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How cells respond to interferons
Abstract
Interferons play key roles in mediating antiviral and antigrowth responses and in modulating immune response. The main signaling pathways are rapid and direct. They involve tyrosine phosphorylation and activation of signal transducers and activators of transcription factors by Janus tyrosine kinases at the cell membrane, followed by release of signal transducers and activators of transcription and their migration to the nucleus, where they induce the expression of the many gene products that determine the responses. Ancillary pathways are also activated by the interferons, but their effects on cell physiology are less clear. The Janus kinases and signal transducers and activators of transcription, and many of the interferon-induced proteins, play important alternative roles in cells, raising interesting questions as to how the responses to the interferons intersect with more general aspects of cellular physiology and how the specificity of cytokine responses is maintained.
... Type I IFN interacts with its receptor, IFN alpha (IFN-α), and beta receptor subunit 1 (IFNAR1), and IFNAR2 on the cell surface to induce heterodimerization of the receptor subunits and activate Janus kinase 1 (JAK1) and tyrosine kinase 2, which phosphorylate and activate signal transducer and activator of transcription 1 (STAT1) and STAT2 (14)(15)(16). Phosphorylated STAT proteins initially form heterodimers and interact with IFN regulatory factor 9 (IRF9) to form IFN-stimulated gene factor 3 (ISGF3), then they bind to the IFN-stimulated responsive element (ISRE) in the nucleus to induce the production of numerous IFN-stimulated genes (ISGs) that inhibit viral propagation (17,18). ...
... The data showed that SVV infection antagonizes the JAK-STAT pathway. The activation of the JAK-STAT signaling pathway induces the transcription of numerous ISGs that exert antiviral functions (17,18). SVV-infected HEK-293T cells were stimulated with IFN-α. ...
... These results indicated that 3C pro suppressed the phosphorylation and expression of STAT1, STAT2, and IRF9 (Fig. 3A). Phosphorylated STAT proteins and IRF9 form the ISGF3 complex that is transported into the nucleus, where it binds ISRE to induce ISGs expression (17,18). To evaluate whether 3C pro inhibited the nuclear translocation of ISGF3, nuclear and cytoplasmic fractiona tion was performed to determine the transportation of the ISGF3 complex in GFP-3C transfected cells (Fig. 3B). ...
The Seneca Valley virus (SVV) is an emerging pathogen that causes vesicular disease in pigs, posing a threat to the pork industry. The type I interferon (IFN) signaling pathway is the major defense mechanism of host innate immune responses, and SVV has developed numerous strategies to antagonize the IFN signaling and promote viral replication. However, the molecular mechanisms by which SVV affects IFN signaling components have not been fully elucidated. Here, we revealed that SVV proteinase (3C pro ) inhibited IFN-α signaling by degrading signal transducer and activator of transcription (STAT) 1, STAT2, and interferon regulatory factor 9 (IRF9) through its protease activity, which is dependent on the caspase pathway. 3C pro was further found to cleave STAT2 at glutamine 758 (Q758) within the transactivation domain, and the cleaved STAT2 products attenuated its ability to activate IFN-stimulated responsive element activity and induce the production of IFN-stimulated genes. Additionally, 3C pro impaired the nuclear import and formation of the IFN-stimulated gene factor 3 complex. 3C pro also induced karyopherin 1 degradation to block STAT1/STAT2 nuclear localization. Collectively, SVV 3C pro is capable of subverting the type I IFN response by targeting STAT1-STAT2-IRF9 and karyopherin α1 signals, which uncovers a novel mechanism exploited by SVV to evade host type I IFN response for efficient replication.
IMPORTANCE
Type I interferon (IFN) signaling plays a principal role in host innate immune responses against invading viruses. Viruses have evolved diverse mechanisms that target the Janus kinase-signal transducer and activator of transcription (STAT) signaling pathway to modulate IFN response negatively. Seneca Valley virus (SVV), an emerging porcine picornavirus, has received great interest recently because it poses a great threat to the global pork industry. However, the molecular mechanism by which SVV evades host innate immunity remains incompletely clear. Our results revealed that SVV proteinase (3C pro ) antagonizes IFN signaling by degrading STAT1, STAT2, and IRF9, and cleaving STAT2 to escape host immunity. SVV 3C pro also degrades karyopherin 1 to block IFN-stimulated gene factor 3 nuclear translocation. Our results reveal a novel molecular mechanism by which SVV 3C pro antagonizes the type I IFN response pathway by targeting STAT1-STAT2-IRF9 and karyopherin α1 signals, which has important implications for our understanding of SVV-evaded host innate immune responses.
... Viral pathogen-associated molecular patterns (PAMPs), encompassing viral RNAs and intermediate RNAs, are promptly recognized by host-pathogen recognition receptors (PRRs). This recognition subsequently initiates intracellular sensing mechanisms, involving key players like the interferon (IFN) regulatory factor (IRF) family members and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [33]. These transcription factors induce the expression of type I interferon (IFN-I), and, subsequently, secreted IFN- were transfected into Marc-145 cells as indicated. ...
... Viral pathogen-associated molecular patterns (PAMPs), encompassing viral RNAs and intermediate RNAs, are promptly recognized by host-pathogen recognition receptors (PRRs). This recognition subsequently initiates intracellular sensing mechanisms, involving key players like the interferon (IFN) regulatory factor (IRF) family members and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [33]. These transcription factors induce the expression of type I interferon (IFN-I), and, subsequently, secreted IFN-I binds to the IFN receptor, activating the downstream expression of IFN-stimulated genes (ISGs) [34][35][36]. ...
Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the Arteriviridae family, represents a persistent menace to the global pig industry, causing reproductive failure and respiratory disease in pigs. In this study, we delved into the role of histone deacetylases (HDAC2) during PRRSV infection. Our findings revealed that HDAC2 expression is downregulated upon PRRSV infection. Notably, suppressing HDAC2 activity through specific small interfering RNA led to an increase in virus production, whereas overexpressing HDAC2 effectively inhibited PRRSV replication by boosting the expression of IFN-regulated antiviral molecules. Furthermore, we identified the virus’s nonstructural protein 11 (nsp11) as a key player in reducing HDAC2 levels. Mutagenic analyses of PRRSV nsp11 revealed that its antagonistic effect on the antiviral activity of HDAC2 is dependent on its endonuclease activity. In summary, our research uncovered a novel immune evasion mechanism employed by PRRSV, providing crucial insights into the pathogenesis of this virus and guiding the development of innovative prevention strategies against PRRSV infection.
... Type I interferons (IFNs) are pluripotent cytokines that play a major role in the host immune response to respiratory viruses, including Respiratory Syncytial Virus (RSV), influenza, and coronaviruses (e.g., SARS-CoV-2), by regulating several hundred genes involved in antiviral defense, immune modulation, and cell growth [1,2]. SARS-CoV-2 infection and the resulting severe coronavirus disease have caused millions of deaths worldwide [3,4]. ...
... Pathogen molecular pat-tern recognition by lung resident immune cells such as dendritic cells and macrophages and the production of type I interferons play an important role in innate immunity to respiratory viruses [1]. The type I interferon effects are pleiotropic with antiviral, antiproliferative, immunomodulatory, and metabolic effects through the modulation of gene expression [2]. Previous bioinformatic studies have revealed an antiviral module composed of transcription factors from the STAT, IRF, and SP families and an inflammatory module composed of STAT1, STAT3, JUN, RELA, and BRCA1 in innate immunity [27,28]. ...
Respiratory viruses are the causative agents responsible for seasonal epidemics and occasional pandemic outbreaks and are a leading cause of death worldwide. Type I interferon (IFNα/β) signaling in the lung epithelial cells plays a major role in the innate immunity to respiratory viruses. Gene signatures are a set of differentially expressed genes in a particular disease or condition and are used to diagnose, monitor, and predict disease progression. These signatures can be used to identify regulatory modules and gene regulatory networks (GRNs) in mammalian signal transduction pathways. Considerable progress has been made in the identification of type I interferon-regulated gene signatures in the host response to respiratory viruses, including antiviral, immunomodulatory, apoptosis, and transcription factor signatures. Respiratory virus infections and host defenses require a dramatic change in the metabolic flux of macromolecules involved in nucleotide, lipid, and protein metabolism. The profiling of IFN-stimulated metabolic genes induced in the host response to several respiratory viruses led to the identification of a common gene signature in human lung epithelial cells and in the lungs of mouse models of respiratory virus infection. The regulation of the metabolic gene signature was correlated with the induction of IFN-beta (IFN-β) and IFN-inducible transcription factors at the RNA level in lung epithelial cells. Furthermore, the gene signature was also detected in response to bacterial lipopolysaccharide-induced acute lung injury. A protein interaction network analysis revealed that metabolic enzymes interact with IFN-regulated transcription factors and members of the unfolded protein response (UPR) to form a module and potentially regulate type I interferon signaling, constituting a feedback loop. In addition, components of the metabolic gene expression signature were differentially regulated in the lung tissues of COVID-19 patients compared with healthy controls. These results suggest that the metabolic gene signature is a potential therapeutic target for the treatment of respiratory virus infections and inflammatory diseases.
