Toll-like Receptors Impact on Safety and Efficacy of Gene Transfer Vectors
Authors: Scott E Hensley, Andrea Amalfitano
Journal: Molecular Therapy (2007)

Innate immune responses are triggered when pattern-recognition receptors recognize specific conserved patterns on pathogens. The most extensively studied pattern-recognition receptors are toll-like receptors (TLRs), which are comprised of 11 different receptors, named TLR1–11. TLRs recognize motifs that are found on a wide range of pathogens, and activation of TLRs results in the production of large amounts of type I interferons and several proinflammatory cytokines. These cytokine responses are important in controlling pathogen replication and they also provide an initiation signal for the adaptive immune response. Although numerous manuscripts have reviewed the important role of TLRs in host defense against wild-type viruses, bacteria, and/or their subcomponents, none have focused on how TLRs recognize commonly utilized gene delivery vehicles such as adenovirus and adeno-associated virus (AAV) vectors. In this review, we discuss our understanding of how TLRs are activated by gene transfer vectors. Innate immune responses are triggered when pattern-recognition receptors recognize specific conserved patterns on pathogens. The most extensively studied pattern-recognition receptors are toll-like receptors (TLRs), which are comprised of 11 different receptors, named TLR1–11. TLRs recognize motifs that are found on a wide range of pathogens, and activation of TLRs results in the production of large amounts of type I interferons and several proinflammatory cytokines. These cytokine responses are important in controlling pathogen replication and they also provide an initiation signal for the adaptive immune response. Although numerous manuscripts have reviewed the important role of TLRs in host defense against wild-type viruses, bacteria, and/or their subcomponents, none have focused on how TLRs recognize commonly utilized gene delivery vehicles such as adenovirus and adeno-associated virus (AAV) vectors. In this review, we discuss our understanding of how TLRs are activated by gene transfer vectors. The Drosophila toll protein was originally identified as a key component in fly embryonic development. Subsequent studies have shown that this protein is also involved in the antifungal response in flies.1Lemaitre B Nicolas E Michaut L Reichhart JM Hoffmann JA The dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in Drosophila adults.Cell. 1996; 86: 973-983Abstract Full Text Full Text PDF PubMed Scopus (3017) Google Scholar Toll-like receptors (TLRs), vertebrate counterparts of the Drosophila toll protein, recognize components of bacteria, viruses, and protozoa in addition to fungi. Only 11 mammalian TLRs have been identified, but it has been proposed that this limited number of receptors may be sufficient in detecting almost all known pathogens as TLRs recognize common molecular components of pathogens.2Akira S TLR signaling.Curr Top Microbiol Immunol. 2006; 311: 1-16PubMed Google Scholar Indeed, some individual TLRs are capable of recognizing very diverse pathogens. For example, TLR2 recognizes peptidoglycan from Gram-positive bacteria, zymosan from yeast cell walls, and glycophosphatidylinositol anchors from Trypanosma cruzi.3Schwandner R Dziarski R Wesche H Rothe M Kirschning CJ Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2.J Biol Chem. 1999; 274: 17406-17409Crossref PubMed Scopus (1437) Google Scholar,4Takeuchi O Hoshino K Kawai T Sanjo H Takada H Ogawa T et al.Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components.Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2797) Google Scholar,5Underhill DM Ozinsky A Hajjar AM Stevens A Wilson CB Bassetti M et al.The toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens.Nature. 1999; 401: 811-815Crossref PubMed Scopus (1195) Google Scholar,6Campos MA Almeida IC Takeuchi O Akira S Valente EP Procopio DO et al.Activation of toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan parasite.J Immunol. 2001; 167: 416-423Crossref PubMed Scopus (459) Google Scholar TLR2 can also recognize bacterial lipoproteins when dimerizing with TLR1 and mycoplasma lipoproteins when dimerizing with TLR6.7Ozinsky A Underhill DM Fontenot JD Hajjar AM Smith KD Wilson CB et al.The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors.Proc Natl Acad Sci USA. 2000; 97: 13766-13771Crossref PubMed Scopus (1692) Google Scholar Some TLRs are thought to be much more specific, but this may simply be a function of being understudied. For example, in the late 1990s, TLR4 was thought to be exclusively a sensor for lipopolysaccharide, but subsequent studies have demonstrated that TLR4 also recognizes mouse retroviruses.8Rassa JC Meyers JL Zhang Y Kudaravalli R Ross SR Murine retroviruses activate B cells via interaction with toll-like receptor 4.Proc Natl Acad Sci USA. 2002; 99: 2281-2286Crossref PubMed Scopus (343) Google Scholar With this in mind, it is easy to imagine that other "specific" TLRs, such as TLR5 that recognizes flagellin,9Hayashi F Smith KD Ozinsky A Hawn TR Yi EC Goodlett DR et al.The innate immune response to bacterial flagellin is mediated by toll-like receptor 5.Nature. 2001; 410: 1099-1103Crossref PubMed Scopus (2835) Google Scholar may also recognize additional ligands that are currently unidentified. Many TLRs recognize viral and bacterial nucleic acids. TLR3 is activated by double-stranded RNA,10Alexopoulou L Holt AC Medzhitov R Flavell RA Recognition of double-stranded RNA and activation of NF-kappaB by toll-like receptor 3.Nature. 2001; 413: 732-738Crossref PubMed Scopus (4956) Google Scholar whereas TLR7 and TLR8 are activated by single-stranded RNA.11Hemmi H Kaisho T Takeuchi O Sato S Sanjo H Hoshino K et al.Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway.Nat Immunol. 2002; 3: 196-200Crossref PubMed Scopus (2078) Google Scholar,12Diebold SS Kaisho T Hemmi H Akira S Reis e Sousa C Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA.Science. 2004; 303: 1529-1531Crossref PubMed Scopus (2752) Google Scholar,13Heil F Hemmi H Hochrein H Ampenberger F Kirschning C Akira S et al.Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8.Science. 2004; 303: 1526-1529Crossref PubMed Scopus (3080) Google Scholar,14Lund JM Alexopoulou L Sato A Karow M Adams NC Gale NW et al.Recognition of single-stranded RNA viruses by toll-like receptor 7.Proc Natl Acad Sci USA. 2004; 101: 5598-5603Crossref PubMed Scopus (1504) Google Scholar,15Jurk M Heil F Vollmer J Schetter C Krieg AM Wagner H et al.Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848.Nat Immunol. 2002; 3: 499Crossref PubMed Scopus (819) Google Scholar TLR9 is activated by unmethylated CpG DNA motifs present in bacterial DNA16Hemmi H Takeuchi O Kawai T Kaisho T Sato S Sanjo H et al.A toll-like receptor recognizes bacterial DNA.Nature. 2000; 408: 740-745Crossref PubMed Scopus (5390) Google Scholar and in viruses such as mouse cytomegalovirus17Krug A French AR Barchet W Fischer JA Dzionek A Pingel JT et al.TLR9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine responses that activate antiviral NK cell function.Immunity. 