Izortze Santin

The Human Pancreatic Islet Transcriptome: Expression of Candidate Genes for Type 1 Diabetes and the Impact of Pro-Inflammatory Cytokines

Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic beta cells are killed by infiltrating immune cells and by cytokines released by these cells. Signaling events occurring in the pancreatic beta cells are decisive for their survival or death in diabetes. We have used RNA sequencing (RNA–seq) to identify transcripts, including splice variants, expressed in human islets of Langerhans under control conditions or following exposure to the pro-inflammatory cytokines interleukin-1β (IL-1β) and interferon-γ (IFN-γ). Based on this unique dataset, we examined whether putative candidate genes for T1D, previously identified by GWAS, are expressed in human islets. A total of 29,776 transcripts were identified as expressed in human islets. Expression of around 20% of these transcripts was modified by pro-inflammatory cytokines, including apoptosis- and inflammation-related genes. Chemokines were among the transcripts most modified by cytokines, a finding confirmed at the protein level by ELISA. Interestingly, 35% of the genes expressed in human islets undergo alternative splicing as annotated in RefSeq, and cytokines caused substantial changes in spliced transcripts. Nova1, previously considered a brain-specific regulator of mRNA splicing, is expressed in islets and its knockdown modified splicing. 25/41 of the candidate genes for T1D are expressed in islets, and cytokines modified expression of several of these transcripts. The present study doubles the number of known genes expressed in human islets and shows that cytokines modify alternative splicing in human islet cells. Importantly, it indicates that more than half of the known T1D candidate genes are expressed in human islets. This, and the production of a large number of chemokines and cytokines by cytokine-exposed islets, reinforces the concept of a dialog between pancreatic islets and the immune system in T1D. This dialog is modulated by candidate genes for the disease at both the immune system and beta cell level.

RNA-sequencing identifies dysregulation of the human pancreatic islet transcriptome by the saturated fatty acid palmitate

Pancreatic β-cell dysfunction and death are central in the pathogenesis of type 2 diabetes (T2D). Saturated fatty acids cause β-cell failure and contribute to diabetes development in genetically predisposed individuals. Here we used RNA sequencing to map transcripts expressed in five palmitate-treated human islet preparations, observing 1,325 modified genes. Palmitate induced fatty acid metabolism and endoplasmic reticulum (ER) stress. Functional studies identified novel mediators of adaptive ER stress signaling. Palmitate modified genes regulating ubiquitin and proteasome function, autophagy, and apoptosis. Inhibition of autophagic flux and lysosome function contributed to lipotoxicity. Palmitate inhibited transcription factors controlling β-cell phenotype, including PAX4 and GATA6. Fifty-nine T2D candidate genes were expressed in human islets, and 11 were modified by palmitate. Palmitate modified expression of 17 splicing factors and shifted alternative splicing of 3,525 transcripts. Ingenuity Pathway Analysis of modified transcripts and genes confirmed that top changed functions related to cell death. Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis of transcription factor binding sites in palmitate-modified transcripts revealed a role for PAX4, GATA, and the ER stress response regulators XBP1 and ATF6. This human islet transcriptome study identified novel mechanisms of palmitate-induced β-cell dysfunction and death. The data point to cross talk between metabolic stress and candidate genes at the β-cell level.

PTPN2, a Candidate Gene for Type 1 Diabetes, Modulates Pancreatic β-Cell Apoptosis via Regulation of the BH3-Only Protein Bim

