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 gammaRII 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.
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-alpha 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.
Activation of TLR7 and TLR9 by endogenous RNA- or DNA-containing ligands, respectively, is thought to contribute to the complicated pathophysiology of systemic lupus erythematosus (SLE). These ...ligands induce the release of type-I interferons by plasmacytoid dendritic cells and autoreactive antibodies by B-cells, both responses being key events in perpetuating SLE. We recently described the development of inhibitory oligonucleotides (INH-ODN), which are characterized by a phosphorothioate backbone, a CC(T)XXX3-5GGG motif and a chemical modification of the G-quartet to avoid the formation of higher order structures via intermolecular G-tetrads. These INH-ODNs were equally or significantly more efficient to impair TLR7- and TLR9-stimulated murine B-cells, macrophages, conventional and plasmacytoid dendritic cells than the parent INH-ODN 2088, which lacks G-modification. Here, we evaluate the inhibitory/therapeutic potential of our set of G-modified INH-ODN on human immune cells. We report the novel finding that G-modified INH-ODNs efficiently inhibited the release of IFN-α by PBMC stimulated either with the TLR7-ligand oligoribonucleotide (ORN) 22075 or the TLR9-ligand CpG-ODN 2216. G-modification of INH-ODNs significantly improved inhibition of IL-6 release by PBMCs and purified human B-cells stimulated with the TLR7-ligand imiquimod or the TLR9-ligand CpG-ODN 2006. Furthermore, inhibition of B-cell activation analyzed by expression of activation markers and intracellular ATP content was significantly improved by G-modification. As observed with murine B-cells, high concentrations of INH-ODN 2088 but not of G-modified INH-ODNs stimulated IL-6 secretion by PBMCs in the absence of TLR-ligands thus limiting its blocking efficacy. In summary, G-modification of INH-ODNs improved their ability to impair TLR7- and TLR9-mediated signaling in those human immune cells which are considered as crucial in the pathophysiology of SLE.
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.
The ability of the host to distinguish between self and foreign nucleic acids is one of the critical factors contributing to the recognition of pathogens by Toll-like receptors (TLRs). Under certain ...circumstances, eukaryotic self-RNA may reach TLR-containing compartments allowing for self-recognition. Specific modifications were previously demonstrated to suppress immune activation when placed at several positions in an immune stimulatory RNA or silencing RNA (siRNA). However, we show that even a simple natural modification such as a single 2′-O-methylation at different nucleotide positions throughout a sequence derived from a self-RNA strongly interferes with TLR-mediated effects. Such a single modification can even have an inhibitory effect in vitro and in vivo when placed in a different than the immune stimulatory RNA strand acting as suppressive RNA. Several safeguard mechanisms appear to have evolved to avoid cellular TLR-mediated activation by self-RNAs that may under other circumstances result in inflammatory or autoimmune responses. This knowledge can be used to include as few as a single 2′-O-methyl modification at a specific position in a siRNA sense or anti-sense strand to avoid TLR immune effects.
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.
Activation of Toll‐like receptors (TLRs) results in stimulation of the innate immune response, including secretion of proinflammatory cytokines, up‐regulation of co‐stimulatory molecules, and secretion of cytokines and chemokines. In our SAR model, the CpG dinucleotide motif of immune stimulatory oligodeoxynucleotides is recognized by TLR9 by the Hoogsteen site of guanine and the C5 corner of cytosine resulting in potent stimulation of the innate immune response.
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.
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