Gene silencing of IL-12 in dendritic cells inhibits autoimmune arthritis

Abstract

Background: We have previously demonstrated that immune modulation can be accomplished by administration of gene silenced dendritic cells (DC) using siRNA. In this study, we demonstrate the therapeutic utilization of shRNA-modified DC as an antigen-specific tolerogenic vaccine strategy for autoimmune arthritis.

Methods: A shRNA that specifically targets IL-12 p35 was designed and cloned into a plasmid vectors (IL-12 shRNA). Bone marrow-derived DC from DBA/1 mice were transfected with the IL-12 shRNA construct in vitro. Mice with collagen II (CII)-induced arthritis (CIA) were treated with the modified DCs expressing the shRNA. Recall response and disease progression were assessed.

Results: After gene silencing of IL-12 in DC, DC were shown to selectively inhibit T cell proliferation on recall responses and in an MLR. In murine CIA, we demonstrated that administration of IL-12 shRNA-expressing DC that were pulsed with CII inhibited progression of arthritis. The therapeutic effects were evidenced by decreased clinical scores, inhibition of inflammatory cell infiltration in the joint, and suppression of T cell and B cell responses to CII.

Conclusion: We demonstrate a novel tolerance-inducing protocol for the treatment of autoimmune inflammatory joint disease in which the target antigen is known, utilizing DNA-directed RNA interference.

Figure 1

Gene silencing of IL-12 shRNA on DC. (A) Gene silencing efficacy of IL-12 shRNA was determined by RT-PCR. DC were cultured for 4 days as described in the methods. On day 5, 10⁶ DC were transfected with 2 μg of IL-12 shRNA using GenePORTER reagent. One day after transfection, DC were harvested and mRNA detected. (B) Gene silencing efficacy of IL-12 shRNA was determined by flow cytometry. DC were transfected as above and collected 48 h after transfection. DC were intracellular stained with FITC labeled anti-IL-12 antibody and followed by flow cytometry analysis. The data presented one of three independent experiments.

Figure 2

Immune modulation by IL-12 shRNA silenced DC. (A) IL-12 silenced DC promote Th2 cytokine production. Control and siRNA-treated DC were cultured with allogeneic T cells for 3 days. Co-cultured T cells were harvested and gene expression of IFNγ, IL-2, IL-4 and IL-10 were detected by RT-PCR. (B) DC silenced with IL-12 shRNA inhibited allogeneic T cell proliferation. DC (1 × 10⁵ cells/well) were co-cultured with allo-T cells (5 × 10⁵ cells/well) in 96-well plate for 72 h. 1 μCi/well of ³H-labelled thymidine was added to the culture for the last 16 h of culture and proliferation was assessed by scintillation counting. Results were expressed as the mean counts per min of triplicate cultures ± SEM. * = P < 0.05.

Figure 3

Inhibition of clinical development of CIA in mice injected with IL-12 shRNA-transfected DC. (A) Index of disease severity of joint after single DC treatment. 12 days after intradermal challenge with CII (200 μg per mouse), DBA/1 LacJ mice were treated with one i.p. injections of 5 × 10⁶ IL-12-silenced DC pulsed with CII (10 μg/ml). Control siRNA -transfected DC pulsed with CII served as a control. All mice were i.p. boosted with the same dose of CII 21 day after priming with the antigen. The animals were observed for 4 weeks since arthritis onset. Each limb was graded on a scale from 0 to 4 and the average clinical score per affected paw was calculated. Each point denotes the score of 5 mice in each group. Results represent 1 of 3 experiments. * = P < 0.05 versus control DC. (B) Histological sections of joints from arthritis mice injected with control DC. H&E stained sagittal sections of proximal interphalangeal joints. Sections from control mice show widespread inflammation cell infiltration, mild edema and congestion. Cartilage surface became uneven due to soft bone damage. (C) Histological sections of joints from arthritis mice injected with IL-12 shRNA-transfected DC. The majority of sections from animals treated with single injection of IL-12-silenced DC pulsed with CII do not show monocyte infiltration, edema and congestion. Cartilage surface appears smooth. Original magnification × 100. Results represent 1 of 15 mice. (D) Index of disease severity of joint after two DC treatments. 7 days before and 12 days after intradermal challenge with CII (200 μg per mouse), DBA/1 LacJ mice were treated with i.p. injections of 5 × 10⁶ IL-12-silenced DC pulsed with CII (10 μg/ml). Mock-transfected DC pulsed with CII served as a control. All mice were i.p. boosted with the same dose of CII 21 day after priming with the antigen. The animals were observed for 4 weeks since arthritis onset. Each limb was graded on a scale from 0 to 4 and the average clinical score per affected paw was calculated. Each point denotes the score of 5 mice in each group. Results represent 1 of 3 experiments. * = P < 0.05 versus control DC.

Figure 4

T cell recall responses to CII in arthritis mice injected with IL-12 shRNA-transfected DC. Experimental and control groups of mice were treated with two injections of IL-12-silenced DC as described in the legend to Figure 3. At the end of clinical assessment of CIA development, the mice were sacrificed and T cells from lymph nodes (A) and spleens (B) were isolated. A CII-specific response from different group of animals was performed by proliferation, as described in Materials and Methods. Lymphocytes were restimulated in vitro with different concentrations of CII, KLH or PBS alone and a ³H-labelled thymidine incorporation was measured. Results represent 1 of 3 similar experiments (n = 4 per group/experiment). * = P < 0.05 versus control PBS-treated DC.

Figure 5

Inhibition of CII-specific antibody production in arthritis mice following injections with IL-12 shRNA-transfected DC. Blood was taken 4 weeks after arthritis onset from experimental and control groups of mice treated with one (A) or two (B) injections of IL-12-silenced DC, as described in the legend to the Figure 2. Serum levels of anti-CII immunoglobulin Fc were determined using ELISA. Results show average levels of antibodies expressed as OD (n = 4 per group/experiment). * = P < 0.05 versus control shRNA-transfected DC.