Massive parallel gene expression profiling of RINm5F pancreatic islet ?-cells stimulated with interleukin-1? (original) (raw)
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F1000 - Post-publication peer review of the biomedical literature, 2012
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-1b (IL-1b) and interferon-c (IFN-c). 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.
Clinical and Experimental Immunology, 2010
Summary Type 1 diabetes (T1D) is caused by the selective destruction of the insulin-producing β 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 regener...
PLoS Genetics, 2012
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-1b (IL-1b) and interferon-c (IFN-c). 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.
Identification of Key β Cell Gene Signaling Pathways Involved in Type 1 Diabetes
Annals of the New York Academy of Sciences, 2004
In type 1 diabetes,  cells die through a process of immunemediated apoptosis. To better understand this process, it has been accepted practice to study  cell or islet apoptosis in vitro in response to a range of immune stimuli, such as interferon gamma, interleukin-1, nitric oxide or free radicals. In particular, much use has been made of immortalized  cell lines for such studies, although it is not clear to what extent the behavior of these cell lines might mimic the behavior of normal  cells in vivo, or freshly isolated  cells ex vivo. To address this question we compared the gene expression of freshly isolated NOD islets in the presence or absence of insulitis, with previously published data examining either islet or  cell gene or protein expression in a range of different species and contexts. There was a high correlation between  cell genes found be to be expressed by mouse and rat islets, by either gene expression or proteomic analysis. There was also a surprisingly high correlation between  cell genes found be to be expressed by islets exposed to insulitis in vivo and islets stimulated with IFN-␥ and IL-1 in vitro, suggesting that these two cytokines as produced by the islet infiltrate are important for priming  cells in vivo. There was a much lower correlation when gene expression was compared between fresh  cells and  cell lines, consistent with the view that  cell lines are very poorly representative of real  cells. Hence, any results obtained using  cell lines should be interpreted with great caution when extrapolating to the behavior of real  cells.
Diabetes, 2005
Different degrees of -cell failure and apoptosis are present in type 1 and type 2 diabetes. It has been recently suggested that high glucose-induced -cell apoptosis in type 2 diabetes shares a final common pathway with type 1 diabetes, involving interleukin-1 (IL-1) production by -cells, nuclear factor-B (NF-B) activation, and death via Fas-FasL. The aim of this study was to test whether human islet exposure to high glucose in vitro, or to the type 2 diabetes environment in vivo, induces IL-1 expression and consequent activation of NF-B-dependent genes. Human islets were isolated from five normoglycemic organ donors. The islets were cultured for 48 h to 7 days at 5.6, 11, or 28 mmol/l glucose. For comparative purposes, islets were also exposed to IL-1. Gene mRNA expression levels were assessed by real-time RT-PCR in a blinded fashion. Culture of the human islets at 11 and 28 mmol/l glucose induced a four-to fivefold increase in medium insulin as compared with 5.6 mmol/l glucose, but neither IL-1 nor IL-1 receptor antagonist (IL-1ra) expression changed. IL-1 and IL-1ra protein release to the medium was also unchanged. Stimulated human monocytes, studied in parallel, released >50-fold more IL-1 than the islets. There was also no glucose-induced islet Fas expression. Expression of the NF-B-dependent genes IB-␣ and monocyte chemoattractant protein (MCP)-1 was induced in human islets by IL-1 but not by high glucose. In a second set of experiments, human islets were isolated from seven type 2 diabetic patients and eight control subjects. The findings on mRNA levels were essentially the same as in the in vitro experiments, namely the in vivo diabetic state did not induce IL-1, Fas, or MCP-1 expression in human islets, and also did not modify IL-1ra expression. The present findings suggest that high glucose in vitro, or the diabetic milieu in vivo, does not induce IL-1 production or NF-B activation in human islets. This makes it unlikely that locally produced IL-1 is an important mediator of glucotoxicity to human islets and argues against the IL-1-NF-B-Fas pathway as a common mediator for -cell death in type 1 and type 2 diabetes.
Diabetologia, 2002
Aims/hypothesis. Type I (insulin-dependent) diabetes mellitus is characterized by selective destruction of the insulin producing beta cells. Interleukin-1β (IL-1β) modulates the beta-cell function, protein synthesis, energy production and causes apoptosis. We have previously shown changes in the expression of 82 out of 1 815 protein spots detected by two dimensional gel electrophoresis in IL-1β exposed diabetes prone Bio Breeding (BB-DP) rat islets of Langerhans in vitro. The aim of this study was to identify the proteins in these 82 spots by mass spectrometry and compare these changes with those seen in IL-1β exposed Wistar Furth (WF) rat islets. Methods. The 82 protein spots, that changed expression after IL-1β exposure, were all re-identified on preparative gels of 200 000 neonatal WF rat islets, cut out and subjected to mass spectrometry for identification.