2004; 21: 107-119Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar and herpes simplex viruses.18Lund J Sato A Akira S Medzhitov R Iwasaki A Toll-like receptor 9-mediated recognition of herpes simplex virus-2 by plasmacytoid dendritic cells.J Exp Med. 2003; 198: 513-520Crossref PubMed Scopus (1014) Google Scholar,19Krug A Luker GD Barchet W Leib DA Akira S Colonna M Herpes simplex virus type 1 activates murine natural interferon-producing cells through toll-like receptor 9.Blood. 2004; 103: 1433-1437Crossref PubMed Scopus (574) Google Scholar Although many TLRs are expressed exclusively on the cell surface, TLR7, TLR8, and TLR9 are primarily located in the endosomal compartments of cells.12Diebold SS Kaisho T Hemmi H Akira S Reis e Sousa C Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA.Science. 2004; 303: 1529-1531Crossref PubMed Scopus (2752) Google Scholar,18Lund J Sato A Akira S Medzhitov R Iwasaki A Toll-like receptor 9-mediated recognition of herpes simplex virus-2 by plasmacytoid dendritic cells.J Exp Med. 2003; 198: 513-520Crossref PubMed Scopus (1014) Google Scholar,20Latz E Schoenemeyer A Visintin A Fitzgerald KA Monks BG Knetter CF et al.TLR9 signals after translocating from the ER to CpG DNA in the lysosome.Nat Immunol. 2004; 5: 190-198Crossref PubMed Scopus (1164) Google Scholar TLR3 is also expressed intracellularly, at least in certain cell types.21Matsumoto M Funami K Tanabe M Oshiumi H Shingai M Seto Y et al.Subcellular localization of toll-like receptor 3 in human dendritic cells.J Immunol. 2003; 171: 3154-3162Crossref PubMed Scopus (622) Google Scholar Uropathogenic bacteria have recently been described as ligands for TLR11,22Zhang D Zhang G Hayden MS Greenblatt MB Bussey C Flavell RA et al.A toll-like receptor that prevents infection by uropathogenic bacteria.Science. 2004; 303: 1522-1526Crossref PubMed Scopus (885) Google Scholar whereas TLR10 still remains without a known ligand. It is possible that additional TLRs will be identified in the future and it has been proposed that mammalian species may have up to 15 TLRs.23Iwasaki A Medzhitov R Toll-like receptor control of the adaptive immune responses.Nat Immunol. 2004; 5: 987-995Crossref PubMed Scopus (3364) Google Scholar For a complete review of TLR ligands, see ref. 24Barton GM Medzhitov R Toll-like receptors and their ligands.Curr Top Microbiol Immunol. 2002; 270: 81-92Crossref PubMed Scopus (254) Google Scholar. TLRs are type 1 transmembrane proteins that contain a leucine-rich repeat extracellular domain and a cytosolic toll-interleukin-1 receptor (TIR) homology domain that is essential for intracellular signaling.2Akira S TLR signaling.Curr Top Microbiol Immunol. 2006; 311: 1-16PubMed Google Scholar Upon activation, TLRs recruit the TIR-containing adaptor molecules, myeloid differentiating factor 88 (MyD88), TIR domain–containing adaptor inducing IFN-beta (TRIF), TIR domain–containing adaptor protein, and TIR domain–containing adaptor inducing IFN-beta-related adaptor molecule (Figure 1). Many studies of TLR signaling have utilized MyD88 knockout mice, as MyD88 is an essential signaling component for most TLRs. Signaling through MyD88 leads to the nuclear translocation of nuclear factor κB (NF-κB), as well as activation of the mitogen-activated protein kinase pathway. As a result, interleukin-6 (IL-6), pro-IL-1β, tumor necrosis factor-α (TNF-α), and IL-12p40 are produced through MyD88-mediated signaling.25Kawai T Adachi O Ogawa T Takeda K Akira S Unresponsiveness of MyD88-deficient mice to endotoxin.Immunity. 1999; 11: 115-122Abstract Full Text Full Text PDF PubMed Scopus (1735) Google Scholar,26Kawai T Takeuchi O Fujita T Inoue J Muhlradt PF Sato S et al.Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes.J Immunol. 