Endosomal Translocation of Vertebrate DNA Activates Dendritic Cells via TLR9-Dependent and -Independent Pathways 1

TLRs discriminate foreign from self via their specificity for pathogen-derived invariant ligands, an example being TLR9 recognizing bacterial unmethylated CpG motifs. In this study we report that endosomal translocation of CpG DNA via the natural endocytotic pathway is inefficient and highly saturable, whereas endosomal translocation of DNA complexed to the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) is not. Interestingly, DOTAP-mediated enhanced endosomal translocation of otherwise nonstimulatory vertebrate DNA or of certain noncanonical CpG motifs triggers robust dendritic cell activation in terms of both up-regulation of CD40/CD69 and cytokine production, such as type I IFN and IL-6. We report that the stimulatory activity of phosphorothioated noncanonical CpG oligodeoxynucleotides is TLR9 dependent, whereas phosphodiester DNA, such as vertebrate DNA, in addition trigger TLR9-independent pathways. We propose that the inefficiency of the natural route for DNA internalization hinders low affinity TLR9 ligands in endosomes to reach threshold concentrations required for TLR9 activation. Endosomal compartmentalization of TLR9 may thus reflect an evolutionary strategy to avoid TLR9 activation by self-DNA.

[1]  S. Akira,et al.  A Toll-like receptor recognizes bacterial DNA , 2000, Nature.

[2]  Jongdae Lee,et al.  Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: Activation of Toll-like receptor 7 , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[3]  M. Capron,et al.  Methylated CpG‐Containing Plasmid Activates the Immune System , 2004, Scandinavian journal of immunology.

[4]  A. Bird,et al.  Functions for DNA methylation in vertebrates. , 1993, Cold Spring Harbor symposia on quantitative biology.

[5]  L. Rönnblom,et al.  Importance of CpG Dinucleotides in Activation of Natural IFN‐α‐Producing Cells by a Lupus‐Related Oligodeoxynucleotide , 2001, Scandinavian journal of immunology.

[6]  S. Akira,et al.  Contribution of Toll-like receptor 9 signaling to the acute inflammatory response to nonviral vectors. , 2004, Molecular therapy : the journal of the American Society of Gene Therapy.

[7]  S. Akira,et al.  Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[8]  R. Kasukawa,et al.  CpG motif-containing DNA fragments from sera of patients with systemic lupus erythematosus proliferate mononuclear cells in vitro. , 1999, The Journal of rheumatology.

[9]  B. Monks,et al.  TLR9 signals after translocating from the ER to CpG DNA in the lysosome , 2004, Nature Immunology.

[10]  P. Payette,et al.  Oligodeoxynucleotides lacking CpG dinucleotides mediate Toll‐like receptor 9 dependent T helper type 2 biased immune stimulation , 2004, Immunology.

[11]  S. Akira,et al.  Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8 , 2004, Science.

[12]  J. M. Rodríguez,et al.  Strong Cytosine-Guanosine-Independent Immunostimulation in Humans and Other Primates by Synthetic Oligodeoxynucleotides with PyNTTTTGT Motifs , 2003, The Journal of Immunology.

[13]  D. Busch,et al.  Cutting Edge: Toll-Like Receptor 9 Expression Is Not Required for CpG DNA-Aided Cross-Presentation of DNA-Conjugated Antigens but Essential for Cross-Priming of CD8 T Cells1 , 2003, The Journal of Immunology.

[14]  V. Hornung,et al.  Identification of CpG oligonucleotide sequences with high induction of IFN‐α/β in plasmacytoid dendritic cells , 2001 .

[15]  F. Schmitz,et al.  Herpes simplex virus type-1 induces IFN-α production via Toll-like receptor 9-dependent and -independent pathways , 2004 .

[16]  S. Akira,et al.  The Toll‐like receptor 7 (TLR7)‐specific stimulus loxoribine uncovers a strong relationship within the TLR7, 8 and 9 subfamily , 2003, European journal of immunology.

[17]  M. Shlomchik,et al.  Chromatin–IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors , 2002, Nature.

[18]  T. Takai Roles of Fc receptors in autoimmunity , 2002, Nature Reviews Immunology.

