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Keywords:

  • Innate immunity;
  • DNA;
  • RNA;
  • CpG motif;
  • TLR;
  • dsRNA

Abstract

  1. Top of page
  2. Abstract
  3. TLR
  4. CpG DNA and TLR9
  5. Self-DNA and TLR9
  6. Suppressive DNA: not just as a TLR9 antagonist
  7. Double stranded DNA: TLR9-independent innate immune activation
  8. Double stranded RNA: TLR3-dependent and independent pathways
  9. Single stranded RNA: TLR7/8, 3 or something else?
  10. Immunostimulatory element of RNA
  11. CONCLUSION
  12. Acknowledgements
  13. REFERENCES

During infection or tissue damage, the innate immune system detects and responds to nucleic acids released from pathogens or damaged host cells. Accumulating evidence has showed that specific sequences, modifications or structures of nucleic acids influence their immunomodulatory activities. Resulting innate immune modulations are regulated by Toll-like receptor (TLR)-dependent or -independent signaling pathways. The first step in host defense against foreign or unwelcome self nucleic acids may play important roles in immune responses against infectious organisms, as well as in clearance of unnecessary tissues, which may be linked to autoimmune diseases and possibly to other immunological disorders. Elucidating mechanisms of innate immune activation by nucleic acids will help future development of more efficient or safer nucleic acid-based immunotherapies and gene therapies. © 2005 Wiley-Liss, Inc.

Nucleic acids such as DNA and RNA are essential components of all living organisms. Accumulating evidence over the last several decades suggested that nucleic acids not only function as essential ultimate units of life, but also stimulate the immune system when they are released from pathogens.1, 2 Such phenomenon had often been ignored, but are now in the limelight after the recent discovery of Toll-like receptors (TLR) (Figs. 1,2).3, 4 Structure- or sequence-dependent immune recognition of nucleic acids by TLR were shown to play an important role in both innate and adaptive immune responses to infectious organisms including bacteria, virus and parasites.5, 6 Novel therapeutics including nucleic acid-based agonists/antagonists via TLR-mediated immuno-modulation are being developed for multiple therapeutic applications to prevent or treat infectious diseases, allergic disorders and cancer.7, 8

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Figure 1. Cell surface Toll-like receptors recognizing proteins and lipids. Immune cells express TLR that specifically recognize proteins and lipids. TLR1 and TLR6 cooperate with TLR2 to discriminate subtle differences between triacyl and diacyl lipopeptides, respectively. TLR4 is the receptor for LPS. TLR5 recognizes flagellin. TLR11 recognizes profilin-like proteins. Generally, TLR stimulation by cognate ligands, the pro-inflammatory response genes including cytokines such as TNFα, IL-6, IL-12 and co-stimulatory molecules are induced via MyD88-dependent activation of NF-κB and MAP kinases, whereas TLR4-mediated, MyD88-independent type-1 IFN and their inducible genes are induced via TBK1 and IRF3.

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Figure 2. Intracellular innate immune recognition system of nucleic acids. Nucleic acids such as DNA and RNA released from pathogens or host cells are taken-up and delivered into endo- or phagocytic vesicles where TLR3, 7(8) and 9 are expressed. Note that TLR9 (possibly TLR7) initially resides in the endoplasmic reticulum, but is believed to form ER/phagosome upon cognate ligand stimulation. TLR7(8) and 9 ligation via cognate ligands such as ssRNA and DNA recruit MyD88, followed by complex formation with IRAK1, IRAK4, TRAF6 resulting in the activation of NF-κB, MAP kinases or IRF7. TLR3 mediates extracellular dsRNA-induced innate immune activation via a TRAM/TRIF dependent pathway, whereas RIG-I seems to mediate intracellular dsRNA generated by direct infection and replication of virus. These 2 distinct dsRNA-induced signals culminate in TBK-1/IKK-i complex to IRF3/7 activation. dsDNA within or out of cells stimulates IFN inducible genes via unknown mechanisms.

