IFN-α/β and autophagy: tug-of-war between HCV and the host

Hepatitis C virus (HCV) infects approximately 130 million people worldwide. The clinical sequelae of this chronic disease include cirrhosis, functional failure and carcinoma of the liver. HCV induces autophagy, a fundamental cellular process for maintaining homeostasis and mediating innate immune response, and also inhibits autophagic protein degradation and suppresses antiviral immunity. In addition to this ploy, the HCV serine protease composed of the viral non-structural proteins 3/4A (NS3/4A) can enzymatically digest two cellular proteins, mitochondria-associated anti-viral signaling protein (MAVS) and Toll/interleukin-1 receptor domain containing adaptor inducing IFN-β (TRIF). Since these two proteins are the adaptor molecules in the retinoic acid-inducible gene I (RIG-I) and TLR3 pathways, respectively, their cleavage has been suggested as a pivotal mechanism by which HCV blunts the IFN-α/β signaling and antiviral responses. Thus far, how HCV perturbs autophagy and copes with IFN-α/β in the liver remains unclear.     

Leukocyte-Derived IFN-α/β and Epithelial IFN-λ Constitute a Compartmentalized Mucosal Defense System that Restricts Enteric Virus Infections

Epithelial cells are a major port of entry for many viruses, but the molecular networks which protect barrier surfaces against viral infections are incompletely understood. Viral infections induce simultaneous production of type I (IFN-α/β) and type III (IFN-λ) interferons. All nucleated cells are believed to respond to IFN-α/β, whereas IFN-λ responses are largely confined to epithelial cells. We observed that intestinal epithelial cells, unlike hematopoietic cells of this organ, express only very low levels of functional IFN-α/β receptors. Accordingly, after oral infection of IFN-α/β receptor-deficient mice, human reovirus type 3 specifically infected cells in the lamina propria but, strikingly, did not productively replicate in gut epithelial cells. By contrast, reovirus replicated almost exclusively in gut epithelial cells of IFN-λ receptor-deficient mice, suggesting that the gut mucosa is equipped with a compartmentalized IFN system in which epithelial cells mainly respond to IFN-λ that they produce after viral infection, whereas other cells of the gut mostly rely on IFN-α/β for antiviral defense. In suckling mice with IFN-λ receptor deficiency, reovirus replicated in the gut epithelium and additionally infected epithelial cells lining the bile ducts, indicating that infants may use IFN-λ for the control of virus infections in various epithelia-rich tissues. Thus, IFN-λ should be regarded as an autonomous virus defense system of the gut mucosa and other epithelial barriers that may have evolved to avoid unnecessarily frequent triggering of the IFN-α/β system which would induce exacerbated inflammation.     

Gamma Interferon (IFN-γ) Receptor Restricts Systemic Dengue Virus Replication and Prevents Paralysis in IFN-α/β Receptor-Deficient Mice

We previously reported that mice lacking alpha/beta and gamma interferon receptors (IFN-α/βR and -γR) uniformly exhibit paralysis following infection with the dengue virus (DENV) clinical isolate PL046, while only a subset of mice lacking the IFN-γR alone and virtually no mice lacking the IFN-α/βR alone develop paralysis. Here, using a mouse-passaged variant of PL046, strain S221, we show that in the absence of the IFN-α/βR, signaling through the IFN-γR confers approximately 140-fold greater resistance against systemic vascular leakage-associated dengue disease and virtually complete protection from dengue-induced paralysis. Viral replication in the spleen was assessed by immunohistochemistry and flow cytometry, which revealed a reduction in the number of infected cells due to IFN-γR signaling by 2 days after infection, coincident with elevated levels of IFN-γ in the spleen and serum. By 4 days after infection, IFN-γR signaling was found to restrict DENV replication systemically. Clearance of DENV, on the other hand, occurred in the absence of IFN-γR, except in the central nervous system (CNS) (brain and spinal cord), where clearance relied on IFN-γ from CD8⁺ T cells. These results demonstrate the roles of IFN-γR signaling in protection from initial systemic and subsequent CNS disease following DENV infection and demonstrate the importance of CD8⁺ T cells in preventing DENV-induced CNS disease.     

