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• W2104630342 abstract "Studies involving Toll-like receptor 3 (TLR3)-deficient mice suggest that this receptor binds double-stranded RNA. In the present study, we analyzed ligand/receptor interactions and receptor-proximal events leading to TLR3 activation. The mutagenesis approach showed that certain cysteine residues and glycosylation in TLR3 amino-terminal leucine-rich repeats were necessary for ligand-induced signaling. Furthermore, inactive mutants had a dominant negative effect, suggesting that the signaling module is a multimer. We constructed a chimeric molecule fusing the amino-terminal ectodomain of TLR3 to the transmembrane and carboxyl terminal domains of CD32a containing an immunoreceptor tyrosine-based motif. Expression of TLR3-CD32 in HEK293T cells and the myeloid cell line U937 resulted in surface localization of the receptor, whereas the nonrecombinant molecule was intracellularly localized. The synthetic double-stranded RNAs poly(I-C) and poly(A-U) induced calcium mobilization in a TLR3-CD32 stably transfected U937 clone but not in control cells transfected with other constructs. An anti-TLR3 antibody also induced Ca2+ flux but only when cross-linked by a secondary anti-immunoglobulin antibody, confirming that multimerization by the ligand is a requirement for signaling. The inhibitors of lysosome maturation, bafilomycin and chloroquine, inhibited the poly(I-C)-induced biological response in immune cells, showing that TLR3 interacted with its ligand in acidic subcellular compartments. Furthermore, TLR3-CD32 activation with poly(I-C) was only observed within a narrow pH window (pH 5.7–6.7), whereas anti-TLR3-mediated Ca2+ flux was pH-insensitive. The importance of an acidic pH for TLR3-ligand interaction becomes critical when using oligomeric poly(I-C) (15–40-mers). These observations demonstrate that engagement of TLR3 by poly(I-C) at an acidic pH, probably in early phagolysosomes or endosomes, induces receptor aggregation leading to signaling. Studies involving Toll-like receptor 3 (TLR3)-deficient mice suggest that this receptor binds double-stranded RNA. In the present study, we analyzed ligand/receptor interactions and receptor-proximal events leading to TLR3 activation. The mutagenesis approach showed that certain cysteine residues and glycosylation in TLR3 amino-terminal leucine-rich repeats were necessary for ligand-induced signaling. Furthermore, inactive mutants had a dominant negative effect, suggesting that the signaling module is a multimer. We constructed a chimeric molecule fusing the amino-terminal ectodomain of TLR3 to the transmembrane and carboxyl terminal domains of CD32a containing an immunoreceptor tyrosine-based motif. Expression of TLR3-CD32 in HEK293T cells and the myeloid cell line U937 resulted in surface localization of the receptor, whereas the nonrecombinant molecule was intracellularly localized. The synthetic double-stranded RNAs poly(I-C) and poly(A-U) induced calcium mobilization in a TLR3-CD32 stably transfected U937 clone but not in control cells transfected with other constructs. An anti-TLR3 antibody also induced Ca2+ flux but only when cross-linked by a secondary anti-immunoglobulin antibody, confirming that multimerization by the ligand is a requirement for signaling. The inhibitors of lysosome maturation, bafilomycin and chloroquine, inhibited the poly(I-C)-induced biological response in immune cells, showing that TLR3 interacted with its ligand in acidic subcellular compartments. Furthermore, TLR3-CD32 activation with poly(I-C) was only observed within a narrow pH window (pH 5.7–6.7), whereas anti-TLR3-mediated Ca2+ flux was pH-insensitive. The