2020 Volume 45 Issue 6 Pages 327-337
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-regulating 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, Interleukin [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.
Hydrolyzed wheat proteins (HWPs) have been widely used in shampoos, conditioners, and moisturizers that are exposed to the skin. However, increasing evidence suggests that certain HWPs in cosmetics can cause contact urticaria or anaphylaxis (Niinimäki et al., 1998; Varjonen et al., 2000; Laurière et al., 2006). In particular, acid-HWP, Glupearl 19S® (GP19S), caused an immediate-type allergy outbreak which was associated with the use of facial soap in Japan (Fukutomi et al., 2011; Yagami et al., 2017). More recently Noguchi et al. (2019) reported that Japanese GP19S allergy strongly associated with HLA-DQ variants but not with FLG loss-of-function mutation in genome-wide association study. Conversely, several comparative studies among GP19S, other HWPs, and native gluten were conducted, and GP19S was found to contain novel epitopes and high molecular weight antigens (Nakamura et al., 2013, 2016; Yokooji et al., 2013). Our previous study, as well as others studies, demonstrated that compared with native gluten, GP19S possess higher allergenic potential than native gluten in mice and guinea pig models (Adachi et al., 2012; Matsunaga et al., 2015). In addition to host susceptibility, higher allergenicity of HWP itself may be vital for GP19S allergy. Although the Cosmetic Ingredient Review Expert Panel concluded that < 3,500 Da HWP was safe for use in cosmetics (Belsito et al., 2014; Burnett et al., 2018), it remains biologically unknown how allergenic HWPs, such as GP19S, evoke immediate-type allergy via the skin route.
Generally, an immediate-type allergy becomes sensitized by the antigen-uptake of antigen-presenting cells and antigen presentation to naïve T cells, leading to type 2 helper T (TH2) cell expansion, development of T follicular helper cells, and IgE production. In the skin, an epithelial cell-derived cytokine, namely thymic stromal lymphopoietin (TSLP), acts as a type 2 immunity promoting factor. High TSLP levels were detected at the lesion sites of patients with atopic dermatitis and asthma, which mediated type 2 immunity (Soumelis et al., 2002; Shikotra et al., 2012). In addition, TSLP receptor signaling in Langerhans cells was essential for percutaneous sensitization with protein allergen and TH2-type immune response in a murine model (Nakajima et al., 2012). We recently reported that highly allergenic proteins, including GP19S, induced the long form of TSLP (loTSLP) in epidermal keratinocytes (Kuroda et al., 2017). TSLP has two transcription variants. loTSLP plays a vital role in type 2 immune response-related allergic diseases, including atopic dermatitis, allergic rhinitis, and asthma. The short form of TSLP (shTSLP) is constitutively expressed at both mRNA and protein levels in the skin of healthy subjects and have anti-inflammatory property (Bjerkan et al., 2015; Fornasa et al., 2015). shTSLP is related to homeostatic functions in the thymus and gut (Tsilingiri et al., 2017). Therefore, specific analysis of loTSLP is essential for detecting inflammatory responses. However the most studies have reported that the regulatory mechanisms of TSLP expression were analyzed as total-TSLP. Entire TSLP induction is regulated by NF-κB and activator protein 1 (AP-1) (Takai, 2012). In airway epithelial cells, IL-1β, and TNF-α-activated TSLP gene expression was promoted by the upstream NF-κB site (Lee and Ziegler, 2007). In the airway’s smooth muscle cells, IgE-induced TSLP promoter activation was mediated by NF-κB and AP-1 (Redhu et al., 2011). Furthermore, TSLP promoter region contained NF-κB, AP-1, STAT, and Smad binding sites in mice (Ganti et al., 2017). Nevertheless, little is known about the mechanisms of loTSLP transcription in epidermal keratinocytes. More recently, Redhu et al. (2020) reported four NF-κB binding sites were located in the human TSLP promoter region, and IL-1α promoted NF-κB recruitment to two active binding sites in human keratinocytes. In same setting, loTSLP mRNA was significantly augmented. Therefore, the binding NF-κB to these binding sites may important for loTSLP mRNA induction.
In this study, we performed comprehensive gene expression analysis to demonstrate the potential for biological predisposition to skin sensitization and induction of loTSLP by allergenic HWP. This study provides mechanistic insights into how highly allergenic proteins acts on epidermal keratinocytes.