... IFN-γ is secreted by adaptive immune cells, such as CD4 + and CD8 + T cells, and signals via JAK1/JAK2/ STAT1 to regulate the expression of many genes, including the transcription factors of the IRF family [13,14]. An IFN-γ signature was found in young islet autoantibodypositive individuals [15]. ...
... The bar plots represent the gene and transcript isoform expression of CD47 comparing non-treated (NT) and TNF-α treatment (24 h). (g, h) Validation of the CD47 isoform usage in EndoC-βH1 cells (g) and dispersed human islets (h); results were expressed as a ratio between ENST00000361309.6 and ENST00000355354.13 isoforms. ...
Aims/hypothesis
The proinflammatory cytokines IFN-α, IFN-γ, IL-1β and TNF-α may contribute to innate and adaptive immune responses during insulitis in type 1 diabetes and therefore represent attractive therapeutic targets to protect beta cells. However, the specific role of each of these cytokines individually on pancreatic beta cells remains unknown.
Methods
We used deep RNA-seq analysis, followed by extensive confirmation experiments based on reverse transcription-quantitative PCR (RT-qPCR), western blot, histology and use of siRNAs, to characterise the response of human pancreatic beta cells to each cytokine individually and compared the signatures obtained with those present in islets of individuals affected by type 1 diabetes.
Results
IFN-α and IFN-γ had a greater impact on the beta cell transcriptome when compared with IL-1β and TNF-α. The IFN-induced gene signatures have a strong correlation with those observed in beta cells from individuals with type 1 diabetes, and the level of expression of specific IFN-stimulated genes is positively correlated with proteins present in islets of these individuals, regulating beta cell responses to ‘danger signals’ such as viral infections. Zinc finger NFX1-type containing 1 (ZNFX1), a double-stranded RNA sensor, was identified as highly induced by IFNs and shown to play a key role in the antiviral response in beta cells.
Conclusions/interpretation
These data suggest that IFN-α and IFN-γ are key cytokines at the islet level in human type 1 diabetes, contributing to the triggering and amplification of autoimmunity.
Graphical Abstract
... Phosphorylation of eIF2α is critical for mounting the integrated cellular stress response [3,4]. Double-stranded RNA produced during viral replication activates PKR, which phosphorylates eIF2α to inhibit overall translation within the cell, resulting in apoptosis of infected cells and thereby preventing virus spread [5]. Interferons induce higher intracellular levels of PKR, rendering PKR an essential mediator of interferon-induced antiviral responses [5]. ...
... Double-stranded RNA produced during viral replication activates PKR, which phosphorylates eIF2α to inhibit overall translation within the cell, resulting in apoptosis of infected cells and thereby preventing virus spread [5]. Interferons induce higher intracellular levels of PKR, rendering PKR an essential mediator of interferon-induced antiviral responses [5]. ...
Specific sequences within RNA encoded by human genes essential for survival possess the ability to activate the RNA-dependent stress kinase PKR, resulting in phosphorylation of its substrate, eukaryotic translation initiation factor-2α (eIF2α), either to curb their mRNA translation or to enhance mRNA splicing. Thus, interferon-γ (IFNG) mRNA activates PKR through a 5′-terminal 203-nucleotide pseudoknot structure, thereby strongly downregulating its own translation and preventing a harmful hyper-inflammatory response. Tumor necrosis factor-α (TNF) pre-mRNA encodes within the 3′-untranslated region (3′-UTR) a 104-nucleotide RNA pseudoknot that activates PKR to enhance its splicing by an order of magnitude while leaving mRNA translation intact, thereby promoting effective TNF protein expression. Adult and fetal globin genes encode pre-mRNA structures that strongly activate PKR, leading to eIF2α phosphorylation that greatly enhances spliceosome assembly and splicing, yet also structures that silence PKR activation upon splicing to allow for unabated globin mRNA translation essential for life. Regulatory circuits resulting in each case from PKR activation were reviewed previously. Here, we analyze mutations within these genes created to delineate the RNA structures that activate PKR and to deconvolute their folding. Given the critical role of intragenic RNA activators of PKR in gene regulation, such mutations reveal novel potential RNA targets for human disease.
... Likewise, the β-IFN-mediated decrease in viable cells was not associated with an increase in cell death, as measured by membrane exclusion of a viability dye (Fig. 6E) or by LDH release to the extracellular environment (Fig. 6F), but rather by a reduction in cellular proliferation, as measured by the dilution of fluorescent dye over time (Fig. 6G). Different cell types that exhibit an antiproliferative response to IFN-I treatment have been found to vary in the specific stage(s) at which cell cycle progression is delayed (43)(44)(45)(46). We performed propidium iodide staining and flow cytometry of untreated and β-IFN-treated Calu-3 cells, finding no significant shift in cell cycle distribution over 4 days of treatment (Fig. S9). ...
... The potential of IFN-I signaling to induce an antiproliferative response has long been recognized (71,72), but the sensitivity of different cell types to these (and other) IFN-I responses varies considerably (43)(44)(45)(46). Our findings suggest that respiratory epithelial cells are highly sensitive to the antiproliferative effect of IFN-I signaling and that during SARS-CoV-2 infection, this response has a greater impact on cell fitness than any concurrent activation of antiviral or regulated cell death pathways. ...
The proliferation of respiratory epithelial cells is crucial to host recovery from acute lung injury caused by SARS-CoV-2 and other viral pathogens, but the molecular pathways that govern this process are poorly understood. We performed a high-throughput CRISPR screen that surprisingly revealed a detrimental effect of specific host response, type I interferon (IFN-I) signaling, on the fitness of SARS-CoV-2-infected Calu-3 cells. While IFN-I signaling has been previously associated with several potential downstream responses, we found this effect to be primarily mediated by an inhibition of Calu-3 cellular proliferation after the early peak of SARS-CoV-2-induced cell death. Our findings provide a plausible mechanism for how sustained IFN-I signaling during SARS-CoV-2 infection might worsen lung pathology by blocking the regeneration of the alveolar epithelium from progenitor cells.
... Phosphorylated STAT1 can form a homodimer known as gamma-interferon activation factor (GAF) after stimulation by type II interferon (IFN) and IL-27 and, to a lesser extent, type I and III IFNs. GAF translocates to the nucleus and binds gammainterferon-activated sites to upregulate or downregulate IFNstimulated and IFN-regulated genes (7). Phosphorylated STAT1 also forms a heterotrimer known as interferonstimulated gene factor 3 (ISGF3) along with STAT2 and interferon regulatory factor 9 after stimulation by type I or III IFNs. ...
... These variants result in enhanced STAT1 phosphorylation, compared to wild-type STAT1, due to impaired nuclear dephosphorylation (8), as well as enhanced STAT1 signaling down-stream of STAT1-dependant cytokines, such as IFN-α/β, IFN-γ, and IL-27, and downstream STAT3-dependent cytokines, such as IL-6 and IL-21, resulting in impaired Th17 cell development (5,8). Accumulated data indicate that these mutations account for approximately half of patients with CMC and are associated with a gain in STAT1 function due to impaired dephosphorylation of STAT1 (7,8). The development of CMC in this disorder may also be attributable to the impaired production of IL-17A and IL-22 (4,5,8,9). ...
A 30-year-old man presented with oral candidiasis and a history of lung abscess. He experienced recurring oral and skin candidiasis in childhood but spent long periods without any infections. Therefore, immunodeficiency was suspected. T and B lymphocyte and natural killer cell counts as well as immunoglobulin levels were normal. Human immunodeficiency virus test results were negative. Therefore, we suspected chronic mucocutaneous candidiasis (CMC). The signal transducer and activator of transcription (STAT) mutation, the leading cause of CMC, was detected by exome sequencing. Most cases of STAT-1 mutations are diagnosed in childhood, but a few are diagnosed in adulthood because Candida infections may not be severe.
... IFN-γ crucially combats viruses and exhibits anti-tumor and anti-proliferative properties [67]. Nuclear factor kappa-B (NF-κB) preferentially binds to +874T allele, perhaps boosting IFN-γ expression and affecting the pathogenesis of tuberculosis [68], and T carriers had statistically increased CML risk in a dominant model (i.e., TT + TA vs. AA) [69]. ...
Tyrosine kinase inhibitor (TKI) drugs have significantly improved chronic myeloid leukemia (CML) outcomes. Neopeptides from CML cells may induce specific immune responses, which are crucial for deep molecular (DMR) and treatment-free remission (TFR). In this study of Ethiopian patients with CML (n = 162), the HLA alleles and single-nucleotide polymorphisms of five cytokines revealed significant associations with clinical outcomes. Clinically unfavorable outcomes correlated with HLA alleles A*03:01/02, A*23:17:01, B*57:01/02/03, and HLA-DRB4*01:01 (p-value = 0.0347, p-value = 0.0285, p-value = 0.037, and p-value = 0.0127, respectively), while HLA-DRB4*01:03:01 was associated with favorable outcomes (p-value = 0.0058). After assigning values for the ‘low,’ ‘intermediate,’ and ‘high’ gene expression of the SNPs’ respective cytokine genes, Kaplan–Meier estimates for relapse-free survival, adjusted for age, treatment duration, and relapse risk among patients after the administration of TKIs, indicated that a gene expression ratio above the overall median of TNF-α, IL-6, and the combination of TGF-β1/IL-10, IFNγ, and IL-6/IL-10 TGF-β1 was correlated with a higher likelihood of treatment failure ((RR: 3.01; 95% CI: 1.1–8.3; p-value = 0.0261) and (RR: 2.4; 95% CI: 1.1–5.2; p-value = 0.022), respectively). Multi-SNPs, surpassing single-SNPs, and HLA allele polymorphisms showed promise in predicting outcomes of patients with CML during TKI treatment, prompting further exploration into their potential utility.
