Double-Stranded RNA Induces Pancreatic β-Cell Apoptosis by Activation of the Toll-Like Receptor 3 and Interferon Regulatory Factor 3 Pathways (original) (raw)
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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.
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.
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.).
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
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.
Global profiling of double stranded RNA and IFN-γ-induced genes in rat pancreatic beta cells
Diabetologia, 2003
Aims/hypothesis Viral infections and local production of IFN- 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- results in beta-cell dysfunction and apoptosis. To elucidate the molecular mechanisms involved
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
Toll-like receptor 3 (TLR3) and the development of type 1 diabetes mellitus
Archives of Endocrinology and Metabolism, 2015
Type 1 diabetes mellitus (T1DM) is a chronic, progressive autoimmune disease characterized by metabolic decompensation often leading to dehydration and ketoacidosis. Viral agents seem to play an important role in triggering the autoimmune destruction that leads to the development of T1DM. Among several viral strains investigated so far, the enterovirus family has been consistently associated with the onset of 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 Toll-like receptor 3 (TLR3) gene codes for an endoplasmic receptor of the pattern-recognition receptors (PRRs) family that recognizes dsRNA, plays an important role in the innate immune response triggered by viral infection. Binding of dsRNA to the TLR3 triggers the release of proinflammatory cytokines, such as interferons, which exhibit potent antiviral action; thus, protecting uninfected cells and inducing apoptosis of infected ones. Therefore, the TLR3 gene is a good candidate for the development of T1DM. Within this context, the objective of the present review was to address the role of the TLR3 gene in the development of T1DM.