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This study clinically evaluated a novel PEGylated form of interferon beta-1a (PEG-IFN beta-1a), a potential first-line treatment for relapsing multiple sclerosis, in healthy volunteers. Two randomized, blinded phase I studies were conducted: a single-dose study (n = 60) comparing subcutaneous or intramuscular PEG-IFN beta-1a (63, 125, or 188 µg) with intramuscular unmodified IFN beta-1a 30 µg and a multiple-dose study (n = 69) comparing subcutaneous PEG-IFN beta-1a dosed once every 2 or 4 weeks with placebo. Assessments included pharmacokinetic and pharmacodynamic (serum neopterin and 2',5'-OAS) measures, exploratory immune assessments, safety, and tolerability. A dose-proportional increase in PEG-IFN beta-1a exposure was observed, with a 4-fold greater exposure at 63 µg (6 million international units [MIU]) of PEG-IFN beta-1a than with 30 µg (6 MIU) intramuscular unmodified IFN beta-1a. Increases in neopterin and 2',5'-OAS levels and changes in T helper cell pathway gene expression and lymphocyte subsets were greater and more sustained with PEG-IFN beta-1a than with unmodified IFN beta-1a. PEG-IFN beta-1a was well tolerated, with only transient reductions in absolute neutrophils and some lymphocytes. Flu-like symptoms were a commonly reported adverse event. These data support the continued clinical development of PEG-IFN beta-1a as a potentially effective treatment for patients with relapsing multiple sclerosis.

IFNs transduce signals by binding to cell surface receptors and activating cellular pathways and regulatory networks that control transcription of IFN-stimulated genes (ISGs) and mRNA translation, leading to generation of protein products that mediate biological responses. Previous studies have shown that type I IFN receptor-engaged pathways downstream of AKT and mammalian target of rapamycin complex (mTORC) 1 play important roles in mRNA translation of ISGs and the generation of IFN responses, but the roles of mTORC2 complexes in IFN signaling are unknown. We provide evidence that mTORC2 complexes control IFN-induced phosphorylation of AKT on serine 473 and their function is ultimately required for IFN-dependent gene transcription via interferon-stimulated response elements. We also demonstrate that such complexes exhibit regulatory effects on other IFN-dependent mammalian target of rapamycin-mediated signaling events, likely via engagement of the AKT/mTORC1 axis, including IFN-induced phosphorylation of S6 kinase and its effector rpS6, as well as phosphorylation of the translational repressor 4E-binding protein 1. We also show that induction of ISG protein expression and the generation of antiviral responses are defective in Rictor and mLST8-KO cells. Together, our data provide evidence for unique functions of mTORC2 complexes in the induction of type I IFN responses and suggest a critical role for mTORC2-mediated signals in IFN signaling.

Human interferon (IFN) β has well established beneficial effects in treating relapsing forms of multiple sclerosis, but current first-line treatment requires frequent (from daily to weekly) parenteral administration. A 20-kDa polyethylene glycol (PEG)-conjugated IFN β-1a (PEG-IFN β-1a) is being developed to decrease the frequency of administration and improve patient convenience and compliance. We present pharmacokinetic (PK) and pharmacodynamic (PD) parameters, immunogenicity, and safety of PEG-IFN β-1a in Rhesus monkeys in support of a phase 1 clinical trial. Two single-dose PK/PD studies and one 5-week repeat-dose toxicity study compliant with good laboratory practice were conducted. The PK of IFN β-1a and PEG-IFN β-1a were modeled with a two-compartment model, and the link between drug concentration and neopterin response (PD marker) was described with an indirect stimulatory model. PEG-IFN β-1a showed greater exposure, longer half-life, lower clearance, and reduced volume of distribution than unmodified IFN β-1a. Consistent with the pharmacology of type I IFNs, PEG-IFN β-1a resulted in the elevation of neopterin concentration, a transient body temperature increase, and a reversible lymphocyte count decrease. As expected, neutralizing antibodies to PEG-IFN β-1a formed in almost all monkeys after 5 weeks of treatment, which resulted in significantly reduced drug exposure and abrogation of neopterin induction. There were no drug-related adverse effects at doses up to 100 μg/kg (11 MIU/kg) given subcutaneously or intramuscularly once weekly for 5 weeks. The no-observed-adverse-effect level was determined to be 100 μg/kg (11 MIU/kg), the highest dose tested.