importance of an acidic pH for TLR3-ligand interaction becomes critical when using oligomeric poly(I-C) (15–40-mers). These observations demonstrate that engagement of TLR3 by poly(I-C) at an acidic pH, probably in early phagolysosomes or endosomes, induces receptor aggregation leading to signaling. Mammalian Toll-like receptors (TLRs) 7The abbreviations used are: TLR, Toll-like receptor; DC, dendritic cell(s); HEK, human embryonic kidney; IL, interleukin; ITAM, immunoreceptor tyrosine-based motif; LRR, leucine-rich repeat; MonoDC, monocyte-derived DC(s); PBMC, peripheral blood mononuclear cell(s); TRIF, Toll/interleukin-1 receptor domain-containing adapter protein; dsRNA, double-stranded RNA. belong to a family of receptors that recognize pathogen-associated molecular patterns. TLRs play a key role in host defense during pathogen infection by regulating and linking the innate and adaptive immune responses (1.Medzhitov R. Janeway Jr., C. Trends Microbiol. 2000; 8: 452-456Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 2.Akira S. Takeda K. Kaisho T. Nat. Immunol. 2001; 2: 675-680Crossref PubMed Scopus (3946) Google Scholar, 3.Takeda K. Kaisho T. Akira S. Annu. Rev. Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4751) Google Scholar). TLRs are expressed in dendritic cells (DC), sentinels of the immune system, endowing them with the capacity to sense pathogen-derived products and to alert the immune system (4.Kaisho T. Akira S. Curr. Mol. Med. 2003; 3: 373-385Crossref PubMed Scopus (159) Google Scholar). Members of the TLR family are also variably expressed on nonhematopoietic cells. TLR-deficient mice and transfected cell lines have been the keys to understanding TLR function. Ligand specificity has been elucidated for most TLRs; thus, TLR2 and TLR4 recognize Gram-positive and Gram-negative bacterial cell wall products, respectively. TLR5 recognizes a structural epitope of bacterial flagellin, and TLR7, TLR8, and TLR9 have been demonstrated to recognize different forms of microbial-derived nucleic acid (5.Beutler B. Nature. 2004; 430: 257-263Crossref PubMed Scopus (1261) Google Scholar). Host-derived ligands for the TLRs have also been identified; in particular, TLR4 recognizes heat shock proteins and pulmonary surfactant (6.Beg A.A. Trends Immunol. 2002; 23: 509-512Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar), and TLR9 recognizes chromatin-IgG complexes (7.Leadbetter E.A. Rifkin I.R. Hohlbaum A.M. Beaudette B.C. Shlomchik M.J. Marshak-Rothstein A. Nature. 2002; 416: 603-607Crossref PubMed Scopus (1614) Google Scholar). TLR3 has been extensively characterized to be a receptor for poly(I-C), a synthetic double-stranded RNA (dsRNA) mimic (8.Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4936) Google Scholar); recently, it has been shown to mediate responses to West Nile virus (9.Wang T. Town T. Alexopoulou L. Anderson J.F. Fikrig E. Flavell R.A. Nat. Med. 2004; 10: 1366-1373Crossref PubMed Scopus (903) Google Scholar) as well as dsRNA derived from the helminth parasite Schistosoma (10.Aksoy E. Zouain C.S. Vanhoutte F. Fontaine J. Pavelka N. Thieblemont N. Willems F. Ricciardi-Castagnoli P. Goldman M. Capron M. Ryffel B. Trottein F. J. Biol. Chem. 2005; 280: 277-283Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Additionally, host-derived mRNA has recently been shown to activate TLR3, suggesting that activation via TLR3 can occur in a variety of situations (11.Kariko K. Ni H. Capodici J. Lamphier M. Weissman D. J. Biol. Chem. 2004; 279: 12542-12550Abstract Full Text Full Text PDF PubMed Scopus (828) Google Scholar). Whereas most TLRs recruit myeloid differentiation factor 88 (12.O'Neill L.A. Fitzgerald K.A. Bowie A.G. Trends Immunol. 