OBJECTIVE Genome-wide association studies allowed the identification of several associations between specific loci and type 1 diabetes (T1D). However, the mechanisms by which most candidate genes predispose to T1D remain unclear. We presently evaluated the mechanisms by which PTPN2, a candidate gene for T1D, modulates β-cell apoptosis after exposure to type I and II interferons (IFNs), cytokines that contribute to β-cell loss in early T1D. RESEARCH DESIGN AND METHODS Small interfering RNAs were used to inhibit PTPN2, STAT1, Bim, and Jun NH2-terminal kinase 1 (JNK1) expression. Cell death was assessed by Hoechst and propidium iodide staining. BAX translocation, Bim phosphorylation, cytochrome c release, and caspases 9 and 3 activation were measured by Western blot or immunofluorescence. RESULTS PTPN2 knockdown exacerbated type I IFN–induced apoptosis in INS-1E, primary rat, and human β-cells. PTPN2 silencing and exposure to type I and II IFNs induced BAX translocation to the mitochondria, cytochrome c release, and caspase 3 activation. There was also an increase in Bim phosphorylation that was at least in part regulated by JNK1. Of note, both Bim and JNK1 knockdown protected β-cells against IFN-induced apoptosis in PTPN2-silenced cells. CONCLUSIONS The present findings suggest that local IFN production may interact with a genetic factor (PTPN2) to induce aberrant proapoptotic activity of the BH3-only protein Bim, resulting in increased β-cell apoptosis via JNK activation and the intrinsic apoptotic pathway. This is the first indication of a direct interaction between a candidate gene for T1D and the activation of a specific downstream proapoptotic pathway in β-cells.

TH17 (and TH1) signatures of intestinal biopsies of CD patients in response to gliadin.

Celiac disease (CD) is an immunological disorder caused by intolerance to ingested gliadin and other cereal prolamins that has been included in the TH1-dominated group of diseases, where IL-12 induced IFNγ is the major proinflamatory signal. Recently, another linage of T cells has been described, namely TH17, characterized by production of IL-17, that differentiate in response to TGFβ and IL-6 and participate in the pathogenesis of several autoimmune diseases. Using RT-PCR analysis of gene expression, we analyzed the presence of TH1 (IL-12 and IFNγ) and TH17 (TGFβ, IL-6, IL-17A, IL-17F and IL-23) related cytokines in intestinal biopsies from CD patients with active disease compared to remission and from treated patients after acute, in vitro re-exposure to gliadin. Potent TH1 and TH17 responses were present in the active stage of the disease, whereas short incubation of normalized biopsies with gliadin did not increase the expression of the effector cytokines, although a tendency of upregulation for both TH1 and TH17 promoting factors was observed, suggestive of a reactivation of proinflammatory pathways. These results place CD into the group of autoimmune disorders in which TH17 cells also participate, although the relative importance of each T cell response and their role in the initial events of the disease need further investigation.

Candidate genes for type 1 diabetes modulate pancreatic islet inflammation and β-cell apoptosis

Genome‐wide association studies (GWAS) have identified more than 50 loci associated with genetic risk of type 1 diabetes (T1D). Several T1D candidate genes have been suggested or identified within these regions, but the molecular mechanisms by which they contribute to insulitis and β‐cell destruction remain to be clarified. More than 60% of the T1D candidate genes are expressed in human pancreatic islets, suggesting that they contribute to T1D by regulating at least in part pathogenic mechanisms at the β‐cell level. Recent studies by our group indicate that important genetically regulated pathways in β‐cells include innate immunity and antiviral activity, involving RIG‐like receptors (particularly MDA5) and regulators of type I IFNs (i.e. PTPN2 and USP18), and genes related to β‐cell phenotype and susceptibility to pro‐apoptotic stimuli (i.e. GLIS3). These observations reinforce the concept that the early pathogenesis of T1D is characterized by a dialogue between the immune system and pancreatic β‐cells. This dialogue is probably influenced by polymorphisms in genes expressed at the β‐cell and/or immune system level, leading to inadequate responses to environmental cues such as viral infections. Further studies are needed to clarify how these disease‐associated variants affect pancreatic β‐cell responses to inflammation and the subsequent triggering of autoimmune responses and progressive β‐cell loss.

