An Acid-Hydrolyzed Wheat Protein Activates the Inflammatory and NF

The Journal of Toxicological Sciences (J. Toxicol. Sci.) 327 Vol.45, No.6, 327-337, 2020 Letter An acid-hydrolyzed wheat protein activates the inflammatory and NF-κB pathways leading to long TSLP transcription in human keratinocytes Yasutaka Kuroda1,2, Takuo Yuki1, Yutaka Takahashi1, Hitoshi Sakaguchi1, Kayoko Matsunaga3 and Hiroshi Itagaki2,4 1Safety Science Research Laboratories, Kao Corporation, 2606, Akabane, Ichikai, Haga, Tochigi, Japan 2Department of Material Science and Engineering, Faculty of Engineering, Yokohama National University, 79-5, Tokiwadai, Hodogaya-ku, Yokohama, Kanagawa, Japan 3Department of Integrative Medical Science for Allergic Disease, Fujita Health University School of Medicine, 3-6-10, Otobashi, Nakagawa-ku, Nagoya, Aichi, Japan 4ITACS Consulting, 5-11-19-2504, Minamidai, Minami-ku, Sagamihara, Kanagawa, Japan (Received August 19, 2019; Accepted March 16, 2020) ABSTRACT — Hydrolyzed wheat proteins (HWPs) contained in cosmetics have occasionally caused immediate-type hypersensitivity following repeated skin exposure. Although the Cosmetic Ingredient Review Expert Panel concluded that < 3,500 Da HWP is safe for use in cosmetics, it remains biologically unknown how allergenic HWPs evoke immediate-type allergy percutaneously. Keratinocyte-derived thymic stromal lymphopoietin (TSLP) induces type 2 immune responses, which play an essential role in the pathogenesis of immediate-type allergy. Previously, we demonstrated that protein allergens in cultured human keratinocytes strongly induced long-form TSLP (loTSLP) transcription. However loTSLP-regulat- ing signaling by HWP is poorly understood. In this study, we performed global gene expression analysis by microarray to investigate how the allergenic HWP acts on epidermal keratinocytes and the induction of loTSLP. Compared to human serum albumin (HSA), allergenic HWP induced a distinct gene expression pattern and preferentially activated various inflammatory pathways (High Mobility Group Box 1, Inter- leukin [IL]-6, IL-8, and acute phase response signaling). We identified 85 genes as potential nuclear factor-kappa B (NF-κB) target genes in GP19S-treated cells, compared with 29 such genes in HSA-treated cells. In addition, HWP specifically altered IL-17 signaling pathways in which transcription factors, NF-κB and activator protein-1, were activated. NF-κB signaling may be an important factor for HWP- induced inflammatory loTSLP transcription via inhibition assay. In conclusion, allergenic HWP caused an easily sensitizable milieu of activated inflammatory pathways and induced NF-κB-dependent loTSLP transcription in keratinocytes. Key words: Hydrolyzed wheat protein, Keratinocytes, Thymic stromal lymphopoietin, Nuclear factor-kappa B INTRODUCTION diate-type allergy outbreak which was associated with the use of facial soap in Japan (Fukutomi et al., 2011; Hydrolyzed wheat proteins (HWPs) have been wide- Yagami et al., 2017). More recently Noguchi et al. (2019) ly used in shampoos, conditioners, and moisturizers that reported that Japanese GP19S allergy strongly associated are exposed to the skin. However, increasing evidence with HLA-DQ variants but not with FLG loss-of-function suggests that certain HWPs in cosmetics can cause con- mutation in genome-wide association study. Conversely, tact urticaria or anaphylaxis (Niinimäki et al., 1998; several comparative studies among GP19S, other HWPs, Varjonen et al., 2000; Laurière et al., 2006). In particu- and native gluten were conducted, and GP19S was found lar, acid-HWP, Glupearl 19S® (GP19S), caused an imme- to contain novel epitopes and high molecular weight anti- Correspondence: Yasutaka Kuroda (E-mail: [email protected]) Vol. 45 No. 6 328 Y. Kuroda et al. gens (Nakamura et al., 2013, 2016; Yokooji et al., 2013). ertheless, little is known about the mechanisms of loTSLP Our previous study, as well as others studies, demon- transcription in epidermal keratinocytes. More recent- strated that compared with native gluten, GP19S pos- ly, Redhu et al. (2020) reported four NF-κB binding sites sess higher allergenic potential than native gluten in mice were located in the human TSLP promoter region, and and guinea pig models (Adachi et al., 2012; Matsunaga IL-1α promoted NF-κB recruitment to two active bind- et al., 2015). In addition to host susceptibility, higher ing sites in human keratinocytes. In same setting, loTSLP allergenicity of HWP itself may be vital for GP19S aller- mRNA was significantly augmented. Therefore, the bind- gy. Although the Cosmetic Ingredient Review Expert ing NF-κB to these binding sites may important for loT- Panel concluded that < 3,500 Da HWP was safe for use SLP mRNA induction. in cosmetics (Belsito et al., 2014; Burnett et al., 2018), In this study, we performed