Abstract
Lipopolysaccharide (LPS)-binding protein (LBP) as an acute-phase protein plays a crucial role in innate host response to bacterial challenge. Our previous study shows that LBP expression in human gingiva is associated with periodontal status. Porphyromonas gingivalis is a keystone periodontopathogen, and its LPS with lipid A structural heterogeneity critically accounts for periodontal pathogenesis. This study investigated the effects of LBP and its interactions with two featured isoforms of P. gingivalis LPS (tetra-acylated LPS1435/1449 and penta-acylated LPS1690) on the expression of pro-inflammatory cytokines in human oral keratinocytes (HOKs), and the involvement of Toll-like receptor (TLR) signaling. HOKs were pre-incubated with recombinant human LBP (rhLBP) at 10ng/ml, 100ng/ml and 1μg/ml for 1 h, followed by the treatment of P. gingivalis LPS1690 or LPS1435/1449 for 3h or 24h respectively. The expression of IL-6 and IL-8, and involvements of TLR2 and TLR4 were analyzed. The genes associated with TLR signaling were assessed by PCR array. Interestingly, rhLBP per se significantly up-regulated the expression of IL-6 and IL-8 in HOKs (p<0.05), which was blocked by TLR2 antibody (p<0.001). LPS1435/1449 down-regulated more significantly rhLBP-induced IL-6 and IL-8 mRNAs with reference to P. gingivalis LPS1690 (approximately 80% vs. 40%, p<0.05; and 90% vs. 36%, p<0.001, respectively). Moreover, rhLBP markedly down-regulated the gene expression of TLRs and their adaptors such as CD180 (-2.44 folds) and MD-1 (-9.62 folds), while the interaction of P. gingivalis LPS1435/1449 with rhLBP greatly up-regulated both transcripts (7.11 and 4.05 folds, respectively). Notably, P. gingivalis LPS1690-rhLBP interaction dramatically up-regulated CD180 transcript (20.86 folds) and significantly down-regulated MD-1 transcript (-6.93 folds). This pioneering study shows that rhLBP enables to enhance the expression of pro-inflammatory cytokines in HOKs through TLR2 signaling pathway. P. gingivalis LPS with different lipid A structures down-regulates to different extents rhLBP-induced cytokine expression, possibly through fine-tuning of the CD180-MD1 complex and relevant TLRs.
Highlights
Lipopolysaccharide (LPS)-binding protein (LBP) as an acute-phase protein is primarily produced by hepatocytes [1]
It was worthy to note that recombinant human LBP (rhLBP) per se at 100ng/ml markedly up-regulated the transcript expression of IL-6 and IL-8 in human oral keratinocytes (HOKs) at 3 h (Figs 1A and 2A) (p
The interaction of rhLBP with P. gingivalis LPS1690 or LPS1435/1449 down-regulated to different extents IL-6 and IL-8 expression, with reference to the controls
Summary
Lipopolysaccharide (LPS)-binding protein (LBP) as an acute-phase protein is primarily produced by hepatocytes [1]. LBP has a classical dual role, namely enhancing LPS-induced cellular activation at a low concentration and neutralizing the effects of bacterial endotoxins at a high concentration [3,4]. It is worthy to note that our early study shows that human gingival epithelia can produce LBP with a well-lined expression at the dentogingival niche, and its expression level in periodontally healthy subjects is significantly higher than that in chronic periodontitis patients. These findings suggest that LBP may be significantly involved in innate response to bacterial LPS, and critically contribute to periodontal pathogenesis [10]. These studies indicate that LBP expression in gingiva instantly acts on bacterial challenge and greatly accounts for periodontal homeostasis
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