2001; 167: 5887-5894Crossref PubMed Scopus (901) Google Scholar,27Kaisho T Takeuchi O Kawai T Hoshino K Akira S Endotoxin-induced maturation of MyD88-deficient dendritic cells.J Immunol. 2001; 166: 5688-5694Crossref PubMed Scopus (432) Google Scholar Activation of the MyD88 pathway can also lead to type I interferon (IFN) induction in certain cell types.28Hemmi H Kaisho T Takeda K Akira S The roles of toll-like receptor 9, MyD88, and DNA-dependent protein kinase catalytic subunit in the effects of two distinct CpG DNAs on dendritic cell subsets.J Immunol. 2003; 170: 3059-3064Crossref PubMed Scopus (300) Google Scholar,29Hoshino K Kaisho T Iwabe T Takeuchi O Akira S Differential involvement of IFN-beta in toll-like receptor-stimulated dendritic cell activation.Int Immunol. 2002; 14: 1225-1231Crossref PubMed Scopus (238) Google Scholar Some TLRs, such as TLR3 and TLR4, can signal independently of MyD88.10Alexopoulou L Holt AC Medzhitov R Flavell RA Recognition of double-stranded RNA and activation of NF-kappaB by toll-like receptor 3.Nature. 2001; 413: 732-738Crossref PubMed Scopus (4956) Google Scholar,26Kawai T Takeuchi O Fujita T Inoue J Muhlradt PF Sato S et al.Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes.J Immunol. 2001; 167: 5887-5894Crossref PubMed Scopus (901) Google Scholar The adaptor molecule, TRIF, is essential for MyD88-independent signaling of TLR3 and TLR4.30Yamamoto M Sato S Hemmi H Hoshino K Kaisho T Sanjo H et al.Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway.Science. 2003; 301: 640-643Crossref PubMed Scopus (2527) Google Scholar,31Hoebe K Du X Georgel P Janssen E Tabeta K Kim SO et al.Identification of Lps2 as a key transducer of MyD88-independent TIR signalling.Nature. 2003; 424: 743-748Crossref PubMed Scopus (1037) Google Scholar Signaling through TRIF activates NF-κB, leading to the production of proinflammatory cytokines. TRIF signaling also leads to the phosphorylation of IFN regulatory factor 3, which results in type I IFN production. Less is known about the adaptor molecules TIR domain–containing adaptor protein and TIR domain–containing adaptor inducing IFN-beta-related adaptor molecule. TIR domain–containing adaptor protein is involved in MyD88-dependent signaling of TLR2 and TLR4,32Yamamoto M Sato S Hemmi H Sanjo H Uematsu S Kaisho T et al.Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4.Nature. 2002; 420: 324-329Crossref PubMed Scopus (820) Google Scholar whereas TIR domain–containing adaptor inducing IFN-beta-related adaptor molecule is involved in MyD88-independent signaling of TLR4.33Yamamoto M Sato S Hemmi H Uematsu S Hoshino K Kaisho T et al.TRAM is specifically involved in the toll-like receptor 4-mediated MyD88-independent signaling pathway.Nat Immunol. 2003; 4: 1144-1150Crossref PubMed Scopus (832) Google Scholar As described above, TLRs are a crucial component in the recognition of diverse pathogens, and activation of TLRs leads to the production of cytokines that are crucial for host defense. It is surprising that a limited number of studies have examined the role of TLR recognition of gene delivery vehicles, and most of these studies have focused only on adenovirus (Ad) vectors. The remainder of this review focuses on these studies and the relationship of TLR signaling and gene delivery. Ad capsids activate the innate immune system through mechanisms that do not involve viral replication or gene expression.34Muruve DA Barnes MJ Stillman IE Libermann TA Adenoviral gene therapy leads to rapid induction of multiple chemokines and acute neutrophil-dependent hepatic injury in vivo.Hum Gene Ther. 1999; 10: 965-976Crossref PubMed Scopus (412) Google Scholar,35Zhang Y Chirmule N Gao GP Qian R Croyle M Joshi B et al.Acute cytokine response to systemic adenoviral vectors in mice is mediated by dendritic cells and macrophages.Mol Ther. 2001; 3: 697-707Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar,36Zaiss AK Liu Q Bowen GP Wong NC Bartlett JS Muruve DA Differential activation of innate immune responses by adenovirus and adeno-associated virus vectors.J Virol. 