Direct exposure of Dermatophagoides pteronyssinus extracts to engineered water nanostructures significantly prevent Der p-induced allergic asthma in mice

Background and Purpose: House dust mites (Dermataphagoides pteronyssinus, Der p) commonly cause asthma; however, effective prevention and treatment methods are limited. This study examined whether direct treatment of Der p with engineered water nanostructures (EWNS) alters the allergenicity of the major Der p allergen Der p 1 in terms of dendritic cell (DC) activation and the development of asthma in a Der p 1-induced mouse model of allergic asthma. Experimental Approach: Der p extracts were treated with EWNS for 1h or 24 h, and the Der p 1 concentration was measured. Then, EWNS-treated Der p 1-induced DC activation was evaluated in vitro using the mouse DC line DC2.4, focusing on Ca2+-influx, dynamics of antigenic expression, and cytokine production. A mouse model of Der p 1-induced allergic asthma was generated by repeated intranasal administration of EWNS-treated or untreated Der p 1. Key Results: EWNS exposure reduced the concentration of Der p 1 in Der p extracts. The Ca2+ influx, dynamics of antigenic expression, inflammatory cytokine production, and gene expression in DC2.4 cells were also inhibited by exposure to EWNS-treated Der p 1. Significantly reduced Der p 1-induced allergic asthma symptoms were observed in mice exposed to EWNS-treated Der p 1, as evidenced by changes in percutaneous oxygen saturation and pro-inflammatory responses. Conclusions and Implications: This is the first study to show that direct exposure to EWNS significantly reduces the allergenicity of house dust mite allergens. Treatment of house dust mite allergens with EWNS during the sensitization and/or challenge phases may be an effective method to prevent asthma development. Direct exposure of Dermatophagoides pteronyssinus extracts to engineered water nanostructures significantly prevent Der p-induced allergic asthma in mice 11 Yuta Kaga 1, Nana Komai 1, Takahiro Suzuki 1, Chiharu Ohira 1, Mao Kaneki 1, Yukari Inaba 1, Aika Hamauzu 1, Tomoki Fukuyama 1,2 Affiliations 1 School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara City, Kanagawa, Japan. 2 Center for Human and Animal Symbiosis Science, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara City, Kanagawa, Japan. Correspondence Tomoki Fukuyama, Laboratory of Veterinary Pharmacology, Azabu University, 1-17-71, Fuchinobe, Chuo-ku, Sagamihara City, Kanagawa, Japan, 252-5201, Japan. E-mail address: [email protected] (T. Fukuyama)

Background and Purpose: House dust mites ( Dermataphagoides pteronyssinus, Der p) commonly cause asthma; however, effective prevention and treatment methods are limited. This study examined whether direct treatment of Der p with engineered water nanostructures (EWNS) alters the allergenicity of the major Der p allergen Der p 1 in terms of dendritic cell (DC) activation and the development of asthma in a Der p 1-induced mouse model of allergic asthma. Experimental Approach: Der p extracts were treated with EWNS for 1h or 24 h, and the Der p 1 concentration was measured. Then, EWNS-treated Der p 1-induced DC activation was evaluated in vitro using the mouse DC line DC2.4, focusing on Ca 2+ -influx, dynamics of antigenic expression, and cytokine production. A mouse model of Der p 1-induced allergic asthma was generated by repeated intranasal administration of EWNS-treated or untreated Der p 1. Key Results: EWNS exposure reduced the concentration of Der p 1 in Der p extracts. The Ca 2+ influx, dynamics of antigenic expression, inflammatory cytokine production, and gene expression in DC2.4 cells were also inhibited by exposure to EWNS-treated Der p 1. Significantly reduced Der p 1-induced allergic asthma symptoms were observed in mice exposed to EWNS-treated Der p 1, as evidenced by changes in percutaneous oxygen saturation and pro-inflammatory responses.

and Implications: This is the first study to show that direct exposure to EWNS significantly reduces the allergenicity of house dust mite allergens. Treatment of house dust mite allergens with EWNS during the sensitization and/or challenge phases may be an effective method to prevent asthma development.

Engineered water nanostructures, Der p 1, dendritic cells, allergic asthma, Ca 2+ signaling.