USP18 is a key regulator of the interferon-driven gene network modulating pancreatic beta cell inflammation and apoptosis

Type 1 diabetes (T1D) is an autoimmune disease targeting pancreatic beta cells. Genome-wide association studies and gene expression analysis identified interferon (IFN)-driven gene networks as crucial pathways in the pathogenesis of T1D. IFNs are linked to the response to viral infections and might contribute to the initiation of the autoimmune process in T1D. We presently analyzed the role of ubiquitin-specific peptidase 18 (USP18), an interferon-stimulated gene 15-specific protease, on IFN-induced pancreatic beta cell inflammation and apoptosis. Our findings indicate that USP18 inhibition induces inflammation by increasing the STAT signaling and exacerbates IFN-induced beta cell apoptosis by the mitochondrial pathway of cell death. USP18 regulates activation of three BH3-only proteins, namely, DP5, Bim and PUMA in pancreatic beta cells, suggesting a direct link between regulators of the type I IFN signaling pathway and members of the BCL-2 family. USP18 depletion increases the expression of the T1D candidate gene MDA5, leading to an upregulation of double-stranded RNA-induced chemokine production. These data suggest a cross talk between the type I IFN signaling pathway and a candidate gene for T1D to increase pro-inflammatory responses in beta cells. The present study shows that USP18 is a key regulator of IFN signaling in beta cells and underlines the importance of this pathway in beta cell inflammation and death.

BACH2, a Candidate Risk Gene for Type 1 Diabetes, Regulates Apoptosis in Pancreatic β-Cells via JNK1 Modulation and Crosstalk With the Candidate Gene PTPN2

Type 1 diabetes is a chronic autoimmune disease characterized by specific destruction of pancreatic β-cells by the immune system. Linkage and genome-wide association studies have identified more than 50 loci across the human genome associated with risk of type 1 diabetes. Recently, basic leucine zipper transcription factor 2 (BACH2) has been associated with genetic risk to develop type 1 diabetes, in an effect ascribed to the immune system. We evaluated whether BACH2 may also play a role in immune-mediated pancreatic β-cell apoptosis. BACH2 inhibition exacerbated cytokine-induced β-cell apoptosis in human and rodent β-cells by the mitochondrial pathway of cell death, whereas BACH2 overexpression had protective effects. BACH2 silencing and exposure to proinflammatory cytokines increased phosphorylation of the proapoptotic protein JNK1 by upregulation of mitogen-activated protein kinase kinase 7 (MKK7) and downregulation of PTPN2. JNK1 increased phosphorylation of the proapoptotic protein BIM, and both JNK1 and BIM knockdown protected β-cells against cytokine-induced apoptosis in BACH2-silenced cells. The present findings suggest that the type 1 diabetes candidate gene BACH2 regulates proinflammatory cytokine–induced apoptotic pathways in pancreatic β-cells by crosstalk with another candidate gene, PTPN2, and activation of JNK1 and BIM. This clarifies an unexpected and relevant mechanism by which BACH2 may contribute to diabetes.

The Transcription Factor C/EBP delta Has Anti-Apoptotic and Anti-Inflammatory Roles in Pancreatic Beta Cells

In the course of Type 1 diabetes pro-inflammatory cytokines (e.g., IL-1β, IFN-γ and TNF-α) produced by islet-infiltrating immune cells modify expression of key gene networks in β-cells, leading to local inflammation and β-cell apoptosis. Most known cytokine-induced transcription factors have pro-apoptotic effects, and little is known regarding “protective” transcription factors. To this end, we presently evaluated the role of the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) on β-cell apoptosis and production of inflammatory mediators in the rat insulinoma INS-1E cells, in purified primary rat β-cells and in human islets. C/EBPδ is expressed and up-regulated in response to the cytokines IL-1β and IFN-γ in rat β-cells and human islets. Small interfering RNA-mediated C/EBPδ silencing exacerbated IL-1β+IFN-γ-induced caspase 9 and 3 cleavage and apoptosis in these cells. C/EBPδ deficiency increased the up-regulation of the transcription factor CHOP in response to cytokines, enhancing expression of the pro-apoptotic Bcl-2 family member BIM. Interfering with C/EBPδ and CHOP or C/EBPδ and BIM in double knockdown approaches abrogated the exacerbating effects of C/EBPδ deficiency on cytokine-induced β-cell apoptosis, while C/EBPδ overexpression inhibited BIM expression and partially protected β-cells against IL-1β+IFN-γ-induced apoptosis. Furthermore, C/EBPδ silencing boosted cytokine-induced production of the chemokines CXCL1, 9, 10 and CCL20 in β-cells by hampering IRF-1 up-regulation and increasing STAT1 activation in response to cytokines. These observations identify a novel function of C/EBPδ as a modulatory transcription factor that inhibits the pro-apoptotic and pro-inflammatory gene networks activated by cytokines in pancreatic β-cells.

