuated skin IL-17A levels in allergen-exposed mice. Skin RNA was isolated from the skin of mice subjected to the chronic AD model shown in Figure 2A after the last patch. Expression of indicated genes was determined by q-PCR (A and B); C. Ly6G staining of neutrophils in the patched skin and D. neutrophil counts; n=6–10 mice/group. Values are expressed as mean±SEM. *P<0.05, **P<0.01, and ***P<0.001. Each experiment was performed a minimum of 3 times. Scale bars, 50μm.

Figure 5

Cytokine production by restimulated skin draining lymph node cells following repeated cutaneous allergen exposure. Skin draining LN cells isolated from the chronic AD model shown in Figure 2A were cultured and re-stimulated with allergen in vitro. IL-4 (A), IL-17A (B) and IFNγ (undetectable) levels in the cultured medium were quantified by ELISA (n=6–10 mice/group). Values are expressed as mean±SEM. *P<0.05. Each experiment was performed a minimum of 3 times.

Figure 6

IL-17A levels are further enhanced in waved-2 mice compared to wild type mice following repeated cutaneous challenge. A. RNA was isolated from patched skin of waved-2 (WD2) and wild type mice after the last patch of the acute AD model shown in Figure 1A. Expression of IL-17A was determined; B. Skin draining LN cells isolated from the same mice were cultured and re-stimulated with allergen in vitro and IL-17A levels in the medium were assessed by ELISA. Values are expressed as mean±SEM; C. Expression of IL-22 was determined by q-PCR. (n=4–6 mice/group). *P<0.05, ***P<0.001. Each experiment was performed a minimum of 3 times.

Figure 7

EGF treatment alters expression of IL-6 and IL-1β but not TGFβ1 or IL-23/p19 in patched skin following cutaneous allergen exposure. RNA was isolated from patched skin from the mice from the chronic AD model shown in Figure 2A and expression of IL-6, IL-1β, TGFβ1, and IL-23/p19 was determined by q-PCR (A, n=6–10 mice/group). RNA was isolated from cultured (B) HaCat cell and (C) primary human keratinocytes treated as indicated and IL-6 expression was determined by q-PCR (n=3). Values are expressed as mean±SEM. *P<0.05, **P<0.01, ***P<0.001. Each experiment was performed a minimum of 3 times.