Figure 3.

Overexpression of A20 increases T and B-cell infiltration in vascular aortic allografts and breaks media immunoprivilege by inhibiting inflammation-induced IDO upregulation. A. Representative photomicrographs show increased numbers of CD3+, CD4+ and CD8+ T cells, as well as B220+ B cells in rAd.A20 as compared to saline and rAd.βgal treated aortic allografts, albeit this increase was only significant for CD8+, and B cells. Graphs depict numbers of CD3+, CD4+, CD8+, B220+ cells/ high power field (HPF). Each bar represents the mean±SE of CD3+, CD4+, CD8+, or B220+ cells/ HPF of 3-4 mice in rAd.A20-treated group and 4 mice in the control group combining 2 saline and 2 rAd.βgal-treated allografts. Breach of media immunoprivilege relates to overexpression of A20 in SMC preventing upregulation of IDO in response to inflammatory insults, as shown in B. HCASMC cultures, and C. in media of aortic allografts in vivo. B. Representative Western blots of cell lysates from non-transduced (NT), rAd.A20, and rAd.βgal transduced SMC stimulated for 24 hours with IL-17 (20 ng/mL), a triple cytokine (TC) cocktail (400 U/ml TNFα, 100 U/ml IL-1β, 400 U/ml IFNγ), or a combination of TC and IL-17, immunoblotted with anti-IDO and anti-GAPDH (loading control) antibodies. Bar graphs represent densitometric quantification of migrating bands from 3 independent experiments, expressed as mean±SE fold induction of control (C=non transduced, untreated cells). C. Representative immunofluorescence staining of IDO demonstrates increased expression in media of saline and rAd.βgal treated, but not rAd.A20-treated aortic allografts. Color codes are as follows: Red-IDO, blue-4’,6-diamidino-2-phenylindole (DAPI) nuclear staining, green-elastic lamina auto fluorescence. I-intima, M-media, and A-adventitia, original magnification X400. *p<0.05, **p<0.01, ***p<0.001.