Untargeted Metabolomics Approach Correlated Enniatin B Mycotoxin Presence in Cereals with Kashin-Beck Disease Endemic Regions of China.

Kashin-Beck disease (KBD) is a multifactorial endemic disease that only occurs in specific Asian areas. Mycotoxin contamination, especially from the Fusarium spp., has been considered as one of the environmental risk factors that could provoke chondrocyte and cartilage damage. This study aimed to investigate whether new mycotoxins could be identified in KBD-endemic regions as a potential KBD risk factor. This was investigated on 292 barley samples collected in Tibet during 2009-2016 and 19 wheat samples collected in Inner Mongolia in 2006, as control, from KBD-endemic and non-endemic areas. The LC-HRMS(/MS) data, obtained by a general mycotoxin extraction technic, were interpreted by both untargeted metabolomics and molecular networks, allowing us to identify a discriminating compound, enniatin B, a mycotoxin produced by some Fusarium spp. The presence of Fusarium spp. DNA was detected in KBD-endemic area barley samples. Further studies are required to investigate the role of this mycotoxin in KBD development in vivo.

Human carbamylome description identifies carbamylated α2-macroglobulin and hemopexin as two novel autoantigens in early rheumatoid arthritis.

OBJECTIVES: Anti-carbamylated protein antibodies (anti-CarPAs) are present in RA sera and have been associated with erosive disease. The exact targets of anti-CarPAs in vivo are currently not well known; we used a proteomic approach on serum and SF of RA patients to assess the human carbamylome and to identify carbamylated autoantigens as potential biomarkers in early RA. METHODS: Mass spectrometry was performed on SF and serum from RA patients. Carbamylated proteins present in both sample types were selected as candidate autoantigens for the establishment of ELISAs. A cohort of early RA patients was tested for positivity for specific anti-CarPAs. RESULTS: Eleven novel carbamylated proteins were identified, and five were selected as potential autoantigens for detection of anti-CarPAs. Among them, antibodies against carbamylated hemopexin (anti-CaHPX) and alpha-2-macroglobulin (anti-CaA2M) showed comparable diagnostic value to the established carbamylated foetal calf serum-based ELISA. A cohort of 189 early RA patients was studied. The combination of these new biomarkers with anti-citrullinated protein antibodies and RF identified 89% of early RA patients in our cohort. There was little correlation between the tested biomarkers, and each one of the tested antigens could identify a different subset of seronegative RA patients. Anti-CaA2M positivity showed clinical potential, being associated with higher disease disability. CONCLUSION: We highlight the detection of novel carbamylated autoantigens in vivo using a combined proteomics approach in the SF and serum of RA patients. Anti-CaHPX and anti-CaA2M are promising clinical biomarkers, especially in seronegative RA.

Serum calprotectin (S100A8/A9): a promising biomarker in diagnosis and follow-up in different subgroups of juvenile idiopathic arthritis.

INTRODUCTION: In the management of juvenile idiopathic arthritis (JIA), there is a lack of diagnostic and prognostic biomarkers. This study assesses the use of serum calprotectin (sCal) as a marker to monitor disease activity, and as a classification and prognosis tool of response to treatment or risk of flares in patients with JIA. METHODS: Eighty-one patients with JIA from the CAP48 multicentric cohort were included in this study, as well as 11 non-paediatric healthy controls. An ELISA method was used to quantify sCal with a commercial kit. RESULTS: Patients with an active disease compared with healthy controls and with patients with inactive disease showed an eightfold and a twofold increased level of sCal, respectively. sCal was found to be correlated with the C-reactive protein (CRP) and even more strongly with the erythrocyte sedimentation rate. Evolution of DAS28 scores correlated well with evolution of sCal, as opposed to evolution of CRP. With regard to CRP, sCal could differentiate forms with active oligoarthritis from polyarthritis and systemic forms. However, sCal brought an added value compared with the CRP as a prognosis marker. Indeed, patients with active disease and reaching minimal disease activity (according to Juvenile Arthritis Disease Activity Score) at 6 months following the test had higher sCal levels, while patients with inactive disease had higher sCal levels if a flare was observed up to 3-9 months following the test. CONCLUSIONS: This study confirms the potential uses of sCal as a biomarker in the diagnosis and follow-up of JIA.

