Recognition of Double-stranded RNA by Human Toll-like Receptor 3 and Downstream Receptor Signaling Requires Multimerization and an Acidic pH

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. Flavell R.A. Nat. Med. 2004; PubMed Scopus Google the B. Full Text Full Text PDF PubMed Scopus Google Scholar). has been shown to suggesting that activation TLR3 in a of K. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). most TLRs myeloid Trends Immunol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), with in the signaling by the of Toll/interleukin-1 adapter and TLR3 only S. S. K. Kaisho T. Takeda K. Akira S. Nat. Immunol. 2003; PubMed Scopus Google Scholar, S. K. K. Takeda K. Akira S. Immunol. 2002; PubMed Scopus Google Scholar, K. B. 2004; PubMed Scopus Google Scholar). and S. K. Kaisho T. Takeda K. Akira S. 2003; PubMed Scopus Google and 3 T. Nat. Immunol. 2003; PubMed Scopus Google and S. S. Nat. Mol. 2004; PubMed Scopus Google Scholar). the signaling of TLRs is the interactions the receptors and the ligands S. S. 2004; PubMed Scopus Google the role of of the TLR family or other surface that or with TLRs and the of interactions the and of the immune response to a TLR2 recognizes ligands with or and this is for signaling A. A.M. L. A. S. A. 2000; PubMed Scopus Google Scholar, L. T. E.A. 2003; PubMed Scopus Google Scholar). in most signaling involving the induces in to signaling R. Janeway Jr., Nature. PubMed Scopus Google Scholar). TLR2 and TLR4 are expressed at the cell TLR7, and the and S. Immunol. 2002; PubMed Scopus Google Scholar, T. Takeda K. Akira S. S. Immunol. 2003; PubMed Scopus Google Scholar, L. S. A. 2003; PubMed Scopus Google Scholar). The of by TLR9 has been shown to in A. A. T. Nat. Immunol. 2004; PubMed Scopus Google Scholar), and interaction was observed at an acidic pH T. S. Immunol. 2004; PubMed Scopus Google Scholar). In of RNA by has been to an acidic L. S. A. 2003; PubMed Scopus Google Scholar, Kaisho T. Akira S. C. 2004; PubMed Scopus Google Scholar). In the of is expressed on myeloid on nonhematopoietic cells T. 2001; PubMed Scopus Google Scholar), cell surface has been in and cells S. K. T. 2002; PubMed Scopus Google Scholar), whereas it is to be expressed in an in monocyte-derived dendritic cells K. A. T. Immunol. 2003; PubMed Scopus Google Scholar). to the subcellular in the interaction TLR3 and its ligand The of chimeric involving different TLRs or TLR and other has been used to TLR signaling R. Janeway Jr., Nature. PubMed Scopus Google Scholar, S. 2004; PubMed Scopus Google Scholar, S. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). We to chimeric to interactions TLR3 and its We constructed a molecule fusing the amino-terminal of TLR3 and the transmembrane and carboxyl of CD32a is a transmembrane molecule an immunoreceptor tyrosine-based activation of a and leading to calcium mobilization Annu. Rev. Immunol. PubMed Scopus Google Scholar). flux are to and and not have the of or in The TLR3-CD32 was expressed in the myeloid cell line We observed cell surface of the and calcium mobilization TLR3 this mutants that a dominant negative effect, we demonstrate that poly(I-C) TLR3 and we a critical role of acidic pH in this activation. Furthermore, we that in immune cells, the interaction is on the of a subcellular of and blood were to blood mononuclear cells were by monocyte-derived were cells on a and for cells were and with human and human was for A. Med. PubMed Scopus Google Scholar). were in with HEK293T and cell lines were in with with of and and of and were at a of and and with different TLR ligands for The were used at of poly(I-C) poly(A-U) by or in and or for and MonoDC, respectively. and bafilomycin were to the of were and for and by are expressed a of and was using 3 and of and and and and were in to for at of molecular poly(I-C) was by by for of poly(I-C) was with RNA pH in a of for at poly(I-C) was to The of were on a of human TLR3 was the of the and was used for of the TLR3 mutants was using mutagenesis of residues in the of human were using the using of were and and and and and and The used to the is the mutants were and other were mutants and were by a of mutagenesis on Expression in HEK293T and cells were to on the of were transfected with by 3 of with of in of for The was to a of an cells were and for cell U937 cells were using the with the and the by the cells were in and in of at and of was to the cell and in an for cells were for with or HEK293T cells were transfected using with of with and of the and TLR3 respectively. to of and TLR3 were also in HEK293T cells. were transfected with of with of or TLR3 In the were using cells were The cells were with poly(I-C) at for and analyzed for were analyzed for using by the were using a was in and was by of the of TLR3 cells were transfected using transfected HEK293T and