Dynamic control of gene expression by ISGF3 and IRF1 during IFNß and IFNγ signaling.

Type I interferons (IFN-I, including IFNß) and IFNγ produce overlapping, yet clearly distinct immunological activities. Recent data show that the distinctness of global transcriptional responses to the two IFN types is not apparent when comparing their immediate effects. By analyzing nascent transcripts induced by IFN-I or IFNγ over a period of 48 h, we now show that the distinctiveness of the transcriptomes emerges over time and is based on differential employment of the ISGF3 complex as well as of the second-tier transcription factor IRF1. The distinct transcriptional properties of ISGF3 and IRF1 correspond with a largely diverse nuclear protein interactome. Mechanistically, we describe the specific input of ISGF3 and IRF1 into enhancer activation and the regulation of chromatin accessibility at interferon-stimulated genes (ISG). We further report differences between the IFN types in altering RNA polymerase II pausing at ISG 5' ends. Our data provide insight how transcriptional regulators create immunological identities of IFN-I and IFNγ.

STAT1-cooperative DNA binding distinguishes type 1 from type 2 interferon signaling.

STAT1 is an indispensable component of a heterotrimer (ISGF3) and a STAT1 homodimer (GAF) that function as transcription regulators in type 1 and type 2 interferon signaling, respectively. To investigate the importance of STAT1-cooperative DNA binding, we generated gene-targeted mice expressing cooperativity-deficient STAT1 with alanine substituted for Phe77. Neither ISGF3 nor GAF bound DNA cooperatively in the STAT1F77A mouse strain, but type 1 and type 2 interferon responses were affected differently. Type 2 interferon-mediated transcription and antibacterial immunity essentially disappeared owing to defective promoter recruitment of GAF. In contrast, STAT1 recruitment to ISGF3 binding sites and type 1 interferon-dependent responses, including antiviral protection, remained intact. We conclude that STAT1 cooperativity is essential for its biological activity and underlies the cellular responses to type 2, but not type 1 interferon.

ISGF3 and STAT2/IRF9 Control Basal and IFN-Induced Transcription through Genome-Wide Binding of Phosphorylated and Unphosphorylated Complexes to Common ISRE-Containing ISGs.

In addition to the canonical ISGF3 and non-canonical STAT2/IRF9 complexes, evidence is emerging of the role of their unphosphorylated counterparts in IFN-dependent and -independent ISG transcription. To better understand the relation between ISGF3 and U-ISGF3 and STAT2/IRF9 and U-STAT2/IRF9 in IFN-I-stimulated transcriptional responses, we performed RNA-Seq and ChIP-Seq, in combination with phosphorylation inhibition and antiviral experiments. First, we identified a group of ISRE-containing ISGs that were commonly regulated in IFNα-treated WT and STAT1-KO cells. Thus, in 2fTGH and Huh7.5 WT cells, early and long-term IFNα-inducible transcription and antiviral activity relied on the DNA recruitment of the ISGF3 components STAT1, STAT2 and IRF9 in a phosphorylation- and time-dependent manner. Likewise, in ST2-U3C and Huh-STAT1KO cells lacking STAT1, delayed IFN responses correlated with DNA binding of phosphorylated STAT2/IRF9 but not U-STAT2/IRF9. In addition, comparative experiments in U3C (STAT1-KO) cells overexpressing all the ISGF3 components (ST1-ST2-IRF9-U3C) revealed U-ISGF3 (and possibly U-STAT2/IRF9) chromatin interactions to correlate with phosphorylation-independent ISG transcription and antiviral activity. Together, our data point to the dominant role of the canonical ISGF3 and non-canonical STAT2/IRF9, without a shift to U-ISGF3 or U-STAT2/IRF9, in the regulation of early and prolonged ISG expression and viral protection. At the same time, they suggest the threshold-dependent role of U-ISFG3, and potentially U-STAT2/IRF9, in the regulation of constitutive and possibly long-term IFNα-dependent responses.

Negative regulation of type I IFN signaling by phosphorylation of STAT2 on T387.