The prevalence of allergic diseases has increased worldwide (Gentry, 2025; Pritchard, Falcone, & Mitchell, 2021). In a recent study, over 260 million people were affected by bronchial asthma, and 400 million suffered from allergic rhinitis (Pawankar, 2014; Pawankar, Canonica, Holgate, & Lockey, 2012; Takao et al., 2025; Q. Xu et al., 2025). The most common therapy for allergic asthma is glucocorticoids; however, asthma often remains poorly controlled despite high-dose corticosteroid therapy or when eosinophil counts exceed 5% (Xie, Zhang, Zhang, Liu, & Che, 2025). Although effective at an early stage, long-term glucocorticoid treatment can lead to various adverse effects and limited benefits (Y. L. Xu et al., 2025). Recently, phenotype-based biological treatments, including omalizumab, mepolizumab, and dupilumab, have become a new alternative therapeutic option (Lee et al., 2025; Sanchez et al., 2025; Shi et al., 2024). Omalizumab is a monoclonal antibody with an anti-IgE effect used in patients with allergic phenotypes and mepolizumab is an anti-IL-5 biologic that reduces eosinophil numbers (Jura-Szoltys, Niemiec-Gorska, Gluck, Branicka, & Gawlik, 2025). While these treatment options are both readily available and effective, in the face of the increasing prevalence of allergic conditions such as asthma the focus must shift to prevention rather than treatment. Allergic conditions, including asthma, allergic rhinitis, atopic dermatitis, and food allergies, are driven by dysregulated immune responses, often involving IgE-mediated mast cell and basophil activation, type 2 helper T cells (Th2) inflammation, and epithelial dysfunction (Vaseghi-Shanjani, Samra, Yousefi, Biggs, & Turvey, 2025). Challenges remain in asthma prediction and treatment, owing to its complex etiology involving genetic and environmental factors (Van Asselt et al., 2025). While genetic factors have recently become clear, controlling environmental factors is discussed more at the government level (Q. Xu et al., 2025). House dust mites are major protein allergens that induce allergic asthma and rhinitis; however, removing these requires considerable time and resources. In this study, we focused on engineered water nanostructures (EWNS) and examined whether exposure to EWNS challenges the allergen surfaces and inactivate their antigenicity. EWNS are produced by collecting invisible water in the air and applying a high voltage (Nomura et al., 2017). EWNS have a unique structure: an electron-rich water shell of 5 to 20 nm in diameter, which contains reactive oxygen species (ROS) that are long-lived and can be delivered to wider areas (Pyrgiotakis, McDevitt, Bordini, et al., 2014). Previous studies have shown that EWNS inactivate several bacterial species and viruses, including the influenza virus and SARS-CoV-2, on surfaces. We anticipated that EWNS might have the potential to inactivate protein allergens such as those of Dermataphagoides pteronyssinus (Der p), which is a widespread risk factor for the development of asthma (Pyrgiotakis, McDevitt, Bordini, et al., 2014; Pyrgiotakis, McDevitt, Gao, et al., 2014; Vaze et al., 2019). The objective of this study was to examine whether direct EWNS treatment of Der p alters the allergenicity of Der p 1, a major Der p allergen, from the perspective of Der p 1-specific antibody binding, dendritic cell (DC) activation, and asthma development in a mouse model.

[1]¿p1 [1]¿m1 2.1. Exposure of Der p extracts to EWNS Der p extracts (ITEA Inc., Tokyo, Japan) were treated with EWNS from a distance of 5 cm for 1–24 h, as shown in Figure 1a. EWNS were generated according to a previous report, as shown in Figure 1a (Pyrgiotakis, McDevitt, Bordini, et al., 2014; Pyrgiotakis, McDevitt, Gao, et al., 2014). Der p extracts without EWNS exposure but handled under the same conditions were used as controls. The concentration of Der p 1 in the EWNS-treated or untreated (control) Der p extracts was measured using a Der p 1 enzyme-linked immunosorbent assay (ELISA; ITEA Inc.) according to the manufacturer’s protocol. Optical density was measured using a microplate reader (Multiskan SkyHigh microplate reader, Thermo Fisher Scientific, Waltham, MA, USA). [1]¿p1 [1]¿m1 2.2. Murine dendritic cell line (DC2.4) The murine dendritic cell line (DC2.4) was obtained from the American Type Culture Collection (Manassas, VA, USA). The DC2.4 cells were cultured in RPMI 1640 medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich Co. LLC., Tokyo, Japan) and penicillin-streptomycin (FUJIFILM Wako Pure Chemical Corporation). DC2.4 cells (1 × 10 5 cells/100 μL) at 70% confluency were seeded in a 3.5 mm diameter glass bottom dish or a 96-well or 12-well culture plate and exposed to 24 h EWNS-treated or control Der p extracts (0.5 μg/mL Der p 1). 2.3. Calcium imaging of DC2.4 cell lines induced by Der p 1 Calcium imaging was performed on cultured DC2.4 cells after 24 h exposure to EWNS-treated or control Der p extract (0.5 μg/mL Der p 1) as previously described, with minor modifications (Fukuyama et al., 2017). Cells were first loaded with 2 μmol/L Fura-2-acetoxymethyl ester for 30 minutes at 37°C in darkness. The glass-bottomed dishes were mounted on perfusion blocks and viewed under an inverted fluorescence microscope (Nikon Solutions Co. Ltd., Tokyo, Japan). The fluorescence was alternately excited by UV light at 340 and 380 nm, and the emitted light was collected every 200 ms using a camera (ORCA-Fusion BT Digital CMOS camera; Hamamatsu Photonics K.K., Shizuoka, Japan). Cells were initially incubated in Locke buffer without CaCl 2 (pH 7.4; 136 mmol/L NaCl, 5.4 mmol/L KCl, 2.5 mmol/L MgCl 2, 10.9 mmol/L D-glucose, 0.6 mmol/l NaHPO 4, and 14.3 mmol/L NaHCO 3 ) and buffer was changed to Locke buffer with CaCl 2 (pH 7.4; 136 mmol/L NaCl, 5.4 mmol/L KCl, 2.9 mmol/L CaCl 2, 2.5 mmol/L MgCl 2, 10.9 mmol/L D-glucose, 0.6 mmol/l NaHPO 4, and 14.3 mmol/L NaHCO 3 ) 3 mins after initiation of setting. The solutions were administered using a temperature-controlled perfusion system. Cells were considered responsive if the ratio increased by greater than 20% of the resting level after chemical application. 