Role of Scaffolds, Subchondral, Intra-Articular Injections of Fresh Autologous Bone Marrow Concentrate Regenerative Cells in Treating Human Knee Cartilage Lesions: Different Approaches and Different Results.

The value of bone marrow aspirate concentrates for treatment of human knee cartilage lesions is unclear. Most of the studies were performed with intra-articular injections. However, subchondral bone plays an important role in the progression of osteoarthritis. We investigated by a literature review whether joint, subchondral bone, or/and scaffolds implantation of fresh autologous bone marrow aspirate concentrated (BMAC) containing mesenchymal stem cells (MSCs) would improve osteoarthritis (OA). There is in vivo evidence that suggests that all these different approaches (intra-articular injections, subchondral implantation, scaffolds loaded with BMAC) can improve the patient. This review analyzes the evidence for each different approach to treat OA. We found that the use of intra-articular injections resulted in a significant relief of pain symptoms in the short term and was maintained in 12 months. However, the clinical trials indicate that the application of autologous bone marrow concentrates in combination with scaffolds or in injection in the subchondral bone was superior to intra-articular injection for long-term results. The tendency of MSCs to differentiate into fibrocartilage affecting the outcome was a common issue faced by all the studies when biopsies were performed, except for scaffolds implantation in which some hyaline cartilage was found. The review suggests also that both implantation of subchondral BMAC and scaffolds loaded with BMAC could reduce the need for further surgery.

Induction of Stearoyl-CoA 9-Desaturase 1 Protects Human Mesenchymal Stromal Cells Against Palmitic Acid-Induced Lipotoxicity and Inflammation.

In bone diseases such as osteonecrosis and osteoporosis, a shift toward a preferential differentiation of mesenchymal stromal cells (MSC) into adipocytes at the expense of the osteoblastic lineage is described, leading to excessive accumulation of adipocytes in the bone marrow of the patients. The influence of cytokines and adipokines secreted by adipocytes on skeletal health is already well-documented but the impact of free fatty acids release on bone cell biology and viability is an emerging concept. We have previously demonstrated that the saturated fatty acid (SFA) palmitate (Palm) is cytotoxic for human MSC (hMSC) and osteoblasts whereas oleate (Ole), a monounsaturated fatty acid (MUFA), has no toxic effect. Moreover, Ole protects cells against lipotoxicity. Our observations led us to propose that the toxicity of the SFA is not correlated to its intracellular accumulation but could rather be related to the intracellular SFA/MUFA ratio, which finally determines the toxic effect of SFA. Therefore, in the present study, we have investigated the potential protective role of the enzyme stearoyl-CoA 9-desaturase 1 (SCD1) against the deleterious effects of Palm. SCD1 is an enzyme responsible for desaturation of SFA to MUFA; its activation could therefore lead to modifications of the intracellular SFA/MUFA ratio. In the present study, we showed that hMSC express SCD1 and liver X receptors (LXRs), transcription factors regulating SCD1 expression. Human MSC treatment with a LXRs agonist triggered SCD1 expression and drastically reduced Palm-induced cell mortality, caspases 3/7 activation, endoplasmic reticulum stress and inflammation. We also observed that, in the presence of Palm, the LXRs agonist provoked lipid droplets formation, augmented the total cellular neutral lipid content but decreased the SFA/MUFA ratio when compared to Palm treatment alone. Addition of an inhibitor of SCD1 activity abrogated the positive effects of the LXRs agonist, suggesting that SCD1 could play a key role in protecting hMSC against lipotoxicity.

Effects of press-fit biphasic (collagen and HA/ßTCP) scaffold with cell-based therapy on cartilage and subchondral bone repair knee defect in rabbits.

