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1.
Ribonucleic nucleic acid recognition by Toll-like receptors (TLRs) induces innate immune responses. However, no comprehensive analysis of gene expression in human blood cells in response to unmodified and 2'-modified immunostimulatory RNAs has been reported. Using oligonucleotide microarrays, we show that around 400 genes were significantly (P<0.001) altered in peripheral blood mononuclear cells (PBMC) in response to either single-stranded (ss) or double-stranded (ds) small interfering RNAs (siRNAs). Most of the upregulated genes encode proteins involved in innate and adaptive immune responses, including proinflammatory cytokines, interferons, chemokines and chemokine receptors. Genes encoding proteins involved in lymphocyte activation (e.g. CD80, CD40, and CD69) and in regulation of the immune responses (e.g. SOCS proteins) were upregulated. Also, genes encoding for antiviral proteins (Mx1, Mx2, TRIM proteins), and interferon regulatory factors (e.g. IRF7) were upregulated. Around 90% of the genes (140 out of 160) affected by R-848, a specific ligand for TLR7 and TLR8, were also affected by ss siRNAs or ds siRNAs, indicating that the signaling pathways activated by R-848 are also activated by immunostimulatory siRNAs. In addition to immunoactivation via TLRs, ss siRNAs and ds siRNAs induced TLR-independent gene alterations. Surprisingly, replacement of only uridine bases with either 2'-fluoro or 2'-O-methyl modified counterparts abrogated all the observed bystander effects. Collectively, these microarray data offer for the first time an insight into human PMBC response to immunostimulatory RNAs such as ss siRNAs and ds siRNAs. The data should help to define strategies to either enhance or avoid the non-specific effects of siRNAs in order to develop safe therapeutics.  相似文献   

2.
Short interfering RNAs (siRNAs) are the processing product originating from long double-stranded RNAs (dsRNAs) that are cleaved by the RNase III-like ribonuclease Dicer. As siRNAs mediate cleavage of specific single-stranded target RNAs, they are essential intermediates of RNA interference (RNAi). When applied in synthetic form, siRNAs likewise can induce the silencing process in the absence of long dsRNAs. Here, we tested variations of a conventional synthetic siRNA that had been used successfully to silence the Drosophila notch gene. The variants had two 3 ' -terminal deoxynucleotides in their protruding single-stranded ends. In one case, the deoxynulceotides would match to the notch mRNA, whereas the other variant had nonmatching deoxy-T residues, representing a widely used siRNA design. siRNAs with different combinations of sense and antisense strands were injected into Drosophila embryos at two different concentrations. We found that the all-ribonucleotide siRNA gave the best inhibition of notch expression. The combination of two modified strands with 3 ' -terminal deoxynucleotides was effective, but if combined with a sense or antisense ribostrand, the efficacy dropped. The siRNAs with nonmatching 3 ' -terminal TT residues showed a reduced silencing potential, which became evident at low concentration. An siRNA with a nonmatching 3 ' -terminal ribonucleotide in the antisense strand retained most of its silencing potential in accordance with the hypothesis that primer extension for generation of ssRNA from single-stranded mRNA does not operate in Drosophila.  相似文献   

3.
The concept of small interfering RNA (siRNA) has been extended to include not only short double-stranded RNA of 19-25bp, but also single-stranded antisense RNA of the same length, since such single-stranded antisense siRNAs were recently found to be able to inhibit gene expression as well. We made comprehensive comparison of double- and single-stranded siRNA functions in RNA interference (RNAi), targeting multiple sites and different mRNAs, measuring RNAi effects at different time-points and in different cell lines, and examining response curves. Duplex siRNAs were found to be more potent than single-stranded antisense siRNAs. This was verified by the observation that single-stranded antisense siRNAs, which were inefficient in some cases when used alone, could be rescued from inefficiency by sequentially transfecting with the sense siRNAs. This result suggests that the structural character of siRNA molecules might be a more important determinant of siRNA efficiency than the cellular persistence of them.  相似文献   