2003; 24: 286-290Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar), with some variation in the signaling profile mediated by the additional recruitment of Toll/interleukin-1 receptor-containing adapter protein and TRIF-related adaptor molecule, TLR3 recruits only TRIF (13.Yamamoto M. Sato S. Hemmi H. Uematsu S. Hoshino K. Kaisho T. Takeuchi O. Takeda K. Akira S. Nat. Immunol. 2003; 4: 1144-1150Crossref PubMed Scopus (827) Google Scholar, 14.Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. J. Immunol. 2002; 169: 6668-6672Crossref PubMed Scopus (1022) Google Scholar, 15.Hoebe K. Beutler B. J. Endotoxin Res. 2004; 10: 130-136Crossref PubMed Scopus (55) Google Scholar). TRIF can activate both NF-κB through TRAF6 and receptor-interacting protein-1 (16.Yamamoto M. Sato S. Hemmi H. Hoshino K. Kaisho T. Sanjo H. Takeuchi O. Sugiyama M. Okabe M. Takeda K. Akira S. Science. 2003; 301: 640-643Crossref PubMed Scopus (2510) Google Scholar) and interferon-regulatory factor 3 through IκB kinase/TANK-binding kinase 1 (17.Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2085) Google Scholar) and phosphoinositide 3-kinase (18.Sarkar S.N. Peters K.L. Elco C.P. Sakamoto S. Pal S. Sen G.C. Nat. Struct. Mol. Biol. 2004; 11: 1060-1067Crossref PubMed Scopus (309) Google Scholar). Although the signaling pathway of TLRs is increasingly well characterized, the parameters controlling interactions between the receptors and the ligands remain poorly documented. A recent review (19.Mukhopadhyay S. Herre J. Brown G.D. Gordon S. Immunology. 2004; 112: 521-530Crossref PubMed Scopus (117) Google Scholar) summarized the role of individual members of the TLR family or other surface antigens that either physically or functionally interact with TLRs and how the cumulative effects of these interactions instruct the nature and outcome of the immune response to a particular pathogen. For example, TLR2 recognizes specific ligands upon association with TLR1 or TLR6, and this heterodimerization is essential for signaling (20.Ozinsky A. Underhill D.M. Fontenot J.D. Hajjar A.M. Smith K.D. Wilson C.B. Schroeder L. Aderem A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13766-13771Crossref PubMed Scopus (1687) Google Scholar, 21.Sandor F. Latz E. Re F. Mandell L. Repik G. Golenbock D.T. Espevik T. Kurt-Jones E.A. Finberg R.W. J. Cell Biol. 2003; 162: 1099-1110Crossref PubMed Scopus (105) Google Scholar). Equally, in most signaling assays involving recombinant CD4-TLR fusions, the CD4 moiety induces dimerization in order to produce constitutive signaling (22.Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4439) Google Scholar). Whereas TLR2 and TLR4 are expressed at the cell surface, TLR7, -8, and -9 traffic between the endoplasmic reticulum and lysosomes (23.Ahmad-Nejad P. Hacker H. Rutz M. Bauer S. Vabulas R.M. Wagner H. Eur. J. Immunol. 2002; 32: 1958-1968Crossref PubMed Scopus (632) Google Scholar, 24.Heil F. Ahmad-Nejad P. Hemmi H. Hochrein H. Ampenberger F. Gellert T. Dietrich H. Lipford G. Takeda K. Akira S. Wagner H. Bauer S. Eur. J. Immunol. 2003; 33: 2987-2997Crossref PubMed Scopus (456) Google Scholar, 25.Lee J. Chuang T.H. Redecke V. She L. Pitha P.M. Carson D.A. Raz E. Cottam H.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6646-6651Crossref PubMed Scopus (518) Google Scholar). The recognition of CpG-containing DNA by TLR9 has been shown to occur in lysosomes (26.Latz E. Schoenemeyer A. Visintin A. Fitzgerald K.A. Monks B.G. Knetter C.F. Lien E. Nilsen N.J. Espevik T. Golenbock D.T. Nat. Immunol. 2004; 5: 190-198Crossref PubMed Scopus (1158) Google Scholar), and optimal interaction was observed at an acidic pH (27.Rutz M. Metzger J. Gellert T. Luppa P. Lipford G.B. Wagner H. Bauer S. Eur. J. Immunol. 