Knockout of toll-like receptor-2 attenuates both the proinflammatory state of diabetes and incipient diabetic nephropathy

Abstract

Objective: Type 1 diabetes (T1DM) is a proinflammatory state and confers an increased risk for vascular complications. Toll-like receptors (TLR) could participate in diabetic vasculopathies. Whether TLR activation contributes to the proinflammatory state of T1DM and the pathogenesis of diabetic nephropathy remains unknown.

Methods and results: We induced T1DM in TLR2 knockout mice (TLR2-/-) and wild-type littermates (C57BL/6J-WT) using streptozotocin (STZ). Fasting blood, peritoneal macrophages, and kidneys were obtained for flow cytometry, Western blot, microscopy, and cytokine assays at 6 and 14 weeks after induction of diabetes. Macrophage TLR2 expression and MyD88-dependent signaling were increased in diabetic mice (WT+STZ) compared with nondiabetic WT mice. These biomarkers were attenuated in diabetic TLR2-/- macrophages. WT+STZ mice showed increased kidney:body weight ratio due to cell hypertrophy, increased albuminuria, decreased kidney nephrin, podocin, and podocyte number and increased transforming growth factor-β and laminin compared with WT mice. Nephrin, podocin, and podocyte number and effacement were restored, and transforming growth factor-β and laminin levels were decreased in TLR2-/-+ STZ mice kidneys versus WT+STZ. Peritoneal and kidney macrophages were predominantly M1 phenotype in WT+STZ mice; this was attenuated in TLR2-/-+STZ mice.

Conclusions: These data support a role for TLR2 in promoting inflammation and early changes of incipient diabetic nephropathy, in addition to albuminuria and podocyte loss.

Figure 1

Figure 1a: Surface expression of TLR2 and TLR4: Peritoneal macrophages were obtained from WT (n=5), TLR2−/− (n=5), WT+STZ (n=20) and TLR2−/−+STZ (n=22) mice at 6 weeks and surface expression of TLR2 and TLR4 were assessed by flow cytometry as described in Methods. Values are expressed as MFI/10,000 cells (mean ± SD). *P<0.001 vs WT and TLR2−/−; †P<0.001 vs WT or WT+STZ; ‡P<0.05 vs WT. Figure 1b: TLR2-MyD88 dependent signaling: MyD88, IRAK-1 phosphorylation, IRF3, Trif, and nuclear p65 protein levels were measured in peritoneal macrophage lysates from WT+STZ (n=20) and TLR2−/−+STZ mice (n=22) at 6 weeks using Western lot assay. Representative blots with densitometric ratios were depicted in the figure. Values are expressed as protein/β-actin ratio (mean± SD). *P<0.001 vs WT+STZ mice. Total IRAK-1 and β-actin were used as internal controls. Figure 1c: Nuclear NF-kB activation in peritoneal macrophages of WT (n=5), TLR2−/− (n=5), WT+STZ (n=20) and TLR2−/−+STZ (n=22) mice at six weeks was determined by Trans-AM activity assay and normalized to nuclear protein as described in Methods. Values are expressed as ng/mg protein (mean ± SD). *P<0.005 vs WT and TLR2−/−; †P<0.001 vs WT+STZ mice.

Figure 1

Figure 1a: Surface expression of TLR2 and TLR4: Peritoneal macrophages were obtained from WT (n=5), TLR2−/− (n=5), WT+STZ (n=20) and TLR2−/−+STZ (n=22) mice at 6 weeks and surface expression of TLR2 and TLR4 were assessed by flow cytometry as described in Methods. Values are expressed as MFI/10,000 cells (mean ± SD). *P<0.001 vs WT and TLR2−/−; †P<0.001 vs WT or WT+STZ; ‡P<0.05 vs WT. Figure 1b: TLR2-MyD88 dependent signaling: MyD88, IRAK-1 phosphorylation, IRF3, Trif, and nuclear p65 protein levels were measured in peritoneal macrophage lysates from WT+STZ (n=20) and TLR2−/−+STZ mice (n=22) at 6 weeks using Western lot assay. Representative blots with densitometric ratios were depicted in the figure. Values are expressed as protein/β-actin ratio (mean± SD). *P<0.001 vs WT+STZ mice. Total IRAK-1 and β-actin were used as internal controls. Figure 1c: Nuclear NF-kB activation in peritoneal macrophages of WT (n=5), TLR2−/− (n=5), WT+STZ (n=20) and TLR2−/−+STZ (n=22) mice at six weeks was determined by Trans-AM activity assay and normalized to nuclear protein as described in Methods. Values are expressed as ng/mg protein (mean ± SD). *P<0.005 vs WT and TLR2−/−; †P<0.001 vs WT+STZ mice.