Primary human neonatal keratinocytes (Thermo Fisher Scientific/Life Technologies, Waltham, MA, USA) were cultured in HuMedia KG2 (Kurabo, Osaka, Japan) and seeded at 4.5 × 104 cells/well in flat-bottomed, 24-well culture plates. Once the cells reached 80% confluence (2-3 days after plating), the medium was replaced with HuMedia KG2, without hydrocortisone, in 24 hr according to Kinoshita et al. (2009)’s procedure. The cells that were passaged once were used for the assays.
GP19S, a product of wheat gluten obtained by partial hydrolysis with hydrogen chloride (HCl) at 95°C for 40 min, was supplied by Katayama Chemical, Inc. (Osaka, Japan). GP19S was suspended in 1 M tris (hydroxymethyl) aminomethane (Tris) solution (pH 11.4). After 24 hr at room temperature, the samples were neutralized with HCl by a previously reported method (Adachi et al., 2012). A control vehicle sample (Tris-HCl) was prepared by neutralizing Tris with HCl. Human serum albumin (HSA; A5843, Sigma, St. Louis, MO, USA), with a confirmed endotoxin limit of 1.0 unit/mg, was dissolved in phosphate-buffered saline (PBS, pH 7.2; Wako Chemical, Osaka). Nuclear factor-kappa B (NF-κB) activation inhibitors, 4-methyl-N1-(3-phenylpropyl)benzene-1,2-diamine (JSH-23; Merck KGaA, Darmstadt, Germany), and (1aR,4E,7aS,10aS,10bR)-2,3,6,7,7a,8,10a,10b-Octahydro-1a,5-dimethyl-8-methylene-oxireno[9,10] cyclodeca[1,2-b]furan-9(1aH)-one (Parthenolide; Sigma) were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) at concentrations of 3 and 10 μM, respectively. Another NF-κB activation inhibitor, ammonium pyrrolidine dithiocarbamate (PDTC; Sigma), was dissolved in PBS at a concentration of 100 μM. The effectiveness of inhibitor concentrations was verified in the paper by Inman et al. (2008), Dommisch et al. (2010), and Galbiati et al. (2011).
The inhibitors or vehicles were added to the cells an hour before sample exposure. Later, the cells placed in 400 μL culture medium were treated with 100 μL of the prepared sample for 3 hr. After removing the medium/test sample, the remaining adherent cells were processed for PCR analyses by the direct addition of 350 μL of RLT buffer (Qiagen, Hilden, Germany) bearing 3.5 μL of 2-mercapto-ethanol (Thermo Fisher Scientific) to each well. The total RNA was extracted from the lysed cells using the RNeasy Micro Kit in combination with QIAshredder (Qiagen) according to the manufacturer’s instructions. After confirming RNA concentration and purity using the NanoDrop 2000 System (Thermo Fisher Scientific), the first-strand of cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher Scientific). These experiments were conducted in triplicate for statistical analysis.
Total RNA quality and quantity was evaluated using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and NanoDrop 2000. An Affymetrix GeneChip Human Gene 2.0 ST Array (Affymetrix, Santa Clara, CA, USA) was used for the microarray experiment according to the manufacturer’s instructions. GeneSpring GX 14.5 (Agilent Technologies) was used to filter and normalize the raw data. RMA16 and a baseline transformation to the medians of all samples were used. For statistical analysis, one-way ANOVA was applied for multiple group comparison, followed by multiple testing correction and setting of the false discovery rate to 0.05 using the Benjamini and Hochberg method. Differentially expressed genes (DEGs) were determined using this statistical analysis and fold change cut-off of 2.0 for GP19S vs. Tris-HCl and HSA vs PBS. A list of DEGs was uploaded to the Ingenuity Pathway Analysis (IPA, Qiagen) software to investigate the biological networks resulting from the altered global gene expression. IPA generated the activation score (Z-score) and the p-value for canonical pathways and upstream regulator analysis.
