Peptidoglycan-associated lipoprotein of Aggregatibacter actinomycetemcomitans induces apoptosis and production of proinflammatory cytokines via TLR2 in murine macrophages RAW 264.7 in vitro

Peptidoglycan-associated lipoprotein (PAL) is a conserved pro-inflammatory outer membrane lipoprotein in Gram-negative bacteria. Compared to systemic pathogens, little is known about the virulence properties of PAL in Aggregatibacter actinomycetemcomitans (AaPAL). The aims of this study were to investigate the cytolethality of AaPAL and its ability to induce pro-inflammatory cytokine production in macrophages. Mouse macrophages were stimulated with AaPAL, and the production of IL-1β, IL-6, TNF-α, and MCP-1 was measured after 6, 24, and 48 h. To investigate which receptor AaPAL employs for its interaction with macrophages, anti-toll-like receptor (TLR)2 and anti-TLR4 antibodies were used to block respective TLRs on macrophages. Metabolic activity and apoptosis of the macrophages were investigated after stimulation with AaPAL. AaPAL induced the production of MCP-1, TNF-α, IL-6, and IL-1β from mouse macrophages in order of decreasing abundance. The pre-treatment of macrophages with an anti-TLR2 antibody significantly diminished cytokine production. Under AaPAL stimulation, the metabolic activity of macrophages decreased in a dose- and time-dependent manner. Furthermore, AaPAL induced apoptosis in 56% of macrophages after 48 h of incubation. Our data suggest that AaPAL can kill macrophages by apoptosis. The results also emphasize the role of AaPAL as a potent pro-inflammatory agent in A. actinomycetemcomitans-associated infections.