[19]  T. Wu,et al.  Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[20]  A. Krieg,et al.  Induction of autoantibody production but not autoimmune disease in HEL transgenic mice vaccinated with HEL in combination with CpG or control oligodeoxynucleotides. , 2004, Vaccine.

[21]  Cevayir Coban,et al.  Genomic DNA Released by Dying Cells Induces the Maturation of APCs1 2 , 2001, The Journal of Immunology.

[22]  J. Metzger,et al.  Toll‐like receptor 9 binds single‐stranded CpG‐DNA in a sequence‐ and pH‐dependent manner , 2004, European journal of immunology.

[23]  S. Akira,et al.  Toll-like Receptor 9–Dependent and –Independent Dendritic Cell Activation by Chromatin–Immunoglobulin G Complexes , 2004, The Journal of experimental medicine.

[24]  D. Klinman,et al.  Suppressive Oligodeoxynucleotides Inhibit Th1 Differentiation by Blocking IFN-γ- and IL-12-Mediated Signaling1 , 2004, The Journal of Immunology.

[25]  H. Wagner The immunobiology of the TLR9 subfamily. , 2004, Trends in immunology.

[26]  Shizuo Akira,et al.  Innate Antiviral Responses by Means of TLR7-Mediated Recognition of Single-Stranded RNA , 2004, Science.

[27]  A. Yamamoto,et al.  Subcellular Localization of Toll-Like Receptor 3 in Human Dendritic Cells , 2003, The Journal of Immunology.

[28]  H. Mischak,et al.  CpG‐DNA‐specific activation of antigen‐presenting cells requires stress kinase activity and is preceded by non‐specific endocytosis and endosomal maturation , 1998, The EMBO journal.

[29]  Y. Baba,et al.  Cationic liposome-mediated gene delivery: biophysical study and mechanism of internalization. , 2003, Archives of biochemistry and biophysics.

[30]  H. Yoshida,et al.  Lethal anemia caused by interferon-β produced in mouse embryos carrying undigested DNA , 2005, Nature Immunology.

[31]  H. Wagner,et al.  Bacterial CpG DNA activates immune cells to signal infectious danger. , 1999, Advances in immunology.

[32]  G. Bishop,et al.  CpG motifs in bacterial DNA trigger direct B-cell activation , 1995, Nature.

[33]  M. Hashida,et al.  Plasmid DNA activates murine macrophages to induce inflammatory cytokines in a CpG motif-independent manner by complex formation with cationic liposomes. , 2002, Biochemical and biophysical research communications.

[34]  J. Ellwart,et al.  Bacterial CpG-DNA Triggers Activation and Maturation of Human CD11c−, CD123+ Dendritic Cells1 , 2001, The Journal of Immunology.

[35]  A. Krieg,et al.  CpG motifs in bacterial DNA and their immune effects. , 2002, Annual review of immunology.

[36]  D. Busch,et al.  Vaccination with Plasmid DNA Activates Dendritic Cells via Toll-Like Receptor 9 (TLR9) but Functions in TLR9-Deficient Mice 1 , 2003, The Journal of Immunology.

[37]  V. Gopal,et al.  Cationic lipids and cationic ligands induce DNA helix denaturation: detection of single stranded regions by KMnO4 probing , 2003, FEBS letters.

[38]  David A. Hume,et al.  The Molecular Basis for the Lack of Immunostimulatory Activity of Vertebrate DNA1 , 2003, The Journal of Immunology.

[39]  R. Vabulas,et al.  Bacterial CpG‐DNA and lipopolysaccharides activate Toll‐like receptors at distinct cellular compartments , 2002, European journal of immunology.

[40]  L. Rönnblom,et al.  Anti-double-stranded DNA antibodies and immunostimulatory plasmid DNA in combination mimic the endogenous IFN-alpha inducer in systemic lupus erythematosus. , 1999, Journal of immunology.

[41]  B. Ramsahoye,et al.  DNA methylation: biology and significance. , 1996, Blood reviews.

[42]  G. Tamura,et al.  B Cells Capturing Antigen Conjugated with CpG Oligodeoxynucleotides Induce Th1 Cells by Elaborating IL-121 , 2002, The Journal of Immunology.