Type I interferons (IFNs) function as the first line of defense against viral infections by modulating cell growth, establishing an antiviral state and influencing the activation of various immune cells. Viruses such as influenza have developed mechanisms to evade this defense mechanism and during infection with influenza A viruses, the non-structural protein 1 (NS1) encoded by the virus genome suppresses induction of IFNs-α/β. Here we show that expression of avian H5N1 NS1 in HeLa cells leads to a block in IFN signaling. H5N1 NS1 reduces IFN-inducible tyrosine phosphorylation of STAT1, STAT2 and STAT3 and inhibits the nuclear translocation of phospho-STAT2 and the formation of IFN-inducible STAT1:1-, STAT1:3- and STAT3:3- DNA complexes. Inhibition of IFN-inducible STAT signaling by NS1 in HeLa cells is, in part, a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. In support of this NS1-mediated inhibition, we observed a reduction in expression of ifnar1 in ex vivo human non-tumor lung tissues infected with H5N1 and H1N1 viruses. Moreover, H1N1 and H5N1 virus infection of human monocyte-derived macrophages led to inhibition of both ifnar1 and ifnar2 expression. In addition, NS1 expression induces up-regulation of the JAK/STAT inhibitors, SOCS1 and SOCS3. By contrast, treatment of ex vivo human lung tissues with IFN-α results in the up-regulation of a number of IFN-stimulated genes and inhibits both H5N1 and H1N1 virus replication. The data suggest that NS1 can directly interfere with IFN signaling to enhance viral replication, but that treatment with IFN can nevertheless override these inhibitory effects to block H5N1 and H1N1 virus infections.

Background: Few data exist concerning conversion to secondary progressive MS in patients treated with disease-modifying therapies. Objective: Determine the proportion of alemtuzumab-treated patients converting from relapsing-remitting to secondary progressive MS during the CARE-MS core and extension studies. Methods: Patients (N = 811) were analyzed post hoc for secondary progressive MS conversion. Optimal conversion definition: Expanded Disability Status Scale (EDSS) score ≥4, pyramidal functional system score ≥2, and confirmed progression over ≥3 months including confirmation within the functional system leading to progression, independent of relapse. Results: Over 6.2 years median follow-up, 20 alemtuzumab-treated patients converted (Kaplan-Meier estimate, 2.7%; 95% confidence interval, 1.8%–4.2%). Sensitivity analysis accounting for dropouts showed similar results (3%), as did analyses using alternative definitions with different EDSS thresholds and/or confirmation periods, and analysis of core study subcutaneous interferon beta-1a-treated patients who received alemtuzumab in the extension. Patients converting to secondary progressive MS were older, and had higher EDSS scores and greater brain lesion volumes at baseline, but did not need additional alemtuzumab or other therapies. Conclusions: The 6-year conversion rate to secondary progressive MS was low for alemtuzumab-treated patients, supporting further study of the role alemtuzumab may play in reducing risk of secondary progression. ClinicalTrials.gov identifiers: NCT00530348, NCT00548405, NCT00930553. [ABSTRACT FROM AUTHOR]

Background: Alemtuzumab is a highly effective therapy for relapsing-remitting multiple sclerosis (RRMS), and immune thrombocytopenia (ITP) has been identified as a risk. Objective: To examine ITP incidence, treatment, and outcomes during the clinical development of alemtuzumab for RRMS and discuss postmarketing experience outside clinical trials. Methods: CAMMS223 and Comparison of Alemtuzumab and Rebif® Efficacy in Multiple Sclerosis (CARE-MS) I and II investigated two annual courses of alemtuzumab 12 mg (or 24 mg in CAMMS223/CARE-MS II) versus subcutaneous interferon beta-1a three times per week. Patients completing core studies could enroll in an extension. Monthly monitoring for ITP continued until 48 months after the last alemtuzumab infusion. Results: Of 1485 alemtuzumab-treated MS patients in the clinical development program, 33 (2.2%) developed ITP (alemtuzumab 12 mg, 24 [2.0%]; alemtuzumab 24 mg, 9 [3.3%]) over median 6.1 years of follow-up after the first infusion; most had a sustained response to first-line ITP therapy with corticosteroids, platelets, and/or intravenous immunoglobulin. All cases occurred within 48 months of the last alemtuzumab infusion. Postmarketing surveillance data suggest that the ITP incidence is not higher in clinical practice than in clinical trials. Conclusion: Alemtuzumab-associated ITP occurs in approximately 2% of patients and is responsive to therapy. Careful monitoring is key for detection and favorable outcomes. [ABSTRACT FROM AUTHOR]

