Development of Type 1 Diabetes Mellitus in Nonobese Diabetic Mice Follows Changes in Thymocyte and Peripheral T Lymphocyte Transcriptional Activity

As early as one month of age, nonobese diabetic (NOD) mice feature pancreatic infiltration of autoreactive T lymphocytes, which destruct insulin-producing beta cells, producing autoimmune diabetes mellitus (T1D) within eight months. Thus, we hypothesized that during the development of T1D, the transcriptional modulation of immune reactivity genes may occur as thymocytes mature into peripheral T lymphocytes. The transcriptome of thymocytes and peripheral CD3+ T lymphocytes from prediabetic or diabetic mice analyzed through microarray hybridizations identified 2,771 differentially expressed genes. Hierarchical clustering grouped mice according to age/T1D onset and genes according to their transcription profiling. The transcriptional activity of thymocytes developing into peripheral T lymphocytes revealed sequential participation of genes involved with CD4+/CD8+ T-cell differentiation (Themis), tolerance induction by Tregs (Foxp3), and apoptosis (Fasl) soon after T-cell activation (IL4), while the emergence of T1D coincided with the expression of cytotoxicity (Crtam) and inflammatory response genes (Tlr) by peripheral T lymphocytes

Transcription profiling of Prss16 (Tssp) can be used to find additional peptidase genes that are candidates for self-peptide generation in the thymus

Positive selection (PS) in the thymus involves the presentation of self-peptides that are bound to MHC class II on the surface of cortical thymus epithelial cells (cTECs). Prss16 gene corresponds to one important element regulating the PS of CD4(+) T lymphocytes, which encodes Thymus-specific serine protease (Tssp), a cTEC serine-type peptidase involved in the proteolytic generation of self-peptides. Nevertheless, additional peptidase genes participating in the generation of self-peptides need to be found. Because of its role in the mechanism of PS and its expression in cTECs, the Prss16 gene might be used as a transcriptional marker to identify new genes that share the same expression profile and that encode peptidases in the thymus. To test this hypothesis, we compared the differential thymic expression of 4,500 mRNAs of wild-type (WT) C57BL/6 mice with their respective Prss16-knockout (KO) mutants by using microarrays. From these, 223 genes were differentially expressed, of which 115 had known molecular/biological functions. Four endopeptidase genes (Casp1, Casp2, Psmb3 and Tpp2) share the same expression profile as the Prss16 gene; i.e., induced in WT and repressed in KO while one endopeptidase gene, Capns1, features opposite expression profile. The Tpp2 gene is highlighted because it encodes a serine-type endopeptidase functionally similar to the Tssp enzyme. Profiling of the KO mice featured down-regulation of Prss16, as expected, along with the genes mentioned above. Considering that the Prss16-KO mice featured impaired PS, the shared regulation of the four endopeptidase genes suggested their participation in the mechanism of self-peptide generation and PS.Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq, Brazil)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), BrazilFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), BrazilFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP, Brazil)Institut National de la Sante et de la Recherche Medicale (INSERM, France)Institut National de la Sante et de la Recherche Medicale (INSERM, France

Age-related deregulation of Aire and peripheral tissue antigen genes in the thymic stroma of non-obese diabetic (NOD) mice is associated with autoimmune type 1 diabetes mellitus (DM-1)

Gene expression of peripheral tissue antigens (PTAs) in stromal medullary thymic epithelial cells (mTECs) is a key process to the negative selection of autoreactive thymocytes. This phenomenon was termed ""promiscuous gene expression"" (PGE), which is partially controlled by the Aire gene. Nevertheless, reasons for the correlation of Aire and PTAs with the emergence of autoimmune diseases are largely unknown, though it may be a result of a chronological effect. Although the effect of Aire mutations in pathogenic autoimmunity is well know, it could not be a unique cause for autoimmunity. Independently of mutations, temporal deregulation of Aire expression may imbalance Aire-dependent PTAs and/or wide PGE. This deregulation may be an early warning sign for autoimmune diseases as it guarantees autoantigen representation in the thymus. To assess this hypothesis, we studied the expression levels of Aire, Aire-dependent (Ins2) and Aire-independent (Gad67 and Col2a1) PTAs using real-time-PCR of the thymic stromal cells of NOD mice during the development of autoimmune type 1 diabetes mellitus (DM-1). Wide PGE was studied by microarrays in which the PTA genes were identified through parallel CD80(+) mTEC 3.10 cell line expression profiling. The results show that Aire gene was down-regulated in young pre-autoimmune (pre-diabetic) NOD mice. PGE and specific PTA genes were down-regulated in adult autoimmune diabetic animals. These findings represent evidence indicating that chronological deregulation of genes important to negative selection may be associated with the development of an autoimmune disease (DM-1) in mice.Fun dacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), BrazilCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Ministry of Education of Brazil (MEC)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq, Brazil

