The role of NLRP3 in regulating gingival epithelial cell responses evoked by Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) has myriads of virulence factors among which leukotoxin provides A. actinomycetemcomitans with the advantage to thrive in the surrounding hostile environ-ment and evade host immune defences. The NLRP3 inflammasome has been associated with periodontal disease development. However, our understanding of the involvement of caspase-1, caspase-4, and NLRP3 in the release of IL-113 and other inflammatory mediators from gingival epithelial cells during a A. actinomycetemcomitans infection is limited. The aim of this study was to investigate how the inflammasome-associated proteins caspase-1, caspase-4 and NLRP3 regulate the immune response of gingival epithelial cells during a A. actinomycetemcomitans infection. Human gingival epithelial cells (Ca9-22) deficient in NLRP3, caspase-1 or caspase-4 were created using CRISPR/Cas9. Gingival epithelial cells were stimulated with the A. actinomycetemcomitans low-leukotoxic strain NCTC9710 or the highly leukotoxic JP2 strain HK 165 for 6, 12 and 24 h. The results showed that the JP2 strain HK1651 induced higher IL-113 and IL-1RA release and mediated more epithelial cell death compared to the NCTC9710 strain. These findings were found to be capsase-1, caspase-4 and NLRP3-dependant. A targeted protein analysis of inflammation-related proteins showed that the expression of 37 proteins were identified as being significantly altered after HK1651 infection compared to unstimulated Cas9 and NLRP3-deficient cells. Of the 37 proteins, 23 of these inflammation-related proteins released by NLRP3- deficient cells differed significantly compared to Cas9 cells after infection. This suggests that NLRP3 has a broad effect on the inflammatory response in gingival epithelial cells.

Automated summary

This study investigated the role of leukotoxin, caspase-1, caspase-4, and NLRP3 in the immune response of gingival epithelial cells during Aggregatibacter actinomycetemcomitans infection. The results showed that the highly leukotoxic strain induced higher release of inflammatory mediators and cell death, which were dependent on caspase-1, caspase-4, and NLRP3. Furthermore, the absence of NLRP3 resulted in significantly different release of inflammatory proteins in infected cells compared to normal cells.