Synthetic Double-stranded RNA Research Articles

Poly I:C Stimulates Sca-1 Expression in Murine Bone Marrow and Increases the Number of Phenotypic Hematopoietic Stem Cells.

Abstract 2504Poster Board II-481Polyinosinic:polycytidlyic acid (poly I:C) is a synthetic double-stranded RNA used to mimic viral infections in order to study immune responses and to activate gene deletion in lox-p systems employing a Cre gene responsive to an Mx-1 promoter. Recent observations made by us and others have suggested hematopoietic stem cells, responding to either poly I:C administration or interferon directly, enter cell cycle. Twenty-two hours following a single 100mg intraperitoneal injection of poly I:C into 10-12 week old male C57Bl/6 mice, the mice were injected with a single pulse of BrdU. Two hours later, bone marrow was harvested from legs and stained for Lineage, Sca-1, ckit, CD48, IL7R, and BrdU. In two independent experiments, each with n = 4, 41 and 33% of Lin- Sca-1+ cKit+ (LSK) IL-7R- CD48- cells from poly I:C-treated mice had incorporated BrdU, compared to 7 and 10% in cells from PBS-treated mice. These data support recently published reports. Total bone marrow cellularity was reduced to 45 and 57% in the two experiments, indicating either a rapid death and/or mobilization of marrow cells. Despite this dramatic loss of hematopoietic cells from the bone marrow of poly I:C treated mice, the number of IL-7R- CD48- LSK cells increased 145 and 308% in the two independent experiments. Importantly, the level of Sca-1 expression increased dramatically in the bone marrow of poly I:C-treated mice. Both the percent of Sca-1+ cells and the expression level of Sca-1 on a per cell basis increased after twenty-four hours of poly I:C, with some cells acquiring levels of Sca-1 that are missing from control bone marrow. These data were duplicated in vitro. When total marrow cells were cultured overnight in media containing either PBS or 25mg/mL poly I:C, percent of Sca-1+ cells increased from 23.6 to 43.7%. Within the Sca-1+ fraction of poly I:C-treated cultures, 16.7% had acquired very high levels of Sca-1, compared to only 1.75% in control cultures. Quantitative RT-PCR was employed to measure a greater than 2-fold increase in the amount of Sca-1 mRNA in poly I:C-treated cultures. Whereas the numbers of LSK cells increased in vivo, CD150+/− CD48- IL-7R- Lin- Sca-1- cKit+ myeloid progenitors almost completely disappeared following poly I:C treatment, dropping to 18.59% of control marrow, a reduction that is disproportionately large compared to the overall loss of hematopoietic cells in the marrow. These cells are normally proliferative, with 77.1 and 70.53% accumulating BrdU during the 2-hour pulse in PBS and poly I:C-treated mice, respectively. Interestingly, when Sca-1 is excluded from the analysis, the percent of Lin- IL7R- CD48- cKit+ cells incorporating BrdU decreases following poly I:C treatment, in keeping with interferon's published role as a cell cycle repressor. One possible interpretation of these data is that the increased proliferation of LSK cells noted by us and others is actually the result of Sca-1 acquisition by normally proliferating Sca-1- myeloid progenitors. This new hypothesis is currently being investigated. Disclosures:No relevant conflicts of interest to declare.

Autophagy Links Pattern Recognition Receptors to Tumor Cell Apoptosis

Pathogen nucleic acids are detected by the infected body’s immune cells via specific innate immune receptors, resulting in reactions such as secretion of T helper type 1 (Th1)-like and proinflammatory cytokines. Two studies have now reported the induction of cell death in melanoma cells, but not healthy skin cells, via stimulation of the cytoplasmic receptor MDA5 by the synthetic double-stranded RNA (dsRNA) agonist poly I:C, and one of the studies elucidated the mechanism for this response. Polyethyleneimine (PEI)-formulated poly I:C engages an intracellular process called autophagy via MDA5 to result in the induction of prodeath programs that ultimately lead to apoptosis of melanoma cells. Together with previous work, these reports demonstrate that immune detection of foreign nucleic acids involves diverse signaling pathways and thus yields different cellular outcomes that may overcome the inherent resistance of cancer cells to current cancer treatments.