TYK2, a candidate gene for type 1 diabetes, modulates apoptosis and the innate immune response in human pancreatic β-cells

Pancreatic β-cells are destroyed by an autoimmune attack in type 1 diabetes. Linkage and genome-wide association studies point to >50 loci that are associated with the disease in the human genome. Pathway analysis of candidate genes expressed in human islets identified a central role for interferon (IFN)-regulated pathways and tyrosine kinase 2 (TYK2). Polymorphisms in the TYK2 gene predicted to decrease function are associated with a decreased risk of developing type 1 diabetes. We presently evaluated whether TYK2 plays a role in human pancreatic β-cell apoptosis and production of proinflammatory mediators. TYK2-silenced human β-cells exposed to polyinosinic-polycitidilic acid (PIC) (a mimick of double-stranded RNA produced during viral infection) showed less type I IFN pathway activation and lower production of IFNα and CXCL10. These cells also had decreased expression of major histocompatibility complex (MHC) class I proteins, a hallmark of early β-cell inflammation in type 1 diabetes. Importantly, TYK2 inhibition prevented PIC-induced β-cell apoptosis via the mitochondrial pathway of cell death. The present findings suggest that TYK2 regulates apoptotic and proinflammatory pathways in pancreatic β-cells via modulation of IFNα signaling, subsequent increase in MHC class I protein, and modulation of chemokines such as CXCL10 that are important for recruitment of T cells to the islets.

IL-17A increases the expression of proinflammatory chemokines in human pancreatic islets

Aims/hypothesisCytotoxic T cells and macrophages contribute to beta cell destruction in type 1 diabetes at least in part through the production of cytokines such as IL-1β, IFN-γ and TNF-α. We have recently shown the IL-17 pathway to be activated in circulating T cells and pancreatic islets of type 1 diabetes patients. Here, we studied whether IL-17A upregulates the production of chemokines by human pancreatic islets, thus contributing to the build-up of insulitis.MethodsHuman islets (from 18 donors), INS-1E cells and islets from wild-type and Stat1 knockout mice were studied. Dispersed islet cells were left untreated, or were treated with IL-17A alone or together with IL-1β+IFN-γ or TNF-α+IFN-γ. RNA interference was used to knock down signal transducer and activator of transcription 1 (STAT1). Chemokine expression was assessed by quantitative RT-PCR, ELISA and histology. Cell viability was evaluated with nuclear dyes.ResultsIL-17A augmented IL-1β+IFN-γ- and TNF-α+IFN-γ-induced chemokine mRNA and protein expression, and apoptosis in human islets. Beta cells were at least in part the source of chemokine production. Knockdown of STAT1 in human islets prevented cytokine- or IL-17A+cytokine-induced apoptosis and the expression of particular chemokines, e.g. chemokine (C-X-C motif) ligands 9 and 10. Similar observations were made in islets isolated from Stat1 knockout mice.Conclusions/interpretationOur findings indicate that IL-17A exacerbates proinflammatory chemokine expression and secretion by human islets exposed to cytokines. This suggests that IL-17A contributes to the pathogenesis of type 1 diabetes by two mechanisms, namely the exacerbation of beta cell apoptosis and increased local production of chemokines, thus potentially aggravating insulitis.