The transcription factor ISGF3, comprised of IRF9 and tyrosine-phosphorylated STATs 1 and 2, transmits the signal from the type I interferon receptor to the genome. We have discovered a novel phosphorylation of STAT2 on T387 that negatively regulates this response. In most untreated cell types, the majority of STAT2 is phosphorylated on T387 constitutively. In response to interferon-ß, the T387A mutant of STAT2 is much more effective than wild-type STAT2 in mediating the expression of many interferon-stimulated genes, in protecting cells against virus infection, and in inhibiting cell growth. Interferon-ß-treated cells expressing wild-type STAT2 contain much less ISGF3 capable of binding to an interferon-stimulated response element than do cells expressing T387A STAT2. T387 lies in a cyclin-dependent kinase (CDK) consensus sequence, and CDK inhibitors decrease T387 phosphorylation. Using CDK inhibitors to reverse the constitutive inhibitory phosphorylation of T387 of U-STAT2 might enhance the efficacy of type I interferons in many different clinical settings.

LMWF5A suppresses cytokine release by modulating select inflammatory transcription factor activity in stimulated PBMC.

BACKGROUND: Dysregulation of transcription and cytokine expression has been implicated in the pathogenesis of a variety inflammatory diseases. The resulting imbalance between inflammatory and resolving transcriptional programs can cause an overabundance of pro-inflammatory, classically activated macrophage type 1 (M1) and/or helper T cell type 1 (Th1) products, such as IFNγ, TNFα, IL1-ß, and IL12, that prevent immune switching to resolution and healing. The low molecular weight fraction of human serum albumin (LMWF5A) is a novel biologic drug that is currently under clinical investigation for the treatment of osteoarthritis and the hyper-inflammatory response associated with COVID-19. This study aims to elucidate transcriptional mechanisms of action involved with the ability of LMWF5A to reduce pro-inflammatory cytokine release. METHODS: ELISA arrays were used to identify cytokines and chemokines influenced by LMWF5A treatment of LPS-stimulated peripheral blood mononuclear cells (PBMC). The resulting profiles were analyzed by gene enrichment to gain mechanistic insight into the biologic processes and transcription factors (TFs) underlying the identified differentially expressed cytokines. DNA-binding ELISAs, luciferase reporter assays, and TNFα or IL-1ß relative potency were then employed to confirm the involvement of enriched pathways and TFs. RESULTS: LMWF5A was found to significantly inhibit a distinct set of pro-inflammatory cytokines (TNFα, IL-1ß, IL-12, CXCL9, CXCL10, and CXCL11) associated with pro-inflammatory M1/Th1 immune profiles. Gene enrichment analysis also suggests these cytokines are, in part, regulated by NF-κB and STAT transcription factors. Data from DNA-binding and reporter assays support this with LMWF5A inhibition of STAT1α DNA-binding activity as well as a reduction in overall NF-κB-driven luciferase expression. Experiments using antagonists specific for the immunomodulatory and NF-κB/STAT-repressing transcription factors, peroxisome proliferator-activated receptor (PPAR)γ and aryl hydrocarbon receptor (AhR), indicate these pathways are involved in the LMWF5A mechanisms of action by reducing LMWF5A drug potency as measured by TNFα and IL-1ß release. CONCLUSION: In this report, we provide evidence that LMWF5A reduces pro-inflammatory cytokine release by activating the immunoregulatory transcription factors PPARγ and AhR. In addition, our data indicate that LMWF5A suppresses NF-κB and STAT1α pro-inflammatory pathways. This suggests that LMWF5A acts through these mechanisms to decrease pro-inflammatory transcription factor activity and subsequent inflammatory cytokine production.

Regulation of interferon stimulated gene expression levels at homeostasis.

Interferon stimulated genes (ISGs), a collection of genes important in the early innate immune response, are upregulated in response to stimulation by extracellular type I interferons. The regulation of ISGs has been extensively studied in cells exposed to significant interferon stimulation, but less is known about ISG regulation in homeostatic regimes in which extracellular interferon levels are low. Using a collection of pre-existing, publicly available microarray datasets, we investigated ISG regulation at homeostasis in CD4, pulmonary epithelial, fibroblast and macrophage cells. We used a linear regression model to predict ISG expression levels from regulator expression levels. Our results suggest significant regulation of ISG expression at homeostasis, both through the ISGF3 molecule and through IRF7 and IRF8 associated pathways. We find that roughly 50% of ISGs have expression levels significantly correlated with ISGF3 expression levels at homeostasis, supporting previous results suggesting that homeostatic IFN levels have broad functional consequences. We find that ISG expression levels varied in their correlation with ISGF3, with epithelial and macrophage cells showing more correlation than CD4 and fibroblast cells. Our analysis provides a novel approach for decomposing and quantifying ISG regulation.

IRAK1 Limits TLR3/4- and IFNAR-Driven IL-27 Production through a STAT1-Dependent Mechanism.