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The innate immune system that fights infection seems to have an important role in clearing unnecessary or abnormal host molecules, including nucleic acids. The innate immune system possesses specialized sets of genes including TLR to facilitate the clearance during trauma, tumor or autoimmune diseases.9, 10 This challenges the widely accepted dogma in which the innate immune system including TLR, discriminates infectious non-self from non-infectious self including nucleic acids. What element(s) within DNA or RNA (i.e., sequence, modification or higher structure) is (are) recognized by the innate immune system has become an important question that needs to be clarified to further understand the immune system. We review recent advances to understand innate immune recognition of nucleic acids and describe the resulting immune modulation through either TLR-dependent or -independent pathways.

TLR

  1. Top of page
  2. Abstract
  3. TLR
  4. CpG DNA and TLR9
  5. Self-DNA and TLR9
  6. Suppressive DNA: not just as a TLR9 antagonist
  7. Double stranded DNA: TLR9-independent innate immune activation
  8. Double stranded RNA: TLR3-dependent and independent pathways
  9. Single stranded RNA: TLR7/8, 3 or something else?
  10. Immunostimulatory element of RNA
  11. CONCLUSION
  12. Acknowledgements
  13. REFERENCES

Eleven members of the TLR family have been reported in mammals (TLR 1–11). TLR family members recognize and respond to diverse molecules containing PAMP including lipids, proteins and nucleic acids. TLR1 and TLR6 cooperate with TLR2 to discriminate subtle differences between triacyl and diacyl lipopeptides, respectively. TLR4 is the receptor for LPS. TLR5 recognizes flagellin. TLR11 recognizes profilin-like protein.11 TLR3, 7(8) or 9 were found to recognize nucleic acids such as double stranded (ds), single stranded (ss) RNA or DNA, respectively. MyD88 is an essential TLR adaptor molecule for TLR 2, 5, 7 and 9, and is involved in innate immune activation including NF-κB dependent cytokine production, upregulation of co-stimulatory molecules as well as production of IFN. TIRAP specifies MyD88 dependent pathway via TLR2 as well as TLR4. TRIF and TRAM, in contrast, mediate MyD88 independent pathway via TLR4. As a result of TLR stimulations by cognate ligands, pro-inflammatory response genes including cytokines such as TNFα, IL-6, IL-12 and co-stimulatory molecules are induced via activation of NF-κB and MAP kinases, whereas Type-1 IFN and their inducible genes are induced via interferon regulatory factors (IRF) 3 or 7 (Figs. 1,2).

CpG DNA and TLR9

  1. Top of page
  2. Abstract
  3. TLR
  4. CpG DNA and TLR9
  5. Self-DNA and TLR9
  6. Suppressive DNA: not just as a TLR9 antagonist
  7. Double stranded DNA: TLR9-independent innate immune activation
  8. Double stranded RNA: TLR3-dependent and independent pathways
  9. Single stranded RNA: TLR7/8, 3 or something else?
  10. Immunostimulatory element of RNA
  11. CONCLUSION
  12. Acknowledgements
  13. REFERENCES

Unmethylated CpG dinucleotides are present in high amounts in bacterial DNA but are suppressed, and methylated in mammalian DNA. The mammalian immune system recognizes and responds to DNA sequence motifs containing unmethylated CpG (CpG motifs) via TLR9.4, 7, 8 CpG DNA binds to, and is taken up by immune cells through endocytic pathways, then co-accumulates with TLR9 in phagosome-like vesicles, a process controlled by PI3 kinase.8 Interaction of CpG DNA with TLR9 triggers the recruitment of the MyD88 adaptor molecule, followed by activation of IRAK1/4, TRAF6 and subsequently the MAP kinase signaling cascade, culminating with nuclear translocation of NF-κB.7 CpG DNA-mediated activation of the innate immune system is characterized by B cell proliferation, dendritic (DC) maturation, NK cell activation and production of pro-inflammatory cytokines (such as IL-6, 12 and Type-I and Type-II IFN), chemokines (such as MCP-1, IP-10, MIP-1α,β) and immunoglobulins. Single stranded oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG ODN) mimic immunostimulatory activity of bacterial DNA. Impressive immunostimulatory activity of CpG ODN is being recorded for future use in a variety of therapeutic purposes.8