2002; 76: 4580-4590Crossref PubMed Scopus (328) Google Scholar,37Liu Q Zaiss AK Colarusso P Patel K Haljan G Wickham TJ et al.The role of capsid-endothelial interactions in the innate immune response to adenovirus vectors.Hum Gene Ther. 2003; 14: 627-643Crossref PubMed Scopus (134) Google Scholar,38Brunetti-Pierri N Palmer DJ Beaudet AL Carey KD Finegold M Ng P Acute toxicity after high-dose systemic injection of helper-dependent adenoviral vectors into nonhuman primates.Hum Gene Ther. 2004; 15: 35-46Crossref PubMed Scopus (228) Google Scholar,39Bowen GP Borgland SL Lam M Libermann TA Wong NC Muruve DA Adenovirus vector-induced inflammation: capsid-dependent induction of the C-C chemokine RANTES requires NF-kappaB.Hum Gene Ther. 2002; 13: 367-379Crossref PubMed Scopus (87) Google Scholar Following infection with ultra violet–inactivated Ad vectors, human peripheral blood mononuclear cells rapidly produce many cytokines, including IL-6, IL-1β, granulocyte monocyte colony stimulating factor, IL-8, and TNF-α.40Higginbotham JN Seth P Blaese RM Ramsey WJ The release of inflammatory cytokines from human peripheral blood mononuclear cells in vitro following exposure to adenovirus variants and capsid.Hum Gene Ther. 2002; 13: 129-141Crossref PubMed Scopus (103) Google Scholar High levels of these cytokines are also found in the serum of mice and non-human primates after Ad vector injection.35Zhang Y Chirmule N Gao GP Qian R Croyle M Joshi B et al.Acute cytokine response to systemic adenoviral vectors in mice is mediated by dendritic cells and macrophages.Mol Ther. 2001; 3: 697-707Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar,36Zaiss AK Liu Q Bowen GP Wong NC Bartlett JS Muruve DA Differential activation of innate immune responses by adenovirus and adeno-associated virus vectors.J Virol. 2002; 76: 4580-4590Crossref PubMed Scopus (328) Google Scholar,41Lozier JN Csako G Mondoro TH Krizek DM Metzger ME Costello R et al.Toxicity of a first-generation adenoviral vector in rhesus macaques.Hum Gene Ther. 2002; 13: 113-124Crossref PubMed Scopus (112) Google Scholar Dendritic cells (DCs) produce large amounts of type I IFN following infection with ultra violet–inactivated Ad vectors, and this directly inhibits transgene product expression.42Hensley SE Giles-Davis W McCoy KC Weninger W Ertl HC Dendritic cell maturation, but not CD8+ T cell induction, is dependent on type I IFN signaling during vaccination with adenovirus vectors.J Immunol. 2005; 175: 6032-6041Crossref PubMed Scopus (63) Google Scholar,43Hensley SE Cun AS Giles-Davis W Li Y Xiang Z Lasaro MO et al.Type I interferon inhibits antibody responses induced by a chimpanzee adenovirus vector.Mol Ther. 2007; 15: 393-403Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Cell types that are not classically thought to be involved in immune recognition, such as kidney and respiratory epithelial cells, also produce large amounts of cytokines and chemokines shortly after infection with Ad vectors.34Muruve DA Barnes MJ Stillman IE Libermann TA Adenoviral gene therapy leads to rapid induction of multiple chemokines and acute neutrophil-dependent hepatic injury in vivo.Hum Gene Ther. 1999; 10: 965-976Crossref PubMed