Long non-coding RNA prostate cancer-associated transcript 6 inhibited gefitinib sensitivity of non-small cell lung cancer by serving as a competing endogenous RNA of miR-326 to up-regulate interferon-alpha receptor 2.

The critical roles of lncRNAs in drug resistance of malignancies have been widely recognized. This investigation aims to study the function of lncRNA PCAT6 in the resistance of non-small cell lung cancer (NSCLC) to gefitinib. In our study, we demonstrated that prostate cancer-associated transcript 6 (PCAT6) was upregulated in gefitinib-resistant NSCLC. PCAT6 knockdown inhibited gefitinib resistance of NSCLC, as indicated by decreased IC50 value, proliferation, and metastasis, and increased cell apoptosis. Besides, PCAT6 could directly target miR-326 in gefitinib-resistant NSCLC cells and augment NSCLC resistance to gefitinib by serving as ceRNA of miR-326. Furthermore, interferon-alpha receptor 2 (IFNAR2) was validated as a downstream target of miR-326 and miR-326 reduced resistance to gefitinib by inhibiting IFNAR2 expression. Our investigation identified that PCAT6 enhanced gefitinib resistance of NSCLC via miR-326/IFNAR2 axis, which might offer a new therapeutic strategy against gefitinib resistance of NSCLC patients.

Microglial responses to peripheral type 1 interferon.

BACKGROUND: Interferon α (IFNα) is a cytokine whose production is increased endogenously in response to viral infection and in autoimmune diseases such as systemic lupus erythematosus (SLE). An elevated IFNα signature has been associated with clinically observed neuro-behavioural deficits such as mild cognitive impairment, fatigue, depression and psychosis in these diseases. However, the mechanisms underlying these neuropsychiatric symptoms remain largely unknown, and it is as yet unclear how IFNα signalling might influence central nervous system (CNS) function. Aberrant microglia-mediated synaptic pruning and function has recently been implicated in several neurodegenerative and neuropsychiatric diseases, but whether and how IFNα modulates these functions are not well defined. METHODS: Using a model of peripheral IFNα administration, we investigated gene expression changes due to IFNAR signalling in microglia. Bulk RNA sequencing on sorted microglia from wild type and microglia-specific Ifnar1 conditional knockout mice was performed to evaluate IFNα and IFNAR signalling-dependent changes in gene expression. Furthermore, the effects of IFNα on microglia morphology and synapse engulfment were assessed, via immunohistochemistry and flow cytometry. RESULTS: We found that IFNα exposure through the periphery induces a unique gene signature in microglia that includes the expected upregulation of multiple interferon-stimulated genes (ISGs), as well as the complement component C4b. We additionally characterized several IFNα-dependent changes in microglial phenotype, including expression of CD45 and CD68, cellular morphology and presynaptic engulfment, that reveal subtle brain region-specific differences. Finally, by specifically knocking down expression of IFNAR1 on microglia, we show that these changes are largely attributable to direct IFNAR signalling on microglia and not from indirect signalling effects through other CNS parenchymal cell types which are capable of IFNα-IFNAR signal transduction. CONCLUSIONS: Peripheral IFNα induces unique genetic and phenotypic changes in microglia that are largely dependent on direct signalling through microglial IFNAR. The IFNα-induced upregulation of C4b could play important roles in the context of aberrant synaptic pruning in neuropsychiatric disease.

A high yielding IFNAR1 ECD mammalian expression process for use in autoimmune disease drug development.

Interferon-alpha receptor 1 (IFNAR1) is a target of interest for recombinant biotherapeutics that block the JAK/STAT pathway. This pathway is believed to play a role in many diseases including Hepatitis B and C, Herpes Simplex, Multiple Sclerosis, and other autoimmune disorders. By using IFNAR1 as a target to block Type I IFN from binding to the JAK/STAT pathway and prevent activation of this target, autoimmune disease progression can be modulated. Current IFNAR1 extracellular domain (ECD) expression and purification protocols are labor intensive with low product yield and limited scalability. In this work, we evaluate three different expression systems (baculovirus, human embryonic kidney 293 (HEK293×), and Chinese hamster ovary (CHO)) to improve expression of IFNAR1 ECD. We demonstrate the benefits of utilizing mammalian CHO cell transient transfection to increase expression titer, as well as an improved two-step purification process performed using immobilized metal affinity chromatography (IMAC) as the capture step and Ceramic Hydroxyapatite (CHT) Type II for HMW impurity removal in flow through mode. This process showed an 20-fold increase in productivity compared to the baseline process as measured by grams purified per liter of cell culture fluid. Lastly, the improved process showed good scalability, enabling efficient purification of 3.6 g of product from a 30 L scale bioreactor.