Recent studies have indicated that PBMC from primates responded to at least 3 structurally and functionally distinct types of CpG ODN: the D-type (also known as A-type), the K- type (also known as B-type)8, 12 and the C-type described recently.13, 14 These 3 types of ODN possess CpG dinucleotides, but their flanking sequences and compositions are different. For example, K-type ODN contain multiple CpG motifs, whereas D-type ODN have one CpG with palindromic flanking sequences. D-, but not K- nor C-type ODN have a poly-G (5-6 bases) tail at the 3′-end, which may account for their distinct activity. K- and C- but not D-type ODN have phosphorothioate linkage between all nucleotides. D-type ODN stimulate plasmacytoid DC (pDC) to secrete large amounts of IFNα, whereas K-type ODN strongly stimulate B cells to proliferate and to secrete IL-6 and IgM. C-type ODN show a combined activity of K- and D-type ODN, but to a lesser extent.

Why K- or D-type ODN lead to such distinct innate immune activation is an interesting question.12 D-, but not K-type ODN form aggregates in PBS that may contribute to D-type specific IFNα production in PBMC.13 Poly-G tail in D-type ODN may account for differential binding to cognate scavenger receptors, thereby leading to distinct uptake and following signaling mechanisms.15 In fact, K-type and D-type ODN are taken up by endocytosis controlled by PI3 kinase, but end up in different vesicles.15, 16 Such differential uptake of K- and D-type ODN into distinct vesicles within cells may contribute to their distinct roles,16 although there remains a possibility for the existence of K- or D-type ODN specific co-receptor(s) on the cell surface. Nevertheless, all immune activations mediated by these ODN entirely depend on TLR9, as cells that lack this receptor are not responsive to either type of ODN.17

Differential innate immune activation by K- and D-type ODN may also contribute to their distinct mode of intracellular signaling. Strong NF-κB activation is induced by K- but not by D-type ODN.12 Excessive NF-κB activation may lead to immediate induction of pro-inflammatory cytokines such as TNFα and maturation of pre-pDC into mature pDC, thereby unable to produce IFNα.18 Recently, several lines of evidence have shown an intimate relationship between D-type ODN-induced IFNα by pDC and interferon regulatory factor 7 (IRF7). Plasmacytoid DC were shown to express high levels of IRF7 compared to other dendritic subsets or immune cells,19, 20 enabling pDC to be in ‘ready to go’ state to produce large amounts of IFNα. Such high expression of IRF7 by pDC may explain why pDC immediately produce large amounts of IFNα in response to D-type ODN. However, results obtained from IFNαβ receptor KO mice suggested that IFN inducible genes including IRF7 were still required for D-type CpG ODN-induced IFNα production by pDC.17, 21 More recently, MyD88, an essential adaptor protein for TLR9-mediated innate immune activation, was shown to form a complex with IRF7, TRAF6, and with IRAK1 and 4.22, 23 This was confirmed in vivo as mice lacking either IRF7, IRAK1 or IRAK4 failed to produce IFNα in response to D-type ODN,23, 24, 25 suggesting that a unique signaling complex possibly initiated from late endo- or phago-lysosomes plays an important role in robust TLR9- (also TLR7) mediated IFNα production.