2.4. Influence of EWNS treatment on Der p 1-induced DC2.4 activation DC2.4 cells were exposed to EWNS-treated or control Der p extracts (0.5 μg/mL Der p 1) for 24 h. After exposure, DC2.4 cell phenotypes (CD11c and major histocompatibility complex [MHC] class II) were analyzed using monoclonal antibodies (anti-mouse CD11c, anti-mouse MHC class II, and DAPI from BioLegend, Inc., Tokyo, Japan). The cells were washed and analyzed using a BD FACSAria III cell sorter (BD Biosciences, Franklin Lakes, NJ, USA). Levels of tumor necrosis factor (TNF)-α in the supernatant of DC2.4 cells 24 h after exposure to EWNS-treated or control Der p extracts (0.5 μg/mL Der p 1) were evaluated using ELISA (DuoSet ELISA kit, R&D Systems, Minneapolis, MN, USA) and a microplate reader. Gene expressions of interleukin ( IL) -1 β, IL - 6, interferon regulatory factor (IRF)5, signal transducers and activator of transcription (Stat)3, transforming growth factor (TGF)β1 and TNFα in DC2.4 cells were measured using qRT-RCR. Total RNA was extracted from the cells using the NucleoSpin® RNA kit (TaKaRa Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. Extracted total RNA (500 ng) was reverse transcribed using the PrimeScript™ RT Master Mix (TaKaRa Bio). Expression levels of each target were assessed using respective specific primers (Takara Bio), PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific), and a qPCR system (CFX Duet Real-Time PCR System, Bio-Rad Laboratories, Inc., Tokyo, Japan). The expression of each target gene was normalized to that of β-actin . 2.5. Mouse model of Der p 1-induced asthma Seven-week-old female BALB/c mice were obtained from Japan SLC Ltd. (Shizuoka, Japan) and used to generate a Der p 1-induced asthma model. A maximum of four mice were housed per cage under controlled conditions (12 h light-dark cycle at 22 ± 3°C and 55 ± 15% humidity). The mice were provided with a certified pellet diet and tap water ad libitum . All the experiments were conducted in accordance with the Animal Care and Use Program of Azabu University (Approval No. 230327-23). A mouse model of asthma was generated using a previously described method with slight modifications (Ando et al., 2023; Ohira et al., 2023). As a dose-range study, a chronic asthma model was generated by daily (5 days/week) intranasal administration of EWNS-treated or control Der p extracts for 0.5 h or 2 h (25 μg/25 μL/mouse) as shown in Supplemental Figure S1A. Based on the results of the dose-range study, the initial setting was performed by intranasal sensitization (25 μg/25 μL/mouse) and intranasal challenge (5 μg/5 μL/mouse) with EWNS-treated or control Der p extracts for 1 h or 24 h as shown in Figure 2a. The second setting was also performed to confirm the reproducibility and determine the effects of EWNS treatment alone on sensitization or challenge. The detailed settings are shown in Figure 5a. The day after the final Der p challenge, serum, bronchoalveolar lavage fluid (BALF), hilar lymph nodes (LNs), and lungs were collected for further analysis. 2.6. Measurement of total and Der p 1-specific IgE levels in mouse serum and BALF Total (BD Biosciences) and Der p 1-specific IgE levels (MyBioSource.com. San Diego, CA, USA) in the serum and BALF were determined using ELISA kits according to the manufacturer’s protocol. 2.7. Isolation of group 2 innate lymphoid cell (ILC2) from lung tissue Single-cell suspensions of lung tissue (left lobe) were prepared using a gentle MACS Dissociator Octo with a Multi-Tissue Dissociation Kit 1 (Miltenyi Biotec K.K., Tokyo, Japan) according to the manufacturer’s protocol. Isolation of group 2 innate lymphoid cell (ILC2) was conducted using monoclonal antibodies (PE-conjugated anti-mouse CD25, APC-Vio770-conjugated anti-mouse CD45.2, PerCP-Vio770-conjugated anti-mouse CD90.2, FITC-conjugated anti-mouse ST2/IL-33, APC-conjugated anti-mouse Lineage Antibody Cocktail, and DAPI; BioLegend, Inc., Miltenyi Biotec K.K., and BD Biosciences) based on a previously described method (Ohira et al., 2023). The cells were then washed and analyzed using a FACSAria III cell sorter (BD Biosciences). 2.8. Histological assessment of lungs The portion of lung corrected from each allergy model mouse was fixed in a 10% formalin solution, embedded in paraffin wax, sectioned to 5 μm thickness, and stained with hematoxylin and eosin. A semi-quantitative histopathological evaluation was performed in a blinded fashion by a pathologist using the following grading system: 0, within normal limits; 1, mild; 2, moderate; and 3, severe. 2.9. BALF analysis BALF was collected by cannulating the trachea and lavaging the lungs twice with 1 mL phosphate-buffered saline containing 0.1% bovine serum albumin (Thermo Fisher Scientific). BALF cell pellets were used to measure the total number of live cells (CellDrop Cell Counting System) and neutrophil, eosinophil, and macrophage differentiation (10,000 events) using a FACSAria III cell sorter (BD Biosciences) with specific monoclonal antibodies (anti-mouse CCR3, anti-mouse CD11c, anti-mouse CD11b, and anti-mouse Gr-1; BioLegend Inc., CA, USA). Cytokine levels (IL-1β, IL-6, TNFα, MCP-1, and eotaxin) in the supernatant of the first BALF fraction were determined using an appropriate ELISA kit as per the manufacturer’s instructions (DuoSet ELISA kit, R&D Systems). 2.10. Cell differentiation of LNs Single-cell suspensions isolated from the LNs were prepared as described previously (Ando et al., 2023; Kaneki et al., 2023; Matsuzaka et al., 2024), and total cell number was measured using a CellDrop™ Cell Counting system (DeNovix Inc., Wilmington, DE, USA). To avoid nonspecific binding in the flow cytometric analysis, 1 × 10 6 cells were first incubated with 1 µg of mouse Fc Block (BioLegend) prior to incubation with monoclonal antibodies (anti-mouse CD3, anti-mouse CD4, anti-mouse CD11b, anti-mouse CD11c, anti-mouse CD19, anti-mouse CD44, anti-mouse CD62L, anti-mouse MHC class II, and DAPI were purchased from BioLegend and anti-mouse IgE from Sony Biotechnology Inc., Tokyo, Japan). The cells were then washed and analyzed using a FACSAria III cell sorter (BD Biosciences). 2.11. Cytokine release assay for LNs Single-cell suspensions of LNs were used to examine cytokine release by T cells. Briefly, single-cell suspensions of LNs (5 × 10 5 cells/well) were incubated with Dynabeads mouse T-Activator CD3/CD28 (Thermo Fisher Scientific) for 24 h. Subsequently, levels of IL-4, IL-13, and TNFα in the supernatant were evaluated using an appropriate ELISA kit as per the manufacturer’s instructions (DuoSet ELISA kit, R&D Systems). 2.13. Statistical analysis All data are expressed as the mean ± standard error of the mean (SEM). We performed analysis of variance (ANOVA) followed by Šídák’s multiple comparison test. Statistical significance was estimated the 5% probability level. Data were analyzed using the GraphPad Prism 10.0 (GraphPad Software, San Diego, CA, USA).