... The results revealed that the DEPs were most associated with interferon (INF)-γ and -α/β signaling pathways ( Figure 3e) in which the transcription factor signal transducer and activator of transcription 1 (STAT1, FC = 1.24) and a deoxynucleotide triphosphate (dNTP) hydrolase sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1, FC = 1.39) were upregulated (Figure 3f,g). INF-γ/α/β signaling plays an important role in modulating immune responses, and its activation could enhance the efficacy of cisplatin [31,32]. STAT1 is a crucial mediator of IFN signaling and is considered a tumor suppressor. ...
Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, promotes the cytotoxicity of the genotoxic anticancer drug cisplatin, yet the underlying mechanism remains poorly understood. Herein, we revealed that TSA at a low concentration (1 μM) promoted the cisplatin-induced activation of caspase-3/6, which, in turn, increased the level of cleaved PARP1 and degraded lamin A&C, leading to more cisplatin-induced apoptosis and G2/M phase arrest of A549 cancer cells. Both ICP-MS and ToF-SIMS measurements demonstrated a significant increase in DNA-bound platinum in A549 cells in the presence of TSA, which was attributable to TSA-induced increase in the accessibility of genomic DNA to cisplatin attacking. The global quantitative proteomics results further showed that in the presence of TSA, cisplatin activated INF signaling to upregulate STAT1 and SAMHD1 to increase cisplatin sensitivity and downregulated ICAM1 and CD44 to reduce cell migration, synergistically promoting cisplatin cytotoxicity. Furthermore, in the presence of TSA, cisplatin downregulated TFAM and SLC3A2 to enhance cisplatin-induced ferroptosis, also contributing to the promotion of cisplatin cytotoxicity. Importantly, our posttranslational modification data indicated that acetylation at H4K8 played a dominant role in promoting cisplatin cytotoxicity. These findings provide novel insights into better understanding the principle of combining chemotherapy of genotoxic drugs and HDAC inhibitors for the treatment of cancers.
... IFNAR engagement results in JAK1 and TYK2 activation leading to the formation of the transcription factor complex IFN-stimulated gene factor 3 (ISGF3), which is composed of signal transducers and activators of transcription (STAT1 and STAT2) and IFN-regulatory factor 9 (IRF9) [13]. This complex translocates to the nucleus to promote transcription of IFNstimulated genes [12,14], whose products establish an antiviral state in the cell [15,16]. Type I IFNs exert antiproliferative and immunomodulatory effects essential to control viral infection. ...
Tyrosine kinase 2 (TYK2) is involved in type I interferon (IFN-I) signaling through IFN receptor 1 (IFNAR1). This signaling pathway is crucial in the early antiviral response and remains incompletely understood on B cells. Therefore, to understand the role of TYK2 in B cells, we studied these cells under homeostatic conditions and following in vitro activation using Tyk2-deficient (Tyk2−/−) mice. Splenic B cell subpopulations were altered in Tyk2−/− compared to wild type (WT) mice. Marginal zone (MZ) cells were decreased and aged B cells (ABC) were increased, whereas follicular (FO) cells remained unchanged. Likewise, there was an imbalance in transitional B cells in juvenile Tyk2−/− mice. RNA sequencing analysis of adult MZ and FO cells isolated from Tyk2−/− and WT mice in homeostasis revealed altered expression of IFN-I and Toll-like receptor 7 (TLR7) signaling pathway genes. Flow cytometry assays corroborated a lower expression of TLR7 in MZ B cells from Tyk2−/− mice. Splenic B cell cultures showed reduced proliferation and differentiation responses after activation with TLR7 ligands in Tyk2−/− compared to WT mice, with a similar response to lipopolysaccharide (LPS) or anti-CD40 + IL-4. IgM, IgG, IL-10 and IL-6 secretion was also decreased in Tyk2−/− B cell cultures. This reduced response of the TLR7 pathway in Tyk2−/− mice was partially restored by IFNα addition. In conclusion, there is a crosstalk between TYK2 and TLR7 mediated by an IFN-I feedback loop, which contributes to the establishment of MZ B cells and to B cell proliferation and differentiation.
... Type I interferons (IFNs) including alpha (IFN-a) and beta (IFN-b), are extracellular cytokines. IFNs function by binding to receptors and activating the Janus tyrosine kinase signal transducer and activator of transcription (JAK-STAT) pathway or other signaling pathways to regulate various genes (3)(4)(5)(6). IFN-b exists in a single form and is expressed in virtually all tissues and cell types. IFN-a has 13 subtypes and is noted to be produced in many types of cells including plasmacytoid dendritic and other immune cells (7)(8)(9). ...
... The compactness of the HBV genome restricts sequence plasticity, minimizing the virus's ability to evade silencing sequences [13]. Moreover, the targeted nature of RNAi, focusing on the viral transcript, offers the potential to minimize undesired side effects and cellular toxicity [14,15]. HBV is differentiated into ten genotypes (A-J), with distinct geographical distribution [16,17]. ...
Chronic hepatitis B (CHB) is a global health challenge that can result in significant liver-related morbidity and mortality. Despite a prophylactic vaccine being available, patients already living with CHB often must engage in lifelong therapy with nucleoside analogues. However, the potential of RNA interference (RNAi) therapeutics as a promising avenue for CHB treatment is being explored. RNAi, particularly using small interfering RNA (siRNA), targets viral RNA that can be used to inhibit hepatitis B virus (HBV) replication. Several candidates are currently being studied and have exhibited varying success in reducing hepatitis B surface antigen (HBsAg) levels, with some showing sustained HBsAg loss after cessation of therapy. The dynamic evolution of RNAi therapy presents a promising trajectory for the development of effective and sustained treatments for CHB. This review highlights recent findings on RNAi therapeutics, including modifications for stability, various delivery vectors, and specific candidates currently in development.
... Interleukin 1 beta is an important pro-inflammatory cytokine that mediates the innate immune response and is primarily produced by activated macrophages (Lee et al., 2015). Interferon gamma, which is produced by T cells and natural killer (NK) cells, plays a crucial role in modulating immune responses, promoting inflammation, and activating macrophages (Stark et al., 1998;Tau and Rothman, 1999). In the NE challenge, we observed a comprehensive inflammatory response in the small intestine, with increased levels of IL1b and IFNg compared to the nonchallenged controls at both 6 and 8 dpi. ...
Chickens have undergone genetic improvements in the past few decades to maximize growth efficiency. However, necrotic enteritis (NE), an enteric disease primarily caused by C. perfringens, remains a significant problem in poultry production. A study investigated the differences in intestinal health between the nonselected meat-type chicken Athens Canadian Random Bred (ACRB) and the modern meat-type Cobb 500 broilers (Cobb) when challenged with experimental NE. The study utilized a 2 × 3 factorial arrangement, consisting of two main effects of chicken strain and NE challenge model (nonchallenged control, NC; NE challenge with 2,500/12,500 Eimeria maxima oocysts + 1 × 10⁹C. perfringens, NE2.5/NE12.5). A total of 432 fourteen-day-old male ACRB and Cobb were used until 22 d (8 d postinoculation with E. maxima on d 14, dpi), and the chickens were euthanized on 6 and 8 dpi for the analysis. All data were statistically analyzed using a two-way ANOVA, and Student's t-test or Tukey's HSD test was applied when P < 0.05. The NE12.5 group showed significant decreases in growth performance and relative growth performance from d 14 to 20, regardless of chicken strain (P < 0.01). The ACRB group exhibited significant decreases in relative body weight and relative body weight gain compared to the Cobb group from d 14 to 22 (P < 0.01). On 6 and 8 dpi, both NE challenge groups showed significant decreases in intestinal villus height to crypt depth ratio, jejunal goblet cell count, and jejunal MUC2 and LEAP2 expression (P < 0.01). Additionally, the NE12.5 group had significantly higher intestinal NE lesion score, intestinal permeability, fecal E. maxima oocyst count, intestinal C. perfringens count, and jejunal IFNγ and CCL4 expression compared to the NC group (P < 0.05). In conclusion, NE negatively impacts growth performance and intestinal health in broilers, parameters regardless of the strain.
... Two prominent downstream effector molecules induced by type 1 IFNs include RNase L and PKR [60]. Previous results reported PKR to be a principal molecule involved in resistance to HSV-1 replication in the trigeminal ganglia of mice transduced with IFN-β, whereas RNase L was responsible for resistance to HSV-1 infection in the cornea [61,62]. ...