Novel role for the double‐stranded RNA‐activated protein kinase PKR: modulation of macrophage infection by the protozoan parasiteLeishmania

The evolution of Leishmania infection depends on the balance between microbicidal and suppressor macrophage functions. Double-stranded RNA (dsRNA)-activated protein kinase R (PKR), a classic antiviral protein, is able to regulate a number of signaling pathways and macrophage functions. We investigated the possible role of PKR in the modulation of Leishmania infection. Our data demonstrated that Leishmania amazonensis infection led to PKR activation and increased PKR levels. Consistently, in macrophages from PKR knockout 129Sv/Ev mice and RAW-264.7 cells stably expressing a dominant-negative (DN) construct of PKR (DN-PKR), L. amazonensis infection was strongly reduced. The treatment of infected macrophages with the synthetic double-stranded RNA poly(I:C), a potent PKR inductor, increased L. amazonensis intracellular proliferation. This effect was reversed by 2-aminopurine (2-AP), a pharmacological inhibitor of PKR, as well as by the expression of DN-PKR. NO release induced by dsRNA treatment was inhibited by L. amazonensis through NF-kappaB modulation. PKR activation induced by dsRNA also resulted in IL-10 production, whose neutralization with specific antibody completely abrogated L. amazonensis proliferation. Our data demonstrated a new role of PKR in protozoan parasitic infection through IL-10 modulation.

Enhanced delivery of siRNA complexes by sonoporation in transgenic rice cell suspension cultures.

Small interfering synthetic double-stranded RNA (siRNA) was applied to suppress the expression of the human cytotoxic- T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) gene transformed in transgenic rice cell cultures. The sequence of the 21-nucleotide siRNA was deliberately designed and synthesized with overhangs to inactivate the expression of hCTLA4Ig. The chemically synthesized siRNA duplex was combined with polyethyleneimine (PEI) at a mass ratio of 1:10 (0.33 microg siRNA:3.3 microg PEI) to produce complexes. The siRNA complexes (siRNA+PEI) were labeled with Cy3 in order to subsequently confirm the delivery by fluorescent microscopy. In addition, the cells were treated with sonoporation at 40 kHz and 419 W for 90 s to improve the delivery. The siRNA complexes alone inhibited the expression of hCTLA4Ig to 45% compared with control. The siRNA complexes delivered with sonoporation downregulated the production of hCTLA4Ig to 73%. Therefore, we concluded that the delivery of siRNA complexes into plant cells could be enhanced successfully by sonoporation.

Optimized Basic Conditions Are Essential for Successful siRNA Transfection into Primary Endothelial Cells

RNA interference (RNAi) is a powerful technique in basic research and has a high potential for therapeutic applications. To realize its clinical applicability, introduction of short double-stranded RNA (dsRNA) has to be carried out under physiological conditions. This study evaluates two cationic liposomal transfection reagents on the efficiency of successful silencing of primary human endothelial cells. Transfection efficiency was investigated under different conditions, for example different media during transfection, duration of transfection, siRNA concentration, and the use of serum and antibiotics. Viability after transfection was examined by CASY and MTT assay. Interferon response was examined by real-time PCR. First we revealed that transfection carried out in the presence of serum and antibiotics caused good knockdown results only by the use of the novel lipid cationic transfection reagent. Both lipid cations had slightly the same transfection efficiency over the range of 10-150 nM siRNA concentration. Examination of interferon response showed increasing OAS1 and STAT1 expression, but not as high as if the transfections were carried out with synthetic polyinosinic-polycytidylic acid double-stranded RNA (poly[IC]). The optimized combination of basic conditions for transfection significantly enhanced the efficiency of the siRNA-mediated knockdown, without causing toxicity or stimulation of the interferon pathway.

Regulation of TLR2 Expression and Function in Human Airway Epithelial Cells

Toll-like receptor (TLR1-6) mRNAs are expressed in normal human bronchial epithelial cells with higher basal levels of TLR3. TLR2 mRNA and plasma membrane protein expression was enhanced by pretreatment with Poly IC, a synthetic double-stranded RNA (dsRNA) known to activate TLR3. Poly IC also enhanced mRNA expression of adaptor molecules (MyD88 and TIRAP) and coreceptors (Dectin-1 and CD14) involved in TLR2 signaling. Additionally, mRNA expression of TLR3 and dsRNA-sensing proteins MDA5 and RIG-I increased following Poly IC treatment. In contrast, basal mRNA expression of TLR5 and TLR2 coreceptor CD36 was reduced by 77% and 62%, respectively. ELISA of apical and basolateral solutions from Poly IC-stimulated monolayers revealed significantly higher levels of IL-6 and GM-CSF compared with the TLR2 ligand PAM(3)CSK(4). Pretreatment with anti-TLR2 blocking antibody inhibited the PAM(3)CSK(4)-induced increase in IL-6 secretion after Poly IC exposure. An increase in IL-6 secretion was also observed in cells stimulated with Alternaria extract after pretreatment with Poly IC. However, IL-6 secretion was not stimulated by zymosan or lipothechoic acid (LTA). These data demonstrated that upregulation of TLR2 following exposure to dsRNA enhances functional responses of the airway epithelium to certain (PAM(3)CSK(4)), but not all (zymosan, LTA) TLR2 ligands and that this is likely due to differences in coreceptor expression.