2004; 34: 2541-2550Crossref PubMed Scopus (456) Google Scholar). In addition, recognition of single-stranded RNA by TLR7 has been reported to require an acidic compartment (25.Lee J. Chuang T.H. Redecke V. She L. Pitha P.M. Carson D.A. Raz E. Cottam H.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6646-6651Crossref PubMed Scopus (518) Google Scholar, 28.Diebold S.S. Kaisho T. Hemmi H. Akira S. Reis e Sousa C. Science. 2004; 303: 1529-1531Crossref PubMed Scopus (2739) Google Scholar). In the case of TLR3, which is expressed on myeloid DC as well as on nonhematopoietic cells (29.Matsumoto M. Seya T. Uirusu. 2001; 51: 209-214Crossref PubMed Scopus (7) Google Scholar), cell surface expression has been described in both fibroblast and epithelial cells (30.Matsumoto M. Kikkawa S. Kohase M. Miyake K. Seya T. Biochem. Biophys. Res. Commun. 2002; 293: 1364-1369Crossref PubMed Scopus (381) Google Scholar), whereas it is reported to be expressed in an intracellular compartment in monocyte-derived dendritic cells (31.Matsumoto M. Funami K. Tanabe M. Oshiumi H. Shingai M. Seto Y. Yamamoto A. Seya T. J. Immunol. 2003; 171: 3154-3162Crossref PubMed Scopus (621) Google Scholar). Thus, to date, the exact subcellular compartment in which the interaction between TLR3 and its ligand occurs remains unknown. The construction of chimeric molecules involving different TLRs or between TLR and other molecules (e.g. CD4) has been used mainly to decipher TLR signaling pathways (22.Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4439) Google Scholar, 32.Hasan U.A. Dollet S. Vlach J. Biochem. Biophys. Res. Commun. 2004; 321: 124-131Crossref PubMed Scopus (24) Google Scholar, 33.Zhang G. Ghosh S. J. Biol. Chem. 2002; 277: 7059-7065Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). We thus decided to develop novel chimeric molecules to study interactions between TLR3 and its ligands. We constructed a recombinant molecule fusing the extracellular amino-terminal of TLR3 and the transmembrane and intracellular carboxyl terminus of CD32a. CD32a is a transmembrane molecule displaying an immunoreceptor tyrosine-based activation motif (ITAM) allowing recruitment of a cytosolic kinase complex, including phosphoinositide 3-kinase and phospholipase Cγ, leading to calcium mobilization from intracellular stores (34.Daeron M. Annu. Rev. Immunol. 1997; 15: 203-234Crossref PubMed Scopus (1046) Google Scholar). Calcium flux assays are immediate, simple to perform, and highly sensitive and do not have the classical toxicity problems of molecules or solvent often found in reporter assays. The TLR3-CD32 construction was expressed in the myeloid cell line U937. We observed cell surface expression of the chimera and calcium mobilization upon TLR3 aggregation. Using this model as well as mutants that produced a dominant negative effect, we demonstrate that poly(I-C) mediates TLR3 cross-linking, and we observe a critical role of acidic pH in mediating this activation. Furthermore, we show that physiologically, in ex vivo immune cells, the TLR3/ligand interaction is dependent on the acidification of a subcellular compartment. Isolation of Immune Cells and Cell Culture—Human blood samples were obtained according to institutional guidelines. Peripheral blood mononuclear cells (PBMC) were purified by Ficoll-Hypaque centrifugation. For monocyte-derived DC (MonoDC) generation, monocytes were further purified as low density cells on a 52% Percoll gradient and plated for adherence. Nonadherent cells were removed, and complete RPMI (Invitrogen) supplemented with 200 ng/ml recombinant human granulocyte-macrophage colony-stimulating factor