Gene expression profiles for the human pancreas and purified islets in type 1 diabetes: new findings at clinical onset and in long-standing diabetes

Abstract

Type 1 diabetes (T1D) is caused by the selective destruction of the insulin-producing beta cells of the pancreas by an autoimmune response. Due to ethical and practical difficulties, the features of the destructive process are known from a small number of observations, and transcriptomic data are remarkably missing. Here we report whole genome transcript analysis validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and correlated with immunohistological observations for four T1D pancreases (collected 5 days, 9 months, 8 and 10 years after diagnosis) and for purified islets from two of them. Collectively, the expression profile of immune response and inflammatory genes confirmed the current views on the immunopathogenesis of diabetes and showed similarities with other autoimmune diseases; for example, an interferon signature was detected. The data also supported the concept that the autoimmune process is maintained and balanced partially by regeneration and regulatory pathway activation, e.g. non-classical class I human leucocyte antigen and leucocyte immunoglobulin-like receptor, subfamily B1 (LILRB1). Changes in gene expression in islets were confined mainly to endocrine and neural genes, some of which are T1D autoantigens. By contrast, these islets showed only a few overexpressed immune system genes, among which bioinformatic analysis pointed to chemokine (C-C motif) receptor 5 (CCR5) and chemokine (CXC motif) receptor 4) (CXCR4) chemokine pathway activation. Remarkably, the expression of genes of innate immunity, complement, chemokines, immunoglobulin and regeneration genes was maintained or even increased in the long-standing cases. Transcriptomic data favour the view that T1D is caused by a chronic inflammatory process with a strong participation of innate immunity that progresses in spite of the regulatory and regenerative mechanisms.

Fig. 1

Distributions of differentially expressed genes in the Type 1 diabetes (T1D) pancreas and islets by functional categories. The numbers of differentially expressed genes in each category for each sample are shown. Only the 15 most represented categories are given. Each stacked bar corresponds to a different sample and is divided into up-regulated genes (red) and down-regulated genes (green). From left to right: pancreas case 1, pancreas case 2, pancreas case 3, pancreas case 4, islets case 1, islets case 4. (a) Up- and down-regulated genes in general categories and (b) up- and down-regulated genes of immune response subcategory.

Fig. 2

Heatmaps of gene expression profiles of the immune system in pancreas and purified islets from Type 1 diabetes (T1D) patients. Rows are for differentially expressed genes and columns are for pancreases and purified islets from T1D patients. Data were transformed to log₂ ratios relative to the mean of the normal controls and subjected to hierarchical clustering. The colour gradient key reflects relative expression on a log₂ scale. The most over-represented immune response subcategories are antigen presentation (a), chemotaxis (b), innate immunity and inflammation (c), complement (d), immunoregulation (e), adhesion molecules (f), interferon responsive (g) and leucocytes (h). P1, pancreas from case 1; P2, pancreas from case 2; P3, pancreas from case 3; P4, pancreas from case 4; I1, islets from case 1; I4, islets from case 4.

Fig. 3

Heatmaps of islet (a) and nervous system (b) gene expression profiles in pancreas and purified islets from Type 1 diabetes (T1D) patients. Rows are for differentially expressed genes and columns are for pancreases and purified islets from T1D patients. Data were transformed to log₂ ratios relative to the mean of the normal controls and subjected to hierarchical clustering. The colour gradient key reflects relative expression on a log₂ scale. P1, pancreas from case 1; P2, pancreas from case 2; P3, pancreas from case 3; P4, pancreas from case 4; I1, islets from case 1; I4, islets from case 4.

Fig. 4

Quantitative real-time polymerase chain reaction results for selected genes in the Type 1 diabetes (T1D) pancreas and purified islets. Gene expression signals were normalized to HPRT and calibrator sample. Results from three independent experiments (mean ± standard error of the mean) (*P < 0·05; **P < 0·01; ***P < 0·001). (a) Whole pancreas. White bars are means of three normal pancreases, clear grey bars are means of three to five blocks from case 1, grey bars are means of three blocks from case 2, dark grey bars are means of three blocks from case 3, black bars are means of three blocks from case 4. (b) Purified islets. White bars are means of four islets samples from different donors, clear grey bars correspond to islets from case 1 and black bars correspond to islets from case 4.

Fig. 5

Double immunofluorescence staining of pancreatic cryostat sections from cases 1 and 4 and control. Left column: control pancreas; middle column: case 1; right column case 4. From top to bottom, haematoxylin and eosin staining (a), insulin (INS)/glucagon (GCG) (b), GCG/CD45 (c), GCG/CD19 (d), GCG/CRP (e), GCG/CD44 (f), glutamic acid decarboxylase (GAD)/human leucocyte antigen E-related (HLA-E) (g), GCG/CD36 (h) and GAD/REG3A (i). INS, GCG and GAD are islet markers. Magnification ×200. Bars = 100 µm.