Interferon alpha receptors and STAT1 as therapy predictors of a sustained virological response in hepatitis C/B co-infection.

OBJECTIVE: : To determine the expression of interferon alpha receptors 1 and 2 along with signal transducer and activator of transcription-1 in peripheral blood mononuclear cells of both hepatitis C mono-infected and hepatitis C and B co-infected patients, and to assess whether these targeted genes predict sustained virological response to interferon therapy. METHODS: This cross-sectional study was carried out at the Army Medical College, Rawalpindi, Pakistan, from January 2012 to December 2015, and comprised hepatitis C mono-infected and hepatitis C and B co-infected patients. The patients were divided into groups 1 and 2. Group-1a and group-2a consisted of mono-infected and co-infected sustained responders, while group-1b and group-2b had mono-infected and co-infected non sustained responders. Peripheral blood mononuclear cells from healthy controls were also quantified for these subunits. Target gene expressions were studied by retro-transcription of respective messenger ribonuclieic acid extracted from the cells followed by polymerase chain reaction amplification. RESULTS: Of the 191 subjects, there were 20(10.5%) in group-1a, 35(18.3%) in group-2a, 65(34%) in group-1b and 51(26.7%) in group-2b. The remaining 20(10.5%) were controls. Overall, 106 (55.5%) were males and 85 (44.5%) were females. Interferon alpha receptor 1 expression in groups 1a and 2a was significantly higher compared to groups 1b (p=0.018) and 2b (p 0.031). Signal transducer and activator of transcription-1 protein expression showed no significant difference (p=0.062 and p=0.519). No difference in expression was measured between the two sets of groups with regard to interferon alpha receptor 2 expression (p=0.278 and p=0.590). CONCLUSIONS: Our results show that levels of IFNAR-1 mRNA expression may be a good predictor for IFN-related anti-viral activity in both HCV mono infected and HCV/HBV co-infected patients.

Decreased CD122 on CD56dim NK associated with its impairment in asymptomatic chronic HBV carriers with high levels of HBV DNA, HBsAg and HBeAg.

AIMS: NK cells play important roles in inhibiting HBV replication and preventing HBV infection. However, NK-cell dysfunction has not been fully studied in asymptomatic chronic HBV carriers (ASC). This study aims to assess decreased expression of CD122 associated with impaired NK cells and the restoration of NK cells with IL-2 and IL-15 stimulation. MAIN METHODS: The experiments were performed by flow cytometer, cytotoxicity assay, ELISA and western blotting. KEY FINDINGS: The reduced CD122 on CD56+ NK cells and CD56dim NK cells is associated with high levels of HBV DNA, HBsAg or HBeAg in ASCs, in which CD56dim NK-cell impairment is observed. Moreover, decreased IFN-γ and degranulation and low cytotoxicity by CD56dim NK cells after CD122 blockade were revealed. IL-2 and/or IL-15 can restore impaired CD56dim NK cells, together with increased p-STAT5, which can be reversed by CD122 blockade. Additionally, IL-2 or IL-15 can enhance IFN-α2-activated CD56dim NK-cell immune responses via up-regulating interferon alpha and beta receptor subunit 2 (IFNAR2). SIGNIFICANCE: Down-regulated CD122 on CD56dim NK cell in ASCs with massive viral antigens and high viremia is associated with its impairment, which can be restored by IL-2 and/or IL-15, or combined with IFN-α2.

Ischemia/Reperfusion Induces Interferon-Stimulated Gene Expression in Microglia.

