The focus of this study was to characterize molecular events in spleen leukocytes of nonobese diabetic (NOD) female mice at the earliest stages of development of autoimmune DM1 (before pathology in the islet of Langerhans is seen). Gene expression was characterized at the mRNA level on microarrays from Affymetrix and at the proteome level by 2D-gel electrophoresis. Samples consisted of pooled spleen leukocytes from 2- and 4-week-old NOD, NON, and C57Bl/6 female mice. The data were analyzed using GeneSpring software. Two-way-ANOVA (age 3 strain) with Bonferroni correction identified 388 genes showing a strain effect and 130 genes showing an age effect (p ≤ .05). Hierarchical clustering of the 388 strain-specific genes showed that much of the transcriptome followed a pattern of one strain showing relatively low expression compared to the other two strains. A subgroup of 112 of the 388 genes had uniquely high or low expression in NOD mice compared to both of the control strains. Proteome analysis indicated that 107 proteins showed strain-specific expression differences. We also conducted an analysis to define genes whose expression showed abnormal patterns of change in NOD mice between age 2 and 4 weeks. Because of substantial differences in gene expression between the two control strains, NON and C57BL/6, we compared NOD mice to each of these strains, separately. NOD mice had "abnormal development " in expression of 247 genes relative to NON mice and 164 genes relative to C57BL/6. Analysis of the proteome data showed similar abnormal development in expression of 55 and 97 proteins, respectively. The gene/protein lists were subjected to pathway analysis (www.ingenuity.com) to reveal specific molecular networks associated with each gene/protein list. The networks produced by this analysis suggested that the basic strain defects in NOD mice were associated with molecular networks centered around TGFB1, KITLG, IFNG, and TNF. The networks created from the lists of genes associated with developmental defects in NOD mice were centered around the same genes and in addition IL1B, TP53, and CASP3. The cytokine IL1B has been shown to induce apoptosis in islets, and it is particularly potent and beta cell specific when combined with INFG and TNF. Our data support a model where an increase in IL1B between 2 and 4 weeks of age combined with preexisting expression of INFG and TNF induces apoptosis and precipitates T lymphocyte-dependent autoimmunity, initiating DM1.
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