IL-27 is a cytokine exerting pleiotropic immunomodulatory effects on a broad spectrum of immune cells. Optimal IL-27 production downstream of TLR3/4 ligand stimulation relies on autocrine type I IFN signaling, defining a first and second phase in IL-27 production. This work shows that IL-1 receptor-associated kinase 1 (IRAK1) limits TLR3/4- and IFNAR-induced IL-27 production. At the mechanistic level, we identified IRAK1 as a novel regulator of STAT1, IRF1, and IRF9. We found hyperactivation of STAT1 together with increased nuclear levels of IRF1 and IRF9 in IRAK1-deficient murine macrophages compared with control cells following stimulation with LPS and poly(I:C). IRAK1-deficient human microglial cells showed higher basal levels of STAT1 and STAT2 compared with control cells. Blocking the kinase activity of TBK1/IKKε in IRAK1 knockdown human microglial cells reduced the high basal levels of STAT1/2, uncovering a TBK1/IKKε kinase-dependent mechanism controlling basal levels of STAT1/2. Stimulating IRAK1 knockdown human microglial cells with IFN-ß led to increased IL-27p28 expression compared with control cells. In IRAK1-deficient murine macrophages, increased IL-27 levels were detected by ELISA following IFN-ß stimulation compared with control macrophages together with increased nuclear levels of p-STAT1, IRF1, and IRF9. Treatment of wild-type and IRAK1-deficient murine macrophages with fludarabine similarly reduced TLR3/4-induced IL-27 cytokine levels. To our knowledge, this work represents the first report placing IRAK1 in the IFNAR pathway and identifies IRAK1 as an important regulator of STAT1, controlling IL-27 production downstream of TLR3/4 and IFNAR signaling pathways.

Condensin II and GAIT complexes cooperate to restrict LINE-1 retrotransposition in epithelial cells.

LINE-1 (L1) retrotransposons can mobilize (retrotranspose) within the human genome, and mutagenic de novo L1 insertions can lead to human diseases, including cancers. As a result, cells are actively engaged in preventing L1 retrotransposition. This work reveals that the human Condensin II complex restricts L1 retrotransposition in both non-transformed and transformed cell lines through inhibition of L1 transcription and translation. Condensin II subunits, CAP-D3 and CAP-H2, interact with members of the Gamma-Interferon Activated Inhibitor of Translation (GAIT) complex including the glutamyl-prolyl-tRNA synthetase (EPRS), the ribosomal protein L13a, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and NS1 associated protein 1 (NSAP1). GAIT has been shown to inhibit translation of mRNAs encoding inflammatory proteins in myeloid cells by preventing the binding of the translation initiation complex, in response to Interferon gamma (IFN-γ). Excitingly, our data show that Condensin II promotes complexation of GAIT subunits. Furthermore, RNA-Immunoprecipitation experiments in epithelial cells demonstrate that Condensin II and GAIT subunits associate with L1 RNA in a co-dependent manner, independent of IFN-γ. These findings suggest that cooperation between the Condensin II and GAIT complexes may facilitate a novel mechanism of L1 repression, thus contributing to the maintenance of genome stability in somatic cells.

Nonconventional initiation complex assembly by STAT and NF-kappaB transcription factors regulates nitric oxide synthase expression.

Transcriptional regulation of the Nos2 gene encoding inducible nitric oxide synthase (iNOS) requires type I interferon (IFN-I) signaling and additional signals emanating from pattern recognition receptors. Here we showed sequential and cooperative contributions of the transcription factors ISGF3 (a complex containing STAT1, STAT2, and IRF9 subunits) and NF-kappaB to the transcriptional induction of the Nos2 gene in macrophages infected with the intracellular bacterial pathogen Listeria monocytogenes. NF-kappaB preceded ISGF3 at the Nos2 promoter and generated a transcriptional memory effect by depositing basal transcription factor TFIIH with the associated CDK7 kinase for serine 5 phosphorylation of the RNA polymerase II (pol II) carboxyterminal domain (CTD). Subsequent to TFIIH deposition by NF-kappaB, ISGF3 attracted the pol II enzyme and phosphorylation at CTD S5 occurred. Thus, STATs and NF-kappaB cooperate through pol II promoter recruitment and the phosphorylation of its CTD, respectively, as a prerequisite for productive elongation of iNOS mRNA.

Type I interferon-regulated gene expression and signaling in murine mixed glial cells lacking signal transducers and activators of transcription 1 or 2 or interferon regulatory factor 9.