Self-DNA and TLR9

  1. Top of page
  2. Abstract
  3. TLR
  4. CpG DNA and TLR9
  5. Self-DNA and TLR9
  6. Suppressive DNA: not just as a TLR9 antagonist
  7. Double stranded DNA: TLR9-independent innate immune activation
  8. Double stranded RNA: TLR3-dependent and independent pathways
  9. Single stranded RNA: TLR7/8, 3 or something else?
  10. Immunostimulatory element of RNA
  11. CONCLUSION
  12. Acknowledgements
  13. REFERENCES

Unmethylated CpG motifs in the genome are observed more frequently in bacterial and certain viral DNA than in vertebrate DNA, and are therefore recognized as a PAMP by the innate immune system via TLR9. Recently, this central dogma has been revisited. Self (host) DNA are normally sequestered from the immune system because they are usually inside cells, e.g., in nuclei or mitochondria or cytoplasm, protected by the plasma membrane. Once cells are damaged and destroyed, these nucleic acids might be released into unusual intra- or extra-cellular environment unless the process results in normal (silent) apoptosis. Some of them are digested immediately by nucleases abundant in serum, but some are taken up mostly by neighboring stromal cells or immune cells such as macrophages and DC. Recent evidence suggests that nucleic acids released from damaged tissues or cells have an ability to modulate innate immune responses.

Self-DNA-immunoglobulin complexes that do not seem to contain any pathogen derived molecules, are known to contribute to pathogenesis of systemic lupus erythematosus (SLE) and other systemic autoimmune diseases. Interestingly, these DNA-immunoglobulin complexes were reported as potent IFNα producers.26 Subsequently, recent studies demonstrated that activation of B cells and DC by DNA-immunoglobulin complexes was TLR9 dependent.27, 28 It should be noted, however, that Fc gamma receptor IIa or III was required for optimal activation by DNA-immunoglobulin complexes and that TLR9-independent activation was still observed.28, 29 These reports suggest that TLR9 not only detects CpG motifs in pathogen DNA, but also recognizes either CpG motifs or yet unknown molecular patterns in host-derived DNA.

Requirement for CpG motifs in TLR9-mediated, DNA-induced immune activation is also being investigated. Many groups have reported recently that ODN containing no CpG-motifs activated the innate immune system via a TLR9 dependent manner.30, 31, 32, 33, 34 These observations became apparent when either nucleosides of CpG dinucleotides were modified, including bicyclic heterobase (1-(2′-deoxy-β-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine or 2′-deoxy-7-deazaguanosine respectively), or when ODN linkages were phosphorothioate modified. These results suggest that TLR9, like other TLR, may not have evolved to recognize not only CpG motifs as a conserved molecular pattern in pathogen DNA, but also abnormal composition, structure or chemical feature of DNA. Nevertheless, what makes TLR9 discriminates between DNA ligands is a topic of increasing interest in the field, and should be clarified in future studies. Although direct binding of TLR9 to ODN has been demonstrated, it remains controversial whether the binding is CpG motif-specific (in parallel to their immunostimulatory activity), or sufficient for specific activation of TLR9 mediated signaling.35, 36, 37 Moreover, heme polymers (so-called hemozoin) produced as heme metabolites during malaria infection in red blood cells were found to be unique non-DNA TLR9 ligands.38 What really determines specificity of TLR9 ligands? Numerous factors remain to be studied: hydrophobicity, charge, secondary structure. Further understanding of TLR9 binding to such diverse molecules or their pattern may require structural analysis.

Suppressive DNA: not just as a TLR9 antagonist

  1. Top of page
  2. Abstract
  3. TLR
  4. CpG DNA and TLR9
  5. Self-DNA and TLR9
  6. Suppressive DNA: not just as a TLR9 antagonist
  7. Double stranded DNA: TLR9-independent innate immune activation
  8. Double stranded RNA: TLR3-dependent and independent pathways
  9. Single stranded RNA: TLR7/8, 3 or something else?
  10. Immunostimulatory element of RNA
  11. CONCLUSION
  12. Acknowledgements
  13. REFERENCES