Not All Polyriboinosinic-polyribocytidylic Acids (Poly I:C) are Equivalent for Inducing Maturation of Dendritic Cells

This study compares the behavior of 2 commercially available polyriboinosinic-polyribocytidylic acids (poly I:C1 and poly I:C2) and the structural analog poly I:C12U in regard to dendritic cell (DC) maturation. When the Toll-like receptor 3 (TLR3) agonists are tested in combination with interferon-alpha, tumor necrosis factor-alpha, interleukin (IL)-1beta, and interferon-gamma (the so-called alpha-type-1 DC), the 3 different cocktails generate phenotypically mature DCs, but with different functional properties. Higher migratory capacity is observed with poly I:C1, the only poly I:C allowing spontaneous release of IL-12p70 by DCs. However, upon CD40 triggering, cocktails containing poly I:C2 or poly I:C12U allow a far higher production of IL-12p70 compared with those containing poly I:C1. Using a TLR signaling pathway reverse transcription profiler polymerase chain reaction to analyze changes in gene expression after treatment of DCs with the agonists alone, we show that 39% of the 84 tested genes are differentially regulated between the 3 conditions. Poly I:C12U induces far fewer regulated genes than the 2 other poly I:Cs. These different behaviors could be due to alternative ways of sensing double-stranded RNA, which do not rely solely on TLR3 but also on other types of receptors, depending on the size of poly I:Cs. As the 2 poly I:Cs tested here have very different molecular weights, this could partly explain the observed differences. In conclusion, neither the poly I:Cs nor their structural analog poly I:C12U have an equivalent behavior. This should be taken into an account not only when they are used in cocktails for DC maturation but also when analyzing signaling pathways with synthetic double-stranded RNA analogs.

Synthetic Double-Stranded RNAs Are Adjuvants for the Induction of T Helper 1 and Humoral Immune Responses to Human Papillomavirus in Rhesus Macaques

Toll-like receptor (TLR) ligands are being considered as adjuvants for the induction of antigen-specific immune responses, as in the design of vaccines. Polyriboinosinic-polyribocytoidylic acid (poly I:C), a synthetic double-stranded RNA (dsRNA), is recognized by TLR3 and other intracellular receptors. Poly ICLC is a poly I:C analogue, which has been stabilized against the serum nucleases that are present in the plasma of primates. Poly I:C12U, another analogue, is less toxic but also less stable in vivo than poly I:C, and TLR3 is essential for its recognition. To study the effects of these compounds on the induction of protein-specific immune responses in an animal model relevant to humans, rhesus macaques were immunized subcutaneously (s.c.) with keyhole limpet hemocyanin (KLH) or human papillomavirus (HPV)16 capsomeres with or without dsRNA or a control adjuvant, the TLR9 ligand CpG-C. All dsRNA compounds served as adjuvants for KLH-specific cellular immune responses, with the highest proliferative responses being observed with 2 mg/animal poly ICLC (p = 0.002) or 6 mg/animal poly I:C12U (p = 0.001) when compared with immunization with KLH alone. Notably, poly ICLC—but not CpG-C given at the same dose—also helped to induce HPV16-specific Th1 immune responses while both adjuvants supported the induction of strong anti-HPV16 L1 antibody responses as determined by ELISA and neutralization assay. In contrast, control animals injected with HPV16 capsomeres alone did not develop substantial HPV16-specific immune responses. Injection of dsRNA led to increased numbers of cells producing the T cell–activating chemokines CXCL9 and CXCL10 as detected by in situ hybridization in draining lymph nodes 18 hours after injections, and to increased serum levels of CXCL10 (p = 0.01). This was paralleled by the reduced production of the homeostatic T cell–attracting chemokine CCL21. Thus, synthetic dsRNAs induce an innate chemokine response and act as adjuvants for virus-specific Th1 and humoral immune responses in nonhuman primates.