Type I interferons (IFN-I) are critical in antimicrobial and antitumor defense. Although IFN-I signal via the interferon-stimulated gene factor 3 (ISGF3) complex consisting of STAT1, STAT2, and IRF9, IFN-I can mediate significant biological effects via ISGF3-independent pathways. For example, the absence of STAT1, STAT2, or IRF9 exacerbates neurological disease in transgenic mice with CNS production of IFN-I. Here we determined the role of IFN-I-driven, ISGF3-independent signaling in regulating global gene expression in STAT1-, STAT2-, or IRF9-deficient murine mixed glial cell cultures (MGCs). Compared with WT, the expression of IFN-α-stimulated genes (ISGs) was reduced in number and magnitude in MGCs that lacked STAT1, STAT2, or IRF9. There were significantly fewer ISGs in the absence of STAT1 or STAT2 versus in the absence of IRF9. The majority of ISGs regulated in the STAT1-, STAT2-, or IRF9-deficient MGCs individually were shared with WT. However, only a minor number of ISGs were common to WT and STAT1-, STAT2-, and IRF9-deficient MGCs. Whereas signal pathway activation in response to IFN-α was rapid and transient in WT MGCs, this was delayed and prolonged and correlated with increased numbers of ISGs expressed at 12 h versus 4 h of IFN-α exposure in all three IFN-I signaling-deficient MGCs. In conclusion, 1) IFN-I can mediate ISG expression in MGCs via ISGF3-independent signaling pathways but with reduced efficiency, with delayed and prolonged kinetics, and is more dependent on STAT1 and STAT2 than IRF9; and 2) signaling pathways not involving STAT1, STAT2, or IRF9 play a minor role only in mediating ISG expression in MGCs.

Anti-Inflammatory and Neuroprotective Role of Natural Product Securinine in Activated Glial Cells: Implications for Parkinson's Disease.

Glial activation and subsequent release of neurotoxic proinflammatory factors are believed to play an important role in the pathogenesis of several neurological disorders including Parkinson's disease (PD). Inhibition of glial activation and inflammatory processes may represent a therapeutic target to alleviate neurodegeneration. Securinine, a major natural alkaloid product from the root of the plant Securinega suffruticosa, has been reported to have potent biological activity and is used in the treatment of neurological conditions such as amyotrophic lateral sclerosis, poliomyelitis, and multiple sclerosis. In this study, we explored the underlying mechanisms of neuroprotection elicited by securinine, particularly its anti-inflammatory effects in glial cells. Our results demonstrate that securinine significantly and dose-dependently suppressed the nitric oxide production in microglia and astrocytic cultures. In addition, securinine inhibited the activation of the inflammatory mediator NF-κB, as well as mitogen-activated protein kinases in lipopolysaccharide- (LPS-) stimulated BV2 cells. Additionally, securinine also inhibited interferon-γ- (IFN-γ-) induced nitric oxide levels and iNOS mRNA expression. Furthermore, conditioned media (CM) from securinine pretreated BV2 cells significantly reduced mesencephalic dopaminergic neurotoxicity compared with CM from LPS stimulated microglia. These findings suggest that securinine may be a potential candidate for the treatment of neurodegenerative diseases related to neuroinflammation.

Impaired T-bet-pSTAT1α and perforin-mediated immune responses in the tumoral region of lung adenocarcinoma.

BACKGROUND: In spite of modern therapies for non-small-cell lung cancer (NSCLC), prognosis for many patients is still poor and survival rates are low. Immunotherapy is the possibility to improve the lung immune response surrounding the tumour. However, this approach requires detailed understanding of the local immune-responses of NSCLC patients. METHODS: We analysed samples from three different regions within the lungs of NSCLC patients, whereas we distinguished between patients suffering from adenocarcinoma and squamous cell carcinoma. Expression of type 1 T helper (Th1)/type 1 cytotoxic (Tc1) factors was assessed by quantitative real-time PCR, western blot analyses or immunohistochemistry. Cytotoxic cell activity of CD8(+) T cells was determined via co-culture with autologous tumour cells and apoptosis assay. RESULTS: We found decreased levels of the transcription factor T-box expressed in T cells (T-bet or Tbx21) and of the downstream activated IFN-γ-dependent pSTAT1α isoform in the lung tumour areas of patients with NSCLC as compared with tumour-free control regions. In these patients, reduced T-bet and pSTAT1α levels were found associated with increased immunosuppressive markers like cytotoxic T lymphocyte-associated protein 4, programmed cell death 1 and with a suppression of the Th1 cell cytokine production and Tc1 cell activity. CONCLUSIONS: These findings confirm a central role of T-bet in targeted immunotherapy for patients with NSCLC.

