Global profiling of double stranded RNA and IFN-γ-induced genes in rat pancreatic beta cells (original) (raw)
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Global profiling of double stranded RNA- and IFN-?-induced genes in rat pancreatic beta cells
Diabetologia, 2003
Aims/hypothesis. Viral infections and local production of IFN-γ might contribute to beta-cell dysfunction/ death in Type 1 Diabetes. Double stranded RNA (dsRNA) accumulates in the cytosol of viral-infected cells, and exposure of purified rat beta cells to dsRNA (tested in the form of polyinosinic-polycytidylic acid, PIC) in combination with IFN-γ results in beta-cell dysfunction and apoptosis. To elucidate the molecular mechanisms involved in PIC + IFN-γ-effects, we determined the global profile of genes modified by these agents in primary rat beta cells. Methods. FACS-purified rat beta cells were cultured for 6 or 24 h in control condition or with IFN-γ, PIC or a combination of both agents. The gene expression profile was analysed in duplicate by high-density oligonucleotide arrays representing 5000 full-length genes and 3000 EST's. Changes of greater than or equal to 2.5-fold were considered as relevant. Results. Following a 6-or 24-h treatment with IFN-γ, PIC or IFN-γ and PIC, we observed changes in the expression of 51 to 189 genes. IFN-γ modified the expression of MHC-related genes, and also of genes in-volved in beta-cell metabolism, protein processing, cytokines and signal transduction. PIC affected preferentially the expression of genes related to cell adhesion, cytokines and dsRNA signal transduction, transcription factors and MHC. PIC and/or IFN-γ up-regulated the expression of several chemokines and cytokines that could contribute to mononuclear cell homing and activation during viral infection, while IFN-γ induced a positive feedback on its own signal transduction. PIC + IFN-γ inhibited insulin and GLUT-2 expression without modifying pdx-1 mRNA expression. Conclusion/interpretation. This study provides the first comprehensive characterization of the molecular responses of primary beta cells to dsRNA + IFN-γ, two agents that are probably present in the beta cell milieu during the course of virally-induced insulitis and Type 1 Diabetes. Based on these findings, we propose an integrated model for the molecular mechanisms involved in dsRNA + IFN-γ induced beta-cell dysfunction and death. [Diabetologia (2003[Diabetologia ( ) 46:1641[Diabetologia ( -1657
Mechanisms of β-Cell Death in Response to Double-Stranded (ds) RNA and Interferon-γ
The American Journal of Pathology, 2001
Viral infection is one environmental factor that has been implicated as a precipitating event that may initiate -cell damage during the development of diabetes. This study examines the mechanisms by which the viral replicative intermediate, double-stranded (ds) RNA impairs -cell function and induces -cell death. The synthetic dsRNA molecule polyinosinicpolycytidylic acid (poly IC) stimulates -cell DNA damage and apoptosis without impairing islet secretory function. In contrast, the combination of poly IC and interferon (IFN)-␥ stimulates DNA damage, apoptosis, and necrosis of islet cells, and this damage is associated with the inhibition of glucose-stimulated insulin secretion. Nitric oxide mediates the inhibitory and destructive actions of poly IC ؉ IFN-␥ on insulin secretion and islet cell necrosis. Inhibitors of nitric oxide synthase, aminoguanidine, and N G-monomethyl-L-arginine, attenuate poly IC ؉ IFN-␥-induced DNA damage to levels observed in response to poly IC alone, prevent islet cell necrosis, and prevent the inhibitory actions on glucose-stimulated insulin secretion. N G-monomethyl-L-arginine fails to prevent poly IC-and poly IC ؉ IFN-␥-induced islet cell apoptosis. PKR, the dsRNA-dependent protein kinase that mediates the antiviral response in infected cells, is required for poly IC-and poly IC ؉ IFN-␥-induced islet cell apoptosis, but not nitric oxide-mediated islet cell necrosis. Alone, poly IC fails to stimulate DNA damage in islets isolated from PKR-deficient mice; how-Supported by National Institutes of Health grants AI44458 (to J. A. C.) and AI42394 (R. J. K.).