and 10 ng/ml recombinant human IL-4 (Schering-Plough Research Institute, Kenilworth, NJ) was added for 5–6 days (35.Sallusto F. Lanzavecchia A. J. Exp. Med. 1994; 179: 1109-1118Crossref PubMed Scopus (4504) Google Scholar). Cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Flow Laboratories, Irvine, UK), 2 mm l-glutamine, 100 μg/ml gentamicin (Schering-Plough, Levallois-Perret, France). HEK293T and HEK293 cell lines (ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium/F-12 with Glutamax (Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum. Activation of PBMC and DC and Detection of Cytokine Secretion—Freshly isolated PBMC and MonoDC were plated at a concentration of 106 cells/ml and 5 × 105 cells/ml, respectively, and activated with different TLR ligands for 18 h. The following activating factors were used at final concentrations of 10 μg/ml poly(I-C) (InvivoGen, San Diego, CA), 10 μg/ml poly(A-U) (Sigma), 5 μg/ml CpG D19 (synthesized by MWG (Courtaboeuf, France)), 1 or 10 μm R-848 (imidazoquinoline, resiquimod synthesized in our laboratory; Schering-Plough), and 1 μg/ml or 10 ng/ml Escherichia coli lipopolysaccharide (Sigma) for PBMC and MonoDC, respectively. Chloroquine and bafilomycin (Sigma) were added 45 min prior to the addition of activators. After 18 h, supernatants were collected and tested for IP-10/CXCL10 and IL-6 secretion by enzyme-linked immunosorbent assay (BD OptiEIA; BD Biosciences). Results are expressed as a mean of triplicates, and statistical analysis was performed using GraphPad Prism 3 (GraphPad Software, Inc., San Diego, CA). Generation and Use of Small Poly(I-C) Fragments—Poly(I) (15-, 30-, and 40-mers) and poly(C) (15-, 30-, and 40-mers) (purchased from Ambion (Huntingdon, Cambridgeshire, UK) and Invitrogen) were mixed in equal proportions according to their size for 15 min at 95 °C. Independently, fragmentation of high molecular weight poly(I-C) was performed by metal-induced hydrolysis as recommended by Affymetrix (Affymetrix protocols for eukaryotic target preparation; Affymetrix, High Wycombe, UK). Briefly, 200 μg of poly(I-C) was incubated with 40 μlof5× RNA fragmentation buffer (200 mm Tris acetate, pH 8.1, 500 mm KOAc, 150 mm MgOAc) (Sigma) in a final volume of 200 μl, for 35 min at 90 °C. Fragmented poly(I-C) was then allowed to reach room temperature before purification. The relative sizes of dsRNA fragments were examined on a 2% agarose gel (NuSieveR 3:1; Bio-Whittaker Molecular Applications, Walkersville, ME). Mutagenesis of Human TLR3—Full-length human TLR3 was subcloned from the TLR3-pUNO plasmid (InvivoGen) into pMET7 (a gift of the DNAX Institute, Schering-Plough Research Institute, Palo Alto, CA), and TLR3-pMET7 was then used for mutagenesis. Generation of the TLR3 mutants was performed using site-directed mutagenesis of residues in the luminal domain of human TLR3. Mutations were inserted using the GeneEditor kit (Invitrogen) using oligonucleotides summarized below. Pairs of oligonucleotides were as follows: C95A, forward (5′-ACTGGAGCCAGAATTCGCCCAGAAACTTCCC-3′) and reverse (5′-AATTCTGGCTCCAGTTTTGAGATGG-3′); C122A, forward (5′-GATAAAACCTTTGCCTTCGCGACGAATTTGA-3′ and reverse (5′-GAAGGCAAAGGTTTTATCAGAAAGTTG-3′); C226A, forward (5′-GAGTTTTCTCCAGGGGCCCTTCACGCAATTGG-3′) and reverse (5′-CCCTGGAGAAAACTCTTTAATTTGATTCG-3′); C242A, forward (5′-GCCTTACAGAGAAGCTTGCGTTGGAATTAGC-3′) and reverse (5′-AGCTTCTCTGTAAGGCTGGGACCCAGCTGG-3′); N196G, forward (5′-GAACTGGATATCTTTGCCGGCTCATCTTT-3′) and reverse (5′-GGCAAAGATATCCAGTTCTTCTTCACTTTTTAGC-3′); N247R, forward (5′-GAAGCTATGTTTGGAATTAGCCGGCACAAGCATTC-3′) and reverse (5′-GCTAATTCCAAACATAGCTTCTCTGTAAGGC-3′). The inserted restriction site used to identify the mutation