IκB kinase epsilon (IKK(epsilon)) regulates the balance between type I and type II interferon responses.

Virus infection induces the production of type I and type II interferons (IFN-I and IFN-II), cytokines that mediate the antiviral response. IFN-I (IFN-α and IFN-ß) induces the assembly of IFN-stimulated gene factor 3 (ISGF3), a multimeric transcriptional activation complex composed of STAT1, STAT2, and IFN regulatory factor 9. IFN-II (IFN-γ) induces the homodimerization of STAT1 to form the gamma-activated factor (GAF) complex. ISGF3 and GAF bind specifically to unique regulatory DNA sequences located upstream of IFN-I- and IFN-II-inducible genes, respectively, and activate the expression of distinct sets of antiviral genes. The balance between type I and type II IFN pathways plays a critical role in orchestrating the innate and adaptive immune systems. Here, we show that the phosphorylation of STAT1 by IκB kinase epsilon (IKKε) inhibits STAT1 homodimerization, and thus assembly of GAF, but does not disrupt ISGF3 formation. Therefore, virus and/or IFN-I activation of IKKε suppresses GAF-dependent transcription and promotes ISGF3-dependent transcription. In the absence of IKKε, GAF-dependent transcription is enhanced at the expense of ISGF3-mediated transcription, rendering cells less resistant to infection. We conclude that IKKε plays a critical role in regulating the balance between the IFN-I and IFN-II signaling pathways.

In vitro assessment of the biologic activity of interferon beta formulations used for the treatment of relapsing multiple sclerosis.

A new formulation (NF) of subcutaneous (sc) interferon (IFN) ß-1a was developed in an attempt to improve injection tolerability and immunogenicity. We compared antiviral and IFNß-stimulated gene (ISG) activities of IFNß-1a sc NF with IFNß-1a sc original formulation and IFNß-1b sc. When equivalent unit amounts were compared, the IFNß formulations demonstrated similar antiviral activity and induced similar levels of ISG mRNA. However, on a weight basis (ng/mL), significantly more IFNß-1b sc was needed to equal the antiviral activity of either IFNß-1a sc formulation, and both IFNß-1a sc formulations induced significantly higher levels of ISG mRNA than IFNß-1b sc.

Porcine deltacoronavirus NS7a antagonizes JAK/STAT pathway by inhibiting the interferon-stimulated gene factor 3 (ISGF3) formation.

The accessory proteins of coronaviruses play a crucial role in facilitating virus-host interactions and modulating host immune responses. Previous study demonstrated that the NS7a protein of porcine deltacoronavirus (PDCoV) partially hindered the host immune response by impeding the induction of IFN-α/ß. However, the potential additional functions of NS7a protein in evading innate immunity have yet to be elucidated. This study aimed to investigate the mechanism of PDCoV NS7a protein regulating the JAK/STAT signaling pathway. We presented evidence that NS7a effectively inhibited ISRE promoter activity and ISGs transcription. NS7a hindered STAT1 phosphorylation, interacted with STAT2 and IRF9, and further impeded the formation and nuclear accumulation of ISGF3. Furthermore, comparative analysis of NS7a across different PDCoV strains revealed that the mutation of Leu4 to Pro4 led to an increase in the molecular weights of NS7a and disrupted its inhibition on the JAK/STAT signaling pathway. This finding implied that NS7a with key amino acids may be an indicator of virulence for PDCoV strains. Taken together, this study revealed a novel role of NS7a in antagonizing the IFN-I signaling pathway.

Recent advances in understanding viral evasion of type I interferon.

The type I interferon (IFN) system mediates a wide variety of antiviral effects and represents an important first barrier to virus infection. Consequently, viruses have developed an impressive diversity of tactics to circumvent IFN responses. Evasion strategies can involve preventing initial virus detection, via the disruption of the Toll-like receptors or the retinoic acid inducible gene I (RIG-I) -like receptors, or by avoiding the initial production of the ligands recognized by these receptors. An alternative approach is to preclude IFN production by disarming or degrading the transcription factors involved in the expression of IFN, such as interferon regulatory factor 3 (IRF3)/IRF7, nuclear factor-κB (NF-κB), or ATF-2/c-jun, or by inducing a general block on host cell transcription. Viruses also oppose IFN signalling, both by disturbing the type I IFN receptor and by impeding JAK/STAT signal transduction upon IFN receptor engagement. In addition, the global expression of IFN-stimulated genes (ISGs) can be obstructed via interference with epigenetic signalling, and specific ISGs can also be selectively targeted for inhibition. Finally, some viruses disrupt IFN responses by co-opting negative regulatory systems, whereas others use antiviral mechanisms to their own advantage. Here, we review recent developments in this field.

