Upregulated expressions of caspase-4 and -5 upon challenging with periodontal microorganisms in human gingival...

Gram-negative periodontal microorganisms may be recognized by caspase-4/-5, lipopolysaccharide sensors, in human gingival epithelial cells (HGECs). This study aimed to determine expressions of inflammasomal biomolecules and to elucidate their signaling in epithelial cells upon challenging with cell wall extract of Porphyromonas gingivalis, Prevotella intermedia, or Fusobacterium nucleatum.

HGECs were stimulated with 1 or 5 μg/ml of each extract for 24 h. Expressions of caspase-1, caspase-4/-5, and gasdermin D were assayed by RT-qPCR and immunoblotting. Interleukin (IL)-1β and IL-18 levels in conditioned medium were measured by ELISA. Apoptosis was analyzed by immunoblotting for caspase-3, BCL-2, and BAX expressions and by flow cytometry for annexin V and propidium iodide staining. Upon stimulation, phosphorylation of receptor-interacting serine/threonine-protein 2 (RIP2) kinase and localization of the p50 and p65 subunits of NF-kappaB were determined.

Significantly upregulated expressions of caspase-4/-5 proteins, but not their gene, upon challenging with each extract were found (p < 0.05). However, gene and protein expressions of caspase-1 or gasdermin D were not significantly altered in the extract-stimulated HGECs, consistent with unchanged levels of IL-1β or IL-18, unaltered expressions of caspase-3, BCL-2, BAX, and non-differences in the percentages of apoptosis. Enhancement of phosphorylated RIP2 kinase and nuclear expression of the p50 and p65 subunits were instead observed in HGECs stimulated with each extract.

The cell wall extract of Porphyromonas gingivalis, Prevotella intermedia, or Fusobacterium nucleatum upregulated expressions of caspase-4/-5 proteins in HGECs and activated RIP2 kinase that led to induced expression of NF-kappaB, without any sign of pyroptotic cell death.