Figure 1.
Transgene expression kinetics.
Every animal received 10 µg of p24GAG equivalent of lentivirus encoding firefly luciferase intravenously. Luciferase activity was determined after injection of luciferine using biophotonic imaging with the IVIS Lumina. Region of interest was placed over abdomen of mice. Kinetics of transgene expression (A) and organ distribution of transduction (B) after lentivirus injection via the retro orbital sinus, n = 6. B). Comparison between retro orbital sinus and tail vein injection (n = 2 in both groups) for abdominal expression (C) and localization of transgene expression in tail vein injected animals (D).
Figure 2.
Dose finding of intravenous lentivirus injection.
Increasing doses of lentivirus encoding GFP were injected via the tail vein and GFP expression was analyzed 10 days after injection. A) Total spleen cells were analyzed by flow cytometry for GFP expression. Statistics: one-way ANOVA with Bonferonni post test compared to PBS injected animals, n = 4 per group. ** p<0.01, *** p<0.001. B) GFP mRNA expression in liver and spleen samples. Relative expression = 2−ΔCt. Data represented as mean±SEM.
Figure 3.
Localization splenic GFP expression and identification of transduced cell types by cell surface marker analysis.
A) Increasing doses of lentivirus encoding GFP were injected via the tail vein and GFP expression was analyzed by immunohistochemistry ten days after virus injection. Representative histological images of experiment (magnification 200×), dotted line outlines white pulpa. W = white pulpa, R = red pulpa. Arrow heads indicate GFP positive cells. B) Flow cytometric analysis of GFP expressing cells 4 and 7 days after virus injection. Data represented as average (±SEM) GFP positive cells in gate indicated on x-axis (n = 4). C) Representative plots of cell marker and GFP FACS analysis.
Figure 4.
Effect of splenic TAK1 targeting on arthritis incidence and knee joint swelling.
A) Arthritis incidence was monitored over time macroscopically. B) Swelling of knee joints macroscopically at time of sacrifice (day 30 of CIA). Data represented as mean±SEM. Statistics macroscopic scoring: Student's t-test, n = 7 per group. ** p<0.01. C–E) Knee joints were isolated and evaluated for pathohistological features. C) Inflammation scores determined on HE stained sections. D) Cartilage erosion scores determined on safranin-O stained sections. E) Bone erosion scores determined on safranin-O stained sections. F-G) Representative histological images for bone erosion of knee joints. Original magnification 100×. Bone erosion indicated with arrow head. P = patella, F = femur. Statistics histology: Student's t-test, n = 8 for GFP, n = 6 for TAK1-K63W. * p<0.05.
Figure 5.
Proinflammatory cytokine expression in synovium of arthritic animals.
Synovium was isolated from arthritic animals and disrupted. Total mRNA was isolated and analyzed for A) cytokines (IL-1β, IL-6, KC) and B) RANK, and RANKL mRNA expression. Data represented as mean±SEM. Relative expression = 2−ΔCt. Statistics: Student's t-test, n = 6. * p<0.05.
Figure 6.
T-cell subsets in spleen during arthritis after TAK1 targeting in splenic APCs.
A) Spleen cells were isolated and stimulated with PMA, ionomycin, and Brefeldin A for 4 hours and subsequently stained for CD4, IFNγ, and IL-17. Percent positive cells from CD4 gate is shown. B) Representative FACS analysis plots. IL-17 and IFNγ positive cells in CD4+ gate shown. Data represented as mean±SEM. Statistics: Student's t-test, two tailed, n = 7. * p<0.05.
Figure 7.
IL-12 and IL-23 mRNA expression decreased in bone marrow derived DCs.
Bone marrow cells were harvested and transduced with either LV-GFP or LV-TAK1-K63W and subsequently differentiated into dendritic cells. After ten day differentiation period, mRNA was collected and analyzed for basal expression of IL-12p35, IL-12p40, and IL-23p19 using RT-PCR as described in Material and Methods. Data represented as mean±SEM, Relative expression = 2−ΔCt, n = 2.