The complement system (CS) contributes to the initial containment of viral and bacterial pathogens and clearance of dying cells in circulation. We previously reported mice deficient in complement component 3 (C3KO mice) were more sensitive than wild-type (WT) mice to ocular HSV-1 infection, as measured by a reduction in cumulative survival and elevated viral titers in the nervous system but not the cornea between days three and seven post infection (pi). The present study was undertaken to determine if complement deficiency impacted virus replication and associated changes in inflammation at earlier time points in the cornea. C3KO mice were found to possess significantly (p < 0.05) less infectious virus in the cornea at 24 h pi that corresponded with a decrease in HSV-1 lytic gene expression at 12 and 24 h pi compared to WT animals. Flow cytometry acquisition found no differences in the myeloid cell populations residing in the cornea including total macrophage and neutrophil populations at 24 h pi with minimal infiltrating cell populations detected at the 12 h pi time point. Analysis of cytokine and chemokine content in the cornea measured at 12 and 24 h pi revealed that only CCL3 (MIP-1α) was found to be different between WT and C3KO mice with >2-fold increased levels (p < 0.05, ANOVA and Tukey’s post hoc t-test) in the cornea of WT mice at 12 h pi. C3KO mouse resistance to HSV-1 infection at the early time points correlated with a significant increase in type I interferon (IFN) gene expression including IFN-α1 and IFN-β and downstream effector genes including tetherin and RNase L (p < 0.05, Mann–Whitney rank order test). These results suggest early activation of the CS interferes with the induction of the type I IFN response and leads to a transient increase in virus replication following corneal HSV-1 infection.
... Interferons (IFNs), a superfamily of cytokines known for their antiviral properties, are crucial in the body's response to viral infections, including HBV (Lindenmann et al., 1957). It has been observed that IFNs not only inhibit virus replication but also play roles in cell proliferation, apoptosis, inflammation, and adaptive immunity (Stark et al., 1998). IFNs, a broad-spectrum antiviral agent, do not directly neutralize the virus. ...
Hepatitis B, a global health concern caused by the hepatitis B virus (HBV), infects nearly 2 billion individuals worldwide, as reported by the World Health Organization (WHO). HBV, a hepatotropic DNA virus, predominantly targets and replicates within hepatocytes. Those carrying the virus are at increased risk of liver cirrhosis and hepatocellular carcinoma, resulting in nearly 900,000 fatalities annually. The HBV X protein (HBx), encoded by the virus’s open reading frame x, plays a key role in its virulence. This protein is integral to viral replication, immune modulation, and liver cancer progression. Despite its significance, the precise molecular mechanisms underlying HBx remain elusive. This review investigates the HBx protein’s roles in HBV replication, interferon signaling regulation, and hepatocellular carcinoma progression. By understanding the complex interactions between the virus and its host mediated by HBx, we aim to establish a solid foundation for future research and the development of HBx-targeted therapeutics.
... The major STATs that are induced by IFN-I are STAT1 and STAT2 that form a complex with IRF9, which is termed IFN-stimulated genes (ISG)-F3 (9,10). These STAT proteins form either homodimers or heterodimers and these complexes bind to IFN-stimulated response element (ISRE) in IFN stimulating genes (ISG) promoters, thereby initiating their transcription (11,12). Mitogen activated protein kinases (MAPK) are also known to mediate IFN I signaling via phosphorylation of p38 and ERK kinases (13). ...
Positive-strand RNA viruses and DNA viruses generate double-stranded RNA (dsRNA) during their replication processes and innate immune responses against viral infections are orchestrated by numerous interferon-stimulating genes, yet the detailed coordination of downstream signaling of anti-viral immune responses is not fully understood. Recent studies suggest 2’-5’- Oligoadenylate Synthetase 1 (OAS1) may have a protective role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections; however, the mechanism regulating OAS1 remains uninvestigated. Our aim is to understand the regulation of OAS1 and its modulation of RNaseL activity, as this has significant implications for responses to RNA viruses, including Vesicular stomatitis virus (VSV) and SARS-CoV-2. We explore the hypothesis that ABCF1 an ATP-binding cassette family member protein, a key regulator of innate immune responses and macrophage polarization and cytokine storm, play a role in regulating the antiviral responses and downstream dsRNA signaling revealed by measuring responses to the synthetic dsRNA analog termed poly (I:C). We utilize ABCF1 haplo-insufficient mice to discover that ABCF1 modulates the amplitude and frequency of VSV-specific Cytolytic T lymphocyte in anti-viral immune responses and suggests that innate immune responses underpin this process. To understand this mechanism, we describe that ABCF1 interacts with 2’-5’-oligoadenylate synthetase 1 (OAS1) which in turn modulates essential proteins that leads to the modulation of RNaseL activity via ABCE1. Furthermore, we find that ABCF1 also influences the production of interferon-α (IFN-α) and interferon-β (IFN-β) in bone marrow-derived macrophages. Overall,, we unexpectedly discovered that ABCF1 acts as a crucial link between innate and adaptive immunity, regulating the development of adaptive Cytolytic T lymphocyte responses and interacting with OAS1, a key regulator of innate immune responses against viral infections. Exploring pharmacological agents that target ABCE1 or ABCF1 may lead to the discovery of novel modalities for countering SARS CoV-2 and other viruses where OAS1 is a crucial innate immune response gene.
... Type 1 IFNs, including IFN-α and beta (IFN-β), share the same receptor components and induce similar biological activities [42][43][44]. They exhibit anti-proliferative, immunostimulatory, and anti-angiogenic activities [45][46][47][48]. ...
Hepatocellular carcinoma (HCC) usually recurs after curative surgical resection. Currently, no approved adjuvant therapy has been shown to reduce HCC recurrence rates. In this study, the in vivo effect of sequential combination treatment with recombinant mouse interferon-alpha (rmIFN-α) and an anti-mouse-PD1 antibody on hepatitis B virus (HBV) clearance in mice was evaluated. A Phase I clinical trial was then conducted to assess the safety, tolerability, and inhibitory activity of sequential therapy with ropeginterferon alfa-2b and nivolumab in patients with HCC recurrence who underwent curative surgery for HBV-related HCC. The animal modeling study showed that HBV suppression was significantly greater with the rmIFN-α and anti-PD1 sequential combination treatment in comparison with sole treatment with rmIFN-α or anti-PD1. In the Phase I study, eleven patients completed the sequential therapy with ropeginterferon alfa-2b every two weeks for six doses at 450 µg, followed by three doses of nivolumab every two weeks up to 0.75 mg/kg. A notable decrease in or clearance of HBV surface antigen was observed in two patients. The dose-limiting toxicity of grade 3 alanine transaminase and aspartate aminotransferase increases was observed in one patient. The maximum tolerated dose was then determined. To date, no HCC recurrence has been observed. The treatment modality was well tolerated. These data support the further clinical development of sequential combination therapy as a post-surgery prophylactic measure against the recurrence of HBV-related HCC.
... In mammals, the JAK family includes four JAKs: JAK1, JAK2, JAK3, and TYK2 (24). To determine whether these molecules contribute to the ability of IL-2/7/33 to induce ILC2 activation, we cotreated murine lung ILC2s with various JAK inhibitors, including AZD1480, which suppresses both JAK1 and JAK2 activation (JAK1/2i) and PF06651600, a JAK3 inhibitor (JAK3i). ...
Steroids are the standard treatment for allergic airway inflammation in asthma, but steroid-refractory asthma poses a challenge. Group 2 innate lymphoid cells (ILC2s), such as T helper 2 (T H 2) cells, produce key asthma-related type 2 cytokines. Recent insights from mouse and human studies indicate a potential connection between ILC2s and steroid-resistant asthma. Here, we highlight that lung ILC2s, rather than T H 2 cells, can develop steroid resistance, allowing them to persist and maintain their disease-driving activity even during steroid treatment. The emergence of multipotent IL-5 ⁺ IL-13 ⁺ IL-17A ⁺ ILC2s is associated with steroid-resistant ILC2s. The Janus kinase 3 (JAK3)/signal transducer and activator of transcription (STAT) 3, 5, and 6 pathways contribute to the acquisition of steroid-resistant ILC2s. The JAK3 inhibitor reduces ILC2 survival, proliferation, and cytokine production in vitro and ameliorates ILC2-driven Alternaria -induced asthma. Furthermore, combining a JAK3 inhibitor with steroids results in the inhibition of steroid-resistant asthma. These findings suggest a potential therapeutic approach for addressing this challenging condition in chronic asthma.
... Type 1 IFNs, including IFN-α and beta (IFN-β), share the same receptor components and induce similar biological activities [37][38][39]. They induce anti-proliferative, immune-stimulatory, and antiangiogenic activities [40][41][42][43]. ...
In hepatocellular carcinoma (HCC), recurrence usually occurs after curative surgical resection. Currently, no approved adjuvant therapy has been shown to reduce recurrence rates. In this report, the in vivo mouse effect of sequential combination treatment with recombinant mouse interferon-alpha (rmIFN-α) and an anti-mouse-PD1 antibody on hepatitis B virus (HBV) clearance was evaluated. A phase I clinical trial was then conducted to assess the safety, tolerability, and inhibitory activity of sequential therapy with ropeginterferon alfa-2b and nivolumab in HCC recurrence in patients who have undergone curative surgery for HBV-related HCC. In animal modeling, HBV suppression was significantly greater with rmIFN-α and anti-PD1 sequential combination treatment than with their treatment alone. In the Phase I study, eleven patients completed the sequential therapy with ropeginterferon alfa-2b every two weeks at the dose of 450 µg for six doses, followed by three doses of nivolumab every two weeks up to 0.75 mg/kg. A notable decrease or clearance of HBV surface antigen was observed in two patients. Dose-limiting toxicity of grade 3 alanine transaminase and aspartate aminotransferase increases was observed in one patient. The maximum tolerated dose was determined. Currently no HCC recurrence has been observed. The treatment modality was well tolerated. The data support further clinical development of sequential combination therapy as a post-surgery prophylactic measure against the recurrence of HBV-related HCC.