Innate immune signaling is important in the pathophysiology of ischemia/reperfusion (stroke)-induced injury and recovery. Several lines of evidence support a central role for microglia in these processes. Recent work has identified Toll-like receptors (TLRs) and type I interferon (IFN) signaling in both ischemia/reperfusion-induced brain injury and ischemic preconditioning-mediated neuroprotection. To determine the effects of "ischemia/reperfusion-like" conditions on microglia, we performed genomic analyses on wild-type (WT) and TLR4-/- cultured microglia after sequential exposure to hypoxia/hypoglycemia and normoxia/normoglycemia (H/H-N/N). We observed increased expression of type 1 IFN-stimulated genes (ISGs) as the predominant transcriptomal feature of H/H-N/N-exposed WT, but not TLR4-/-, microglia. Microarray analysis on ex vivo sorted microglia from ipsilateral male mouse cortex after a transient in vivo ischemic pulse also demonstrated robust expression of ISGs. Type 1 IFNs, including the IFN-αs and IFN-ß, activate the interferon-α/ß receptor (IFNAR) complex. We confirmed both in vitro H/H-N/N- and in vivo ischemia/reperfusion-induced microglial ISG responses by quantitative real-time PCR and demonstrated that both were dependent on IFNAR1. We characterized the effects of hypoxia/hypoglycemia on phosphorylation of signal transducer and activator of transcription 1 (STAT1), release of type 1 IFNs, and surface expression of IFNAR1 in microglia. We demonstrated that IFN-ß induces dose-dependent secretion of ISG chemokines in cultured microglia and robust ISG expression in microglia both in vitro and in vivo Finally, we demonstrated that the microglial ISG chemokine responses to TLR4 agonists were dependent on TLR4 and IFNAR1. Together, these data suggest novel ischemia/reperfusion-induced pathways for both TLR4-dependent and -independent, IFNAR1-dependent, type 1 IFN signaling in microglia.SIGNIFICANCE STATEMENT Stroke is the fifth leading cause of death in the United States and is a leading cause of serious long-term disability worldwide. Innate immune responses are critical in stroke pathophysiology, and microglia are key cellular effectors in the CNS response to ischemia/reperfusion. Using a transcriptional analysis approach, we identified a robust interferon (IFN)-stimulated gene response within microglia exposed to ischemia/reperfusion in both in vitro and in vivo experimental paradigms. Using a number of complementary techniques, we have demonstrated that these responses are dependent on innate immune signaling components including Toll-like receptor-4 and type I IFNs. We have also elucidated several novel ischemia/reperfusion-induced microglial signaling mechanisms.

Type-I interferon signalling through IFNAR1 plays a deleterious role in the outcome after stroke.

Neuroinflammation contributes significantly to the pathophysiology of stroke. Here we test the hypothesis that the type I interferon receptor (IFNAR1) plays a critical role in neural injury after stroke by regulating the resultant pro-inflammatory environment. Wild-type and IFNAR1-/- primary murine neurons and glia were exposed to oxygen glucose deprivation (OGD) and cell viability was assessed. Transient cerebral ischemia/reperfusion injury was induced by mid-cerebral artery occlusion (MCAO) in wild-type and IFNAR1-/- and IFNAR2-/- mice in vivo, and infarct size, and molecular parameters measured. To block IFNAR1 signalling, wild-type mice were treated with a blocking monoclonal antibody directed to IFNAR1 (MAR-1) and MCAO was performed. Quantitative PCR confirmed MCAO in wild-type mice induced a robust type-I interferon gene regulatory signature. Primary cultured IFNAR1-deficient neurons were found to be protected from cell death when exposed to OGD in contrast to primary cultured IFNAR1-deficient glial cells. IFNAR1-/- mice demonstrated a decreased infarct size (24.9 ± 7.1 mm3 n = 8) compared to wild-type controls (65.1 ± 4.8 mm3 n = 8). Western blot and immunohistochemistry showed alterations in Akt and Stat-3 phosphorylation profiles in the IFNAR1-/- brain. MAR-1 injection into WT mice (i.v. 0.5 mg 60 min prior to MCAO) resulted in a 60% decrease in infarct size when compared to the IgG control. IFNAR2-/- mice failed to display the neuroprotective phenotype seen in IFNAR1-/- mice after MCAO. Our data proposes that central nervous system signalling through IFNAR1 is a previously unrecognised factor that is critical to neural injury after stroke.