Diabetes, 2008
OBJECTIVE— Viral infections contribute to the pathogenesis of type 1 diabetes. Viruses, or viral products such as double-stranded RNA (dsRNA), affect pancreatic β-cell survival and trigger autoimmunity by unknown mechanisms. We presently investigated the mediators and downstream effectors of dsRNA-induced β-cell death. RESEARCH DESIGN AND METHODS— Primary rat β-cells and islet cells from wild-type, toll-like receptor (TLR) 3, type I interferon receptor (IFNAR1), or interferon regulatory factor (IRF)-3 knockout mice were exposed to external dsRNA (external polyinosinic-polycytidylic acid [PICex]) or were transfected with dsRNA ([PICin]). RESULTS— TLR3 signaling mediated PICex-induced nuclear factor-κB (NF-κB) and IRF-3 activation and β-cell apoptosis. PICin activated NF-κB and IRF-3 in a TLR3-independent manner, induced eukaryotic initiation factor 2α phosphorylation, and triggered a massive production of interferon (IFN)-β. This contributed to β-cell death, as islet cells from IFNAR...
Journal of Biological Chemistry, 1999
Viral infection has been implicated as a triggering event that may initiate -cell damage during the development of autoimmune diabetes. In this study, the effects of the viral replicative intermediate, doublestranded RNA (dsRNA) (in the form of synthetic polyinosinic-polycytidylic acid (poly IC)) on islet expression of inducible nitric oxide synthase (iNOS), production of nitric oxide, and islet function and viability were investigated. Treatment of rat islets with poly(IC) ؉ interferon-␥ (IFN-␥) stimulates the time-and concentrationdependent expression of iNOS and production of nitrite by rat islets. iNOS expression and nitrite production by rat islets in response to poly(IC) ؉ IFN-␥ correlate with an inhibition of insulin secretion and islet degeneration, effects that are prevented by the iNOS inhibitor aminoguanidine (AG). We have previously shown that poly(IC) ؉ IFN-␥ activates resident macrophages, stimulating iNOS expression, nitric oxide production and interleukin-1 (IL-1) release. In addition, in response to tumor necrosis factor-␣ (TNF-␣) ؉ lipopolysaccharide, activated resident macrophages mediate -cell damage via intraislet IL-1 release followed by IL-1-induced iNOS expression by -cells. The inhibitory and destructive effects of poly(IC) ؉ IFN-␥, however, do not appear to require resident macrophages. Treatment of macrophage-depleted rat islets for 40 h with poly(IC) ؉ IFN-␥ results in the expression of iNOS, production of nitrite, and inhibition of insulin secretion. The destructive effects of dsRNA ؉ IFN-␥ on islets appear to be mediated by a direct interaction with -cells. Poly IC ؉ IFN-␥ stimulates iNOS expression and inhibits insulin secretion by primary -cells purified by fluorescence-activated cell sorting. In addition, AG prevents the inhibitory effects of poly(IC) ؉ IFN-␥ on glucose-stimulated insulin secretion by -cells. These results indicate that dsRNA ؉ IFN-␥ interacts directly with -cells stimulating iNOS expression and inhibiting insulin secretion in a nitric oxide-dependent manner. These findings provide biochemical evidence for a novel mechanism by which viral infection may directly mediate the initial destruction of -cells during the development of autoimmune diabetes.
Auto-immunity Highlights, 2021
Background: Type 1 diabetes (T1D) is an autoimmune disease characterized by the progressive destruction of pancreatic beta cells. Interferon-α (IFNα), an antiviral cytokine, is expressed in the pancreatic islets in early T1D, which may be secondary to viral infections. However, not all patients harboring a type I IFN signature present signals of viral infection, suggesting that this response might be initiated by other "danger signals". Accumulation of mitochondrial double-stranded RNA (mtdsRNA; a danger signal), secondary to silencing of members of the mitochondrial degradosome, PNPT1 and SUV3, has been described to activate the innate immune response. Methods: To evaluate whether mtdsRNA represents a "danger signal" for pancreatic beta cells in the context of T1D, we silenced PNPT1 and/or SUV3 in slowly proliferating human insulin-secreting EndoC-βH1 cells and in non-proliferating primary human beta cells and evaluated dsRNA accumulation by immunofluorescence and the type I IFN response by western blotting and RT-qPCR. Only the simultaneous silencing of PNPT1/SUV3 induced dsRNA accumulation in EndoC-βH1 cells but not in dispersed human islets, and there was no induction of a type I IFN response. By contrast, silencing of these two genes individually was enough to induce dsRNA accumulation in fibroblasts present in the human islet preparations. Conclusions: These data suggest that accumulation of endogenous mtdsRNA following degradosome knockdown depends on the proliferative capacity of the cells and is not a mediator of the type I IFN response in human pancreatic beta cells.
