Acid exposure induces pyroptosis in esophageal epithelial cells

Gastroesophageal reflux disease (GERD) is a common digestive disorder featuring epithelial hyperplasia and may lead to esophageal malignancy. Acid reflux associated with GERD has been blamed for this consequence. Thus, acid suppression has been a primary clinical strategy for treating GERD. Is acid the real cause of esophageal hyperplasia?

This study dissected the role of acid in esophageal cell proliferation versus cell death by in vitro experimentation using esophageal epithelial cell lines. Cells were exposed to pH 4.5 medium either acutely or chronically, and then assessed for cell viability, expression and localization of proliferating cell nuclear antigen (PCNA), p21, caspase 1/3/4/7, Gasdermin-D (GSDMD), interleukin-1β/18 (IL-1β/18), and mixed lineage kinase domain-like (MLKL) by Western blotting, immunoprecipitation, enzyme-linked immunosorbent assay, and immunofluorescence.

Although PCNA was upregulated in both nuclei and cytoplasm of esophageal epithelial cells, acid alone mainly induced cytoplasmic PCNA accumulation. Moreover, acid was found to induce pyroptotic cell death, rather than cell proliferation or other types of cell death such as apoptosis and necroptosis, characterized by CASP1/GSDMD cleavage rather than CASP3/7 cleavage, IL-1β/18 activation and release, and GSDMD rather than MLKL membrane localization. Inhibition of pyroptosis by either blocking CASP1 activation with Z-YVAD-FMK or knockdown of GSDMD reversed the antiproliferative effect of acid, rescued cells from death by 22.2% and 51.5% after acute and chronic acid exposure, respectively, while inhibition of apoptosis with Z-IETD-FMK or Z-LEHD-FMK or necroptosis with Necrosulfonamide did not.

Altogether, this study demonstrated that acid exposure induces pyroptotic cell death rather than cell proliferation in esophageal epithelial cells, suggesting that the cause of esophageal epithelial hyperplasia in GERD may be more complex than just acid exposure.