Poly (I:C) induces shedding of soluble TNFR1 from human airway epithelial cells via a TLR3-RIP1-ERK signaling pathway (38.9)

Abstract Soluble tumor necrosis factor receptors (sTNFR) modulate TNF bioactivity by binding TNF. The type 1 TNF receptor (TNFR1), a key receptor for TNF signaling, is generated by either inducible cleavage and shedding of TNFR1 ectodomains or constitutive release of TNFR1 exosome-like vesicles. The goal of this study was to identify Toll-like receptor (TLR) signaling pathways that induce shedding of sTNFR1. We demonstrated that poly (I:C), a synthetic double-strand RNA (dsRNA) analog which signals via TLR3, induced sTNFR1 shedding from human airway epithelial (NCI-H292) cells in a dose-dependent manner over 2h, whereas ligands for TLR2, TLR4, TLR7, and NOD2 (nucleotide-binding oligomerization domain containing 2) did not. RNA interference experiments showed that poly (I:C)-induced sTNFR1 shedding was mediated via TLR3 and receptor interacting protein1 (RIP1). To further define the TLR3 signaling pathways that mediate sTNFR1 release, we showed that a specific, cell-permeable peptide inhibitor of ERK activation significantly attenuated sTNFR1 release by 55%. In contrast, neither dual oxidase 1 or 2, nor reactive oxygen species (ROS) mediated poly (I:C)-triggered sTNFR1 shedding. Furthermore, the pan-caspase inhibitor z-VAD-fmk attenuated sTNFR1 shedding by 28%, which suggests that caspase-dependent pathways also participate in this process. In conclusion, we show that poly (I:C), a viral dsRNA analog, induces sTNFR1 shedding from human airway epithelial cells primarily via a TLR3-RIP1-ERK signaling pathway, with a minor contribution from caspase-dependent pathways. This identifies a mechanism by which airway epithelial cells can modulate viral-induced airway inflammation. This work was funded by the Division of Intramural Research in NHLBI/NIH.

Suppression of CD8 T cell survival by the synthetic adjuvant poly (I:C) at low dosage (83.3)

Abstract The synthetic double-stranded RNA (dsRNA) poly I:C is commonly used as an adjuvant to boost CD8 T cell functions through type I IFN production but is also effective at suppressing disease development in certain autoimmunity models. It is unclear how a single adjuvant achieves two distinct immunoregulatory roles. By utilizing the transgenic T cell OTI adoptive transfer model, we found that low dose poly I:C suppresses costimulation-enhanced CD8 T cell survival. Perhaps surprisingly, early expansion and effector differentiation were not significantly impacted. Reduction in CD8 T cell survival was rescued by increasing the poly I:C dosage and is dependent on the precursor frequency of antigen-specific CD8 T cells. We hypothesize that the opposing effects of poly I:C on CD8 T cells at low versus high doses depend on differential activation of multiple dsRNA receptors. Compared to the intracellular dsRNA receptors such as MDA5 and RIG-I, TLR3 may have better access to poly I:C in soluble form due to its strategic location in the endosomal compartment. Hence we postulate that TLR3 is responsible for suppression of CD8 T cell survival when low dose poly I:C is administered. Indeed, a minor subset of splenic B cells that express CD5 and CD21 surface markers produce IL-10 in response to poly I:C. Interleukin-10 synthesis was inhibited in TLR3-/- B cells, although these cells were capable of producing IL-10 in response to PMA and ionomycin. The relationship of IL-10 and reduced T cell survival is being explored, as is the anatomical location of the CD5+CD21+ B cells. Consistent with other reports on the immunoregulatory role of IL-10-producing B cells on adaptive immunity, our findings further suggest the feasibility of fine-tuning such regulatory balance by altering the dosage of TLR ligands.

Fish immunization using a synthetic double-stranded RNA Poly(I:C), an interferon inducer, offers protection against RGNNV, a fish nodavirus

Viral nervous necrosis (VNN), caused by a fish nodavirus, is one of the most serious fish diseases worldwide. Here we report a unique vaccination method in sevenband grouper Epinephelus septemfasciatus using a synthetic double-stranded RNA polyinosinic polycytidylic acid (Poly(I:C)), an interferon inducer, followed by challenge with a live fish nodavirus. Fish injected with Poly(I:C) at 200 microg fish(-1) were highly protected from artificial challenge with red-spotted grouper nervous necrosis virus (RGNNV) (relative percentage survival, RPS: 100%), and specific antibodies against RGNNV were detected in sera from survivors. Moreover, the surviving fish were protected from rechallenge with RGNNV (relative percent survival RPS: 100%). Thus, it was confirmed that specific immunity against RGNNV was established in sevenband grouper by injection with live RGNNV following Poly(I:C) administration. Antiviral state was induced in fish by injection with Poly(I:C) at > or = 50 microg fish(-1), but no toxic response was observed in the fish even if Poly(I:C) was injected at a dose of 200 microg fish . In fish injected with Poly(I:C) at 200 pg fish(-1), a high level of antiviral state of > 90% RPS against RGNNV challenge lasted for at least 4 d after Poly(I:C) injection. However, no curative effect by Poly(I:C) injection was observed in fish already infected with RGNNV. It is considered that the present immunization method using Poly(I:C) followed by a live virus injection could offer protection against various viral infections in a broader range of fish species.

