Atsutoshi Yoshimura

Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide

Lipopolysaccharide (LPS) is the main inducer of shock and death in Gram-negative sepsis. Recent evidence suggests that LPS-induced signal transduction begins with CD14-mediated activation of 1 or more Toll-like receptors (TLRs). The lipid A analogues lipid IVa and Rhodobacter sphaeroides lipid A (RSLA) exhibit an uncommon species-specific pharmacology. Both compounds inhibit the effects of LPS in human cells but display LPS-mimetic activity in hamster cells. We transfected human TLR4 or human TLR2 into hamster fibroblasts to determine if either of these LPS signal transducers is responsible for the species-specific pharmacology. RSLA and lipid IVa strongly induced NF-kappaB activity and IL-6 release in Chinese hamster ovary fibroblasts expressing CD14 (CHO/CD14), but these compounds antagonized LPS antagonists in CHO/CD14 fibroblasts that overexpressed human TLR4. No such antagonism occurred in cells overexpressing human TLR2. We cloned TLR4 from hamster macrophages and found that human THP-1 cells expressing the hamster TLR4 responded to lipid IVa as an LPS mimetic, as if they were hamster in origin. Hence, cells heterologously overexpressing TLR4 from different species acquired a pharmacological phenotype with respect to recognition of lipid A substructures that corresponded to the species from which the TLR4 transgene originated. These data suggest that TLR4 is the central lipid A-recognition protein in the LPS receptor complex.

Lipopolysaccharides from Periodontopathic Bacteria Porphyromonas gingivalis and Capnocytophaga ochracea Are Antagonists for Human Toll-Like Receptor 4

ABSTRACT Toll-like receptors (TLRs) 2 and 4 have recently been identified as possible signal transducers for various bacterial ligands. To investigate the roles of TLRs in the recognition of periodontopathic bacteria by the innate immune system, a Chinese hamster ovary (CHO) nuclear factor-κB (NF-κB)-dependent reporter cell line, 7.7, which is defective in both TLR2- and TLR4-dependent signaling pathways was transfected with human CD14 and TLRs. When the transfectants were exposed to freeze-dried periodontopathic bacteria, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Capnocytophaga ochracea, and Fusobacterium nucleatum, and a non-oral bacterium, Escherichia coli, all species of the bacteria induced NF-κB-dependent CD25 expression in 7.7/huTLR2 cells. Although freeze-dried A. actinomycetemcomitans, F. nucleatum, and E. coli also induced CD25 expression in 7.7/huTLR4 cells, freeze-dried P. gingivalis did not. Similarly, lipopolysaccharides (LPS) extracted from A. actinomycetemcomitans, F. nucleatum, and E. coli induced CD25 expression in 7.7/huTLR4 cells, but LPS from P. gingivalis and C. ochracea did not. Furthermore, LPS from P. gingivalis and C. ochracea attenuated CD25 expression in 7.7/huTLR4 cells induced by repurified LPS from E. coli. LPS from P. gingivalis and C. ochracea also inhibited the secretion of interleukin-6 (IL-6) from U373 cells, the secretion of IL-1β from human peripheral blood mononuclear cells, and ICAM-1 expression in human gingival fibroblasts induced by repurified LPS from E. coli. These findings indicated that LPS from P. gingivalis and C. ochracea worked as antagonists for human TLR4. The antagonistic activity of LPS from these periodontopathic bacteria may be associated with the etiology of periodontal diseases.

Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides and their recognition by toll-like receptors 2 and 6.

ABSTRACT The lipopeptide FSL-1 [S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe, Pam2CGDPKHPKSF] synthesized on the basis of the N-terminal structure of a Mycoplasma salivarium lipoprotein capable of activating normal human gingival fibroblasts to induce the cell surface expression of ICAM-1 revealed an activity to induce production of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-8. FSL-1 also activated macrophages to produce tumor necrosis factor alpha as the Mycoplasma fermentans-derived lipopeptide MALP-2 (Pam2CGNNDESNISFKEK), a potent macrophage-activating lipopeptide, did. The level of the activity of FSL-1 was higher than that of MALP-2. This result suggests that the difference in the amino acid sequence of the peptide portion affects the activity because the framework structure other than the amino acid sequence of the former is the same as that of the latter. To determine minimal structural requirements for the activity of FSL-1, the diacylglyceryl Cys and the peptide portions were examined for this activity. Both portions did not reveal the activity. A single amino acid substitution from Phe to Arg and a fatty acid substitution from palmitic acid to stearic acid drastically reduced the activity. Similar results were obtained in measuring the NF-κB reporter activity of FSL-1 to human embryonic kidney 293 cells transfected with Toll-like receptor 2 and 6, together with a NF-κB-dependent luciferase reporter plasmid. These results suggest that both the diacylglyceryl and the peptide portions of FSL-1 are indispensable for the expression of biological activities and for the recognition by Toll-like receptors 2 and 6 and that the recognition of FSL-1 by Toll-like receptors 2 and 6 appears to be hydrophobic.