Real-time PCR analysis was performed using the TaqMan® Universal PCR Master Mix and Applied Biosystems® 7500 Fast Real-Time PCR System (Thermo Fisher Scientific). The total-TSLP (toTSLP) and β-actin transcription levels were analyzed using PCR primer sets recommended by Thermo Fisher Scientific (Hs00263639_m1 and Hs01060665_g1). The loTSLP transcription was analyzed using a specific long form probe set (Hs01572933_m1). The primer and probe sets for shTSLP were designed and synthesized by TaqMan MGB probe service (Applied Biosystems) from the shTSLP-specific region of human TSLP, transcript variant 2, mRNA (NM_138551.4), according to Kuroda et al. (2017). The relative gene expression levels were normalized to β-actin, and the differences, when compared to control CT values, were calculated using 2-ΔΔCt method (Livak and Schmittgen, 2001). Unpaired student’s t-test (2-tailed) was used for statistical analyses. p < 0.05 indicated statistical significance.
According to our previous work, GP19S, unlike HSA, induced potent loTSLP transcription (Supplementary Fig. 1). To elucidate this difference, the gene expression profiles of keratinocytes exposed to 3 mg/mL GP19S and 66 mg/mL HSA were compared. The microarray data were deposited into Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/), with accession number GSE138725. A total of 513 DEGs were identified between GP19S-exposed condition and the solvent control, including 404 upregulated and 109 downregulated genes. On the other hand, 187 DEGs between HSA-exposed condition and solvent control, including 117 upregulated and 70 downregulated genes, were identified (Fig. 1A). GP19S induced 2.7-times more DEGs than HSA. GP19S induced 396 non-overlapped DEGs (Fig. 1B) and completely different expression patterns when compared to HSA and solvent control (Fig. 1C).
Comparison of DEGs between GP19S-treated and HSA-treated epidermal keratinocytes. (A) The numbers of upregulated and downregulated DEGs in the GP19S-treated and HSA-treated epidermal keratinocytes were determined using statistical analysis and a 2.0 fold change cut-off for GP19S vs Tris-HCl and HSA vs PBS. (B) A Venn diagram depicts the overlap of DEGs in the GP19S-treated and HSA-treated epidermal keratinocytes. (C) A heat map depicting hierarchical clustering of DEGs in the GP19S-treated, HSA-treated, and solvent-treated samples (green = upregulation, red = downregulation).
To investigate which canonical pathways were preferentially activated in GP19S-exposed keratinocytes, the expression profiles of 513 and 187 DEGs that were altered in GP19S and HSA-treated keratinocytes, respectively, were applied to pathway analyses. The inflammation-related pathways, such as High Mobility Group Box 1 (HMGB1), Interleukin (IL)-6, IL-8, and acute phase response signaling, were identified as preferentially GP19S-activated (Fig. 2). A more detailed analysis of the components of these pathways revealed that c-Fos, nuclear factor-kappa B (NF-κB), and the target genes (IL-6, Tumor Necrosis Factor [TNF]-α, and IL-1) were upregulated (Tables 1 and 2). Moreover, upstream analysis used to predict the regulators confirmed more GP19S activated putative inflammatory regulators than HSA (Fig. 3A). For instance, upstream analysis identified 85 potential NF-κB target genes in GP19S-treated cells when compared with 29 such genes in HSA-treated cells (Fig. 3B, Supplementary Table 1).
Comparison analysis of canonical signaling pathways activated by GP19S and HSA treatment. A list of top 10 canonical pathways of DEGs in GP19S or HSA-treated samples is shown with Z-scores.
Upstream regulator analysis of DEGs in epidermal keratinocytes after GP19S and HSA treatments. (A) A list of top 11 upstream regulators of DEGs in GP19S or HSA-treated samples is shown with Z-scores. (B) The numbers of putative downstream factors regulated by NF-κB (complex) in the GP19S-treated and HSA-treated epidermal keratinocytes.
To clarify the influence of GP19S but not HSA, the specifically induced factors of 396 DEGs, which were altered only in GP19S-treated keratinocytes, were applied to the canonical pathway analyses. Several IL-17 related pathways were identified (Fig. 4). A more detailed investigation of the IL-17 signaling pathway components revealed that the components of NF-κB, AP-1, and CCAAT/enhancer binding protein (C/EBPβ) were upregulated (Table 3).
Canonical pathway analysis of GP19S-specific DEGs in epidermal keratinocytes. A list of top 17 canonical pathways of DEGs, specifically affected by GP19S, are shown with p-values. Orange squares indicated the ratio of the number of DEGs that map to a given pathway divided by the total number of genes that are involved in the pathway.