... Interferons are another group of immunomodulatory and anti-proliferative cytokines (184). IFN-γ as a critical innate and adaptive immune cytokine promotes MHC-II expression that is produced by NKT and Th1 cells (185). ...
The immune system has a critical tumor suppressor function via the detection and elimination of the tumor cells. Tumor cells' immune escaping is commonly observed during neoplastic transformations. Immune response suppression or aberrations facilitate immune escape that promotes tumor progression in distant or primary locations via epithelial-mesenchymal transition and angiogenesis, respectively. It has been reported that there is a rising trend of cancer incidence among the Iranian population. Since aberrant immune responses are involved in tumorigenesis; immunotherapeutic methods can be efficient for tumor cell elimination. In the present review, we discussed all of the immune-related genes that have been associated with tumor progression among the Iranian population to clarify the genetics of immune deficiency during tumor progression in this population. T regulatory and T helper related genes were the most frequently deregulated genes during tumor progression in the Iranian population. This review paves the way to suggest an immune-specific panel of genetic markers for diagnostic and immunotherapeutic purposes among Iranian cancer patients.
... [8][9][10][11][12] Hence, IFNγ activity must be finely balanced to elicit appropriate immune responses. 7,13,14 IFNγ-induced immune responses are mainly mediated via STAT1. [15][16][17] Binding of IFNγ activates the cell-surface receptor-associated tyrosine kinases, Janus kinase1 (JAK1), and JAK2, which then phosphorylate STAT1 at a conserved tyrosine residue (Y701), leading to its activation. ...
Signaling desensitization is key to limiting signal transduction duration and intensity. Signal transducer and activator of transcription 1 (STAT1) can mediate type II interferon (IFNγ)‐induced immune responses, which are enhanced and inhibited by STAT1 phosphorylation and sumoylation, respectively. Here, we identified an N‐MYC interacting protein, NMI, which can enhance STAT1 phosphorylation and STAT1‐mediated IFNγ immune responses by binding and sequestering the E2 SUMO conjugation enzyme, UBC9, and blocking STAT1 sumoylation. NMI facilitates UBC9 nucleus‐to‐cytoplasm translocation in response to IFNγ, thereby inhibiting STAT1 sumoylation. STAT1 phosphorylation at Y701 and sumoylation at K703 are mutually exclusive modifications that regulate IFNγ‐dependent transcriptional responses. NMI could not alter the phosphorylation level of sumoylation‐deficient STAT1 after IFNγ treatment. Thus, IFNγ signaling is modulated by NMI through sequestration of UBC9 in the cytoplasm, leading to inhibition of STAT1 sumoylation. Hence, NMI functions as a switch for STAT1 activation/inactivation cycles by modulating an IFNγ‐induced desensitization mechanism.
... Pretreatment of BV-2 cells with IFNγ decreased MNoV CW3 (norovirus herein after) replication ~5,000-fold (Fig. 1A) and cytopathicity by 50% (Fig. 1B). Stat1 and Irf1 encode transcription factors essential for IFNγ responses (23) and are required for robust IFNγ-induced inhibition of norovirus replication both in vitro in primary macrophages and in vivo (24)(25)(26). To determine whether these transcription factors were required for IFNγ-induced inhibition of norovirus replication in BV-2 cells we generated two independent clonal Stat1 or Irf1 knockout BV-2 cell lines (Stat1 −/− , Irf1 −/− ) ( Fig. 1A and B). ...
Genes required for the lysosomal degradation pathway of autophagy play key roles in topologically distinct and physiologically important cellular processes. Some functions of ATG genes are independent of their role in degradative autophagy. One of the first described of these ATG gene-dependent, but degradative autophagy independent, processes is the requirement for a subset of ATG genes in interferon-γ (IFNγ)-induced inhibition of norovirus and Toxoplasma gondii replication. Herein, we identified additional genes that are required for, or that negatively regulate, this innate immune effector pathway. Enzymes in the UFMylation pathway negatively regulated IFNγ-induced inhibition of norovirus replication via effects of Ern1 . IFNγ-induced inhibition of norovirus replication required Gate-16 (also termed GabarapL2 ), Wipi2b , Atg9a , Cul3 , and Klhl9 but not Becn1 (encoding Beclin 1), Atg14 , Uvrag , or Sqstm1 . The phosphatidylinositol-3-phosphate and ATG16L1-binding domains of WIPI2B, as well as the ATG5-binding domain of ATG16L1, were required for IFNγ-induced inhibition of norovirus replication. Other members of the Cul3 , Atg8 , and Wipi2 gene families were not required, demonstrating exquisite specificity within these gene families for participation in IFNγ action. The generality of some aspects of this mechanism was demonstrated by a role for GATE-16 and WIPI2 in IFNγ-induced control of Toxoplasma gondii infection in human cells. These studies further delineate the genes and mechanisms of an ATG gene-dependent programmable form of cytokine-induced innate intracellular immunity.
IMPORTANCE
Interferon-γ (IFNγ) is a critical mediator of cell-intrinsic immunity to intracellular pathogens. Understanding the complex cellular mechanisms supporting robust interferon-γ-induced host defenses could aid in developing new therapeutics to treat infections. Here, we examined the impact of autophagy genes in the interferon-γ-induced host response. We demonstrate that genes within the autophagy pathway including Wipi2 , Atg9 , and Gate-16 , as well as ubiquitin ligase complex genes Cul3 and Klhl9 are required for IFNγ-induced inhibition of murine norovirus (norovirus hereinafter) replication in mouse cells. WIPI2 and GATE-16 were also required for IFNγ-mediated restriction of parasite growth within the Toxoplasma gondii parasitophorous vacuole in human cells. Furthermore, we found that perturbation of UFMylation pathway components led to more robust IFNγ-induced inhibition of norovirus via regulation of endoplasmic reticulum (ER) stress. Enhancing or inhibiting these dynamic cellular components could serve as a strategy to control intracellular pathogens and maintain an effective immune response.
... The complex network of cytokines and other immunoregulatory factors that trigger innate or specific immune responses against viral infections are primarily mediated by the recruitment and activation of effector immune cells [28,29]. However, with the intrinsic ability of viruses to interfere with cytokine-induced signaling pathways, most of them can alter downstream effector functions, facilitating their self-survival and replication [30,31]. ...
Background
The conventional means of controlling the recurring pandemics of Type A Influenza Virus (IAV) infections remain challenging primarily because of its high mutability and increasing drug resistance. As an alternative to control IAV infections, the prophylactic use of cytokines to drive immune activation of multiple antiviral host factors has been progressively recognized. Among them, Type III Interferons (IFNs) exhibit a pivotal role in inducing potent antiviral host responses by upregulating the expression of several antiviral genes, including the Interferon-Stimulated Genes (ISGs) that specifically target the virus replication machinery. To harness the immuno-adjunctive potential, we examined whether pre-treatment of IFNλ3, a Type III IFN, can activate antiviral host responses against IAV infections.
Methods
In the present study, we bioengineered a food-grade lactic acid-producing bacteria (LAB), Lactococcus lactis (L. lactis), to express and secrete functional murine IFNλ3 (MuIFNλ3) protein in the extracellular milieu. To test the immune-protective potential of MuIFNλ3 secreted by recombinant L. lactis (rL. lactis), we used murine B16F10 cells as an in vitro model while mice (BALB/c) were used for in vivo studies.
Results
Our study demonstrated that priming with MuIFNλ3 secreted by rL. lactis could upregulate the expression of several antiviral genes, including Interferon Regulatory Factors (IRFs) and ISGs, without exacerbated pulmonary or intestinal inflammatory responses. Moreover, we also showed that pre-treatment of B16F10 cells with MuIFNλ3 can confer marked immune protection against mice-adapted influenza virus, A/PR/8/1934 (H1N1) infection.
Conclusion
Since the primary target for IAV infections is the upper respiratory and gastrointestinal tract, immune activation without affecting the tissue homeostasis suggests the immune-adjunctive potential of IFNλ3 against IAV infections.
... The innate immune system is the first line of the host defense program against pathogens and harmful substances. The activated innate immune system produces IFNs and cytokines that perform antiviral functions to eliminate invading viruses [39][40][41][42]. However, during the coevolution with their host, viruses have developed diverse strategies to evade host antiviral defense programs to benefit their replications [43][44][45][46]. ...