4.
Oligodeoxynucleotides containing unmethylated CpG motifs (CpG DNAs) can function as powerful immune adjuvants by activating APC. Compared with conventional phosphorothioate-backbone CpG DNAs, another type of CpG DNAs, called an A or D type (A/D-type), possesses higher ability to induce IFN-alpha production. Conventional CpG DNAs can exert their activity through Toll-like receptor 9 (TLR9) signaling, which depends on a cytoplasmic adapter, MyD88. However, it remains unknown how A/D-type CpG DNAs exhibit their immunostimulatory function. In this study we have investigated murine dendritic cell (DC) responses to these two distinct CpG DNAs. Not only splenic, but also in vitro bone marrow-derived, DCs could produce larger amounts of IFN-alpha in response to A/D-type CpG DNAs compared with conventional CpG DNAs. This IFN-alpha production was mainly due to the B220(+) DC subset. On the other hand, the B220(-) DC subset responded similarly to both CpG DNAs in terms of costimulatory molecule up-regulation and IL-12 induction. IFN-alpha, but not IL-12, induction was dependent on type I IFN. However, all activities of both CpG DNAs were abolished in TLR9- and MyD88-, but were retained in DNA-PKcs-deficient DCs. This study demonstrates that the TLR9-MyD88 signaling pathway is essential for all DC responses to both types of CpG DNAs.  相似文献   

5.
Epithelial cells of the lung are the primary targets for respiratory viruses. Virus-carried single-stranded RNA (ssRNA) can activate Toll-like receptors (TLRs) 7 and 8, whereas dsRNA is bound by TLR3 and a cytoplasmic RNA helicase, retinoic acid-inducible protein I (RIG-I). This recognition leads to the activation of host cell cytokine gene expression. Here we have studied the regulation of influenza A and Sendai virus-induced alpha interferon (IFN-alpha), IFN-beta, interleukin-28 (IL-28), and IL-29 gene expression in human lung A549 epithelial cells. Sendai virus infection readily activated the expression of the IFN-alpha, IFN-beta, IL-28, and IL-29 genes, whereas influenza A virus-induced activation of these genes was mainly dependent on pretreatment of A549 cells with IFN-alpha or tumor necrosis factor alpha (TNF-alpha). IFN-alpha and TNF-alpha induced the expression of the RIG-I, TLR3, MyD88, TRIF, and IRF7 genes, whereas no detectable TLR7 and TLR8 was seen in A549 cells. TNF-alpha also strongly enhanced IKK epsilon mRNA and protein expression. Ectopic expression of a constitutively active form of RIG-I (deltaRIG-I) or IKK epsilon, but not that of TLR3, enhanced the expression of the IFN-beta, IL-28, and IL-29 genes. Furthermore, a dominant-negative form of RIG-I inhibited influenza A virus-induced IFN-beta promoter activity in TNF-alpha-pretreated cells. In conclusion, IFN-alpha and TNF-alpha enhanced the expression of the components of TLR and RIG-I signaling pathways, but RIG-I was identified as the central regulator of influenza A virus-induced expression of antiviral cytokines in human lung epithelial cells.  相似文献   

6.
A20 is an ubiquitin-editing enzyme that ensures the transient nature of inflammatory signaling pathways induced by cytokines like TNF-α and IL-1 or pathogens via Toll-like receptor (TLR) pathways. It has been identified as a negative regulator of dendritic cell (DC) maturation and attenuator of their immunostimulatory properties. Ex vivo A20-depleted dendritic cells showed enhanced expression of pro-inflammatory cytokines and costimulatory molecules, which resulted in hyperactivation of tumor-infiltrating T lymphocytes and inhibition of regulatory T cells. In the present study, we demonstrate that a synthetic molecule consisting of a CpG oligonucleotide TLR9 agonist linked to A20-specific siRNAs silences its expression in TLR9+ mouse dendritic cells in vitro and in vivo. In the B16 mouse melanoma tumor model, silencing of A20 enhances the CpG-triggered induction of NFκB activity followed by elevated expression of IL-6, TNF-α and IL-12. This leads to potentiated antitumor immune responses manifested by increased numbers of tumor-specific cytotoxic T cells, high levels of tumor cell apoptosis and delayed tumor growth. Our findings confirm the central role of A20 in controlling the immunostimulatory potency of DCs and provide a strategy for simultaneous A20 silencing and TLR activation in vivo.  相似文献   

7.
RNA polymerase III (Pol III) expression systems for short hairpin RNAs (U6 shRNAs or chimeric VA1 shRNAs) or individually expressed sense/antisense small interfering RNA (siRNA) strands have been used to trigger RNA interference (RNAi) in mammalian cells. Here we show that individually expressed siRNA expression constructs produce 21-nucleotide siRNAs that strongly accumulate as duplex siRNAs in the nucleus of human cells, exerting sequence-specific silencing activity similar to cytoplasmic siRNAs derived from U6 or VA1-expressed hairpin precursors. In contrast, 29-mer siRNAs separately expressed as sense/antisense strands fail to elicit RNAi activity, despite accumulation of these RNAs in the nucleus. Our findings delineate different intracellular accumulation patterns for the three expression strategies and suggest the possibility of a nuclear RNAi pathway that requires 21-mer duplexes.  相似文献   