LIPOPOLYSACCHARIDE RECOGNITION, CD14, AND LIPOPOLYSACCHARIDE RECEPTORS

The ability of a host to sense invasion by a pathogenic organism, and to respond appropriately to control infection, is paramount to survival. To that end, an array of receptors and binding proteins has evolved as part of the innate immune system to detect Gram-negative bacteria. This article reviews the role of CD14, other LPS binding proteins, and the Toll family of receptors in the innate recognition of bacterial lipopolysaccharide.

Biological Activities of Bacteroides forsythus Lipoproteins and Their Possible Pathological Roles in Periodontal Disease

ABSTRACT Bacteroides forsythus is a gram-negative, anaerobic, fusiform bacterium and is considered to be an etiological agent in periodontal disease. A lipoprotein fraction prepared from B. forsythus cells by Triton X-114 phase separation (BfLP) activated human gingival fibroblasts and a human monocytic cell line, THP-1, to induce interleukin-6 production and tumor necrosis factor alpha production. BfLP was found to be capable of inducing nuclear factor-κB translocation in human gingival fibroblasts and THP-1 cells. By using Chinese hamster ovary K1 cells transfected with Toll-like receptor genes together with a nuclear factor-κB-dependent CD25 reporter plasmid, it was found that signaling by BfLP was mediated by Toll-like receptor 2 but not by CD14 or Toll-like receptor 4. BfLP induced apoptotic cell death in human gingival fibroblasts, KB cells (an oral epithelial cell line), HL-60 cells (a human myeloid leukemia cell line), and THP-1 cells but not in MOLT4 cells (a T-cell leukemia cell line). Caspase-8, an initiator caspase in apoptosis, was found to be activated in these cells in response to BfLP stimulation. Thus, this study suggested that BfLP plays some etiological roles in oral infections, especially periodontal disease, by induction of cell activation or apoptosis.

A Single Nucleotide Polymorphism in 3′-Untranslated Region Contributes to the Regulation of Toll-like Receptor 4 Translation

Background: Genetic variation of SNP rs11536889 in 3′-UTR of TLR4 is implicated in certain diseases, including periodontitis, gastric atrophy, and prostate cancer. Results: The G allele of rs11536889 inhibited translation, but not transcription, of TLR4. Conclusion: Genetic variation of rs11536889 regulates TLR4 expression. Significance: Polymorphism in rs11536889 could be an excellent genetic marker for the diseases caused by TLR4-ligands. We have previously shown that a single nucleotide polymorphism rs11536889 in the 3′-untranslated region (UTR) of TLR4 was associated with periodontitis. In this study the effects of this single nucleotide polymorphism on Toll-like receptor (TLR) 4 expression were investigated. Monocytes from subjects with the C/C genotype expressed higher levels of TLR4 on their surfaces than those from subjects with the other genotypes. Peripheral blood mononuclear cells (PBMCs) from the C/C and G/C subjects secreted higher levels of IL-8 in response to lipopolysaccharide (LPS), a TLR4 ligand, than the cells from the G/G subjects. However, there was no significant difference in TLR4 mRNA levels in PBMCs from the subjects with each genotype. After stimulation with tripalmitoylated CSK4 (Pam3CSK4), TLR4 mRNA levels increased in PBMCs from both the C/C and G/G subjects, whereas TLR4 protein levels increased in PBMCs from the C/C but not G/G subjects. Transient transfection of a series of chimeric luciferase constructs revealed that a fragment of 3′-UTR containing rs11536889 G allele, but not C allele, suppressed luciferase activity induced by LPS or IL-6. Two microRNAs, hsa-miR-1236 and hsa-miR-642a, were predicted to bind to rs11536889 G allele. Inhibition of these microRNAs reversed the suppressed luciferase activity. These microRNA inhibitors also up-regulated endogenous TLR4 protein on THP-1 cells (the G/G genotype) after LPS stimulation. Furthermore, mutant microRNAs that bind to the C allele inhibited the luciferase activity of the construct containing the C allele. These results indicate that genetic variation of rs11536889 contributes to translational regulation of TLR4, possibly by binding to microRNAs.