Immune response genes receptors expression and polymorphisms in relation to multiple sclerosis susceptibility and response to INF-ß therapy.

Interferon (IFN)-ß is one of the disease modifying drugs used in the treatment of multiple sclerosis. A predictive marker that indicates good or poor response to the treatment is highly desirable. We aimed to investigate the relation between the immune response genes receptors (IFNAR1, IFNAR2, and CCR5) expression and their polymorhic variants and multiple sclerosis (MS) susceptibility as well as the response to IFN-ß therapy. The immune response genes receptors expression and genotyping were analyzed in 80 patients with MS, treated with IFN-ß and in 110 healthy controls. There was a significant decrease of IFNAR1 and IFNAR2 mRNA expression and a significant increase of CCR5 mRNA expression in MS patients compared with the control group. Also, the level of IFNAR1, IFNAR2, and CCR5 mRNA expression was found to be significantly lower in the responders than nonresponders. Carriers of IFNAR1 18417 C/C genotype and C allele had an increased risk of developing MS. There was a significant relation between CCR5 Δ32 allele and IFN-ß treatment response in MS patients. Our results highlighted the significance of IFNAR and CCR5 genes in multiple sclerosis risk and the response to IFN-ß therapy. © 2016 IUBMB Life, 68(9):727-734, 2016.

Ischemic Preconditioning in White Matter: Magnitude and Mechanism.

Ischemic preconditioning (IPC) is a robust neuroprotective phenomenon whereby brief ischemic exposure confers tolerance to a subsequent ischemic challenge. IPC has not been studied selectively in CNS white matter (WM), although stroke frequently involves WM. We determined whether IPC is present in WM and, if so, its mechanism. We delivered a brief in vivo preconditioning ischemic insult (unilateral common carotid artery ligation) to 12- to 14-week-old mice and determined WM ischemic vulnerability [oxygen-glucose deprivation (OGD)] 72 h later, using acutely isolated optic nerves (CNS WM tracts) from the preconditioned (ipsilateral) and control (contralateral) hemispheres. Functional and structural recovery was assessed by quantitative measurement of compound action potentials (CAPs) and immunofluorescent microscopy. Preconditioned mouse optic nerves (MONs) showed better functional recovery after OGD than the non-preconditioned MONs (31 ± 3 vs 17 ± 3% normalized CAP area, p < 0.01). Preconditioned MONs also showed improved axon integrity and reduced oligodendrocyte injury compared with non-preconditioned MONs. Toll-like receptor-4 (TLR4) and type 1 interferon receptor (IFNAR1), key receptors in innate immune response, are implicated in gray matter preconditioning. Strikingly, IPC-mediated WM protection was abolished in both TLR4(-/-) and IFNAR1(-/-) mice. In addition, IPC-mediated protection in WM was also abolished in IFNAR1(fl/fl) LysM(cre), but not in IFNAR1(fl/fl) control, mice. These findings demonstrated for the first time that IPC was robust in WM, the phenomenon being intrinsic to WM itself. Furthermore, WM IPC was dependent on innate immune cell signaling pathways. Finally, these data demonstrated that microglial-specific expression of IFNAR1 plays an indispensable role in WM IPC. SIGNIFICANCE STATEMENT: Ischemic preconditioning (IPC) has been studied predominantly in gray matter, but stroke in humans frequently involves white matter (WM) as well. Here we describe a novel, combined in vivo/ex vivo mouse model to determine whether IPC occurs in WM. It does. Using genetically altered mice, we identified two innate immune cell receptors, Toll-like receptor 4 and type 1 interferon receptor (IFNAR1), that are required for IPC-mediated protection in WM. Furthermore, using microglia-targeted IFNAR1 knockdown, we demonstrate that interferon signaling specifically in microglia is essential for this protection. The discovery of IPC as an intrinsic capability of WM is novel and important. This is also the first in vivo demonstration that cell-type-specific expression of an individual gene plays an indispensable role in IPC-mediated protection.