To date there are no approved antiviral drugs for the treatment of Ebola virus disease (EVD). Based on our in vitro evidence of antiviral activity of interferon (IFN)-ß activity against Ebola virus, we conducted a single arm clinical study in Guinea to evaluate the safety and therapeutic efficacy of IFN β-1a treatment for EVD. Nine individuals infected with Ebola virus were treated with IFN β-1a and compared retrospectively with a matched cohort of 21 infected patients receiving standardized supportive care only during the same time period at the same treatment unit. Cognizant of the limitations of having treated only 9 individuals with EVD, the data collected are cautiously considered. When compared to supportive care only, IFN β-1a treatment seemed to facilitate viral clearance from the blood and appeared associated with earlier resolution of disease symptoms. Survival, calculated from the date of consent for those in the trial and date of admission from those in the control cohort, to the date of death, was 19% for those receiving supportive care only, compared to 67% for those receiving supportive care plus IFN β-1a. Given the differences in baseline blood viremia between the control cohort and the IFN-treated cohort, an additional 17 controls were included for a subset analysis, from other treatment units in Guinea, matched with the IFN-treated patients based on age and baseline blood viremia. Subset analyses using this expanded control cohort suggests that patients without IFN β-1a treatment were ~ 1.5–1.9 fold more likely to die than those treated. Viewed altogether the results suggest a rationale for further clinical evaluation of IFN β-1a. [ABSTRACT FROM AUTHOR]

The contributions of IFN regulatory factor (IRF) 3/7 and the type I IFNs IFN-α/β to the innate host defense have been extensively investigated; however, their role in thymic development is less clear. In this study, we show that mice lacking the type I IFN receptor IFN-α/β receptor (IFNAR) or the downstream transcription factor STAT1 harbor a significant reduction in self-Ag-presenting, autoimmune regulator (AIRE)+ medullary thymic epithelial cells (mTECs). Constitutive IFNAR signaling occurs in the thymic medulla in the absence of infection or inflammation. Receptor activator for NF-κB (RANK) ligand stimulation results in IFN-β upregulation, which in turn inhibits RANK signaling and facilitates AIRE expression in mTECs. Finally, we find that IRF7 is required for thymic IFN-β induction, maintenance of thymic architecture, and mTEC differentiation. We conclude that spatially and temporally coordinated cross talks between the RANK ligand/RANK and IRF7/IFN-β/IFNAR/STAT1 pathways are essential for differentiation of AIRE+ mTECs. [ABSTRACT FROM AUTHOR]

An ability to sense pathogens by a number of specialized cell types including the dendritic cells plays a central role in host's defenses. Activation of these cells through the stimulation of the pathogen-recognition receptors induces the production of a number of cytokines including Type I interferons (IFNs) that mediate the diverse mechanisms of innate immunity. Type I IFNs interact with the Type I IFN receptor, composed of IFNAR1 and IFNAR2 chains, to mount the host defense responses. However, at the same time, Type I IFNs elicit potent anti-proliferative and pro-apoptotic effects that could be detrimental for IFN-producing cells. Here, we report that the activation of p38 kinase in response to pathogen-recognition receptors stimulation results in a series of phosphorylation events within the IFNAR1 chain of the Type I IFN receptor. This phosphorylation promotes IFNAR1 ubiquitination and accelerates the proteolytic turnover of this receptor leading to an attenuation of Type I IFN signaling and the protection of activated dendritic cells from the cytotoxic effects of autocrine or paracrine Type I IFN. In this paper we discuss a potential role of this mechanism in regulating the processes of innate immunity. [ABSTRACT FROM AUTHOR]