8.
Polyinosinic acid is a ligand for toll-like receptor 3   总被引:3,自引:0,他引:3  
Innate immune responses are critical in controlling viral infections. Viral proteins and nucleic acids have been shown to be recognized by pattern recognition receptors of the Toll-like receptor (TLR) family, triggering downstream signaling cascades that lead to cellular activation and cytokine production. Viral DNA is sensed by TLR9, and TLRs 3, 7, and 8 have been implicated in innate responses to RNA viruses by virtue of their ability to sense double-stranded (ds) RNA (TLR3) or single-stranded RNA (murine TLR7 and human TLR8). Viral and synthetic dsRNAs have also been shown to be a potent adjuvant, promoting enhanced adaptive immune responses, and this property is also dependent on their recognition by TLR3. It has recently been shown that mRNA that is largely single-stranded is a ligand for TLR3. Here we have investigated the ability of single-stranded homopolymeric nucleic acids to induce innate responses by murine immune cells. We show for the first time that polyinosinic acid (poly(I)) activates B lymphocytes, dendritic cells, and macrophages and that these responses are dependent on the expression of both TLR3 and the adaptor molecule, Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF). We therefore conclude that TLR3 is able to sense both single-stranded RNA and dsRNA.  相似文献   

9.
Human TLR7 and 8 (hTLR7/8) have been implicated in the sequence-dependent detection of RNA oligonucleotides in immune cells. Although hTLR7 sequence-specific sensing of short RNAs has been inferred from studies of murine TLR7, this has yet to be established for hTLR7. We found that different short ssRNA sequences selectively induced either TNF-alpha or IFN-alpha in human PBMCs. The sequence-specific TNF-alpha response to ssRNAs observed in PBMCs could be replicated in activated human macrophage-like (THP-1) cells pretreated with IFN-gamma. Surprisingly, suppression of hTLR7 expression by RNA interference in this model reduced sensing of all immunostimulatory ssRNAs tested. Modulation of the relative expression ratio of hTLR7 to hTLR8 in THP-1 cells correlated with differential sensing of immunostimulatory sequences. Furthermore, the sequence-specific IFN-alpha induction profile in human PBMCs was accurately modeled by a sequence-specific activation of murine TLR7 in mouse macrophages. Thus, we demonstrate for the first time that hTLR7 is involved in sequence-specific sensing of ssRNAs. We establish a novel cell model for the prediction of TNF-alpha induction by short RNAs in human macrophages. Our results suggest that differential sequence-specific sensing of RNA oligonucleotides between human and mouse macrophages is due to the modulation of TLR7 sensing by human TLR8.  相似文献   

10.
Phospholipase D1 plays a key role in TNF-alpha signaling   总被引:1,自引:0,他引:1  
The primary characteristic features of any inflammatory or infectious lesions are immune cell infiltration, cellular proliferation, and the generation of proinflammatory mediators. TNF-alpha is a potent proinflammatory and immuno-regulatory cytokine. Decades of research have been focused on the physiological/pathophysiological events triggered by TNF-alpha. However, the signaling network initiated by TNF-alpha in human leukocytes is still poorly understood. In this study, we report that TNF-alpha activates phospholipase D1 (PLD1), in a dose-dependent manner, and PLD1 is required for the activation of sphingosine kinase and cytosolic calcium signals. PLD1 is also required for NFkappaB and ERK1/2 activation in human monocytic cells. Using antisense oligonucleotides to reduce specifically the expression of PLD isozymes showed PLD1, but not PLD2, to be coupled to TNF-alpha signaling and that PLD1 is required to mediate receptor activation of sphingosine kinase and calcium transients. In addition, the coupling of TNF-alpha to activation of the phosphorylation of ERK1/2 and the activation of NFkappaB were inhibited by pretreating cells with antisense to PLD1, but not to PLD2; thus, demonstrating a specific requirement for PLD1. Furthermore, use of antisense oligonucleotides to reduce expression of PLD1 or PLD2 demonstrated that PLD1 is required for TNF-alpha-induced production of several important cytokines, such as IL-1beta, IL-5, IL-6, and IL-13, in human monocytes. These studies demonstrate the critical role of PLD1 in the intracellular signaling cascades initiated by TNF-alpha and its functional role for coordinating the signals to inflammatory responses.  相似文献   

