Induction of toll-like receptor expression by Porphyromonas gingivalis.

BACKGROUND: Toll-like receptors (TLRs) play pivotal roles in host immune responses and have been suggested to be involved in the development of many infectious diseases. In this study, the mRNA expression levels of TLR2, TLR4, and TLR9 and their relationship with periodontopathic bacteria in periodontal tissue are examined. Furthermore, the mechanism of TLR induction by Porphyromonas gingivalis is investigated in human gingival fibroblasts (HGFs). METHODS: Gingival tissue and subgingival plaque samples were collected from 19 patients with chronic periodontitis (CP) and 16 control individuals without periodontitis. Gene expression levels in the tissues and in HGFs were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The numbers of periodontopathic bacteria were determined by quantitative real-time PCR. RESULTS: The expression levels of TLR2 and TLR9 were significantly higher in the tissues of patients with CP compared to the tissues of control individuals. The mRNA levels of TLR2 and TLR9, but not TLR4, were positively correlated with the number of P. gingivalis in subgingival plaque. P. gingivalis sonicated extract, P. gingivalis lipopolysaccharide, P. gingivalis DNA, and tumor necrosis factor-α(TNF-α) could significantly upregulate the mRNA expression of TLR2 in HGFs. Furthermore, P. gingivalis-mediated TLR2 expression was suppressed by TNF-α antibody. CONCLUSIONS: This study suggests that P. gingivalis infection induces TLR2 and TLR9 upregulation in patients with CP. P. gingivalis-induced TLR2 expression in HGFs is partially dependent on TNF-α and may lead to sensitization of HGFs to bacterial components encountered in the periodontal microenvironment.

Modulation of Wnt5a expression by periodontopathic bacteria.

Wingless proteins, termed Wnt, are involved in embryonic development, blood cell differentiation, and tumorigenesis. In mammalian hematopoiesis, Wnt signaling is essential for stem-cell homeostasis and lymphocyte differentiation. Recent studies have suggested that these molecules are associated with cardiovascular diseases, rheumatoid arthritis, and osteoarthritis. Furthermore, Wnt5a signaling is essential for the general inflammatory response of human macrophages. Periodontitis is a chronic inflammatory disease caused by gram-negative periodontopathic bacteria and the resultant host immune response. Periodontitis is characterized by loss of tooth-supporting structures and alveolar bone resorption. There have been no previous reports on Wnt5a expression in periodontitis tissue, and only few study reported the molecular mechanisms of Wnt5a expression in LPS-stimulated monocytic cells. Using RT-PCR, we demonstrated that Wnt5a mRNA expression was up-regulated in chronic periodontitis tissue as compared to healthy control tissue. P. gingivalis LPS induced Wnt5a mRNA in the human monocytic cell line THP-1 with a peak at 4 hrs after stimulation. P. gingivalis LPS induced higher up-regulation of Wnt5a mRNA than E. coli LPS. The LPS receptors TLR2 and TLR4 were equally expressed on the surface of THP-1 cells. P. gingivalis LPS induced IκBα degradation and was able to increase the NF-κB binding activity to DNA. P. gingivalis LPS-induced Wnt5a expression was inhibited by NF-κB inhibitors, suggesting NF-κB involvement. Furthermore, IFN-γ synergistically enhanced the P. gingivalis LPS-induced production of Wnt5a. Pharmacological investigation and siRNA experiments showed that STAT1 was important for P. gingivalis LPS-induced Wnt5a expression. These results suggest that the modulation of Wnt5a expression by P. gingivalis may play an important role in the periodontal inflammatory process and serve a target for the development of new therapies.

RANKL upregulation associated with periodontitis and Porphyromonas gingivalis.