INTRODUCTION: Human spontaneous osteonecrosis of the knee (SPONK) is still challenging as the current treatments do not allow the production of hyaline cartilage tissue. The aim of the present study was to explore the therapeutic potential of cartilage regeneration using a new biphasic scaffold (type I collagen/hydroxyapatite) previously loaded or not with concentrated bone marrow cells. MATERIAL AND METHODS: Female rabbits were operated of one knee to create articular lesions of the trochlea (three holes of 4 × 4mm). The holes were left empty in the control group or were filled with the scaffold alone or the scaffold previously loaded with concentrated bone marrow cells. After two months, rabbits were sacrificed and the structure of the newly formed tissues were evaluated by macroscopic, MRI, and immunohistochemistry analyses. RESULTS: Macroscopic and MRI evaluation of the knees did not show differences between the three groups (p > 0.05). However, histological analysis demonstrated that a higher O'Driscoll score was obtained in the two groups treated with the scaffold, as compared to the control group (p < 0.05). The number of cells in treated area was higher in scaffold groups compared to the control group (p < 0.05). There was no difference for intensity of collagen type II between the groups (p > 0.05) but subchondral bone repair was significantly thicker in scaffold-treated groups than in the control group (1 mm for the control group vs 2.1 and 2.6 mm for scaffold groups). Furthermore, we observed that scaffolds previously loaded with concentrated bone marrow were more reabsorbed (p < 0.05). CONCLUSION: The use of a biphasic scaffold previously loaded with concentrated bone marrow significantly improves cartilage lesion healing.

Non traumatic osteonecrosis of the femoral head is associated with low bone mass.

OBJECTIVE: Osteoporosis (OP) and osteonecrosis of the femoral head (ONFH) share common clinical and pathophysiological features we sought to determine whether ONFH was associated with an increased prevalence of OP and whether the increased prevalence of OP was related to the stage of ONFH at diagnosis. METHODS: We included 243 patients with ONFH and 399 age and sex-matched healthy controls. Data was gathered including demography, risk factors, ARCO staging of ONFH and bone mineral density (BMD). RESULTS: Overall, BMD (defined by the T-score) was significantly lower in the ONFH group at both the femoral head (-0.96±1.11) and the lumbar spine (-1.22±1.47) compared to the control group (-0.55±0.97 and -0.73±1.31) (p<0.01). The ONFH group depicted a significantly higher proportion of osteopenia (50.39% vs 40.87%, p=0.027) and of OP (18.78% vs 7.33%, p<0.001) relative to the control group. Stage 1 and 2 ONFH patients (53.86%, p=0.0203; OR=1.54 (95% CI: [1.04; 2.29])) were at a higher risk of osteopenia than the control group (40.88%), but not stages 3 or 4 (48.47%, p=0.2569; OR=1.27 (95% CI: [0.78; 2.06]). Patients with stage 3 or 4 ONFH (25.31%, p<0.001; OR=3.93 (95% CI: [1.63; 10.96])) were at a higher risk of osteoporosis than patients in the stage 1 and 2 ONFH (7.24%), and compared to the control group (7.33%, adj. p-value<0.001; OR=4.89 (95% CI: [2.77; 8.76]). CONCLUSIONS: Non-traumatic osteonecrosis of the femoral heads is associated with low bone mineral density. This study showed that fractural stages ONFH were associated with a 5-fold risk of osteoporosis.

Osteonecrosis of the Femoral Head: Lipotoxicity Exacerbation in MSC and Modifications of the Bone Marrow Fluid.