Endocrinology, 2002
Viral infections may trigger the autoimmune assault leading to type 1 diabetes mellitus. Double-stranded RNA (dsRNA) is produced by many viruses during their replicative cycle. The dsRNA, tested as synthetic poly(IC) (PIC), in synergism with the proinflammatory cytokines interferon-␥ (IFN-␥) and/or IL-1, results in nitric oxide production, Fas expression, -cell dysfunction, and death. Activation of the transcription nuclear factor-B (NF-B) is required for PIC-induced inducible nitric oxide synthase expression in -cells, and we hypothesized that this transcription factor may also participate in PIC-induced Fas expression and -cell apoptosis. This hypothesis, and the possibility that PIC induces expression of additional chemokines and cytokines (previously reported as NF-B dependent) in pancreatic -cells, was investigated in the present study. We observed that the PIC-responsive region in the Fas promoter is located between nucleotides ؊223 and ؊54. Site-directed mutations at the NF-B and CCAAT/ enhancer binding protein-binding sites prevented PICinduced Fas promoter activity. Increased Fas promoter ac-tivity was paralleled by enhanced susceptibility of PIC ؉ cytokine-treated -cells to apoptosis induced by Fas ligand.
Human Molecular Genetics, 2009
b-Cell destruction in type 1 diabetes (T1D) is at least in part consequence of a 'dialog' between b-cells and immune system. This dialog may be affected by the individual's genetic background. We presently evaluated whether modulation of MDA5 and PTPN2, two candidate genes for T1D, affects b-cell responses to doublestranded RNA (dsRNA), a by-product of viral replication. These genes were selected following comparison between known candidate genes for T1D and genes expressed in pancreatic b-cells, as identified in previous array analysis. INS-1E cells and primary fluorescence-activated cell sorting-purified rat b-cells were transfected with small interference RNAs (siRNAs) targeting MDA5 or PTPN2 and subsequently exposed to intracellular synthetic dsRNA (polyinosinic-polycitidilic acid-PIC). Real-time RT-PCR, western blot and viability assays were performed to characterize gene/protein expression and viability. PIC increased MDA5 and PTPN2 mRNA expression, which was inhibited by the specific siRNAs. PIC triggered apoptosis in INS-1E and primary b-cells and this was augmented by PTPN2 knockdown (KD), although inhibition of MDA5 did not modify PICinduced apoptosis. In contrast, MDA5 silencing decreased PIC-induced cytokine and chemokine expression, although inhibition of PTPN2 induced minor or no changes in these inflammatory mediators. These findings indicate that changes in MDA5 and PTPN2 expression modify b-cell responses to dsRNA. MDA5 regulates inflammatory signals, whereas PTPN2 may function as a defence mechanism against pro-apoptotic signals generated by dsRNA. These two candidate genes for T1D may thus modulate b-cell apoptosis and/or local release of inflammatory mediators in the course of a viral infection by acting, at least in part, at the pancreatic b-cell level.
Diabetologia, 1999
Type I (insulin-dependent) diabetes mellitus is an autoimmune disease, where T-cells and macrophages invade the islets of Langerhans and lead to specific destruction of pancreatic beta cells . Cytokines such as interleukin-1b (IL-1b) and interferon-g are released by these infiltrating mononuclear cells and can contribute to beta-cell death . Recent observations indicate that genes and proteins activated by the beta cells are crucial to determine whether these cells will survive or undergo apoptosis when faced with immune-mediated cell damage [4±6]. We and others have previously adopted the ªcandidate geneº approach to characterize beta-cell responses to immune-mediated assaults and observed that cytokines induce the expression of different genes in rodent and human pancreatic islets, encoding both proteins involved in beta-cell damage and Diabetologia (1999) 42: 1199±1203
Regulation and function of the cytosolic viral RNA sensor RIG-I in pancreatic beta cells
Biochimica et biophysica acta, 2009
Enteroviral infections are associated with type I diabetes. The mechanisms by which viruses or viral products such as double-stranded RNA (dsRNA) affect pancreatic beta cell function and survival remain unclear. We have shown that extracellular dsRNA induces beta cell death via Toll-like receptor-3 (TLR3) signaling whereas cytosolic dsRNA triggers the production of type I interferons and apoptosis via a TLR3-independent process. We presently examined expression of the intracellular viral RNA sensors, the RNA helicases RIG-I and MDA5, and documented the functionality of RIG-I in pancreatic beta cells. FACS-purified rat beta cells and islet cells from wild-type or TLR3 −/− mice were cultured with or without the RIG-I-specific ligand 5′-triphosphate single-stranded RNA (5′triP-ssRNA), the synthetic dsRNA polyI:C (PIC) or 5′OH-ssRNA (negative control); the RNA compounds were added in the medium or