Coronaviruses (CoVs) are a family of the largest RNA viruses that typically cause respiratory, enteric, and hepatic diseases in animals and humans, imposing great threats to the public safety and animal health. Porcine deltacoronavirus (PDCoV), a newly emerging enteropathogenic coronavirus, causes severe diarrhea in suckling piglets all over the world and poses potential risks of cross-species transmission. Here, we use PDCoV as a model of CoVs to illustrate the reciprocal regulation between CoVs infection and host antiviral responses. In this study, downregulation of DNA polymerase delta interacting protein 3 (POLDIP3) was confirmed in PDCoV infected IPEC-J2 cells by isobaric tags for relative and absolute quantification (iTRAQ) and Western blotting analysis. Overexpression of POLDIP3 inhibits PDCoV infection, whereas POLDIP3 knockout (POLDIP3 -/- ) by CRISPR-Cas9 editing significantly promotes PDCoV infection, indicating POLDIP3 as a novel antiviral regulator against PDCoV infection. Surprisingly, an antagonistic strategy was revealed that PDCoV encoded nonstructural protein 5 (nsp5) was responsible for POLDIP3 reduction via its 3C-like protease cleavage of POLDIP3 at the glutamine acid 176 (Q176), facilitating PDCoV infection due to the loss of antiviral effects of the cleaved fragments. Consistent with the obtained data in IPEC-J2 cell model in vitro , POLDIP3 reduction by cleavage was also corroborated in PDCoV infected-SPF piglets in vivo . Collectively, we unveiled a new antagonistic strategy evolved by PDCoV to counteract antiviral innate immunity by nsp5-mediated POLDIP3 cleavage, eventually ensuring productive virus replication. Importantly, we further demonstrated that nsp5s from PEDV and TGEV harbor the conserved function to cleave porcine POLDIP3 at the Q176 to despair POLDIP3-mediated antiviral effects. In addition, nsp5 from SARS-CoV-2 also cleaves human POLDIP3. Therefore, we speculate that coronaviruses employ similar POLDIP3 cleavage mechanisms mediated by nsp5 to antagonize the host antiviral responses to sustain efficient virus infection.
... Among these cytokines, the expression of Type I interferons (IFNα/β) is frequently associated with the inhibition of viral replication and the induction of a cellular anti-viral environment within infected but also neighbouring cells [181,184,221,227]. ...
Ex vivo organ cultures (EVOC) of tracheal explants with an air interface system have been successfully developed and used in the study of both human and animal respiratory pathogens. Such systems reproduce, to a great extent, the physiological conditions and the cellular complexity of the respiratory mucosa of the host in a highly controlled setting. In this study, we developed an EVOC system using pig trachea to quantify the infection dynamics of influenza A viruses.
Pig tracheal explants maintained the structure and function present in the living host preserving the intricate complexity of the respiratory mucosa during the first four days of the organ culture.
Inoculation with the swine virus A/swine/England/435/06 (H1N1) resulted in productive viral infection in a dose-dependent fashion. Infected explants showed histopathology of influenza infection. Viral titres peaked at day 2 post-inoculation and ciliary activity was abrogated shortly after. Explants collected from different sections of the trachea showed significant differences in the production of infectious virus.
The host innate immunity induction was evaluated by quantifying the mRNA levels of the cytokines IFN-β, IFN-α, TNF-α, IL-1α, IL-6 and the chemokine IL-8 during influenza infection of tracheal explant. In uninfected explants, IFN-β IFN-α and TNF-α mRNA expression levels did not change while cytokines associated with the inflammatory response - IL-1α, IL-6 and IL-8 - were significantly elevated throughout the organ culture. In influenza-infected explants the elevation in IFN-β expression appeared to be the predominant early response to influenza infection. IFN-α and TNF-α levels were not significantly altered whereas IL-1α, IL-6 and IL-8 expression showed a marked decrease in expression when the viral genome copy numbers peaked.
Productive infection was also observed in explants infected with the 2009 influenza pandemic virus A/England/195/09 (H1N1) but not with laboratory-adapted strain A/Puerto Rico/8/34 (H1N1) and not related with the induction of the innate immune system in tracheal explants.
RNA-based vaccines have sparked a paradigm shift in the treatment and prevention of diseases by nucleic acid medicines. There has been a notable surge in the development of nucleic acid therapeutics and vaccines following the global approval of the two messenger RNA-based COVID-19 vaccines. This growth is fueled by the exploration of numerous RNA products in preclinical stages, offering several advantages over conventional methods, i.e., safety, efficacy, scalability, and cost-effectiveness. In this chapter, we provide an overview of various types of RNA and their mechanisms of action for stimulating immune responses and inducing therapeutic effects. Furthermore, this chapter delves into the varying delivery systems, particularly emphasizing the use of nanoparticles to deliver RNA. The choice of delivery system is an intricate process involved in developing nucleic acid medicines that significantly enhances their stability, biocompatibility, and site-specificity. Additionally, this chapter sheds light on the current landscape of clinical trials of RNA therapeutics and vaccines against intracellular pathogens.
Introduction:
Non-melanoma skin cancer is the most common tumor. The combination of IFN-alpha 2b and IFN-gamma has been used as a new therapeutic opportunity to treat basal cell carcinomas and cutaneous squamous cell carcinomas. The aim of this report is to record prospectively the recurrence and new lesions rates in patients participating in phase II clinical trials.
Methods
Phase II clinical trials (double-blind randomized one center study, InCarbacel-III, in patients with basal cell carcinoma; and open, non-randomized multicenter study, CECIN, in patients with cutaneous squamous cell carcinomas, with the use of the combination of IFN-alpha 2b and IFN-gamma) were conducted to evaluate the efficacy, safety and the 5-year duration of clinical responses. Both studies were approved by institutional ethics committees, from the Hermanos Ameijeiras (INCARBACEL-III) and Amalia Simoni (INCARBACEL-III and CECIN) hospitals, in Havana and Camaguey respectively. All patients gave their written informed consent to participate in them.
The investigational treatment was administered, peri- or intralesionally, three times per week, during 3 weeks. Clinical (RECIST 1.0) responses were evaluated three months after the end of treatment.
Results
The combination of IFNs in InCarbacel-III study showed the best clinical response (complete response of 64.3%, overall response of 85.7%) with the highest doses (10.5 MIU); without patient’s recurrence at 5 years follow-up (3.5 MUI and 10.5 MUI groups). The frequency of new lesions decreased in the treated patients 8 times. In the CECIN study 14 patients achieved complete response and 4 partial responses (overall response rate 67%). Up to the 5-year follow-up none of the patients with complete response had recurrence or new lesion. In both studies the cosmetic results were excellent and the reported adverse events were mostly of mild intensity.
Conclusions:
The use of the combination of IFN-alpha 2b and IFN-gamma showed efficacy in basal cell carcinoma and cutaneous squamous cell carcinoma promoting a long term response for at least 5 years and decreasing the rate of new lesions, safely and with excellent cosmetic effects.
Introduction:
Non-melanoma skin cancer is the most common tumor. The combination of IFN-alpha 2b and IFN-gamma has been used as a new therapeutic opportunity to treat basal cell carcinomas and cutaneous squamous cell carcinomas. The aim of this report is to record prospectively the recurrence and new lesions rates in patients participating in phase II clinical trials.
Methods
Phase II clinical trials (double-blind randomized one center study, InCarbacel-III, in patients with basal cell carcinoma; and open, non-randomized multicenter study, CECIN, in patients with cutaneous squamous cell carcinomas, with the use of the combination of IFN-alpha 2b and IFN-gamma) were conducted to evaluate the efficacy, safety and the 5-year duration of clinical responses. Both studies were approved by institutional ethics committees, from the Hermanos Ameijeiras (INCARBACEL-III) and Amalia Simoni (INCARBACEL-III and CECIN) hospitals, in Havana and Camaguey respectively. All patients gave their written informed consent to participate in them.
The investigational treatment was administered, peri- or intralesionally, three times per week, during 3 weeks. Clinical (RECIST 1.0) responses were evaluated three months after the end of treatment.
Results
The combination of IFNs in InCarbacel-III study showed the best clinical response (complete response of 64.3%, overall response of 85.7%) with the highest doses (10.5 MIU); without patient’s recurrence at 5 years follow-up (3.5 MUI and 10.5 MUI groups). The frequency of new lesions decreased in the treated patients 8 times. In the CECIN study 14 patients achieved complete response and 4 partial responses (overall response rate 67%). Up to the 5-year follow-up none of the patients with complete response had recurrence or new lesion. In both studies the cosmetic results were excellent and the reported adverse events were mostly of mild intensity.
Conclusions:
The use of the combination of IFN-alpha 2b and IFN-gamma showed efficacy in basal cell carcinoma and cutaneous squamous cell carcinoma promoting a long term response for at least 5 years and decreasing the rate of new lesions, safely and with excellent cosmetic effects.
At present, there are a variety of different approaches to the targeted regulation of gene expression. However, most approaches are devoted to the activation of gene transcription, and the methods for gene silencing are much fewer in number. In this review, we describe the main systems used for the targeted suppression of gene expression (including RNA interference (RNAi), chimeric transcription factors, chimeric zinc finger proteins, transcription activator-like effectors (TALEs)-based repressors, optogenetic tools, and CRISPR/Cas-based repressors) and their application in eukaryotes—plants and animals. We consider the advantages and disadvantages of each approach, compare their effectiveness, and discuss the peculiarities of their usage in plant and animal organisms. This review will be useful for researchers in the field of gene transcription suppression and will allow them to choose the optimal method for suppressing the expression of the gene of interest depending on the research object.
