Toxoplasma gondii Triggers Phosphorylation and Nuclear Translocation of Dendritic Cell STAT1 while Simultaneously Blocking IFNγ-Induced STAT1 Transcriptional Activity

The protozoan Toxoplasma gondii actively modulates cytokine-induced JAK/STAT signaling pathways to facilitate survival within the host, including blocking IFNγ-mediated STAT1-dependent proinflammatory gene expression. We sought to further characterize inhibition of STAT1 signaling in infected murine dendritic cells (DC) because this cell type has not previously been examined, yet is known to serve as an early target of in vivo infection. Unexpectedly, we discovered that T. gondii infection alone induced sustained STAT1 phosphorylation and nuclear translocation in DC in a parasite strain-independent manner. Maintenance of STAT1 phosphorylation required active invasion but intracellular parasite replication was dispensable. The parasite rhoptry protein ROP16, recently shown to mediate STAT3 and STAT6 phosphorylation, was not required for STAT1 phosphorylation. In combination with IFNγ, T. gondii induced synergistic STAT1 phosphorylation and binding of aberrant STAT1-containing complexes to IFNγ consensus sequence oligonucleotides. Despite these findings, parasite infection blocked STAT1 binding to the native promoters of the IFNγ-inducible genes Irf-1 and Lrg47, along with subsequent gene expression. These results reinforce the importance of parasite-mediated blockade of IFNγ responses in dendritic cells, while simultaneously showing that T. gondii alone induces STAT1 phosphorylation.     

Marburg Virus Evades Interferon Responses by a Mechanism Distinct from Ebola Virus

Previous studies have demonstrated that Marburg viruses (MARV) and Ebola viruses (EBOV) inhibit interferon (IFN)-α/β signaling but utilize different mechanisms. EBOV inhibits IFN signaling via its VP24 protein which blocks the nuclear accumulation of tyrosine phosphorylated STAT1. In contrast, MARV infection inhibits IFNα/β induced tyrosine phosphorylation of STAT1 and STAT2. MARV infection is now demonstrated to inhibit not only IFNα/β but also IFNγ-induced STAT phosphorylation and to inhibit the IFNα/β and IFNγ-induced tyrosine phosphorylation of upstream Janus (Jak) family kinases. Surprisingly, the MARV matrix protein VP40, not the MARV VP24 protein, has been identified to antagonize Jak and STAT tyrosine phosphorylation, to inhibit IFNα/β or IFNγ-induced gene expression and to inhibit the induction of an antiviral state by IFNα/β. Global loss of STAT and Jak tyrosine phosphorylation in response to both IFNα/β and IFNγ is reminiscent of the phenotype seen in Jak1-null cells. Consistent with this model, MARV infection and MARV VP40 expression also inhibit the Jak1-dependent, IL-6-induced tyrosine phosphorylation of STAT1 and STAT3. Finally, expression of MARV VP40 is able to prevent the tyrosine phosphorylation of Jak1, STAT1, STAT2 or STAT3 which occurs following over-expression of the Jak1 kinase. In contrast, MARV VP40 does not detectably inhibit the tyrosine phosphorylation of STAT2 or Tyk2 when Tyk2 is over-expressed. Mutation of the VP40 late domain, essential for efficient VP40 budding, has no detectable impact on inhibition of IFN signaling. This study shows that MARV inhibits IFN signaling by a mechanism different from that employed by the related EBOV. It identifies a novel function for the MARV VP40 protein and suggests that MARV may globally inhibit Jak1-dependent cytokine signaling.     

Interferon Alpha Signalling and Its Relevance for the Upregulatory Effect of Transporter Proteins Associated with Antigen Processing (TAP) in Patients with Malignant Melanoma

Introduction

Interferon alpha (IFNα) is routinely used in the clinical practice for adjuvant systemic melanoma therapy. Understanding the molecular mechanism of IFNα effects and prediction of response in the IFNα therapy regime allows initiation and continuation of IFNα treatment for responder and exclusion of non-responder to avoid therapy inefficacy and side-effects. The transporter protein associated with antigen processing-1 (TAP1) is part of the MHC class I peptide-loading complex, and important for antigen presentation in tumor and antigen presenting cells. In the context of personalized medicine, we address this potential biomarker TAP1 as a target of IFNα signalling.

