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The diacylated lipopeptide FSL-1 enhances phagocytosis of bacteria by macrophages through Toll-like receptor 2- Title mediated signaling pathway Mae, Masako; Iyori, Mitsuhiro; Yasuda, Motoaki; Shamsul, Haque; Kataoka, Hideo; Kiura, Kazuto; Hasebe, Akira; Author(s) Totsuka, Yasunori; Shibata, Ken-ichiro FEMS immunology and medical microbiology, 49, 398-409 Citation https://doi.org/10.1111/j.1574-695X.2007.00218.x Issue Date 2007-02-22 Doc URL http://hdl.handle.net/2115/43975 Type article (author version) File Information FEMStext mae huscup.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP The diacylated lipopeptide FSL-1 enhances phagocytosis of bacteria by macrophages through Toll-like receptor 2-mediated signaling pathway Masako Mae1,2*, Mitsuhiro Iyori1*, Motoaki Yasuda1, Haque Mohammad Shamsul1, Hideo Kataoka1, Kazuto Kiura1, Akira Hasebe1, Yasunori Totsuka2 and Ken-ichiro Shibata1 Laboratories of Oral Molecular Microbiology1 and Oral & Maxillofacial Surgery2, Department of Oral Pathobiological Science, Hokkaido University Graduate School of Dental Medicine, Nishi 7, Kita 13, Kita-ku, Sapporo 060-8586, Japan. Running title: Enhancement of phagocytosis by a lipopeptide Keywords: Toll-like receptor 2, the lipopeptide FSL-1, phagocytosis, macrophage *: Masako Mae and Mitsuhiro Iyori are equally contributed to this work. Correspondence: Ken-ichiro Shibata, Laboratory of Oral Molecular Microbiology, Department of Oral Pathobiological Science, Hokkaido University Graduate School of Dental Medicine, Nishi 7, Kita 13, Kita-ku, Sapporo 060-8586, Japan. Phone: 81-11-706-4240. Fax: 81-11-706-4901. E-mail: [email protected] Abstract Enormous lines of evidence have been accumulated that Toll-like receptors (TLRs) function as sensors for microbial invasion. However, less is known about how signaling triggered by TLRs leads to phagocytosis of pathogens. This study was designed to determine whether stimulation of TLR2 with mainly the lipopeptide FSL-1 plays a role in phagocytosis of pathogens by macrophages. FSL-1 markedly enhanced phagocytosis of E. coli more strongly than that of S. aureus, but did not enhance phagocytosis of latex beads. FSL-1 stimulation resulted in enhanced phagocytosis of bacteria by macrophages from TLR2+/+ mice but not those from TLR2-/- mice. Chinese hamster ovary cells stably expressing TLR2 failed to phagocytose these bacteria, but the cells expressing CD14 did. FSL-1 induced upregulation of the expression of phagocytic receptors including MSR1, CD36, DC-SIGN and Dectin-1 in THP-1 cells. Human embryonic kidney 293 cells transfected with DC-SIGN and MSR1 phagocytosed these bacteria. These results suggest that the FSL-1-induced enhancement of phagocytosis of bacteria by macrophages may be explained partially by the upregulation of scavenger receptors and the C-type lectins through TLR2-mediated signaling pathways and that TLR2 by itself does not function as a phagocytic receptor. Introduction Detection of bacterial invasion and the uptake and killing of bacteria are a key function of the innate immune system. Phagocytes, such as macrophages, neutrophils and dendritic cells, play important roles in combating bacterial infection. These phagocytes are able to detect bacterial invasion through various pattern recognition receptors such as Toll-like receptors (TLRs) before uptake and degradation of bacteria. To date, more than ten TLRs have been identified and have been shown to be critical for signaling by pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), peptidoglycan (PGN), lipoteichoic acid (LTA), and lipoprotein/lipopeptide (LP/LPT) (Takeda et al., 2003). The activation of innate immunity by TLRs also leads to the development of antigen-specific adaptive immunity. Thus, 1 TLRs also play a key role in bridging between innate immunity and adaptive immunity (Takeda et al., 2003). Therefore, it is thought that TLRs not only detect bacterial invasion but also play essential roles in activating signal transduction pathways leading to the killing and clearance of pathogens, since the events after detection of bacterial invasion are uptake and degradation of pathogens for presentation of antigens in the context of MHC to T cells. There have been only a few reports of TLRs playing roles in phagocytosis of bacteria by phagocytic cells. Doyle et al. (Doyle et al., 2004) have recently demonstrated that numerous TLR ligands specifically enhance phagocytosis of bacteria by macrophages. Blander and Medzhitov et al. (Blander and Medzhitov, 2004) have also suggested that TLR-mediated signaling regulates bacterial phagocytosis. However, it is not fully understood how signaling triggered by TLRs leads to endocytosis