comprehensive gene it remains biologically unknown how allergenic HWPs, expression analysis to demonstrate the potential for bio- such as GP19S, evoke immediate-type allergy via the logical predisposition to skin sensitization and induction skin route. of loTSLP by allergenic HWP. This study provides mech- Generally, an immediate-type allergy becomes sen- anistic insights into how highly allergenic proteins acts on sitized by the antigen-uptake of antigen-presenting cells epidermal keratinocytes. and antigen presentation to naïve T cells, leading to type 2 helper T (TH2) cell expansion, development of T fol- MATERIALS AND METHODS licular helper cells, and IgE production. In the skin, an epithelial cell-derived cytokine, namely thymic strom- Cells al lymphopoietin (TSLP), acts as a type 2 immunity pro- Primary human neonatal keratinocytes (Thermo Fisher moting factor. High TSLP levels were detected at the Scientific/Life Technologies, Waltham, MA, USA) were lesion sites of patients with atopic dermatitis and asthma, cultured in HuMedia KG2 (Kurabo, Osaka, Japan) and which mediated type 2 immunity (Soumelis et al., 2002; seeded at 4.5 × 104 cells/well in flat-bottomed, 24-well Shikotra et al., 2012). In addition, TSLP receptor sign- culture plates. Once the cells reached 80% confluence aling in Langerhans cells was essential for percutaneous (2-3 days after plating), the medium was replaced with sensitization with protein allergen and TH2-type immune HuMedia KG2, without hydrocortisone, in 24 hr accord- response in a murine model (Nakajima et al., 2012). We ing to Kinoshita et al. (2009)’s procedure. The cells that recently reported that highly allergenic proteins, includ- were passaged once were used for the assays. ing GP19S, induced the long form of TSLP (loTSLP) in epidermal keratinocytes (Kuroda et al., 2017). TSLP has Preparation of the exposure samples two transcription variants. loTSLP plays a vital role in GP19S, a product of wheat gluten obtained by par- type 2 immune response-related allergic diseases, includ- tial hydrolysis with hydrogen chloride (HCl) at 95°C for ing atopic dermatitis, allergic rhinitis, and asthma. The 40 min, was supplied by Katayama Chemical, Inc. short form of TSLP (shTSLP) is constitutively expressed (Osaka, Japan). GP19S was suspended in 1 M tris at both mRNA and protein levels in the skin of healthy (hydroxymethyl) aminomethane (Tris) solution (pH 11.4). subjects and have anti-inflammatory property (Bjerkan After 24 hr at room temperature, the samples were neutral- et al., 2015; Fornasa et al., 2015). shTSLP is related to ized with HCl by a previously reported method (Adachi et homeostatic functions in the thymus and gut (Tsilingiri al., 2012). A control vehicle sample (Tris-HCl) was pre- et al., 2017). Therefore, specific analysis of loTSLP is pared by neutralizing Tris with HCl. Human serum albu- essential for detecting inflammatory responses. However min (HSA; A5843, Sigma, St. Louis, MO, USA), with a the most studies have reported that the regulatory mech- confirmed endotoxin limit of 1.0 unit/mg, was dissolved in anisms of TSLP expression were analyzed as total-TSLP. phosphate-buffered saline (PBS, pH 7.2; Wako Chemical, Entire TSLP induction is regulated by NF-κB and acti- Osaka). Nuclear factor-kappa B (NF-κB) activa- vator protein 1 (AP-1) (Takai, 2012). In airway epitheli- tion inhibitors, 4-methyl-N1-(3-phenylpropyl)ben- al cells, IL-1β, and TNF-α-activated TSLP gene expres- zene-1,2-diamine (JSH-23; Merck KGaA, Darmstadt, sion was promoted by the upstream NF-κB site (Lee and Germany), and (1aR,4E,7aS,10aS,10bR)- Ziegler, 2007). In the airway’s smooth muscle cells, 2,3,6,7,7a,8,10a,10b-Octahydro-1a,5-dimethyl-8-meth- IgE-induced TSLP promoter activation was mediated ylene-oxireno[9,10] cyclodeca[1,2-b]furan-9(1aH)-one by NF-κB and AP-1 (Redhu et al., 2011). Furthermore, (Parthenolide; Sigma) were dissolved in dimethyl sul- TSLP promoter region contained NF-κB, AP-1, STAT, foxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) and Smad binding sites in mice (Ganti et al., 2017). Nev- at concentrations of 3 and 10 μM, respectively. Another Vol. 45 No. 6 329 HWP activates inflammatory and NF-kappaB pathways leading to loTSLP NF-κB activation inhibitor, ammonium pyrrolidine dithi- Real-time quantitative PCR analysis ocarbamate (PDTC; Sigma), was dissolved in PBS at a Real-time PCR analysis was performed using the concentration of 100 μM. The effectiveness of inhibi- TaqMan® Universal PCR Master Mix and Applied tor concentrations was verified in the paper by Inman et Biosystems® 7500 Fast Real-Time PCR System al. (2008), Dommisch et al. (2010), and Galbiati et al. (Thermo Fisher Scientific). The total-TSLP (toTSLP) (2011). and β-actin transcription levels were analyzed using

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