Diabetes, 2001
Type 1 diabetes is an autoimmune disease resulting from the selective destruction of insulin-producing -cells. Cytokines may contribute to pancreatic -cell death in type 1 diabetes. -cell exposure to interleukin (IL)-1 induces functional impairment, whereas -cell culture for 6-9 days in the presence of IL-1 and interferon (INF)-␥ leads to apoptosis. To clarify the mechanisms involved in these effects of cytokines, we studied the general pattern of cytokine-induced gene expression in -cells. Primary rat -cells were fluorescenceactivated cell sorter-purified and exposed for 6 or 24 h to control condition, IL-1 ؉ INF-␥, or IL-1 alone (24 h only). Gene expression profile was analyzed in duplicate by oligonucleotide arrays. Nearly 3,000 transcripts were detected in controls and cytokine-treated -cells. Of these, 96 and 147 displayed changes in expression after 6 and 24 h, respectively, of exposure to IL-1 ؉ INF-␥, whereas 105 transcripts were modified after a 24-h exposure to IL-1. The cytokine-responsive genes were clustered according to their biological functions. The major clusters observed were metabolism, signal transduction, transcription factors, protein synthesis/ processing, hormones, and related receptors. These modifications in gene expression may explain some of the cytokine effects in -cells, such as decreased protein biosynthesis and insulin release. In addition, there was induction of diverse cytokines and chemokines; this suggests that -cells may contribute to mononuclear cell homing during insulitis. Several of the cytokine-induced genes are potentially regulated by the transcription factor NF-B. Clarification of the function of the identified cytokine-induced gene patterns may unveil some of the mechanisms involved in -cell damage and repair in type 1 diabetes. Diabetes 50:909-920, 2001
Interleukin-1� induced changes in the protein expression of rat islets: A computerized database
Electrophoresis, 1997
The cytokine interleukin 1 inhibits insulin release and is selectively cytotoxic to B-cells in isolated pancreatic rat isiets [l]. Active protein synthesis is a crucial part of P-cell destruction, defense, and repair after insults such as cytokines. The free radical nitric oxide (NO) has been demonstrated to be an important mediator of the deleterious effects of cytokines on islet B-cells [2-41. Thus, analogues of L-arginine, the substrate for NO production, prevent the deleterious effects of interleukin 16 (IL-lj3) [2-41, and mRNA for the cytokine-inducible isoform of NO synthase (iNOS) is induced by IL-Ifl in B-, but not a-cells [5]. We recently cloned iNOS from neonatal rat islets and demonstrated the expression of the recom-Correspondence: Prof. Jorn Nerup, D . MSc., Steno Diabetes Center, Niels Steensens vej 2, DK-2820 Gentofte, Denmark (Tel: +45-44-43-93-89; Fax: +45-44-43-82-32. Nonsíandard ebbrevistions: CV, coeficient of variation; DB, database; HBSS, Hanks' balanced salt solution; IL-16, interleukin; ¡NOS, cytokine-inducible isoform of NO synthase; VoIOD, percentage of integrated optical density; NHS, normal human serum
Diabetologia, 2002
Aims/hypothesis. Type I (insulin-dependent) diabetes mellitus is characterized by selective destruction of the insulin producing beta cells. Interleukin-1β (IL-1β) modulates the beta-cell function, protein synthesis, energy production and causes apoptosis. We have previously shown changes in the expression of 82 out of 1 815 protein spots detected by two dimensional gel electrophoresis in IL-1β exposed diabetes prone Bio Breeding (BB-DP) rat islets of Langerhans in vitro. The aim of this study was to identify the proteins in these 82 spots by mass spectrometry and compare these changes with those seen in IL-1β exposed Wistar Furth (WF) rat islets. Methods. The 82 protein spots, that changed expression after IL-1β exposure, were all re-identified on preparative gels of 200 000 neonatal WF rat islets, cut out and subjected to mass spectrometry for identification.
Interleukin-1β induced changes in the protein expression of rat islets: A computerized database
Electrophoresis, 1997
The cytokine interleukin 1 inhibits insulin release and is selectively cytotoxic to B-cells in isolated pancreatic rat isiets [l]. Active protein synthesis is a crucial part of P-cell destruction, defense, and repair after insults such as cytokines. The free radical nitric oxide (NO) has been demonstrated to be an important mediator of the deleterious effects of cytokines on islet B-cells [2-41. Thus, analogues of L-arginine, the substrate for NO production, prevent the deleterious effects of interleukin 16 (IL-lj3) [2-41, and mRNA for the cytokine-inducible isoform of NO synthase (iNOS) is induced by IL-Ifl in B-, but not a-cells [5]. We recently cloned iNOS from neonatal rat islets and demonstrated the expression of the recom-Correspondence: Prof. Jorn Nerup, D . MSc., Steno Diabetes Center, Niels Steensens vej 2, DK-2820 Gentofte, Denmark (Tel: +45-44-43-93-89; Fax: +45-44-43-82-32. Nonsíandard ebbrevistions: CV, coeficient of variation; DB, database; HBSS, Hanks' balanced salt solution; IL-16, interleukin; ¡NOS, cytokine-inducible isoform of NO synthase; VoIOD, percentage of integrated optical density; NHS, normal human serum