APC-independent activation of NK cells by the Toll-like receptor 3 agonist double-stranded RNA

Toll-like receptors (TLRs) play a fundamental role in the recognition of bacteria and viruses. TLR3 is activated by viral dsRNA and polyinosinic-polycytidylic acid (poly(I:C)), a synthetic mimetic of viral RNA. We show that NK cells, known for their capacity to eliminate virally infected cells, express TLR3 and up-regulate TLR3 mRNA upon poly(I:C) stimulation. Treatment of highly purified NK cells with poly(I:C) significantly augments NK cell-mediated cytotoxicity. Poly(I:C) stimulation also leads to up-regulation of activation marker CD69 on NK cells. Furthermore, NK cells respond to poly(I:C) by producing proinflammatory cytokines like IL-6 and IL-8, as well as the antiviral cytokine IFN-gamma. The induction of cytokine production by NK cells was preceded by activation of NF-kappaB. We conclude that the ability of NK cells to directly recognize and respond to viral products is important in mounting effective antiviral responses.     

Toll-like receptor, RIG-I-like receptors and the NLRP3 inflammasome: key modulators of innate immune responses to double-stranded RNA viruses

Double-stranded RNA (dsRNA), the genetic material for many RNA viruses, induces robust host immune responses via pattern recognition receptors, which include Toll-like receptor 3 (TLR3), retinoic acid-inducible gene-I-like receptors (RLRs) and the multi-protein NLRP3 inflammasome complex. The engagement of dsRNA receptors or inflammasome activation by viral dsRNA initiates complex intracellular signaling cascades that play essential roles in inflammation and innate immune responses, as well as the resultant development of adaptive immunity. This review focuses on signaling pathways mediated by TLR3, RLRs and the NLRP3 inflammasome, as well as the potential use of agonists and antagonists that target these pathways to treat disease.     

Both triggering and amplifying pathways contribute to fuel-induced insulin secretion in the absence of sulfonylurea receptor-1 in pancreatic beta-cells

In normal beta-cells glucose induces insulin secretion by activating both a triggering pathway (closure of K(ATP) channels, depolarization, and rise in cytosolic [Ca(2+)](i)) and an amplifying pathway (augmentation of Ca(2+) efficacy on exocytosis). It is unclear if and how nutrients can regulate insulin secretion by beta-cells lacking K(ATP) channels (Sur1 knockout mice). We compared glucose- and amino acid-induced insulin secretion and [Ca(2+)](i) changes in control and Sur1KO islets. In 1 mm glucose (non-stimulatory for controls), the triggering signal [Ca(2+)](i) was high (loss of regulation) and insulin secretion was stimulated in Sur1KO islets. This "basal" secretion was decreased or increased by imposed changes in [Ca(2+)](i) and was dependent on ATP production, indicating that both triggering and amplifying signals are involved. High glucose stimulated insulin secretion in Sur1KO islets, by an unsuspected, transient increase in [Ca(2+)](i) and a sustained activation of the amplifying pathway. Unlike controls, Sur1KO islets were insensitive to diazoxide and tolbutamide, which rules out effects of either drug at sites other than K(ATP) channels. Amino acids potently increased insulin secretion by Sur1KO islets through both a further electrogenic rise in [Ca(2+)](i) and a metabolism-dependent activation of the amplifying pathway. After sulfonylurea blockade of their K(ATP) channels, control islets qualitatively behaved like Sur1KO islets, but their insulin secretion rate was consistently lower for a similar or even higher [Ca(2+)](i). In conclusion, fuel secretagogues can control insulin secretion in beta-cells without K(ATP) channels, partly by an unsuspected influence on the triggering [Ca(2+)](i) signal and mainly by the modulation of a very effective amplifying pathway.     

Heparin inhibits IP3-induced Ca2+ release in permeabilized pancreatic beta-cells

Heparin was found to inhibit the Ca2+ release induced by inositol 1,4,5-trisphosphate (IP3) in permeabilized pancreatic beta-cells obtained from obese hyperglycemic mice. The effect of heparin was dose-dependent and not due to inhibition of Ca2+ uptake into the IP3-sensitive pool. The effect appeared specific for heparin and was not reproduced by other polysaccharides such as chondroitin sulfates. Heparin might consequently be a useful tool when investigating the molecular mechanism whereby IP3 mobilizes Ca2+.     

Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic beta-cells

The effect of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibition on the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) was studied in primary insulin-releasing pancreatic beta-cells isolated from mice, rats and human subjects as well as in clonal rat insulinoma INS-1 cells. In Ca(2+)-deficient medium the individual primary beta-cells reacted to the SERCA inhibitor cyclopiazonic acid (CPA) with a slow rise of [Ca(2+)](i) followed by an explosive transient elevation. The [Ca(2+)](i) transients were preferentially observed at low intracellular concentrations of the Ca(2+) indicator fura-2 and were unaffected by pre-treatment with 100 microM ryanodine. Whereas 20mM caffeine had no effect on basal [Ca(2+)](i) or the slow rise in response to CPA, it completely prevented the CPA-induced [Ca(2+)](i) transients as well as inositol 1,4,5-trisphosphate-mediated [Ca(2+)](i) transients in response to carbachol. In striking contrast to the primary beta-cells, caffeine readily mobilized intracellular Ca(2+) in INS-1 cells under identical conditions, and such mobilization was prevented by ryanodine pre-treatment. The results indicate that leakage of Ca(2+) from the endoplasmic reticulum after SERCA inhibition is feedback-accelerated by Ca(2+)-induced Ca(2+) release (CICR). In primary pancreatic beta-cells this CICR is due to activation of inositol 1,4,5-trisphosphate receptors. CICR by ryanodine receptor activation may be restricted to clonal beta-cells.     

Expression and function of the extracellular calcium-sensing receptor in pancreatic beta-cells

The extracellular calcium-sensing receptor (CaR) was first identified in tissues involved in systemic Ca2+ homeostasis, where it acts to sense changes in circulating Ca2+. It has since been reported that the CaR is expressed in many tissues that are not associated with Ca2+ homeostasis, including the endocrine cells in pancreatic islets of Langerhans. In the present study we have used an insulin-secreting pancreatic beta-cell line (MIN6) to investigate the expression and function of CaR, using the calcimimetic A568, a CaR agonist that activates the CaR at physiological concentrations of extracellular Ca2+ ([Ca2+]o). Immunocytochemistry, Western blotting and RT-PCR confirmed the expression of CaR in MIN6 cells. CaR activation was associated with rapid and transient increases in [Ca2+]o, which were accompanied by the initiation of a marked but transient insulin secretory response. Stimulation of beta-cell secretory activity had no detectable effect on CaR mRNA levels, but CaR mRNA was markedly reduced by configuring MIN6 cells into islet- like structures. Our data are consistent with an important function for the beta-cell CaR in cell - cell communication within islets to co-ordinate insulin secretory responses.     

VP3, a structural protein of infectious pancreatic necrosis virus, interacts with RNA-dependent RNA polymerase VP1 and with double-stranded RNA

Infectious pancreatic necrosis virus (IPNV) is a bisegmented, double-stranded RNA (dsRNA) virus of the Birnaviridae family that causes widespread disease in salmonids. Its two genomic segments are encapsulated together with the viral RNA-dependent RNA polymerase, VP1, and the assumed internal protein, VP3, in a single-shell capsid composed of VP2. Major aspects of the molecular biology of IPNV, such as particle assembly and interference with host macromolecules, are as yet poorly understood. To understand the infection process, analysis of viral protein interactions is of crucial importance. In this study, we focus on the interaction properties of VP3, the suggested key organizer of particle assembly in birnaviruses. By applying the yeast two-hybrid system in combination with coimmunoprecipitation, VP3 was proven to bind to VP1 and to self-associate strongly. In addition, VP3 was shown to specifically bind to dsRNA in a sequence-independent manner by in vitro pull-down experiments. The binding between VP3 and VP1 was not dependent on the presence of dsRNA. Deletion analyses mapped the VP3 self-interaction domain within the 101 N-terminal amino acids and the VP1 interaction domain within the 62 C-terminal amino acids of VP3. The C-terminal end was also crucial but not sufficient for the dsRNA binding capacity of VP3. For VP1, the 90 C-terminal amino acids constituted the only dispensable part for maintaining VP3-binding ability. Kinetic analysis revealed the presence of VP1-VP3 complexes prior to the formation of mature virions in IPNV-infected CHSE-214 cells, which indicates a role in promoting the assembly process.     