Type I interferon receptors in goose: molecular cloning, structural identification, evolutionary analysis and age-related tissue expression profile.

The cDNAs encoding two distinct type I interferon receptors were firstly cloned from the spleen of white goose (the Chinese goose, Anser cygnoides). The cDNA of goose IFNAR1 consisted of 1616 bp and encoded 406 amino acids with a predicted molecular weight of 46.4 kDa, while the cDNA of goose IFNAR2 consisted of 1525 bp and encoded 294 amino acids with a predicted molecular weight of 32.6 kDa. The IFNAR1 shared 85.4% identity in deduced amino acid sequence with duck IFNAR1, while IFNAR2 amino acid sequence showed 86% identity with that of duck IFNAR2. The age-related analysis of gene expression revealed that goose IFNα and IFNARs were all highly transcribed in pancreas, which may due to a reasonable amount of dendritic cells aggregated in pancreas. And goose IFNα and its cognate receptors had different structural features and tissue expression patterns during the period from embryonic goose to adult goose, suggesting that IFNα and IFNARs may maintain a developmental dynamic immune competence in unstimulated states. The data provided in this study may contribute to future understanding of the interaction between interferon and interferon receptors in immune mechanism. And it also helps us to understand the age-related susceptibility to pathogens in birds better.

Analysis of the crow lung transcriptome in response to infection with highly pathogenic H5N1 avian influenza virus.

The highly pathogenic avian influenza (HPAI) H5N1 virus, currently circulating in Asia, causes severe disease in domestic poultry as well as wild birds like crow. However, the molecular pathogenesis of HPAIV infection in crows and other wild birds is not well known. Thus, as a step to explore it, a comprehensive global gene expression analysis was performed on crow lungs, infected with HPAI H5N1 crow isolate (A/Crow/India/11TI11/2011) using high throughput next generation sequencing (NGS) (GS FLX Titanium XLR70). The reference genome of crow is not available, so RNA seq analysis was performed on the basis of a de novo assembled transcriptome. The RNA seq result shows, 4052 genes were expressed uniquely in noninfected, 6277 genes were expressed uniquely in HPAIV infected sample and of the 6814 genes expressed in both samples, 2279 genes were significantly differentially expressed. Our transcriptome profile data allows for the ability to understand the molecular mechanism behind the recent lethal HPAIV outbreak in crows which was, until recently, thought to cause lethal infections only in gallinaceous birds such as chickens, but not in wild birds. The pattern of differentially expressed genes suggest that this isolate of H5N1 virus evades the host innate immune response by attenuating interferon (IFN)-inducible signalling possibly by down regulating the signalling from type I IFN (IFNAR1 and IFNAR2) and type II IFN receptors, upregulation of the signalling inhibitors suppressor of cytokine signalling 1 (SOCS1) and SOCS3 and altering the expression of toll-like receptors (TLRs). This may be the reason for disease and mortality in crows.

Valproic acid enhances the anti-tumor effect of pegylated interferon-α towards pancreatic cancer cell lines.

BACKGROUND: Valproic acid (VPA) acts as a specific inhibitor of class I HDACs and it use has been proven to be safe since a long time. MATERIALS AND METHODS: In the present study, we investigated the effect of VPA in the combination with pegylated interferon-α (PEG-IFNα) in inhibition of cell proliferation of human pancreatic cancer cell lines. RESULTS: VPA enhanced the effect of PEG-IFNα, and the effect was decreased by the caspase inhibitor. VPA alone and VPA in combination with PEG-IFNα increased the expression of interferon-α receptor and interferon regulatory factor 8. CONCLUSION: The combination of VPA and PEG-IFNα can be useful for the treatment of pancreatic cancer.

Inteleukin-23 promotes interferon-α responsiveness in hepatitis C virus/HIV-coinfected patients.