Glycogen synthase kinase-3β controls autophagy during myocardial ischemia and reperfusion

Autophagy is a catabolic process that degrades long-lived proteins, pathogens and damaged organelles. Autophagy is active in the heart at baseline and is further stimulated by stresses, such as nutrient starvation, ischemia/reperfusion (I/R) and heart failure. Baseline autophagy plays an adaptive role in the heart, and contributes to the maintenance of cardiac structure and function and the inhibition of age-associated abnormalities, by achieving quality control of proteins and organelles. Activation of autophagy during ischemia is beneficial because it improves cell survival and cardiac function. However, excessive autophagy with robust upregulation of BECN1 during reperfusion appears to enhance cell death, which is detrimental to the heart. We have shown recently that autophagy during prolonged ischemia and I/R is critically regulated by glycogen synthase kinase-3β (GSK-3β), a ubiquitously expressed serine/threonine kinase, in a phase-dependent manner. Here we discuss the role of GSK-3β in mediating autophagy in the heart.     

Melatonin attenuates MPTP-induced neurotoxicity via preventing CDK5-mediated autophagy and SNCA/α-synuclein aggregation

Autophagy is involved in the pathogenesis of neurodegenerative diseases including Parkinson disease (PD). However, little is known about the regulation of autophagy in neurodegenerative process. In this study, we characterized aberrant activation of autophagy induced by neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and demonstrated that melatonin has a protective effect on neurotoxicity. We found an excessive activation of autophagy in monkey brain tissues and C6 cells, induced by MPTP, which is mediated by CDK5 (cyclin-dependent kinase 5). MPTP treatment significantly reduced total dendritic length and dendritic complexity of cultured primary cortical neurons and melatonin could reverse this effect. Decreased TH (tyrosine hydroxylase)-positive cells and dendrites of dopaminergic neurons in the substantia nigra pars compacta (SNc) were observed in MPTP-treated monkeys and mice. Along with decreased TH protein level, we observed an upregulation of CDK5 and enhanced autophagic activity in the striatum of mice with MPTP injection. These changes could be salvaged by melatonin treatment or knockdown of CDK5. Importantly, melatonin or knockdown of CDK5 reduced MPTP-induced SNCA/α-synuclein aggregation in mice, which is widely thought to trigger the pathogenesis of PD. Finally, melatonin or knockdown of CDK5 counteracted the PD phenotype in mice induced by MPTP. Our findings uncover a potent role of CDK5-mediated autophagy in the pathogenesis of PD, and suggest that control of autophagic pathways may provide an important clue for exploring potential target for novel therapeutics of PD.     

Liprin-mediated large signaling complex organization revealed by the liprin-α/CASK and liprin-α/liprin-β complex structures

Liprins are highly conserved scaffold proteins that regulate cell adhesion, cell migration, and synapse development by binding to diverse target proteins. The molecular basis governing liprin/target interactions is poorly understood. The liprin-α2/CASK complex structure solved here reveals that the three SAM domains of liprin-α form an integrated supramodule that binds to the CASK kinase-like domain. As supported by biochemical and cellular studies, the interaction between liprin-α and CASK is unique to vertebrates, implying that the liprin-α/CASK interaction is?likely to regulate higher-order brain functions in mammals. Consistently, we demonstrate that three recently identified X-linked mental retardation mutants of CASK are defective in binding to liprin-α. We also solved the liprin-α/liprin-β SAM domain complex structure, which uncovers the mechanism underlying liprin heterodimerizaion. Finally, formation of the CASK/liprin-α/liprin-β ternary complex suggests that liprins can mediate assembly of target proteins into large protein complexes capable of regulating numerous cellular activities.     

Urolithin A suppresses glucolipotoxicity-induced ER stress and TXNIP/NLRP3/IL-1β inflammation signal in pancreatic β cells by regulating AMPK and autophagy

BackgroundPancreatic inflammation plays a key role in diabetes pathogenesis and progression. Urolithin A (UA), an intestinal flora metabolite of pomegranate, has anti-diabetic, anti-inflammatory and kidney protection effects among others. However, its effects on pancreatic inflammation and the potential mechanisms have not been clearly established.PurposeThis study aimed at investigating the molecular mechanisms of UA anti-pancreatic inflammation under a diabetic environment.MethodsDiabetes induction in male C57BL/6 mice was achieved by a high fat diet and intraperitoneal streptozotocin injections. Then, diabetic mice were orally administered with UA for 8 weeks. In vitro, endoplasmic reticulum stress and MIN6 pancreatic β cell inflammation were induced using 25 mM glucose and 0.5 mM palmitic acid. The effects of UA were evaluated by immunohistochemistry, Western blot, and enzyme linked immunosorbent assays. Finally, the underlying mechanisms were elucidated using an autophagy inhibitor (chloroquine, CQ) and an AMPK inhibitor (dorsomorphin dihydrochloride).ResultsUA significantly inhibited IL-1β secretion and TXNIP/NLRP3 expression in the pancreas of diabetic mice and in MIN6 pancreatic cells. UA downregulated the ER stress protein, p-PERK, and promoted AMPK phosphorylation. UA activated autophagy to inhibit TXNIP/NLRP3 IL-1β inflammatory signal, an effect that was reversed by CQ. Dorsomorphin 2HCL, reversed the autophagy-activation and anti-inflammatory effects of UA. Verapamil, clinically applied as an antiarrhythmic drug, is a TXNIP inhibitor for prevention of beta cell loss and diabetes development, but limited by its cardiac toxicity. In this study, verapamil (as positive control) inhibited NLRP3 /IL-1β signaling in MIN6 cells. Inhibitory effects of UA on TXNIP and IL-1β were weaker than those of verapamil (both at 50 μM, p < 0.05, p < 0.01). Conversely, inhibitory effects of UA on p62 were stronger, relative to those of verapamil (p < 0.05), and there were no differences in AMPK activation and LC3 enhancement effects between UA and verapamil.ConclusionUA is a potential anti-pancreatic inflammation agent that activates AMPK and autophagy to inhibit endoplasmic reticulum stress associated TXNIP/NLRP3/IL-1β signal pathway.     

