PAR silencing affects bacterial stimulation of NOD in gingival epithelia

Protease-activated receptors (PARs) are seven-transmembrane domains that mediate cellular responses to extracellular proteinases, including proteases secreted by perio-pathogens. Nucleotide-binding oligomerization domain (NOD) receptors are intracellular pattern-recognition molecules that play a role in innate immunity. PAR2 is thought to be involved in NOD- and TLR-dependent activation of cytokines in monocytic cells, but little is known about how these receptors cross-communicate in epithelial innate immune responses. Objectives: The goal of this study was to investigate how silencing PARs affects NOD and TLR expression upon exposure to various oral bacteria. Methods: Human gingival epithelial cells (GECs) from multiple donors were transfected with siRNA specific for PAR1 or PAR2 for 48h, then subsequently stimulated for 16h with periopathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, or non-pathogens Fusobacterium nucleatum and Streptococcus gordonii. Unstimulated GECs and cells transfected with non-silencing siRNA were used as controls. QRT-PCR was performed in duplicate to analyze the effect of PAR knock-down on the expression of NOD1, 2, 4 and TLR2, 4 in the presence of oral bacteria. Results: NOD1, 2 and 4 expression was increased in GECs stimulated with F. nucleatum (p<0.01). Increased expression of NOD1 was observed when PAR1 was knocked-down and GECs were subsequently stimulated with A. actinomycetemcomitans (p<0.005), while NOD2 expression increased with A. actinomycetemcomitans stimulation after PAR2 silencing (p<0.01), compared to cells stimulated with bacteria only. NOD4 expression was increased in PAR2-silenced GECs subsequently stimulated with any bacteria (p<0.01). No significant change in TLR expression was observed in the samples transfected with siRNA and stimulated with bacteria compared to controls. Conclusion: Our data suggest NOD activity increases to compensate for blocked PAR activation during epithelial response to bacteria. Further studies may lead to better understanding of how various receptors balance their responses in epithelial innate immunity.

Supported by NIDCR grants DE16961-01, DE015812 and DE13573.