Intermittent hypoxia aggravates asthma inflammation via NLRP3/IL-1β-dependent pyroptosis mediated by HIF-1α signalling pathway

Abstract

Background: Asthma is a common chronic inflammatory airway disease and intermittent hypoxia is increasingly recognized as a factor that may impact disease progression. The present study investigated whether intermittent hypoxia (IH) could aggravate asthma by promoting hypoxia-inducible factor-1α (HIF-1α)/nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain-containing protein 3 (NLRP3)/interleukin (IL)-1β-dependent pyroptosis and the inflammatory response and further elucidated the underlying molecular mechanisms involved.

Methods: A total of 49 patients diagnosed with severe bronchial asthma and diagnosed by polysomnography were enrolled at Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, between January 2022 and December 2022, and their general data and induced sputum were collected. BEAS-2B cells were treated with IL-13 and subjected to IH. An ovalbumin (OVA)-treated mouse model was also used to assess the effects of chronic intermittent hypoxia (CIH) on asthma. Pyroptosis, the inflammatory response, and related signalling pathways were assessed in vivo and in vitro .

Results: In this study, as the apnoea and hypopnea index (AHI) increased, the proportion of patients with uncontrolled asthma increased. The proportions of neutrophils and the levels of IL-6, IL-8, HIF-1α and NLRP3 in induced sputum were related to the AHI. NLRP3-mediated pyroptosis, which could be mediated by the HIF-1α signalling pathway, was activated in IL-13 plus IH-treated BEAS-2B cells and in the lungs of OVA/CIH mice. HIF-1α downregulation significantly reduced lung pyroptosis and ameliorated neutrophil inflammation by modulating the NLRP3/IL-1β pathway both in vitro and in vivo . Similarly, pretreatment with LW6, an inhibitor of HIF-1α, effectively blocked the generation of inflammatory cytokines in neutrophils. In addition, administration of the NLRP3 activator nigericin obviously increased lung neutrophil inflammation.

Conclusions: Obstructive sleep apnoea-hypopnea syndrome (OSAHS) is a risk factor for asthma exacerbation. IH aggravates neutrophil inflammation in asthma via NLRP3/IL-1β-dependent pyroptosis mediated by the HIF-1α signalling pathway, which should be considered a potential therapeutic target for the treatment of asthma with OSAHS.

Keywords: Asthma; Hypoxia-inducible factor-1α (HIF-1α); Inflammation; Interleukin-1β; Intermittent hypoxia; NOD-like receptor pyrin domain-containing protein 3 (NLRP3).

Figure 1

Classification of induced sputum cells by Liu’s staining and patient cytokine analysis. (A) Proportion of macrophages. (B) Proportion of eosinophils. (C) Proportion of leukomonocytes. (D) Proportion of neutrophils. (E) Expression of IL-6. (F) Expression of IL-8. (G) Expression of HIF-1α. (H) Expression of NLRP3. (I) Expression of CRP. ^*P <0.05. Data are presented as points, the horizontal line within each plot represents the mean value of the data. CRP: C-reactive protein; HIF-1α: Hypoxia-inducible factor 1α; IL-6: Interleukin-6; IL-8: Interleukin-8; NLRP3: NOD-like receptor pyrin domain-containing protein 3; OSAHS: Obstructive sleep apnoea hypopnea syndrome.

