J Arthropod-Borne Dis, June 2019, 13(2): 177–184 SY Liu et al.: Effects of Diatomaceous … 177 http://jad.tums.ac.ir Published Online: June 24, 2019 Original Article Effects of Diatomaceous Earth on House Dust Mite Sensitization in BALB/c Mice Sung-Yuan Liu1; Yu-Hui Chang2; Hui-Ru Ji3; *Cheng-Di Chiu3,4,5 1Renai Biotechnology Limited, Taichung, Taiwan 2Green Polymer Corporation, Taichung, Taiwan 3Graduate Institute of Biomedical Science, China Medical University, Taichung, Taiwan 4School of Medicine, China Medical University, Taichung, Taiwan 5Neurosurgical Department, China Medical University Hospital, Taichung, Taiwan (Received 2 Nov 2017; accepted 28 Apr 2019) Abstract Background: House Dust Mite (HDM) is associated with hypersensitivity such as asthma. Patients with asthma benefit from improved living environment by reducing HDM exposure. In this study, we examined the effects of commercialized diatomaceous earth product, Casaggia® used as construction materials, on hypersensitivity in HDM-sensitized mice. Methods: Male mice were sensitized with house dust mite extract for 7d and then housed in diatomaceous earth (DE)-coated cages for 14 days at Animal Center of the Taichung Veterans General Hospital, Taichung, Taiwan in 2014. Levels of cytokine were determined using ELISA. White blood cell counts were recorded over 21d. Histological anal- ysis was conducted to determine the remodeling of respiratory tract. Results: Exposure to DE resulted in a suppression in elevated eosinophilia induced by HDM in mice. In addition, elevated serum IgE responding to HDM sensitization were restored in the presence of DE. DE ameliorated the in- flammation progression in airway. Conclusion: Environmental exposure to DE is suggested to benefit patients with hypersensitivity through relieving inflammatory symptoms. In a sense of prevention, DE represents a potential material against development of asthma. Keywords: Asthma; Diatomaceous earth; House dust mite; Allergy; Environmental exposure Introduction Asthma known as respiratory allergic dis- ease is characterized by excessive inflamma- tory response, structural remodeling, and ob- struction in the airway. The prevalence of asth- ma has increased worldwide in past few dec- ades, ranging from 1–18% (1, 2). Development of asthma is attributed to repeated exposure to aeroallergens including pollens and House Dust Mite (HDM) (3, 4). HDM is known as the most common cause of sensitization in asth- matic patients that approximately 85% of asth- matics are HDM allergic (5). HDM are found in dust and products with woven material or stuffing which provide suitable habitat for HDM. In addition, moisture that plays a vital role in natural habitation of HDM has been shown to contribute to development of aller- gic asthma. Despite the relevance of climatic factors including humidity to increase in asth- ma, the effects of manipulating climatic fac- tor on HDM sensitization are still unclear. Diatomaceous earth (DE) consists of pre- dominately fossilized remains of diatoms which is a type of hard-shelled algae. It is common- ly used for in a broad spectrum of purposes based on its chemical and structural proper- ties such as filtration, abrasive and construc- tion. DE has highly porous surface that makes it an ideal material for humidity control. Use of DE has been demonstrated to effectively reduce the growth of insects (6-8). In addi- tion, as organic in origin, DE has been used *Corresponding author: Dr Cheng-Di Chiu, E- mail: cdchiu4046@gmail.com mailto:cdchiu4046@gmail.com J Arthropod-Borne Dis, June 2019, 13(2): 177–184 SY Liu et al.: Effects of Diatomaceous … 178 http://jad.tums.ac.ir Published Online: June 24, 2019 to control internal parasites in livestock (9, 10). However, the effects of DE on moisture control associated with human disorder such as asthma is sketchy. In the present study, we hypothesized that use of DE ameliorates HDM induced allergic responses. We examined physiological effects of DE at HDM-sensitized hypersensitivity in animal model. The changes of subtypes of blood cells and serum IgE levels after HDM challenge and pathological changes of lung tissue were analyzed. Materials and Methods Mice To dispel the possibility that gender may influence allergic hypersensitivity, we chose male BALB/c mice aged 6 to 8-wk and weigh- ing 20–25g as