Osteonecrosis of the femoral head (ON) is a multifactorial bone disease that can evolve to a progressive destruction of the hip joint. Different pathogenic processes have been proposed, among them, an increase of bone marrow (BM) fat resulting from adipocyte accumulation. Marrow adipocytes are active BM residents that influence the microenvironment by releasing cytokines, adipokines, and free fatty acids (FA). We explored the impact of palmitate (Palm) and oleate on function and survival of BM-derived mesenchymal stromal cells (MSC) of osteonecrotic patients (ONMSC) and healthy volunteers. Moreover, we analyzed the FA profile of the serum and the BM supernatant fluid (BMSF). We demonstrated that exposure to the saturated FA Palm favored MSC differentiation through the adipogenic lineage at the expense of the osteoblastic phenotype. Moreover, adipogenesis was intensified in ONMSC. The susceptibility to Palm toxicity was aggravated in ONMSC concomitantly with a greater activation of the proapoptotic extracellular signal-regulated kinase pathway. Moreover, cellular mechanisms implicated in the protection against lipotoxicity, such as stearoyl-coenzyme A desaturase 1 and carnitine palmitoyl transferase 1 expression, were dysregulated in ONMSC. Palm-induced interleukin (IL)-6 and IL-8 secretion was also exacerbated in ONMSC. Our results established that, in the serum, the FA profiles were comparable in ON and healthy subjects. However, both the concentrations and the FA composition were modified in the BMSF of ON patients, highlighting a drastic change of the BM microenvironment in ON patients. Altogether, our work suggests that marrow adipocyte enlargement could affect the process of bone remodeling and, therefore, play a role in the pathogenesis of ON.

Adenosine triphosphate prevents serum deprivation-induced apoptosis in human mesenchymal stem cells via activation of the MAPK signaling pathways.

Human mesenchymal stem cells (hMSC) are multipotent cells derived from various sources including adipose and placental tissues as well as bone marrow. Owing to their regenerative and immunomodulatory properties, their use as a potential therapeutic tool is being extensively tested. However, one of the major hurdles in using cell-based therapy is the use of fetal bovine serum that can trigger immune responses, viral and prion diseases. The development of a culture medium devoid of serum while preserving cell viability is therefore a major challenge. In this study, we demonstrated that adenosine triphosphate (ATP) restrained serum deprivation-induced cell death in hMSC by preventing caspases 3/7 activation and modulating ERK1/2 and p38 MAPK signaling pathways. We also showed that serum deprivation conditions triggered dephosphorylation of the proapoptotic protein Bad leading to cell death. Adjunction of ATP restored the phosphorylation state of Bad. Furthermore, ATP significantly modulated the expression of proapoptopic and antiapoptotic genes, in favor of an antiapoptotic profile expression. Finally, we established that hMSC released a high amount of ATP in the extracellular medium when cultured in a serum-free medium. Collectively, our results demonstrate that ATP favors hMSC viability in serum deprivation conditions. Moreover, they shed light on the cardinal role of the MAPK pathways, ERK1/2 and p38 MAPK, in promoting hMSC survival.

Potential involvement of the IL-33-ST2 axis in the pathogenesis of primary Sjogren's syndrome.

OBJECTIVES: To investigate the role of the interleukin (IL)-33-ST2 axis in the pathophysiology of primary Sjögren's syndrome (pSS). METHODS: Serum levels of IL-33 and sST2 were determined by ELISA. The expression of IL-33 and ST2 was investigated in salivary glands (SG) by immunohistochemistry. PBMC were isolated and stimulated with IL-33, IL-12 and IL-23 and the cytokine profile response was examined by flow cytometry. Intracellular cytokine detection of IFNγ and IL-17 was performed by flow cytometry. RESULTS: Serum IL-33 and sST2 levels were increased in pSS patients compared with controls and patients with systemic lupus erythematosus. Expression of IL-33 was upregulated in SG with Chisholm scores of 2 and 3 of pSS patients but comparable with controls for SG with Chisholm score of 4. ST2 expression in SG was downregulated in pSS patients. IL-33 at different concentrations did not increase the secretion of pro-inflammatory cytokines but acted synergistically with IL-12 and IL-23 to promote IFNγ production. NK and NKT cells were identified as main producers of IFNγ in vitro and were found in SG of pSS patients. CONCLUSIONS: IL-33 is released in pSS, and acts with IL-12 and IL-23 to favour the secretion of IFNγ by NK and NKT cells.