Scopus (412) Google Scholar,44Amin R Wilmott R Schwarz Y Trapnell B Stark J Replication-deficient adenovirus induces expression of interleukin-8 by airway epithelial cells in vitro.Hum Gene Ther. 1995; 6: 145-153Crossref PubMed Scopus (68) Google Scholar Many studies have focused on the intracellular signaling pathways involved during the innate immune response to Ad vectors (for review see ref. 45Liu Q Muruve DA Molecular basis of the inflammatory response to adenovirus vectors.Gene Ther. 2003; 10: 935-940Crossref PubMed Scopus (370) Google Scholar). Extracellular signal–regulated kinase (ERK) signaling has been implicated in Ad vector induction of IL-8 in HeLa cells,46Bruder JT Kovesdi I Adenovirus infection stimulates the Raf/MAPK signaling pathway and induces interleukin-8 expression.J Virol. 1997; 71: 398-404Crossref PubMed Google Scholar and both p38 and extracellular signal–regulated kinase signaling are crucial for the expression of IP-10 following Ad vector infection of renal epithelial cells.47Tibbles LA Spurrell JC Bowen GP Liu Q Lam M Zaiss AK et al.Activation of p38 and ERK signaling during adenovirus vector cell entry lead to expression of the C-X-C chemokine IP-10.J Virol. 2002; 76: 1559-1568Crossref PubMed Scopus (111) Google Scholar NF-κB, which is activated by Ad vectors following infection of DCs, is required for Ad vector–induced DC maturation.48Morelli AE Larregina AT Ganster RW Zahorchak AF Plowey JM Takayama T et al.Recombinant adenovirus induces maturation of dendritic cells via an NF-kappaB-dependent pathway.J Virol. 2000; 74: 9617-9628Crossref PubMed Scopus (206) Google Scholar NF-κB is also required for the production of RANTES and IP-10 following infection of HeLa and renal epithelial cells with Ad vectors.39Bowen GP Borgland SL Lam M Libermann TA Wong NC Muruve DA Adenovirus vector-induced inflammation: capsid-dependent induction of the C-C chemokine RANTES requires NF-kappaB.Hum Gene Ther. 2002; 13: 367-379Crossref PubMed Scopus (87) Google Scholar,49Borgland SL Bowen GP Wong NC Libermann TA Muruve DA Adenovirus vector-induced expression of the C-X-C chemokine IP-10 is mediated through capsid-dependent activation of NF-kappaB.J Virol. 2000; 74: 3941-3947Crossref PubMed Scopus (132) Google Scholar Despite our increasing knowledge of these pathways, it has remained unclear how these signaling pathways are initially activated. Recent data have indicated that TLRs are involved in this process.50Iacobelli-Martinez M Nemerow GR Preferential activation of toll-like receptor nine by CD46-utilizing adenoviruses.J Virol. 2007; 81: 1305-1312Crossref PubMed Scopus (97) Google Scholar,51Basner-Tschakarjan E Gaffal E O'Keeffe M Tormo D Limmer A Wagner H et al.Adenovirus efficiently transduces plasmacytoid dendritic cells resulting in TLR9-dependent maturation and IFN-alpha production.J Gene Med. 2006; 8: 1300-1306Crossref PubMed Scopus (94) Google Scholar,52Hartman ZC Black EP Amalfitano A Adenoviral infection induces a multi-faceted innate cellular immune response that is mediated by the toll-like receptor pathway in A549 cells.Virology. 2007; 358: 357-372Crossref PubMed Scopus (73) Google Scholar,53Hartman ZC Kiang A Everett RS Serra D Yang XY Clay TM et al.Adenovirus infection triggers a rapid, MyD88 regulated, transcriptome response critical to acute phase and adaptive immune responses in vivo.J Virol. 2007; 81: 1796-1812Crossref PubMed Scopus (127) Google Scholar,54Zhu J Huang X Yang Y Innate immune response to adenoviral vectors is mediated by both toll-like receptor-dependent and -independent pathways.J Virol. 