Targeted delivery of small interfering RNA to human dendritic cells to suppress dengue virus infection and associated proinflammatory cytokine production

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 (siFvE(D)) 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-alpha), 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-2Rgamma(-/-) 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-alpha effectively suppressed poly(I:C)-induced TNF-alpha 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.

FIG. 1.

DC3-9dR peptide delivers FITC siRNA specifically to MDDCs. (A) siRNA was incubated with DC3-9dR peptide at the indicated molar ratios for 15 min and electrophoretic mobility tested by agarose gel electrophoresis. (B) MDDCs were incubated with FITC-siRNA (200 pmol) complexed with the indicated molar ratios of DC3-9dR, and siRNA uptake was assayed 16 h later by flow cytometry. (C) Representative results for MDDCs treated with FITC-siRNA complexed with Lipofectamine, DC3-9dR, or 9dR at a 1:10 molar ratio. (D and E) PHA-stimulated T lymphocytes (D) or indicated cell lines (E) were transfected or transduced with FITC-labeled siRNA using Lipofectamine or DC3-9dR peptide, respectively, and analyzed 16 h later for siRNA uptake. The shaded histogram in the overlay in panel E represents Lipofectamine transfection, and the open histogram represents DC3-9dR transduction.

FIG. 2.

DC3-9dR-delivered siRNA silences gene expression specifically in dendritic cell subsets. Human MDDCs or PHA-stimulated T cells (A) (n = 4), blood dendritic cells (B) (n = 2), and CD34-derived dendritic cells (C) (n = 3) were transfected with CyPB siRNA using Lipofectamine or transduced with DC3-9dR-complexed CyPB siRNA, and specific gene knockdown was assayed by quantitating mRNA levels by real time-PCR 24 h later. Error bars indicate standard deviations. *, P < 0.5.

FIG. 3.

FvE^D siRNA potently suppresses dengue virus replication in BHK-21 cells. (A and B) BHK-21 cells were transfected with the indicated siRNAs using Lipofectamine, and after 24 h cells were challenged with DEN-2 (MOI of 0.5 or 1.5), followed by another round of siRNA transfection 24 h postinfection. Viral replication was monitored at 72 h (B, top panel) or 96 h (B, bottom panel) postinfection by flow cytometry. Representative results after 72 h of infection (MOI of 1.5) are shown in panel A, and cumulative data from three independent experiments are shown in panel B. (C) BHK21 cells were first infected with DEN-2 (MOI of 1.5) and 24 h or 48 h later were transfected with control siLuci or FvE^D siRNA. Infection levels were monitored at 72 h postinfection. Error bars indicate standard deviations.

FIG. 4.

DC3-9dR-mediated delivery of FvED suppresses DEN-2 infection in MDDCs. (A) MDDCs were infected with DEN-2 at the indicated MOIs and viral replication monitored after 72 h by flow cytometry. (B, C, and D) MDDCs were transduced with DC3-9dR-complexed siFvE^D twice on two consecutive days before challenge with DEN-2 at an MOI of 2. Viral replication was monitored at 72 h postinfection either by flow cytometry after intracellular staining (B) or by quantitating viral positive-strand RNA load by real-time RT-PCR (C) or detection of DEN-2 negative strand RNA by the tagged RT-PCR method using primers targeting the NS3 region of the genome (D). (E and F) BHK-21 cells were infected with serially diluted supernatants used for panel B, and infection was monitored after 96 h by plaque assay (E) or flow cytometry (F). The results shown in panel F are for the 1:100 dilution. Error bars indicate standard deviation. *, P < 0.5.

FIG. 5.

The DC3-9dR peptide can deliver siRNAs to M-CSF-treated and DEN-2-infected MDMs. (A) Monocytes were cultured in the absence (left panel) or presence (right panel) of M-CSF (20 ng/ml). Five days later MDMs were transfected/transduced with Lipofectamine (blue histogram)-, DC3-9dR (red histogram)-, or 9dR (green histogram)-complexed FITC-labeled siRNA, and uptake was quantitated 16 h later. (B) M-CSF-treated or untreated MDMs were transduced with siCyPB/siLuci using Lipofectamine or DC39dR and assayed for gene knockdown by quantitative PCR 24 h later. Expression relative to mock and irrelevant siRNA-treated cells is shown. (C) MDMs were infected with DEN-2 (MOI of 2) previously complexed with anti-prM antibody (0.1 ng/ml) and tested for infection by flow cytometric analysis at 72 h postinfection. (D and E) Dengue-infected-MDMs were transduced with the DC3-9dR/FvE^D siRNA complex at 24 h postinfection and viral replication monitored 48 h later by quantitation of positive-strand DEN-2 RNA by real-time RT-PCR (D) or negative-strand DEN-2 by a tag amplification method using primers targeting the NS3 region (E). (F and G) BHK-21 cells were infected with serial dilutions of the viral supernatants from panel D and infection monitored after 96 h by plaque assay (F) or by flow cytometry (G). The results shown in panel G are for the 1:100 dilution. Error bars indicate standard deviations. *, P < 0.5.

FIG. 6.

DC3-9dR-mediated siRNA delivery inhibits DEN-2-induced TNF-α expression without increasing viral replication. (A and B) MDDCs were infected with DEN-2 (MOI of 2) and TNF-α mRNA (A, left panel), and protein levels (A, right panel) as well as viral RNA loads (B) were assayed at the indicated time points. Results from three experiments (means ± standard deviations) are shown. (C) MDDCs were transfected with siRNAs targeting the human TNF-α gene and after 24 h were treated with LPS (100 ng/ml). After overnight culture, TNF-α mRNA levels were measured by quantitative PCR. The percent reduction in TNF-α mRNA relative to those in irrelevant siRNA-treated cells is shown. (D and E) MDDCs were transduced with siLuci or siTNFα using Lipofectamine or DC3-9dR twice on two consecutive days before infection with DEN-2 (MOI of 2). Ten hours later TNF-α mRNA (D, left panel) and TNF-α protein (D, right panel) levels were assayed. At 48 hours postinfection, viral copy numbers (E) were measured by quantitative PCR. (F and G) MDDCs were treated with TNF-α and antiviral FvE^D siRNA singly or in combination and infected with DEN-2 as for panel D. TNF-α mRNA levels (F) and viral loads (G) were quantitated as described above. Error bars indicate standard deviations. *, P < 0.5.

FIG. 7.

Intravenous treatment of siRNA complexed to DC3-9dR prevents poly(I:C)-induced TNF-α production in HuHSC mice. (A) Human monocytes were differentiated into MDDCs or MDMs as described in Materials and Methods. At day 5, poly(I:C) (50 μg/ml) was added to the cell cultures, and TNF-α levels in the supernatants collected at the indicated times were assayed by ELISA. (B) HuHSC mice were injected with recombinant human GM-CSF and IL-4 (10 μg/mouse each) daily for 5 days for dendritic cell mobilization. On day 5, mice were intravenously injected thrice (at 6-h intervals) with DC3-9DR/siLuci or siTNFα complex. At 16 hours after the third injection, mice were injected intraperitoneally with poly(I:C) (300 μg/ml), and after the indicated times serum levels of human TNF-α were measured by ELISA (n = 3). ns, not significant. *, P < 0.5.