Classical and Alternative Activation of Rat Microglia Treated with Ultrapure Porphyromonas gingivalis Lipopolysaccharide In Vitro

Abstract

The possible relationship between periodontal disease resulting from the infection of gingival tissue by the Gram-negative bacterium <i>Porphyromonas gingivalis</i> (<i>P. gingivalis</i>) and the development of neuroinflammation remains under investigation. Recently, <i>P. gingivalis</i> lipopolysaccharide (LPS) was reported in the human brain, thus suggesting it might activate brain microglia, a cell type participating in neuroinflammation. We tested the hypothesis of whether in vitro exposure to ultrapure <i>P. gingivalis</i> LPS may result in classical and alternative activation phenotypes of rat microglia, with the concomitant release of cytokines and chemokines, as well as superoxide anion (O₂^-), thromboxane B₂ (TXB₂), and matrix metalloprotease-9 (MMP-9). After an 18-h exposure of microglia to <i>P. gingivalis</i> LPS, the concentration-dependent responses were the following: 0.1-100 ng/mL <i>P. gingivalis</i> LPS increased O₂^- generation, with reduced inflammatory mediator generation; 1000-10,000 ng/mL <i>P. gingivalis</i> LPS generated MMP-9, macrophage inflammatory protein 1α (MIP-1α/CCL3), macrophage inflammatory protein-2 (MIP-2/CXCL2) release and significant O₂^- generation; 100,000 ng/mL <i>P. gingivalis</i> LPS sustained O₂^- production, maintained MMP-9, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) release, and triggered elevated levels of MIP-1α/CCL3, MIP-2/CXCL2, and cytokine-induced neutrophil chemoattractant 1 (CINC-1/CXCL-1), with a very low release of lactic dehydrogenase (LDH). Although <i>P. gingivalis</i> LPS was less potent than <i>Escherichia coli</i> (<i>E. coli</i>) LPS in stimulating TXB₂, MMP-9, IL-6 and interleukin 10 (IL-10) generation, we observed that it appeared more efficacious in enhancing the release of O₂^-, TNF-α, MIP-1α/CCL3, MIP-2/CXCL2 and CINC-1/CXCL-1. Our results provide support to our research hypothesis because an 18-h in vitro stimulation with ultrapure <i>P. gingivalis</i> LPS resulted in the classical and alternative activation of rat brain microglia and the concomitant release of cytokines and chemokines.