Marion Jurk

Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities.

Oligodeoxynucleotides (ODN) with unmethylated deoxycytidyl‐deoxyguanosine (CpG) dinucleotides (CpG ODN) mimic the immunostimulatory activity of bacterial DNA and are recognized by the Toll‐like receptor 9 (TLR9). CpG ODN of the B‐Class stimulate strong B cell and NK cell activation and cytokine production. The highest degrees of NK stimulation as well as IFN‐α secretion by plasmacytoid DC were found to occur only with A‐Class ODN. A third class of CpG ODN combines the immune effects of A‐ and B‐Class CpG ODN. C‐Class ODN strongly stimulate B cell or NK cell activation and IFN‐α production. In contrast to the A‐Class, the C‐Class is wholly phosphorothioate, has no poly‐G stretches, but has palindromic sequences combined with stimulatory CpG motifs. All classes stimulate TLR9‐dependent signaling, but with strikingly different dose‐response relationships that are quite in contrast to those observed for IFN‐α. Effects similar to those on human cells were observed on mouse splenocytes. In contrast, splenocytes from TLR9‐deficient mice did not show any response to the three CpG ODN classes. In vivo studies demonstrate that C‐Class ODN are very potent Th1adjuvants. C‐Class ODN may represent new therapeutic drugs that combine the effects of A‐ and B‐Class ODN for broad applications in infectious disease or cancer therapy.

Immune stimulation mediated by autoantigen binding sites within small nuclear RNAs involves Toll-like receptors 7 and 8

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies to certain cellular macromolecules, such as the small nuclear ribonucleoprotein particles (snRNPs), which had been considered to be passive targets of the autoimmune response. SLE is also characterized by the increased expression of type I interferon (IFN), which appears to be associated with the development and severity of disease. Here, we show that specific, highly conserved RNA sequences within snRNPs can stimulate Toll-like receptors (TLRs) 7 and 8 as well as activate innate immune cells, such as plasmacytoid dendritic cells (pDCs), which respond by secreting high levels of type I IFN. SLE patient sera containing autoantibodies to snRNPs form immune complexes that are taken up through the Fc receptor γRII and efficiently stimulate pDCs to secrete type I IFNs. These results demonstrate that a prototype autoantigen, the snRNP, can directly stimulate innate immunity and suggest that autoantibodies against snRNP may initiate SLE by stimulating TLR7/8.

Identification of RNA Sequence Motifs Stimulating Sequence-Specific TLR8-Dependent Immune Responses

The TLRs 7, 8, and 9 stimulate innate immune responses upon recognizing pathogen nucleic acids. U-rich RNA sequences were recently discovered that stimulate human TLR7/8-mediated or murine TLR7-mediated immune effects. In this study we identified single-stranded RNA sequences containing defined sequence motifs that either preferentially activate human TLR8-mediated as opposed to TLR7- or TLR7/8-mediated immune responses. The identified TLR8 RNA motifs signal via TLR8 and fail to induce IFN-α from TLR7-expressing plasmacytoid dendritic cells but induce the secretion of Th1-like and proinflammatory cytokines from TLR8-expressing immune cells such as monocytes or myeloid dendritic cells. In contrast, RNA sequences containing the TLR7/8 motif signal via TLR7 and TLR8 and stimulate cytokine secretion from both TLR7- and TLR8-positive immunocytes. The TLR8-specific RNA sequences are able to trigger cytokine responses from human and bovine but not from mouse, rat, and porcine immune cells, suggesting that these species lack the capability to respond properly to TLR8 RNA ligands. In summary, we describe two classes of single-stranded TLR7/8 and TLR8 RNA agonists with diverse target cell and species specificities and immune response profiles.