A Toll-like receptor 2 ligand, Pam3CSK4, augments interferon-γ-induced nitric oxide production via a physical association between MyD88 and interferon-γ receptor in vascular endothelial cells.

The effect of Pam3CSK4, a Toll-like receptor 2 (TLR2) ligand, on interferon-γ (IFN-γ) -induced nitric oxide (NO) production in mouse vascular endothelial END-D cells was studied. Pre-treatment or post-treatment with Pam3CSK4 augmented IFN-γ-induced NO production via enhanced expression of an inducible NO synthase (iNOS) protein and mRNA. Pam3CSK4 augmented phosphorylation of Janus kinase 1 and 2, followed by enhanced phosphorylation of signal transducer and activator of transcription 1 (STAT1) at tyrosine 701. Subsequently, the enhanced STAT1 activation augmented IFN-γ-induced IFN-regulatory factor 1 expression leading to the iNOS expression. Pam3CSK4 also induced the activation of p38 and subsequent phosphorylation of STAT1 at serine 727. A pharmacological p38 inhibitor abolished the augmentation of IFN-γ-induced NO production by Pam3CSK4. Surprisingly, Pam3CSK4 enhanced a physical association of MyD88 and IFN-γ receptor. Together, these findings suggest that Pam3CSK4 up-regulates IFN-γ signalling in vascular endothelial cells via the physical association between MyD88 and IFN-γ receptor α, and p38-dependent serine 727 STAT1 phosphorylation.

TNF- α and IFN-s-dependent muscle decay is linked to NF-κB- and STAT-1α-stimulated Atrogin1 and MuRF1 genes in C2C12 myotubes.

TNF-α was shown to stimulate mitogenicity in C2C12 myoblasts. Selected cytokines TNF-α, IFNα, or IFNγ reduced the expression of myosin heavy chain (MyHC IIa) when given together. Molecular mechanisms of cytokine activities were controlled by NF-κB and JAK/STAT signaling pathways, as metabolic inhibitors, curcumin and AG490, inhibited some of TNF-α and IFNα/IFNγ effects. Insulin was hardly antagonistic to TNF-α - and IFNα/IFNγ-dependent decrease in MyHC IIa protein expression. Cytokines used individually or together also repressed myogenesis of C2C12 cells. Moreover, TNF-α - and IFNα/IFNγ-dependent effects on C2C12 myotubes were associated with increased activity of Atrogin1 and MuRF1 genes, which code ubiquitin ligases. MyHC IIa gene activity was unaltered by cytokines. Inhibition of NF-κB or JAK/STAT with specific metabolic inhibitors decreased activity of Atrogin1 and MuRF1 but not MyHC IIa gene. Overall, these results suggest cooperation between cytokines in the reduction of MyHC IIa protein expression level via NF-κB/JAK/STAT signaling pathways and activation of Atrogin1 and MuRF1 genes as their molecular targets. Insulin cotreatment or pretreatment does not protect against muscle decay induced by examined proinflammatory cytokines.

How cancer cells make and respond to interferon-I.

Acute exposure of cancer cells to high concentrations of type I interferon (IFN-I) drives growth arrest and apoptosis, whereas chronic exposure to low concentrations provides important prosurvival advantages. Tyrosine-phosphorylated IFN-stimulated gene (ISG) factor 3 (ISGF3) drives acute deleterious responses to IFN-I, whereas unphosphorylated (U-)ISGF3, lacking tyrosine phosphorylation, drives essential constitutive prosurvival mechanisms. Surprisingly, programmed cell death-ligand 1 (PD-L1), often expressed on the surfaces of tumor cells and well recognized for its importance in inactivating cytotoxic T cells, also has important cell-intrinsic protumor activities, including dampening acute responses to cytotoxic high levels of IFN-I and sustaining the expression of the low levels that benefit tumors. More thorough understanding of the newly recognized complex roles of IFN-I in cancer may lead to the identification of novel therapeutic strategies.