Protection of rainbow trout from infectious hematopoietic necrosis (IHN) by injection of infectious pancreatic necrosis virus (IPNV) or Poly(I:C)

It was recently reported that prophylaxis against infectious hematopoietic necrosis virus (IHNV) in fish was induced by pre-exposure to the infectious pancreatic necrosis virus (IPNV). Here the establishment of IHNV immunity in rainbow trout Oncorhynchus mykiss was investigated by IHNV challenge following non-lethal pre-infection with IPNV. Also, synthetic double-stranded RNA polyinosinic polycytidylic acid, Poly(I:C), an inducer for interferon (IFN), was evaluated as a substitute for IPNV induction of the non-specific antiviral state and subsequent IHNV-specific immunity in fish. Rainbow trout pre-infected with IPNV were protected from IHNV challenge 7 d later (relative percentage survival, RPS: 68.8%), and IHNV-specific antibodies were detected in sera from the survivors. Moreover, these surviving fish showed 91.6% RPS when re-challenged with IHNV 28 d after the primary IHNV challenge. Thus, fish appear to acquire IHNV-specific immunity through the IHNV challenge following pre-injection with IPNV. Fish pre-injected with Poly(I:C) were also highly protected from IHNV challenge 2 d later (RPS: 95.2%), and IHNV-specific antibodies were also detected amongst survivors. The survivors showed a 100% survival rate following re-challenge with IHNV both 21 and 49 d after the primary IHNV challenge. Thus, IHNV immunity in rainbow trout is induced by challenge with live IHNV following pre-injection with either IPNV or Poly(I:C). The use of Poly(I:C) to induce an anti-viral state protecting rainbow trout from an otherwise lethal vaccination dose of IHNV may have application to a wider range of fish species and fish pathogenic viruses.

Ampligen: A potential toll-like 3 receptor adjuvant for immunotherapy of cancer

Therapeutic vaccination against cancer-associated antigens represents an attractive option for cancer therapy in view of its potential efficacy, the possibility of long-lasting immunity against the cancer, and safety profile. Cancer patients are however usually immunosuppressed and most cancer-associated antigens are ‘self-antigens’, making it a significant challenge to vaccinate patients against a cancer-associated antigen. Various immunostimulant adjuvants are therefore under investigation in an effort to boost the immune system to overcome the tolerance to tumour associated self-antigens and the ambient immunosuppressory influence of cytokines and regulatory T-cells (Tregs). Many adjuvants appear to be specific ligands for toll-like receptors (TLR) which are potent activators of innate immune responses [Kwissa M, Kasturi SP, Pulendran B. Expert Rev Vaccines. The Science of Adjuvants 2007 6(October(5)):673–84] [1], activating dendritic cell (DC) maturation and inflammatory cytokine secretion, inducing an increase in the cross talk between the innate and adaptive immune systems, and thereby driving the expansion of CTLs that can destroy cancer cells. However, recently some TLR agonists such as CpG have been shown to generate parallel immunosuppressive and inflammatory responses in innate immune cells capable of induction and expansion of Tregs [Conroy H, Marshall NA, Mills KH. TLR ligand suppression or enhancement of Treg cells? A double-edged sword in immunity to tumours. Oncogene 2008 27(January 7(2)):168–80 [2]; Huang B, Zhao J, Unkeless JC, Feng ZH, Xiong H TLR signaling by tumor and immune cells: a double-edged sword. Oncogene 2008;27(2):218–24] [3]. In this context it is noteworthy that poly(I:C), a synthetic double-stranded RNA polymer and a TLR3 agonist, has been shown in mouse models to be capable of enhancing the T cell response to nonimmunogenic self-antigen linked dendritic cell based vaccine strategy, and stimulating diabetic insulitis in the presence of Tregs [Hornum L, Lundsgaard D, Markholst H. PolyI:C induction of diabetes is controlled by Iddm4 in rats with a full regulatory T cell pool. Ann N Y Acad Sci 2007;1110:65–72] [4]. Ampligen poly(I:C 12U) is a GMP-grade synthetic analogue of poly(I:C) and therefore suitable for human use. Its effects in a preclinical setting have been recently tested to be similar to poly(I:C) [Hornum L, Lundsgaard D, Markholst H. PolyI:C induction of diabetes is controlled by Iddm4 in rats with a full regulatory T cell pool. Ann N Y Acad Sci 2007;1110:65–72]. In particular, it potently induces in vitro maturation of human monocyte derived DC with sustained bioactive IL12 production. In addition human monocyte derived DC primed with tumour lysate and matured with Ampligen are capable of generating Th1 specific anti-cancer responses in peripheral blood T-cells derived from cancer patients in the presence of ascites medium containing immunosuppressory cytokines. In this review the key findings are summarised and discussed with a view to offering a rationale for the use of Ampligen as a potentially safe adjuvant capable of overcoming the tumour-related immune tolerance mechanisms in a clinical setting.

