Culture medium used during small interfering RNA (siRNA) transfection determines the maturation status of dendritic cells

High-throughput screening of RNAi libraries has become an essential part of functional analysis in academic and industrial settings. The transition of a cell-based RNAi assay into a 384-well format requires several optimization steps to ensure the phenotype being screened is appropriately measured and that the signal-to-background ratio is above a certain quantifiable threshold. Methods currently used to assess small interfering RNA (siRNA) efficacy after transfection, including quantitative PCR or branch DNA analysis, face several technical limitations preventing the accurate measurement of mRNA levels in a 384-well format. To overcome these difficulties, the authors developed an approach using a viral-based transfection system that measures siRNA efficacy in a standardized 384-well assay. This method allows measurement of siRNA activity in a phenotypically neutral manner by quantifying the knockdown of an exogenous luciferase gene delivered by a lentiviral vector. In this assay, the efficacy of a luciferase siRNA is compared to a negative control siRNA across many distinct assay parameters including cell type, cell number, lipid type, lipid volume, time of the assay, and concentration of siRNA. Once the siRNA transfection is optimized as a 384-well luciferase knockdown, the biologically relevant phenotypic analysis can proceed using the best siRNA transfection conditions. This approach provides a key technology for 384-well assay development when direct measurement of mRNA knockdown is not possible. It also allows for direct comparison of siRNA activity across cell lines from almost any mammalian species. Defining optimal conditions for siRNA delivery into mammalian cells will greatly increase the speed and quality of large-scale siRNA screening campaigns. ( Journal of Biomolecular Screening 2007:546-559)

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CD83 Knockdown in Monocyte-Derived Dendritic Cells by Small Interfering RNA Leads to a Diminished T Cell Stimulation

The Journal of Immunology ◽

10.4049/jimmunol.178.9.5454 ◽

2007 ◽

Vol 178 (9) ◽

pp. 5454-5464 ◽

Cited By ~ 94

Author(s):

Alexander T. Prechtel ◽

Nadine M. Turza ◽

Alexandros A. Theodoridis ◽

Alexander Steinkasserer

Keyword(s):

Dendritic Cells ◽

T Cell ◽

Small Interfering Rna ◽

Cell Stimulation ◽

T Cell Stimulation ◽

Interfering Rna

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Silencing of CD86 in dendritic cells by small interfering RNA regulates cytokine production in T cells from patients with allergic rhinitis in�vitro

Molecular Medicine Reports ◽

10.3892/mmr.2019.10638 ◽

2019 ◽

Author(s):

Rong Sun ◽

Yang Yang ◽

Zheng Gu ◽

Xinye Tang ◽

Cheng Zhang ◽

...

Keyword(s):

T Cells ◽

Allergic Rhinitis ◽

Dendritic Cells ◽

Small Interfering Rna ◽

Cytokine Production ◽

Interfering Rna

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Efficient Inhibition of Avian and Seasonal Influenza A Viruses by a Virus-Specific Dicer-Substrate Small Interfering RNA Swarm in Human Monocyte-Derived Macrophages and Dendritic Cells

Journal of Virology ◽

10.1128/jvi.01916-18 ◽

2018 ◽

Vol 93 (4) ◽

Cited By ~ 2

Author(s):

Miao Jiang ◽

Pamela Österlund ◽

Veera Westenius ◽

Deyin Guo ◽

Minna M. Poranen ◽

...

Keyword(s):

