Expression profile, molecular regulation and immuno-inflammatory function of LPS-binding protein in human oral keratinocytes

• Main Authors: Ding, Peihui., 丁佩惠.
• Other Authors: Jin, L
• Language: English
• Published: The University of Hong Kong (Pokfulam, Hong Kong) 2013
• Subjects: Keratinocytes.; Endotoxins.
• Online Access: http://hdl.handle.net/10722/180959

Lipopolysaccharide (LPS)-binding protein (LBP) functions as a crucial molecule in innate immune responses to bacterial challenge. Our recent study shows the expression of LBP in human gingiva and its significant association with periodontal condition. Porphyromonas gingivalis is a keystone periodontopathogen with its LPS as a major virulence factor strongly involved in periodontal pathogenesis. Recent study has discovered that P. gingivalis LPS displays a significant lipid A structural heterogeneity. The present study investigated i) the expression profile of LBP in human oral keratinocytes (HOKs) stimulated by P. gingivalis LPS with penta-acylated (LPS1690) and tetra- (LPS1435/1449) lipid A structures as well as E. coli LPS; ii) the involvement of toll-like receptors (TLRs) and downstream signaling mechanisms in LBP expression; and iii) the effects of LBP and its crosstalk with the two isoforms of P. gingivalis LPS on the expression of cytokines and human β-defensins (hBD-2) in HOKs. The expression of LBP mRNA and peptide was significantly up-regulated by P. gingivalis LPS1690 and E. coli LPS, while not by P. gingivalis LPS1435/1449. P. gingivalis LPS1690-induced LBP expression was through both TLR2 and TLR4, and the relevant down-stream signaling mechanisms were then further investigated. Western blot results showed that P. gingivalis LPS1690 activated the phosphorylation of IκBα, p65, p38 MAPK and SAPK/JNK, whereas E. coli LPS phosphorylated IκBα, p38 MAPK and SAPK/JNK. A nuclear translocation of NF-κB transcription factor was confirmed upon stimulation by both forms of LPS. Further blocking assay revealed that P. gingivalis LPS1690 induction of LBP was through NF-κB and p38 MPAK pathways, while E. coli LPS induction of LBP was mediated by NF-κB, p38 MPAK and JNK pathways. The effects of LBP and its crosstalk with P. gingivalis LPS1690 or LPS1435/1449 on the expression of cytokines and hBD-2 were further investigated. Interestingly, recombinant human LBP (rhLBP) per se could significantly up-regulate the expression of IL-6, IL-8 and TNF-α, while down-regulate hBD-2 expression. P. gingivalis LPS1690 or LPS1435/1449 modulated to different extents the rhLBP-induced cytokine expression. Notably, P. gingivalis LPS1690 significantly down-regulated rhLBP-induced IL-8 expression; whereas, P. gingivalis LPS1435/1449 down-regulated IL-8 expression more intensively (around 80% vs. 40% reduction). The key mediators of TLRs and their adaptors like CD180 and MD-1 were significantly down-regulated by rhLBP (fold changes: -2.44 and -9.62, respectively). Both CD180 and MD-1 mRNAs were up-regulated by P. gingivalis LPS1435/1449 (7.11 and 4.05 folds, respectively); while these two genes were reversely modulated by P. gingivalis LPS1690 (20.86 and -6.93 folds, respectively). The present study demonstrates that P. gingivalis LPS with a lipid A structural heterogeneity differentially modulates LBP expression in HOKs. P. gingivalis LPS1690 promotes LBP expression in HOKs through TLR2 and TLR4 as well as NF-κB and p38 MAPK pathways in a way different from E. coli LPS. rhLBP per se significantly up-regulates the expression of IL-6, IL-8 and TNF-α, while down-regulates hBD-2 expression. P. gingivalis LPS with different lipid A structures down-regulates to different extents the rhLBP-induced expression of cytokines in HOKs, likely through fine-tuning of the CD180-MD1 complex and the relevant TLRs. === published_or_final_version === Dentistry === Doctoral === Doctor of Philosophy