Figure 4

Immune response in NOD mice treated with an IHoP- siRNA construct. (a) Immunofluorescence staining for glucagon (green) and MHC Class II (red) was performed on normal and post-onset NOD islets. Nuclei were counterstained with DAPI (blue) Scale bars, 20 μm. (b) IHC showing MHC Class II expression in normal, post-onset NOD and IHoP-siRNA-treated NOD mice islets. Nuclei were counterstained with hematoxylin (blue). Scale bars, 40 μm. (c) Adoptive transfer of splenocytes produced the following three cohorts: (1) control; NOD/LtSz-scid recipient alone. (2) Diabetic ATx; diabetic NOD/shiLtj donor into NOD/LtSz-scid recipient. (3) IHoP-siRNA ATx; pre-diabetic NOD/shiLtj at 7 weeks post IHoP-siRNA injection donor into NOD/LtSz-scid recipient. Center panel: when control splenocytes were given to NOD mice, the recipient mice developed diabetes and showed massive immunologic response within islets (Diabetic ATx). Right panel: in mice that received IHoP- siRNA donor splenocytes, immune cells became activated and surrounded the islet but did not invade (IHoP-siRNA ATx). Hematoxylin and eosin staining from tissue collected 4 weeks post transplantation. Scale bars, 20 μm. (d) To confirm the results seen in (c), blood glucose levels of all three cohorts were tested. At 4 weeks post transplantation, glucose levels in IHoP-siRNA adoptive transferred animals (IHoP-siRNA ATx) were comparable to those of control mice. In contrast, non-treated animals became hyperglycemic. *P<0.05. (e) Insulitis in adoptive transferred mice. Control mice showed no insulitis and were scored as 0, the mice that received diabetic donor splenocytes scored 3.25 and IHoP-siRNA mouse-derived splenocyte recipients scored 1.13. (f) Glucagon-synthesizing mouse αTC1.9 cells were used to determine the effects of IHoP-siRNA transduction on MHC Class II gene expression in vitro. In agreement with in vivo results (b), αTC1.9 cells transducing the IHoP-siRNA construct (IHoP-siRNA) downregulated MHC Class II expression. β-Actin was used as loading control.