From the above results, NF-κB signaling appeared to be an important factor serving as a GP19S-preferred and GP19S-specific activator. Therefore, we investigated whether inducing loTSLP mRNA by GP19S was influenced by 3 NF-κB activation inhibitors, namely JSH-23 (selective blocker of the nuclear translocation of RelA), parthenolide (inhibitors of IκBα degradation and RelA nuclear translocation), and PDTC (antioxidant). As the keratinocytes were pretreated with each NF-κB activation inhibitor, loTSLP mRNA induction by sufficient amounts of GP19S (100 and 500 μg/mL) was almost completely blocked (Fig. 5). In contrast, shTSLP mRNA induction by 100 μg/mL GP19S was not significantly blocked, and shTSLP mRNA induction by 500 μg/mL GP19S was significantly blocked, but not as much as loTSLP mRNA. The cytotoxic effects of all NF-κB activation inhibitors were not observed at the tested concentration (data not shown). The obtained data suggest that NF-κB activation plays pivotal roles in loTSLP transcription and partial shTSLP transcription induced by protein allergens.
Impact of NF-κB activation inhibitors on short and long TSLP transcription induced by GP19S. Short and long TSLP transcription, induced by 100 and 500 μg/mL GP19S, was affected by NF-κB activation inhibitors (JSH-23, Parthenolide, and PDTC) in epidermal keratinocytes. *p < 0.01 t-test vs. DMSO + GP19S group.
For preventing allergic outbreaks, it is important to elucidate the underlying mechanisms of percutaneous sensitization by highly allergenic HWPs. This study revealed the highly allergenic HWP, GP19S, induced a distinct gene expression pattern and preferentially various inflammatory pathways compared to HSA in epidermal keratinocytes. In these pathways, NF-κB, AP-1, and C/EBPβ were activated. In addition, NF-κB signaling seems to be an important factor in HWP-induced inflammatory loTSLP transcription.
It was reported that the ability of food allergens, such as ovalbumin, to pass through the skin to the draining lymph node was quite limited and did not result in specific IgE development when the protein was applied on intact skin (Dioszeghy et al., 2011; Li et al., 2012). However, percutaneous application of the macromolecular allergenic HWP, dissolved in aqueous buffer, induced systemic type 2 immune response and immediate-type hypersensitivity in guinea pigs (Matsunaga et al., 2015) and mice (Adachi et al., 2012). These findings imply that external proteins usually minimally penetrate into the epidermis and are therefore insufficient to cause sensitization; nonetheless, allergic HWP activates the epidermal cells and results in epidermal immune cell activation and/or enhanced skin permeability. In epidermal keratinocytes, highly allergenic HWP induced 2.7-times as many DEGs than the low allergenic HSA. In pathway analyses, the HWP-specific DEGs were linked to various inflammatory cytokines (IL-6, IL-1, and TNF) that stimulate dendritic cells (Toebak et al., 2009). HWP-activated keratinocytes may provide an environmental niche for efficient sensitization. We previously reported that IL-17 impaired the tight junction barrier (Yuki et al., 2016). Although IL-17 was basically supplied by the T cells and did not exist in our experimental system, other HWP-activated signaling pathways may cross talk with IL-17 signaling and be relevant to skin permeability. In the case of anaphylaxis caused by HWP-containing facial soap in Japan, surfactants may have enhanced the sensitivity of the inflammatory response to HWP. In the preliminary data (n = 2), the pathway analysis of DEGs (fold change > 2) altered in 0.5 mg/mL wheat gluten-treated keratinocytes showed that IL-6 and some inflammatory signaling pathways were detected in the top 10 canonical pathways (Fig. 6A), whereas that altered in 68 mg/mL ovalbumin (low endotoxin)-treated keratinocytes showed that inflammatory signaling pathways were not preferentially activated (Fig. 6B). Because wheat gluten, but not ovalbumin, can be sensitized by application to intact skin, the findings of HWP in this study will expand to other protein allergens which can be sensitized via the skin route.
Canonical pathway analysis activated by wheat gluten and ovalbumin treatment. The lists of top 10 canonical pathways of DEGs affected by wheat gluten (A) or ovalbumin (B) are shown with p-values. Orange squares indicated the ratio of the number of DEGs that map to a given pathway divided by the total number of genes that are involved in the pathway.