Figure 2

IH activates the HIF-1α/NLRP3/GSDMD pathway to promote IL-13-induced pyroptosis and inflammation in vitro. BEAS-2B cells were treated with IL-13 (4 ng/mL) and/or IH for 24 hours when the cell density reached 70–80%. (A) Immunoblot analysis of the protein expression of HIF-1α in BEAS-2B cells. (B) Immunoblot analysis of the protein expression of NLRP3, GSDMD, IL-1β, cleaved caspase-1, and IL-18 in BEAS-2B cells. (C) Flow cytometric analysis of apoptosis in BEAS-2B cells. (D) ELISA was used to measure LDH activity. (E) ELISA was used to measure the levels of TNF-α, IL-6, MMP-9, and IL-8. (F) qPCR was used to assess the mRNA expression of NLRP3, GSDMD, IL-1β, cleaved caspase-1, and IL-18 in BEAS-2B cells. Data are presented as mean ± SEM. ^*P <0.05, ^†P <0.01, ^‡P <0.001, ^§P <0.0001. ELISA: Enzyme-linked immunosorbent assay; FITC-A: Fluorescein Isothiocyanate-A; GSDMD: Gasdermin D; H1-LL: Histogram1-lower left; H1-LR: Histogram1-lower right; H1-UL: Histogram1-upper left; H1-UR: Histogram1-upper right; HIF-1α: Hypoxia-inducible factor-1α; IH: Intermittent hypoxia; LDH: Lactate dehydrogenase; IL: Interleukin; MMP-9: Matrix metalloproteinase-9; mRNA: Messenger RNA; NLRP3: Nucleotide-binding oligomerization domain-like receptor protein 3; PE-A: Phycoerythrin-A; qPCR: Quantitative polymerase chain reaction; SEM: Standard error of the mean; TNF-α: Tumor necrosis factor-α; MW: Molecular weight.

Figure 3

HIF-1α downregulation alleviates IL-13-induced pyroptosis and inflammation under IH by inhibiting the NLRP3/GSDMD pathway in vitro. Lipofectamine 3000 and HIF-1α-siRNA or negative control (NC) siRNA were transfected when the cell density reached 70–80%. Twenty-four hours after transfection, IL-13 (4 ng/mL) and/or IH were used to treat BEAS-2B cells for 24 hours. (A–B) Immunoblot analysis of HIF-1α protein expression in BEAS-2B cells. (C) Immunoblot analysis of the protein expression of NLRP3, GSDMD, IL-1β, cleaved caspase-1, and IL-18 in BEAS-2B cells. (D–E) Flow cytometric analysis of BEAS-2B apoptosis. (F) ELISA was used to measure LDH activity. (G) ELISA was used to measure the levels of TNF-α, IL-6, MMP-9, and IL-8. Data are presented as mean ± SEM. ^*P <0.05, ^†P <0.01, ^‡P <0.001, ^§P <0.0001. ELISA: Enzyme-linked immunosorbent assay; FITC-A: Fluorescein isothiocyanate-A; GSDMD: Gasdermin D; H1-LL: Histogram1-lower left; H1-LR: Histogram1-lower right; H1-UL: Histogram1-upper left; H1-UR: Histogram1-upper right; HIF-1α: Hypoxia-inducible factor-1α; IH: Intermittent hypoxia; IL: Interleukin; LDH: Lactate dehydrogenase; MMP-9: Matrix metalloproteinase-9; NC: Negative control; NLRP3: Nucleotide-binding oligomerization domain-like receptor protein 3; PE-A: Phycoerythrin-A; SEM: Standard error of the mean; siRNA: Small interfering RNA; TNF-α: Tumor necrosis factor-α; MW: Molecular weight.

Figure 4

Activation of NLRP3 reverses IL-13 plus IH-induced pyroptosis and inflammation that are alleviated by downregulation of HIF-1α in vitro. Lipofectamine 3000 and HIF-1α-siRNA or negative control (NC) siRNA were transfected when the cell density reached 70–80%. Twenty-four hours after transfection, IL-13 (4 ng/mL) and IH and/or nigericin (10 μmol/L) were used to treat BEAS-2B cells for 24 hours. (A) Western blot analysis of the protein expression of HIF-1α, NLRP3, GSDMD, IL-1β, cleaved caspase-1, and IL-18 in BEAS-2B cells. (B) ELISA was used to measure TNF-α, IL-6, MMP-9 and IL-8 levels. Data are presented as mean ± SEM. ^*P <0.05, ^†P <0.01, ^‡P <0.001, ^§P <0.0001. ELISA: Enzyme-linked immunosorbent assay; FITC-A: Fluorescein isothiocyanate-A; GSDMD: Gasdermin D; HIF-1α: Hypoxia-inducible factor-1α; IH: Intermittent hypoxia; IL: Interleukin; MMP-9: Matrix metalloproteinase-9; NLRP3: Nucleotide-binding oligomerization domain-like receptor protein 3; SEM: Standard error of the mean; siRNA: Small interfering RNA; TNF-α: Tumor necrosis factor-α; MW: Molecular weight.