Results

We could show that IFNα upregulates TAP1 expression in peripheral blood mononuclear cells (PBMCs) of patients with malignant melanoma receiving adjuvant high-dose immunotherapy. IFNα also induced expression of TAP1 in mouse blood and tumor tissue and suppressed the formation of melanoma metastasis in an in vivo B16 tumor model. Besides its expression, TAP binding affinity and transport activity is induced by IFNα in human monocytic THP1 cells. Furthermore, our data revealed that IFNα clearly activates phosphorylation of STAT1 and STAT3 in THP1 and A375 melanoma cells. Inhibition of Janus kinases abrogates the IFNα-induced TAP1 expression. These results suggest that the JAK/STAT pathway is a crucial mediator for TAP1 expression elicited by IFNα treatment.

Conclusion

We suppose that silencing of TAP1 expression provides tumor cells with a mechanism to escape cytotoxic T-lymphocyte recognition. The observed benefit of IFNα treatment could be mediated by the shown dual effect of TAP1 upregulation in antigen presenting cells on the one hand, and of TAP1 upregulation in ‘silent’ metastatic melanoma cells on the other hand. In conclusion, this work contributes to a better understanding of the mode of action of IFNα which is essential to identify markers to predict, assess and monitor therapeutic response of IFNα treatment in the future.     

The Induction of Lipocalin-2 Protein Expression in Vivo and in Vitro

Lipocalin-2 (LCN2) is secreted from adipocytes, and its expression is up-regulated in obese and diabetic mice and humans. LCN2 expression and secretion have been shown to be induced by two proinflammatory cytokines, IFNγ and TNFα, in cultured murine and human adipocytes. In these studies, we demonstrated that IFNγ and TNFα induced LCN2 expression and secretion in vivo. Although we observed a strong induction of LCN2 expression and secretion from white adipose tissue (WAT) depots, the induction of LCN2 varied among different insulin-sensitive tissues. Knockdown experiments also demonstrated that STAT1 is required for IFNγ-induced lipocalin-2 expression in murine adipocytes. Similarly, knockdown of p65 in adipocytes demonstrated the necessity of the NF-κB signaling pathway for TNFα-mediated effects on LCN2. Activation of ERKs by IFNγ and TNFα also affected STAT1 and NF-κB signaling through modulation of serine phosphorylation. ERK activation-induced serine phosphorylation of both STAT1 and p65 mediated the additive effects of IFNγ and TNFα on LCN2 expression. Our results suggest that these same mechanisms occur in humans as we observed STAT1 and NF-κB binding to the human LCN2 promoter in chromatin immunoprecipitation assays performed in human fat cells. These studies substantially increase our knowledge regarding the requirements and mechanisms used by proinflammatory cytokines to induce LCN2 expression.     

Serine Residues in the Cytosolic Tail of the T-cell Antigen Receptor α-Chain Mediate Ubiquitination and Endoplasmic Reticulum-associated Degradation of the Unassembled Protein

The T-cell antigen receptor (TCR) α-chain (TCRα) is a type I integral membrane protein that becomes ubiquitinated and targeted to the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway when it fails to assemble into the heteromeric TCR complex. Remarkably, TCRα has a cytosolic tail of only five amino acid residues (i.e. RLWSS), none of which is the conventional ubiquitin acceptor, lysine. Herein we report that substitution of two conserved serine residues in the cytosolic tail of TCRα to alanine decreased ubiquitination, whereas placement of additional serine residues enhanced it. Moreover, replacement of the cytosolic serine residues by other ubiquitinatable residues (i.e. cysteine, threonine, or lysine) allowed ubiquitination to take place. Serine-dependent ubiquitination perfectly correlated with targeting of TCRα for ERAD. We also found that this ubiquitination was mediated by the ER-localized ubiquitin ligase, HRD1. These findings indicate that serine-dependent, HRD1-mediated ubiquitination targets TCRα to the ERAD pathway.     