cells were in containing pH and In of by was used for and with anti-TLR3 antibody proteins were with secondary and of the TLR3-CD32 and the Expression of human TLR3 was the using the and The transmembrane and domains of CD32a were using the and the an was the of TLR3 and of CD32a leading to the of a at of The chimeric was the and of the for were and for TLR3 and respectively. was on a Expression in U937 cells were with at a of of an expressed in cells a to of the was at a of was in at were by cell with the TLR3 antibody were with of anti-TLR3 antibody or a control antibody was at a of was using were with at 3 in for at and at in cells were in of with were in a were when cells were in the at a of are expressed a of not the pH was at to different pH in a to was in by the of or and cells were in the the calcium flux and antibody was at a of to the cells within the for aggregation was by the of at cells transfected with TLR3-CD32 was to the with the of at or antibody at for the by the of with the was with at by the was for a was when the had the line or 3 the when calcium flux had been and of TLR3 for the of the TLR3 leucine-rich repeats in and We that the TLR3 of and and in leucine-rich The of TLR3 suggest an and but not to a for and PubMed Scopus Google Scholar, S. A. PubMed Scopus Google Scholar). we and that were of to were to on the of the leucine-rich the mutants of of the and were and TLR3 were transfected HEK293T cells with a containing when the of The TLR3 were expressed to a the TLR3 shown by using anti-TLR3 and be in the of the cysteine the glycosylation mutants a with to In the a with a be that of glycosylation for human TLR3 in cell cells. of was for the glycosylation suggesting that the molecule is to the other and in response to poly(I-C) at not suggesting that have most of ligand whereas mutants and not of of TLR3 a on TLR3 the HEK293T cell cells TLR3 and to In we using signaling when cells were with poly(I-C), of TLR3 not We of this to the of the different mutants to the of We observed a of poly(I-C)-induced in cells transfected with mutants and that the of was not to an of dominant negative of or multimerization was by of the mutants with TLR3 at different in HEK293T cells and of the dominant were in cells by TLR5 Full Text Full Text PDF PubMed Scopus Google not These observations that TLR3 multimerization was a requirement for and that this multimerization was on the of cysteine residues certain glycosylation present on the Expression of the TLR3-CD32 at the of to the interactions TLR3 and its we constructed a TLR3-CD32 chimeric CD32a is a cell surface receptor induced by interaction with its ligand a in calcium PubMed Scopus Google Scholar, L. Immunol. Google Scholar). CD32a signaling has been to a in the of the containing a the M.J. PubMed Google that of L. Immunol. Google Scholar). We and K. A. T. Immunol. 2003; PubMed Scopus Google have observed that TLR3 is not expressed at the cell surface in in the TLR3-CD32 chimeric we the transmembrane of CD32a in to cell surface of the the cell lines HEK293T and U937 resulted in a of cell surface of the chimeric receptor using anti-TLR3 and was on the cell surface of cells or on cells transfected with the and or in cells transfected with a for a not The that the TLR3 molecule induced TLR3 in the and not on the cell surface of transfected HEK293T cells and We of U937 cells transfected with were on cell surface of TLR3 clone These to and be for in of of the of the TLR3-CD32 by and Ca2+ in U937 cell line U937 was used in Ca2+ mobilization We observed that this cell line and we were to that of this receptor by by with a secondary antibody induces calcium mobilization in cells We the of multimerization of the chimeric TLR3-CD32 receptor using anti-TLR3 In the TLR3-CD32 stably transfected cell line in anti-TLR3 induced but with a secondary antibody induced a Ca2+ flux and were with a anti-TLR3 and and a anti-TLR3 and response to anti-TLR3 with or by a secondary antibody was on U937 cells or on cells transfected with a not In of with an and secondary antibody induced Ca2+ flux These observations showed in the the expressed the TLR3-CD32 multimerization for induces Ca2+ flux on U937 cells stably transfected with the TLR3-CD32 flux is induced by poly(I-C) on the TLR3-CD32 stably transfected clone but not on the cells with a induced a of poly(I-C) a of poly(I-C) and of poly(A-U) induced a Ca2+ flux that the response to poly(I-C) and The response to poly(I-C) on the clone with the on that the Ca2+ flux the was the TLR3 response in immune We used poly(I-C), a ligand of to the response of the TLR3-CD32 chimeric a Ca2+ flux on the U937 cells stably the TLR3-CD32 and but not on U937 cells transfected with a We also a Ca2+ flux of using a molecule that in to poly(I-C) but to has not been shown to TLR3 We the response to poly(I-C) for