The innate host response to lipopolysaccharide (LPS) obtained from Porphyromonas gingivalis is unusual in that different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) as well as an antagonist or agonist for TLR4. In this report it is shown that P. gingivalis LPS is highly heterogeneous, containing more lipid A species than previously described. In addition, purification of LPS can preferentially fractionate these lipid A species. It is shown that an LPS preparation enriched for lipid A species at m/z 1,435 and 1,450 activates human and mouse TLR2, TLR2 plus TLR1, and TLR4 in transiently transfected HEK 293 cells coexpressing membrane-associated CD14. The HEK cell experiments further demonstrated that cofactor MD-2 was required for functional engagement of TLR4 but not of TLR2 nor TLR2 plus TLR1. In addition, serum-soluble CD14 effectively transferred P. gingivalis LPS to TLR2 plus TLR1, but poorly to TLR4. Importantly, bone marrow cells obtained from TLR2؊/؊ and TLR4 ؊/؊ mice also responded to P. gingivalis LPS in a manor consistent with the HEK results, demonstrating that P. gingivalis LPS can utilize both TLR2 and TLR4. No response was observed from bone marrow cells obtained from TLR2 and TLR4 double-knockout mice, demonstrating that P. gingivalis LPS activation occurred exclusively through either TLR2 or TLR4. Although the biological significance of the different lipid A species found in P. gingivalis LPS preparations is not currently understood, it is proposed that the presence of multiple lipid A species contributes to cell activation through both TLR2 and TLR4.

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Odontoblasts (OBs) are cells lining the inner surface of the tooth. Their potential role in host defenses within the tooth is suggested by their production of antimicrobial beta-defensins, but their role needs confirmation. The present study sought to define the roles of human OBs in microbial recognition and innate host responses. Toll-like receptor 2 (TLR2) and TLR4, as well as CCR6, were immunolocalized in human OBs and their dentinal processes in situ. To examine OB function we used organotypic tooth crown cultures to maintain human OBs within their dentin scaffold. Cells in the OB layer of cultured and non-cultured crown preparations expressed mRNA for several markers of innate immunity including chemokine CCL20, chemokine receptor CCR6, TLR2, TLR4 and the OB marker dentin sialophosphoprotein (DSPP). Expression of human beta-defensin 1 (hBD1), hBD2, hBD3, interleukin-8 (IL-8), and CCL20 increased with time in culture. Tooth crown odontoblast (TcOB) cultures were stimulated with agonist that was specific for TLR2 (Pam3CSK4) or TLR4 [Escherichia coli lipopolysaccharide (LPS)]. Nuclear factor-kappaB assays confirmed the TLR2 activity of Pam3CSK4 and the TLR4 activity of LPS. LPS up-regulated IL-1beta, tumor necrosis factor-alpha (TNF-alpha), CCL20, hBD2, IL-8, TLR2 and TLR4; however, Pam3CSK4 down-regulated these mRNAs. IL-1beta, TNF-alpha, CCL20 were also up-regulated from six-fold to 30-fold in TcOB preparations from decayed teeth. Our results show for the first time that OBs express microbial pattern recognition receptors in situ, thus allowing differential responses to gram-positive and gram-negative bacteria, and suggest that pro-inflammatory cytokines and innate immune responses in decayed teeth may result from TLR4 signaling.