BACKGROUND: Receptor activator of nuclear factor-kappa B (NF-kappaB) ligand (RANKL) and osteoprotegerin (OPG) are critical for homeostatic control of osteoclast activity, suggesting their vital roles in the progression of bone loss in periodontitis. In this study, the expression of RANKL and OPG mRNA and the relationship between these factors and periodontopathic bacteria in periodontal tissue were studied. METHODS: Gingival tissue and subgingival plaque samples were collected from 15 patients with chronic periodontitis and 15 periodontally healthy subjects. RNA was extracted from the tissue and subjected to reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for RANKL or OPG. Beta-actin was amplified as a control to ensure equal loading. The intensity of RT-PCR products was analyzed by a densitometer in proportion to the intensity of beta-actin. The numbers of Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans were determined by quantitative real-time PCR. RESULTS: Our results showed increased levels of RANKL mRNA in chronic periodontitis tissues. The RANKL/OPG expression ratio was significantly higher in the periodontitis group compared to the healthy control group (P = 0.001). Interestingly, the expression of RANKL (r = 0.64; P <0.001), but not OPG (r = -0.24; P = 0.20), was significantly correlated with increased numbers of P. gingivalis. A. actinomycetemcomitans was detected in only 6.7% of all sites. CONCLUSIONS: Chronic periodontitis was associated with RANKL mRNA upregulation and increased RANKL/OPG mRNA expression ratio. In addition, our data showed for the first time to our knowledge an association between upregulated RANKL levels and the number of P. gingivalis in clinically obtained periodontal tissues.

TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src.

Interleukin 1 (IL-1) has been implicated in the reorganization of the actin cytoskeleton. An expression vector encoding a PKB/Akt pleckstrin-homology domain fused to a fluorescent protein was used to detect phosphoinositide 3-kinase (PI 3-kinase) products. It was observed that PI 3-kinase was activated either by treatment with IL-1 or by expression of either TRAF6, Src, MyD88 or dominant-positive PI 3-kinase, and resulted in the formation of long filopodia-like cellular protrusions that appeared to branch at membrane sites consisting of clusters of phosphoinositide. This depended upon a TRAF6 polyproline motif and Src catalytic activity, and was blocked by inhibitors of PI 3-kinase, Src and Ras. Using both conventional and split fluorescent protein probes fused to expressed TRAF6 and Src in living cells, the polyproline sequence of TRAF6 and the Src-homology 3 (SH3) domain of Src were shown to be required for interaction between these two proteins. Interaction occurred within the cytoplasm, and not at either the cell membrane or cytoplasmic sequestosomes. In addition, co-transfection of vectors expressing fluorescent-protein-fused TRAF6 and non-fluorescent MyD88, IRAK1 and IRAK2 revealed an inverse correlation between increased sequestosome formation and activation of both PI 3-kinase and NF-kappaB. Although a key factor in TRAF6-dependent activation of PI 3-kinase, ectopic expression of Src was insufficient for NF-kappaB activation and, in contrast to NF-kappaB, was not inhibited by IRAK2.

Role of macrophages in LPS-induced osteoblast and PDL cell apoptosis.

In periradicular lesions and periodontal disease, bacterial invasion leads to chronic inflammation resulting in disruption of the structural integrity of the periodontal ligament and progressive alveolar bone destruction. The pathogenesis of these conditions has been attributed not only to bacterial-induced tissue destruction but also to a defect in periodontal tissue repair. Accumulated data have also shown that lipopolysaccharide (LPS) can directly induce cell death or apoptosis in many cell types, including macrophages, osteoblasts, vascular endothelial cells, hepatocytes and myocytes. The present study hypothesized that bacterial LPS-induced apoptosis in osteoblasts and periodontal ligament fibroblasts (PDL cells) is an important contributing factor to the defect in periodontal tissue repair in periodontal and periapical disease. Macrophages have been shown to respond to bacterial LPS by increasing the production of proinflammatory cytokines. In addition, large numbers of macrophages are present in inflamed periodontal tissue. We speculated that macrophages were a potential candidate cell for mediating apoptosis in osteoblasts and PDL cells in response to bacteria-derived LPS. The macrophage-like cell line, RAW 264.7, was stimulated with LPS, and the conditioned medium was used to treat osteoblasts and PDL cells. Bacterial LPS had no direct apoptotic effect on mouse osteoblasts or PDL cells, whereas the conditioned medium from LPS-activated macrophages was able to induce apoptosis in these cells. To evaluate the contribution of tumor necrosis factor-alpha (TNF-alpha) released from macrophages on osteoblast and PDL cell apoptosis, cells were incubated with conditioned medium from LPS-treated macrophages in the presence and absence of anti-TNF-alpha neutralizing antibodies. TNF-alpha neutralizing antibody pretreatment inhibited the effect of conditioned medium from LPS-treated macrophages on osteoblast and PDL cell apoptosis in a dose-dependent manner. These results suggest that LPS could indirectly induce apoptosis in osteoblasts and PDL cells through the induction of TNF-alpha release from macrophages. These studies provide insight into a potential mechanism by which bacterial-derived LPS could contribute to defective periodontal and bone tissue repair in periodontal and periapical disease.