D-glucose­ and 3-O-methyl-D-glucose-induced upregulation of selected genes in rat hepatocytes and INS1E cells: re­evaluation of the possible role of hexose phosphorylation.

The biochemical events involved in the upregulation of selected glucose­responsive genes by 3­O­methyl­D­glucose (3­MG) remain to be elucidated. The present study mainly aimed to re­evaluate the possible role of 3­MG phosphorylation in the upregulation of the thioredoxin interacting protein (TXNIP) and liver pyruvate kinase (LPK) genes in rat hepatocytes and INS1E cells. TXNIP and LPK transcription was assessed in rat liver and INS1E cells exposed to a rise in D­glucose concentration, 2­deoxy­D­glucose (2­DG), 3­MG and, when required, D­mannoheptulose. The phosphorylation of D­[U­14C]glucose and 3­O­[14C]methyl­D­glucose (14C-labeled 3-MG) was measured in rat liver, INS1E cell and rat pancreatic islet homogenates. The utilization of D­[5­3H]glucose by intact INS1E cells was also measured. In rat hepatocytes, a rise in the D­glucose concentration increased the TXNIP/hypoxanthine­guanine phosphoribosyl transferase (HPRT) and LPK/HPRT ratios, while 2­DG and 3­MG also increased the TXNIP/HPRT ratio, but not the LPK/HPRT ratio. In INS1E cells, the TXNIP/HPRT and LPK/HPRT ratios were increased in response to the addition of D­glucose, 2­DG and 3­MG. Furthermore, D­mannoheptulose abolished the response to D­glucose and 2­DG, but not to 3­MG, in these cells. Liver cell homogenates catalyzed the phosphorylation of 3­MG to a modest extent, whilst INS1E and rat pancreatic islet cell homogenates did not. Moreover, 3­MG marginally decreased D­glucose phosphorylation in INS1E cell homogenates but not in liver cell homogenates. D­[5­3H]glucose utilization by intact INS1E cells was decreased by 2­DG, but not by 3­MG. These findings reinforce the view that the upregulation of the TXNIP and LPK genes induced by 3­MG is not attributable to its phosphorylation or any favorable effect on D­glucose metabolism.

Decreased pool of mesenchymal stem cells is associated with altered chemokines serum levels in atrophic nonunion fractures.

Nonunion fractures can cause severe dysfunction and are often difficult to treat mainly due to a poor understanding of their physiopathology. Although many aspects of impaired fracture healing have been extensively studied, little is known about the cellular and molecular mechanisms leading to atrophic nonunion. Therefore, the aim of the present study was to assess the pools and biological functions of bone marrow-derived mesenchymal stem cells (hMSCs) and circulating endothelial progenitor cells (EPCs) in atrophic nonunion patients compared to healthy subjects, and the systemic levels of growth factors involved in the recruitment, proliferation and differentiation of these cells. In nonunions, the pool of hMSCs was decreased and their proliferation delayed. However, once committed, hMSCs from nonunions were able to proliferate, differentiate into osteoblastic cells and mineralize in vitro as efficiently as hMSCs from healthy subjects. In parallel, we found altered serum levels of chemokines and growth factors involved in the chemotaxis and proliferation of hMSCs such as leptin, interleukin-6 (IL-6) and its soluble receptor, platelet-derived growth factor-BB (PDGF-BB), stem cell factor (SCF) and insulin-like growth factor-1 (IGF-1). Moreover, we showed that the number of EPCs and their regulating growth factors were not affected in nonunion patients. If nonunion is generally attributed to a vascular defect, our results also support a role for a systemic mesenchymal and osteogenic cell pool defect that might be related to alterations in systemic levels of factors implicated in their chemotaxis and proliferation.

Regulation of human papillomavirus type 16 early gene expression in trophoblastic and cervical cells.