animal subjects to induce HDM sensitization. Mice were purchased from the BioLASCO Taiwan Co., Ltd and housed in environmentally controlled conditions (22 °C, a 12h light/dark cycle) with ad libitum access to standard laboratory chow and water. Prior to experiments, mice were habituated to new environment for one week. All experiments were performed at animal center of the Tai- chung Veterans General Hospital, Taichung, Taiwan in 2014. The study protocol was reviewed and ap- proved by the Research Ethics Committee of the Taichung Veterans General Hospital and all animal experiments were performed accord- ing to the institutional and state guidelines on the care and use of animals for experimental purposes. Overall, 66 rats were used in the present study and allocated into 3 groups: mice with- out treatment (sham group), mice housed in a normal cage undergo HDM sensitization pro- cess (HDM group) and mice housed in a DE- coated cage undergo HDM sensitization pro- cess (HDM+DE group). Among them, 48 mice were assigned for histological examination and 18 mice were used for blood sampling at 4 different time points. Preparation of house dust mite crude extract HDME was prepared by dissolving 1mg lyophilized HDM (Allergon AB, Angelholm, Sweden) in 1.0mL normal saline. Resulting solution was mixed with rotation for 90min and then centrifuged at 13000rpm for 15min. The supernatant was used as a crude extract of HDM (HDME). The protein concentrations of HDM were measured using a commercial- ly available BCA protein assay kit (Pierce, USA) and adjusted to 100μg protein/mL for further use. Sensitization and aerosol challenge proce- dure The mouse sensitization and aerosol chal- lenge algorithm were performed (11). In brief, for mouse sensitization, 100μg/ml HDME was absorbed to 1mg/ml aluminum hydroxide [Al (OH)3] (Merck). The animals were adminis- tered intraperitoneally with 100μL HDM (100 μg/mL) and Al(OH)3 (1mg/mL) (n=6, the HDM group, and n=6, the DE group). After 7d post-administration, mice in the control, HDM+DE group and HDM group were housed separately in plastic cages of which plastic cag- es for DE group were coated with commercial- ized DE product (Casaggia®, GREEN POLY- MER CORPORATION, Taichung, Taiwan). Animals were challenged by repeated expo- sures for 30min each day to an aerosol of HDM (100μg/mL) delivered at 0.5mL/min by a SUMO V-16 nebulizer (Japan) for 3d post- separation. White blood cell counts in mouse serum Blood samples were obtained via the ret- ro-orbital venous plexus on Day 1, 5, 7, and 14 post-exposure to HDM aerosol. Complete blood count was conducted using KX-21-He- matology Analyzer. ELISA Total serum IgE was measured using a J Arthropod-Borne Dis, June 2019, 13(2): 177–184 SY Liu et al.: Effects of Diatomaceous … 179 http://jad.tums.ac.ir Published Online: June 24, 2019 mouse IgE ELISA kit (BD Biosciences, USA) according to the instruction provided by the manufacturer. The total IgE concentration in each serum (ng/mL) sample was estimated using a standard sample from the manufac- turer’s kit. Histological examination The mice (n=4 in each group) were sacri- ficed on Day 1, 5, 7 and 14 post-exposure to HDM aerosol. Infusion was performed via the trachea with 4% paraformaldehyde. Lungs were excised and immersed in fresh fixative over- night. Lung tissue sections with a thickness of 2μm were obtained and stained with he- matoxylin and eosin (H an E). Pulmonary le- sions were categorized according to Shackel- ford et al. into five severity grades as follow- ing, 1=minimal (<1%), 2=slight (1–25%, +), 3= moderate (26–50%, ++), 4=moderate/severe (51–75%, +++), 5=severe/high (76–100%, ++++). Statistical analysis Statistical comparisons were performed by analysis of variance (ANOVA) and turkey’s HSD post hoc tests. We used nonparametric statistics (Wilcoxon rank sum test) to test da- ta differences between two groups if the data distribution is not corresponding Gaussian dis- tribution (Kolmogorov-Smirnov test is signif- icant). All values are shown as mean±standard deviation (SD). P< 0.05 was regarded as sig- nificant. All statistical analyses were per- formed using SPSS software (ver. 18.0, IBM Corp., Armonk, NY, USA). Results Effects of DE on differential WBC counts in HDM-sensitized mice To investigate whether DE abrogates HDM-induced eosinophils, peripheral total