3.1. Inhibition of allergenicity of Der p 1 by exposure to EWNS Initially, the direct inhibition of Der p 1 allergenicity was evaluated using a Der p 1 specific ELISA. The ELISA results showed that the allergenicity of Der p extract was dramatically reduced by exposure to EWNS. One hour of EWNS exposure resulted in an approximately 70.4% reduction in Der p 1 concentration compared to the baseline value, and 24 h of EWNS exposure resulted in a 99.6% reduction in Der p 1 concentration compared to the baseline value (Figure 1c). 3.2. Exposure to EWNS-treated Der p extract significantly reduces dendritic cell activation in vitro The effect of EWNS treatment on antigen-presenting cells was examined using DC2.4 cells. EWNS-treated Der p extracts were directly applied to DC2.4 cells to further elucidate whether the dendritic cell response could be affected by EWNS treatment. As previously published, house dust mites can mediate the increase in intracellular calcium concentrations by activating protease-activated receptor 2 and Ca 2+ release channels (Ouyang et al., 2024). Therefore, we examined whether EWNS treatment of Der p extract could influence the Ca 2+ influx of DC2.4 cells. Our findings indicate that exposure of DC2.4 cells to control Der p extract significantly increased activated DCs compared to the untreated (medium only) DCs, whereas Ca 2+ influx induced by EWNS-treated Der p extract was significantly reduced compared to that in the control Der p extract exposure group, and almost at the same level as that of untreated control cells (Figure 1d). The MHC class II antigenic expression were also significantly enhanced by control Der p extract, and this enhancement was suppressed by EWNS treatment (Figure 1e). Production of TNFα was significantly enhanced by exposure to control Der p extract, whereas TNFα production was significantly suppressed in the EWNS-treated Der p extract group compared to that in the control Der p extract group (Figure 1f). These results were validated using RNA analysis of factors including IL-1β, IL-6, IRF5, Stat3, TGFβ1, and TNFα in DC2.4 cells (Figure 1g–l). 3.2. Influence of EWNS-treated Der p in a mouse model of chronic asthma Next, we explored the effects of 0.5 h and 2 h treatment with EWNS-treated or control Der p extracts in a mouse model of chronic asthma. EWNS-treated Der p extracts did not affect body weight during the experimental period (Figure S1b). Exposure to control Der p extract significantly reduced the SpO 2 level in mice compared to that in control mice. Exposure to EWNS-treated Der p extract for 0.5 h significantly ameliorated the reduction in SpO 2 level compared to that of the control Der p group (Figure S1c). Serum levels of total and Der p 1-specific IgE, which play a central role in the development of asthma symptoms, were significantly elevated in mice exposed to control Der p compared to those in untreated mice, and EWNS-treated Der p 1 extract significantly decreased IgE levels (Figure S1d and e). The ratio of ILC2s in the lung tissue was also significantly higher in the control Der p extract group than in the control group, whereas a significant reduction in the number of ILC2s was observed in the EWNS-treated Der p extract group compared to the control Der p extract group (Figure S1f). Semi-quantitative histopathological evaluation of the lungs confirmed a significant increase in asthma symptoms in mice exposed to control Der p extract compared to control mice, and peribronchial inflammation was significantly ameliorated in mice exposed to EWNS-treated Der p compared to that in the control Der p extract group (Table S1, Figure S1g). Neutrophil and eosinophil influx and IL-1β, IL-6, and TNFα levels in BALF were also increased in mice exposed to control Der p extract compared to untreated mice, and all indices except for eosinophil infiltration were significantly decreased in mice exposed to EWNS-treated Der p (Figure S2). Cellular infiltration into the LN was increased in mice exposed to control Der p extract compared to control mice and was significantly decreased in mice exposed to EWNS-treated Der p (Figure S3a-d). However, the effect of EWNS exposure on cytokine production by T cells was not as pronounced as that on cellular infiltration into the LN. There was no significant change in the production of proinflammatory cytokines (Figure S3e–g). 3.3. Influence of long-term (24 h) EWNS-treated Der p in a mouse model of moderate asthma As slight effects of short-term (0.5 h and 2 h) exposure to EWNS-treated Der p extracts were observed in a mouse model of chronic asthma, long-term (24 h) exposure to EWNS-treated Der p in a mouse model of weak to moderate asthma was used in the next step. Exposure to control Der p (1 h and 24 h) significantly reduced the SpO 2 level in mice compared to that in control mice, and exposure to EWNS-treated Der p (1 h and 24 h) significantly ameliorated the reduction in SpO 2 levels induced by control Der p exposure (Figure 2b). Total and Der p 1-specific IgE levels in serum and the number of ILC2s in lung tissue were significantly elevated in mice exposed to control Der p (1 h and 24 h) compared to those in control mice, and significant downregulation was observed at both 1 and 24 h in the EWNS-treated Der p group (Figure 2c–e). Semi-quantitative histopathological evaluation of the lungs confirmed a significant increase in asthma symptoms in mice exposed to control Der p compared to those in control mice, and asthmatic symptoms were significantly ameliorated in both 1 h and 24 h EWNS-treated Der p exposure groups (Table 1, Figure 2f). Cellular influx and IL-1β, IL-6, TNFα, MCP-1, eotaxin, and total IgE levels in BALF were also increased in mice exposed to control Der p compared to untreated mice, and they were significantly decreased in the EWNS-treated Der p group (Figure 3a-j). Cellular infiltration into LN and pro-inflammatory cytokine production were increased in mice exposed to control Der p and untreated mice, and the immune hyperreaction induced by Der p was significantly inhibited by EWNS treatment even after 1 h (Figure 4a-g). 