Comprehensive Survey of miRNA-mRNA Interactions Reveals That Ccr7 and Cd247 (CD3 zeta) are Posttranscriptionally Controlled in Pancreas Infiltrating T Lymphocytes of Non-Obese Diabetic (NOD) Mice

<div><p>In autoimmune type 1 diabetes mellitus (T1D), auto-reactive clones of CD4⁺ and CD8⁺ T lymphocytes in the periphery evolve into pancreas-infiltrating T lymphocytes (PILs), which destroy insulin-producing beta-cells through inflammatory insulitis. Previously, we demonstrated that, during the development of T1D in non-obese diabetic (NOD) mice, a set of immune/inflammatory reactivity genes were differentially expressed in T lymphocytes. However, the posttranscriptional control involving miRNA interactions that occur during the evolution of thymocytes into PILs remains unknown. In this study, we postulated that miRNAs are differentially expressed during this period and that these miRNAs can interact with mRNAs involved in auto-reactivity during the progression of insulitis. To test this hypothesis, we used NOD mice to perform, for the first time, a comprehensive survey of miRNA and mRNA expression as thymocytes mature into peripheral CD3⁺ T lymphocytes and, subsequently, into PILs. Reconstruction of miRNA-mRNA interaction networks for target prediction revealed the participation of a large set of miRNAs that regulate mRNA targets related to apoptosis, cell adhesion, cellular regulation, cellular component organization, cellular processes, development and the immune system, among others. The interactions between miR-202-3p and the Ccr7 chemokine receptor mRNA or Cd247 (Cd3 zeta chain) mRNA found in PILs are highlighted because these interactions can contribute to a better understanding of how the lack of immune homeostasis and the emergence of autoimmunity (e.g., T1D) can be associated with the decreased activity of Ccr7 or Cd247, as previously observed in NOD mice. We demonstrate that these mRNAs are controlled at the posttranscriptional level in PILs.</p></div

Modulated miRNAs according to cell type during the transition from pre-diabetic to diabetic NOD mice, their potential mRNA targets as predicted by the GenMir++ algorithm, the number of miRNA-mRNA interactions and GO biological processes of predicted target mRNAs.

<p>(↓) Down-regutated, (↑) Up-regulated.</p

Transcriptome (mRNA) expression profiles.

<p>The samples analyzed were as follow: Thymocytes (n = 3) or pancreas-infiltrating lymphocytes (PILs) derived from pre-diabetic (1 mo) (n = 3) or peripheral CD3⁺ T lymphocytes derived from pre-diabetic [1 mo (n = 2) or 7 mo (n = 3)] NOD mice. These cell types featured unique transcriptome expression signature. The dendrograms and heat map were obtained using cluster and tree-view algorithms through the Agilent GeneSpring platform. Heat map legend: red = up-regulation, green = down-regulation, black = unmodulated (Pearson correlation metrics).</p

Mirnome (miRNA) expression profiles.

<p>The samples analyzed were as follow: Thymocytes (n = 2) or pancreas-infiltrating lymphocytes (PILs) (n = 2) derived from pre-diabetic (1 mo) or peripheral CD3⁺ T lymphocytes derived from pre-diabetic (1 mo n = 2 or 7 mo n = 3) NOD mice. These cell types featured unique mirnome expression signature. The dendrograms and heat map were obtained using cluster and tree-view algorithms through the Agilent GeneSpring platform. Heat map legend: red = up-regulation, green = down-regulation, black = unmodulated (Pearson correlation metrics).</p