During infection or tissue damage, inflammation must be waned and terminated with tissue remodeling and healing. In this case, a negative feedback system of innate immune activation occurs via several inhibitory signals. In particular, CpG-DNA driven immune activation may have deleterious consequences, such as exacerbating inflammatory tissue damage, or increasing sensitivity to autoimmune diseases or toxic shock. Recent evidence suggested that host DNA contained some antagonistic elements to the immunostimulatory effect in their DNA or against pathogen derived CpG-rich DNA, possibly suppressing DNA-driven immunostimulation.39 Neutralizing or suppressive motifs can selectively block CpG-mediated immune stimulation.40 Suppressive motifs are rich in poly-G or GC sequences, and optimal motifs are surprisingly identical to telomere sequences (with a repeat of TTAGGG), which are present in DNA of mammals, but not in bacteria.41 Suppressive activity of ODN also correlates with their ability to form higher structures such as G-tetrads.41 Recent studies indicated that suppressive ODN did not interfere with binding or uptake of CpG ODN.42 Rather, they blocked either TLR9 binding or assembling of CpG DNA or the signaling cascade initiated by CpG DNA upstream of NF-κB translocation to the nucleus.41,42 This effect was reproduced in vivo in lung inflammation induced by bacterial DNA or when CpG ODN were blocked by simultaneous administration of suppressive ODN.43 In an experimental model in which inflammatory arthritis was induced by intra-articular CpG ODN injection, pre-treatment of mice with suppressive ODN significantly reduced CpG-induced inflammatory arthritis.44 Whether suppressive ODN affected other inflammatory events that are TLR9 independent has been explored. Suppressive ODN were shown to bind STAT1 and STAT4, thereby inhibiting their downstream signaling cascade that is independent of TLR9 signaling, resulting in reduced incidence of LPS-induced lethality and Th2 biased adaptive immune responses.45, 46 It is quite interesting that suppressive sequences in self-DNA may play a role in neutralizing exacerbating inflammation or modulating both innate and adaptive immune responses in a TLR9 independent manner, thereby providing potential therapeutic uses as natural anti-inflammatory agents or Th2 inducing adjuvants.

Double stranded DNA: TLR9-independent innate immune activation

  1. Top of page
  2. Abstract
  3. TLR
  4. CpG DNA and TLR9
  5. Self-DNA and TLR9
  6. Suppressive DNA: not just as a TLR9 antagonist
  7. Double stranded DNA: TLR9-independent innate immune activation
  8. Double stranded RNA: TLR3-dependent and independent pathways
  9. Single stranded RNA: TLR7/8, 3 or something else?
  10. Immunostimulatory element of RNA
  11. CONCLUSION
  12. Acknowledgements
  13. REFERENCES

When DNA forms a double strand, DNA may become immunologically active unless it is digested immediately by nucleases. DNase-I, which is the major nuclease in serum may contribute to eliminate extracellular free DNA in sera, and may suppress deleterious effects of DNA to further development of SLE-like autoimmune diseases as in DNase-I KO mice,47 and as DNase-I mutation was observed in SLE patients.48 DNase-II, which is the major DNase in phagosomes in macrophages also plays a major role in clearing DNA from phagocytosed apoptotic cells or debris. Without clearance of DNA in their phagosomes, macrophages start producing IFNβ leading to subsequent inflammatory events.49 Moreover, DNase-III KO mice develop inflammatory myocarditis,50 suggesting the importance of clearing unnecessary DNA from the body to prevent inflammatory responses that may otherwise result in autoimmune disorders.51 Experimentally, double stranded, but not single stranded DNA derived from both pathogens and host, when introduced into the cytoplasm, was shown to activate immune cells (macrophages and dendritic cells) and non-immune cells (thyroid cells, fibroblasts, muscle cells), characterized by upregulation of IFN-inducible genes including MHC Class-I, -II, TAP1, LMP2, CD40, 80 and 86.52, 53 Type-I IFN was induced by double stranded DNA via a TLR9 independent pathway,54 suggesting that this DNA-induced innate immune activation is independent of residual CpG motifs in host DNA. These results implicate that double stranded DNA forming a complex with chromatin, immunoglobulins, lipids or other cross-linking agents may contribute to pathogenesis or defense mechanisms of inflammatory diseases. Furthermore, DNA-based gene therapies should pay an attention to such ‘non-specific’ inflammatory DNA properties. Immunotherapies using DNA, such as DNA vaccines, need to consider both TLR9 dependent and independent immunostimulatory activities of DNA vectors as ‘built-in’ adjuvants.