11.
Human plasmacytoid precursor dendritic cells (ppDC) are a major source of type I IFN upon exposure to virus and bacteria, yet the stimulus causing their maturation into DCs is unknown. After PBMC activation with immunostimulatory bacterial DNA sequences (CpG-DNA) we found that ppDC are the primary source of IFN-alpha. In fact, either CpG-DNA or dsRNA (poly(I:C)) induced IFN-alpha from purified ppDC. Surprisingly, only CpG-DNA triggered purified ppDC survival, maturation, and production of TNF, GM-CSF, IL-6, and IL-8, but not IL-10 or IL-12. Known DC activators such as CD40 ligation triggered ppDC maturation, but only IL-8 production, while bacterial LPS was negative for all activation criteria. An additional finding was that only CpG-DNA could counteract IL-4-induced apoptosis in ppDC. Therefore, CpG-DNA represents a pathogen-associated molecular pattern for ppDC. In contrast to these finding, CpG-DNA, like LPS, caused TNF, IL-6, and IL-12 release from PBMC and purified monocytes; however, differentiation of monocytes into DCs with GM-CSF and IL-4 unexpectedly resulted in refractoriness to CpG-DNA, but not LPS. Taken together, these results suggest that within a DC subset a multiplicity of responses can be generated by distinct environmental stimuli and that responses to a given stimulus may be dissimilar between DC subsets.  相似文献   

12.
PBMC cocultured with HIV-infected monocytes for 12 to 48 h released high levels of IFN activity. IFN titers were directly dependent upon time after virus infection and level of HIV replication in infected cells. IFN induction in PBMC was evident with HIV-infected monocytes and PBMC and with myeloid and lymphoblastoid cell lines with at least three different HIV strains. In HIV-infected cell line pairs in which virus infection occurs in both productive and restricted forms, IFN induction in PBMC occurred only with productive infection. IFN activity was acid stable and completely neutralized by antibodies against IFN-alpha. Induction of IFN required cell-cell contact between HIV-infected cells and PBMC, but was independent of MHC compatibility. With PBMC co-cultured with autologous HIV-infected monocytes, IFN induction was highly selective: IL-1 beta, IL-6, or TNF-alpha activity and mRNA were not detected. Cell surface determinants on HIV-infected monocytes that induced IFN in PBMC remained active after fixation in 4% paraformaldehyde. Both adherent and nonadherent PBMC produced IFN after coculture with HIV-infected monocytes. Ability to produce IFN by PBMC was not affected by depletion of T cell, NK cell, B cell, or monocyte subpopulations. The IFN activity produced by PBMC cocultured with HIV-infected cells was about 20-fold less active than equal quantities of rIFN-alpha 2b for inhibition of HIV replication in monocytes and at low concentrations enhanced virus growth. Clinical studies with HIV-infected patients and parallel findings in animal lentivirus disease suggest an adverse role for IFN in disease progression. Conditions for induction of IFN in the culture system described in this report may mimic those in the HIV-infected patient. Defining the molecular basis for IFN induction, the cells that produce IFN, and the altered biologic activity of this important cytokine may provide insight into the pathogenesis of HIV disease.  相似文献   

13.
RNAi-mediated gene silencing is a recent, powerful tool to investigate gene function. Controlling for experimental factors such as transfection efficiencies, siRNA concentration, gene suppression levels, gene suppression kinetics, or non-specific effects is key to robust results. In this methods paper, we compare the efficiencies of different transfection reagents in primary human chondrocytes (PHCs). We investigated TAK1 gene suppression efficiencies and kinetics on the mRNA and protein level depending on the siRNA concentration used. Furthermore, we evaluated PKR, IL-6, and TNF-alpha induction, as well as IkappaB degradation and NFkappaB activation as control parameters of non-specific siRNA effects. PKR and IL-6 proved to be appropriate markers of cellular inflammatory responses resulting from siRNA transfection. In addition, we compared different siRNAs (silencing, non-silencing, classic 21-mer, and 25-mer stealth siRNA) with respect to their capacity to induce cellular inflammatory responses. We found the occurrence of cellular responses in PHCs to be a function of the specific siRNA sequence in use. Hence, it is essential to analyze and to compare gene silencing siRNAs and control siRNAs with respect to their off-target effects prior to any functional gene validation.  相似文献   