We compared the outcome of different cellular and viral factors on the regulation of the HPV-16 early viral gene expression in trophoblastic and cervical cancer cells. A high variability of the long control (LCR) activity was observed, prompting us to evaluate the role of secreted factors in the control of the early gene expression in trophoblastic cell lines. Endogenous progesterone and exogenous dexamethasone were found to activate LCR driven transcriptional activity. Since host cells express HPV early proteins to regulate LCR activity, we investigated the effect of the combined HPV-16 early proteins on the LCR driven transcription and the possible involvement of E2. A physiological level of HPV-16 early proteins expression strongly induced the LCR driven reporter activity. According to mutational analysis, E1 and E2 proteins, indispensable for viral replication, were not involved in LCR extrachromosomal transcriptional regulation. This suggests that E5 and/or E6 and/or E7, consequently, activated viral transcription.

Regulation and function of the cytosolic viral RNA sensor RIG-I in pancreatic beta cells.

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-kappaB and IFN-beta 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-kappaB and IFN-beta promoter activation were prevented by the DN-RIG-I. The RIG-I-specific ligand 5'triP-ssRNA induced IFN-beta 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.

Detailed transcriptome atlas of the pancreatic beta cell.

BACKGROUND: Gene expression patterns provide a detailed view of cellular functions. Comparison of profiles in disease vs normal conditions provides insights into the processes underlying disease progression. However, availability and integration of public gene expression datasets remains a major challenge. The aim of the present study was to explore the transcriptome of pancreatic islets and, based on this information, to prepare a comprehensive and open access inventory of insulin-producing beta cell gene expression, the Beta Cell Gene Atlas (BCGA). METHODS: We performed Massively Parallel Signature Sequencing (MPSS) analysis of human pancreatic islet samples and microarray analyses of purified rat beta cells, alpha cells and INS-1 cells, and compared the information with available array data in the literature. RESULTS: MPSS analysis detected around 7600 mRNA transcripts, of which around a third were of low abundance. We identified 2000 and 1400 transcripts that are enriched/depleted in beta cells compared to alpha cells and INS-1 cells, respectively. Microarray analysis identified around 200 transcription factors that are differentially expressed in either beta or alpha cells. We reanalyzed publicly available gene expression data and integrated these results with the new data from this study to build the BCGA. The BCGA contains basal (untreated conditions) gene expression level estimates in beta cells as well as in different cell types in human, rat and mouse pancreas. Hierarchical clustering of expression profile estimates classify cell types based on species while beta cells were clustered together. CONCLUSION: Our gene atlas is a valuable source for detailed information on the gene expression distribution in beta cells and pancreatic islets along with insulin producing cell lines. The BCGA tool, as well as the data and code used to generate the Atlas are available at the T1Dbase website (T1DBase.org).

Initiation and execution of lipotoxic ER stress in pancreatic beta-cells.

Free fatty acids (FFA) cause apoptosis of pancreatic beta-cells and might contribute to beta-cell loss in type 2 diabetes via the induction of endoplasmic reticulum (ER) stress. We studied here the molecular mechanisms implicated in FFA-induced ER stress initiation and apoptosis in INS-1E cells, FACS-purified primary beta-cells and human islets exposed to oleate and/or palmitate. Treatment with saturated and/or unsaturated FFA led to differential ER stress signaling. Palmitate induced more apoptosis and markedly activated the IRE1, PERK and ATF6 pathways, owing to a sustained depletion of ER Ca(2+) stores, whereas the unsaturated FFA oleate led to milder PERK and IRE1 activation and comparable ATF6 signaling. Non-metabolizable methyl-FFA analogs induced neither ER stress nor beta-cell apoptosis. The FFA-induced ER stress response was not modified by high glucose concentrations, suggesting that ER stress in primary beta-cells is primarily lipotoxic, and not glucolipotoxic. Palmitate, but not oleate, activated JNK. JNK inhibitors reduced palmitate-mediated AP-1 activation and apoptosis. Blocking the transcription factor CHOP delayed palmitate-induced beta-cell apoptosis. In conclusion, saturated FFA induce ER stress via ER Ca(2+) depletion. The IRE1 and resulting JNK activation contribute to beta-cell apoptosis. PERK activation by palmitate also contributes to beta-cell apoptosis via CHOP.

