<i>Porphyromonas gingivalis</i> –Derived Virulence Lipids Accelerate Osteoclastogenesis Independently of High Mobility Group Box Protein‐1 Canonical Signaling

Periodontal bacterial pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) accelerate inflammatory osteoclastogenesis, resulting in alveolar bone loss. The core PAMP and DAMP prototype molecules are periodontal bacterium Porphyromonas gingivalis-derived virulence lipids, for example, phosphoglycerol dihydroceramide (PGDHC) and lipopolysaccharide (LPS Pg), and the host non-histone alarmin high mobility group box protein-1 (HMGB1), respectively. Although it was reported that extracellularly released HMGB1 is critical for the promotion of sepsis inflammation in response to non-periodontal bacterial LPS, our understanding of the crosstalk between HMGB1 and P. gingivalis-derived virulence lipids remains limited. Therefore, we used Hmgb1^fl/fl LysM-Cre⁺ mice with ablated HMGB1 mRNA and littermate Hmgb1^fl/fl LysM-Cre^- controls. We observed limited Hmgb1^fl/fl LysM-Cre⁺ osteoclastogenesis compared to Hmgb1^fl/fl in response to RANKL in vitro. Furthermore, recombinant HMGB1 protein restored osteoclast formation in Hmgb1^fl/fl LysM-Cre⁺ cells, indicating the pivotal role of extracellular HMGB1 in osteoclastogenesis in vitro. Using bulk RNA-sequencing, we identified the diminished osteoclastogenesis in Hmgb1^fl/fl LysM-Cre⁺ cells are linked to accelerated expression of canonical osteoclast-suppressing interferon genes. We surprisingly detected that PGDHC and LPS Pg accelerate osteoclastogenesis in Hmgb1^fl/fl LysM-Cre⁺ cells in vitro. Using bulk RNA-sequencing and real-time PCR assays, we confirmed that PGDHC diminishes the expression patterns of different interferon-inducible guanylate-binding proteins (GBP 3, 4, 5, 9). At the same time, LPS Pg accelerates the expression of osteoclast-promoting matrix metalloproteases (MMP 8 and 12) mRNAs. The results suggest that the RANKL-primed osteoclastogenesis accelerated by P. gingivalis-derived virulence lipids is mediated by different MMP or GBP signaling pathways independently from canonical HMGB1 signaling.