Double stranded RNA: TLR3-dependent and independent pathways

  1. Top of page
  2. Abstract
  3. TLR
  4. CpG DNA and TLR9
  5. Self-DNA and TLR9
  6. Suppressive DNA: not just as a TLR9 antagonist
  7. Double stranded DNA: TLR9-independent innate immune activation
  8. Double stranded RNA: TLR3-dependent and independent pathways
  9. Single stranded RNA: TLR7/8, 3 or something else?
  10. Immunostimulatory element of RNA
  11. CONCLUSION
  12. Acknowledgements
  13. REFERENCES

Double stranded (ds) RNA is generated in host cells during replication of most viruses.55 The host innate immune system thus recognizes dsRNA as a PAMP resulting in robust immune responses characterized by production of Type-I IFN and proinflammatory cytokines. Whereas poly (I:C) synthetic dsRNA analogues are widely used as IFN inducers in many research and clinical applications, specific receptor-like molecules that recognize poly(I:C) have not been fully characterized. Double stranded RNA-dependent protein kinase (PKR) has long been believed to be a sole receptor for dsRNA (poly(I:C)). However, cells derived from PKR-deficient (PkR−/−) mice still responded to poly(I:C), indicating the existence of another receptor, which recognizes poly(I:C). Alexopoulou et al.56 demonstrated that TLR3 conferred strong dsRNA-induced NF-κB activation in 293 cells when ectopically expressed, and that TLR3 KO mice displayed reduced responses to dsRNA including poly(I:C). Yet, dsRNA still stimulated dendritic cells from TLR3 KO mice, especially when administered directly into the cytosol by transfection.57 This suggests that there is an unknown intracellular detection system that is independent of TLR3 (Fig. 2).

Two key molecules that play critical roles in both TLR3 dependent and independent signaling pathways have been identified. TANK-binding kinase 1 (TBK-1) and IKK epsilon (IKK-e, also referred to as IKK-i) were shown to be required for TLR3-dependent and independent poly(I:C)-mediated innate immune activations.58, 59 Results obtained using embryonic fibroblasts (EF) derived from mice lacking TBK-1 or IKK-i confirmed their critical but differential roles in dsRNA-induced innate immune activation.60, 61, 62 Interestingly, although marked reduction of IFN-inducible genes was observed in ds-RNA-stimulated TBK-1 KO fibroblasts, TBK KO macrophages responded normally to poly (I:C), suggesting that there was a TBK-independent pathway for poly(I:C)-induced macrophage (or other immune cells) activation that was possibly compensated by IKK-i.61 Thus, although further in vivo studies are required, TBK-1 and IKK-i complexes may play important roles in TLR3-dependent and independent innate activation by poly(I:C) as well as in viral infection. Yoneyama et al.63 demonstrated recently that RIG-I, a DExD/H box RNA helicase containing caspase recruitment domain (CARD) bound to dsRNA, and was required for dsRNA-induced IFNβ induction through IRF3 phosphorylation. This intriguing study showed a distinct mode of RNA recognition system compared to the TLR3-dependent pathway. Yoneyama et al.63 also described other RNA helicases (Lgp2 and Mda5) that displayed considerable homology to RIG-I whereas Lgp2 was completely devoid of a CARD. Results obtained using RIG-I deficient mice showed that virus-induced Type-I IFNs were abrogated in EF and conventional CD11c−hi, and B220 DC; but not in plasmacytoid CD11−lo, B220+ DC, suggesting that cell type specific anti-viral response distinct signaling pathways similar to those that have been observed in TBK KO cells should be considered as well.64 It would be interesting to examine whether these molecules are involved in viral or dsRNA-induced innate immune activation or suppression (Fig. 2). Having investigated molecular mechanisms by which these 2 distinct receptors and signaling pathways merge into TBK-1 or IKK-i mediated pathways for Type-I IFN induction, a report demonstrated that FADD or RIP-1 deficient fibroblasts had a defect in Type-I IFN production by dsRNA. These results suggest that FADD and RIP-1 may be important players upstream of the TBK-1/IKK-i complex and downstream of the TLR3-independent pathway.65 Further studies will clarify the molecular mechanism(s) underlying TLR3-dependent and independent innate immune activation against dsRNA during viral infection.