and digestion of pathogens in the endosome. LP is a bacterial cell wall component involved in the systemic inflammatory response caused by gram-negative and gram-positive bacteria as well as in chronic inflammatory disorders (Zhang et al., 1998; Zhang et al., 1997; Brandt et al., 1990; Chamberlain et al., 1989). Recently, we have purified and characterized mycoplasmal LP44 responsible for activation of macrophages and fibroblasts (Shibata et al., 2000; Dong et al., 1999) and synthesized the diacylated LPT called FSL-1 [S-(2,3-bispalmitoyloxypropyl) CGDPKHPKSF] on the basis of the structure of LP44. In addition, we have found that FSL-1 is recognized by TLR2 and/or TLR6 (Okusawa et al., 2004; Fujita et al., 2003). Therefore, this study was designed to determine whether stimulation of TLR2 with FSL-1 plays a role in phagocytosis of pathogens by macrophages. Esherichia coli LPS and Staphylococcus aureus PGN were also used as TLR stimulants for comparative studies. Materials and methods Antibodies and Reagents Polyclonal antibodies (pAbs) to phospho-phosphoinositide 3-kinase (PI3-K) p85 binding motif (#3821), nonphosphorylated PI3-K (#4292) and Akt/PKB kinase (#9272) and monoclonal antibodies (mAbs) to phospho-Akt/PKB (Ser473) kinase (#4051) were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-CD36 mAb was obtained from Abcam (Stockholm, Sweden), and phycoerythrin (PE)-conjugated anti-human TLR2 and PE-conjugated mouse IgG2a isotype control were purchased from eBioscience (San Diego, CA, USA). mAbs to the phosphorylated and nonphosphorylated mitogen-activated protein kinases (MAPKs) ERK1/2, SAPK/JNK and p38 were purchased from BD Pharmingen (San Diego, CA). E. coli LPS O55:B5, wortmannin, cytochalasin-D and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). S. aureus PGN was obtained from Fluka Chemie GmbH. FSL-1 derived from Mycoplasma salivarium was synthesized according to the method described previously (Shibata et al., 2000). SB203580 was purchased from Calbiochem-Novabiochem Co. (La Jolla, CA, USA). pUNO-hDC-SIGN1a (human DC-specific intercellular adhesion molecule-grabbing nonintegrin1a) and pUNO-hDectin1b were purchased from InvivoGen (San Diego, CA, USA). All of the other chemicals were obtained from commercial sources and were of analytical or reagent grade. Gene cloning and transfection The cDNAs of human TLR2, CD36 and macrophage scavenger receptor 1 (MSR1) were obtained by RT-PCR of RNA isolated from THP-1 cells, a human monocytic cell line. The cDNA of TLR2 was cloned into a pEF6/V5-His TOPO vector (hereafter referred to as pEF-TLR2) (Invitrogen Co., Carlsbad, CA, USA) and other cDNAs were cloned into a pcDNA3.1-His-TOPO (pcDNA3.1-CD36 and pcDNA3.1-MSR1) (Invitrogen). CHO K1 cells grown in F-12 medium containing fetal bovine serum (FBS) (Gibco BRL, Rockville, MD, USA), penicillin G (100 units mL-1) and streptomycin (100 μg mL-1) were transfected with pEF-TLR2 by Fugene 6 Transfection Reagent (Roche Molecular Biochemicals, 2 Indianapolis, IN, USA), and stable transfectants (CHO/TLR2) were selected in the presence of 10 μg mL-1 blasticidin (Invitrogen). The expression of TLR2 was detected by polyclonal antibody to TLR2 prepared in our laboratory (Fujita et al., 2003). CHO cells expressing CD14 (CHO/CD14) and CHO/CD14/TLR2 cells were kindly provided by Dr. D. G. Golenbock, Department of Medicine, Division of Infectious Diseases & Immunology, University of Massachusetts Medical School, Boston. Human embryonic kidney (HEK) 293 cells obtained from ATCC (CRL-1573) were maintained in DMEM containing 10% FBS, penicillin G (100 units mL-1) and streptomycin (100 μg mL-1). HEK293 cells were transiently transfected with pUNO-hDC-SIGN1a, pUNO-hDectin1b, pcDNA3.1-CD36 or pcDNA3.1-MSR1 and are referred to as 293/DC-SIGN, 293/Dectin1, 293/CD36 or 293/MSR1, respectively. Mice and peritoneal macrophages (PM) Sex-matched C57BL/6 mice (TLR2+/+ mice) were purchased from Japan Clea (Tokyo, Japan). TLR2-deficient mice (TLR2-/- mice) generated by gene targeting on the same genetic background were kindly provided by Dr. Shizuo Akira, Department of Host Defense, Research Institute for Microbial Diseases, Osaka University (Osaka, Japan). All mice were maintained in specific pathogen-free conditions at the animal facility at Hokkaido University, and all experiments were approved by the regulations of Hokkaido University Animal Care and Use Committee. Peritoneal exudate cells were harvested by lavage with 6 ml of cold Hank’s balanced salt solution. They were washed twice in RPMI 1640 medium without serum and antibiotics (RPMI 1640 base medium) and then resuspended in RPMI 1640 medium containing 10% (vol/vol) FBS, penicillin G (100 units mL-1) and streptomycin (100 μg mL-1) (RPMI 1640