Type I interferons (IFNs) play a pivotal role in immune response modulation, yet dysregulation is implicated in various disorders. Therefore, it is crucial to develop tools that facilitate the understanding of their mechanism of action and enable the development of more effective anti-IFN therapeutic strategies. In this study, we isolated, cloned, and characterized anti-IFN-α and anti-IFN-β antibodies (Abs) from peripheral blood mononuclear cells of individuals treated with IFN-α or IFN-β, harboring confirmed neutralizing Abs. Clones AH07856 and AH07857 were identified as neutralizing anti-IFN-α-specific with inhibition against IFN-α2a, -α2b, and -αK subtypes. Clones AH07859 and AH07866 were identified as neutralizing anti-IFN-β1a-specific signaling, and able to block Lipopolysaccharide or S100 calcium binding protein A14-induced IFN-β signaling effects. Cloned Abs bind rhesus but not murine IFNs. The specificity of inhibition between IFN-α and IFN-β suggests potential for diverse research and clinical applications.
BACKGROUND: The innate immune response, particularly the interferon system, plays a crucial role in defending the host against viral pathogens. Interferon signaling induces the expression of specific antiviral proteins known as interferon-stimulated genes, which inhibit viral replication through various mechanisms. AIM: This study aimed to develop a quantitative PCR system to assess the molecular regulation of human interferon-stimulated genes MxA, OAS1, and PKR, and to determine their expression in blood leukocytes in response to RNA-containing viruses. MATERIALS AND METHODS: Leukocytes were isolated from patients with laboratory-confirmed influenza and COVID-19 infections 3–4 days after symptom onset. Ex vivo viral infection was induced using influenza viruses A/California/07/09pdm (H1N1pdm09), B/Malaysia/2506/04 (Vic), strain A2 respiratory syncytial virus, and SARS-CoV-2 HCoV-19/Russia/SPE-RII-3524V/2020. RESULTS: A multiplex qPCR assay was developed for analyzing human MxA, OAS1, and PKR gene expression, with high amplification efficiency. The test system was used to study the molecular regulation of these genes in leukocytes in influenza and COVID-19 patients. The expression levels of MxA, OAS1, and PKR genes were significantly increased in blood leukocytes of hospitalized patients 3–4 days after symptom onset. Stimulation of leukocytes by influenza A, influenza B, and respiratory syncytial virus led to increased mRNA levels of these genes, while stimulation by SARS-CoV-2 did not result in changes in gene expression. CONCLUSIONS: The multiplex test system can be used to characterize the expression of antiviral effector interferon-stimulated genes, aiding in the study of virus evasion from the innate immune response.
IRF1 is a transcription factor well known for its role in IFN signaling. Although IRF1 was initially identified for its involvement in inflammatory processes, there is now evidence that it provides a function in carcinogenesis as well. IRF1 has been shown to affect several important antitumor mechanisms, such as induction of apoptosis, cell cycle arrest, remodeling of tumor immune microenvironment, suppression of telomerase activity, suppression of angiogenesis and others. Nevertheless, the opposite effects of IRF1 on tumor growth have also been demonstrated. In particular, the “immune checkpoint” molecule PD-L1, which is responsible for tumor immune evasion, has IRF1 as a major transcriptional regulator. These and several other properties of IRF1, including its proposed association with response and resistance to immunotherapy and several chemotherapeutic drugs, make it a promising object for further research. Numerous mechanisms of IRF1 regulation in cancer have been identified, including genetic, epigenetic, transcriptional, post-transcriptional, and post-translational mechanisms, although their significance for tumor progression remains to be explored. This review will focus on the established tumor-suppressive and tumor-promoting functions of IRF1, as well as the molecular mechanisms of IRF1 regulation identified in various cancers.
Heavy metal (i.e. lead (Pb)) is one of the environmental issues
recently due to its danger for human health. Therefore,
strategy for removing Pb from waste water treatment is
important. One of the prospective methods to remove Pb is
membrane biofilter. Here, the purpose of this study was to
prepare the membrane biofilter for Pb removal process. In
this study, membrane biofilter was produced from banana
stem. Banana stem was selected because of its abundant
availability in Indonesia. And, for somewhat, this banana
stem can be environmental problems (become waste) since
Indonesia is one of the top producers in the world. In short of
the experimental procedure, we conducted three steps of
experiments: (1) Preparation of microbial cellulose using
Acetobacter xylinum using banana stem for a main source; (2)
Synthesis of cellulose acetate; and (3) Preparation of
biomembrane from obtained cellulose acetate. To produce
membrane biofilter, the cellulose acetate was dissolved into
dichloromethane to form a dope solution. Then, the doped
solution was printed in Petri dish. Some biomembrane
properties were characterized for identification, i.e. infrared
spectra, electron microscope, and elemental analysis.
Experimental results showed that we succeeded to prepare
biomembrane with a pore size of 5 μm. The filtration
efficiency of our prepared membrane was 93.7% of Pb when
using Pb with a concentration of 10 ppm.
Simple Summary
Despite recent improvements in survival rates, colorectal cancer is still responsible for millions of premature deaths worldwide. A better understanding of the steps that lead from chronic inflammation to cancer progression could offer new therapeutic options and save many lives. The present study aimed to investigate the role of the transcription factor signal transducer and activator of transcription 2 (STAT2) in colorectal cancer by taking advantage of experimental mouse models and three-dimensional tumoroids. Our results indicate that STAT2 promotes colorectal cancer by various mechanisms and that anti-cancer drugs could easily kill tumor cells lacking STAT2. It is conceivable that future therapeutic strategies for colorectal cancer might try to mitigate the actions of STAT2 to improve the treatment outcome in affected patients.
Abstract
Colorectal cancer (CRC) is a significant socioeconomic burden in modern society and is accountable for millions of premature deaths each year. The role of signal transducer and activator of transcription 2 (STAT2)-dependent signaling in this context is not yet fully understood, and no therapies targeting this pathway are currently being pursued. We investigated the role of STAT2 in CRC using experimental mouse models coupled with RNA-sequencing (RNA-Seq) data and functional assays with anti-cancer agents in three-dimensional tumoroids. Stat2−/− mice showed greater resistance to the development of CRC in both inflammation-driven and inflammation-independent experimental CRC models. In ex vivo studies, tumoroids derived from Stat2−/− mice with the multiple intestinal neoplasia (Min) mutant allele of the adenomatous polyposis coli (Apc) locus exhibited delayed growth, were overall smaller and more differentiated as compared with tumoroids from ApcMin/+ wildtype (WT) mice. Notably, tumoroids from ApcMin/+ Stat2−/− mice were more susceptible to anti-cancer agents inducing cell death by different mechanisms. Our findings clearly indicated that STAT2 promotes CRC and suggested that interventions targeting STAT2-dependent signals might become an attractive therapeutic option for patients with CRC.
We present the case of a 15-year-old girl, with a fifth cystic progression of an adamantinomatous craniopharyngioma after multiple surgeries and previous local radiotherapy. She had severe visual impairment, panhypopituitarism including diabetes insipidus, and several components of hypothalamic damage, including morbid obesity and severe fatigue. To prevent further late effects hampering her quality of survival, she was treated biweekly with intravenous tocilizumab, an anti-interleukin-6 agent, which stabilized the cyst for a prolonged time. Based on the biology of adamantinomatous craniopharyngioma, this immune-modulating treatment seems promising for the treatment of this cystic tumor in order to reduce surgery and delay or omit radiotherapy.
Promiscuous binding of different Janus kinases (JAKs) to class I/II cytokine receptors has been reported, yet its role in signaling is unclear. To systematically explore
JAK pairing in type I interferon (IFN-I) signaling, we generated an artificial IFN-I receptor (AIR) by replacing the extracellular domains of IFNAR1 and IFNAR2 with anti
mEGFP and mCherry nanobodies. The heterodimeric AIR restored near-native IFN-I activity, while the homomeric variant of IFNAR2 (AIR-dR2) initiated much weaker signaling despite harboring docking sites for signal transducer and activator of transcription (STAT) proteins. AIR-dR1 was signaling inactive, yet, pulldown uncovered
its ICD to bind both TYK2 and JAK1. To further investigate the roles of JAKs on the receptors, knockout (KO) JAK1, JAK2, TYK2, and JAK2/TYK2 were generated. JAK1 KO led to complete loss of IFN-I signaling, which was partially restored by TYK2 overexpression. TYK2 KO cells retained partial activity, which was elevated by JAK1 overexpression, suggesting both JAKs to partially substitute each other. Conversely, JAK2 KO only moderately impacted the biological activity of IFN-Is, even in
JAK2/TYK2 KO cells. Live cell micropatterning confirmed promiscuous binding of JAK1, JAK2 and TYK2 to IFNAR1 and IFNAR2, in line with an AlphaFold model that shows JAKs interchangeability on IFNAR ICDs. Similar promiscuity of JAK binding was observed for TPOR and GHR but not EPOR, accompanied by different downstream signaling activity. The competitive binding of JAKs to cytokine receptors together with the highly diverse absolute and relative JAK expression levels can account for cell typedependent signaling pleiotropy observed for cytokine receptors.
