Mechanism underlying human airway epithelial cell inflammation triggered by dust mite allergen Der f 1

doi:10.12140/j.issn.1000-7423.2025.06.012

Abstract

Abstract:

Objective To decipher the mechanisms underlying human airway epithelial cell inflammation induced by the major allergen Der f 1 of dust mite. Methdos BEAS-2B cells were seeded onto 96-well plates at a density of 5 × 10³ cells per well and divided into three groups, including the Der f 1 group, Der f 1 + ferrostatin-1 (Fer-1) group, and negative control group. Cells in the Der f 1 group were treated with 40 μl of Der f 1 (100 μg/ml), and cells in the Der f 1 + Fer-1 group was pretreated with 4 μl Fer-1 (1 000 μmol/ml) 2 hours prior to Der f 1 treatment, while cells in the negative control group were given an equivalent volume of nuclease-free water. After 24 hours of incubation, cell viability was assessed in the Der f 1 group, Der f 1 + Fer-1 group and negative control group using the CCK-8 assay, and concentrations of interleukin-6 (IL-6), thymic stromal lymphopoietin (TSLP), and IL-33 were measured in the cell culture supernatants with enzyme-linked immunosorbent assay (ELISA). For Western blotting assay, BEAS-2B cells were seeded onto 6-well plates at a density of 5 × 10⁵ cells per well, and assigned into three groups, including the Der f 1 group, Der f 1 + Fer-1 group, and negative control group. Total protein was extracted from BEAS-2B cells, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and probed with primary antibodies against glutathione peroxidase 4 (GPX4, 1︰2 000 dilution) and acyl-CoA synthetase long-chain family member 4 (ACSL4, 1︰2 000 dilution), followed by incubation into horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1︰10 000 dilution). The GPX4 and ACSL4 protein expression was determined in cells using Western blotting assay. To assess ferroptosis-related indicators, BEAS-2B cells were seeded onto 24-well plates at a density of 5 × 10⁴ cells per well and assigned into three groups, including the Der f 1 group, Der f 1 + Fer-1 group, and negative control group. Intracellular fluorescence intensity of Fe²⁺ and reactive oxygen species (ROS) were measured under a fluorescence microscope, and the ratio of red to green fluorescence intensity was estimated following lipid peroxidation (LPO) staining. For colorimetric assay, BEAS-2B cells were seeded onto 6-well plates at a density of 5 × 10⁵ cells per well and assigned into three groups, including the Der f 1 group, Der f 1 + Fer-1 group, and negative control group, and malondialdehyde (MDA) content was measured using a colorimetric assay. In addition, BEAS-2B cells were seeded onto 6-well plates at a density of 5 × 10⁶ cells per well and assigned into three groups, including the Der f 1 group, Der f 1 + Fer-1 group, and negative control group, and mitochondrial morphology was examined under a transmission electron microscope. All statistical analyses were performed using the software GraphPad Prism 8.0.1, and differences of means among three groups were tested for statistical significance with one-way analysis of variance (ANOVA). Results The cell viability was significantly lower in the Der f 1 group (0.79 ± 0.03) than in the negative control (1.07 ± 0.08) (t = 6.663, P < 0.01), and co-treatment with Fer-1 restored cell viability to 0.94 ± 0.03 (t = 6.694, P < 0.01). ELISA showed that the IL-6 concentration in the supernatant of the Der f 1 group was (117.30 ± 