2007; 81: 3170-3180Crossref PubMed Scopus (296) Google Scholar,55Cerullo V Seiler MP Mane V Brunetti-Pierri N Clarke C Bertin TK et al.Toll-like receptor 9 triggers an innate immune response to helper-dependent adenoviral vectors.Mol Ther. 2007; 15: 378-385Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar Microarray studies revealed that several TLR-associated genes are up-regulated in mouse embryonic fibroblasts following infection with Ad vectors.52Hartman ZC Black EP Amalfitano A Adenoviral infection induces a multi-faceted innate cellular immune response that is mediated by the toll-like receptor pathway in A549 cells.Virology. 2007; 358: 357-372Crossref PubMed Scopus (73) Google Scholar In these studies, the Ad vectors were verified to be lipopolysaccharide free. In human respiratory epithelial cells (A549 cells), the adaptor molecules MyD88 and TRIF are required for IL-6 production following Ad vector infection.52Hartman ZC Black EP Amalfitano A Adenoviral infection induces a multi-faceted innate cellular immune response that is mediated by the toll-like receptor pathway in A549 cells.Virology. 2007; 358: 357-372Crossref PubMed Scopus (73) Google Scholar Similar results were obtained when microarray studies were completed with MyD88−/− mice.53Hartman ZC Kiang A Everett RS Serra D Yang XY Clay TM et al.Adenovirus infection triggers a rapid, MyD88 regulated, transcriptome response critical to acute phase and adaptive immune responses in vivo.J Virol. 2007; 81: 1796-1812Crossref PubMed Scopus (127) Google Scholar Compared with wild-type mice, serum levels of CXC1, granulocyte-colony stimulating factor, IL-6, and monocyte chemotactic protein-1 are decreased in MyD88−/− mice 1 hour after Ad vector injection and levels of monocyte chemotactic protein-1, macrophage-inflammatory protein a (MIPa), IL-5, IL-6, IL-12(p40), granulocyte-colony stimulating factor, granulocyte monocyte colony stimulating factor, and RANTES are decreased in MyD88−/− mice 6 hours after Ad vector injection.53Hartman ZC Kiang A Everett RS Serra D Yang XY Clay TM et al.Adenovirus infection triggers a rapid, MyD88 regulated, transcriptome response critical to acute phase and adaptive immune responses in vivo.J Virol. 2007; 81: 1796-1812Crossref PubMed Scopus (127) Google Scholar The individual TLRs that are involved in this process remain only partially characterized. TLR9 expression is required for the production of type I IFN following infection of plasmacytoid DCs with Ad vectors.50Iacobelli-Martinez M Nemerow GR Preferential activation of toll-like receptor nine by CD46-utilizing adenoviruses.J Virol. 2007; 81: 1305-1312Crossref PubMed Scopus (97) Google Scholar,51Basner-Tschakarjan E Gaffal E O'Keeffe M Tormo D Limmer A Wagner H et al.Adenovirus efficiently transduces plasmacytoid dendritic cells resulting in TLR9-dependent maturation and IFN-alpha production.J Gene Med. 2006; 8: 1300-1306Crossref PubMed Scopus (94) Google Scholar,54Zhu J Huang X Yang Y Innate immune response to adenoviral vectors is mediated by both toll-like receptor-dependent and -independent pathways.J Virol. 2007; 81: 3170-3180Crossref PubMed Scopus (296) Google Scholar As described above, TLR9 recognizes unmethylated CpG DNA motifs, which are disproportionately present in the genomes of bacteria and large DNA viruses, such as Ad. Subgroup B Ad vectors induce higher levels of type I IFN compared with subgroup C Ad vectors following infection of HeLa cells.50Iacobelli-Martinez M Nemerow GR Preferential activation of toll-like receptor nine by CD46-utilizing adenoviruses.J Virol. 