Impact of dietary fat on Th1/Th2 cytokine gene expression in the pancreas and gut of diabetes-prone BB rats.

The effect of dietary n-3 or n-6 polyunsaturated fatty acids on the development of autoimmune insulitis was analysed in diabetes-prone BB rats. Litter-matched groups of rats received a standard open formula NIH-07 (National Institutes of Health, NIH) diet enriched with 10% fish oil, 10% flaxseed oil or with 10% palm oil plus 2% cholesterol during the period of insulitis onset (50-70 days of age). Analysis of cytokine gene expression in pancreatic RNA revealed an increase of IFN-gamma and a decrease of IL-10 mRNA with onset of insulitis. When compared to unsupplemented NIH, none of the three fat-enriched diets depressed the rise of IFN-gamma gene expression or the influx of leukocytes into islets. However, all of the fat-enriched diets led to significantly higher IL-10 mRNA levels. Although a specific anti-inflammatory effect of fish oil was not seen in the pancreas, a clear shift of the Th1/Th2 cytokine mRNA ratio towards Th2 was seen in the gut-associated immune system. We conclude that diets high in fat support IL-10 without suppressing IFN-gamma gene expression in islet inflammation. A special anti-inflammatory effect of fish oil was not seen in pancreatic lesions of BB rats, although there was strong modulation of the IFN-gamma/IL-10 mRNA ratio in the gut associated immune system.

Insulin therapy of prediabetes suppresses TH1 associated gene expression in BB rat pancreas.

Subcutaneous insulin treatment of young diabetes prone BB rats has been shown previously to suppress the development of autoimmune diabetes. In this study the hypothesis was tested that exogenous insulin may deviate the autoimmune process by acting on the Th1/Th2 cytokine balance in the pancreas. BB rats were implanted with pellets which continuously released insulin, at 50 d of age. Three weeks later cytokine mRNA expression in the pancreas and insulitis score were determined. While in control BB rats high levels of IFNgamma mRNA were detectable by RT-PCR, insulin treatment almost completely suppressed IFNgamma mRNA levels without concomitant upregulation of counterregulatory IL-10 and TGFbeta gene expression. Insulin also suppressed gene expression of inducible nitric oxide synthase. Mean insulitis scores were decreased after insulin treatment. We conclude that the protective effects of insulin treatment may not be due to the induction of protective Th2 immune reactivity but to general downregulation of immune activation in the pancreas, and hence also of Th1 autoimmunity.

Cytokine gene expression in the BB rat pancreas: natural course and impact of bacterial vaccines.

In diabetes prone BB rat pancreas the Th1/ Th2 cytokine balance and the expression of inducible nitric oxide synthase (iNOS) was determined by mRNA analysis before and after the onset of insulitis. Specific mRNA was amplified by reverse transcriptase polymerase chain reaction, quantitated with radiolabelled probes by phosphoimaging and calibrated with the amount of co-amplified beta-actin mRNA. At 50 days of age, prior to recognizable insulitis, there was already significantly enhanced expression of both, Th1 and Th2 cytokines, and of iNOS mRNA, when compared to Wistar rat pancreas (p < 0.001). This supports the concept of an inconspicuous early phase of islet infiltration by single immunocytes, called single cell insulitis. At 70 days of age mononuclear infiltration of islets had begun and was associated with upregulation of interferon gamma (IFN gamma) and iNOS, but downregulation of interleukin-10 and transforming growth factor beta mRNA (p < 0.001). These findings correlate the onset of insulitis with a shift of the Th1/Th2 cytokine balance towards Th1 cell reactivity. Indeed there was a close correlation of the Th1/Th2 cytokine ratio but not of absolute IFN gamma mRNA levels with the insulitis score. Vaccination at day 50 with tetanus toxoid did not affect cytokine gene expression while diphtheria toxoid and even more strongly BCG administration induced a shift towards Th2 reactivity (p < 0.001) while iNOS mRNA was decreased (p < 0.01). Oral dosing with immunostimulatory components of Escherichia coli also changed the quality of inflammation. Oral lipopolysaccharide (LPS) from E. coli and OM-89, an endotoxin free extract containing immunostimulatory glycolipopeptides and heat shock protein (hsp) 65, both downregulated IFN gamma mRNA while only OM-89 in addition suppressed iNOS mRNA and enhanced Th2 cytokine gene expression (p < 0.001). We conclude that the onset of insulitis is associated with a shift towards Th1 cytokine and iNOS gene expression. Diphtheria toxoid and BCG vaccination stimulates Th2 reactivity but does not downregulate Th1. The latter can be achieved through oral administration of LPS or a glycopeptide fraction (OM-89) from E. coli.