Type I interferons (IFNs) function as the first line of defense against viral infections by modulating cell growth, establishing an antiviral state and influencing the activation of various immune cells. Viruses such as influenza have developed mechanisms to evade this defense mechanism and during infection with influenza A viruses, the non-structural protein 1 (NS1) encoded by the virus genome suppresses induction of IFNs-α/β. Here we show that expression of avian H5N1 NS1 in HeLa cells leads to a block in IFN signaling. H5N1 NS1 reduces IFN-inducible tyrosine phosphorylation of STAT1, STAT2 and STAT3 and inhibits the nuclear translocation of phospho-STAT2 and the formation of IFN-inducible STAT1:1-, STAT1:3- and STAT3:3- DNA complexes. Inhibition of IFN-inducible STAT signaling by NS1 in HeLa cells is, in part, a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. In support of this NS1-mediated inhibition, we observed a reduction in expression of ifnar1 in ex vivo human non-tumor lung tissues infected with H5N1 and H1N1 viruses. Moreover, H1N1 and H5N1 virus infection of human monocyte-derived macrophages led to inhibition of both ifnar1 and ifnar2 expression. In addition, NS1 expression induces up-regulation of the JAK/STAT inhibitors, SOCS1 and SOCS3. By contrast, treatment of ex vivo human lung tissues with IFN-a results in the up-regulation of a number of IFN-stimulated genes and inhibits both H5N1 and H1N1 virus replication. The data suggest that NS1 can directly interfere with IFN signaling to enhance viral replication, but that treatment with IFN can nevertheless override these inhibitory effects to block H5N1 and H1N1 virus infections. [ABSTRACT FROM AUTHOR]

Abstract: Influenza viruses are important human pathogens, associated throughout history with worldwide outbreaks and pandemics. The antiviral effects of interferon (IFN)-αs/β against influenza virus infections are well recognized, yet the mechanisms whereby IFNs exert their immunomodulatory effects on an anti-influenza response remain ill-defined. Here, we describe the effects of IFN-β treatment on the immune response during a respiratory influenza (A/WSN/33) A virus infection of mice. A single dose of IFN-β (1×105 U) enhanced DC migration into the draining lymph node (DLN) on day 3 post-intranasal infection, and subsequently inhibited the migration from the lungs into the DLN of a newly identified late activator antigen-presenting cell population associated with type 2 immunity, LAPC. IFN-β treatment polarized the immune response towards a type 1 immune response, eliciting enhanced TH1 effector and cytolytic T cell responses, but diminished TH2 effector T cell responses in both the DLN and lung tissues of influenza virus-infected mice. Associated with the polarization towards a type 1 immune response, IFN-β treatment of mice resulted in accelerated viral clearance and diminished pulmonary eosinophilia in infected lung tissues. [ABSTRACT FROM AUTHOR]

Phosphorylation of the degron of the IFNAR1 chain of the type I interferon (IFN) receptor triggers ubiquitination and degradation of this receptor and, therefore, plays a crucial role in negative regulation of IFN-α/β signaling. Besides the IFN-stimulated and Jak activity-dependent pathways, a basal ligand-independent phosphorylation of IFNAR1 has been described and implicated in downregulating IFNAR1 in response to virus-induced endoplasmic reticulum (ER) stress. Here we report purification and characterization of casein kinase 1α (CK1α) as a bona fide major IFNAR1 kinase that confers basal turnover of IFNAR1 and cooperates with ER stress stimuli to mediate phosphorylation-dependent degradation of IFNAR1. Activity of CK1α was required for phosphorylation and downregulation of IFNAR1 in response to ER stress and viral infection. While many forms of CK1 were capable of phosphorylating IFNAR1 in vitro, human CK1α and L-CK1 produced by the protozoan Leishmania major were also capable of increasing IFNAR1 degron phosphorylation in cells. Expression of leishmania CK1 in mammalian cells stimulated the phosphorylation-dependent downregulation of IFNAR1 and attenuated its signaling. Infection of mammalian cells with L. major modestly decreased IFNAR1 levels and attenuated cellular responses to IFN-α in vitro. We propose a role for mammalian and parasite CK1 enzymes in regulating IFNAR1 stability and type I IFN signaling. [ABSTRACT FROM AUTHOR]