2007; 81: 1305-1312Crossref PubMed Scopus (97) Google Scholar It is interesting that this is not due to differences in cell entry efficiencies, but rather, differences in the type of receptor-mediated cell entry pathways utilized by each vector.50Iacobelli-Martinez M Nemerow GR Preferential activation of toll-like receptor nine by CD46-utilizing adenoviruses.J Virol. 2007; 81: 1305-1312Crossref PubMed Scopus (97) Google Scholar Subgroup B Ads, which bind to CD46, may traffic differentially compared to subgroup C Ads that bind to the coxsackie and Ad receptor, and therefore interact differentially with TLR9, which is found in endosomal compartments.20Latz E Schoenemeyer A Visintin A Fitzgerald KA Monks BG Knetter CF et al.TLR9 signals after translocating from the ER to CpG DNA in the lysosome.Nat Immunol. 2004; 5: 190-198Crossref PubMed Scopus (1164) Google Scholar In addition to unmethylated CpG motifs within the Ad genome, it is possible that capsid components of Ad vectors activate TLRs. The hexon protein of Ad is a potent adjuvant when co-injected with suboptimal doses of lipopetides56Molinier-Frenkel V Lengagne R Gaden F Hong SS Choppin J Gahery-Segard H et al.Adenovirus hexon protein is a potent adjuvant for activation of a cellular immune response.J Virol. 2002; 76: 127-135Crossref PubMed Scopus (143) Google Scholar and the fiber protein57Molinier-Frenkel V Prevost-Blondel A Hong SS Lengagne R Boudaly S Magnusson MK et al.The maturation of murine dendritic cells induced by human adenovirus is mediated by the fiber knob domain.J Bio Chem. 2003; 278: 37175-37182Crossref PubMed Scopus (49) Google Scholar and the penton protein58Philpott NJ Nociari M Elkon KB Falck-Pedersen E Adenovirus-induced maturation of dendritic cells through a PI3 kinase-mediated TNF-alpha induction pathway.Proc Natl Acad Sci USA. 2004; 101: 6200-6205Crossref PubMed Scopus (95) Google Scholar trigger DC maturation. Additional studies need to be completed to determine if the hexon, fiber, and penton proteins are recognized by TLRs. It is important to note that not all innate immune responses to Ad vectors are triggered by TLRs. Ad vector induction of type I IFN and TNF-α in bone marrow–derived DC occurs independently of TLR signaling.42Hensley SE Giles-Davis W McCoy KC Weninger W Ertl HC Dendritic cell maturation, but not CD8+ T cell induction, is dependent on type I IFN signaling during vaccination with adenovirus vectors.J Immunol. 2005; 175: 6032-6041Crossref PubMed Scopus (63) Google Scholar,58Philpott NJ Nociari M Elkon KB Falck-Pedersen E Adenovirus-induced maturation of dendritic cells through a PI3 kinase-mediated TNF-alpha induction pathway.Proc Natl Acad Sci USA. 2004; 101: 6200-6205Crossref PubMed Scopus (95) Google Scholar It has been proposed that these TLR-independent pathways are initiated through penton–integrin interactions. In addition to TLR9, the double-stranded DNA genome of Ad vectors can also be recognized by a TLR-independent nucleic acid sensing mechanism, which when activated, leads to IFN regulatory factor 3 phosphorylation.59Nociari M Ocheretina O Schoggins JW Falck-Pedersen E Sensing infection by adenovirus: TLR-independent viral DNA recognition signals activation of the IRF3 master regulator.J Virol. 2007; 81: 4145-4157Crossref PubMed Scopus (134) Google Scholar It is clear that there is redundancy in at least some of the innate immune responses triggered by Ad vectors, which may be a testament to their importance. In agreement with this assertion, MyD88−/− mice mount intact, albeit less robust, adaptive immune responses following Ad vector injection.53Hartman ZC Kiang A Everett RS Serra D Yang XY Clay TM et al.Adenovirus infection triggers a rapid, MyD88 regulated, transcriptome response critical to acute phase and ada