The TrxG Complex Mediates Cytokine Induced De Novo Enhancer Formation in Islets

To better understand how β-cells respond to proinflammatory cytokines we mapped the locations of histone 3 lysine 4 monomethylation (H3K4me1), a post-translational histone modification enriched at active and poised cis-regulatory regions, in IFNγ, Il-1β, and TNFα treated pancreatic islets. We identified 96,721 putative cis-regulatory loci, of which 3,590 were generated de novo, 3,204 had increased H3K4me1, and 5,354 had decreased H3K4me1 in IFNγ, Il-1β, and TNFα exposed islets. Roughly 10% of the de novo and increased regions were enriched for the repressive histone modification histone 3 lysine 27 trimethylation (H3K27me3) in untreated cells, and these were frequently associated with chemokine genes. We show that IFNγ, Il-1β, and TNFα exposure overcomes this repression and induces chemokine gene activation in as little as three hours, and that this expression persists for days in absence of continued IFNγ, Il-1β, and TNFα exposure. We implicate trithorax group (TrxG) complexes as likely players in the conversion of these repressed loci to an active state. To block the activity of these complexes, we suppressed Wdr5, a core component of the TrxG complexes, and used the H3K27me3 demethylase inhibitor GSK-J4. We show that GSK-J4 is particularly effective in blunting IFNγ, Il-1β, and TNFα-induced chemokine gene expression in β-cells; however, it induced significant islet-cell apoptosis and β-cell dysfunction. Wdr5 suppression also reduced IFNγ, Il-1β, and TNFα induced chemokine gene expression in β-cells without affecting islet-cell survival or β-cell function after 48hrs, but did begin to increase islet-cell apoptosis and β-cell dysfunction after four days of treatment. Taken together these data suggest that the TrxG complex is potentially a viable target for preventing cytokine induced chemokine gene expression in β-cells.     

Hemoglobin C Trait in Saskatchewan

In the beta chain of normal adult hemoglobin, the amino acid residues in positions six and seven are glutamic acid. In hemoglobin C, lysine replaces the glutamic acid residue in position six. Two hemoglobin variants have been described in which lysine replaces the glutamic acid residue in position seven.During a search for abnormalities of hemoglobin synthesis detectable by conventional electrophoretic techniques, some 20,000 blood samples were screened from the population of Saskatchewan. Two specimens gave an electrophoretic pattern of hemoglobins A and C. The abnormal hemoglobin was purified and “finger printed” and found to be the same as classical hemoglobin C (i.e. α₂β₂ 6 glu → lys). Both specimens had originated from healthy blood donors, one a Ghanaian student and the other a young white man of immigrant Irish-Scottish parents living near Saskatoon.     

IFN‐β rescues neurodegeneration by regulating mitochondrial fission via STAT5, PGAM5, and Drp1

Mitochondrial homeostasis is essential for providing cellular energy, particularly in resource‐demanding neurons, defects in which cause neurodegeneration, but the function of interferons (IFNs) in regulating neuronal mitochondrial homeostasis is unknown. We found that neuronal IFN‐β is indispensable for mitochondrial homeostasis and metabolism, sustaining ATP levels and preventing excessive ROS by controlling mitochondrial fission. IFN‐β induces events that are required for mitochondrial fission, phosphorylating STAT5 and upregulating PGAM5, which phosphorylates serine 622 of Drp1. IFN‐β signaling then recruits Drp1 to mitochondria, oligomerizes it, and engages INF2 to stabilize mitochondria–endoplasmic reticulum (ER) platforms. This process tethers damaged mitochondria to the ER to separate them via fission. Lack of neuronal IFN‐β in the Ifnb ^–/– model of Parkinson disease (PD) disrupts STAT5‐PGAM5‐Drp1 signaling, impairing fission and causing large multibranched, damaged mitochondria with insufficient ATP production and excessive oxidative stress to accumulate. In other PD models, IFN‐β rescues dopaminergic neuronal cell death and pathology, associated with preserved mitochondrial homeostasis. Thus, IFN‐β activates mitochondrial fission in neurons through the pSTAT5/PGAM5/^S622Drp1 pathway to stabilize mitochondria/ER platforms, constituting an essential neuroprotective mechanism.     

Comparison of the Regulation of β-Catenin Signaling by Type I,Type II and Type III Interferons in Hepatocellular Carcinoma Cells

Background/Objective

IFNs are a group of cytokines that possess potent antiviral and antitumor activities, while β-catenin pathway is a proliferative pathway involved in carcinogenesis. Interaction between these two pathways has not been well elaborated in hepatocellular carcinoma (HCC).

Methods

HCC cell lines, HepG2 and Huh7, were used in this study. β-catenin protein levels and corresponding signaling activities were observed by flow cytometry and luciferase assay, respectively. Cell proliferation was quantified by counting viable cells under microscope, and apoptosis by TUNEL assay. DKK1 and GSK3β levels were determined by flow cytometry. Secreted DKK1 was tested by ELISA. FLUD, S3I and aDKK1 were used to inhibit STAT1, STAT3 and DKK1 activities, respectively.