14.
Microbial infections trigger a multiplicity of responses in the host via innate immune sensors, including the Toll-like receptors (TLRs). TLR7 and TLR8, located in endosomes, detect pathogen-derived RNA, which can be mimicked by synthetic single-stranded oligoribonucleotides (ORNs). Detailed analysis of the immunostimulatory properties of numerous silencing RNAs (siRNAs) revealed that almost all tested siRNAs with a phosphodiester backbone actively stimulated cytokine production in human peripheral blood immune cells, but not all of them did contain previously described guanosine/uridine TLR7 or adenosine/uridine TLR8 motifs. By analysis of sequence variants of these siRNAs (as single- or double-strands), we were able to identify a new immunostimulatory, non-uridine-rich TLR7 motif that is present in many published siRNAs. Interestingly, the activity of this motif is dependent on the backbone chemistry. Phosphorothioate ORNs containing the motif did not stimulate immune activation, whereas phosphodiester ORNs of the same sequence induced a strong TLR7-biased immune response with high amounts of interferon-alpha. Using TLR7- and Myd88-deficient mice, we demonstrated that stimulation by ORNs containing this motif was TLR7 dependent. Our findings are of therapeutic relevance as this motif is present in many siRNA sequences and will to contribute to the immunostimulatory properties of unmodified siRNAs.  相似文献   

15.
Short interfering RNA (siRNA) is used in RNA interference technology to avoid non-target-related induction of type I interferon (IFN) typical for long double-stranded RNA. Here we show that in plasmacytoid dendritic cells (PDC), an immune cell subset specialized in the detection of viral nucleic acids and production of type I IFN, some siRNA sequences, independent of their GU content, are potent stimuli of IFN-alpha production. Localization of the immunostimulatory motif on the sense strand of a potent IFN-alpha-inducing siRNA allowed dissection of immunostimulation and target silencing. Injection into mice of immunostimulatory siRNA, when complexed with cationic liposomes, induced systemic immune responses in the same range as the TLR9 ligand CpG, including IFN-alpha in serum and activation of T cells and dendritic cells in spleen. Immunostimulation by siRNA was absent in TLR7-deficient mice. Thus sequence-specific TLR7-dependent immune recognition in PDC needs to be considered as an additional biological activity of siRNA, which then should be termed immunostimulatory RNA (isRNA).  相似文献   

16.
Patients with active systemic lupus erythematosus (SLE) have signs of an ongoing IFN-alpha production, that may be of pathogenic significance in the disease. We previously showed that SLE patients have an IFN-alpha-inducing factor in blood, probably consisting of complexes containing anti-DNA Abs and immunostimulatory DNA. The DNA component could be derived from apoptotic cells, because SLE patients have been reported to have both increased apoptosis and reduced clearance of apoptotic cell material. In the present study, we therefore investigated whether apoptotic cells, together with IgG from SLE patients, could act as an IFN-alpha inducer in normal PBMC in vitro. We found that apoptotic cells of the myeloid leukemia cell line U937 as well as four other cell lines (MonoMac6, H9, Jurkat, U266) could induce IFN-alpha production in PBMC when combined with IgG from SLE patients. The IFN-alpha production by PBMC was much enhanced when PBMC were costimulated by IFN-alpha2b. The ability of IgG from different SLE patients to promote IFN-alpha induction by apoptotic U937 cells was associated with the presence of anti-ribonucleoprotein Abs, but not clearly with occurrence of anti-DNA Abs. These results suggest that apoptotic cells in the presence of autoantibodies can cause production of a clearly immunostimulatory cytokine, which is IFN-alpha. This mechanism for induction of IFN-alpha production could well be operative also in vivo, explain the IFN-alpha production seen in SLE patients, and be important in the pathogenesis of SLE.  相似文献   

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19.
Type I interferons (IFN) (IFN-alpha/beta) are recognized as both inhibitors and effectors of autoimmune disease. In multiple sclerosis, IFN-beta therapy appears beneficial, in part, due to its suppression of autoimmune inflammatory Th cell responses. In contrast, in systemic lupus erythematosus (SLE) triggering of plasmacytoid DC (pDC) Toll-like receptors (TLRs) by autoimmune complexes (autoICs) results in circulating type I IFN that appear to promote disease by driving autoantigen presentation and autoantibody production. To investigate how pDC-derived type I IFN might regulate Th cells in SLE, we examined a model in which sustained pDC stimulation by autoICs is mimicked by pretreating normal human PBMC with TLR9 agonist, CpG-A. Subsequently, PBMC Th cells are activated with superantigen, and APC are activated with CD40L. The role of CpG-A/TLR9-induced type I IFN in regulating PBMC is determined by blocking with virus-derived soluble type I IFN receptor, B18R. In summary, pretreatment with either rhIFN-alpha/beta or CpG-A inhibits PBMC secretion of superantigen-induced IFN-gamma and IL-17, and CD40L-induced IL-12p70 and IL-23. B18R prevents these effects. Data indicate that CpG-A-induced type I IFN inhibit IL-12p70-dependent PBMC IFN-gamma secretion by enhancing IL-10. Our results suggest that in SLE, circulating type I IFN may potentially act to inhibit inflammatory cytokine secretion.  相似文献   

20.
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