is underlined. After mutagenesis, the mutants were entirely sequenced, and no other mutations were detected. Double mutants (CC1A and NN3A) were created by a second round of mutagenesis on selected plasmids. Transient Expression in HEK293T and U937—HEK293T cells were seeded into 24-well plates to give 50–70% confluence on the day of transfection. Cells were transfected with TLR3-pMET7 by incubating 3 μl of Fugene 6 (Roche Applied Science, Mannheim, Germany) with 1 μg of plasmid in 100 μl of Dulbecco's modified Eagle's medium/F-12 for 30 min. The mixture was divided to give a final transfection amount of 250 ng/well. After an 18-h incubation, cells were recovered and harvested for fluorescence-activated cell sorting analysis. U937 cells were nucleofected using the Nucleofector™ technology (Amaxa Biosystems, Cologne, Germany) with the program T14 and the nucleofector solution V, as recommended by the manufacturer. Briefly, 6–8 × 106 cells were washed in PBS and resuspended extemporaneously in 100 μl of supplemented solution V at room temperature, and 6 μg of plasmid was added to the cell suspension and transferred in an appropriate cuvette for nucleoporation impulsion. After 18 h, cells were harvested for assays. NF-κB Reporter Assay—Tissue culture plates (6-well) with HEK293 or HEK293T cells were transiently transfected using Fugene 6 as described above, with 500 ng of NF-κB luciferase reporter plasmid (Invitrogen) together with constant and increasing amounts of the wild type and mutant TLR3 expression vectors, respectively. Controls to test reporter activity of wild type and mutant TLR3 constructions were also tested in HEK293T cells. Cells were transiently transfected with 500 ng of NF-κB luciferase plasmid together with 250 ng of wild type or mutant TLR3 pMET7 vectors. In all transfections, the total DNA amounts were kept constant using empty vector (pMET7). Six hours post-transfection, cells from each transfection were divided into 96-well plates. The following day, cells were stimulated with poly(I-C) at 5 μg/ml for 6 h and analyzed for luciferase activity. Luciferase Assay—Cells were analyzed for firefly luciferase activity using Promega Steady-Glo reporter assay reagents as described by the manufacturer (Promega, Charbonnières, France). Luciferase units were measured using a Fusion α detection apparatus (Packard Instrument Co., Meriden, CT). Each experiment was performed in triplicate and was repeated three times; results generally deviated by less than 10% of the mean value. Western Blot of TLR3 Constructs—HEK293T cells were transiently transfected using Fugene 6 as described above. 48 h post-transfection, transfected HEK293T and nontransfected HEK293 cells were lysed in mild lysis buffer containing 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Triton X-100, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 1% aprotinin, 20 mm NaF, and 0.3 mm ortho-sodium vanadate. In general, 30 μg of total cellular protein (determined by Bradford assay; Bio-Rad) was used for SDS-NuPAGE and immunoblotting (Invitrogen). After incubation with anti-TLR3 antibody (clone 40C1285; Imgenex, Sorrento Valley, CA), reactive proteins were detected with peroxidase-conjugated anti-mouse secondary antibodies (Jackson Immuno-Research Laboratories, Inc., Soham, Cambridgeshire, UK) and ECL (Amersham Biosciences). Construction of the TLR3-CD32 Chimera and Cloning into the Expression Vector pMET7—The luminal domain of human TLR3 was amplified from the TLR3-pUNO plasmid using the primers TLR3-SalI forward (5′-ACGCGTCGACGATCATGAGACAGACTTTGCC-3′) and TLR3-AsnI reverse (5′-TAGCATTAATAGTTCAAAGGGGGCACTGTC-3′). The transmembrane and intracellular domains of CD32a were