Synergistic Activation of Innate Immunity by Double-Stranded RNA and CpG DNA Promotes Enhanced Antitumor Activity

Double-stranded RNA (dsRNA) and unmethylated CpG sequences in DNA are pathogen-associated molecular patterns of viruses and bacteria that activate innate immunity. To examine whether dsRNA and CpG DNA could combine to provide enhanced stimulation of innate immune cells, murine macrophages were stimulated with poly-rI:rC (pIC), a dsRNA analog, and CpG-containing oligodeoxynucleotides (CpG-ODN). Combined treatments demonstrated synergy in nitric oxide, interleukin (IL)-12, tumor necrosis factor α, and IL-6 production. Studies using neutralizing antibodies for type I interferons (IFNs), IFN-α and IFN-β, indicated that nitric oxide synthase synergism is mediated by paracrine/autocrine effects of IFN-β. In contrast, enhanced cytokine production occurred independent of type I IFN and was maintained in macrophages from IFN-α/β receptor knockout mice. Cotransfection of human Toll-like receptors 3 and 9 (receptors for dsRNA and CpG DNA, respectively) into 293T cells supported synergistic activation of an IL-8 promoter reporter construct by pIC, indicating interaction of the signaling pathways in driving the synergy response. In vivo stimulation of mice with pIC and CpG-ODN demonstrated synergy for serum IL-6 and IL-12p40 levels that correlated with an enhanced antitumor effect against established B16-F10 experimental pulmonary metastases. Treatment of tumor-bearing mice with pIC and CpG-ODN in combination resulted in enhanced nitric oxide synthase expression in lung tissue and enhanced up-regulation of class I major histocompatibility complex on splenic dendritic cells relative to treatments with either agent alone. In conclusion, the combined detection of viral pathogen-associated molecular patterns, i.e., dsRNA and CpG DNA, may mimic definitive viral recognition, resulting in an enhanced innate immune response that could be used for tumor vaccination or immunotherapy.

[1]  A. Sher,et al.  Interferon Consensus Sequence Binding Protein–deficient Mice Display Impaired Resistance to Intracellular Infection Due to a Primary Defect in Interleukin 12 p40 Induction , 1997, The Journal of experimental medicine.

[2]  M. Mathews,et al.  Interactions between the double-stranded RNA binding motif and RNA: definition of the binding site for the interferon-induced protein kinase DAI (PKR) on adenovirus VA RNA. , 1995, RNA.

[3]  S. Akira,et al.  Immune Cell Activation by Bacterial Cpg-DNA through Myeloid Differentiation Marker 88 and Tumor Necrosis Factor Receptor–Associated Factor (Traf)6 , 2000, The Journal of experimental medicine.

[4]  B. Williams,et al.  Poly(dI·dC)-induced Toll-like Receptor 3 (TLR3)-mediated Activation of NFκB and MAP Kinase Is through an Interleukin-1 Receptor-associated Kinase (IRAK)-independent Pathway Employing the Signaling Components TLR3-TRAF6-TAK1-TAB2-PKR* , 2003, The Journal of Biological Chemistry.

[5]  R. Khokha,et al.  Inhibition of VLA-4 and up-regulation of TIMP-1 expression in B16BL6 melanoma cells transfected with MHC class I genes , 1998, Clinical & Experimental Metastasis.

[6]  A. Khar,et al.  Cytokine-induced production of NO by macrophages induces apoptosis and immunological rejection of AK-5 histiocytic tumor , 1998, Apoptosis.

[7]  R. Locksley,et al.  The Instructive Role of Innate Immunity in the Acquired Immune Response , 1996, Science.

[8]  Ashok Kumar,et al.  Dexamethasone Inhibits IL-12p40 Production in Lipopolysaccharide-Stimulated Human Monocytic Cells by Down-Regulating the Activity of c-Jun N-Terminal Kinase, the Activation Protein-1, and NF-κB Transcription Factors1 , 2004, The Journal of Immunology.

[9]  M. J. Cody,et al.  TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/beta-dependent gene expression in macrophages. , 2002, Nature immunology.

[10]  S. Akira,et al.  Recognition of CpG DNA is mediated by signaling pathways dependent on the adaptor protein MyD88 , 2000, Current Biology.

[11]  G. Stark,et al.  IRF-3-dependent, NFkappa B- and JNK-independent activation of the 561 and IFN-beta genes in response to double-stranded RNA. , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[12]  M. J. Cody,et al.  TLR4, but not TLR2, mediates IFN-β–induced STAT1α/β-dependent gene expression in macrophages , 2002, Nature Immunology.

[13]  D. Hume,et al.  IFN-gamma primes macrophage responses to bacterial DNA. , 1998, Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research.

[14]  A. Aderem,et al.  Toll-like receptors in the induction of the innate immune response , 2000, Nature.