A novel autophagy modulator 6-Bio ameliorates SNCA/α-synuclein toxicity

Parkinson disease (PD) is a life-threatening neurodegenerative movement disorder with unmet therapeutic intervention. We have identified a small molecule autophagy modulator, 6-Bio that shows clearance of toxic SNCA/α-synuclein (a protein implicated in synucleopathies) aggregates in yeast and mammalian cell lines. 6-Bio induces autophagy and dramatically enhances autolysosome formation resulting in SNCA degradation. Importantly, neuroprotective function of 6-Bio as envisaged by immunohistology and behavior analyses in a preclinical model of PD where it induces autophagy in dopaminergic (DAergic) neurons of mice midbrain to clear toxic protein aggregates suggesting that it could be a potential therapeutic candidate for protein conformational disorders.     

Impact of IFN-β and LIF overexpression on human adipose-derived stem cells properties

Adipose-derived stem cells (ADSCs) are a subset of mesenchymal stem cells that their therapeutic effects in various diseases make them an interesting tool in cell therapy. In the current study, we aimed to overexpress interferon-β (IFN-β) and leukemia inhibitory factor (LIF) cytokines in human ADSCs to evaluate the impact of this overexpression on human ADSCs properties. Here, we designed a construct containing IFN-β and LIF and then, transduced human adipose-derived stem cells (hADSCs) by this construct via a lentiviral vector (PCDH-513B). We assessed the ability of long-term expression of the transgene in transduced cells by western blot analysis and enzyme-linked immunosorbent assay techniques on Days 15, 45, and 75 after transduction. For the evaluation of stem cell properties, flow cytometry and differentiation assays were performed. Finally, the MTT assay was done to assess the proliferation of transduced cells compares to controls. Our results showed high-efficiency transduction with highest expression rates on Day 75 after transduction which were 70 pg/ml for IFN-β and 77.9 pg/ml for LIF in comparison with 25.60 pg/ml and 27.63 pg/ml, respectively, in untransduced cells (p = .0001). Also, transduced cells expressed a high level of ADSCs surface markers and successfully differentiated into adipocytes, chondrocytes, neural cells, and osteocytes besides the preservation rate of proliferation near untreated cells (p = .88). All in all, we successfully constructed an hADSC population stably overexpressed IFN-β and LIF cytokines. Considering the IFN-β and LIF anti-inflammatory and neuroprotective effects as well as immune-regulatory properties of hADSCs, the obtained cells of this study could be subjected for further evaluations in experimental autoimmune encephalomyelitis mice model.     

MAPK14/p38α-dependent modulation of glucose metabolism affects ROS levels and autophagy during starvation

Increased glycolytic flux is a common feature of many cancer cells, which have adapted their metabolism to maximize glucose incorporation and catabolism to generate ATP and substrates for biosynthetic reactions. Indeed, glycolysis allows a rapid production of ATP and provides metabolic intermediates required for cancer cells growth. Moreover, it makes cancer cells less sensitive to fluctuations of oxygen tension, a condition usually occurring in a newly established tumor environment. Here, we provide evidence for a dual role of MAPK14 in driving a rearrangement of glucose metabolism that contributes to limiting reactive oxygen species (ROS) production and autophagy activation in condition of nutrient deprivation. We demonstrate that MAPK14 is phosphoactivated during nutrient deprivation and affects glucose metabolism at 2 different levels: on the one hand, it increases SLC2A3 mRNA and protein levels, resulting in a higher incorporation of glucose within the cell. This event involves the MAPK14-mediated enhancement of HIF1A protein stability. On the other hand, MAPK14 mediates a metabolic shift from glycolysis to the pentose phosphate pathway (PPP) through the modulation of PFKFB3 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase 3) degradation by the proteasome. This event requires the presence of 2 distinct degradation sequences, KEN box and DSG motif Ser273, which are recognized by 2 different E3 ligase complexes. The mutation of either motif increases PFKFB3 resistance to starvation-induced degradation. The MAPK14-driven metabolic reprogramming sustains the production of NADPH, an important cofactor for many reduction reactions and for the maintenance of the proper intracellular redox environment, resulting in reduced levels of ROS. The final effect is a reduced activation of autophagy and an increased resistance to nutrient deprivation.     

