Tetra- and Penta-Acylated Lipid A Structures of Porphyromonas gingivalis LPS Differentially Activate TLR4-Mediated NF-κB Signal Transduction Cascade and Immuno-Inflammatory Response in Human Gingival Fibroblasts

Abstract

Background Porphyromonas gingivalis is a major pathogen of periodontal disease that affects a majority of adults worldwide. Increasing evidence shows that periodontal disease is linked to various systemic diseases like diabetes and cardiovascular disease, by contributing to increased systemic levels of inflammation. Lipopolysaccharides (LPS), as a key virulent attribute of P. gingivalis, possesses significant amount of lipid A heterogeneity containing tetra- (LPS1435/1449) and penta-acylated (LPS1690) structures. Hitherto, the exact molecular mechanism of P. gingivalis LPS involved in periodontal pathogenesis remains unclear, due to limited understanding of the specific receptors and signaling pathways involved in LPS-host cell interactions.Methodology/Principal FindingsThis study systematically investigated the effects of P. gingivalis LPS1435/1449 and LPS1690 on the expression of TLR2 and TLR4 signal transduction and the activation of pro-inflammatory cytokines IL-6 and IL-8 in human gingival fibroblasts (HGFs). We found that LPS1435/1449 and LPS1690 differentially modulated TLR2 and TLR4 expression. NF-κB pathway was significantly activated by LPS1690 but not by LPS1435/1449. In addition, LPS1690 induced significant expression of NF-κB and p38 MPAK pathways-related genes, such as NFKBIA, NFKB1, IKBKB, MAP2K4 and MAPK8. Notably, the pro-inflammatory genes including GM-CSF, CXCL10, G-CSF, IL-6, IL-8 and CCL2 were significantly upregulated by LPS1690 while down-regulated by LPS1435/1449. Blocking assays confirmed that TLR4-mediated NF-κB signaling was vital in LPS1690-induced expression of IL-6 and IL-8 in HGFs.Conclusions/SignificanceThe present study suggests that the tetra- and penta-acylated lipid A structures of P. gingivalis LPS differentially activate TLR4-mediated NF-κB signaling pathway, and significantly modulate the expression of IL-6 and IL-8 in HGFs. The ability to alter the lipid A structure of LPS could be one of the strategies carried-out by P. gingivalis to evade innate host defense in gingival tissues, thereby contributing to periodontal pathogenesis.

Importance of heterogeneity in Porhyromonas gingivalis lipopolysaccharide lipid A in tissue specific inflammatory signalling

“…This is further achieved by changing the host’s defence signalling mechanisms through its heterogeneous A-LPS lipid A structures [31,44]. For example, Herath et al [4] observed that the NF-ĸB signalling pathway was noticeably activated in human gingival fibroblasts (HGFs) by LPS 1 , 690 but not by LPS 1 , 435/1 , 449 . The heterogeneity displayed in the A-LPS also modulated the secretion of a different profile of the pro-inflammatory cytokine expression such as IL-6 and IL-8 in HGFs [45].…”

“…In human monocytes, the A-LPS induced production of IL-1α, IL-1β, IL-6, and IL-8, although at a lower rate than that induced by lipid A obtained from total A-LPS [26]. In addition, pro-inflammatory genes significantly up regulated by LPS 1 , 690 ( GM-CSF, CXCL10, G-CSF, IL-6, IL-8 , and CCL2 ) were down regulated by LPS 1 , 435/1 , 449 [4] (see Table 1). HGFs matrix metalloproteinase (MMP)-3 and its protein were strikingly up regulated by penta-acylated P. gingivalis LPS 1 , 690 and hexa-acylated Escherichia coli LPS but not by tetra-acylated P. gingivalis LPS 1 , 435/1 , 449 [30], suggesting plausible crosstalk between LPS signalling from Gram-negative pathogens during mixed infections.…”

“…Furthermore, human beta-defensins, hBD-1, hBD-2, and hBD-3 mRNAs, were significantly up regulated in human epithelia by P. gingivalis LPS 1 , 690 but down regulated by LPS 1 , 435/1 , 449 [46]. Differential signalling pathway activation in various cell types [4] helps explain (at least in part) the contradictory results reported by earlier studies [28,29] and demonstrates tissue-specific modes of inflammatory signals initiated essentially by the same immunogen. The advantage to the keystone bacterium would likely be in suppressing, some signalling pathways for adapting to different tissue environments.…”

“…LPS is located in the outer membrane of Gram-negative bacteria and is a potent stimulator of host’s innate immune signal transduction pathways in a tissue/cell-specific manner [3]. This is seen in bone, epithelial cell barrier breakdown, and keratinocytes [4,5]. Historically, LPS has been thought to consist of three variable and conserved regions [6,7].…”

“…Of the LPS macromolecule, lipid A is responsible for the endotoxic activities, and its recognition by host cells leads to differential immuno-inflammatory responses [4,7–9]. From LPS, it is the heterogeneity within the A-LPS which represents the major virulence factors that promote inflammation and bone loss.…”