Pg-lipid-A Structure Determines the Differential Inflammatory Response of Human Monocytes

Objectives: Porphyromonas gingivalis (Pg) is a keystone periodontopathogen and its lipopolysacharide (LPS) is strongly associated with periodontal disease. Pg-LPS displays a remarkable heterogeneity with both tetra- (LPS_1435/1449) and penta-acylated (LPS₁₆₉₀) lipid A structures. Human monocytes play a pivotal role in the pathogenesis of periodontitis. In the present study we investigated the inflammatory response of human monocytes to heterogeneous Pg-LPS, an area that has not been investigated thus far. Methods: Human monocytes were isolated from the blood of healthy donors using Ficoll density centrifugation and CD14 selection. Purified total monocytes were then treated with either PgLPS₁₄₃₅ or PgLPS₁₆₉₀ 100 ng/ml for 0.5-24 h.  E. coliLPS was used as a positive control. The expression of inflammatory genes, both at the RNA and protein levels were assessed by real-time qPCR from the cellular fraction and ELISA from culture supernatant respectively. Results: Heterogeneous Pg-LPS evoked a strikingly different inflammatory response in human monocytes. PgLPS₁₆₉₀ and E.coli LPS induced the expression of pro-inflammatory cytokines IL-6, IL-8, TNF-α, IL-1β, IL-12, IFN-β, GM-CSF and chemokines CCL-2, CCL-3, CXCL-10 significantly as compared to PgLPS₁₄₃₅, which did not induce the expression of these genes. In contrast, the expression of IL-10 transcript appeared to be delayed in response to E. coli LPS and PgLPS₁₆₉₀. Furthermore, PgLPS₁₆₉₀significantly up-regulated the protein levels of IL-6, IL-8, TNF-α, which is in line with the gene-expression results. Conclusion: The present study is the first report to unravel the inflammatory response of human monocytes to the heterogeneous Pg-LPS structures. It appeared that lipid A heterogeneity is critical in determining the pro-inflammatory response of monocytes, which has a direct implication in the pathogenesis of periodontitis. Hence, data generated from this study will provide a better insight of the pathogenic mechanism behind periodontal disease and in future, could be translated into clinical benefits for patients.