Dendritic Cells ◽

Influenza Virus ◽

Small Interfering Rna ◽

Influenza A ◽

Seasonal Influenza ◽

Influenza Viruses ◽

Influenza A Viruses ◽

Primary Monocyte ◽

Prevention And Treatment ◽

Interfering Rna

ABSTRACTInfluenza A viruses (IAVs) are viral pathogens that cause epidemics and occasional pandemics of significant mortality. The generation of efficacious vaccines and antiviral drugs remains a challenge due to the rapid appearance of new influenza virus types and antigenic variants. Consequently, novel strategies for the prevention and treatment of IAV infections are needed, given the limitations of the presently available antivirals. Here, we used enzymatically produced IAV-specific double-stranded RNA (dsRNA) molecules andGiardia intestinalisDicer for the generation of a swarm of small interfering RNA (siRNA) molecules. The siRNAs target multiple conserved genomic regions of the IAVs. In mammalian cells, the produced 25- to 27-nucleotide-long siRNA molecules are processed by endogenous Dicer into 21-nucleotide siRNAs and are thus designated Dicer-substrate siRNAs (DsiRNAs). We evaluated the efficacy of the above DsiRNA swarm at preventing IAV infections in human primary monocyte-derived macrophages and dendritic cells. The replication of different IAV strains, including avian influenza H5N1 and H7N9 viruses, was significantly inhibited by pretransfection of the cells with the IAV-specific DsiRNA swarm. Up to 7 orders of magnitude inhibition of viral RNA expression was observed, which led to a dramatic inhibition of IAV protein synthesis and virus production. The IAV-specific DsiRNA swarm inhibited virus replication directly through the RNA interference pathway although a weak induction of innate interferon responses was detected. Our results provide direct evidence for the feasibility of the siRNA strategy and the potency of DsiRNA swarms in the prevention and treatment of influenza, including the highly pathogenic avian influenza viruses.IMPORTANCEIn spite of the enormous amount of research, influenza virus is still one of the major challenges for medical virology due to its capacity to generate new variants, which potentially lead to severe epidemics and pandemics. We demonstrated here that a swarm of small interfering RNA (siRNA) molecules, including more than 100 different antiviral RNA molecules targeting the most conserved regions of the influenza A virus genome, could efficiently inhibit the replication of all tested avian and seasonal influenza A variants in human primary monocyte-derived macrophages and dendritic cells. The wide antiviral spectrum makes the virus-specific siRNA swarm a potentially efficient treatment modality against both avian and seasonal influenza viruses.

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Cholesterol Modification of p40-Specific Small Interfering RNA Enables Therapeutic Targeting of Dendritic Cells

The Journal of Immunology ◽

10.4049/jimmunol.1402989 ◽

2015 ◽

Vol 195 (5) ◽

pp. 2216-2223 ◽

Cited By ~ 14

Author(s):

Jürgen Brück ◽

Steve Pascolo ◽

Kerstin Fuchs ◽

Christina Kellerer ◽

Ivana Glocova ◽

...

Keyword(s):

Dendritic Cells ◽

Small Interfering Rna ◽

Therapeutic Targeting ◽

Cholesterol Modification ◽

Interfering Rna

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Targeted Delivery of Small Interfering RNA to Human Dendritic Cells To Suppress Dengue Virus Infection and Associated Proinflammatory Cytokine Production

Journal of Virology ◽

10.1128/jvi.02105-08 ◽

2009 ◽

Vol 84 (5) ◽

pp. 2490-2501 ◽

Cited By ~ 73

Author(s):

Sandesh Subramanya ◽

Sang-Soo Kim ◽

Sojan Abraham ◽

Jiahong Yao ◽

Mukesh Kumar ◽

...

Keyword(s):