[15]  B. Rouse,et al.  DNA containing CpG motifs induces angiogenesis , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[16]  S. Paludan,et al.  Virus-Cell Interactions Regulating Induction of Tumor Necrosis Factor Alpha Production in Macrophages Infected with Herpes Simplex Virus , 2001, Journal of Virology.

[17]  C. Nathan,et al.  Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells , 1989, The Journal of experimental medicine.

[18]  S. Akira,et al.  Cutting Edge: A Novel Toll/IL-1 Receptor Domain-Containing Adapter That Preferentially Activates the IFN-β Promoter in the Toll-Like Receptor Signaling1 , 2002, The Journal of Immunology.

[19]  B K Slinker,et al.  The statistics of synergism. , 1998, Journal of molecular and cellular cardiology.

[20]  A. Krieg,et al.  Bacterial DNA induces NK cells to produce IFN-gamma in vivo and increases the toxicity of lipopolysaccharides. , 1996, Journal of immunology.

[21]  G. Ghadge,et al.  Activation of Interferon-inducible 2′5′ Oligoadenylate Synthetase by Adenoviral VAI RNA (*) , 1995, The Journal of Biological Chemistry.

[22]  F. Marincola,et al.  Cancer therapy using a self-replicating RNA vaccine , 1999, Nature Medicine.

[23]  P. Angel,et al.  NFkappaB activation is required for interferon regulatory factor-1-mediated interferon beta induction. , 1999, European journal of biochemistry.

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

[25]  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.

[26]  H. Kirchner,et al.  Analysis of the interferons induced in mice in vivo and in macrophages in vitro by Newcastle disease virus and by polyinosinic-polycytidylic acid. , 1986, Journal of interferon research.

[27]  C. Hunter,et al.  Regulatory pathways involved in the infection-induced production of IFN-γ by NK cells , 2002 .

[28]  R. Flavell,et al.  Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3 , 2001, Nature.

[29]  M. Manns,et al.  Apoptotic, but not necrotic, tumor cell vaccines induce a potent immune response in vivo , 2003, International journal of cancer.

[30]  G. Trinchieri,et al.  Interleukin-12 in anti-tumor immunity and immunotherapy. , 2002, Cytokine & growth factor reviews.

[31]  G. Cheng,et al.  Nitric oxide sensitizes prostate carcinoma cell lines to TRAIL-mediated apoptosis via inactivation of NF-κB and inhibition of Bcl-xL expression , 2004, Oncogene.

[32]  B. Williams,et al.  Blockade of Interferon Induction and Action by the E3L Double-Stranded RNA Binding Proteins of Vaccinia Virus , 2002, Journal of Virology.

[33]  B. Williams,et al.  Alphavirus-based DNA vaccine breaks immunological tolerance by activating innate antiviral pathways , 2003, Nature Medicine.

[34]  S Karlin,et al.  Why is CpG suppressed in the genomes of virtually all small eukaryotic viruses but not in those of large eukaryotic viruses? , 1994, Journal of virology.

[35]  P. Angel,et al.  NFκB activation is required for interferon regulatory factor-1-mediated interferon β induction , 1999 .

[36]  S. Wessler,et al.  Neisseria gonorrhoeae Epithelial Cell Interaction Leads to the Activation of the Transcription Factors Nuclear Factor κB and Activator Protein 1 and the Induction of Inflammatory Cytokines , 1997, The Journal of experimental medicine.

[37]  L. Overbergh,et al.  Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR. , 1999, Cytokine.

[38]  R. Herberman.  Cancer immunotherapy with natural killer cells. , 2002, Seminars in oncology.

[39]  B. Williams,et al.  Deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the PKR gene: role of IRF‐1 and NF‐κB , 1997, The EMBO journal.

[40]  G. Stark,et al.  IRF-3-dependent, NFκB- and JNK-independent activation of the 561 and IFN-β genes in response to double-stranded RNA , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[41]  S. Karlin,et al.  Over- and under-representation of short oligonucleotides in DNA sequences. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[42]  T. Chou,et al.  Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. , 1984, Advances in enzyme regulation.

[43]  B. Williams,et al.  Effect of deficiency of the double-stranded RNA-dependent protein kinase, PKR, on antiviral resistance in the presence or absence of ribonuclease L: HSV-1 replication is particularly sensitive to deficiency of the major IFN-mediated enzymes. , 2000, Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research.

[44]  S. Akira,et al.  Herpes simplex virus type 1 activates murine natural interferon-producing cells through toll-like receptor 9. , 2004, Blood.

[45]  J. Kinoshita.  Women Fight Uphill Battle for Equity , 1996, Science.

[46]  M. Mathews,et al.  Interaction of adenovirus VA RNAI with the protein kinase DAI: Nonequivalence of binding and function , 1990, Cell.

[47]  S. Lo,et al.  Lipid Extract of Mycoplasma penetransProteinase K-Digested Lipid-Associated Membrane Proteins Rapidly Activates NF-κB and Activator Protein 1 , 1999, Infection and Immunity.

[48]  Lewis L Lanier,et al.  Natural killer cells and cancer. , 2003, Advances in cancer research.