Figure 5

CIH promotes pyroptosis and inflammation by activating HIF-1α/NLRP3/GSDMD signalling in a mouse model of asthma. (A) Evaluation of lung tissue histopathology by H&E staining (original magnification×80). (B) Liu’s staining of bronchoalveolar lavage fluid (BALF) cell smears from mouse BALF showing neutrophils (blue arrows), eosinophils (yellow arrows), and macrophages (red arrows) (original magnification×40). (C) Evaluation of lung tissue histopathology by PAS staining (original magnification×80). (D) Classification of BALF cells. (E) Assessment of airway inflammatory cell scores based on lung tissue H&E staining. (F) Histopathological evaluation of the number of PAS-positive cells in lung tissue. (G) Measurement of TNF-α, IL-1β, IL-6, IL-8, and MMP-9 levels in BALF using ELISA. (H) Immunoblot analysis of HIF-1α, NLRP3, GSDMD, cleaved caspase-1, and IL-18 in lung tissue. (I) Immunofluorescence double staining to detect the expression of HIF-1α (green) and NLRP3 (red) in lung tissue (original magnification×40). (J) Immunofluorescence double staining to detect the expression of GSDMD (green) and IL-18 (red) in lung tissue (original magnification×40). Data are presented as mean ± SEM. ^*P <0.05, ^†P <0.01, ^‡P <0.001, ^§P <0.0001. CIH: Chronic intermittent hypoxia; DAPI: 4′,6-Diamidino-2-phenylindole; ELISA: Enzyme-linked immunosorbent assay; GSDMD: Gasdermin D; H&E: Hematoxylin and eosin; HIF-1α: Hypoxia-inducible factor-1α; IL: Interleukin; MMP-9: Matrix metalloproteinase-9; NLRP3: Nucleotide-binding oligomerization domain-like receptor protein 3; OVA: Ovalbumin; PAS: Periodic acid-Schiff; SEM: Standard error of the mean; TNF-α: Tumor necrosis factor-α; VEH: Vehicle; MW: Molecular weight.

Figure 6

Downregulation of HIF-1α alleviates inflammation and pyroptosis in mice induced by CIH plus OVA. (A) Assessment of pulmonary tissue histopathology by H&E staining (original magnification×40). (B) Airway inflammatory scores based on pulmonary tissue H&E staining and BALF cell classification counts. (C) ELISA measurements of TNF-α, IL-1β, IL-6, IL-8, and MMP-9 levels in BALF. (D) Immunoblot analysis of the expression of HIF-1α, NLRP3, GSDMD, and IL-18 in pulmonary tissue. (E) Immunofluorescence double staining to assess the expression of HIF-1α (green) and NLRP3 (red) in pulmonary tissue (original magnification×40). Data are presented as mean ± SEM. ^*P <0.05, ^†P <0.01, ^‡P <0.001, ^§P <0.0001. BALF: Bronchoalveolar lavage fluid; CIH: Chronic intermittent hypoxia; DAPI: 4′,6-Diamidino-2-phenylindole; ELISA: Enzyme-linked immunosorbent assay; GSDMD: Gasdermin D; H&E: Hematoxylin and eosin; HIF-1α: Hypoxia-inducible factor-1α; IL: Interleukin; MMP-9: Matrix metalloproteinase-9; NLRP3: Nucleotide-binding oligomerization domain-like receptor protein 3; OVA: Ovalbumin; SEM: Standard error of the mean; TNF-α: Tumor necrosis factor-α; VEH: Vehicle; MW: Molecular weight.

Figure 7

Intermittent hypoxia aggravates asthma inflammation via NLRP3/IL-1β-dependent pyroptosis mediated by the HIF-1α signalling pathway. ASC: Apoptosis-associated speck-like protein containing a CARD; GSDMD: Gasdermin D; HIF-1α: Hypoxia-inducible factor-1α; IH: Intermittent hypoxia; IL: Interleukin; MMP-9: Matrix metalloproteinase-9; NE: Neutrophil elastase; NLRP3: Nucleotide-binding oligomerization domain-like receptor protein 3; OSAHS: Obstructive sleep apnea hypopnea syndrome; OVA: Ovalbumin; TNF-α: Tumor necrosis factor-α.