transfected in the cells using lipofectamine. RIG-I and MDA5 expression were determined by real-time RT-PCR. NF-κB and IFN-β promoter activation were studied in the presence or absence of a dominant-negative form of RIG-I (DN-RIG-I). Both extracellular (PICex) and intracellular (PICin) PIC increased expression of RIG-I and MDA5 in pancreatic beta cells. TLR3 deletion abolished PICex-induced up-regulation of the helicases in beta cells but not in dendritic cells. PICin-induced NF-κB and IFN-β promoter activation were prevented by the DN-RIG-I. The RIG-I-specific ligand 5′triP-ssRNA induced IFN-β promoter activation and beta cell apoptosis. Our results suggest that the RIG-I pathway is present and active in beta cells and could contribute to the induction of insulitis by viral RNA intermediates. j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a m c r
PLoS Pathogens, 2011
The rise in type 1 diabetes (T1D) incidence in recent decades is probably related to modifications in environmental factors. Viruses are among the putative environmental triggers of T1D. The mechanisms regulating beta cell responses to viruses, however, remain to be defined. We have presently clarified the signaling pathways leading to beta cell apoptosis following exposure to the viral mimetic double-stranded RNA (dsRNA) and a diabetogenic enterovirus (Coxsackievirus B5). Internal dsRNA induces cell death via the intrinsic mitochondrial pathway. In this process, activation of the dsRNA-dependent protein kinase (PKR) promotes eIF2a phosphorylation and protein synthesis inhibition, leading to downregulation of the antiapoptotic Bcl-2 protein myeloid cell leukemia sequence 1 (Mcl-1). Mcl-1 decrease results in the release of the BH3-only protein Bim, which activates the mitochondrial pathway of apoptosis. Indeed, Bim knockdown prevented both dsRNA-and Coxsackievirus B5-induced beta cell death, and counteracted the proapoptotic effects of Mcl-1 silencing. These observations indicate that the balance between Mcl-1 and Bim is a key factor regulating beta cell survival during diabetogenic viral infections.
Endocrinology, 2001
Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by progressive destruction of insulin-producing pancreatic -cells. Both viral infections and the cytokines interleukin-1 (IL-1) and interferon-␥ (IFN-␥) have been suggested as potential mediators of -cell death in early T1DM. We presently investigated whether the viral replicative intermediate double stranded RNA [here used as synthetic polyinosinic-polycytidylic acid (PIC)] modifies the effects of IL-1 and IFN-␥ on gene expression and viability of rat pancreatic -cells. For this purpose, fluorescence-activated cell sorting-purified rat -cells were exposed for 6-16 h (study of gene expression by RT-PCR) or 6-9 days (study of viability by nuclear dyes) to PIC and/or IL-1 and IFN-␥. PIC increased the expression of Fas and Mn superoxide dismutase messenger RNAs by 5-to 10-fold. IL-1 and a combination of PIC and IFN-␥ (but not PIC or IFN-␥ alone) induced expression of inducible nitric oxide (NO) synthase (iNOS) and consequent NO production. Induction of iNOS expression by PIC and
Involvement of long non-coding RNAs in beta cell failure at the onset of type 1 diabetes in NOD mice
Diabetologia, 2015
Aims/hypothesis Exposure of pancreatic beta cells to cytokines released by islet-infiltrating immune cells induces alterations in gene expression, leading to impaired insulin secretion and apoptosis in the initial phases of type 1 diabetes. Long non-coding RNAs (lncRNAs) are a new class of transcripts participating in the development of many diseases. As little is known about their role in insulin-secreting cells, this study aimed to evaluate their contribution to beta cell dysfunction. Methods The expression of lncRNAs was determined by microarray in the MIN6 beta cell line exposed to proinflammatory cytokines. The changes induced by cytokines were further assessed by real-time PCR in islets of control and NOD mice. The involvement of selected lncRNAs modified by cytokines was assessed after their overexpression in MIN6 cells and primary islet cells. Results MIN6 cells were found to express a large number of lncRNAs, many of which were modified by cytokine treatment. The changes in the level of selected lncRNAs were confirmed in mouse islets and an increase in these lncRNAs was also seen in prediabetic NOD mice. Overexpression of these lncRNAs in MIN6 and mouse islet cells, either alone or in combination with cytokines, favoured beta cell apoptosis without affecting insulin production or secretion. Furthermore, overexpression of lncRNA-1 promoted nuclear translocation of nuclear factor of κ light polypeptide gene enhancer in B cells 1 (NF-κB). Conclusions/interpretation Our study shows that lncRNAs are modulated during the development of type 1 diabetes in NOD mice, and that their overexpression sensitises beta cells to apoptosis, probably contributing to their failure during the initial phases of the disease.