Oligodeoxynucleotides lacking CpG dinucleotides mediate Toll-like receptor 9 dependent T helper type 2 biased immune stimulation

Oligodeoxynucleotides (ODN) with unmethylated CpG dinucleotides mimic the immune stimulatory activity of bacterial DNA in vertebrates and are recognized by Toll‐like receptor 9 (TLR9). It is also possible to detect immune activation with certain phosphorothioate sequences that lack CpG motifs. These ODN are less potent than CpG ODN and the mechanism by which they stimulate mammalian leucocytes is not understood. We here provide several lines of evidence demonstrating that the effects induced by non‐CpG ODN are mediated by TLR9. First, non‐CpG ODN could not stimulate cytokine secretion from the splenocytes of TLR9‐deficient (TLR9–/–) mice. Second, immunization of TLR9+/+ but not TLR9–/– mice with non‐CpG ODN enhanced antigen‐specific antibody responses, although these were T helper type 2 (Th2)‐biased. Third, reactivity to non‐CpG ODN could be reconstituted by transfection of human TLR9 into non‐responsive cells. In addition, we define a new efficient immune stimulatory motif aside from the CpG dinucleotide that consists of a 5′‐TC dinucleotide in a thymidine‐rich background. Non‐CpG ODN containing this motif induced activation of human B cells, but lacked stimulation of Th1‐like cytokines and chemokines. Our study indicates that TLR9 can mediate either efficient Th1‐ or Th2‐dominated effects depending on whether it is stimulated by CpG or certain non‐CpG ODN.

Modulating responsiveness of human TLR7 and 8 to small molecule ligands with T-rich phosphorothiate oligodeoxynucleotides

Toll‐like receptors (TLR) 7 and 8 are closely related members of the TLR family of pathogen‐associated molecular pattern recognition receptors and have an important function in activation of innate immune responses upon viral infection. TLR7 can be activated selectively by the guanosine analogue loxoribine, whereas the imidazoquinoline derivative Resiquimod (R‐848) activates both TLR7 and TLR8. We demonstrate that co‐incubation of R‐848 with thymidine homopolymer oligodeoxynucleotides (ODN) significantly increased activity of R‐848 on TLR8‐expressing HEK 293 cells, but abolished TLR7‐mediated signaling. Similarly, the combination of loxoribine and thymidine ODN redirected the stimulatory effect of loxoribine away from TLR7, and toward TLR8. This alteration in ligand specificity was demonstrated both in TLR‐transfected HEK cells, and also in human PBMC, with a corresponding change in cytokine production away from IFN‐α secretion by TLR7‐expressing plasmacytoid DC and toward IL‐12, TNF‐α and IFN‐γ secretion by TLR8‐expressing monocytes and NK cells. These results demonstrate an unexpected plasticity in the ligand specificities of TLR7 and TLR8, and suggest a novel sequence‐selective interaction between these receptors and synthetic phosphorothioate ODN.

Therapeutic applications of synthetic CpG oligodeoxynucleotides as TLR9 agonists for immune modulation.

Vertebrate toll-like receptors (TLRs) sense invading pathogens by recognizing bacterial and viral structures and, as a result, activate innate and adaptive immune responses. Ten human functional TLRs have been reported so far; three of these (TLR7, 8, and 9) are expressed in intracellular compartments and respond to single-stranded nucleic acids as natural ligands. The pathogen structure selectively recognized by TLR9 in bacterial or viral DNA was identified to be CpG dinucleotides in specific sequence contexts (CpG motifs). Short phosphorothioate-stabilized oligodeoxynucleotides (ODNs) containing such motifs are used as synthetic TLR9 agonists, and different classes of ODN TLR9 agonists have been identified with distinct immune modulatory profiles. The TLR9-mediated activation of the vertebrate immune system suggests using such TLR9 agonists as effective vaccine adjuvants for infectious disease, and for the treatment of cancer and asthma/allergy. Immune activation by CpG ODNs has been demonstrated to be beneficial in animal models as a vaccine adjuvant and for the treatment of a variety of viral, bacterial, and parasitic diseases. Antitumor activity of CpG ODNs has also been established in numerous mouse models. In clinical vaccine trials in healthy human volunteers or in immunocompromised HIV-infected patients, CpG ODNs strongly enhanced vaccination efficiency. Most encouraging results in the treatment of cancers have come from human phase I and II clinical trials using CpG ODNs as a tumor vaccine adjuvant, monotherapy, or in combination with chemotherapy. Therefore, CpG ODNs represent targeted immune modulatory drugs with a broad range of potential applications.

