Akira Hasebe

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.

Stimulation of human Toll-like receptor (TLR) 2 and TLR6 with membrane lipoproteins of Mycoplasma fermentans induces apoptotic cell death after NF-κB activation

Mycoplasmal membrane diacylated lipoproteins not only initiate proinflammatory responses through Toll‐like receptor (TLR) 2 and TLR6 via the activation of the transcriptional factor NF‐κB, but also initiate apoptotic responses. The aim of this study was to clarify the apoptotic machineries. Mycoplasma fermentans lipoproteins and a synthetic lipopeptide, MALP‐2, showed cytocidal activity towards HEK293 cells transfected with a TLR2‐encoding plasmid. The activity was synergically augmented by co‐expression of TLR6, but not by co‐expression of other TLRs. Under the condition of co‐expression of TLR2 and TLR6, the lipoproteins could induce maximum NF‐κB activation and apoptotic cell death in the cells 6 h and 24 h after stimulation respectively. Dominant‐negative forms of MyD88 and FADD, but not IRAK‐4, reduced the cytocidal activity of the lipoproteins. In addition, both dominant‐negative forms also downregulated the activation of both NF‐κB and caspase‐8 in the cells. Additionally, the cytocidal activity was sufficiently attenuated by a selective inhibitor of p38 MAPK. These findings suggest that mycoplasmal lipoproteins can trigger TLR2‐ and TLR6‐mediated sequential bifurcate responses: NF‐κB activation as an early event, which is partially mediated by MyD88 and FADD; and apoptosis as a later event, which is regulated by p38 MAPK as well as by MyD88 and FADD.

The N-terminal lipopeptide of a 44-kDa membrane-bound lipoprotein of Mycoplasma salivarium is responsible for the expression of intercellular adhesion molecule-1 on the cell surface of normal human gingival fibroblasts.

The activities to induce TNF-α production by a monocytic cell line, THP-1, and ICAM-1 expression and IL-6 production by human gingival fibroblasts were detected in plural membrane lipoproteins of Mycoplasma salivarium. Although SDS-PAGE of the lipoproteins digested by proteinase K did not reveal any protein bands with molecular masses higher than approximately10 kDa, these activities were detected in the front of the gel. A lipoprotein with a molecular mass of 44 kDa (Lp44) was purified. Proteinase K did not affect the ICAM-1 expression-inducing activity of Lp44, but lipoprotein lipase abrogated the activity. These results suggested that the proteinase K-resistant and low molecular mass entity, possibly the N-terminal lipid moiety, played a key role in the expression of the activity. The N-terminal lipid moiety of Lp44 was purified from Lp44 digested with proteinase K by HPLC. Judging from the structure of microbial lipopeptides as well as the amino acid sequence and infrared spectrum of Lp44, the structure of the N-terminal lipid moiety of Lp44 was speculated to be S-(2, 3-bisacyloxypropyl)-cysteine-GDPKHPKSFTEWV-. Its analogue, S-(2, 3-bispalmitoyloxypropyl)-cysteine-GDPKHPKSF, was synthesized. The lipopeptide was similar to the N-terminal lipid moiety of Lp44 in the infrared spectrum and the ICAM-1 expression-inducing activity. Thus, this study suggested that the active entity of Lp44 was its N-terminal lipopeptide moiety, the structure of which was very similar to S-(2, 3-bispalmitoyloxypropyl)-cysteine-GDPKHPKSF.

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.

Mycoplasmal Lipoproteins Induce Toll‐Like Receptor 2‐ and Caspases‐Mediated Cell Death in Lymphocytes and Monocytes

