Utilization of Protease-Activated Receptor signaling pathways in oral innate immune regulation

Abstract

Protease Activated Receptors (PARs) are G-protein coupled receptors and they play an active role in host defense. PAR1 and PAR2 are the most highly expressed members of the PAR family in gingival epithelial cells (GECs). This thesis is focused on the role of these two receptors. Objective: This study investigated how PAR receptors regulate downstream signaling pathways and subsequent cytokines secretion in response to specific bacteria in gingival epithelial innate immunity. Methods: Human GECs were transfected with small interfering RNA (siRNA) specific for PAR1 or PAR2. The cells were then stimulated with perio-pathogenic Porphyromonas gingivalis (PG), Aggregatibacter actinomycetemcomitans (AA) or non-pathogenic bacteria Streptococcus gordonii (SG). We used quantitative real-time polymerase chain reaction (QRT-PCR) to detect the messenger RNA (mRNA) expression level of the different innate immune markers; in addition, we used Multi-ELISA ARRAY to measure the protein level of select cytokines and ELISA to analyze intercellular proteins involved in two signal transduction pathways. Results: PAR1 and PAR2 knock-down affected the signaling pathways of both PI3K and IKBkB. When PAR1 was knocked down, we observed up-regulation of IKBKB (p ¡Ü 0.05) following either AA+SG or PG+SG stimulation, and PI3K up-regulation (p ¡Ü 0.001) following AA+SG stimulation. When PAR2 was knocked down, we observed up-regulation of PI3K (P ¡Ü 0.05) and IKBKB (p ¡Ü 0.05) following SG stimulation. Also our results showed that the secretion levels of innate immune markers varied. IL-8 was down-regulated (p ¡Ü 0.05) with AA+SG, PG+SG, or SG stimulation when PAR1 was knocked down. When PAR2 was knocked down, IL-8 secretion decreased after PG+SG stimulation, and increased with SG stimulation (p ¡Ü 0.05) compared to bacteria-only controls. TNF-¦Á protein secretion level increased with AA+SG, PG+SG, or SG stimulation (p ¡Ü 0.05) and PAR1 knock-down, while with PAR2 knock-down and PG+SG stimulation the protein level of TNF-¦Á decreased compared to controls with the same conditions and no receptors knocked down. IL-1¦Â was down-regulated with PAR2 knock-down with AA+SG or AA+SG stimulation (p ¡Ü 0.05). IL-6 was up-regulated in the un-stimulated group and with AA+SG stimulation when PAR2 was knocked down (p ¡Ü 0.05), while when PAR1 was knocked down, IL-6 down-regulated following AA+SG stimulation (p ¡Ü 0.001). IL-1¦Á was down-regulated when PAR2 was knocked down following AA+SG or SG stimulation (p ¡Ü 0.05). Conclusion: These results suggest that PAR1 and PAR2 receptors conduct their signal through IKBKB and PI3K. Moreover, IL-8, TNF-¦Á, IL-6, IL-1¦Á and IL-1¦Â secretion are modulated by PARs in response to bacterial stimulations. We foresee the information gained from this study will be a stage to further study how different PAR receptors induce appropriate innate immune responses, and also how PAR receptors might work through different signaling pathways to achieve appropriate innate immune response.