Single stranded RNA: TLR7/8, 3 or something else?

  1. Top of page
  2. Abstract
  3. TLR
  4. CpG DNA and TLR9
  5. Self-DNA and TLR9
  6. Suppressive DNA: not just as a TLR9 antagonist
  7. Double stranded DNA: TLR9-independent innate immune activation
  8. Double stranded RNA: TLR3-dependent and independent pathways
  9. Single stranded RNA: TLR7/8, 3 or something else?
  10. Immunostimulatory element of RNA
  11. CONCLUSION
  12. Acknowledgements
  13. REFERENCES

In contrast to dsRNA, which is a well known viral signature thereby being a typical PAMP, single stranded (ss) RNA is abundant not only in pathogens but also in most host cells. It has long been believed that ssRNA is an inert molecule in our immune system. Recent evidence has suggested, however, that ssRNA is immunostimulatory when administered with a transfecting reagent into pDC.66, 67, 68 Early works identified imiquimod and R-848 as small potent antiviral agonist compounds for TLR7.69 A natural ligand for TLR7 was identified as GU rich ssRNA derived from HIV genome stimulating plasmacytoid dendritic cells.67 Influenza ssRNA genome also activated pDC through TLR7 in which the nucleotide preference of such RNA was attributed to poly U but not G, C, A.66 Although long dsRNA is recognized by TLR3, a recent report suggested that short interfering (si) RNA forming double strands with no GU preference activated pDC via TLR7.70 In contrast, another report suggested that TLR3 mediated siRNA-induced sequence independent gene silencing as well as innate immune activation.71 These results may not conflict if different cell types have distinct recognition mechanisms by which pDC utilize TLR7 to recognize ss or short dsRNA whereas myeloid (conventional) DC utilize the same TLR3 as ssRNA do. In fact, human myeloid DC that express TLR3 but not TLR7, are activated by U rich single stranded RNA.72 Monocytes, that express TLR8 but not TLR3, 7 or 9 were shown to respond to ssRNA containing unmethylated CpG sequences plus poly-G, but not -A, -U, or -C tails although ectopic expression of TLR8 in 293 cells did not confer induced immune activation to such ssRNA.73 Further studies are required to elucidate molecular mechanism(s) of cell type-specific recognition of ssRNA by distinct TLR.

Immunostimulatory element of RNA

  1. Top of page
  2. Abstract
  3. TLR
  4. CpG DNA and TLR9
  5. Self-DNA and TLR9
  6. Suppressive DNA: not just as a TLR9 antagonist
  7. Double stranded DNA: TLR9-independent innate immune activation
  8. Double stranded RNA: TLR3-dependent and independent pathways
  9. Single stranded RNA: TLR7/8, 3 or something else?
  10. Immunostimulatory element of RNA
  11. CONCLUSION
  12. Acknowledgements
  13. REFERENCES

