Kazuto Kiura

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.

CD14 directly binds to triacylated lipopeptides and facilitates recognition of the lipopeptides by the receptor complex of Toll-like receptors 2 and 1 without binding to the complex

It has demonstrated that the recognition of triacylated lipopeptides by Toll‐like receptor (TLR) 2 requires TLR1 as a coreceptor. In the NF‐κB reporter assay system in which human embryonic kidney 293 cells were transfected with TLR2 and TLR1 together with an NF‐κB luciferase reporter gene, S‐(2,3‐bispalmitoyloxypropyl)‐N‐palmitoyl‐Cys‐Lys‐Lys‐Lys‐Lys (Pam3CSK4) and Pam3CSSNA were recognized by TLR2/TLR1, but the recognition level was unexpectedly very low. However, cotransfection of CD14 drastically enhanced the recognition of triacylated lipopeptides by TLR2/TLR1. The CD14‐induced enhancement did not occur without cotransfection of TLR1. Both CD14dS39‐A48, a mutant with deletion of the part of possible N‐terminal ligand‐binding pocket, and anti‐CD14 monoclonal antibody reduced the CD14‐induced enhancement. Transfection of a TIR domain‐deficient mutant of TLR2 (TLR2dE772‐S784) or TLR1 (TLR1dQ636‐K779) completely abrogated the CD14‐induced enhancement. Soluble recombinant CD14 added extracellularly enhanced the recognition of Pam3CSSNA by TLR2/TLR1. Immunoprecipitation analysis demonstrated that CD14 was not associated with TLR2 but that TLR1 was associated with TLR2. In addition, surface plasmon resonance‐based assay demonstrated that CD14 binds to Pam3CSK4 at a dissociation constant of 5.7 µM. This study suggests that CD14 directly binds to triacylated lipopeptides and facilitates recognition of the lipopeptides by the TLR2/TLR1 complex without binding to the receptor complex.

Roles of N-linked glycans in the recognition of microbial lipopeptides and lipoproteins by TLR2

Details of roles of carbohydrates attached to Toll‐like receptors (TLRs) in the recognition of pathogen‐associated molecular patterns and in the formation of the functional receptor complex still remain unknown. This study was designed to determine whether the glycans linked at Asn114, Asn199, Asn414 and Asn442 residues of TLR2 ectodomain were involved in the recognition of diacylated lipopeptide and lipoprotein. Single and multiple mutants were transfected into human embryonic kidney (HEK) 293 cells together with a NF‐κB luciferase reporter plasmid. All of these mutants were expressed on the surface. SDS‐PAGE of the transfectants demonstrated that these mutants migrated lower than wild‐type TLR2 and their molecular masses decreased as the number of mutated Asn residues increased. TLR2N114A, TLR2N199A and TLR2N414A as well as wild‐type TLR2 induced NF‐κB activation when stimulated with these ligands, whereas TLR2N442A failed to induce NF‐κB activation. All of triple and quadruple mutants failed to induce NF‐κB activation, but were associated with both wild‐type TLR2 and TLR6 in the transfectants. TLR2N114A,N199A, TLR2N114A,N414A and, to a lesser extent, TLR2N114A,N442A, in which two N‐linked glycans are speculated to be exposed to the concave surface of TLR2 solenoid, not only induce NF‐κB activation but also are associated with wild‐type TLR2 and TLR6. These results suggest that the glycan at Asn442 and at least two N‐linked glycans speculated to be exposed to the concave surface of TLR2 solenoid are involved in the recognition of ligands by TLR2 and/or in formation or maturation of a functional TLR2 receptor complex.

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.

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.

In vivo anti- and pro-tumour activities of the TLR2 ligand FSL-1

TLR ligands as Th1 inducers have been investigated as potential anti-tumour agents. However, few attempts have been made to investigate the anti-tumour activity of TLR ligands as Th2 inducers. This study, therefore, was carried out to determine whether the TLR2 ligand FSL-1 as a Th2 inducers affects the growth of a QRsP tumour, a fibrosarcoma derived from the C57BL/6 (TLR2(+/+)) mouse in vivo. Tumour volumes in TLR2(+/+) mice immunized with both FSL-1 and tumour-associated antigens were significantly smaller than those in control mice. Immunization with both FSL-1 and tumour-associated antigens increased the survival rate of TLR2(+/+) mice. However, surprisingly, immunization with FSL-1 alone significantly enhanced the growth of tumour. Both anti- and pro-tumour activities of FSL-1 were not observed in TLR2(-/-) mice. Immunization of both FSL-1 and tumour-associated antigens induced tumour-associated antigen-specific cytolytic T cells, antibody-dependent cell-mediated cytotoxicity of natural killer cells by production of the tumour-specific antibodies, tumour lysis by complement activation and reduction of the number of regulatory T cells in the draining lymph node. Immunization with FSL-1 alone increased the number of regulatory T cells in the draining lymph node, and in vivo administration of anti-CD25 antibody into mice abrogated the pro-tumour activity of FSL-1, suggesting that regulatory T cells are involved in the pro-tumour activity. This study demonstrated that FSL-1 exhibited TLR2-mediated anti- and pro-tumour activities when immunized with and without tumour-associated antigens, respectively.