3.3. Effects of EWNS only in the sensitization or challenge phase in a mouse model of asthma The temporal usage of EWNS, for example, only during the sensitization or challenge phase, is more realistic than permanent exposure to EWNS. Therefore, the effects of EWNS treatment limited to the sensitization or challenge phases were examined in a mouse model of asthma. Exposure to control Der p significantly reduced the SpO 2 level, and exposure to EWNS-treated Der p in both the sensitization and challenge phases significantly ameliorated the reduction SpO 2 level (Figure 5b). Exposure to EWNS-treated Der p in the sensitization phase also significantly ameliorated the reduction in SpO 2 levels, whereas there was no significant change in mice exposed to EWNS-treated Der p in this phase (Figure 5b). Total and Der p 1-specific IgE levels in serum and the number of ILC2s in lung tissue were significantly elevated in mice exposed to control Der p, and were significantly downregulated in mice exposed to EWNS-treated Der p, both in the sensitization and challenge phases (Figure 5c-e). Exposure to EWNS-treated Der p in the sensitization and challenge phases also resulted in a significant inhibition of IgE levels and the number of ILC2s in the lungs (Figure 5c–e). Semi-quantitative histopathological evaluation of the lungs showed a trend similar to that of serum IgE levels. A significant increase in asthma symptoms was observed in mice exposed to control Der p compared to control mice, and asthma symptoms were significantly ameliorated in all EWNS treatment groups (Table 2, Figure 5f). Interestingly, cellular influx, pro-inflammatory cytokine/chemokine levels, and total IgE levels in BALF (Figure 6a-h) and/or LNs (Figure 7a-f) were also increased in mice exposed to control Der p compared to those in control mice. However, all EWNS treatment conditions showed significant inhibition of inflammatory responses in the BALF and LNs (Figures 6 and 7).

The bactericidal and antiviral effects of EWNS have been previously reported; however, this is the first report to demonstrate the direct effect of EWNS on allergenicity (Pyrgiotakis, McDevitt, Bordini, et al., 2014; Pyrgiotakis, McDevitt, Gao, et al., 2014). In the present study, exposure to EWNS for 1 h resulted in a 70.4% reduction in Der p 1 concentration. Pyrgiotakis, McDevitt, Gao, et al. (2014) indicated that the EWNS can interact with and inactivate airborne mycobacteria, reducing their concentration levels significantly. They also demonstrated the mechanism of EWNS in mycobacterial inactivation and reported that EWNS effectively deliver the ROS encapsulated during the electrospray process to the bacteria, oxidizing their cell membranes, and ultimately inactivating them. In this study, we did not observe the mechanism by which the EWNS inactivated Der p and Der p 1, but we hypothesize that the ROS generated by the EWNS may have acted in a similar fashion, as the method to generate EWNS was almost the same as that in the previous study. Using a similar mechanism to generate the ROS, the cold atmospheric pressure plasma (CAP) has been widely used for bactericidal and antiviral applications (Wu et al., 2014). The inactivation effects of CAP on aerosolized allergens, including Der p 1, Derf1, Aspf1, Alta1, and Canf1, as well as those from indoor and outdoor environments, have been investigated (Wu et al., 2014). As CAP and EWNS have similar mechanisms, the effects of EWNS on the concentration of Der p 1 were considered to be due to its direct interaction with the cell membrane of Der p 1 via ROS. The advantage of the EWNS is that there is no need for gases such as nitrogen and argon to produce energy, which means that the EWNS is much safer than CAP or ultraviolet exposure systems. Demonstrating the reduction in Der p 1 concentrations as determined by ELISA is not definitive in demonstrating the inhibition of the allergenicity of Der p and Der p 1 in the human body. The cell surface of Der p should be broken down upon EWNS exposure, and the ELISA determines the concentration of the allergen by attaching the allergen onto the primary antibody-coated ELISA plate. Even if the cell surface is broken down, the allergenicity of Der p 1 potentially exists and may influence the development and exacerbation of allergies. Therefore, the allergenicity of Der p exposed to EWNS was evaluated in vitro using the DC line DC2.4. DCs are antigen-presenting cells that play a key role in recognizing and spreading allergen information and transferring it to naïve T cells via MHC class II molecules (Xiang et al., 2024; Zhang et al., 2021). We have previously investigated the toxicological and therapeutic immunosuppressive potential of several chemicals, such as mycotoxins (Matsuzaka et al., 2024). In this study, the influence of EWNS exposure on the allergenicity of Der p was examined by focusing on the calcium influx, MHC class II surface molecule expression, TNFα production, and related gene expression. House dust mites, including Der p, mediate the increase in intracellular calcium concentrations by activating protease-activated receptor 2 and Ca 2+ release channels (Ouyang et al., 2024). This triggers subsequent cellular activation and pro-inflammatory responses (Figure 1b). Therefore, Ca 2+ influx induced by Der p 1 was first monitored to evaluate the inhibition of Der p 1 allergenicity imbued by EWNS exposure. Our results indicated that Der p 1-induced Ca 2+ influx was significantly downregulated by EWNS