activation of the chimeric receptor with that for in and The of poly(I-C) were for Ca2+ flux and for the of chimeric is and and an to the receptor-proximal events to the interaction of TLR3 with its the cells were with an of poly(I-C) or we were to a flux on the cells with poly(I-C) and or with cross-linked anti-TLR3 and of the anti-TLR3 or the cells to a response to poly(I-C) and of secondary probably the specificity of poly(I-C) and poly(A-U) for The TLR3-CD32 and TLR3 the of TLR3-CD32 molecule is expressed at the cell surface and Ca2+ mobilization the of The of poly(I-C) or poly(A-U) to mediate a Ca2+ mobilization with that with of anti-TLR3 that the of of poly(I-C) showed that the was of molecular not the receptors by to TLR3 we the of synthetic poly(I-C) to Ca2+ flux on the TLR3-CD32 These were by of and of and The of the double-stranded RNA was on a shown in were to Ca2+ mobilization in transfected U937 cells. of were to of an with that of molecular poly(I-C) in response the different of poly(I-C) not be in Ca2+ of by of the the in response to poly(I-C) and an acidic of poly(I-C) of was used and in was with molecular poly(I-C) Ca2+ mobilization responses were observed with poly(I-C) and poly(A-U) in are to the of the different ligands to mediate TLR3 multimerization and suggest that of poly(I-C) receptor aggregation and activation. The of and to TLR3 TLR3 in an TLR3 has been a receptor, at in monocyte-derived DC, and interaction with its ligand in subcellular within the K. A. T. Immunol. 2003; PubMed Scopus Google Scholar). These an acidic pH, a demonstrated requirement for the interaction TLR9 and and response by human with only a on the response by or also poly(I-C)-induced responses and by human and suggesting that the interaction is within an acidic subcellular the response induced by by and was by bafilomycin but not by is in with that was to L. S. A. 2003; PubMed Scopus Google Scholar), the of to Immunol. 2002; PubMed Scopus Google Scholar), the and The of to and TLR3-CD32 on an the of the pH on the Ca2+ flux the cell surface by poly(I-C) observations showed a Ca2+ flux by poly(I-C) or the cross-linked at pH but showed that pH the poly(I-C) and poly(A-U) responses were the antibody response We the response a of pH observations showed that an acidic pH to was for an response to poly(I-C), with a pH to and that a pH of the In this of pH had on the using anti-TLR3 and not of in this oligomeric poly(I-C) were the on acidic pH was critical an a pH of was for TLR3-CD32 activation by Full activation at pH was characterized by a Ca2+ flux of and the of a secondary response to molecular poly(I-C), of the pH and the of was whereas the response to molecular poly(I-C) at this pH, not by the of to a poly(I-C) These observations showed that pH has a on the interaction poly(I-C) and TLR3 and that interactions in the acidic of subcellular compartments. In the present a mutagenesis approach and of chimeric we that or multimerization of the receptor is a requirement for TLR3 signaling and that TLR3 interaction with its ligands is on an acidic The of TLR3 has been and that of the TLR3 ectodomain with the ligand poly(I-C) have been we observed that and that are in and have been shown to PubMed Scopus Google Scholar, S. A. PubMed Scopus Google Scholar), were for poly(I-C)-induced TLR3 signaling. TLR3 is the only TLR to have cysteine in the leucine-rich and have a of ligand different to other The of this the of the receptor to the ligand to the for of a of and that not to within the TLR3 molecule had on ligand on the of and glycosylation PubMed Scopus Google other the ligand not TLR3 in the of the ectodomain that is by and is not that ligand on the of the in of was to the of the a was to the of we observed that a to on the of the leucine-rich the was critical for signaling. was not in of the in not of the were and in the glycosylation the were The in glycosylation and are probably to the that PubMed Scopus Google and S. A. PubMed Scopus Google to the TLR3 ectodomain in cells. in the of glycosylation different cell lines have been been A. Annu. 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Rev. 2004; PubMed Scopus Google Scholar). a that glycosylation at a of a is for and 2004; PubMed Scopus Google Scholar), to observations with TLR3 we an role for in TLR3 function. we also mutants of the in to the and These mutants to poly(I-C) and in HEK293T cells, and we not extensively the in showed a in in with the TLR3 a dominant negative not with mutants suggest that the and glycosylation of the amino-terminal are critical for and signaling and to be with the structural and for understanding of the ligand of that the transmembrane and are for the localization of TLR T. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, K. T. A. T. 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