Alpha interferon (IFN-α) controls homeostasis of hematopoietic stem cells, regulates antiviral resistance, inhibits angiogenesis, and suppresses tumor growth. This cytokine is often used to treat cancers and chronic viral infections. The extent of cellular responses to IFN-α is limited by the IFN-induced ubiquitination and degradation of the IFN-α/β receptor chain 1 (IFNAR1) chain of the cognate receptor. IFNAR1 ubiquitination is facilitated by the βTrcp E3 ubiquitin ligase that is recruited to IFNAR1 upon its degron phosphorylation, which is induced by the ligand. Here we report identification of protein kinase D2 (PKD2) as a kinase that mediates the ligand-inducible phosphorylation of IFNAR1 degron and enables binding of βTrcp to the receptor. Treatment of cells with IFN-α induces catalytic activity of PKD2 and stimulates its interaction with IFNAR1. Expression and kinase activity of PKD2 are required for the ligand-inducible stimulation of IFNAR1 ubiquitination and endocytosis and for accelerated proteolytic turnover of IFNAR1. Furthermore, inhibition or knockdown of PKD2 robustly augments intracellular signaling induced by IFN-α and increases the efficacy of its antiviral effects. The mechanisms of the ligand-inducible elimination of IFNAR1 are discussed, along with the potential medical significance of this regulation. [ABSTRACT FROM AUTHOR]

Tyk2, a member of the Jak family of protein tyrosine kinases, is critical for the biological actions of alpha/beta interferon (IFN-α/ß). Although Tyk2-/- mice are phenotypically normal, they exhibit abnormal responses to inflammatory challenges in a variety of cells isolated from Tyk2-/- mice. The reported phenotypic alterations in both Tyk2-null cells and mice are consistent with the possibility that the expression of this tyrosine kinase may regulate mitochondrial function. We report here that Tyk2-null pro-B cells are markedly deficient in basal oxygen consumption and exhibit a significant decrease in steady-state cellular ATP levels compared to wild-type cells. Tyk2-null cells also exhibit impaired complex I, III, and IV function of the mitochondrial electron transport chain. Reconstitution of Tyk2-null pro-B cells with either the wild type or a kinase-inactive mutant of Tyk2 restores basal mitochondrial respiration. By contrast, the kinase activity of Tyk2 is required for maintenance of both complex I-dependent mitochondrial respiration as well as induction of apoptosis in cells incubated with IFN-ß. Consistent with the role of Tyk2 in the regulation of tyrosine phosphorylation of Stat3, expression of a constitutively active Stat3 can restore the mitochondrial respiration in Tyk2-null cells treated with IFN-ß. Finally, Tyk2-/- mice show decreased exercise tolerance compared to wild-type littermates. Our results implicate a novel role for Tyk2 kinase and Stat3 phosphorylation in mitochondrial respiration. [ABSTRACT FROM AUTHOR]

Type I interferons activate cellular responses by forming a ternary complex with two receptor components, IFNAR1 and IFNAR2. While the binding of the IFNAR2 receptor to interferon is of high affinity and well characterized, the binding to IFNAR1 is weak, transient, and poorly understood. Here, we mapped the complete binding region of IFNAR1 on IFNα2 by creating a panel of 21 single alanine mutant proteins, and determined their binding affinities. The IFNAR1 binding site on IFNα2 maps to the center of the B and C helices, opposite to the binding site for IFNAR2. No hot spots for binding were found in the interface, with individual mutations having an up to fivefold effect on binding. Of the nine residues that affected binding, three adjacent conserved residues, located on the B helix, conferred an increase in the binding affinity to IFNAR1, as well as an increase in the biological activity of the interferon mutant. This suggests that binding of alpha interferons to the IFNAR1 receptor is sub-optimal. A correlation between binding affinity and biological activity was found, albeit not across the whole range of affinities. In WISH cells, but not DAUDI cells, the anti-proliferative activity was markedly affected by fluctuations in the IFNα2 affinity towards the IFNAR1 receptor. On the other hand, the antiviral activity of interferons on WISH cells seems to change in accordance to the binding affinity towards IFNAR1 only as long as the binding affinity is not beyond twofold of the wild-type. In accordance, the biological roles of the two interferon-receptor subunits are discussed. [Copyright &y& Elsevier]

