Molecular mechanism of interleukin-17A regulating airway epithelial cell ferroptosis based on allergic asthma airway inflammation

Abstract

Interleukin-17A (IL-17A) levels are elevated in patients with asthma. Ferroptosis has been identified as the non-apoptotic cell death type associated with asthma. Data regarding the relation of ferroptosis with asthma and the effect of IL-17A on modulating ferroptosis in asthma remain largely unclear. The present work focused on investigating the role of IL-17A in allergic asthma-related ferroptosis and its associated molecular mechanisms using public datasets, clinical samples, human bronchial epithelial cells, and an allergic asthma mouse model. We found that IL-17A was significantly upregulated within serum in asthma cases. Adding IL-17A significantly increased ferroptosis within human bronchial epithelial cells (BEAS-2B). In ovalbumin (OVA)-induced allergic asthmatic mice, IL-17A regulated and activated lipid peroxidation induced ferroptosis, whereas IL-17A knockdown effectively inhibited ferroptosis in vivo by protection of airway epithelial cells via the xCT-GSH-GPX4 antioxidant system and reduced airway inflammation. Mouse mRNA sequencing results indicated that the tumor necrosis factor (TNF) pathway was the differential KEGG pathway in the OVA group compared to healthy controls and the OVA group compared to the IL-17A knockout OVA group. We further used N-acetylcysteine (TNF inhibitor) to inhibit the TNF signaling pathway, which was found to protect BEAS-2B cells from IL-17A induced lipid peroxidation and ferroptosis damage. Our findings reveal a novel mechanism for the suppression of ferroptosis in airway epithelial cells, which may represent a new strategy for the use of IL-17A inhibitors against allergic asthma.

Keywords: Airway inflammation; Asthma; Bronchial epithelial cells; Ferroptosis; Interleukin-17A.

Fig. 1

Ferroptosis and Th17 cell differentiation are related with asthma. (A) KEGG analysis of patients with asthma compared to healthy controls using the GDS4896 dataset. (B) Relative expressions of differential ferroptosis-related genes of patients with asthma compared to healthy controls using the GDS4896 dataset. (C) PPI networks analysis of differential ferroptosis-related genes in patients with asthma compared to healthy controls using the GDS4896 dataset. (D) Differentially expressed genes associated with ferroptosis obtained in GSE4896 were intersected with differential genes taken from GSE198683. (E) IL-17A mRNA levels were increased in SA compared with the healthy controls in the GDS4896 dataset. (F) IL-5, IL-13, and IL-17A levels in serum samples from patients with asthma and healthy children. (G) Fe²⁺, Fe³⁺, Fe²⁺+Fe³⁺, and Fe²⁺/Fe³⁺ levels in serum samples from patients with acute asthma attack and healthy children. All experiments were repeated at least three times.

Fig. 2

IL-17A disrupts iron homeostasis and induces ferroptosis in BEAS-2B cells. (A) CCK8 proliferation curve of BEAS-2B cells after addition of different concentrations of erastin. (B) IL-17A mRNA level increased with addition of erastin. (C) The MDA levels increased with increased concentrations of IL-17A. (D) Electron microscopy images of BEAS-2B cell after adding IL-17A. Magnification: 20000 × , bar = 500 μm. (E, F) The Fe²⁺ (red fluorescence) and LPO (green fluorescence) levels were photographed via confocal microscopy and arbitrary units were counted. (G) The proportion of cellular ROS increased with the addition of IL-17A and erastin. (H) A similar effect was observed for SLC7A11 and GPX4 mRNA levels of erastin and IL-17A. (I) A similar effect was observed for protein xCT and GPX4 of erastin and IL-17A. All experiments were repeated at least three times. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

IL-17A knockout suppresses asthma-induced lung inflammation. (A) Sketch map showing the construction of the OVA-mediated asthma model used in this study. (B) HE staining (magnification: 200 × , bar = 100 μm) to observe infiltration of perivascular and peribronchial inflammatory cells and PAS staining (magnification: 400 × , bar = 50 μm) to observe mucus production in the epithelial layer in Sham, OVA, IL-17A−/− sham, and IL-17A−/− OVA groups. (D) The mouse serum in the sham and OVA groups. (E) The mouse serum IgE, IL-4, IL-5, IL-13, and IL-17A levels in the four groups. All experiments were repeated at least three times.

Fig. 4

Knockdown of IL-17A regulated iron homeostasis and decreased ferroptosis in the OVA asthma mice model. (A) Electron microscopy images of sham, OVA, IL-17A−/− sham, and IL-17A−/− OVA groups to observe the size and morphology of mitochondria. Magnification: 20,000 × , bar = 500 μm. (B) Perls staining shows Fe³⁺ in lung tissue. (C) Fe²⁺, Fe³⁺, Fe²⁺+Fe³⁺, and Fe²⁺/Fe³⁺ levels in mouse lung tissue. (D) MDA level in lung tissue in the four groups.(E) ROS level in lung tissue by flow cytometry. (F) PTGS2, FACL4, and FHC mRNA levels by qRT-PCR. All experiments were repeated at least three times.

Fig. 5

Inhibition of IL-17A protects airway epithelial cells from ferroptosis via the xCT-GSH-GPX4 antioxidant system (A) The GSSG/GSH ratio in four groups. (B) SLC7A11 and GPX4 mRNA levels by qRT-PCR. (C) The xCT- GSH-GPX4 antioxidant system-related protein levels. All experiments were repeated at least three times.

Fig. 6

IL-17A disrupted iron homeostasis and induced ferroptosis in BEAS-2B cells. (A–C) The Fe²⁺ (red fluorescence) and LPO (green fluorescence) levels were photographed via confocal microscopy and arbitrary units were counted. (D) The proportion of cellular ROS increased with the addition of IL-17A and erastin and decreased with the addition of IL-17A inhibitor and NAC. (F) The MDA levels increased with the addition of IL-17A and erastin and decreased with the addition of IL-17A inhibitor and ferroptosis inhibitor NAC. (G) The Western blot result showed that the TNF signaling pathway was effected by IL-17A inhibitor and ferroptosis inhibitor NAC. All experiments were repeated at least three times. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Schematic diagram showing the effect of IL-17A on regulating ferroptosis during asthma.

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