Aims/hypothesis: Exposure of pancreatic beta-cells to cytokines released by islet-infiltrating immune cells induces alterations in gene expression, leading to impaired insulin secretion and apoptosis in the initial phases of Type 1 diabetes. Long non-coding RNAs (lncRNAs) are a new class of transcripts participating in the development of many diseases. Since little is known about their role in insulin-secreting cells, this study aimed at evaluating their contribution to beta-cell dysfunction. Methods: The expression of lncRNAs was determined by microarray in the MIN6 beta-cell line exposed to proinflammatory cytokines. The changes induced by cytokines were further assessed by real-time PCR in islets of control and NOD mice. The involvement of selected lncRNAs modified by cytokines was assessed after their overexpression in MIN6 cells and primary islet cells. Results: MIN6 cells were found to express a large number of lncRNAs, many of which were modified by cytokine treatment. The changes in the level of selected lncRNAs were confirmed in mouse islets and an increase of these lncRNAs was also seen in pre-diabetic NOD mice. Overexpression of these lncRNAs in MIN6 and mouse islet cells, either alone or in combination with cytokines, favored beta-cell apoptosis without affecting insulin production or secretion. Furthermore, overexpression of lncRNA-1 promoted nuclear translocation of NF-B. Conclusions/interpretations: Our study shows that lncRNAs are modulated during the development of type 1 diabetes in NOD mice, and that their overexpression sensitizes beta cells to apoptosis, likely contributing to their failure during the initial phases of the disease.
Journal of Biological Chemistry, 2005
Viral infections and local production of cytokines probably contribute to the pathogenesis of Type 1 diabetes. The viral replicative intermediate double-stranded RNA (dsRNA, tested in the form of polyinosinic-polycytidylic acid, PIC), in combination with the cytokine interferon-␥ (IFN-␥), triggers -cell apoptosis. We have previously observed by microarray analysis that PIC induces expression of several mRNAs encoding for genes downstream of Toll-like receptor 3 (TLR3) signaling pathway. In this report, we show that exposure of -cells to dsRNA in combination with IFN-␣,-, or-␥ significantly increases apoptosis. Moreover, dsRNA induces TLR3 mRNA expression and activates NF-B and the IFN- promoter in a TRIF-dependent manner. dsRNA also induces an early (1 h) and sustained increase in IFN- mRNA expression, and blocking IFN- with a specific antibody partially prevents PIC plus IFN-␥-induced -cell death. On the other hand, dsRNA plus IFN-␥ does not induce apoptosis in INS-1E cells, and expression of TLR3 and type I IFNs mRNAs is not detected in these cells. Of note, disruption of the STAT-1 signaling pathway protects -cells against dsRNA plus IFN-␥-induced -cell apoptosis. This study suggests that dsRNA plus IFN-␥ triggers -cell apoptosis by two complementary pathways, namely TLR3-TRIF-NF-B and STAT-1.
The Journal of Immunology, 2005
Coxsackievirus (CV) is an important human pathogen that has been linked to the development of autoimmunity. An intact pancreatic β cell IFN response is critical for islet cell survival and protection from type 1 diabetes following CV infection. In this study, we show that IFNs trigger an antiviral state in β cells by inducing the expression of proteins involved in intracellular antiviral defense. Specifically, we demonstrate that 2′,5′-oligoadenylate synthetases (2-5AS), RNase L, and dsRNA-dependent protein kinase (PKR) are expressed by pancreatic islet cells and that IFNs (IFN-α and IFN-γ) increase the expression of 2-5AS and PKR, but not RNase L. Moreover, our in vitro studies uncovered that these pathways play important roles in providing unique and complementary antiviral activities that critically regulate the outcome of CV infection. The 2-5AS/RNase L pathway was critical for IFN-α-mediated islet cell resistance from CV serotype B4 (CVB4) infection and replication, whereas an ...