Knockdown of Severe Acute Respiratory Syndrome Corona Virus (SARS-CoV) Genes by Small Interfering RNA (siRNA) Using siRNA-expression Vectors and Synthetic Double-stranded RNA (dsRNA) as a Model for siRNA Design

While offering the promise of therapeutic use in the future, RNA interference (RNAi) technology is useful to knock down genes posttranscriptionally. We attempted to knock down severe acute respiratory syndrome (SARS)-corona virus (Cov) genes using small interfering RNA (siRNA) employing siRNA-expression vectors and synthetic double-stranded RNA (dsRNA) as a model for effective siRNA design. First, we selected three target sites without mutations among 15 SARS-Cov strains using a prediction algorithm and constructed three siRNA-expression vectors. Using a pGL3 vector, we constructed three corresponding model target vectors to the firefly luciferase gene (Fluc), to which the model targets were connected. Using Renilla luciferase gene (Rluc) as the internal control, the three siRNA vectors knocked down the targets, providing effective target sequences. Almost identical results were obtained when Rluc was integrated into the pGL3-Fluc target vector. Next, effective structures of synthetic double-stranded RNA (dsRNA) were investigated using two targets. In all, six RNAs per target were synthesized: complementary sense and antisense 19-mer core RNAs; sense 21-mer RNAs having a 2-nucleotide (nt) match or unmatch overhang at the 3'-end; and antisense 21-mer RNAs having a 2-nt match or unmatch overhang at the 3'-end. The six RNAs provided nine species of dsRNAs (a blunt 19-mer duplex, a total of 4 19-mer/21-mer duplexes with a match or unmatch 2-nt overhang at the 3'- end of the sense or antisense strand, 4 21-mer/21-mer duplexes with match or unmatch 2-nt overhang at both ends) in combination. Targets were sense or antisense sequences. Generally, 19-mer/21-mer dsRNA with a match 2-nt overhang at the 3'- end of the antisense strand showed the highest activity, irrespective of the thermodynamic stabilities at terminal ends, suggesting that the 2-nt overhang is more critical than thermodynamic stabilities to select the antisense strand to the RNA-induced silencing complex (RISC).

