Gingipains Induce Microglia Migration Through Activation of Protease-activated Receptor-2 (PAR-2)

Objectives: Despite a positive relationship between periodontitis and cognitive decline associated with Alzheimer’s disease (AD) has been demonstrated, the precise mechanism by which periodontitis affects the cognitive functions remains unclear. We hypothesize that diffusible factors secreted from P. gingivalis may induce activation of microglia in the brain, because microglia-mediated neuroinflammation plays a critical role in the cognitive impairment. On the other hand, gingipains, cysteine proteases secreted by P. gingivalis, are known as important virulence factors of periodontal diseases. In the present study, I have thus examined possible roles of gingipains as activators of microglia.
Methods: Boyden chambers were used to test the cell migration of MG6 microglial cells. Immunofluorescent staining using anti-cleaved PAR-2 antibody was performed to visualize the activated form of PAR-2. Texas Red-X phalloidin was used to visualize membrane ruffling. Western blotting using anti-phosphorylated Akt was conducted to examine the activation of Akt.
Results: P. gingivalis infection significantly increased the cell migration of MG6 microglial cells. PAR-2 expressed in MG6 microglial cells was activated following infection with P. gingivalis. P. gingivalis infection-induced cell migration and PAR-2 activation of MG6 microglial cells were significantly inhibited by KYT-1 or KYT-36, a selective inhibitor of Arg-gingipain and Lys-gingipain, respectively. Furthermore, P. gingivalis infection-induced increase in the cell migration and the Akt phosphorylation of MG6 microglial cells were significantly inhibited by siRNA or neutralizing antibody of PAR-2. Moreover, P. gingivalis infection-induced increase in the cell migration was significantly inhibited by PI3K/Akt signaling pathway inhibitors.
Conclusions: These observations suggest that Arg- and Lys-gingipains play critical roles in the cell migration of microglia through the activation of PAR-2. The activation of PAR-2 further activates PI3K/Akt pathway, leading to the actin polymerization and subsequent membrane ruffling. Involvement of gingipains in P. gingivalis infection-induced cell migration of microglia is now further evaluating in in vivo system.