and differential WBC counts in HDM-sensitized mice were conducted. The peripheral total WBCs at 4-time points in 3 groups of mice with or without exposure to DE were com- parable. A significant difference in total WBC counts was observed between HDM and DE groups on Day 5 post-exposure to HDM aer- osol (P< 0.05) (Fig. 1). In addition, there were no significant differences in cell counts and morphological changes among neutrophil, mon- ocyte, and lymphocyte (Fig. 2). Our data showed that eosinophil count in HDM+DE groups reached a peak on Day 7 post-sensitization, whereas both HDM and HDM+DE groups ex- hibited a peak on Day 5. The differential WBC counts revealed that HDM-sensitized mice exhibited a significantly higher number of eo- sinophils in the peripheral blood compared with those of HDM+DE groups at Day1 and Day 5 (P< 0.05) (Fig. 3). Effects of DE on IgE levels in HDM-sensi- tized mice We next examined the effect of DE on HDM-induced increase in serum IgE levels. Exposure of mice to HDM led to markedly high serum IgE levels compared with those of the controls at 4 designated time points (P< 0.001) (Fig. 4). A significant difference in levels of serum IgE between HDM+DE and HDM groups on Day 1, 5 and 7 (P< 0.001). Interestingly, the levels of IgE in HDM+DE mice increased in a time-dependent manner. DE reduced HDM-induced lung inflam- mation To investigate the mechanism by which DE was associated with decreased eosino- philia, we histologically assessed lung tis- sues of mice sensitized by HDM. HDM sen- sitization led to a slight inflammatory cell in- filtration, mainly neutrophils and lymphocytes, fairly eosinophils and slight mucilage in the bronchial goblet cells, along with minimal mac- rophage infiltration in the alveolar space (Fig. 5). In HDM+DE mice, HDM-induced inflam- matory cell infiltration and pathological ap- pearance were observed on Day 7 post-sensi- J Arthropod-Borne Dis, June 2019, 13(2): 177–184 SY Liu et al.: Effects of Diatomaceous … 180 http://jad.tums.ac.ir Published Online: June 24, 2019 tization with minor degree of severity com- pared with that of HDM mice (Table 1). Un- expectedly, the inflammatory response in lung tissue was absent in HDM-treated mice on Day 14 post sensitization. Table 1. Summary of pathological incidence of lung after mite-induced mice Pathology/Degree1 Day/Groups (number, mice) Day5 Day7 Day14 C H+D H C H+D H C H+D H Inflammatory cell, perivascular and peribroncheal, focal (minimal to slight) - - 2 - 2 2 - - - Infiltration, macrophage, alveolar, focal (minimal) - - 1 - 1 1 - - - Mucification, goblet cell, bronchial, foal (slight to moderate) - - 2 - 2 3 - - - Degree of lesions was graded from one to five depending on severity: 1= minimal (< 1%), 2= slight (1– 25%, +), 3= moderate (26–50%, ++) Fig. 1. The effects of DE on peripheral total WBC counts. Blood samples were obtained from the HDM-sensitized mice on Day 1, 5, 7 and 14 post-sensitization (N=6). Data are presented as the mean ± SD. *P< 0.05 Fig. 2. Subtypes of WBCs found in HDM-induced mice (1000x). It denotes (A) eosinophil, 0–4%, responsive for allergy, asthma, parasite; (B) monocyte, 3–8%, (C) neutrophil, 10–40%, (D) lymphocyte, 55–90%. J Arthropod-Borne Dis, June 2019, 13(2): 177–184 SY Liu et al.: Effects of Diatomaceous … 181 http://jad.tums.ac.ir Published Online: June 24, 2019 Fig. 3. The effects of DE on peripheral eosinophil counts. Blood samples were obtained from the HDM-sensitized mice on Day 1, 5, 7 and 14 post-sensitization (N=6). Data are presented as the mean ± SD. *P< 0.05 Fig. 4. Blood IgE levels in mice after HDM sensitization. Blood samples were obtained from the HDM-sensitized mice on Day 1, 5, 7 and 14 post-sensitization. (ng/mL, n=6) Data are presented as the mean ± SD. ***P< 0.001 Fig. 5. Pathological examination of inflammation in lung tissue among mice group of NS, HMD group and HMD+DE (H and E stain, 400x) J Arthropod-Borne Dis, June 2019, 13(2): 177–184 SY Liu et al.: Effects of Diatomaceous … 182 http://jad.tums.ac.ir Published Online: June 24, 2019 Discussion We demonstrated that environmental ex- posure to DE leads to a reduction in degrees of HDM-induced hypersensitivity in vivo. Re- duced eosinophilia was associated with the presence of DE. Moreover, the HDM-induced hypersensitive responses were ameliorated in the mice exposed to DE. Asthma is