Patients coinfected with HIV and hepatitis C virus (HCV) have poor to modest rates of response with interferon-based therapies, which remain a backbone of the treatment in HIV/HCV-coinfected patients. The mechanisms responsible for poor responsiveness to interferon are not well described. In this study a targeted proteomic analysis of plasma from 42 patients infected with both HIV and HCV and undergoing therapy for HCV with peginterferon and ribavirin was performed. Higher baseline plasma levels of interleukin (IL)-23 were associated with sustained virologic response. Further investigation of how IL-23 facilitates interferon (IFN) responsiveness, as evidenced by a >2-fold increase in most interferon-stimulated genes (ISGs), revealed that IL-23 indirectly enhances IFN signaling in peripheral blood mononuclear cells and HCV continuous culture system by preventing the down-regulation of the IFNAR2 receptor after exposure to IFN-α. These findings suggest a unique role of the IL-23 pathway in enhancing host response to type I interferons, thereby facilitating eradication of HCV. Low levels of IL-23 present in plasma of nonresponders may reflect an impaired immune state that in the case of HIV/HCV-coinfected subjects could potentially lead to disruption of TH17 CD4(+) T cells. This study suggests a major role for HIV-associated immune dysregulation present in HIV-infected subjects that subsequently determines the overall responsiveness to exogenous interferon-α-based HCV therapy.

Effect of all-trans-retinoic acid on enterovirus 71 infection in vitro.

Our previous studies have shown that vitamin A (VA) status is associated with antiviral immunity and pathogenic conditions in enterovirus 71 (EV71)-infected children. In the present study, we established an in vitro model to investigate the effects and potential mechanism of the antiviral activity of VA. Human monocytic U937 cells were cultured in vitro and infected with EV71. All-trans-retinoic acid (ATRA), the active metabolite of VA, and Ro 41-5253, a retinoic acid receptor-α (RAR-α) antagonist, were used as the experimental treatment agents. The percentage of EV71-infected cells and apoptosis induced by EV71 were determined using flow cytometry. The level of interferon-α (IFN-α) in the supernatants of the cultures was detected using ELISA. The expression of retinoid-induced gene I (RIG-I) and its downstream genes was examined with real-time quantitative PCR. The results indicated that ATRA reduced the percentage of EV71-infected cells and protected cells against EV71-induced apoptosis. Correspondingly, ATRA increased the production of IFN-α one of the most important antiviral cytokines, at both mRNA and protein levels in EV71-infected cells. In addition, the expression of RIG-I mRNA and its downstream genes was up-regulated by ATRA in EV71-infected cells. Ro 41-5253 abrogated the inhibitory effects of ATRA on EV71. The present findings suggest that ATRA is an interferon-inducing agent with antiviral activity against EV71 in vitro and that its actions are mediated at least in part by RAR-α activity and the RIG-I signalling pathway.

The MyD88 pathway in plasmacytoid and CD4+ dendritic cells primarily triggers type I IFN production against measles virus in a mouse infection model.

Infection by measles virus (MV) induces type I IFN via the retinoic acid-inducible gene I/melanoma differentiation-associated gene 5/mitochondrial antiviral signaling protein (MAVS) pathway in human cells. However, the in vivo role of the MAVS pathway in host defense against MV infection remains undetermined. CD150 transgenic (Tg) mice, which express human CD150, an entry receptor for MV, with the disrupting IFNR gene (Ifnar(-/-)), are susceptible to MV and serve as a model for MV infection. In this study, we generated CD150Tg/Mavs(-/-) mice and examined MV permissiveness compared with that in CD150Tg/Ifnar(-/-) mice. MV replicated mostly in the spleen of i.p.-infected CD150Tg/Ifnar(-/-) mice. Strikingly, CD150Tg/Mavs(-/-) mice were not permissive to MV in vivo because of substantial type I IFN induction. MV barely replicated in any other organs tested. When T cells, B cells, and dendritic cells (DCs) isolated from CD150Tg/Mavs(-/-) splenocytes were cultured with MV in vitro, only the DCs produced type I IFN. In vitro infection analysis using CD150Tg/Mavs(-/-) DC subsets revealed that CD4(+) and plasmacytoid DCs, but not CD8α(+) and CD8α(-)CD4(-) double negative DCs, were exclusively involved in type I IFN production in response to MV infection. Because CD150Tg/Mavs(-/-) mice turned permissive to MV by anti-IFNAR Ab, type I IFN produced by CD4(+) DCs and plasmacytoid DCs plays a critical role in antiviral protection for neighboring cells expressing IFNAR. Induction of type I IFN in these DC subsets was abolished by the MyD88 inhibitory peptide. Thus, production of type I IFN occurs via the MyD88-dependent and MAVS-independent signaling pathway during MV infection.