21.32) pg/ml, which was higher than that of the negative control group (50.07 ± 5.82) pg/ml (t = 5.279, P < 0.01). The IL-6 concentration in the supernatant of the Der f 1 + Fer-1 group was (50.31 ± 12.28) pg/ml, which was lower than that of the Der f 1 group (t = 4.721, P < 0.01). The TSLP concentration in the supernatant of the Der f 1 group was (10.00 ± 2.37) pg/ml, which was higher than that of the negative control group (3.81 ± 0.92) pg/ml (t = 4.223, P < 0.05). The TSLP concentration in the supernatant of the Der f 1 + Fer-1 group was (4.41 ± 1.59) pg/ml, which was lower than that of the Der f 1 group (t = 3.399, P < 0.05). The IL-33 concentration in the supernatant of the Der f 1 group was (24.18 ± 2.53) pg/ml, which was higher than that of the negative control group (12.09 ± 2.08) pg/ml (t = 6.39, P < 0.01). The IL-33 concentration in the supernatant of the Der f 1 + Fer-1 group was (15.76 ± 1.39) pg/ml, which was lower than that of the Der f 1 group (t = 5.045, P < 0.01). Western blotting determined lower relative GPX4 protein expression in the Der f 1 group (0.38 ± 0.08) than in the negative control group (1.00 ± 0.00) (t = 13.21, P < 0.01), and higher GPX4 protein expression in the Der f 1 + Fer-1 group (0.72 ± 0.08) than in the Der f 1 group (t = 5.122, P < 0.01). Conversely, ACSL4 protein expression was upregulated in the Der f 1 group (1.74 ± 0.12 vs. 1.00 ± 0.00; t = 10.65, P < 0.01) and reduced by Fer-1 treatment (1.33 ± 0.13, t = 4.094, P < 0.05). Fluorescence microscopy showed increased intracellular fluorescence intensity of Fe²⁺ in the Der f 1 group (37.19 ± 5.42 vs. 11.93 ± 0.54; t = 8.035, P < 0.01), which was significantly lowered by Fer-1 treatment (13.16 ± 1.89, t = 7.253, P < 0.01). The intracellular ROS fluorescence intensity in the Der f 1 group was 13.48 ± 3.36, which was higher than that in the negative control group (6.80 ± 0.60, t = 3.386, P < 0.05). The fluorescence intensity in the Der f 1 + Fer-1 group was 7.35 ± 0.42, which was lower than that in the Der f 1 group (t = 3.134, P < 0.05). The intracellular LPO fluorescence intensity in the Der f 1 group was 3.91 ± 1.65, which was higher than that in the negative control group (0.31 ± 0.13, t = 3.775, P < 0.05). The LPO fluorescence intensity in the Der f 1 + Fer-1 group was 0.80 ± 0.15, which was lower than that in the Der f 1 group (t = 3.262, P < 0.05). The colorimetric assay results showed that the intracellular MDA content in the Der f 1 group was (5.57 ± 1.66) nmol/mg, which was higher than that in the negative control group (2.18 ± 0.51) nmol/mg (t = 3.393, P < 0.05). The MDA content in the Der f 1 + Fer-1 group was (2.24 ± 0.38) nmol/mg, which was lower than that in the Der f 1 group (t = 3.4, P < 0.05). Transmission electron microscopy displayed well mitochondrial status, clear mitochondrial boundary and many mitochondrial cristae in the negative control group, and Der f 1 exposure led to mitochondrial shrinkage, membrane thickening, and reduction or loss of cristae. These structural alterations were markedly attenuated in the Der f 1 + Fer-1 group, including weakening of mitochondrial shrinkage, membrane thickening and reduced numbers of mitochondrial cristae. Conclusion Der f 1 induces ferroptosis in human airway epithelial cells through disrupting iron homeostasis, suppressing antioxidant defense, and promoting lipid peroxidation, thereby potentiating the release of IL-6, TSLP, and IL-33 to exacerbate airway inflammation.