Size Matters: Differential Effects of RNA and Polyphosphate on Blood Clotting

Introduction: Recently, both inorganic polyphosphate (Smith et al., PNAS 103:903-8, 2006) and RNA (Kannemeier et al., PNAS 104:6388–93, 2007) have been proposed as (patho)physiologic activators of the contact pathway in blood clotting. We also recently showed that polyphosphate of the size secreted by human platelets (approximately 75-mer) acts at two other points in the blood clotting cascade: it accelerates factor V activation and it enhances fibrin clot structure (Smith & Morrissey, Blood, in press). We now compare the ability of RNA and polyphosphate of varying chain lengths to modulate the blood clotting cascade at these three critical points: initiation, factor V activation, and fibrin polymerization.Methods: Polyphosphate was size-fractionated and its procoagulant activities were compared to those of polyinosinic acid, a synthetic singlestranded RNA (ssRNA); polyinosinic acid:polycytidylic acid, a synthetic double-stranded RNA (dsRNA); yeast tRNA or kaolin. Clotting assays were performed using purified fibrinogen, pooled normal plasma, or factor V-deficient plasma to which factor Va was added. Clotting was initiated by CaCl2 (contact pathway), factor Xa, or thrombin.Results: Long-chain polyphosphate (100-mer to 800-mer) triggered the contact pathway with a potency similar to kaolin and was about 30-fold more potent than ssRNA and some 3000- fold more potent than dsRNA or tRNA. Medium-chain polyphosphate (20-mer to 100-mer) and ssRNA both shortened factor Xa clotting times with similar potency, but dsRNA and yeast tRNA were without effect. Replacing plasma factor V with Va blocked the ability of either polyphosphate or ssRNA to shorten factor Xa clotting times, suggesting that both polymers accelerate factor V activation. And finally, when purified fibrinogen was clotted with thrombin, adding either ssRNA or polyphosphate yielded fibrin clots that were about threefold more turbid, indicating that both polymers enhance fibrin clot formation, while dsRNA and yeast tRNA were without effect. (We previously documented that polyphosphate enhances fibrin turbidity by dramatically increasing fibril diameter.) Interestingly, ssRNA significantly shortened the thrombin clotting time of purified fibrinogen, while polyphosphate had no effect on thrombin clotting times. Furthermore, the ability of polyphosphate to enhance fibrin clot structure was calcium-dependent, while ssRNA enhancement of fibrin clotting by thrombin was metal ion-independent.Conclusions: This study shows that RNA modulates critical downstream clotting functions in addition to its previously identified role in triggering the contact pathway. Long-chain polyphosphate (i.e., the size that accumulate in microorganisms, but not the size secreted by platelets) is substantially more potent than RNA in triggering the contact pathway of blood clotting. We therefore propose that polyphosphate may play an important role in host responses to pathogens by triggering the contact pathway. Polyphosphate of the size secreted by platelets had similar potency to ssRNA in accelerating factor V activation. And finally, polyphosphate of the size secreted by platelets had similar potency to RNA in enhancing fibrin clot structure, although the metal ion-dependencies of the two differed, as did their effects on thrombin clotting time. In general, ssRNA was far more potent than dsRNA or tRNA in modulating the blood clotting system.

Activation of Toll-Like Receptor 3 Augments Myofibroblast Differentiation

Airway remodeling is observed in the airways of patients with asthma, and differentiation of fibroblasts to myofibroblasts plays a critical role in the progress of airway remodeling. Viral infection induces not only the disease development and exacerbations but also airway remodeling. The aim of this study was to evaluate whether the activation of Toll-like receptor 3 (TLR3) can affect the differentiation of fibroblasts to myofibroblasts and the extracellular matrix (ECM) protein production. Human fetal lung fibroblasts (HFL-1) and adult lung fibroblasts were treated with a synthetic double-stranded RNA, polyinosine-polycytidylic acid (poly[I:C]) and the expression of alpha-smooth muscle actin (alpha-SMA), a marker of myofibroblast differentiation, was evaluated. The release of transforming growth factor-beta(1) (TGF-beta(1)) and ECM protein production were assessed. The effect of anti-TGF-beta antibody on the alpha-SMA and ECM production was also assessed. Poly(I:C) significantly augmented the alpha-SMA expression (P < 0.01) and release of TGF-beta(1) (P < 0.01) compared with control. Bafilomycin, an inhibitor of TLR3 signaling, diminished poly(I:C)-augmented TGF-beta(1) release. Anti-TGF-beta(1) antibody inhibited the poly(I:C)-augmented alpha-SMA expression. Poly(I:C) enhanced translocation of nuclear factor-kB (NF-kappaB) and interferon regulatory factor-3 (IRF-3) into the nucleus. Poly(I:C)-augmented TGF-beta(1) release was almost completely blocked by NF-kappaB inhibitors, but not by silencing IRF-3. The production of fibronectin and collagen I expression were significantly increased by poly(I:C) (P < 0.01) and they were inhibited by anti-TGF-beta antibody. These results suggest that activation of TLR3 can affect the differentiation to myofibroblasts and enhance ECM production via the NF-kappaB-TGF-beta(1)-dependent pathway.

Acute myeloid leukemic cell lines loaded with synthetic dsRNA trigger IFN-γ secretion by human NK cells

Natural killer (NK) cells are key players of innate immunity. Besides their major cytotoxic function, NK cells can also produce inflammatory cytokines such as interferon (IFN)-γ. In this way, NK cells can shape adaptive immune responses through activation of dendritic cells (DC), thereby promoting the bidirectional cross-talk between NK cells and DC. Including this helper function of NK cells in cancer vaccination might be important for the induction of effective T cell responses. Here, we explored the capacity of purified human NK cells to produce IFN-γ upon two-signal stimulation using different types of acute myeloid leukemia (AML) cells and type I IFN. Based on our previous findings that AML cells produce IFN-α upon electroporation with the synthetic double-stranded (ds)RNA polyriboinosinic polyribocytidylic acid (poly(I:C)), we hypothesized that dsRNA-loaded tumor cells provide both signals to elicit an NK cell-driven IFN-γ production. Our results show that in vitro, NK cells become strong IFN-γ-secreting cells upon stimulation with specific AML cells and IFN-α, with a variable responsiveness against different AML cell lines. Importantly, loading of AML cells with poly(I:C) is an elegant method to provide NK cells with both signals, a feature that could have important clinical implications because it obviates the side effects of systemic cytokine administration. Moreover, in addition to our previous findings that DC become activated upon phagocytosis of poly(I:C)-electroporated AML cells, these data strongly encourage future research on the potential of dsRNA-transfected AML cells and their effect to favor NK-DC cross-talk for the design of leukemia vaccines.