What is (are) the responsible element(s) for immunostimulatory activity of RNA in TLR mediated innate immune recognition? Sequence preference that may confer such activity initially seemed attractive, as the presence of GU or U in ssRNA may explain some of the differences between stimulatory and inert ssRNA.66, 67 Other evidence suggests that mRNA derived from bacteria without poly-A tail, but not from vertebra mRNA that contains a poly-A tail, displays immunostimulatory activity on human myeloid dendritic cells.72 This explains why bacterial but not host mRNA is stimulatory. Similarly, CpG motifs, observed more frequently in bacterial and certain viral DNA may be mirrored in mRNA expressed by such pathogens. In fact, ssRNA with unmethylated CpG motifs was immunostimulatory and its activity was abrogated if 5′-C was methylated, similar to that of CpG DNA.73 Such specific sequences or motifs observed in pathogens, but not in the host ssRNA may also have an impact on the immunostimulatory activity of ssRNA. Interestingly, not only 5′-C methylation in CpG motif but also methylation of 2′-O portion of any nucleotides in the ssRNA abrogated the immunostimulatory effects.73 Furthermore, there are numerous evidence of RNA methylation in other portions of nucleotides (e.g., 2′-O methylation of rRNA and small nuclear [sn] RNA, 7′G cap methylation of mRNA),74, 75, 76, 77 suggesting that such modifications of RNA species in the host may alter immunostimulatory activity of ssRNA. It is attractive to hypothesize that hosts might have evolved to sequester their immunostimulatory element of ssRNA from their own innate immune system by modification (e.g., methylation) of nucleotides. It is of interest if abnormal modifications or absence either in pathogen or host derived ssRNA may occur during infection or tissue injury, which may turn on innate immune response. This may also explain why RNA derived from not only pathogens but also host cells stimulate the innate immune system. Although the innate immune system is thought to have evolved to recognize pathogen associated molecular patterns (PAMP) such as dsRNA, unmethylated CpG DNA, hosts may have evolved to modify or sequester their own default, immunostimulatory molecules to inert RNA as ‘host associated molecular patterns (HAMP)’ by modifications such as methylation to avoid unnecessary innate immune activation. Yet, there should be more studies to clarify the requirement for precise molecular features of immunostimulatory RNA including their sequences and resulting higher structures.

CONCLUSION

  1. Top of page
  2. Abstract
  3. TLR
  4. CpG DNA and TLR9
  5. Self-DNA and TLR9
  6. Suppressive DNA: not just as a TLR9 antagonist
  7. Double stranded DNA: TLR9-independent innate immune activation
  8. Double stranded RNA: TLR3-dependent and independent pathways
  9. Single stranded RNA: TLR7/8, 3 or something else?
  10. Immunostimulatory element of RNA
  11. CONCLUSION
  12. Acknowledgements
  13. REFERENCES

We have described recent advances in immune recognition of nucleic acids. Discovery of TLR and their signaling pathways has further helped our understanding of which innate and adaptive immune system recognize DNA and RNA derived not only from pathogens, but also from damaged host. Some of the immunological alteration by DNA and RNA were shown to be independent of TLR. It will be of interest to investigate the physiological roles of TLR-dependent or -independent immunological activity of nucleic acids especially in case of intracellular pathogens, autoimmune diseases and cancer. Abnormal release (defect in clearance) or aberrant modification of nucleic acids may have an impact on their immunomodulatory effects. Introducing the immunomodulatory DNA or RNA described above may increase the efficacy of gene or cell based immunotherapy or vaccine against variety of disease including cancer. In addition to TLR ligands, our hope is that TLR-independent recognition of nucleic acids may lead to discoveries of novel ligands, receptors, signaling pathways and further the understanding of their physiological roles and therapeutic potentials.

REFERENCES

  1. Top of page
  2. Abstract
  3. TLR
  4. CpG DNA and TLR9
  5. Self-DNA and TLR9
  6. Suppressive DNA: not just as a TLR9 antagonist
  7. Double stranded DNA: TLR9-independent innate immune activation
  8. Double stranded RNA: TLR3-dependent and independent pathways
  9. Single stranded RNA: TLR7/8, 3 or something else?
  10. Immunostimulatory element of RNA
  11. CONCLUSION
  12. Acknowledgements
  13. REFERENCES
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