exposure to a level similar to that in the control group. Subsequent activation including surface antigen expression of MHC class II, TNFα production, and several related gene expressions were all inhibited by EWNS pretreatment of Der p extract. In the preliminary test with EWNS-treated cell medium without Der p 1, no reactions were observed in DC2.4 cells (data not shown); therefore, above all, inhibitory patterns are considered to be associated with the direct inhibition of Der p 1 allergenicity. In vitro experiments have only focused on the activation of DCs, and there is no information on the connection between the epithelium and T cells, which play a pivotal role in the development of all types of allergies. In vivo responses to EWNS-treated Der p were confirmed using a mouse model of asthma. Initially, asthmatic responses induced by exposure of Der p 1 to EWNS for 0.5 and 2 h were evaluated using frequent sensitization and challenge protocols to generate the chronic asthma model (Figure S1a). EWNS and frequent EWNS application did not alter the general condition of mice (body weight). Der p 1-induced asthmatic responses, including effects on SpO 2, serum IgE levels, ILC2 in lung tissue, and cellular infiltration into LN, were significantly inhibited by exposure to EWNS-treated Der p even for a short period of time (0.5 h and 2 h). In contrast, histological evaluation, eosinophil influx into BALF, and pro-inflammatory cytokine production in T cells were not affected by EWNS treatment. The exposure length and intensity of asthmatic symptoms may be considered as the reasons. Therefore, a longer treatment with EWNS for weak-to-moderate asthmatic symptoms will be the focus of the next trial. In the weak-to-moderate asthma model (Figure 2a), exposure of Der p 1 to EWNS for 1 and 24 h significantly inhibited all asthmatic responses, including histological evaluation, eosinophil influx into the BALF, and pro-inflammatory cytokine production in T cells, which were not influenced in the chronic model. Interestingly, the inhibitory profile between 1 h and 24 h of exposure did not show any difference. Exposure to EWNS for 1 h resulted in a 70% reduction in Der p 1 concentration in the in vitro study, which might be sufficient to inhibit asthmatic responses in vivo . In this setting, EWNS-treated Der p 1 was applied to mice in both the sensitization and challenge phases. However, when the applicability of EWNS in real life is considered, spot usage of EWNS, such as only in the sensitization or challenge phases, is more realistic than continuous usage. Next, the application of EWNS only in the sensitization or challenge phase was investigated. When comparing the inhibitory pattern in mice exposed to full-time EWNS-treated Der p 1, amelioration of SpO 2 was weaker in mice exposed only to sensitization or challenge with EWNS-treated Der p 1; however, other asthmatic responses were prevented to a similar extent by all EWNS treatment conditions. It is reprehensible that there are no responses to Der p 1 if the sensitization was not developed by inhibition of EWNS treatment, however, it is interesting to observe that there were also no responses to EWNS-treated Der p 1 after immunization was developed by normal Der p 1 exposure. Higher challenge phase frequency and shorter exposure to EWNS (e.g., shorter than 0.5 h) will be examined in future research. Taken together, this is the first study to demonstrate the novel abilities of EWNS exposure against the house dust mite allergen Der p 1. Even with 1 h exposure of Der p extracts to EWNS, the concentration of Der p 1 dramatically decreased and degranulation led to an inhibition of Ca 2+ influx and pro-inflammatory responses in DCs. In an in vivo study, EWNS-treated Der p 1 prevented the development of immunity and inhibited the onset of symptoms in the challenge phase. Our study suggests that EWNS can be used as both a preventive and therapeutic option for known allergen-related allergies. However, there are limitations to this study, including inactivated properties in the actual atmosphere, which have not been clarified. In addition, interactions with other allergens should be examined. Both limitations are currently under investigation using one of the pollen allergens, Cryj 1, and a larger exposure system. AUTHOR CONTRIBUTIONS T. Fukuyama : Conceptualization (lead), data curation (supporting), formal analysis (supporting), investigation (supporting), methodology (supporting), supervision (lead), validation (supporting), visualization (supporting), writing the original draft (lead), and writing the review and editing (lead). Y. Kaga : Conceptualization (supporting), data curation (lead), formal analysis (lead), investigation (lead), methodology (lead), supervision (supporting), visualization (supporting), writing the original draft (supporting), and writing the review and editing (supporting). N. Komai, T. Suzuki, C. Ohira, M. Kaneki, Y. Inaba and A. Hamauzu : Data curation (supporting), investigation (supporting), methodology (supporting), writing, review, and editing (supporting).

We would like to thank Editage (www.editage.jp) for English language editing. CONFLICT OF INTEREST STATEMENT We have no conflicts of interest arising from relationships with commercial/corporate interests to declare. DATA AVAILABILITY STATEMENT The data supporting the findings of this study are available from the corresponding author upon reasonable request. Some data may not be available due to privacy or ethical restrictions. DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigor of preclinical research, as stated in the BJP guidelines for Design and Analysis, and as recommended by funding agencies, publishers, and other organizations engaged in supporting research. FUNDING This work was self-funded.