Timing of Ca2+ response in pancreatic beta-cells is related to mitochondrial mass

The timing and magnitude of calcium response are cell-specific in individual beta-cells. This may indicate that the cells have different roles in the intact islet. It is unknown what mechanisms determine these characteristics. We previously found that the mechanisms setting cell-specific response timing are disturbed in beta-cells from hyperglycemic mice and one of the causes is likely to be an altered mitochondrial metabolism. Mitochondria play a key role in the control of nutrient-induced insulin secretion. Here, we used confocal microscopy with the fluorescent probe MitoTracker Red CMXRos and Fluo-3 to study how the amount of active mitochondria is related to the lag-time and the magnitude of calcium response to 20mM glucose in isolated beta-cells and in cells within intact lean and ob/ob mouse islets. Results show that the mitochondrial mass is inversely correlated with the lag-times for calcium response both in lean and ob/ob mouse beta-cells (r=-0.73 and r=-0.43, respectively, P<0.05). Thus, the state of mitochondria may determine the timing of calcium response.     

Cellular microRNAs contribute to HIV-1 latency in resting primary CD4+ T lymphocytes

The latency of human immunodeficiency virus type 1 (HIV-1) in resting primary CD4+ T cells is the major barrier for the eradication of the virus in patients on suppressive highly active antiretroviral therapy (HAART). Even with optimal HAART treatment, replication-competent HIV-1 still exists in resting primary CD4+ T cells. Multiple restriction factors that act upon various steps of the viral life cycle could contribute to viral latency. Here we show that cellular microRNAs (miRNAs) potently inhibit HIV-1 production in resting primary CD4+ T cells. We have found that the 3' ends of HIV-1 messenger RNAs are targeted by a cluster of cellular miRNAs including miR-28, miR-125b, miR-150, miR-223 and miR-382, which are enriched in resting CD4+ T cells as compared to activated CD4+ T cells. Specific inhibitors of these miRNAs substantially counteracted their effects on the target mRNAs, measured either as HIV-1 protein translation in resting CD4+ T cells transfected with HIV-1 infectious clones, or as HIV-1 virus production from resting CD4+ T cells isolated from HIV-1-infected individuals on suppressive HAART. Our data indicate that cellular miRNAs are pivotal in HIV-1 latency and suggest that manipulation of cellular miRNAs could be a novel approach for purging the HIV-1 reservoir.     

DARPP-32 and inhibitor-1 are expressed in pancreatic beta-cells

Insulin secretion from pancreatic beta-cells has to be tightly regulated to ensure accurate glucose homeostasis. The capacity of beta-cells to respond to extracellular stimulation is determined by several signaling pathways. One important feature of these pathways is phosphorylation and subsequent dephosphorylation of a wide range of cellular substrates. Protein phosphatase 1 (PP1) is a major eukaryotic serine/threonine protein phosphatase that controls a multitude of physiological processes. We have investigated the expression and cellular distribution of two endogenous inhibitors of PP1 activity in beta-cells. RT-PCR, Western blotting, and immunohistochemistry showed that DARPP-32 and inhibitor-1 are present in insulin-secreting endocrine beta-cells. Subcellular fractionation of mouse islets revealed that both PP1 inhibitors predominantly localized to cytosol-enriched fractions. Inhibitor-1 was also present in fractions containing plasma membrane-associated proteins. These data indicate a potential role for DARPP-32 and inhibitor-1 in the regulation of PP1 activity in pancreatic beta-cell stimulus-secretion coupling.     

Crucial roles of neuronatin in insulin secretion and high glucose-induced apoptosis in pancreatic beta-cells

Neuronatin (Nnat) was initially identified as a selectively-expressed gene in neonatal brains, but its expression has been also identified in pancreatic beta-cells. Therefore, to investigate the possible functions that Nnat may serve in pancreatic beta-cells, two Nnat isotypes (alpha and beta) were expressed using adenoviruses in murine MIN6N8 pancreatic beta-cells, and the cellular fates and the effects of Nnat on insulin secretion, high glucose-induced apoptosis, and functional impairment were examined. Nnatalpha and Nnatbeta were primarily localized in the endoplasmic reticulum (ER), and their expressions increased insulin secretion by increasing intracellular calcium levels. However, under chronic high glucose conditions, the Nnatbeta to Nnatalpha ratio gradually increased in proportion to the length of exposure to high glucose levels. Moreover, adenovirally-expressed Nnatbeta was inclined to form aggresome-like structures, and we found that Nnatbeta aggregation inhibited the function of the proteasome. Therefore, when glucose is elevated, the expression of Nnatbeta sensitizes MIN6N8 cells to high glucose stress, which in turn, causes ER stress. As a result, expression of Nnatbeta increased hyperglycemia-induced apoptosis. In addition, the expression of Nnatbeta under high glucose conditions decreased the expression of genes important for beta-cell function, such as glucokinase (GCK), pancreas duodenum homeobox-1 (PDX-1), and insulin. Collectively, Nnat may play a critical factor in normal beta-cell function, as well as in the pathogenesis of type 2 diabetes.     