Figure 1

Figure 1a: Surface expression of TLR2 and TLR4: Peritoneal macrophages were obtained from WT (n=5), TLR2−/− (n=5), WT+STZ (n=20) and TLR2−/−+STZ (n=22) mice at 6 weeks and surface expression of TLR2 and TLR4 were assessed by flow cytometry as described in Methods. Values are expressed as MFI/10,000 cells (mean ± SD). *P<0.001 vs WT and TLR2−/−; †P<0.001 vs WT or WT+STZ; ‡P<0.05 vs WT. Figure 1b: TLR2-MyD88 dependent signaling: MyD88, IRAK-1 phosphorylation, IRF3, Trif, and nuclear p65 protein levels were measured in peritoneal macrophage lysates from WT+STZ (n=20) and TLR2−/−+STZ mice (n=22) at 6 weeks using Western lot assay. Representative blots with densitometric ratios were depicted in the figure. Values are expressed as protein/β-actin ratio (mean± SD). *P<0.001 vs WT+STZ mice. Total IRAK-1 and β-actin were used as internal controls. Figure 1c: Nuclear NF-kB activation in peritoneal macrophages of WT (n=5), TLR2−/− (n=5), WT+STZ (n=20) and TLR2−/−+STZ (n=22) mice at six weeks was determined by Trans-AM activity assay and normalized to nuclear protein as described in Methods. Values are expressed as ng/mg protein (mean ± SD). *P<0.005 vs WT and TLR2−/−; †P<0.001 vs WT+STZ mice.

Figure 2

Circulating levels of pro-inflammatory cytokines and chemokines in sera of WT (n=5), TLR2−/− (n=5), WT+STZ (n=20) and TLR2−/−+STZ (n=22) mice at 6 weeks were examined by multiplex assays as described in Methods. Values are expressed as pg/ml (mean ± SD). *P<0.001 vs WT and TLR2−/−; †P<0.001 vs to WT or WT+STZ.

Figure 3

Figure 3a: Phenotype of Peritoneal (Upper Panel) and Kidney (Lower Panel) Macrophages were assessed by flow cytometry as described in Methods. M1 Phenotype was characterized by positivity for Ly6C, CCR2 and IL-6 while M2 phenotype was characterized by CD206 and CD 163.. *P<0.001 vs WT and TLR2−/−; †P<0.001 vs. WT or WT+STZ. Figure 3b: Podocytes in glomeruli of diabetic wild type (WT) and TLR2−/− mice kidneys at 14 weeks were examined by WT-1 immunochemical staining as described in methods (n=11/group). 2 Representative photographs of WT-1 staining (brown spots in glomeruli) are shown for the 2 groups.

Figure 3

Figure 3a: Phenotype of Peritoneal (Upper Panel) and Kidney (Lower Panel) Macrophages were assessed by flow cytometry as described in Methods. M1 Phenotype was characterized by positivity for Ly6C, CCR2 and IL-6 while M2 phenotype was characterized by CD206 and CD 163.. *P<0.001 vs WT and TLR2−/−; †P<0.001 vs. WT or WT+STZ. Figure 3b: Podocytes in glomeruli of diabetic wild type (WT) and TLR2−/− mice kidneys at 14 weeks were examined by WT-1 immunochemical staining as described in methods (n=11/group). 2 Representative photographs of WT-1 staining (brown spots in glomeruli) are shown for the 2 groups.

Figure 4

Figure 4a: Slit diaphragm proteins Nephrin, Podocin and Extracellular matrix (ECM) proteins TGF-β, Laminin, MyD88, and IRAK-1 phosphorylation in kidney lysates of WT (n=5), TLR2−/− (n=5), WT+STZ (n=14) and TLR2−/−+STZ (n=16) at 6 weeks after diabetes were determined using Western blot. Representative blots with densitometric ratios were shown in the figure. Total IRAK-1 and β-actin were used as internal controls. Values are expressed as protein/β-actin ratio (mean ± SD). *P<0.005 vs WT; †P<0.02 vs WT+STZ. Figure 4b: TLR2, Nephrin, Podocin, TGF-β, Laminin, MyD88, and IRAK-1 phosphorylation in kidney lysates of WT, TLR2−/−, WT+STZ, and TLR2−/−+STZ (n=11/gr) at 14 weeks after diabetes were measured in kidney tissue lysates using Western blot assay. Representative blots with densitometric ratios were depicted in the figure. Total IRAK-1 and β-actin were used as internal controls. Values are expressed as protein/β-actin ratio (mean ± SD). *P<0.001 vs WT mice. †P<0.05 vs WT+STZ.

Figure 4

Figure 4a: Slit diaphragm proteins Nephrin, Podocin and Extracellular matrix (ECM) proteins TGF-β, Laminin, MyD88, and IRAK-1 phosphorylation in kidney lysates of WT (n=5), TLR2−/− (n=5), WT+STZ (n=14) and TLR2−/−+STZ (n=16) at 6 weeks after diabetes were determined using Western blot. Representative blots with densitometric ratios were shown in the figure. Total IRAK-1 and β-actin were used as internal controls. Values are expressed as protein/β-actin ratio (mean ± SD). *P<0.005 vs WT; †P<0.02 vs WT+STZ. Figure 4b: TLR2, Nephrin, Podocin, TGF-β, Laminin, MyD88, and IRAK-1 phosphorylation in kidney lysates of WT, TLR2−/−, WT+STZ, and TLR2−/−+STZ (n=11/gr) at 14 weeks after diabetes were measured in kidney tissue lysates using Western blot assay. Representative blots with densitometric ratios were depicted in the figure. Total IRAK-1 and β-actin were used as internal controls. Values are expressed as protein/β-actin ratio (mean ± SD). *P<0.001 vs WT mice. †P<0.05 vs WT+STZ.

Figure 5

Cytokine and chemokine concentrations in the kidney lysates of WT, TLR2−/−, WT+STZ and TLR2−/−+STZ mice (n=11/gr) was measured by Multiplex assay as described in Methods at 14 weeks. Values are expressed as pg/mg cell protein (mean ± SD). *P<0.001 vs WT and TLR2−/−; †P<0.01 vs WT+STZ.