Genetically associated CD16(+)56(-) natural killer cell interferon (IFN)-αR expression regulates signaling and is implicated in IFN-α-induced hepatitis C virus decline.

BACKGROUND: Natural killer (NK) cells likely contribute to outcome of acute hepatitis C virus (HCV) infection and interferon (IFN)-induced control of chronic HCV infection. We previously observed IFN-αR and NKp30 expression associated with IFN-α-dependent NK cell activity. METHODS: Here, we examined CD16(+)56(-), CD16(+)56(+), and CD16(-)56(+) NK cell subset IFN-αR and NKp30 expression in relation to magnitude of HCV genotype 1 decrease during pegylated IFN-α plus ribavirin therapy. RESULTS: We observed greater baseline IFN-αR and NKp30 expression on CD16(+)56(+) and CD16(-)56(+) NK subsets in HCV-infected patients than in healthy control subjects. Baseline CD16(+)56(-) NK IFN-αR expression was associated with IFN-α-induced pSTAT1, and both were associated with magnitude of HCV decrease during pegylated IFN-α plus ribavirin therapy. Baseline CD16(+)56(-) NK IFN-αR expression was associated with race and interleukin 28B genotype, negatively associated with aspartate aminotransferase-to platelet ratio index, and positively associated with increase in NKp30 expression after in vivo IFN-α exposure. Finally, in vitro IFN-α2a-activated NK cytolysis of HCV-infected target cells was in part dependent on NKp30, and CD16(+)56(-) NK cell IFN-αR expression correlated with cytolytic activity. CONCLUSIONS: IFN-αR expression on CD16(+)56(-) NK cells during chronic HCV infection may in part be genetically determined, and level of expression regulates IFN-α signaling, which in turn may contribute to control of HCV infection.

Type I IFN-dependent T cell activation is mediated by IFN-dependent dendritic cell OX40 ligand expression and is independent of T cell IFNR expression.

Type I IFNs are important for direct control of viral infection and generation of adaptive immune responses. Recently, direct stimulation of CD4(+) T cells via type I IFNR has been shown to be necessary for the formation of functional CD4(+) T cell responses. In contrast, we find that CD4(+) T cells do not require intrinsic type I IFN signals in response to combined TLR/anti-CD40 vaccination. Rather, the CD4 response is dependent on the expression of type I IFNR (IFNαR) on innate cells. Further, we find that dendritic cell (DC) expression of the TNF superfamily member OX40 ligand was dependent on type I IFN signaling in the DC, resulting in a reduced CD4(+) T cell response that could be substantially rescued by an agonistic Ab to the receptor OX40. Taken together, we show that the IFNαR dependence of the CD4(+) T cell response is accounted for exclusively by defects in DC activation.

Mechanism of HCV's resistance to IFN-α in cell culture involves expression of functional IFN-α receptor 1.