Interferon-α/β upregulate IL-15 expression in vitro and in vivo: analysis in human hepatocellular carcinoma cell lines and in chronic hepatitis C patients during interferon-α/β treatment

Type I interferon (IFN) possesses antiviral and antitumor activities and also having an immune regulatory effect, activating cellular immune response and upregulating several cytokines. Recent study has shown that type I IFN upregurates the dendritic cell production of IL-15 capable of activating natural killer cells and CD8⁺ memory T lymphocytes. However, it is still unknown if type I IFN induces IL-15 production in non-immune cells and if type I IFN affects IL-15 production in vivo. The present study investigated the effect of type I IFNs on IL-15 expression in hepatocellular carcinoma (HCC) cell lines in vitro and in patients with chronic hepatitis C in vivo. When three HCC cell lines, Huh7, HepG2, and JHH4 were cultured in vitro, IFN upregulation of IL-15 expression was observed at both the mRNA and protein levels. In experiments using Huh7 cells, upregulation of IL-15 expression occurred within 24 h of the start of IFN stimulation, and both IFN-α and -β dose-dependently increased IL-15 production in the range from 100 U/ml to 10,000 U/ml of concentration. IFN-β showed stronger activity in IL-15 production induction in vitro than IFN-α. For in vivo examination, sera were obtained from 21 chronic hepatitis C patients treated with IFN and 29 healthy individuals, and the serum IL-15 level was quantified by ELISA. The serum IL-15 level of chronic hepatitis C patients before IFN treatment was similar to that of the healthy controls and significantly increased only during the IFN administration period. These results confirm that IFN-α/β induce IL-15 production and also suggest that IL-15 may be associated with type I IFN-induced immune response.     

PRMT2β suppresses autophagy and glycolysis pathway in human breast cancer MCF-7 cell lines

Autophagy as a novel therapeutic target can inhibit or increase treatment efficacy in various types of breast cancer in a cell-type-dependent manner [1,2].Several studies have revealed that the coordination between Akt and the glycolytic pathway plays an indispensable role in mediating autophagy and caspase-dependent apoptosis,suggesting that a new regulatory mechanism for the process [3,4].Protein arginine N-methyltransferases(PRMTs)are eukaryotic enzymes that catalyze the transfer of methyl groups from S-adenosylmethionine to arginine residues of numerous PRMT substrates [5,6].PRMT2(also known as HRMT1L1)belongs to the arginine methyltransferase family [7].PRMT2β is a novel PRMT2 splice variant isolated from breast cancer cell [8].It occurs at the 3′ end of the PRMT2,resulting in loss of exons 7–9 and downstream frame-shifting [9].PRMT2β possesses 83 new amino acids at the C-terminus and its size is 301 amino acids.Our previous study reported that PRMT2β has potential antitumor effect by suppressing cyclin D1 expression [10].However,little is known about whether PRMT2β could regulate autophagy and glycolysis of MCF-7 cells.     

The problem of pyridinyl imidazole class inhibitors of MAPK14/p38α and MAPK11/p38β in autophagy research

In addition to its established role in inflammation, the stress-activated p38 MAP kinase pathway plays major roles in the regulation of cell cycle, senescence, and autophagy. Robust studies could establish mechanistic links between MAPK11-MAPK14/p38 signaling and macroautophagy converging at ATG9-trafficking and BECN1 phosphorylation. However, several reports seem to monitor MAPK11-MAPK14/p38-dependence of autophagy exclusively by the use of the SB203580/SB202190 class of MAPK14/MAPK11/p38α/β inhibitors. In this “Letter to the editor” we present data to support our claim that these inhibitors interfere with autophagic flux in a MAPK11-MAPK14/p38-independent manner and hence should no longer be used as pharmacological tools in the analysis of MAPK11-MAPK14/p38-dependence of autophagy. We propose a general guideline from Autophagy with regard to this issue to avoid such misinterpretations in the future.     