Results

Our findings show that all three types of IFNs, IFNα, IFNγ and IFNλ, are capable of inhibiting β-catenin signaling activity in HepG2 and Huh7 cells, where IFNγ was the strongest (p<0.05). They expressed suppression of cellular proliferation and induced apoptosis. IFNγ expressed greater induction ability when compared to IFNα and IFNλ (p<0.05). All tested IFNs could induce DKK1 activation but not GSK3β in HepG2 and Huh7 cells. IFNs induced STAT1 and STAT3 activation but by using specific inhibitors, we found that only STAT3 is vital for IFN-induced DKK1 activation and apoptosis. In addition, DKK1 inhibitor blocked IFN-induced apoptosis. The pattern of STAT3 activation by different IFNs is found consistent with the levels of apoptosis with the corresponding IFNs (p<0.05).

Conclusions

In hepatocellular carcinoma, all three types of IFNs are found to induce apoptosis by inhibiting β-catenin signaling pathway via a STAT3- and DKK1-dependent pathway. This finding points to a cross-talk between different IFN types and β-catenin signaling pathways which might be carrying a biological effect not only on HCC, but also on processes where the two pathways bridge.     

SH2 Modified STAT1 Induces HLA-I Expression and Improves IFN-γ Signaling in IFN-α Resistant HCV Replicon Cells

Background

We have developed multiple stable cell lines containing subgenomic HCV RNA that are resistant to treatment with interferon alpha (IFN-α. Characterization of these IFN-α resistant replicon cells showed defects in the phosphorylation and nuclear translocation of STAT1 and STAT2 proteins due to a defective Jak-STAT pathway.

Methodology/Principal Findings

In this study, we have developed an alternative strategy to overcome interferon resistance in a cell culture model by improving intracellular STAT1 signaling. An engineered STAT1-CC molecule with double cysteine substitutions in the Src-homology 2 (SH2) domains of STAT1 (at Ala-656 and Asn-658) efficiently phosphorylates and translocates to the nucleus of IFN-resistant cells in an IFN-γ dependent manner. Transfection of a plasmid clone containing STAT1-CC significantly activated the GAS promoter compared to wild type STAT1 and STAT3. The activity of the engineered STAT1-CC is dependent upon the phosphorylation of tyrosine residue 701, since the construct with a substituted phenylalanine residue at position 701 (STAT1-CC-Y701F) failed to activate GAS promoter in the replicon cells. Intracellular expression of STAT1-CC protein showed phosphorylation and nuclear translocation in the resistant cell line after IFN-γ treatment. Transient transfection of STAT1-CC plasmid clone into an interferon resistant cell line resulted in inhibition of viral replication and viral clearance in an IFN-γ dependent manner. Furthermore, the resistant replicon cells transfected with STAT1-CC constructs significantly up regulated surface HLA-1 expression when compared to the wild type and Y to F mutant controls.

Conclusions

These results suggest that modification of the SH2 domain of the STAT1 molecule allows for improved IFN-γ signaling through increased STAT1 phosphorylation, nuclear translocation, HLA-1 surface expression, and prolonged interferon antiviral gene activation.     

Epstein-Barr Virus Interferes with the Amplification of IFNα Secretion by Activating Suppressor of Cytokine Signaling 3 in Primary Human Monocytes

Background

Epstein-Barr virus is recognized to cause lymphoproliferative disorders and is also associated with cancer. Evidence suggests that monocytes are likely to be involved in EBV pathogenesis, especially due to a number of cellular functions altered in EBV-infected monocytes, a process that may affect efficient host defense. Because type I interferons (IFNs) are crucial mediators of host defense against viruses, we investigated the effect of EBV infection on the IFNα pathway in primary human monocytes.

Methodology/Principal Findings

Infection of monocytes with EBV induced IFNα secretion but inhibited the positive feedback loop for the amplification of IFNα. We showed that EBV infection induced the expression of suppressor of cytokine signaling 3 (SOCS3) and, to a lesser extent, SOCS1, two proteins known to interfere with the amplification of IFNα secretion mediated by the JAK/STAT signal transduction pathway. EBV infection correlated with a blockage in the activation of JAK/STAT pathway members and affected the level of phosphorylated IFN regulatory factor 7 (IRF7). Depletion of SOCS3, but not SOCS1, by small interfering RNA (siRNA) abrogated the inhibitory effect of EBV on JAK/STAT pathway activation and significantly restored IFNα secretion. Finally, transfection of monocytes with the viral protein Zta caused the upregulation of SOCS3, an event that could not be recapitulated with mutated Zta.

Conclusions/Significance

We propose that EBV protein Zta activates SOCS3 protein as an immune escape mechanism that both suppresses optimal IFNα secretion by human monocytes and favors a state of type I IFN irresponsiveness in these cells. This immunomodulatory effect is important to better understand the aspects of the immune response to EBV.