Uptake, Efficacy, and Systemic Distribution of Naked, Inhaled Short Interfering RNA (siRNA) and Locked Nucleic Acid (LNA) Antisense

Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.

CpG oligodeoxynucleotides stimulate IFN-γ-inducible protein-10 production in human B cells

Several classes of CpG oligodeoxynucleotides (ODNs) with different immune stimulatory profiles were recently identified: the A-, B- and C-classes. In this study, we investigated the CpG-dependent stimulation of IFN-γ-inducible protein 10 (IP-10 or CXCL10) in different human immune cell types. CpG ODNs induced IP-10 in monocytes, pDCs and in B cells. Purified B cells as well as RPMI 8226 cells responded to CpG stimulation by IP-10 production. Treatment with exogenous IFN-α2b sensitized PBMCs, purified B cells as well as RPMI 8226 cells to respond more efficiently to stimulation with CpG ODNs by IP-10 production. IP-10 signaling could be directly stimulated via TLR9 in CpG-unresponsive HEK293 cells transfected with human TLR9 and an IP-10 reporter construct. Therefore, CpG-mediated IP-10 production is stimulated through IFN-α in cells that express the IFN-α receptor, a second pathway for IP-10 induction exists in TLR9-expressing B cells and pDCs where IP-10 is stimulated directly upon CpG-mediated TLR9 signaling. Our data provide a better understanding of the mechanisms through which CpG ODNs induce efficient Th1 responses.

Structure–Activity Relationship Studies on the Immune Stimulatory Effects of Base‐Modified CpG Toll‐Like Receptor 9 Agonists

Synthetic oligodeoxynucleotides containing unmethylated deoxycytidylyl‐deoxyguanosine dinucleotide (CpG) motifs are able to stimulate potent immune responses through a signaling pathway involving Toll‐like receptor 9 (TLR9). We have investigated the structure–activity relationship (SAR) of base‐modified CpG oligonucleotides with TLR9 by measuring TLR9 activation by 20‐mer oligonucleotides having just a single human recognition motif (5′‐GTCGTT‐3′) in functional cell‐based TLR9 assays. Substitution of guanine by hypoxanthine and 6‐thioguanine resulted in activity similar to the unmodified parent molecule, whereas purine, 2‐aminopurine, 2,6‐diaminopurine, and 8‐oxo‐7,8‐dihydroguanine substitution resulted in approximately 40–60 % reduction in activity, and 7‐deazaguanine substitution led to the strongest (80 %) reduction in TLR9 stimulation. Furthermore, none of the investigated modifications at C5 and N4 of cytosine were well tolerated with respect to human TLR9 stimulation. Our results are compatible with a SAR model in which guanine is recognized by the Hoogsteen site, and C5 is most critical for recognition of cytosine. In addition, we found significant species‐specific differences between human and murine TLR9 recognition, which demonstrates the importance of choosing appropriate assay systems for SAR studies.

Immunopharmacology of CpG Oligodeoxynucleotides and Ribavirin

ABSTRACT To investigate their potential mechanisms of action, the nucleoside analogue ribavirin and a TLR9 agonist were compared. The CpG oligodeoxynucleotides (ODN) demonstrated strong TLR9-related Th1-type effects, and ribavirin appeared only to mediate signaling in TLR-transfected cells. CpG ODN represent a promising new type of therapeutic drug for hepatitis C or other infectious diseases.