Dendritic Cells ◽

Dengue Virus ◽

Virus Replication ◽

Targeted Delivery ◽

Small Interfering Rna ◽

Envelope Gene ◽

Hematopoietic Stem ◽

Tnf Α ◽

Interfering Rna

ABSTRACT Dengue is a common arthropod-borne flaviviral infection in the tropics, for which there is no vaccine or specific antiviral drug. The infection is often associated with serious complications such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), in which both viral and host factors have been implicated. RNA interference (RNAi) is a potent antiviral strategy and a potential therapeutic option for dengue if a feasible strategy can be developed for delivery of small interfering RNA (siRNA) to dendritic cells (DCs) and macrophages, the major in vivo targets of the virus and also the source of proinflammatory cytokines. Here we show that a dendritic cell-targeting 12-mer peptide (DC3) fused to nona-d-arginine (9dR) residues (DC3-9dR) delivers siRNA and knocks down endogenous gene expression in heterogenous DC subsets, (monocyte-derived DCs [MDDCs], CD34+ hematopoietic stem cell [HSC])-derived Langerhans DCs, and peripheral blood DCs). Moreover, DC3-9dR-mediated delivery of siRNA targeting a highly conserved sequence in the dengue virus envelope gene (siFvED) effectively suppressed dengue virus replication in MDDCs and macrophages. In addition, DC-specific delivery of siRNA targeting the acute-phase cytokine tumor necrosis factor alpha (TNF-α), which plays a major role in dengue pathogenesis, either alone or in combination with an antiviral siRNA, significantly reduced virus-induced production of the cytokine in MDDCs. Finally to validate the strategy in vivo, we tested the ability of the peptide to target human DCs in the NOD/SCID/IL-2Rγ−/− mouse model engrafted with human CD34+ hematopoietic stem cells (HuHSC mice). Treatment of mice by intravenous (i.v.) injection of DC3-9dR-complexed siRNA targeting TNF-α effectively suppressed poly(I:C)-induced TNF-α production by DCs. Thus, DC3-9dR can deliver siRNA to DCs both in vitro and in vivo, and this delivery approach holds promise as a therapeutic strategy to simultaneously suppress virus replication and curb virus-induced detrimental host immune responses in dengue infection.

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Immune Modulation by Silencing IL-12 Production in Dendritic Cells Using Small Interfering RNA

The Journal of Immunology ◽

10.4049/jimmunol.171.2.691 ◽

2003 ◽

Vol 171 (2) ◽

pp. 691-696 ◽

Cited By ~ 88

Author(s):

Jonathan A. Hill ◽

Thomas E. Ichim ◽

Kornel P. Kusznieruk ◽

Mu Li ◽

Xuyan Huang ◽

...

Keyword(s):

Dendritic Cells ◽

Small Interfering Rna ◽

Immune Modulation ◽

Interfering Rna

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Inhibition of MMP-2 gene expression with small interfering RNA in rabbit vascular smooth muscle cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00517.2007 ◽

2007 ◽

Vol 293 (6) ◽

pp. H3593-H3601 ◽

Cited By ~ 16

Author(s):

Hanna Hlawaty ◽

Aurélie San Juan ◽

Marie-Paule Jacob ◽

Roger Vranckx ◽

Didier Letourneur ◽

...

Keyword(s):

Gene Expression ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Small Interfering Rna ◽

Ex Vivo ◽

Gelatin Zymography ◽

Sirna Transfection ◽

Interfering Rna

Matrix metalloproteinase-2 (MMP-2) is constitutively expressed in vascular smooth muscle cells (VSMCs). Using small interfering RNA (siRNA), we evaluated the effect of MMP-2 inhibition in VSMCs in vitro and ex vivo. Rabbit VSMCs were transfected in vitro with 50 nmol/l MMP-2 siRNA or scramble siRNA. Flow cytometry and confocal microscopy showed cellular uptake of siRNA in ∼80% of VSMCs. MMP-2 mRNA levels evaluated by real-time RT-PCR, pro-MMP-2 activity from conditioned culture media evaluated by gelatin zymography, and VSMC migration were reduced by 44 ± 19%, 43 ± 14%, and 36 ± 14%, respectively, in MMP-2 siRNA-transfected compared with scramble siRNA-transfected VSMCs ( P < 0.005 for all). Ex vivo MMP-2 siRNA transfection was performed 2 wk after balloon injury of hypercholesterolemic rabbit carotid arteries. Fluorescence microscopy showed circumferential siRNA uptake in neointimal cells. Gelatin zymography of carotid artery culture medium demonstrated a significant decrease of pro-MMP-2 activity in MMP-2 siRNA-transfected compared with scramble siRNA-transfected arteries ( P < 0.01). Overall, our results demonstrate that in vitro MMP-2 siRNA transfection in VSMCs markedly inhibits MMP-2 gene expression and VSMC migration and that ex vivo delivery of MMP-2 siRNA in balloon-injured arteries reduces pro-MMP-2 activity in neointimal cells, suggesting that siRNA could be used to modify arterial biology in vivo.

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