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(b) Strategy to identify the effects of EWNS exposure on dendritic cell (DC) activation. (c) Inhibitory pattern of Der p 1 concentration after EWNS exposure. (d) Control Der p (0.5 μg/mL) significantly induced DC2.4 cell activation, however, Der p 1-induced activation was significantly inhibited by EWNS exposure for 24 h as determined by effects on calcium influx (%, mean ± SEM, n = 1201–1265). Representative Ca 2+ signals and images in DC2.4 cells loaded with the fluorescent Ca 2+ indicator Fura-2-acetoxymethyl ester are shown. (e) MHC class II expression on the cell surface (mean ± SEM, n = 6), (f) TNFα production in the supernatant (pg/mL, mean ± SEM, n = 6) and gene expressions (mean ± SEM, n = 6) of (g) IL - 1β, (h) IL - 6, (i) IRF5, (j) stat3, (k) TGFβ1, and (l) TNFα in DC2.4 cells were significantly upregulated by control Der p 1 (0.5 μg/mL). The upregulating effects of Der p 1 were significantly inhibited by EWNS treatment. Figure 2 Inhibitory effects of EWNS treatment on Der p extract in a mouse model of asthma. (a) Experimental protocol used in initial setting in vivo study. (b) Percutaneous oxygen saturation (SpO 2 ) was significantly decreased by exposure to control Der p and this decrease was recovered by EWNS treatment on Der p extract (%, mean ± SEM, n = 4–7). (c) Total and (d) Der p 1-sepecific IgE levels in serum (pg/mL, mean ± SEM, n = 4-7) were also significantly enhanced by control Der p. These increases in IgE levels were significantly reversed by EWNS treatment on Der p extract. (e) Ratio of ILC2 cells in lung tissue (%, mean ± SEM, n = 4–7) was increased by control Der p and significantly reduced by EWNS treatment on Der p extract. Representative FACS images of ILC2 expression in each group. (f) Representative microscopy images of lungs in mice from each group (Scale bar = 100 μm). Figure 3 Impact of EWNS treatment on BALF analysis in a mouse model of asthma. Ratio of (a) neutrophils, (b) eosinophils and (c) macrophages in BALF (%, mean ± SEM, n = 4–7) was significantly altered by control Der p and those changes were significantly ameliorated by EWNS treatment on Der p extract. (d) Representative FACS images of eosinophils and macrophages expression in BALF from each group. Levels (pg/mL, mean ± SEM, n = 4-7) of (e) IL-1β, (f) IL-6, (g) TNFα, (h) MCP-1, (i) eotaxin, and (j) total IgE in BALF were significantly upregulated by control Der p but were significantly inhibited by EWNS treatment on Der p extract. Figure 4 Impact of exposure to EWNS on local immune function in a mouse model of asthma. Exposure to EWNS-treated Derp extract (both 1 h and 24 h) significantly decreased the number of (a) CD11c + MHC class II + DCs, (b) CD3 + CD4 + CD44 + CD62L - T cells, and (c) CD19 + IgE + B cells in LN (mean ± SEM, n = 4–7) compared to those in control Der p exposure groups. Representative FACS images of CD19 + IgE + B cells in LNs from each group in LN are shown. Control Der p extract significantly increased the production of (d) IL-4, (e) IL-13, (f) IL-17, and (g) TNFα from CD3/CD28-stimulated T cells in LNs (pg/mL, mean ± SEM, n = 4-7) but these increased were significantly reversed with EWNS treatment on Der p extract. Figure 5 Several conditions of EWNS exposure on asthmatic symptoms in a mouse model of asthma Experimental protocol used in second setting of the in vivo study. (b) Percutaneous oxygen saturation (SpO 2 ) was significantly decreased by control Der p and this decrease were significantly recovered by EWNS exposure to Der p extract in both sensitization and challenge phases, and EWNS only in the sensitization phase (%, mean ± SEM, n = 4–7). (c) Total and (d) Der p 1-sepecific IgE levels in serum (pg/mL, mean ± SEM, n = 4–7) were significantly enhanced by control Der p and those enhanced IgE levels were significantly reduced by all EWNS treatment conditions. (e) Ratio of ILC2 cells in lung tissue (%, mean ± SEM, n = 4–7) was also increased by control Der p and that increase was significantly decreased by all EWNS treatment conditions. Representative FACS images of ILC2 expression in each group. (f) Representative images of the lungs in mice from each group (Scale bar = 100 μm). Figure 6 Several conditions of EWNS exposure on BALF analysis in a mouse model of asthma Ratio of (a) eosinophils and (b) neutrophils in BALF (%, mean ± SEM, n = 4–7) was significantly influenced by control Der p and those changes were significantly ameliorated by all EWNS treatment conditions on Der p extract. Representative FACS images of neutrophil expression in BALF from each group. Levels (pg/mL, mean ± SEM, n = 4–7) of (e) IL-1β, (f) IL-6, (g) TNFα, (h) MCP-1, (i) eotaxin, and (j) total IgE in BALF were significantly upregulated by control Der p these upregulations were significantly inhibited by all EWNS treatment conditions. Figure 7 Impact of exposure to EWNS on local immune function in a mouse model of asthma. Exposure to all conditions of EWNS significantly decreased the number of (a) CD11c + MHC class II + DCs, (b) CD3 + CD4 + CD44 + CD62L - T cells. and (c) CD19 + IgE + B cells in LN (mean ± SEM, n = 4–7) compared to those significantly increased by control Der p exposure compared to untreated group. Control Der p significantly increases the production of (d) IL-4, (e) IL-13, and (f) TNFα from CD3/CD28-stimulated T cells in LNs (pg/mL, mean ± SEM, n = 4–7) but Der p-induced upregulation was significantly inhibited by all conditions of EWNS treatment. Figure S1 Impact of short-term EWNS treatment in a mouse model of Der p-induced chronic asthma. (a) Experimental protocol used to generate the chronic asthma model. (b) Body weight was not affected by any types of Der p treatment. (c) Percutaneous oxygen saturation (SpO 2 ) was significantly decreased by control Der p and those decreases were recovered by 0.5 h EWNS exposure to Der p extract (%, mean ± SEM, n = 5–8). (d) Total and (e) Der p 1-sepecific IgE levels in serum (pg/mL, mean ± SEM, n = 5–8) were also significantly enhanced by control Der p and those enhanced IgE levels were significantly reduced by EWNS treatment. (f) Ratio of ILC2 cells in lung tissue (%, mean ± SEM, n = 5–8) was increased by untreated Der p and that increase was significantly decreased by EWNS treatment. (g) Representative images of the lungs in mice from each group (Scale bar = 100 μm). Figure S2 Impact of short-term EWNS treatment on BALF analysis in a mouse model of Der p-induced chronic asthma. Ratio of (a) neutrophils, (b) eosinophils, and (c) macrophages in BALF (%, mean ± SEM, n = 5–8) was significantly altered by control Der p but only the effects on neutrophils was significantly ameliorated by EWNS treatment. Levels (pg/mL, mean ± SEM, n = 5–8) of (d) IL-1β, (e) IL-6, and (f) TNFα in BALF were significantly upregulated by control Der p but these changes were significantly reversed by EWNS treatment. Figure S3 Impact of exposure to EWNS on local immune function in a mouse model of Der p-induced chronic asthma. Exposure to EWNS (both 0.5 h and 2 h) significantly decreased the number of (a) CD11c + MHC class II + DCs, (b) CD3 + CD4 + CD44 + CD62L - T cells, (c) CD3 + CD4 + CD44 + CD62L + T cells, and (d) CD19 + IgE + B cells in LN (mean ± SEM, n = 5–8) compared to those significantly increased by control Der p exposure. Control Der p significantly increased the production of (e) IL-4, (f) IL-13, and (g) TNFα in CD3/CD28-stimulated T cells in LNs (pg/mL, mean ± SEM, n = 5–8) but EWNS treatment did not influence the upregulation of these cytokines. Table 1 Histological evaluation of lungs in a mouse model of Der p-induced asthma (EWNS exposure time range study). A histological score (0, within normal limits; 1, mild; 2, moderate; and 3, severe) was assigned to each observation. Results are expressed as mean ± SEM. n = 4-7 per group. Dunn’s multiple comparisons tests were assessed as untreated cells vs Control Der p 1 (green) and Control Der p 1 vs EWNS Der p 1 (red). [1]¿p1 [1]¿m1 | Peribranchial inflammation | 0.00 ± 0.00 | 0.71 ± 0.18 | 0.00 ± 0.00 0.018 | 0.71 ± 0.18 | 0.00 ± 0.00 0.018 | | Alveolar macrophage infiltration | 0.00 ± 0.00 | 1.00 ± 0.00 <0.01 | 0.00 ± 0.00 <0.01 | 1.00 ± 0.00 <0.01 | 0.00 ± 0.00 <0.01 | | Perivascular inflammation | 0.00 ± 0.00 | 1.29 ± 0.18 <0.01 | 0.14 ± 0.14 <0.01 | 1.14 ± 0.14 0.012 | 0.14 ± 0.14 <0.01 | Table 2 Histological evaluation of lungs in a mouse model of Der p-induced asthma (efficacy of EWNS on sensitization and challenge phase study). A histological score (0, within normal limits; 1, mild; 2, moderate; and 3, severe) was assigned to each observation. Results are expressed as mean ± SEM. n = 4-7 per group. Dunn’s multiple comparisons tests were assessed as untreated vs Control Der p 1 + Control Der p 1 (green) and Control Der p 1 + Control Der p 1 vs other Der p 1 treated groups (red). | Peribranchial inflammation | 0.00 ± 0.00 | 1.14 ± 0.14 <0.01 | 0.00 ± 0.00 <0.01 | 0.14 ± 0.14 <0.01 | 0.00 ± 0.00 <0.01 | | Alveolar macrophage infiltration | 0.00 ± 0.00 | 1.00 ± 0.00 <0.01 | 0.00 ± 0.00 <0.01 | 0.14 ± 0.14 <0.01 | 0.29 ± 0.18 <0.01 | | Perivascular inflammation | 0.00 ± 0.00 | 1.14 ± 1.14 <0.01 | 0.00 ± 0.00 <0.01 | 0.00 ± 0.00 <0.01 | 0.00 ± 0.00 <0.01 | [1]¿p1 [1]¿m1 TABLE S1 Histological evaluation of lungs in a mouse model of Der p-induced chronic asthma. A histological score (0, within normal limits; 1, mild; 2, moderate; and 3, severe) was assigned to each observation. Results are expressed as mean ± SEM. n = 5-8 per group. Dunn’s multiple comparisons tests were assessed as untreated vs Control Der p 1 (green) and Control Der p 1 vs other Der p 1 treated groups (red). | Bronchial thickening | 0.00 ± 0.00 | 1.00 ± 0.00 <0.01 | 1.00 ± 0.00 | 0.88 ± 0.13 | | Peribranchial Inflammation | 0.00 ± 0.00 | 2.00 ± 0.00 <0.01 | 1.25 ± 0.25 | 0.88 ± 0.23 <0.01 | | Alveolar macrophage infiltration | 0.00 ± 0.00 | 1.00 ± 0.00 <0.01 | 0.63 ± 0.18 | 0.50 ± 0.19 | | Perivascular Inflammation | 0.00 ± 0.00 | 2.00 ± 0.00 <0.01 | 2.00 ± 0.00 | 1.50 ± 0.19 | Information & Authors Information Version history Copyright This work is licensed under a Non Exclusive No Reuse License.

Authors Metrics & Citations Metrics Article Usage 414views 214downloads Citations Download citation Yudai Kaga, Nana Komai, Takao Suzuki, et al. Direct exposure of Dermatophagoides pteronyssinus extracts to engineered water nanostructures significantly prevent Der p-induced allergic asthma in mice. Authorea. 25 March 2025. DOI: https://doi.org/10.22541/au.174291888.80849645/v1 DOI: https://doi.org/10.22541/au.174291888.80849645/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.