Direction of immune system in T1D pathogenesis – analysis by using gene microarray and pathway analysis (GeneGo, MetaCore)

Z. Halbhuber¹, M. Hubáčková², M. Krivjanská¹, K. Štechová²

¹ Central European Biosystems s.r.o., Praha

² Pediatrická klinika UK 2.LF a FN Motol, Praha

Introduction

Diabetes mellitus is a disorder of metabolism that manifests itself as type I or type II. The Type I (T1D, the object of this study) is caused by destruction of the insulin-producing beta-islet cells of the pancreas. It results in development of well known disorder of carbohydrate metabolism which leads to life-lasting necessity of insulin injection administration. It seriously impact not only the patient itself but it has also strong socioeconomic consequences. Nowadays that situation becomes worse in the Czech Republic due to increase in T1D occurrence among infants and children.

Aim

The aim of our study was to monitor in vitro changes in gene expression (mRNA transcription) in peripheral blood mononuclear cells (PBMCs) after stimulation with diabetogenic autoantigens within one family related to different manifestation of the T1D with respect to early detection of developing destructive insulinitis.

Method

PBMCs were divided into equal halves and one half was stimulated using derived syntetic peptides such as glutamic acid decarboxylase 65 (GAD65), IA2 (islet antigen-2, tyrosinephosphatase) and proinzulin. After 72hours incubation totalRNA was isolated from both groups - stimulated and intact which represents basal gene expression.

After RNA isolation, amplification and fluorescent labeling, the samples were competitively hybridize on the high density whole-genome microarray HOA (PhalanxBiotech). The microarray contains over 30 000 genomic probes. Hybridized slides were scanned and analyzed using GeneSpring GX (Agilent) and Pathway analasis software MetaCore (GeneGo). The microarray part was done as a service in microarray facility of Central European Biosystems s.r.o..

Results and conclusion

We identified more than 100 genes which belong mainly to regulatory factors and transcription factors. In accordance with previously published results we found out the similar expression pattern between basal expression and expression in stimulated samples depending on the diabetes development. As a promising results seems especially a decline in the expression of some cytokinines and chemokines in prediabetic phase. Important increase in gene expression after specific stimulation was observed for following genes in this study group (p<0.05): IFN-gamma, IL-1,-2,-6,-13,-22,-31, GATA-3, JUNB, IL-6R, STAT-6, TGF-beta. Moreover probably the most important difference was observed for IL-23R which was importantly downregulated in this group (12 fold difference) and also in T1D patients group (23 fold). In T1D patients we also observed an important activation of IL-2,- IL-33, JUNB genes after specific stimulation but strong downregulation of IL-4. STAT-6 and GATA-3 genes were also slightly downregulated in this group.

The study confirms that there can be find differential expression pattern and markers which preveal risk of diabetes.

This work was supported by grant NPVII 2B06019 from The Ministry of Education, Youth and Sports of the Czech Republic.