E. coli lipopolysaccharide (LPS) induces cytokine and adhesion molecule expression via the toll-like receptor 4 (TLR4) signaling complex in human endothelial cells. In the present study, we investigated the mechanism by which Porphyromonas gingivalis LPS antagonizes E. coli LPS-dependent activation of human endothelial cells. P. gingivalis LPS at 1 g/ml inhibited both E. coli LPS (10 ng/ml) and Mycobacterium tuberculosis heat shock protein (HSP) 60.1 (10 g/ml) stimulation of E-selectin mRNA expression in human umbilical vein endothelial cells (HUVEC) without inhibiting interleukin-1 beta (IL-1␤) stimulation. P. gingivalis LPS (1 g/ml) also blocked both E. coli LPS-dependent and M. tuberculosis HSP60.1-dependent but not IL-1␤-dependent activation of NF-B in human microvascular endothelial (HMEC-1) cells, consistent with antagonism occurring upstream from the TLR/IL-1 receptor adaptor protein, MyD88. Surprisingly, P. gingivalis LPS weakly but significantly activated NF-B in HMEC-1 cells in the absence of E. coli LPS, and the P. gingivalis LPS-dependent agonism was blocked by transient expression of a dominant negative murine TLR4. Pretreatment of HUVECs with P. gingivalis LPS did not influence the ability of E. coli LPS to stimulate E-selectin mRNA expression. Taken together, these data provide the first evidence that P. gingivalis LPS-dependent antagonism of E. coli LPS in human endothelial cells likely involves the ability of P. gingivalis LPS to directly compete with E. coli LPS at the TLR4 signaling complex.The role that Porphyromonas gingivalis plays in the development of periodontal disease likely involves its ability to invade the gingiva and modulate innate host inflammatory responses via proteinases and lipopolysaccharide (LPS) (28,32,46,47). Previous studies have demonstrated that P. gingivalis disrupts the ability of gingival epithelial cells to produce interleukin-8 (IL-8) (8). These data suggest that such "chemokine paralysis" suppresses the host's ability to recruit and localize neutrophils to gingival sites of the infection via an IL-8 gradient (48). Gingival fibroblasts are likely to figure prominently in inflammatory responses to P. gingivalis. For example, P. gingivalis LPS has been shown to stimulate the production of a variety of cytokines, including IL-1, IL-6, and IL-8, in gingival fibroblasts, and it is chronic and excessive cytokine production that is believed to participate in tissue destruction during the course of periodontal disease (49). On the other hand, monocytes and human endothelial cells exhibit a low responsiveness to P. gingivalis LPS compared to E. coli LPS (7, 9, 30). In addition, in vivo studies demonstrated the low biological activity of P. gingivalis LPS in stimulating cytokine and adhesion molecule expression in mice (38).Another key property of P. gingivalis LPS is that it not only fails to stimulate E-selectin expression or p38 mitogen-activated protein kinase activation in human umbilical endothelial cells (HUVEC), but can potently antagonize the ability of E. coli LPS...

We have demonstrated previously that tetra-acylated LPS derived from the oral bacterium, Porphyromonas gingivalis, and penta-acylated msbB LPS derived from a mutant strain of Escherichia coli can antagonize the ability of canonical hexa-acylated E. coli LPS to signal through the TLR4 signaling complex in human endothelial cells. Activation of the TLR4 signaling complex requires the coordinated function of LPS binding protein (LBP), CD14, MD-2, and TLR4. To elucidate the specific molecular components that mediate antagonism, we developed a recombinant human TLR4 signaling complex that displayed efficient LPS-dependent antagonism of E. coli LPS in HEK293 cells. Notably, changes in the expression levels of TLR4 in HEK293 cells modulated the efficiency of antagonism by P. gingivalis LPS. Both soluble (s) CD14 and membrane (m) CD14 supported efficient P. gingivalis LPS-dependent and msbB LPS-dependent antagonism of E. coli LPS in the recombinant TLR4 system. When cells expressing TLR4, MD-2, and mCD14 were exposed to LPS in the absence of serum-derived LBP, efficient LPS-dependent antagonism of E. coli LPS was still observed indicating that LPS-dependent antagonism occurs downstream of LBP. Experiments using immunoprecipitates of sCD14 or sMD-2 that had been pre-exposed to agonist and antagonist indicated that LPS-dependent antagonism occurs partially at sCD14 and potently at sMD-2. This study provides novel evidence that expression levels of TLR4 can modulate the efficiency of LPS-dependent antagonism. However, MD-2 represents the principal molecular component that tetra-acylated P. gingivalis LPS and penta-acylated msbB LPS use to antagonize hexa-acylated E. coli LPS at the TLR4 signaling complex.