Interleukin 1 activates STAT3/nuclear factor-kappaB cross-talk via a unique TRAF6- and p65-dependent mechanism.

Interleukins (IL) 1 and 6 are important cytokines that function via the activation, respectively, of the transcription factors NF-kappaB and STAT3. We have observed that a specific type of kappa B DNA sequence motif supports both NF-kappaB p65 homodimer binding and cooperativity with non-tyrosine-phosphorylated STAT3. This activity, in contrast to that mediated by kappaB DNA motifs that do not efficiently bind p65 homodimers, is shown to be uniquely dependent upon signal transduction through the carboxyl terminus of TRAF6. Furthermore, STAT3 and p65 are shown to physically interact, in vivo, and this interaction appears to inhibit the function of "classical" STAT3 GAS-like binding sites. The distinct p50 form of NF-kappaB is also shown to interact with STAT3. However, in contrast to p65, p50 cooperates with STAT3 bound to GAS sites. These data argue for a novel transcription factor cross-talk mechanism that may help resolve inconsistencies previously reported regarding the mechanism of IL-1 inhibition of IL-6 activity during the acute-phase response.

Characterization of a cascade of protein interactions initiated at the IL-1 receptor.

Prior studies have identified molecules involved in IL-1 signaling that transmit the extracellular stimulus to downstream kinase molecules causing altered transcriptional activity. Many of these investigations have relied heavily on ligand induced protein-protein interactions detected by immuno-coprecipitation to map the cascade of events from receptor binding to activation of downstream signaling intermediates. Direct protein interactions have not been commonly reported. An in vitro study was undertaken to better define the direct protein-protein interactions involved in IL-1 signaling. Results indicate that IRAK2 is capable of direct association with either IL-1R(I) or IL-1R(AcP). IRAK2 is also capable of associating directly with MyD88 by distinct regions. Finally, IRAK2 interactions with TRAF6 were mapped and demonstrate differences from more proximal signaling intermediates. A model is presented that reflects the specific molecular interactions responsible for recruiting signaling intermediates to the IL-1 receptor cytoplasmic domains.

Novel signal transduction pathway utilized by extracellular HSP70: role of toll-like receptor (TLR) 2 and TLR4.

Recent studies have initiated a paradigm shift in the understanding of the function of heat shock proteins (HSP). It is now clear that HSP can and do exit mammalian cells, interact with cells of the immune system, and exert immunoregulatory effects. We recently demonstrated that exogenously added HSP70 possesses potent cytokine activity, with the ability to bind with high affinity to the plasma membrane, elicit a rapid intracellular Ca(2+) flux, activate NF-kappaB, and up-regulate the expression of pro-inflammatory cytokines in human monocytes. Here for the first time, we report that HSP70-induced proinflammatory cytokine production is mediated via the MyD88/IRAK/NF-kappaB signal transduction pathway and that HSP70 utilizes both TLR2 (receptor for Gram-positive bacteria) and TLR4 (receptor for Gram-negative bacteria) to transduce its proinflammatory signal in a CD14-dependent fashion. These studies now pave the way for the development of highly effective pharmacological or molecular tools that will either up-regulate or suppress HSP70-induced functions in conditions where HSP70 effects are desirable (cancer) or disorders where HSP70 effects are undesirable (arthritis and arteriosclerosis).