Osteopontin Deficiency Impacts the Pancreatic Th1/Th2 Cytokine Profile Following Multiple Low Dose Streptozotocin-Induced Diabetes

Abstract

Osteopontin (OPN) is a phosphorylated acidic glycoprotein that causes chemotaxis of macrophages and downregulation of nitric oxide synthesis. OPN has been shown to be involved in the pathogenesis of autoimmune diseases. Here, we tested the hypothesis that increased expression of pancreatic OPN in experimental diabetes has a protective role. The immune response phenotype associated with the induction of diabetes was evaluated in male OPN knockout (KO) and wild type (WT) mice. Multiple low dose streptozotocin (STZ) (MLDS), 40 mg/kg, was injected intraperitoneally for 5 days to establish a model for autoimmune diabetes. Glucose levels and body weight were evaluated in the vehicle and STZ treated groups. ELISA assay was used to monitor OPN serum levels in the WT diabetic mice. Histological studies evaluated insulitis development and Western blot analysis was employed to evaluate the expression levels of Th1 cytokines (TNF-alpha and IFN-gamma) and Th2 cytokines (IL-10 and IL-4). Immunohistochemistry was employed to localize IL-4 in the diabetic WT pancreata. Both WT and KO mice developed diabetes. In the WT, OPN serum levels were significantly upregulated 1 day after STZ injection. Pancreatic islets appeared larger in the KO group. Mild lymphocytic infiltrate and apoptosis were detected in the WT diabetic islets, while no signs of inflammation were detected in the KO group. WT diabetics showed upregulation of both Th1 and Th2 cytokines, whereas in the diabetic KO a mild upregulation of Th1 cytokines was detected with significant downregulation of IL-4. In the diabetic WT mice, IL-4 was localized in the interlobular connective tissue. Our studies show that the pancreatic immune response to MLDS diabetes is balanced between the Th1 and Th2 in the WT animals. KO mice show mild polarization towards the Th1 response. Although OPN is a known promoter for Th1 responses, it appears to have a regulatory control over the Th2 response in MLDS.

Expression and regulation of osteopontin in type 1 diabetes

“…18 Using an OPN knockout model, we showed that OPN is essential for polarizing the islet cytokine immune response towards the protective Th2 profile. 19 We also demonstrated that OPN regulates NO signaling in the islets and b-cells via an RGD-mediated feedback regulation of IL-1b-induced nuclear factor-kB (NFkB) activation with consequent downregulation of induced nitric oxide synthase activity and increased b-cell survival. 20 In this study, we used different age groups of the NOD mice, which spontaneously develop a form of type I diabetes and shares many features of the human disease.…”

“…Based upon previous data supporting the protective and pro survival role of OPN in various tissues 10,11,36 and in the pancreatic islets and b-cells, 18,19,20 we propose that the high levels of islet OPN during the early stages of autoimmune diabetes might isolated from pancreata, islets or b-cells using Trizol reagent (Life Technologies, Gaitherburg, MD), according to the manufacturer's protocol. RNAs were quantified and input amounts were optimized for each amplicon.…”

Osteopontin activates the diabetes-associated potassium channel TALK-1 in pancreatic β-cells

“…Furthermore, it has been well documented that low level inflammation associated with the early stages of T2D leads to upregulation of OPN mRNA and iOPN protein expression in pancreatic islets [42, 44, 47, 53]. Elevation of iOPN serves a protective role in pancreatic islets where it acts as a negative feedback regulator of induced NO synthase (iNOS) and as a promoter of a protective Th2 immune response [42, 44, 47, 54]. Interestingly, TALK-1 channels are activated by NO.…”