Interferons (IFNs) have important antiviral and antineoplastic properties, but the precise mechanisms required for generation of these responses remain to be defined. We provide evidence that during engagement of the Type I IFN receptor (IFNR), there is up-regulation of expression of Sprouty (Spry) proteins 1, 2, and 4. Our studies demonstrate that IFN-inducible up-regulation of Spry proteins is Mnk kinase-dependent and results in suppressive effects on the IFN-activated p38 MAP kinase (MAPK), the function of which is required for transcription of interferon-stimulated genes (ISGs). Our data establish that ISG15 mRNA expression and IFN-dependent antiviral responses are enhanced in Spry1,2,4 triple knock-out mouse embryonic fibroblasts, consistent with negative feedback regulatory roles for Spry proteins in IFN-mediated signaling. In other studies, we found that siRNA-mediated knockdown of Spry1, Spry2, or Spry4 promotes IFN-inducible antileukemic effects in vitro and results in enhanced suppressive effects on malignant hematopoietic progenitors from patients with polycythemia vera. Altogether, our findings demonstrate that Spry proteins are potent regulators of Type I IFN signaling and negatively control induction of Type I IFN-mediated biological responses. [ABSTRACT FROM AUTHOR]

Constitutive activity of Bcr-abl fusion protein kinase causes chronic myeloid leukemia (CML). Inhibitors of Bcr-abl such as imatinib mesylate (IM) have replaced the cytokine interferon alpha (IFNα) as the primary treatment for the management of patients with this malignancy. We found that pre-treatment of CML cells with IM augments the anti-growth effects of IFNα. Furthermore, introduction of Bcr-abl into non-CML cells inhibits the cellular responses to IFNα. This inhibition is mediated via a mechanism that involves activation of protein kinase D2 (PKD2). The latter promotes an accelerated phosphorylation-dependent degradation of the interferon α/β receptor 1 chain of the type I interferon receptor, leading to attenuation of IFNα signaling. We discuss the relationship between Bcr-abl activity and IFNα signaling as a molecular basis of the combination of inhibitors of Bcr-abl and IFNα for CML treatment. [ABSTRACT FROM AUTHOR]

Angiogenesis is stimulated by vascular endothelial growth factor (VEGF) and antagonized by type 1 interferons, including IFN-α/β. On engaging their respective receptors (VEGFR2 and IFNAR), both stimuli activate protein kinase D2 (PKD2) and type 1 IFNs require PKD2 activation and recruitment to IFNAR1 to promote the phosphorylation-dependent ubiquitination, down-regulation, and degradation of the cognate receptor chain, IFNAR1. Data reveal that PKD2 activity is dispensable for VEGF-stimulated down-regulation of VEGFR2. Remarkably, VEGF treatment promotes the recruitment of PKD2 to IFNAR1 as well as ensuing phosphorylation, ubiquitination, and degradation of IFNAR1. In cells exposed to VEGF, phosphorylation-dependent degradation of IFNAR1 leads to an inhibition of type 1 IFN signaling and is required for efficient VEGF-stimulated angiogenesis. Importance of this mechanism for proangiogenic or antiangiogenic responses in cells exposed to counteracting stimuli and the potential medical significance of this regulation are discussed. [ABSTRACT FROM AUTHOR]

Phosphorylation-dependent ubiquitination and degradation of the IFNAR1 chain of type I interferon (IFN) receptor is a robust and specific mechanism that limits the magnitude and duration of IFNα/β signaling. Besides the ligand-inducible IFNAR1 degradation, the existence of an "inside-out" signaling that accelerates IFNAR1 turnover in the cells undergoing the endoplasmic reticulum (ER) stress and activated unfolded protein responses has been recently described. The latter pathway does not require either presence of ligands (IFNα/β) or catalytic activity of Janus kinases (JAK). Instead, this pathway relies on activation of the PKR-like ER kinase (PERK) and ensuing specific priming phosphorylation of IFNAR1. Here, we describe studies that identify the stress activated p38 protein kinase as an important regulator of IFNAR1 that acts downstream of PERK. Results of the experiments using pharmacologic p38 kinase inhibitors, RNA interference approach, and cells from p38α knock-out mice suggest that p38 kinase activity is required for priming phosphorylation of IFNAR1 in cells undergoing unfolded protein response. We further demonstrate an important role of p38 kinase in the ligand-independent stimulation of IFNAR1 ubiquitination and degradation and ensuing attenuation of IFNα/β signaling and anti-viral defenses. We discuss the distinct importance of p38 kinase in regulating the overall responses to type I IFN in cells that have been already exposed to IFNα/β versus those cells that are yet to encounter these cytokines. [ABSTRACT FROM AUTHOR]

