Erdr1 Attenuates Dermatophagoides farina Body Extract-Induced Atopic Dermatitis in NC/Nga Mice

Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by a defective skin barrier and dysregulation of the immune system in terms of T helper type 1 (Th1) and Th2 cell imbalance (Peng and Novak, 2015Peng W. Novak N. Pathogenesis of atopic dermatitis.Clin Exp Allergy. 2015; 45: 566-574Crossref PubMed Scopus (136) Google Scholar). Th2 cytokines, such as IL-4, IL-13, and thymic stromal lymphopoietin, disrupt skin barrier and immune system function and induce inflammation in the lesional skin. Thymic stromal lymphopoietin is significantly increased and activates dendritic cells to produce Th2 chemoattractants, including chemokine (C-C motif) ligand 17 (CCL17) and CCL22, resulting in an increased Th2-skewed inflammatory responses in the lesional skin (Soumelis et al., 2002Soumelis V. Reche P.A. Kanzler H. Yuan W. Edward G. Homey B. et al.Human epithelial cells trigger dendritic cell mediated allergic inflammation by producing TSLP.Nat Immunol. 2002; 3: 673-680Crossref PubMed Scopus (343) Google Scholar). Previously, erythroid differentiation regulator 1 (Erdr1) was shown to have a negative correlation with IL-18, which is closely associated with AD severity (Kim et al., 2016aKim K.E. Houh Y. Lee J. Kim S. Cho D. Park H.J. Downregulation of erythroid differentiation regulator 1 (Erdr1) plays a critical role in psoriasis pathogenesis.Exp Dermatol. 2016; 25: 570-572Crossref PubMed Scopus (10) Google Scholar, Trzeciak et al., 2011Trzeciak M. Glen J. Bandurski T. Sokolowska-Wojdylo M. Wilkowska A. Roszkiewicz J. Relationship between serum levels of interleukin-18, IgE and disease severity in patients with atopic dermatitis.Clin Exp Dermatol. 2011; 36: 728-732Crossref PubMed Scopus (25) Google Scholar). Erdr1 is first discovered in WEHI-3 cell line and released under stress conditions, modulating growth and survival of cells (Dormer et al., 2004aDormer P. Spitzer E. Frankenberger M. Kremmer E. Erythroid differentiation regulator (EDR), a novel, highly conserved factor I. Induction of haemoglobin synthesis in erythroleukaemic cells.Cytokine. 2004; 26: 231-242Crossref PubMed Scopus (27) Google Scholar, Dormer et al., 2004bDormer P. Spitzer E. Moller W. EDR is a stress-related survival factor from stroma and other tissues acting on early haematopoietic progenitors (E-Mix).Cytokine. 2004; 27: 47-57Crossref PubMed Scopus (26) Google Scholar). Although functions and mechanisms of action remain unclear, recent studies show that Erdr1 displays anti-inflammatory and anticancer properties on various skin diseases, including psoriasis, rosacea, and melanoma (Jung et al., 2011Jung M.K. Park Y. Song S.B. Cheon S.Y. Park S. Houh Y. et al.Erythroid differentiation regulator 1, an interleukin 18-regulated gene, acts as a metastasis suppressor in melanoma.J Invest Dermatol. 2011; 131: 2096-2104Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, Kim et al., 2015Kim M. Kim K.E. Jung H.Y. Jo H. Jeong S.W. Lee J. et al.Recombinant erythroid differentiation regulator 1 inhibits both inflammation and angiogenesis in a mouse model of rosacea.Exp Dermatol. 2015; 24: 680-685Crossref PubMed Scopus (41) Google Scholar, Kim et al., 2016aKim K.E. Houh Y. Lee J. Kim S. Cho D. Park H.J. Downregulation of erythroid differentiation regulator 1 (Erdr1) plays a critical role in psoriasis pathogenesis.Exp Dermatol. 2016; 25: 570-572Crossref PubMed Scopus (10) Google Scholar, Kim et al., 2016bKim K.E. Houh Y. Park H.J. Cho D. Therapeutic effects of erythroid differentiation regulator 1 on imiquimod-induced psoriasis-like skin inflammation.Int J Mol Sci. 2016; 17: 224Crossref PubMed Scopus (134) Google Scholar). Therefore, we hypothesized that Erdr1 would exert preventive effects on AD through its anti-inflammatory functions, similar to its effects on psoriasis and rosacea. Here, we compared the expression level of Erdr1 between AD lesional skin and normal skin from humans and mice. To determine Erdr1 expression in AD lesional skin