Diabetologia, 2018
Aims/hypothesis The initial stages of type 1 diabetes are characterised by an aberrant islet inflammation that is in part regulated by the interaction between type 1 diabetes susceptibility genes and environmental factors. Chromosome 16p13 is associated with type 1 diabetes and CLEC16A is thought to be the aetiological gene in the region. Recent gene expression analysis has, however, indicated that SNPs in CLEC16A modulate the expression of a neighbouring gene with unknown function named DEXI, encoding dexamethasone-induced protein (DEXI). We therefore evaluated the role of DEXI in beta cell responses to 'danger signals' and determined the mechanisms involved. Methods Functional studies based on silencing or overexpression of DEXI were performed in rat and human pancreatic beta cells. Beta cell inflammation and apoptosis, driven by a synthetic viral double-stranded RNA, were evaluated by real-time PCR, western blotting and luciferase assays. Results DEXI-silenced beta cells exposed to a synthetic double-stranded RNA (polyinosinic:polycytidylic acid [PIC], a byproduct of viral replication) showed reduced activation of signal transducer and activator of transcription (STAT) 1 and lower production of proinflammatory chemokines that was preceded by a reduction in IFNβ levels. Exposure to PIC increased chromatin-bound DEXI and IFNβ promoter activity. This effect on IFNβ promoter was inhibited in DEXI-silenced beta cells, suggesting that DEXI is implicated in the regulation of IFNβ transcription. In a mirror image of knockdown experiments, DEXI overexpression led to increased levels of STAT1 and proinflammatory chemokines. Conclusions/interpretation These observations support DEXI as the aetiological gene in the type 1 diabetes-associated 16p13 genomic region, and provide the first indication of a link between this candidate gene and the regulation of local antiviral immune responses in beta cells. Moreover, our results provide initial information on the function of DEXI.
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Molecular & Cellular Proteomics, 2007
Cytokines released by islet-infiltrating immune cells play a crucial role in -cell dysfunction and apoptotic cell death in the pathogenesis of type 1 diabetes and after islet transplantation. RNA studies revealed complex pathways of genes being activated or suppressed during this -cell attack. The aim of the present study was to analyze protein changes in insulin-producing INS-1E cells exposed to inflammatory cytokines in vitro using two-dimensional DIGE. Within two different pH ranges we observed 2214 ؎ 164 (pH 4 -7) and 1641 ؎ 73 (pH 6 -9) spots. Analysis at three different time points (1, 4, and 24 h of cytokine exposure) revealed that the major changes were taking place only after 24 h. At this time point 158 proteins were altered in expression (4.1%, n ؍ 4, p < 0.01) by a combination of interleukin-1 and interferon-␥, whereas only 42 and 23 proteins were altered by either of the cytokines alone, giving rise to 199 distinct differentially expressed spots. Identification of 141 of these by MALDI-TOF/TOF revealed proteins playing a role in insulin secretion, cytoskeleton organization, and protein and RNA metabolism as well as proteins associated with endoplasmic reticulum and oxidative stress/defense. We investigated the interactions of these proteins and discovered a significant interaction network (p < 1.27e؊05) containing 42 of the identified proteins. This network analysis suggests that proteins of different pathways act coordinately in a -cell dysfunction/apoptotic -cell death interactome. In addition the data suggest a central role for chaperones and proteins playing a role in RNA metabolism. As many of these identified proteins are regulated at the protein level or undergo post-translational modifications, a proteomics approach, as performed in this study, is required to provide adequate insight into the mechanisms leading to -cell dysfunction and apoptosis. The present findings may open new avenues for the understanding and prevention of -cell loss in type 1 diabetes. Molecular & Cellular Proteomics 6:2180 -2199, 2007.