Abstract: Secondary degeneration leads to an expansion of the initial tissue damage sustained during a spinal cord injury (SCI). Dampening the cellular inflammatory response that contributes to this progressive tissue damage is one possible strategy for neuroprotection after acute SCI. We initially examined whether treatment with a PEGylated form of rat interferon-beta (IFN-β) would modulate the expression of several markers of inflammation and neuroprotection at the site of a unilateral cervical level 5 contusion injury. Adult female Sprague–Dawley rats were injured using the Infinite Horizon Impactor at a force of 200 kdyn (equivalent to a severe injury) and a mean displacement of 1600–1800 μm. A single dose (5×106 units) of PEGylated IFN-β or vehicle was administered 30 min following SCI. Here we demonstrate temporal changes in pro- and anti-inflammatory cytokine levels and the expression of heat shock proteins and iNOS (involved in neuroprotection) at the lesion epicenter and one segment caudally after SCI and PEG IFN-β treatment. The results suggested a potential therapeutic treatment strategy for modulation of secondary damage after acute SCI. Therefore, we examined whether acute treatment with PEG IFN-β would improve forelimb function alone or when combined with forced exercise (Ex). Animals began the Ex paradigm 5 days post SCI and continued for 5 days/week over 8 weeks. Locomotion (forelimb locomotor scale [FLS], hindlimb BBB, and TreadScan) and sensorimotor function (grid walking) was tested weekly. Additional outcome measures included lesion size and glial cell reactivity. Significant FLS improvements occurred at 1 week post SCI in the PEGylated IFN-β-treated group but not at any other time point or with any other treatment approaches. These results suggest that this acute neuroprotective treatment strategy does not translate into long term behavioral recovery even when combined with forced exercise. [Copyright &y& Elsevier]

Phosphorylation-dependent ubiquitination and ensuing down-regulation and lysosomal degradation of the interferon α/β receptor chain 1 (IFNAR1) of the receptor for Type I interferons play important roles in limiting the cellular responses to these cytokines. These events could be stimulated either by the ligands (in a Janus kinase-dependent manner) or by unfolded protein response (UPR) inducers including viral infection (in a manner dependent on the activity of pancreatic endoplasmic reticulum kinase). Both ligand-dependent and -independent pathways converge on phosphorylation of Ser535 within the IFNAR1 degron leading to recruitment of β-Trcp E3 ubiquitin ligase and concomitant ubiquitination and degradation. Casein kinase 1α (CK1α) was shown to directly phosphorylate Ser535 within the ligand-independent pathway. Yet given the constitutive activity of CK1α, it remained unclear how this pathway is stimulated by UPR. Here we report that induction of UPR promotes the phosphorylation of a proximal residue, Ser532, in a pancreatic endoplasmic reticulum kinase-dependent manner. This serine serves as a priming site that promotes subsequent phosphorylation of IFNAR1 within its degron by CK1α. These events play an important role in regulating ubiquitination and degradation of IFNAR1 as well as the extent of Type I interferon signaling. [ABSTRACT FROM AUTHOR]

Abstract: Ligand-specific negative regulation of cytokine-induced signaling relies on down regulation of the cytokine receptors. Down regulation of the IFNAR1 sub-unit of the Type I interferon (IFN) receptor proceeds via lysosomal receptor proteolysis, which is triggered by ubiquitination that depends on IFNAR1 serine phosphorylation. While IFN-inducible phosphorylation, ubiquitination, and degradation requires the catalytic activity of the Tyk2 Janus kinase, here we found the ligand- and Tyk2-independent pathway that promotes IFNAR1 phosphorylation, ubiquitination, and degradation when IFNAR1 is expressed at high levels. A major cellular kinase activity that is responsible for IFNAR1 phosphorylation in vitro does not depend on either ligand or Tyk2 activity. Inhibition of ligand-independent IFNAR1 degradation suppresses cell proliferation. We discuss the signaling events that might lead to ubiquitination and degradation of IFNAR1 via ligand-dependent and independent pathways and their potential physiologic significance. [Copyright &y& Elsevier]

Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)--clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-α/β receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCF[sup βTrcp] (Skp1--Cullin1--F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process. [ABSTRACT FROM AUTHOR]

Abstract: Although the antiviral actions of interferons (IFNs) are observed in most types of cells, the antiproliferative effects of IFNα/β are variable as are the mechanisms of growth inhibition that may or may not be due to the induction of apoptosis. To understand more about the mechanisms that are responsible for IFNα/β-stimulated apoptosis, we have characterized a new human Jurkat T cell variant named H123 where IFNα activates programmed cell death (PCD). No differences in IFNα-stimulated, Stat-dependent gene expression were detected between H123 cells and the parental Jurkat cells, which are growth inhibited, but do not undergo apoptosis with IFNα. Although IFNα stimulates the activity of both caspase 3 and 9 in H123 cells, the general caspase inhibitor Z-VAD only partially reverses the apoptotic actions of IFNα. Induction of apoptosis by IFNα occurs through a mitochondrial-dependent pathway in H123 cells, as demonstrated by the release of cytochrome C from the mitochondria. Furthermore, IFNα treatment of H123 cells stimulates the release of the serine protease HtrA2/Omi from the mitochondria, suggesting that it plays a role in the apoptotic actions of this cytokine. These results provide evidence for a novel type 1 IFN-mediated pathway that regulates apoptosis of T cells through a mitochondrial-dependent and caspase-dependent and independent pathway. [Copyright &y& Elsevier]

The growth-inhibitory effects of type 1 interferons (IFNs) (LFNα/β) are complex, and the role of apoptosis in their antigrowth effects is variable and not well understood. We have examined primary murine interleukin-7-dependent bone marrow-derived pro-B cells, where IFNβ, but not IFNα, induces programmed cell death (PCD). IFNα-stimulated apoptosis is the same in pro-B cells derived from wild type and Stat1-/- mice. However, in pro-B cells from Tyk2-/- mice, where there is normal activation of Stat1 and Stat2, IFNα-stimulated PCD is not observed. Loss of B cells in lymphocytic choriomeningitis virus-infected mice has been shown to be mediated through the expression of IFNα/β (1). In wild type mice infected with lymphocytic choriomeningitis virus, there is a greater loss of B cells in the bone marrow and spleen than in Tyk2-/- mice infected with the virus, suggesting that the expression of this kinase plays an in vivo role in IFNα/β-mediated PCD. In contrast to IFNβ-stimulated tyrosine phosphorylation of Stat 1 and Stat2, Stat3 tyrosine phosphorylation is defective in Tyk2-/- pro-B cells, suggesting that this Stat family member is required for apoptosis. In support of this hypothesis, inhibition of Stat3 activation in wild type B cells reverses the apoptotic effects of IFNβ. Furthermore, expression of a constitutively active form of Stat3 in Tyk2-/- B cells partially restores IFNβ-stimulated PCD. These results demonstrate an important role of Tyk2-mediated tyrosine phosphorylation of Stat3 in the ability of IFNα to stimulate apoptosis of primary pro-B cells. [ABSTRACT FROM AUTHOR]

Interferons (IFNs) play critical roles in host defense by modulating the expression of various genes via tyrosine phosphorylation of STAT transcription factors. IFN-α/β activates another important transcription factor, nuclear factor-κB (NF-κB), but its role in IFN-mediated activity is poorly understood. Sensitivity to the antiviral and gene-inducing effects of IFN was examined in normal fibroblasts and in NF-κB knockout fibroblasts from p50. and p65-null mice. Antiviral assays demonstrated that NF-κB knockout fibroblasts were sensitized to the antiviral action of IFN. Moreover, analysis of IFNstimulated gene expression by real-time PCR demonstrated selective effects of NF-κB on gene expression. Our results demonstrate that a subset of IFN-stimulated genes is regulated through an NF-κB-dependent pathway and that NF-κB may regulate the sensitivity of cells to IFN-mediated antiviral activity. [ABSTRACT FROM AUTHOR]