amplified from PBMC cDNA using the primers CD32-AsnI forward (5′-ATTAATGGGGATCATTGTGGC-3′) and CD32-NotI reverse (5′-TAATGCGGCCGCTGGCATAACGTTACTCTTTAG-3′). For the construction, an AsnI restriction site was created between the luminal domain of TLR3 and methionine 215 of CD32a (Fig. 2A), leading to the insertion of a leucine at base pair 2113 of TLR3. The chimeric construction was cloned into the SalI and NotI sites of the mammalian expression vector pMET7. Oligonucleotides designed for PCR amplification were derived from GenBank™ sequences NM003265 and M31932 for TLR3 and CD32a, respectively. PCR amplification was performed on a PerkinElmer Life Sciences 480 Thermal-Cyler. Stable Expression in U937 Cells—U937 cells were nucleofected with plasmids at a ratio of one copy of pcDNA3.1(+)Hygro (Invitrogen), which contains an hygromycin resistance gene expressed in mammalian cells as a selective marker, to 10 copies of TLR3-CD32/pMET7, following the procedure described above. 24 h later, Hygromycin B (50 mg/ml; Sigma) was added at 150 μg/ml. After a 4-week culture period of selection, cloning was performed in round bottom plates, at approximately 1 cell/well. Clones were screened by fluorescence-activated cell sorting staining with the goat anti-human TLR3 antibody (R&D Systems, Minneapolis, MN). Fluorescence-activated Cell Sorting Analysis—Cells (105/assay) were incubated with 2 μg of anti-TLR3 antibody (goat anti-human TLR3-specific IgG; R&D Systems) or a goat IgG1 control (Sigma). After washes, phytoerythrin-conjugated rabbit anti-goat antibody (Sigma) was added at a final dilution of 1:50. Before intracytoplasmic staining, permeabilization was performed using Cytofix/Cytoperm™ kit (BD Biosciences). Calcium Flux Assay—Cells (107/ml) were loaded with Indo I at 3 μg/ml (Molecular Probes, Inc., Eugene, OR), in RPMI plus 10% fetal bovine serum for 40 min at 37 °C, washed twice, and resuspended at 107 cells/ml in Hanks' balanced salt solution (0.44 mm potassium phosphate, 5.37 mm potassium chloride, 0.34 mm dibasic sodium phosphate, 136.89 mm sodium chloride, 5.55 mm d-glucose), 10 mm Hepes. For assays, 106 cells were diluted in 2 ml of Hanks' balanced salt solution, 10 mm Hepes, supplemented with 1.6 mm CaCl2 (complete Hanks' balanced salt solution). Measurements were made in a temperature-controlled Deltascan spectrofluorometer (Photon Technology International, South Brunswick, NJ). Injections were performed when cells were completely stabilized in the cuvette at a volume of 20 μl. Results are expressed as a ratio of Indo I emission 405/485 nm (calcium-Indo I/free Indo I ratio). When not specified, the pH was kept at 6.6. Hanks' balanced salt solution buffer adjusted to different pH in a gradient from 5.5 to 7.5 was prepared in advance by the addition of either HCl or NaOH, and cells were suspended in the buffer immediately before the calcium flux assay. Cross-linking and Desensitization Conditions—Anti-CD32 (FcγRII) antibody (Stem Cell Technologies) was added at a final concentration of 0.5 μg/ml to the cells within the cuvette for 3–5 min. CD32 aggregation was effective by the addition of goat anti-mouse IgG (H + L) F(ab′)2 fragment, (Immunotech, Beckman Coulter, Marseille, France) at 10 μg/ml final concentration. For cells transfected with TLR3-CD32 chimera, either 0.5 μg/ml goat anti-human TLR3-specific IgG (R&D Systems) was added prior to the cross-linking performed with the addition of biotinylated rabbit anti-goat IgG (Dako, Glostrup, Denmark) at 1:100 final concentration or mouse anti-TLR3.7 antibody (eBiosciences) at 5 μg/ml final concentration for the first incubation, followed by the addition of 10 μg/ml goat anti-mouse IgG (H + L) F(ab′)2 fragment (Immunotech; Beckman-Coulter). For experiments with the TLR2-CD32 chimera, cross-linking was performed with mouse anti-TLR2.1 (eBiosciences, San Diego, CA) at 2.5 μg/ml followed by the goat anti-mouse IgG. Desensitization was performed as described above for cross-linking; briefly, a second injection was performed when the ratio 405/485 had retrieved the base line or 3 min after the latest injection, when no calcium flux had been induced. Cysteine Residues and Glycosylation of TLR3 Leucine-rich Repeats Are Necessary for Ligand-induced Signaling—We compared the sequences of the TLR3 leucine-rich repeats in humans, mice, and rats. We noted that the TLR3 molecules contain two conserved pairs of cysteines (Cys95 and Cys122; Cys226 and Cys242) in adjacent leucine-rich repeats. The recently published crystal structures of TLR3 suggest an intramolecular disulfide bridge between Cys95 and Cys122 but do not allow us to predict a function for Cys226 and Cys242 (36.Choe J. Kelker M.S. Wilson I.A. Science. 2005; 309: 581-585Crossref PubMed Scopus (499) Google Scholar, 37.Bell J.K. Botos I. Hall P.R. Askins J. Shiloach J. Segal D.M. Davies D.R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10976-10980Crossref PubMed Scopus (317) Google Scholar). Equally, we identified two conserved potential N-glycosylation (NX(S/T)) sites (Asn196 and Asn247) that were of particular interest to us, since they were predicted to lie on the same side of the leucine-rich repeat solenoid as the cysteines. Site-specific substitution mutants of all of the four cysteines and these two N-glycosylation sites were produced (Fig. 1E). Wild type and mutant TLR3 molecules were transfected into TLR3-negative HEK293T cells (Fig. 1A) together with a reporter plasmid containing consensus NF-κB sites, which when activated directed the expression of firefly luciferase. The mutant TLR3 molecules were expressed to a similar extent as the native TLR3 molecule, as shown by Western blot using anti-TLR3 (Fig. 1, B and C). Although no difference can be seen in the expression of the cysteine mutants, the glycosylation site mutants show a size difference with respect to native TLR3. In fact, the double mutant (NN3A) shows a size compatible with a less glycosylated molecule. It can thus be supposed that these two sites represent important sites of N-linked glycosylation for human TLR3 in mammalian cell lines, such as HEK293 cells. A consistently lower level of expression was seen for the double glycosylation site mutant, suggesting that the molecule is subject to faster degradation than the other mutants. Mutants C95A, C122A, CC1A (C95A/C122A), N247R, and NN3A (N196G/N247R), in response to poly(I-C) at 25 μg/ml, did not induce luciferase expression (Fig. 1D), suggesting that they have lost most of their ligand recognition activity, whereas mutants N196G, C226A, and C242A did not show significant loss of activity. Loss of Function TLR3 Mutants Have a Dominant Negative Effect on TLR3 Signaling—Unlike the HEK293T cell line, HEK293 cells constitutively express TLR3 (Fig. 1A) and respond to poly(I-C). In addition, we performed experiments using TRIF small interfering RNA, which blocked NF-κB signaling when HEK293 cells were activated with poly(I-C), thereby demonstrating involvement of TLR3 (data not shown). We took advantage of this expression to measure the ability of the different mutants to block the activity of wild type endogenous TLR3. We observed a nearly complete abrogation of poly(I-C)-induced luciferase activity in cells transfected with all nonfunctional mutants (Fig. 2, A and B), establishing that the loss of function was not due to a" @default.
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