Key words: Dust mite, Dermatophagoides farinae allergen 1, Airway epithelial cell, Ferroptosis, Inflammation

CLC Number:

R384.4

JIANG Shangde, ZANG Xiao, MEI Wei, MA Lianzheng, RAO Lina, HONG Shanchao, WANG Wei. Mechanism underlying human airway epithelial cell inflammation triggered by dust mite allergen Der f 1[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2025, 43(6): 827-834.

Figures/Tables 4

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References 24

[1]	World Health Organization. Allergic diseases and asthma: A global public health concern[R]. Geneva: World Health Organization, 2003.
[2]	Hang YQ, Piao X, Wu J, et al. The epidemiology of asthma and its attributable risk factors from 1990 to 2021: A systematic analysis based on the global burden of disease study 2021 and mendelian randomization studies[J]. Public Health, 2025, 243: 105731.
[3]	Liu JX, Zhang Y, Yuan HY, et al. The treatment of asthma using the Chinese Materia Medica[J]. J Ethnopharmacol, 2021, 269: 113558.
[4]	Yang L, Zhu RF. Immunotherapy of house dust mite allergy[J]. Hum Vaccin Immunother, 2017, 13(10): 2390-2396.
[5]	李启松, 杨李, 腾飞翔, 等. 屋尘螨过敏原Der p 4的克隆、表达、免疫原性鉴定及生物信息学分析[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(1): 42-47.
	Li QS, Yang L, Teng FX, et al. Cloning, expression, immunogenicity identification and bioinformatics analysis of Dermatophagoides pteronyssinus allergen Der p 4[J]. Chin J Parasitol Parasit Dis, 2024, 42(1): 42-47. (in Chinese)
[6]	万极硕, 卢山, 杨凯楠. 北京市昌平地区5 068例过敏原特异性IgE抗体检测结果分析[J]. 标记免疫分析与临床, 2021, 28(3): 385-390, 394.
	Wan JS, Lu S, Yang KN. Analysis of detection results of allergen-specific IgE antibody in 5 068 cases in Changping district of Beijing[J]. Labeled Immunoass Clin Med, 2021, 28(3): 385-390, 394. (in Chinese)
[7]	Witt C, Stuckey MS, Woolcock AJ, et al. Positive allergy prick tests associated with bronchial histamine responsiveness in an unselected population[J]. J Allergy Clin Immunol, 1986, 77(5): 698-702.
[8]	Jacquet A. Innate immune responses in house dust mite allergy[J]. ISRN Allergy, 2013, 2013: 735031.
[9]	Liu S, Cai ZL, Liu JC, et al. The novel house dust mite allergen Der p 39 exacerbates atopic dermatitis-like inflammation in mice by inducing skin barrier dysfunction[J]. World Allergy Organ J, 2025, 18(3): 101036.
[10]	Kl?ditz K, Fadeel B. Three cell deaths and a funeral: Macrophage clearance of cells undergoing distinct modes of cell death[J]. Cell Death Discov, 2019, 5: 65.
[11]	Deng LY, He SS, Guo NQ, et al. Molecular mechanisms of ferroptosis and relevance to inflammation[J]. Inflamm Res, 2023, 72(2): 281-299.
[12]	He YX, Wang JY, Ying CM, et al. The interplay between ferroptosis and inflammation: Therapeutic implications for cerebral ischemia-reperfusion[J]. Front Immunol, 2024, 15: 1482386.
[13]	Zeng ZJ, Huang HH, Zhang JM, et al. HDM induce airway epithelial cell ferroptosis and promote inflammation by activating ferritinophagy in asthma[J]. FASEB J, 2022, 36(6): e22359.
[14]	Park M, Park S, Choi Y, et al. The mechanism underlying correlation of particulate matter-induced ferroptosis with inflammasome activation and iron accumulation in macrophages[J]. Cell Death Discov, 2024, 10(1): 144.
[15]	Huang HJ, Sarzsinszky E, Vrtala S. House dust mite allergy: The importance of house dust mite allergens for diagnosis and immunotherapy[J]. Mol Immunol, 2023, 158: 54-67.
[16]	Jinson S, Zhang ZY, Lancaster GI, et al. Iron, lipid peroxidation, and ferroptosis play pathogenic roles in atherosclerosis[J]. Cardiovasc Res, 2025, 121(1): 44-61.
[17]	Murdaca G, Greco M, Tonacci A, et al. IL-33/IL-31 axis in immune-mediated and allergic diseases[J]. Int J Mol Sci, 2019, 20(23): 5856.
[18]	Han MY, Rajput C, Hong JY, et al. The innate cytokines IL-25, IL-33, and TSLP cooperate in the induction of type 2 innate lymphoid cell expansion and mucous Metaplasia in rhinovirus-infected immature mice[J]. J Immunol, 2017, 199(4): 1308-1318.
[19]	Spiric J, Reuter A, Rabin RL. Mass spectrometry to complement standardization of house dust mite and other complex allergenic extracts[J]. Clin Exp Allergy, 2017, 47(5): 604-617.
[20]	王晓艳, 普晓瑜, 陈力嘉, 等. 尘螨致敏蛋白组分在变应性鼻炎和哮喘中的差异及其临床意义[J]. 临床耳鼻咽喉头颈外科杂志, 2022, 36(8): 576-581.
	Wang XY, Pu XY, Chen LJ, et al. Difference of allergenic protein components of dust mite in allergic rhinitis and asthma and its clinical significance[J]. J Clin Otorhinolaryngol Head Neck Surg, 2022, 36(8): 576-581. (in Chinese)
[21]	Hsin L, Varese N, Aui PM, et al. Accurate determination of house dust mite sensitization in asthma and allergic rhinitis through cytometric detection of Der p 1 and Der p 2 binding on basophils (CytoBas)[J]. J Allergy Clin Immunol, 2024, 153(5): 1282-1291.
[22]	Wan H, Winton HL, Soeller C, et al. Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions[J]. J Clin Invest, 1999, 104(1): 123-133.
[23]	Gong ZH, Wang YJ, Li L, et al. Cardamonin alleviates chondrocytes inflammation and cartilage degradation of osteoarthritis by inhibiting ferroptosis via p53 pathway[J]. Food Chem Toxicol, 2023, 174: 113644.
[24]	Duo H, Yang YW, Luo J, et al. Correction: Modulatory role of radioprotective 105 in mitigating oxidative stress and ferroptosis via the HO-1/SLC7A11/GPX4 axis in sepsis-mediated renal injury[J]. Cell Death Discov, 2025, 11(1): 409.