Double-stranded RNA induces pancreatic beta-cell apoptosis by activation of the toll-like receptor 3 and interferon regulatory factor 3 pathways.

OBJECTIVE: Viral infections contribute to the pathogenesis of type 1 diabetes. Viruses, or viral products such as double-stranded RNA (dsRNA), affect pancreatic beta-cell survival and trigger autoimmunity by unknown mechanisms. We presently investigated the mediators and downstream effectors of dsRNA-induced beta-cell death. RESEARCH DESIGN AND METHODS: Primary rat beta-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-kappaB (NF-kappaB) and IRF-3 activation and beta-cell apoptosis. PICin activated NF-kappaB and IRF-3 in a TLR3-independent manner, induced eukaryotic initiation factor 2 alpha phosphorylation, and triggered a massive production of interferon (IFN)-beta. This contributed to beta-cell death, as islet cells from IFNAR1(-/-) or IRF-3(-/-) mice were protected against PICin-induced apoptosis. CONCLUSIONS: PICex and PICin trigger beta-cell apoptosis via the TLR3 pathway or IRF-3 signaling, respectively. Execution of PICin-mediated apoptosis depends on autocrine effects of type I IFNs.

SHIP2 controls PtdIns(3,4,5)P(3) levels and PKB activity in response to oxidative stress.

Reactive oxygen species (ROS) are known to be involved in redox signalling pathways that may contribute to normal cell function as well as disease progression. The tumour suppressor PTEN and the inositol 5-phosphatase SHIP2 are critical enzymes in the control of PtdIns(3,4,5)P(3) level. It has been reported that oxidants, including those produced in cells such as macrophages, can activate downstream signalling via the inactivation of PTEN. The present study evaluates the potential impact of SHIP2 on phosphoinositides in cells exposed to sodium peroxide. We used a model of SHIP2 deficient mouse embryonic fibroblasts (MEFs) stimulated by H(2)O(2): at 15 min, PtdIns(3,4,5)P(3) was markedly increased in SHIP2 -/- cells as compared to +/+ cells. In contrast, no significant increase in PtdIns(3,4)P(2) could be detected at 15 or 120 min incubation of the cells with H(2)O(2) (0.6 mM). PKB activity was also upregulated in SHIP2 -/- cells as compared to +/+ cells in response to H(2)O(2). SHIP2 add back experiments in SHIP2 -/- cells confirm its critical role as a lipid phosphatase in the control of PtdIns(3,4,5)P(3) level in response to H(2)O(2). We conclude that SHIP2 lipid phosphatase activity plays an important role in the metabolism PtdIns(3,4,5)P(3) which is demonstrated in oxygen stressed cells.

Toll-like receptor 3 and STAT-1 contribute to double-stranded RNA+ interferon-gamma-induced apoptosis in primary pancreatic beta-cells.

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-gamma (IFN-gamma), triggers beta-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 beta-cells to dsRNA in combination with IFN-alpha, -beta, or -gamma significantly increases apoptosis. Moreover, dsRNA induces TLR3 mRNA expression and activates NF-kappaB and the IFN-beta promoter in a TRIF-dependent manner. dsRNA also induces an early (1 h) and sustained increase in IFN-beta mRNA expression, and blocking IFN-beta with a specific antibody partially prevents PIC plus IFN-gamma-induced beta-cell death. On the other hand, dsRNA plus IFN-gamma 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 beta-cells against dsRNA plus IFN-gamma-induced beta-cell apoptosis. This study suggests that dsRNA plus IFN-gamma triggers beta-cell apoptosis by two complementary pathways, namely TLR3-TRIF-NF-kappaB and STAT-1.