a preva- lent chronic respiratory disorder in children and adolescents populations. HDM allergens are considered as important indoor allergens for humans inducing allergic diseases. Pres- ence of HDM is suggested to be a risk factor for development of allergic asthma. HDM sensitization has been shown to contribute to the pathogenesis, progression, and severity of asthma. Use of HDM as model allergen for sen- sitization in animal models has been shown to be more ideal and logical than the other mod- els such as ovalbumin (12). HDM-sensitized animal models display many features of al- lergic asthma including respiratory inflamma- tion, airway remodeling and increased eosin- ophilia (13). We employed a HDM sensitiza- tion to establish an allergic airway inflamma- tion model that exhibited typical features of allergic inflammation. HDM-associated increas- es in systemic eosinophilia were restored in presence of DE. Systemic and local eosinophilic inflamma- tion has been associated with severe asthma and poorer asthma control (14). Eosinophilia in blood is a good marker for asthma (15-17) and been used as a measurement of effects of anti-hypersensitive modalities. Airway eosino- philia is considered as a vital characteristic of early-onset allergic asthma, as well as the oc- currence of late-onset non-allergic asthma (16). Our finding showed that DE reduced elevated systemic eosinophilia suggests that use of DE has the potential to improve the symptoms of allergic asthma. Unexpectedly, bronchial eo- sinophilia remained unaffected in the experi- mental setting. The finding is supported by the results of previous research, HDM-induced airway hypersensitiveness is not related to ei- ther eosinophil influx or allergen-specific se- rum IgE. In addition, a retrospective study has reported the absence of airway eosinophilia in patients with allergic asthma (18). Howev- er, further studies are required to determine the differences in the phenomenon of eosin- ophil counts in HDM-sensitization modal. Increasing evidence has highlighted the po- tential use of DE for insect control (19-21). Re- cent studies have demonstrated the insecticidal effect of DE against different species of mites in animal (10, 22). The insecticidal effects of DE have been postulated to be attributed to rapid and lethal dehydration of insect. We ex- plored the use of Casaggia®, a new DE formu- lation, for improving the hypersensitive re- sponse induced by HDM. Animals of HDM+ DE group exhibited relatively lower eosino- phil counts and levels of total IgE in blood compared with those of mice sensitized with HDM, suggesting that asthmatics can benefit from avoidance of environmental risk factor using DE. However, in this study, animals were sensitized with HDM extract and housed in DE-coated cages without direct exposure to dust mites. Moreover, increased eosinophilia in HDM-sensitized rats declined slightly over time. A possible explanation for the anti-hy- persensitive effect in presence of DE is that use of DE leads to a moisture loss of surround- ing environment, which inhibits the growth of microorganisms acting as challenge to HDM- sensitized animals. Nevertheless, further stud- ies are necessary to elucidate the mechanism underlying the effect of DE on hypersensitiv- ity. Long-term occupational exposure of work- ers in the crystalline silica was found to have an increased risk of silicosis (23). Despite cur- rent commercial, DE product contains most- ly amorphous silica but no crystalline silica, there still are doubts for long term usage. Fur- ther investigation for the effect of commer- cial DE products to human lung is necessary. J Arthropod-Borne Dis, June 2019, 13(2): 177–184 SY Liu et al.: Effects of Diatomaceous … 183 http://jad.tums.ac.ir Published Online: June 24, 2019 Conclusion Exposure to DE ameliorates elevated eo- sinophilia and total IgE in animals sensitized with HDM. DE represents a potential ap- proach to prevent occurrence of hypersensi- tivity and to improve the symptoms of aller- gic asthma. Acknowledgements The authors declare that there is no con- flict of interests. References 1. Yangzong Y, Shi Z, Nafstad P, Haheim LL, Luobu O, Bjertness E (2012) The prevalence of childhood asthma in Chi- na: a systematic review. BMC Public Health. 12: 860. 2. 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