Lipoproteins of Mycoplasma salivarium and Mycoplasma fermentans preferentially induced necrotic cell death in lymphocytic cell lines, MOLT‐4 and Raji, and in one monocytic cell line, THP‐1, whereas they preferentially induced apoptotic cell death in another monocytic cell line, HL‐60. These findings were also supported by ultrastructural observations by the use of scanning and transmission electron microscopes and by agarose gel electrophoresis of the genomic DNA. The lipoproteins activated caspase‐3 in both MOLT‐4 and HL‐60 cells, which was assessed by the cleavage of the synthetic substrate DEVD‐pNA and the endogenous substrate poly(ADP‐ribose) polymerase. The cytotoxicity to MOLT‐4 and HL‐60 cells was inhibited by various caspase inhibitors, Ac‐DMQD‐CHO, Ac‐IETD‐CHO, and Z‐VAD‐FMK. The cytotoxicity was also partially suppressed by the monoclonal antibody to Toll‐like receptor 2. Thus this study demonstrated that mycoplasmal lipoproteins induce caspases‐dependent necrotic and apoptotic cell death in lymphocytes and monocytes/macrophages, which is partially induced by TLR2‐mediated signaling.

Signaling Pathways Induced by Lipoproteins Derived from Mycoplasma salivarium and a Synthetic Lipopeptide (FSL-1) in Normal Human Gingival Fibroblasts

Lipoproteins derived from Mycoplasma salivarium and a synthetic lipopeptide (FSL‐1) activate human gingival fibroblasts to induce IL‐6 production and ICAM‐1 expression. Human gingival fibroblasts were treated with lipoproteins or FSL‐1 and then examined for the activation of mitogen‐activated protein kinases (MAPKs), ERK1/2, p38, and SAPK/JNK, and transcription factors, AP‐1 and NF‐κB. Western blotting indicated that p38 and SAPK/JNK were activated in response to the stimulators, but the activation of ERK1/2 could not be discriminated because ERK1/2 was activated in the absence of stimulators. The p38 inhibitor SB 203580 also suppressed their IL‐6 production‐inducing activities, whereas the ERK1/2‐activating MAPK kinase (MEK1) inhibitor PD 98059 did not suppress their activities. Moreover, they were capable of inducing the activation of AP‐1 and NF‐κB. NF‐κB activation was also confirmed by the phosphorylation of IκB‐α. On the basis of these results, it was concluded that lipoproteins of M. salivarium and FSL‐1 are capable of activating the MAPKs p38 and SAPK/JNK and the transcriptional factors AP‐1 and NF‐κB in human gingival fibroblasts.

The Toll‐like receptor 2 (TLR2) ligand FSL‐1 is internalized via the clathrin‐dependent endocytic pathway triggered by CD14 and CD36 but not by TLR2

Little is known of how Toll‐like receptor (TLR) ligands are processed after recognition by TLRs. This study was therefore designed to investigate how the TLR2 ligand FSL‐1 is processed in macrophages after recognition by TLR2. FSL‐1 was internalized into the murine macrophage cell line, RAW264.7. Both chlorpromazine and methyl‐β‐cyclodextrin, which inhibit clathrin‐dependent endocytosis, reduced FSL‐1 uptake by RAW264.7 cells in a dose‐dependent manner but nystatin, which inhibits caveolae‐ and lipid raft‐dependent endocytosis, did not. FSL‐1 was co‐localized with clathrin but not with TLR2 in the cytosol of RAW264.7 cells. These results suggest that internalization of FSL‐1 is clathrin dependent. In addition, FSL‐1 was internalized by peritoneal macrophages from TLR2‐deficient mice. FSL‐1 was internalized by human embryonic kidney 293 cells transfected with CD14 or CD36 but not by the non‐transfected cells. Also, knockdown of CD14 or CD36 in the transfectants reduced FSL‐1 uptake. In this study, we suggest that (i) FSL‐1 is internalized into macrophages via a clathrin‐dependent endocytic pathway, (ii) the FSL‐1 uptake by macrophages occurs irrespective of the presence of TLR2, and (iii) CD14 and CD36 are responsible for the internalization of FSL‐1.