The mechanisms underlying the Hepatitis C virus (HCV) resistance to interferon alpha (IFN-α) are not fully understood. We used IFN-α resistant HCV replicon cell lines and an infectious HCV cell culture system to elucidate the mechanisms of IFN-α resistance in cell culture. The IFN-α resistance mechanism of the replicon cells were addressed by a complementation study that utilized the full-length plasmid clones of IFN-α receptor 1 (IFNAR1), IFN-α receptor 2 (IFNAR2), Jak1, Tyk2, Stat1, Stat2 and the ISRE-luciferase reporter plasmid. We demonstrated that the expression of the full-length IFNAR1 clone alone restored the defective Jak-Stat signaling as well as Stat1, Stat2 and Stat3 phosphorylation, nuclear translocation and antiviral response against HCV in all IFN-α resistant cell lines (R-15, R-17 and R-24) used in this study. Moreover RT-PCR, Southern blotting and DNA sequence analysis revealed that the cells from both R-15 and R-24 series of IFN-α resistant cells have 58 amino acid deletions in the extracellular sub domain 1 (SD1) of IFNAR1. In addition, cells from the R-17 series have 50 amino acids deletion in the sub domain 4 (SD4) of IFNAR1 protein leading to impaired activation of Tyk2 kinase. Using an infectious HCV cell culture model we show here that viral replication in the infected Huh-7 cells is relatively resistant to exogenous IFN-α. HCV infection itself induces defective Jak-Stat signaling and impairs Stat1 and Stat2 phosphorylation by down regulation of the cell surface expression of IFNAR1 through the endoplasmic reticulum (ER) stress mechanisms. The results of this study suggest that expression of cell surface IFNAR1 is critical for the response of HCV to exogenous IFN-α.

Expanded potential for recombinant trisegmented lymphocytic choriomeningitis viruses: protein production, antibody production, and in vivo assessment of biological function of genes of interest.

The recombinant engineering of trisegmented lymphocytic choriomeningitis virus (LCMV) to express two genes of interest was recently reported. We used this technology to efficiently express green fluorescent protein (GFP) and the immunoregulatory gene product interleukin-10 (IL-10) in vitro, assess IL-10 function in vivo during viral meningitis, and generate specific, robust monoclonal antibody responses to IL-10. Tripartite viruses were attenuated in wild-type and TLR7(-/-) mice. However, IFNAR1(-/-) mice sustained systemic viral replication when 2 nucleotide substitutions from a persistent LCMV variant were present. These findings demonstrate the utility of tripartite LCMV in vitro and in vivo to study genes in the context of a well-defined model system.

Quantitative analysis of interferon alpha receptor subunit 1 and suppressor of cytokine signaling 1 gene transcription in blood cells of patients with chronic hepatitis C.

BACKGROUND: Interferon (IFN)-α receptor 1 (ifnar1) and suppressor of cytokine signaling 1 (socs1) transcription levels were quantified in peripheral blood mononuclear cells (PBMC) of 59 patients infected with hepatitis C virus (HCV) and 17 non-infected individuals. Samples were obtained from patients infected with HCV that were either untreated or treated with IFN-α2 plus ribavirin for 1 year and divided into responders and non-responders based on viral load reduction 6 months after treatment. Ifnar1 and socs1 transcription was quantified by real-time RT-PCR, and the fold difference (2(⁻ΔΔCT)) with respect to hprt housekeeping gene was calculated. RESULTS: Ifnar1 transcription increased significantly in HCV-infected patients either untreated (3.26 ± 0.31), responders (3.1 ± 0.23) and non-responders (2.18 ± 0.23) with respect to non-infected individuals (1 ± 0.34; P = 0.005). Ifnar1 transcription increased significantly (P = 0.003) in patients infected with HCV genotypes 1a (4.74 ± 0.25) and 1b (2.81 ± 0.25) but not in 1a1b (1.58 ± 0.21). No association was found of Ifnar1 transcription with disease progress, initial viral load or other clinical factors. With respect to socs1 transcription, values were similar for non-infected individuals (1 ± 0.28) and untreated patients (0.99 ± 0.41) but increased in responders (2.81 ± 0.17) and non-responder patients (1.67 ± 0.41). Difference between responder and non-responder patients was not statistically significant. Socs1 transcription increased in patients infected with HCV genotypes 1a and 1b (2.87 ± 0.45 and 2.22 ± 0.17, respectively) but not in 1a1b (1.28 ± 0.40). Socs1 transcript was absent in three patients infected with HCV genotype 1b. A weak correlation between ifnar1 and socs1 transcription was found, when Spearman's correlation coefficient was calculated. CONCLUSION: Our results suggest that HCV infection may up-regulate ifnar1 transcription. HCV genotypes differ in their capacity to affect ifnar1 and socs1 transcription, as well as in the ability to evade the antiviral response.