Activation of extracellular signal-regulated kinase (ERK)1/2 contributes to cytokine-induced apoptosis in purified rat pancreatic beta-cells

Cytokines may contribute to beta-cell apoptosis in the early stages of type 1 diabetes mellitus. It has been reported recently that interleukin-1 beta (IL-1 beta) induces activation of the mitogen-activated protein kinases (MAPK) p38 and ERK1/2 in neonatal rat islets. Since these kinases may participate in cytokine-induced apoptosis, we evaluated whether cytokines induce activation of MAPKs in FACS-purified primary rat beta-cells, and whether blockers of p38 and/or ERK1/2 prevent beta-cell death. IL-1 beta, but not interferon-gamma (IFN-gamma), caused phosphorylation of the substrates Elk-1, ATF-2 and hsp25, and the phosphorylation of both Elk-1 and hsp25 were decreased by the p38 blocker SB203580 (p38i) and the MAPK/ERK blocker PD 098059 (MEKi). When added together, p38i and MEKi decreased IL-1 beta-induced nitrite production over 24 hours by 60%, but did not affect IL-1 beta-induced manganese superoxide dismutase (MnSOD) mRNA expression. To test the effects of MAPK inhibitors on beta-cell death by necrosis or apoptosis, these cells were exposed for 6 or 9 days to IL-1 beta + IFN-gamma. This treatment induced cell death, mostly by apoptosis. The MEKi, but not the p38i, significantly decreased cytokine-induced apoptosis, thus decreasing the total number of dead cells. This protection was only partial, suggesting that ERK1/2 activation is not the only mechanism by which cytokines induce beta-cell apoptosis. We conclude that IL-1 beta induces activation of both p38 and ERK1/2, and that ERK1/2 contributes to the pro-apoptotic effects of the cytokine in primary beta-cells.     

Apoptosis signal regulating kinase-1 and NADPH oxidase mediate human amylin evoked redox stress and apoptosis in pancreatic beta-cells

Misfolded toxic human islet amyloid polypeptide or amylin (hA) and plasma membrane-associated redox complex, NADPH oxidase (NOX), have been implicated in the islet β-cell demise associated with type-2 diabetes mellitus (T2DM). Studies show that hA accumulation is stressful to β-cells and that misfolding of human amylin evokes redox stress and activates mitogen activated protein (MAP) kinases, p38 MAPK and c-Jun N-terminal (JNK) kinase. However, the molecular link and causality between hA-evoked redox stress, NOX activity and MAP kinases signaling in pancreatic β-cells is incompletely understood. Here, we show that in the process of activating JNK, aggregation prone hA also activates an upstream apoptosis signal regulating kinase-1 (ASK1) with concomitant decrease in intracellular levels of reduced glutathione. Inhibition of ASK1 kinase activity, either by specific ASK1 inhibitor, NQDI1 or by thiol antioxidants reduces human amylin-evoked ASK1 and JNK activation and consequently human amylin toxicity in rat insulinoma Rin-m5F cells and human islets. β-cell specific overexpression of human amylin in mouse islets elicited ASK1 phosphorylation and activation in β-cells but not in other rodent's islet or exocrine cells. This ASK1 activation strongly correlated with islet amyloidosis and diabetes progression. Cytotoxic human amylin additionally stimulated pro-oxidative activity and expressions of plasma membrane bound NADPH oxidase (NOX) and its regulatory subunits. siRNA mediated NOX1 knockdown and selective NOX inhibitors, ML171 and apocynin, significantly reduced hA-induced mitochondrial stress in insulinoma beta-cells. However, NOX inhibitors were largely ineffective against hA-evoked redox stress and activation of cytotoxic ASK1/JNK signaling complex. Thus, our studies suggest that NOX1 and ASK1 autonomously mediate human amylin-evoked redox and mitochondrial stress in pancreatic β-cells.