Synergic effects of mycoplasmal lipopeptides and extracellular ATP on activation of macrophages

ABSTRACT Mycoplasmal lipopeptides S-(2,3-bispalmitoyloxypropyl)-CGDPKHSPKSF and S-(2,3-bispalmitoyloxypropyl)-CGNNDESNISFKEK activated a monocytic cell line, THP-1 cells, to produce tumor necrosis factor alpha. The activity of the lipopeptides was augmented by ATP in a dose-dependent manner. In addition, the level of expression of mRNAs for tumor necrosis factor alpha and interleukin-1β, -6, and -8 was also upregulated by the lipopeptides and/or extracellular ATP, but that of interleukin-10 was not. The P2X purinergic receptor antagonists pyridoxal phosphate 6-azophenyl 2′,4′-disulfonic acid and periodate-oxidized ATP suppressed the activity of ATP to augment the activation of THP-1 cells by the lipopeptides, suggesting that P2X receptors play important roles in the activity of ATP. The nuclear factor κB inhibitor dexamethasone also suppressed the activity, suggesting that the activity of ATP is dependent upon the nuclear factor κB. Thus, these results suggest that the interaction of extracellular ATP with the P2X receptors is attributed to the activity of ATP to augment the activation of THP-1 cells by mycoplasmal lipopeptides.

Resveratrol Modulates Phagocytosis of Bacteria through an NF-κB-Dependent Gene Program

ABSTRACT Many studies have shown that the pharmacological effects of resveratrol, a phytoalexin polyphenolic compound, include protective effects against cancer and inflammation as well as enhancement of stress resistance. In this study, we examined whether resveratrol affected the phagocytosis of bacteria by macrophages and the activation of the transcription factor NF-κB after stimulation with or without the ligand FSL-1 for Toll-like receptor 2 (TLR2). Phagocytosis of Escherichia coli and of Staphylococcus aureus by THP-1 cells and RAW264.7 cells was inhibited by resveratrol in a dose-dependent manner regardless of stimulation with FSL-1. The NF-κB activity in HEK293 cells stably expressing TLR2 was also inhibited by resveratrol after stimulation with FSL-1. Resveratrol also inhibited both the translocation of p65 of NF-κB into nuclei in the transfectant and tumor necrosis factor alpha production by THP-1 cells or RAW264.7 cells. It has recently been reported that TLR-mediated signaling pathways lead to the upregulation of mRNAs of phagocytic receptors, including scavenger receptors and C-type lectin receptors. This study also demonstrated that FSL-1 induced the upregulation of mRNAs of phagocytic receptors such as macrophage scavenger receptor-1, CD36, DC-SIGN, and Dectin-1 and that the FSL-1-induced upregulation of their mRNAs was inhibited by resveratrol. In addition, it was found that the expression of DC-SIGN in HEK293 cells stably expressing DC-SIGN was reduced by resveratrol and that the phagocytic activity was significantly inhibited by resveratrol. Thus, this study suggests that resveratrol inhibited bacterial phagocytosis by macrophages by downregulating the expression of phagocytic receptors and NF-κB activity.

The TLR2 ligand FSL-1 is internalized via clathrin-dependent endocytic pathway triggered by CD14 and CD36 but not by TLR2

Little is known of how Toll‐like receptor (TLR) ligands are processed after recognition by TLRs. This study was therefore designed to investigate how the TLR2 ligand FSL‐1 is processed in macrophages after recognition by TLR2. FSL‐1 was internalized into the murine macrophage cell line, RAW264.7. Both chlorpromazine and methyl‐β‐cyclodextrin, which inhibit clathrin‐dependent endocytosis, reduced FSL‐1 uptake by RAW264.7 cells in a dose‐dependent manner but nystatin, which inhibits caveolae‐ and lipid raft‐dependent endocytosis, did not. FSL‐1 was co‐localized with clathrin but not with TLR2 in the cytosol of RAW264.7 cells. These results suggest that internalization of FSL‐1 is clathrin dependent. In addition, FSL‐1 was internalized by peritoneal macrophages from TLR2‐deficient mice. FSL‐1 was internalized by human embryonic kidney 293 cells transfected with CD14 or CD36 but not by the non‐transfected cells. Also, knockdown of CD14 or CD36 in the transfectants reduced FSL‐1 uptake. In this study, we suggest that (i) FSL‐1 is internalized into macrophages via a clathrin‐dependent endocytic pathway, (ii) the FSL‐1 uptake by macrophages occurs irrespective of the presence of TLR2, and (iii) CD14 and CD36 are responsible for the internalization of FSL‐1.