J Rhinol.  2024 Jul;31(2):106-113. 10.18787/jr.2024.00021.

Particulate Matter Induces NLRP3 Inflammasome-Mediated Pyroptosis in Human Nasal Epithelial Cells

• Choi H
• Choi H
• Oh JM
• Lee DC

• KMID: 2558237
• DOI: http://doi.org/10.18787/jr.2024.00021

Abstract

Background and Objectives
Air pollution, particularly particulate matter (PM), has a variety of adverse effects on human health. PM is known to induce cell death through various pathways, including pyroptosis. Despite its significance, research on PM-induced pyroptosis in nasal epithelial cells remains limited. This study aimed to explore PM-induced pyroptosis in cultured human nasal epithelial cells.
Methods
For the in vitro experiments, human nasal epithelial cells were cultured. Cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while cell death was evaluated through propidium iodide (PI) staining and lactate dehydrogenase (LDH) release measurement. Protein expression levels related to pyroptosis were examined via western blot using antibodies against NOD-like receptor family, pyrin domain containing 3 (NLRP3), cleaved caspase-1 (CASP1 P20), gasdermin D (GSDMD)-N, and glyceraldehyde phosphate dehydrogenase. Immunofluorescent staining with a CASP1 P20 antibody was conducted to visualize cellular localization. Enzyme-linked immunosorbent assay was utilized to quantify interleukin (IL)-1β and IL-18 protein levels.
Results
Treatment with PM resulted in decreased cell viability, elevated LDH release, and intensified PI staining, indicating cell death. Pyroptosis was confirmed by the elevated expression of NLRP3, CASP1 P20, and GSDMD-N, along with increased levels of IL-1β and IL-18. Inhibiting the NLRP3 inflammasome with MCC950 reduced the PM-induced effects on protein expression and cytokine release, highlighting the role of the NLRP3 inflammasome in PM-triggered pyroptosis in human nasal epithelial cells.
Conclusion
We showed that PM triggers pyroptosis in human nasal epithelial cells, driven by NLRP3 inflammasome-dependent signaling pathways.

Keyword

Particulate matter; Pyroptosis; Inflammasomes; Nasal mucosa

Figure

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  Fig. 1. PM induced cell death. A: Cell viability was assessed using the MTT assay after treating the cells with different concentrations of PM (0, 25, 50, or 100 μg/mL) for 24 hours. The MTT assay showed a significant decrease in cell viability with increasing concentrations of PM exposure. B: Cell death was evaluated using the LDH assay after exposing the cells to PM at varying concentrations for 24 hours. The LDH assay indicated a notable induction of cell death in response to PM exposure. C: Cell death was further confirmed using PI staining. After 24 hours of exposure to PM, the cells were stained with PI to visualize dead cells. The PI staining results confirmed that PM exposure induced cell death in human nasal epithelial cells. The experiment was performed three times. For all experiments, data are presented as mean±SEM of the results from three individual experiments. *p<0.05 compared to the untreated control; Scale bar= 50 μm. PM, particulate matter; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; LDH, lactate dehydrogenase; PI, propidium iodide.

• 

  Fig. 2. PM exposure induced pyroptosis in human nasal epithelial cells. The cells were treated with varying concentrations of PM (0, 25, 50, or 100 μg/mL) for 24 hours. A: Western blot analysis assessed the expression levels of key proteins involved in NLRP3 inflammasome activation, including NLRP3, cleaved caspase-1, and GSDMD-N. B-D: The western blot signals were quantified, and the relative intensity of protein levels compared to the GAPDH control was determined. At PM concentrations of 50 μg/mL or higher, the levels of NLRP3, cleaved caspase-1, and GSDMD-N significantly increased compared to the control. E: Immunofluorescence staining was used to assess the expression levels of cleaved caspase-1, a key protein in NLRP3 inflammasome activation. Nuclei were counterstained with DAPI. The expression levels of cleaved caspase-1 increased proportionally with the PM concentration. F and G: ELISA was conducted to measure the release of IL-1β and IL-18, two pro-inflammatory cytokines, in the cell culture supernatant. The release of IL-1β and IL-18 increased compared to the control following PM application. For all experiments, data are presented as mean±SEM from three individual experiments. *p<0.05 compared to the untreated control; Scale bar=50 μm. PM, particulate matter; NLRP3, NOD-like receptor family, pyrin domain containing 3; CASP1 P20, cleaved caspase-1; GSDMD, gasdermin D; GAPDH, glyceraldehyde phosphate dehydrogenase; DAPI, 4',6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; IL, interleukin.

• 

  Fig. 3. PM induced pyroptosis in human nasal epithelial cells in an NLRP3 inflammasome-dependent manner. Cells were pretreated with MCC950 at a concentration of 1 μM for 2 hours before the addition of PM at a concentration of 50 μg/mL. Following PM exposure, the cells were incubated for an additional 24 hours. A: Western blot analysis was conducted to assess the expression levels of NLRP3, cleaved caspase- 1, and GSDMD-N. B-D: The western blot signals were quantified, showing that MCC950 pretreatment resulted in reduced expression levels of NLRP3, cleaved caspase-1, and GSDMD-N compared to treatment with PM alone. E: Immunofluorescence staining was used to evaluate the expression levels of cleaved caspase-1, with nuclei counterstained using DAPI. The expression levels of cleaved caspase-1 were lower following MCC950 pretreatment than with PM treatment alone. F and G: ELISA was utilized to measure the release of IL-1β and IL-18. MCC950 pretreatment also led to a reduction in the release of IL-1β and IL-18 compared to PM treatment alone. For all experiments, data are presented as mean ± SEM from three individual experiments. *p<0.05 compared to the untreated control and †p<0.05 compared to the PM alone-treated group; scale bar=50 μm. PM, particulate matter; NLRP3, NOD-like receptor family, pyrin domain containing 3; CASP1 P20, cleaved caspase-1; GSDMD, gasdermin D; GAPDH, glyceraldehyde phosphate dehydrogenase; DAPI, 4',6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; IL, interleukin.

Reference

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