from human subjects, skin tissues were collected from normal control (n = 5) and patients with AD (n = 11). This experiment using human subjects was approved by the ethical committee of the Catholic University of Korea, and all human subjects provided written informed consent. Also, all experimental procedures with mice were approved by the Institutional Animal Care and Use Committee of Sookmyung Women’s University (SM-IACUC-2013-0726-022). As shown in Supplementary Figure S1a and b online, Erdr1 expression was significantly lower in AD lesional skin than normal skin, and effective treatments restored expression, indicating that Erdr1 may play a critical role in AD. To investigate the role of Erdr1 in AD, Dermatophagoides farina body ointment was topically applied to the ears of NC/Nga mice to induce AD-like skin inflammation. Then, 10 μg/kg of recombinant Erdr1 (rErdr1) or 2 mg/kg of dexamethasone as a positive control was administered by intraperitoneal injection. As shown in Figure 1a, rErdr1 significantly improved the external symptoms of AD-like skin inflammation compared with the group injected with the vehicle, phosphate buffered saline. Histological analysis also showed that rErdr1 administration resulted in decreased inflammatory cell infiltration, such as eosinophils and mast cells, and hyperproliferation in the epidermis. To determine the severity of AD, symptoms of redness, edema, scaling, and excoriation were scored, and the overall score was evaluated by the sum of each category. The scores were considerably decreased by rErdr1 administration compared with those in the vehicle-injected group (Figure 1b and Supplementary Figure S2 online). In addition, ear thickness and ear weight indicative of edema were also examined. rErdr1 treatment markedly reduced both ear thickness and weight, showing decreased hyperproliferation in the epidermis and edema in the ear skin (Figure 1c and d). The effects of rErdr1 on AD were further confirmed using 2,4-dinitrochlorobenzene-induced AD in BABL/C mice. rErdr1 alleviated epidermis thickness and mast cell infiltration in 2,4-dinitrochlorobenzene-induced AD lesional skin (Supplementary Figure S3a and b online). Although AD pathogenesis remains unclear, general characteristics of AD include Th2 polarization and high levels of serum IgE (Peng and Novak, 2015Peng W. Novak N. Pathogenesis of atopic dermatitis.Clin Exp Allergy. 2015; 45: 566-574Crossref PubMed Scopus (136) Google Scholar). To determine whether rErdr1 administration could suppress IgE and IL-4 production in the AD mouse model, levels of IgE and IL-4 were measured. Serum IgE levels were significantly increased in mice induced to have AD that were injected with vehicle (Figure 1e). Administration of rErdr1 considerably reduced the serum IgE level in the AD-induced mice, and the reduction range was similar to the dexamethasone-injected group, showing that this dose of Erdr1 has similar preventive potential to dexamethasone. Expression of IL-4 both in the lesional skin and splenocytes also increased on AD induction, and this increase in expression was significantly smaller in the rErdr1-injected group, implying that the distribution of Th2 cells was reduced in rErdr1-injected mice (Figure 1f). During AD pathogenesis, Th2 chemoattractants, CCL17 and CCL22, are increased to recruit the Th2 cells to AD lesional skin (Gros et al., 2009Gros E. Bussmann C. Bieber T. Forster I. Novak N. Expression of chemokines and chemokine receptors in lesional and nonlesional upper skin of patients with atopic dermatitis.J Allergy Clin Immunol. 2009; 124: 753-760.e1Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). As shown in Figure 2a, Tslp mRNA expression in AD lesional skin was significantly decreased in the rErdr1-injected group compared with the vehicle-injected group. In accordance with the decreased level of Tslp, both the mRNA and protein levels of CCL17 and CCL22 were also downregulated by rErdr1 injection in the AD lesional skin. Keratinocytes and activated dendritic cells are known to be the main producers of CCL17 and CCL22, respectively, in AD (Soumelis et al., 2002Soumelis V. Reche P.A. Kanzler H. Yuan W. Edward G. Homey B. et al.Human epithelial cells trigger dendritic cell mediated allergic inflammation by producing TSLP.Nat Immunol. 