Molecular cloning and expression analysis of three Mx isoforms of rock bream, Oplegnathus fasciatus

Complementary DNAs (cDNAs) corresponding to three isoforms of rock bream ( Oplegnathus fasciatus) Mx (RbMx1, RbMx2 and RbMx3) were cloned using RACE reactions. Analysis of deduced amino acid sequences revealed that the tripartite GTP-binding domain, the dynamine family signature and the leucine zipper repeat were present in all three rock bream Mx isoforms. Cloning of genomic DNA sequence and Southern blot analysis showed that three rock bream Mx isoforms were encoded by different genomic loci, and they were not alternative splicing variants, although some alternative splicing variants were found in RbMx1 and RbMx2. When comparing amino acid sequence identity, RbMx1 shares about 60–70% identities with other fish Mx proteins, whereas both RbMx2 and RbMx3 share slightly high identity of 70–90%. As a result of expression analysis using RT-PCR, RbMx1 was constitutively expressed in the spleen and kidney of rock bream yearling, but RbMx2 and RbMx3 were rarely detected in both organs. When injected with synthetic double-stranded RNA polyinosinic:polycytidylic acid (poly I:C), expression of all rock bream Mx isoforms was up-regulated in spleen and head kidney. RbMx1 was continuously up-regulated throughout experimental period of 72 h but RbMx2 and RbMx3 were down-regulated to almost non-detectable level at 48 h post-injection.

Function of TRADD in tumor necrosis factor receptor 1 signaling and in TRIF-dependent inflammatory responses

Tumor necrosis factor receptor 1 (TNFR1) and Toll-like receptors (TLRs) regulate immune and inflammatory responses. Here we show that the TNFR1-associated death domain protein (TRADD) is critical in TNFR1, TLR3 and TLR4 signaling. TRADD deficiency abrogated TNF-induced apoptosis, prevented recruitment of the ubiquitin ligase TRAF2 and ubiquitination of the adaptor RIP1 in the TNFR1 signaling complex, and considerably inhibited but did not completely abolish activation of the transcription factor NF-kappaB and mitogen-activated protein kinases 'downstream' of TNFR1. TRIF-dependent cytokine production induced by the synthetic double-stranded RNA poly(I:C) and lipopolysaccharide was lower in TRADD-deficient mice than in wild-type mice. Moreover, TRADD deficiency inhibited poly(I:C)-mediated RIP1 ubiquitination and activation of NF-kappaB and mitogen-activated protein kinase signaling in fibroblasts but not in bone marrow macrophages. Thus, TRADD is an essential component of TNFR1 signaling and has a critical but apparently cell type-specific function in TRIF-dependent TLR responses.

Toll-Like Receptor 3 Activation Induces Antiviral Immune Responses in Mouse Sertoli Cells1

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and elicit antimicrobial immune responses. In the testis, viruses can induce pathological conditions, such as orchitis, and may participate in the etiology of testicular cancer; however, the molecular mechanisms involved remain under investigation. It has been suggested that because they constitutively express interferon (IFN)-inducible antiviral proteins, Sertoli cells participate in the testicular antiviral defense system. Previously, we demonstrated a key function of mouse Sertoli cells in the bactericidal testicular defense mechanism mediated by a panel of TLRs. To better characterize the potential role of Sertoli cells in the response against testicular viral infections, we investigated the TLR3 expression and function in these cells. Sertoli cells express TLR3, and under stimulation with the synthetic double-stranded RNA analogue poly (I:C), they produce the proinflammatory molecule ICAM1 and secrete functionally active CCL2 chemokine. Using both pharmacological and genetic approaches, we found that these effects are TLR3-dependent. Moreover, using ELISA, we found that IFNA is constitutively produced and not further inducible, whereas IFNB1 is absent and dramatically induced only by transfected poly (I:C), indicating different control mechanisms underlying IFNA and IFNB1 production. To conclude, poly (I:C) elicits both inflammatory and antiviral responses in Sertoli cells.