Arquivos Brasileiros de Endocrinologia & Metabologia, 2013
Type 1 diabetes mellitus (T1DM) is a chronic, progressive, autoimmune disease characterized by metabolic decompensation frequently leading to dehydration and ketoacidosis. Viral pathogens seem to play a major role in triggering the autoimmune destruction that leads to the development of T1DM. Among several viral strains investigated so far, enteroviruses have been consistently associated with T1DM in humans. One of the mediators of viral damage is the double-stranded RNA (dsRNA) generated during replication and transcription of viral RNA and DNA. The IFIH1 gene encodes a cytoplasmic receptor of the pattern-recognition receptors (PRRs) family that recognizes dsRNA, playing a role in the innate immune response triggered by viral infection. Binding of dsRNA to this PRR triggers the release of proinflammatory cytokines, such as interferons (IFNs), which exhibit potent antiviral activity, protecting uninfected cells and inducing apoptosis of infected cells. The IFIH1 gene appears to play a major role in the development of some autoimmune diseases, and it is, therefore, a candidate gene for T1DM. Within this context, the objective of the present review was to address the role of IFIH1 in the development of T1DM. Arq Bras Endocrinol Metab. 2013;57(9):667-76
Cytokines activate genes of the endocytotic pathway in insulin-producing RINm5F cells
Diabetologia, 2004
Aims/hypothesis. Cytokines are important humoral mediators of beta cell destruction in autoimmune diabetes. The aim of this study was to identify novel cytokine-induced genes in insulin-producing RINm5F cells, which may contribute to beta cell death or survival. Methods. A global gene expression profile in cytokine-exposed insulin-producing RINm5F cells was achieved by automated restriction fragment differential display PCR. The expression of selected candidate genes was confirmed by real-time RT-PCR analysis. Results. Exposure of RINm5F cells to IL-1β or to a cytokine mixture (IL-1β, TNF-α, IFN-γ) for 6 h resulted in the differential expression of a functional gene cluster. Apart from the well-known up-regulation of the cytokine-responsive genes iNOS, NF-κB, MnSOD and Hsp70, several genes that belong to the functional cluster of the endocytotic pathway were identified. These endocytotic genes comprised: clathrin, megalin, synaptotagmin and calcineurin, which were up-regulated by IL-1β or the cytokine mixture.
Proceedings of the National Academy of Sciences, 2005
We have previously described rat insulinoma INS-1-derived cell lines with robust or poor glucose-stimulated insulin secretion (GSIS). In the current study, we have further resolved these lines into three classes: class 1, glucose-unresponsive͞glucagon-expressing; class 2, glucose-unresponsive͞glucagon-negative; and class 3, glucose-responsive͞glucagon-negative. The transcription factor Nkx2.2 was expressed with relative abundance of 3.3, 1.0, and 1.0 in class 1, class 2, and class 3 cells, respectively, whereas Nkx6.1 expression had the opposite trend: 1.0, 2.6, and 6.4 in class 1, class 2, and class 3 cells, respectively. In class 1 cells, overexpressed Nkx6.1 suppressed glucagon expression but did not affect the levels of several other prominent beta cell transcription factors.
Immunopathology of the human pancreas in type-I diabetes
Seminars in Immunopathology, 2011
Type 1 diabetes is a chronic autoimmune disease characterised by the selective destruction of pancreatic beta (β) cells. The understanding of the aetiology of this disease has increased dramatically in recent years by the study of tissue recovered from patients, from analysis of the responses of isolated islet and β-cells in tissue culture and via the use of animal models. However, knowledge of the immunopathology of type 1 diabetes in humans is still relatively deficient due largely to the difficulty of accessing appropriate samples. Here we review the state of current knowledge in relation to the histopathological features of the disease in humans. We focus specifically on recentonset type 1 diabetes cases since in such patients, evidence of the ongoing disease process is still present. We chart the progression of the disease by describing the characteristic features of the pancreas, consider the sequence of immune cell infiltration and discuss the abnormalities of MHC antigen expression. The possibility that these changes might derive from a persistent enteroviral infection of the islet beta cells is examined. Keywords Insulitis . Beta-cell death . Enterovirus . Recent-onset type 1 diabetes . MHC . Chemokine . This article is published as part of the Special Issue on Immunopathology of the pancreas in type 1 diabetes.
In silico identification of NF-kappaB-regulated genes in pancreatic beta-cells
BMC bioinformatics, 2007
Pancreatic beta-cells are the target of an autoimmune attack in type 1 diabetes mellitus (T1DM). This is mediated in part by cytokines, such as interleukin (IL)-1beta and interferon (IFN)-gamma. These cytokines modify the expression of hundreds of genes, leading to beta-cell dysfunction and death by apoptosis. Several of these cytokine-induced genes are potentially regulated by the IL-1beta-activated transcription factor (TF) nuclear factor (NF)-kappaB, and previous studies by our group have shown that cytokine-induced NF-kappaB activation is pro-apoptotic in beta-cells. To identify NF-kappaB-regulated gene networks in beta-cells we presently used a discriminant analysis-based approach to predict NF-kappaB responding genes on the basis of putative regulatory elements. The performance of linear and quadratic discriminant analysis (LDA, QDA) in identifying NF-kappaB-responding genes was examined on a dataset of 240 positive and negative examples of NF-kappaB regulation, using stratifi...