Green tea catechin inhibits lipopolysaccharide‐induced bone resorption in vivo

BACKGROUND AND OBJECTIVE Bone resorption is positively regulated by receptor activator of nuclear factor-kappaB ligand (RANKL). Pro-inflammatory cytokines, such as interleukin (IL)-1beta, promote RANKL expression by stromal cells and osteoblasts. Green tea catechin (GTC) has beneficial effects on human health and has been reported to inhibit osteoclast formation in an in vitro co-culture system. However, there has been no investigation of the effect of GTC on periodontal bone resorption in vivo. We therefore investigated whether GTC has an inhibitory effect on lipopolysaccharide (LPS)-induced bone resorption. MATERIAL AND METHODS Escherichia coli (E. coli) LPS or LPS with GTC was injected a total of 10 times, once every 48 h, into the gingivae of BALB/c mice. Another group of mice, housed with free access to water containing GTC throughout the experimental period, were also injected with LPS in a similar manner. RESULTS The alveolar bone resorption and IL-1beta expression induced by LPS in gingival tissue were significantly decreased by injection or oral administration of GTC. Furthermore, when GTC was added to the medium, decreased responses to LPS were observed in CD14-expressing Chinese hamster ovary (CHO) reporter cells, which express CD25 through LPS-induced nuclear factor-kappaB (NF-kappaB) activation. These findings demonstrated that GTC inhibits nuclear translocation of NF-kappaB activated by LPS. In addition, osteoclasts were generated from mouse bone marrow macrophages cultured in a medium containing RANKL and macrophage colony-stimulating factor with or without GTC. The number of osteoclasts was decreased in dose-dependent manner when GTC was added to the culture medium. CONCLUSION These results suggest that GTC suppresses LPS-induced bone resorption by inhibiting IL-1beta production or by directly inhibiting osteoclastogenesis.

Assessment of the Plasma/Serum IgG Test to Screen for Periodontitis

Chronic periodontitis is a silent infectious disease prevalent worldwide and affects lifestyle-related diseases. Therefore, efficient screening of patients is essential for general health. This study was performed to evaluate prospectively the diagnostic utility of a blood IgG antibody titer test against periodontal pathogens. Oral examination was performed, and IgG titers against periodontal pathogens were measured by ELISA in 1,387 individuals. The cut-off value of the IgG titer was determined in receiver operating characteristic curve analysis, and changes in periodontal clinical parameters and IgG titers by periodontal treatment were evaluated. The relationships between IgG titers and severity of periodontitis were analyzed. The best cut-off value of IgG titer against Porphyromonas gingivalis for screening periodontitis was 1.682. Both clinical parameters and IgG titers decreased significantly under periodontal treatment. IgG titers of periodontitis patients were significantly higher than those of healthy controls, especially in those with sites of probing pocket depth over 4 mm. Multiplied cut-off values were useful to select patients with severe periodontitis. A blood IgG antibody titer test for Porphyromonas gingivalis is useful to screen hitherto chronic periodontitis patients (ClinicalTrials.gov number NCT01658475).

NOD1 and NOD2 Mediate Sensing of Periodontal Pathogens

In bacterial infection, Nucleotide-binding Oligomerization Domain (NOD) 1 and NOD2 induce innate immune responses by recognizing fragments of the bacterial component peptidoglycan (PGN). To determine the roles of these receptors in detection of periodontal pathogens, we stimulated human embryonic kidney cells expressing NOD1 or NOD2 with heat-killed Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum or their soluble PGNs (sPGNs). All bacteria and their sPGNs could stimulate activation of NF-κB. However, there were differences in NOD1- and NOD2-stimulatory activities among the species of bacteria. P. gingivalis showed weaker NOD1- and NOD2-stimulatory activities than did other bacteria. These differences in activities were confirmed by production of interleukin-8 from oral epithelial cells stimulated with sPGNs. These findings indicate that both NOD1 and NOD2 might be involved in the recognition of periodontal pathogens, and that the weak NOD-stimulatory property of P. gingivalis might be helpful for survival in the periodontal pocket.

Lack of Toll-like receptor 4 decreases lipopolysaccharide-induced bone resorption in C3H/HeJ mice in vivo

INTRODUCTION Few in vivo studies have demonstrated whether Toll-like receptor 4 (TLR4) is indispensable for lipopolysaccharide (LPS)-induced bone resorption and little is known about the receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) expression induced by LPS under conditions of lack of TLR4. METHODS We compared bone resorption histomorphometrically in C3H/HeN and C3H/HeJ mice that were repeatedly injected with Actinobacillus actionmycetemcomitans LPS into their gingiva every 48 h. RANKL-, interleukin-1beta- and OPG-positive cells in the connective tissue were also compared immunohistochemically. RESULTS Bone resorption in C3H/HeJ mice in the fourth, seventh, and tenth injection groups was significantly less than that C3H/HeN mice (P < 0.05). The number of RANKL-positive cells in C3H/HeJ mice in the 10th injection group was significantly smaller than that in C3H/HeN mice (P < 0.05). The numbers of interleukin-1beta-positive cells in C3H/HeJ mice in the seventh and tenth injection groups were significantly decreased compared with those in C3H/HeN mice (P < 0.05). The numbers of OPG-positive cells in C3H/HeN and C3H/HeJ mice gradually increased, but there was no significant difference between the two strains of mice. CONCLUSION TLR4 is indispensable for LPS-induced bone resorption in vivo.