2002; 3: 673-680Crossref PubMed Scopus (343) Google Scholar, Vulcano et al., 2001Vulcano M. Albanesi C. Stoppacciaro A. Bagnati R. D'Amico G. Struyf S. et al.Dendritic cells as a major source of macrophage-derived chemokine/CCL22 in vitro and in vivo.Eur J Immunol. 2001; 31: 812-822Crossref PubMed Scopus (223) Google Scholar). In the vehicle-injected group, CCL17 and CCL22 were expressed primarily in the epidermis and dermis, respectively, and expression was decreased by rErdr1 injection (Figure 2b). Next, we determined the effect of Erdr1 on angiogenesis. It has been reported that a representative angiogenic factor vascular endothelial growth factor expression is significantly higher in the stratum corneum of AD than normal stratum corneum, suggesting that keratinocytes in AD lesional skin produce greater amounts of vascular endothelial growth factor. Also, vascular endothelial growth factor levels are highly correlated with AD scores, especially redness and edema (Amarbayasgalan et al., 2012Amarbayasgalan T. Takahashi H. Dekio I. Morita E. Content of vascular endothelial growth factor in stratum corneum well correlates to local severity of acute inflammation in patients with atopic dermatitis.Int Arch Allergy Immunol. 2012; 157: 251-258Crossref PubMed Scopus (23) Google Scholar). Figure 2c shows that expression of vascular endothelial growth factor in the lesional skin, especially keratinocytes in the epidermis, was significantly inhibited by rErdr1 injection. CD31 immunofluorescence staining data indicated that angiogenesis was induced in the vehicle-injected group but was significantly decreased by rErdr1 injection (Figure 2d). This study suggests that rErdr1 has preventive effects on AD. Th2 chemoattractants and IL-4 expression were decreased by rErdr1 in an AD-induced mouse model. Erdr1 was previously reported to also have a therapeutic effect on psoriasis, which is a Th1-dominant skin disease (Kim et al., 2016bKim K.E. Houh Y. Park H.J. Cho D. Therapeutic effects of erythroid differentiation regulator 1 on imiquimod-induced psoriasis-like skin inflammation.Int J Mol Sci. 2016; 17: 224Crossref PubMed Scopus (134) Google Scholar). These studies imply that rErdr1 might regulate both Th1 and Th2 responses. Recent studies have shown that several therapeutic agents improve the symptoms of AD by increasing generation of Treg. The treatment significantly increases the number of Treg cells, leading to suppression of Th1 and Th2 responses along with reduction of serum IgE in AD (Haitz and Anandasabapathy, 2015Haitz K.A. Anandasabapathy N. Docosahexaenoic Acid alleviates atopic dermatitis in mice by generating T regulatory cells and m2 macrophages.J Invest Dermatol. 2015; 135: 1472-1474Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, Han et al., 2015Han S.C. Koo D.H. Kang N.J. Yoon W.J. Kang G.J. Kang H.K. et al.Docosahexaenoic acid alleviates atopic dermatitis by generating Tregs and IL-10/TGF-beta-modified macrophages via a TGF-beta-dependent mechanism.J Invest Dermatol. 2015; 135: 1556-1564Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, Li et al., 2016Li H. Li C. Zhang H. Zhang L. Cheng R. Li M. et al.Effects of lidocaine on regulatory T cells in atopic dermatitis.J Allergy Clin Immunol. 2016; 137: 613-617.e5Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Therefore, further investigation is needed to determine whether Erdr1 suppresses both Th1 and Th2 responses by enhancing generation of Treg. The authors state no conflict of interest. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2013R1A1A2062797) and Creative Materials Discovery Program through the NRF funded by the Ministry of Science, ICT and Future Planning (2016M3D1A1021387). Download .pdf (3.11 MB) Help with pdf files Supplementary Data