In this study, among the various HWP materials, we focused on allergenic GP19S, which was acid-hydrolyzed for a short duration (40 min). Our research, as well as other works, has demonstrated that GP19S stimulated anaphylactic response by percutaneous sensitization in animal models and induced inflammatory signaling and NF-κB-dependent loTSLP transcription in epidermal keratinocytes (Matsunaga et al., 2015, Kuroda et al., 2017). Such potentials of other HWP materials, which were hydrolyzed by acid, alkali, or enzymatic treatment, are largely unknown. Wheat gluten, when acid-hydrolyzed for 9 hr, induced slightly weaker anaphylactic reaction in guinea pig model than the compound acid-hydrolyzed for merely 0.5 hr (Matsunaga et al., 2015). Moreover, when wheat gluten was acid-hydrolyzed for 6 hr, it induced lower loTSLP transcription and TSLP protein release in keratinocytes than the one acid-hydrolyzed for 0.5 hr (Kuroda et al., 2017). Since the wheat gluten acid-hydrolyzed for 9 hr had a lower molecular weight than the one acid-hydrolyzed for 0.5 hr, we predicted that small HWPs do not induce inflammatory signaling and NF-κB-dependent loTSLP transcription in keratinocytes. However, further work is needed to investigate whether other safely used and allergenic HWPs induce inflammatory signaling and NF-κB-dependent loTSLP transcription in epidermal keratinocytes.
We proved that HWP activated NF-κB, AP-1, and C/EBPβ. Harada et al. (2009) reported that poly(I:C)-induced loTSLP transcription was regulated by the AP-1 binding site in human bronchial epithelial cells. More recently, Li et al. (2018) proposed that human metapneumovirus infection specifically induced loTSLP transcription, which depended on NF-κB activation in human airway epithelial cell lines. Despite the cell source differences, these findings are consistent with our results that allergenic HWPs induced loTSLP mRNA via NF-κB signaling pathway. In our preliminary data (n = 2), upstream regulator analysis estimated that wheat gluten and ovalbumin, which are known loTSLP inducers, significantly activated NF-κB (Tables 4 and 5). Two NF-κB activation inhibitors (JSH-23 and parthenolide) blocked the nuclear translocation of RelA (p65). Vu et al. (2011) demonstrated that RelA small interfering RNA transfection inhibited poly(I:C)-induced TSLP mRNA augmentation in epidermal keratinocytes. Apart from us, other researchers also established that poly(I:C) could induce loTSLP mRNA (Xie et al., 2012; Kuroda et al., 2017). Therefore, RelA is a pivotal factor for inducing loTSLP transcription. In our system, two blockers of the nuclear translocation of RelA (JSH-23 and parthenolide) inhibited loTSLP mRNA upregulation by HWP. We conclude that NF-κB, especially RelA, strictly associated with HWP-inducing loTSLP transcription. Although AP-1 can influence loTSLP induction by protein allergens, further study is needed to clarify its role.
Further, little is known regarding shTSLP transcription. Tsilingiri et al. (2017) mentioned that shTSLP was upregulated by Vitamin D in epithelial cells and in silico analysis revealed that Vitamin D receptor binding site was identified in the upstream of the short isoform open reading frame. Martin Mena et al. (2017) reported that shTSLP mRNA expression was decreased by the peroxisome proliferator activated receptor-gamma knockdown or this antagonist in epithelial cells. To our knowledge, this is the first study to demonstrate that NF-κB may regulate the inducible shTSLP transcription. ShTSLP exon is shared with loTSLP exon, although each promoter was different. Therefore, shTSLP transcription may be influenced by loTSLP-regulating factors like NF-κB. On the other hand, shTSLP mRNA expression was not changed when NF-κB was recruited to binding sites of human TSLP promoter by IL-1α in human keratinocytes (Redhu et al., 2020). In our inhibition assay, the inhibitory effects of NF-κB inhibitors on shTSLP upregulation was much weaker than that on loTSLP induction. Accordingly, NF-κB components without RelA may need for shTSLP induction.
In conclusion, an allergenic HWP induced an easily sensitizable milieu by activating several inflammatory pathways and induced NF-κB-dependent loTSLP transcription in epidermal keratinocytes. Comprehensive identification of inflammatory pathways and loTSLP transcriptional data can help estimate the allergen’s epidermal sensitizing potency as well as the sensitizing mechanisms of type 2 immune response and immediate-type hypersensitivity.
Kayoko Matsunaga is the principle investigator of the laboratory funded by Hoyu Co., Ltd. and received research fund from Yuuka Co., Ltd.. These funders had no role in the study design, conduct of the study, data collection, data interpretation or preparation of the manuscript.