[49]  T. Dubensky,et al.  Enhancement of tumor-specific immune response with plasmid DNA replicon vectors. , 2000, Cancer research.

[50]  C. Hunter,et al.  Regulatory pathways involved in the infection-induced production of IFN-gamma by NK cells. , 2002, Microbes and infection.

[51]  S. Snyder,et al.  Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[52]  D. Kwiatkowski,et al.  Interaction of AP-1 with a cluster of NF-kappa B binding elements in the human TNF promoter region. , 2001, Biochemical and biophysical research communications.

[53]  G. Koretzky,et al.  Differential regulation of the IL-12 p40 promoter and of p40 secretion by CpG DNA and lipopolysaccharide. , 1999, Journal of immunology.

[54]  R. Steinman,et al.  Dendritic Cells Charged with Apoptotic Tumor Cells Induce Long-Lived Protective CD4+ and CD8+ T Cell Immunity against B16 Melanoma 1 , 2003, The Journal of Immunology.

[55]  V. Hearing,et al.  Inhibition of melanoma-associated antigen expression and ecotropic retrovirus production in B16BL6 melanoma cells transfected with major histocompatibility complex class I genes. , 1996, Cancer research.

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

[57]  G. Trinchieri,et al.  Interleukin-12 and the regulation of innate resistance and adaptive immunity , 2003, Nature Reviews Immunology.

[58]  S. Force,et al.  Cationic lipid:bacterial DNA complexes elicit adaptive cellular immunity in murine intraperitoneal tumor models. , 2000, Cancer research.

[59]  G. Trinchieri,et al.  Type I interferons and IL‐12: convergence and cross‐regulation among mediators of cellular immunity , 2001, European journal of immunology.

[60]  Leaf Huang,et al.  Systemic administration of LPD prepared with CpG oligonucleotides inhibits the growth of established pulmonary metastases by stimulating innate and acquired antitumor immune responses , 2001, Cancer Immunology, Immunotherapy.

[61]  E. Chiang,et al.  Correction of Defects Responsible for Impaired Qa-2 Class Ib MHC Expression on Melanoma Cells Protects Mice from Tumor Growth 1 , 2003, The Journal of Immunology.

[62]  D. Levy,et al.  IRF3 and IRF7 Phosphorylation in Virus-infected Cells Does Not Require Double-stranded RNA-dependent Protein Kinase R or IκB Kinase but Is Blocked by Vaccinia Virus E3L Protein* , 2001, The Journal of Biological Chemistry.

[63]  R. Scheule.  The role of CpG motifs in immunostimulation and gene therapy. , 2000, Advanced drug delivery reviews.

[64]  S. Pleschka,et al.  Influenza Virus-induced AP-1-dependent Gene Expression Requires Activation of the JNK Signaling Pathway* , 2001, The Journal of Biological Chemistry.

[65]  F. Sallusto,et al.  Origin, maturation and antigen presenting function of dendritic cells. , 1997, Current opinion in immunology.

[66]  I. Mohr,et al.  Characterization of RNA Determinants Recognized by the Arginine- and Proline-Rich Region of Us11, a Herpes Simplex Virus Type 1-Encoded Double-Stranded RNA Binding Protein That Prevents PKR Activation , 2002, Journal of Virology.

[67]  G. Stark,et al.  How cells respond to interferons. , 1998, Annual review of biochemistry.

[68]  E. Borden,et al.  Suppression of NF-κB Survival Signaling by Nitrosylcobalamin Sensitizes Neoplasms to the Anti-tumor Effects of Apo2L/TRAIL* , 2003, Journal of Biological Chemistry.

[69]  D. Morrison,et al.  Cutting edge: bacterial DNA and LPS act in synergy in inducing nitric oxide production in RAW 264.7 macrophages. , 1999, Journal of immunology.

[70]  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.

[71]  R. Silverman,et al.  Interferon action and apoptosis are defective in mice devoid of 2′,5′‐oligoadenylate‐dependent RNase L , 1997, The EMBO journal.

[72]  J. Fay,et al.  Dendritic cells: controllers of the immune system and a new promise for immunotherapy. , 2003, Novartis Foundation symposium.

[73]  T. Whiteside,et al.  Therapeutic Activity of NK Cells against Tumors , 2001, International reviews of immunology.

[74]  C. Nathan,et al.  Nitric oxide and macrophage function. , 1997, Annual review of immunology.

[75]  Y. Ohmori,et al.  Requirement for STAT1 in LPS‐induced gene expression in macrophages , 2001, Journal of leukocyte biology.

[76]  S. Akira,et al.  Toll-like Receptor 9–mediated Recognition of Herpes Simplex Virus-2 by Plasmacytoid Dendritic Cells , 2003, The Journal of experimental medicine.

[77]  S. Akira,et al.  Role of Adaptor TRIF in the MyD88-Independent Toll-Like Receptor Signaling Pathway , 2003, Science.