Diabetes Prevention by Immunomodulatory FTY720 Treatment in the LEW.1AR1-iddm Rat Despite Immune Cell Activation

Abstract

The prevention of diabetes by the immunomodulatory agent FTY720 (fingolimod) was studied in the LEW.1AR1-iddm (IDDM) rat, an animal model of human type 1 diabetes. Immune cell subtypes and cytokine profiles in pancreatic islets, secondary lymphoid tissue, and serum were analyzed for signs of immune cell activation. Animals were treated with FTY720 (1 mg/kg body weight) for 40 d starting on d 50 of life. Changes in gene and protein expression of cytokines, CD8 markers, monocyte chemoattractant protein-1, inducible NO synthase, and caspase 3 were evaluated. Treatment with FTY720 prevented diabetes manifestation and islet infiltration around d 60 of life, the usual time of spontaneous diabetes development. On d 120, 30 d after the end of FTY720 therapy, diabetes prevention persisted. However, six of 12 treated animals showed increased gene expression of IL-1beta, TNF-alpha, and CD8 markers in pancreas-draining lymph nodes, indicating immune cell activation. In parallel, serum concentrations of these proinflammatory cytokines were increased. These six animals also showed macrophage infiltration without proinflammatory cytokine expression in a small minority (2-3%) of islets. Interestingly, regulatory T lymphocytes were significantly increased in the efferent vessels of the pancreas-draining lymph nodes only in animals without signs of immune cell activation but not in the rats with immune cell activation. This provides an indication for a lack of protective capacity in the animals with activated immune cells. Thus, FTY720 treatment prevented the manifestation of diabetes by promoting the retention of activated immune cells in the lymph nodes, thereby avoiding islet infiltration and beta-cell destruction by proinflammatory cytokines.

Fasting and meal-stimulated residual beta cell function is positively associated with serum concentrations of proinflammatory cytokines and negatively associated with anti-inflammatory and regulatory cytokines in patients with longer term type 1 diabetes

“…Therefore, we conclude that systemic IL-6 and TNF-α probably reflect ongoing islet inflammation. It has been shown in NOD mice and rat models of insulin-dependent diabetes mellitus that pancreatic cytokine levels correlate with systemic cytokine concentrations and the degree of insulitis or islet destruction [26,27]. Interestingly, findings in mice show that IL-6 can enhance insulin secretion by increasing glucagon-like peptide-1 secretion from L-cells and alpha-cells [28].…”

TNF-α Antibody Therapy in Combination With the T-Cell–Specific Antibody Anti-TCR Reverses the Diabetic Metabolic State in the LEW.1AR1-iddmRat

“…Biopsies of tissue (30 mg) were obtained from the pancreas tail of each animal under isoflurane anesthesia as previously described (36). To identify changes in the pancreatic islets, one biopsy was collected on the day of T1D manifestation, immediately before the start of therapy, and another at the end of therapy; the remaining pancreas, comprising head and tail, pancreas-draining lymph nodes, and serum, were collected 60 days after the end of therapy (25).…”

“…To identify changes in the pancreatic islets, one biopsy was collected on the day of T1D manifestation, immediately before the start of therapy, and another at the end of therapy; the remaining pancreas, comprising head and tail, pancreas-draining lymph nodes, and serum, were collected 60 days after the end of therapy (25). Tissue specimens were fixed for microscopic analyses (25,36). Blood glucose concentrations were determined daily (Glucometer Elite; Bayer, Leverkusen, Germany).…”

“…Blood glucose concentrations were determined daily (Glucometer Elite; Bayer, Leverkusen, Germany). Serum C-peptide was analyzed with a rat-specific ELISA (Mercodia, Uppsala, Sweden) and serum cytokine protein concentrations with a multiplex immunoassay kit (BioRad, Munich, Germany) (25,36).…”

“…Serial sections were stained with the avidin-biotin complex method or the double-immunofluorescence technique with primary antibodies for b-cells and immune cells (3,36). The antibodies against TNF-a and T cells recognized epitopes other than those targeted by the antibodies used for therapy.…”