Simultaneous quantification of GABAergic 3α,5α/3α,5β neuroactive steroids in human and rat serum

The 3α,5α- and 3α,5β-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone enhance GABAergic neurotransmission and produce inhibitory neurobehavioral and anti-inflammatory effects. Despite substantial information on the progesterone derivative (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone), the physiological significance of the other endogenous GABAergic neuroactive steroids has remained elusive. Here, we describe the validation of a method using gas chromatography–mass spectrometry to simultaneously identify serum levels of the eight 3α,5α- and 3α,5β-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone. The method shows specificity, sensitivity and enhanced throughput compared to other methods already available for neuroactive steroid quantification. Administration of pregnenolone to rats and progesterone to women produced selective effects on the 3α,5α- and 3α,5β-reduced neuroactive steroids, indicating differential regulation of their biosynthetic pathways. Pregnenolone administration increased serum levels of 3α,5α-THP (+1488%, p < 0.001), (3α,5α)-3,21-dihydroxypregnan-20-one (3α,5α-THDOC, +205%, p < 0.01), (3α,5α)-3-hydroxyandrostan-17-one (3α,5α-A, +216%, p < 0.001), (3α,5α,17β)-androstane-3,17-diol (3α,5α-A-diol, +190%, p < 0.01). (3α,5β)-3-hydroxypregnan-20-one (3α,5β-THP) and (3α,5β)-3-hydroxyandrostan-17-one (3α,5β-A) were not altered, while (3α,5β)-3,21-dihydroxypregnan-20-one (3α,5β-THDOC) and (3α,5β,17β)-androstane-3,17-diol (3α,5β-A-diol) were increased from undetectable levels to 271 ± 100 and 2.4 ± 0.9 pg ± SEM, respectively (5/8 rats). Progesterone administration increased serum levels of 3α,5α-THP (+1806%, p < 0.0001), 3α,5β-THP (+575%, p < 0.001), 3α,5α-THDOC (+309%, p < 0.001). 3α,5β-THDOC levels were increased by 307%, although this increase was not significant because this steroid was detected only in 3/16 control subjects. Levels of 3α,5α-A, 3α,5β-A and pregnenolone were not altered. This method can be used to investigate the physiological and pathological role of neuroactive steroids and to develop biomarkers and new therapeutics for neurological and psychiatric disorders.     

IFN-β differentially regulates the function of T cell subsets in MS and EAE

Multiple sclerosis (MS) is considered as a T cell mediated autoimmune disease of the CNS, although a pathogenic role has also been attributed to other immune cell types as well as to environmental and genetic factors. Considering that T cells are interesting from an immunopathogenic point of view and consequently from a therapeutic perspective, various T cell targeted therapies have been approved for MS. Interferon beta (IFN-β) is widely used as first-line intervention for modulating T cell responses, although its pleiotropic and multifaceted activities influence its effectiveness on the disease development, with mechanisms that are not yet fully understood. Since different T cell populations, including pro-inflammatory and regulatory T cells, might affect the course of MS, the effects of IFN-β become even more complex.This review will summarize recent findings regarding the T cell targeted effect of IFN-β in MS and its animal model EAE, with emphasis on the direct actions of endogenous and exogenous IFN-β on each T cell subpopulation involved in CNS autoimmunity.Delineating how IFN-β exerts its action on different T cell types may eventually contribute to the designing of therapeutic strategies aiming to improve the effectiveness of this drug for MS treatment.     

Molecular cloning and expression analysis of IFN-β promoter stimulator 1 in Tibetan pigs

IFN-β promoter stimulator 1 (IPS-1) is an important adaptor protein linking RIG-I/MDA5 to the downstream signaling molecules and plays the pivotal role in type I interferons induction. In this study, we cloned and characterized Tibetan porcine IPS-1, investigated the tissue distribution, compared different messenger RNA expression for IPS-1 between Tibetan and Crossbred (Duroc × Yorkshire × Landrace) pigs (DLY). The Tibetan porcine IPS-1 gene was first cloned from spleen. The entire open reading frame (ORF) of the IPS-1 is 1,575 bp and encodes for 524 amino acid residues, has 1 putative transmembrane domains, with a higher degree of sequence similarity with common pig (99.37%) and cattle (81.23%) than with human (70.20%) or mouse (63.44%). Real-time quantitative PCR analysis indicated that Tibetan porcine IPS-1 mRNA was most abundant in the liver and kidney. The expression of IPS-1 of Tibetan pigs in most tissues was higher than DLY pigs.