Stunned Silence: Gene Expression Programs in Human Cells Infected with Monkeypox or Vaccinia Virus
PLoS ONE, 2011
Poxviruses use an arsenal of molecular weapons to evade detection and disarm host immune responses. We used DNA microarrays to investigate the gene expression responses to infection by monkeypox virus (MPV), an emerging human pathogen, and Vaccinia virus (VAC), a widely used model and vaccine organism, in primary human macrophages, primary human fibroblasts and HeLa cells. Even as the overwhelmingly infected cells approached their demise, with extensive cytopathic changes, their gene expression programs appeared almost oblivious to poxvirus infection. Although killed (gamma-irradiated) MPV potently induced a transcriptional program characteristic of the interferon response, no such response was observed during infection with either live MPV or VAC. Moreover, while the gene expression response of infected cells to stimulation with ionomycin plus phorbol 12-myristate 13-acetate (PMA), or poly (I-C) was largely unimpaired by infection with MPV, a cluster of pro-inflammatory genes were a notable exception. Poly(I-C) induction of genes involved in alerting the innate immune system to the infectious threat, including TNF-alpha, IL-1 alpha and beta, CCL5 and IL-6, were suppressed by infection with live MPV. Thus, MPV selectively inhibits expression of genes with critical roles in cell-signaling pathways that activate innate immune responses, as part of its strategy for stealthy infection.
PLoS ONE, 2013
Autoimmune destruction of insulin producing pancreatic b-cells is the hallmark of type I diabetes. One of the key molecules implicated in the disease onset is the immunoproteasome, a protease with multiple proteolytic sites that collaborates with the constitutive 19S and the inducible 11S (PA28) activators to produce immunogenic peptides for presentation by MHC class I molecules. Despite its importance, little is known about the function and regulation of the immunoproteasome in pancreatic b-cells. Of special interest to immunoproteasome activation in b-cells are the effects of IFNb, a type I IFN secreted by virus-infected cells and implicated in type I diabetes onset, compared to IFNc, the classic immunoproteasome inducer secreted by cells of the immune system. By qPCR analysis, we show that mouse insulinoma MIN6 cells and mouse islets accumulate the immune proteolytic b1 i , b2 i and b5 i , and 11S mRNAs upon exposure to IFNb or IFNc. Higher concentrations of IFNb than IFNc are needed for similar expression, but in each case the expression is transient, with maximal mRNA accumulation in 12 hours, and depends primarily on Interferon Regulatory Factor 1. IFNs do not alter expression of regular proteasome genes, and in the time frame of IFNb-mediated response, the immune and regular proteolytic subunits co-exist in the 20S particles. In cell extracts with ATP, these particles have normal peptidase activities and degrade polyubiquitinated proteins with rates typical of the regular proteasome, implicating normal regulation by the 19S activator. However, ATP depletion rapidly stimulates the catalytic rates in a manner consistent with levels of the 11S activator. These findings suggest that stochastic combination of regular and immune proteolytic subunits may increase the probability with which unique immunogenic peptides are produced in pancreatic b-cells exposed to IFNb, but primarily in cells with reduced ATP levels that stimulate the 11S participation in immunoproteasome function.
BMC bioinformatics, 2005
Type 1 diabetes mellitus (T1DM) is a autoimmune disease caused by a long-term negative balance between immune-mediated beta-cell damage and beta-cell repair/regeneration. Following immune-mediated damage the beta-cell fate depends on several genes up- or down-regulated in parallel and/or sequentially. Based on the information obtained by the analysis of several microarray experiments of beta-cells exposed to pro-apoptotic conditions (e.g. double stranded RNA (dsRNA) and cytokines), we have developed a spotted rat oligonucleotide microarray, the APOCHIP, containing 60-mer probes for 574 genes selected for the study of beta-cell apoptosis. The APOCHIP was validated by a combination of approaches. First we performed an internal validation of the spotted probes based on a weighted linear regression model using dilution series experiments. Second we profiled expression measurements in ten dissimilar rat RNA samples for 515 genes that were represented on both the spotted oligonucleotide c...