White spot syndrome virus (WSSV), a double-stranded DNA virus, is harmful in aquaculture. The signal transducer and activator of transcription (STAT) has been shown to play a role during host infection with the virus, but the exact mechanism by which it acts is unclear. In this study, three STAT isoforms (MnSTAT1, MnSTAT2, and MnSTAT3) were identified in Macrobrachium nipponense. The full-length sequence of MnSTAT1 was 3336 bp, with 2259 bp open reading frame (ORF), encoding a 852 amino acids protein. The full-length sequence of MnSTAT2 was 2538 bp, and the ORF was 2391 bp, encoding 796 amino acids. The full-length sequence of MnSTAT3 sequence was 2618 bp, and the ORF was 2340 bp, encoding 779 amino acids. MnSTAT1-3 is produced by alternative last exon. MnSTAT1-3 all contain a STAT_int, a STAT_alpha, a STAT_bind, and a SH2 structure. MnSTAT1-3 are widely expressed in various tissues tested. The expression levels of MnSTAT1-3 in the intestine of M. nipponense were upregulated at multiple time points following WSSV stimulation. The expression of seven anti-lipopolysaccharide factors (ALFs) was significantly reduced with the knockdown of MnSTATs during WSSV infection. Results showed that MnSTATs regulated the expression of intestinal ALFs and was involved in the innate immunity against WSSV of M. nipponense.
Purpose of review:
Systemic sclerosis (SSc) is a multisystem autoimmune connective tissue disease characterized by early inflammation followed by excessive fibrosis in the skin and internal organs. Enhancing our comprehension of SSc pathogenesis is essential to develop effective therapeutic strategies. Animal models that mimic one or more aspects of SSc have been proven to be a valuable resource for investigating disease mechanisms. This review aims to provide an updated overview of the existing SSc animal models and the potentially relevant pathways to SSc pathogenesis.
Recent findings:
This review focuses on the most recently generated and investigated animal models, which delve into novel pathways beyond existing models or employ genetic technologies to gain a deeper understanding of SSc pathogenesis including activation of early type I interferon (IFN) signaling pathway, immune cell function and pulmonary artery hypertension (PAH).
Summary:
While no single animal model can fully replicate SSc, a combination of different models can offer valuable insights into the pathways involved in the onset and advancement of the SSc. These insights can prove animal models as a crutial preclinical tool for developing effective treatments for SSc.
Neurons require physiological IFN-γ signaling to maintain central nervous system (CNS) homeostasis, however, pathological IFN-γ signaling can cause CNS pathologies. The downstream signaling mechanisms that cause these drastically different outcomes in neurons has not been well studied. We hypothesized that different levels of IFN-γ signaling in neurons results in differential activation of its downstream transcription factor, signal transducer and activator of transduction 1 (STAT1), causing varying outcomes. Using primary cortical neurons, we showed that physiological IFN-γ elicited brief and transient STAT1 activation, whereas pathological IFN-γ induced prolonged STAT1 activation, which primed the pathway to be more responsive to a subsequent IFN-γ challenge. This is an IFN-γ specific response, as other IFNs and cytokines did not elicit such STAT1 activation nor priming in neurons. Additionally, we did not see the same effect in microglia or astrocytes, suggesting this non-canonical IFN-γ/STAT1 signaling is unique to neurons. Prolonged STAT1 activation was facilitated by continuous janus kinase (JAK) activity, even in the absence of IFN-γ. Finally, although IFN-γ initially induced a canonical IFN-γ transcriptional response in neurons, pathological levels of IFN-γ caused long-term changes in synaptic pathway transcripts. Overall, these findings suggest that IFN-γ signaling occurs via non-canonical mechanisms in neurons, and differential STAT1 activation may explain how neurons have both homeostatic and pathological responses to IFN-γ signaling.
Objective:
To investigate the effect of Porphyromonas gingivalis (Pg) infection on IFNGR1 palmitoylation and biological behaviors of esophageal squamous cell carcinoma (ESCC) cells and the clinical implications.
Methods:
The expression levels of IFNGR1 protein in ESCC cell lines KYSE30 and KYSE70 were detected using Western blotting at 24 and 48 h after Pg infection, and 2-BP was used to detect IFNGR1 palmitoylation in the cells. KYSE70 cells with wild-type IFNGR1 (IFNGR1-WT cells) and with IFNGR1-C122A palmitoylation site mutation induced by site-specific mutagenesis (IFNGR1-C122A cells) were both infected with Pg, and the changes in palmitoylation of IFNGR1-C122A were analyzed using immunofluorescence and Click-iT assays. The changes in proliferation, migration and invasion ability of the infected cells were evaluated using plate cloning assay, scratch assay and Transwell assay, and IFNGR1 co-localization with lysosomal marker LAMP2 was dected using immunofluorescence assay. Immunohistochemistry was used to detect Pg infection and IFNGR1 protein expression in 50 ESCC tissues, and their correlation with the clinicopathological characteristics and survival outcomes of the patients was analyzed.
Results:
Pg infection down-regulated the protein expression of IFNGR1 in ESCC and promoted IFNGR1 palmitoylation at site 122. In IFNGR1-WT cells, Pg infection significantly enhanced cell proliferation, migration and invasion (P < 0.05). Similarly, Pg also significantly promoted proliferation, migration and invasion of IFNGR1-C122A cells, but to a lesser extent as compared with the wild-type cells (P < 0.05). Immunofluorescence assay showed that Pg and ZDHHC3 promoted IFNGR1 degradation within the lysosome. Immunohistochemical studies of the ESCC tissue samples showed a negative correlation between IFNGR1 and Pg expression, and a reduced IFNGR1 expression was correlated with a poorer survival outcome of the patient.
Conclusion:
Pg infection enhances IFNGR1 palmitoylation to promote progression of ESCC, and elimination of Pg and inhibiting IFNGR1 palmitoylation may effectively control ESCC progression.
Background
STAT1 is an intracellular signaling molecule that is crucially involved in the regulation of the innate immune system by activation of defense mechanisms against microbial pathogens. Phosphorylation-dependent activation of the STAT1 transcription factor is associated with a conversion from an antiparallel to parallel dimer configuration, which after nuclear import binds to DNA. However, not much is known about the specific intermolecular interactions that stabilize unphosphorylated, antiparallel STAT1 complexes prior to activation.
Results
In this study, we identified a previously unknown interdimeric interaction site, which is involved in the termination of STAT1 signaling. Introduction of the glutamic acid-to-alanine point mutation E169A in the coiled-coil domain (CCD) by site-directed mutagenesis led to increased tyrosine phosphorylation as well as accelerated and prolonged nuclear accumulation in transiently transfected cells. In addition, DNA-binding affinity and transcriptional activity were strongly enhanced in the substitution mutant compared to the wild-type (WT) protein. Furthermore, we have demonstrated that the E169 residue in the CCD mediates the release of the dimer from the DNA in an auto-inhibitory manner.
Conclusion
Based on these findings, we propose a novel mechanism for the inactivation of the STAT1 signaling pathway, assigning the interface with the glutamic acid residue 169 in the CCD a crucial role in this process.
Hepatocellular carcinoma (HCC) caused by HBV, HCV infection, and other factors is one of the most common malignancies in the world. Although, percutaneous treatments such as surgery, ethanol injection, radiofrequency ablation, and transcatheter treatments such as arterial chemoembolization are useful for local tumor control, they are not sufficient to improve the prognosis of patients with HCC. External interferon agents that induce interferon-related genes or type I interferon in combination with other drugs can reduce the recurrence rate and improve survival in HCC patients after surgery. Therefore, in this review, we focus on recent advances in the mechanism of action of type I interferons, emerging therapies, and potential therapeutic strategies for the treatment of HCC using IFNs.
We studied the phosphorylation of the alpha and beta subunits of the Type I interferon (IFN) receptor induced by Type I IFNs in the human U-266 and MOLT-4 cell lines. Both IFN-alpha and IFN-beta induced tyrosine phosphorylation of the beta subunit of the receptor. The Type I IFN-induced tyrosine phosphorylation of the beta subunit was rapid and transient, being detectable within 1 min of Type I IFN treatment and gradually diminishing to almost base-line levels by 60 min. All Type I IFNs studied were found to induce tyrosine phosphorylation of the alpha subunit of the Type I IFN receptor, the p135tyk2 and JAK-1 tyrosine kinases, and the ISGF3 alpha components. Interestingly, IFN-beta, but not IFN-alpha or IFN-omega, induced tyrosine phosphorylation of an alpha subunit-associated protein with an apparent molecular mass of approximately 100 kDa (p100). These data suggest the existence of a common signaling pathway(s) for Type I IFNs involving the alpha and beta subunits of the receptor, the tyrosine kinases p135tyk2 and JAK-1, and the ISGF3 alpha components. However, differences between the signaling pathways of different Type I IFNs exist, as suggested by tyrosine phosphorylation of an alpha subunit-associated protein only in response to IFN-beta.
We report preliminary characterization of a gene designated β-R1, which is selectively expressed in response to interferon
β (IFN-β) compared with IFN-α. In human astrocytoma cells, β-R1 was induced to an equivalent extent by 10 IU/mL IFN-β or 2500
IU/mL IFN-α2. To address the mechanism of this differential response, we analyzed induction of the β-R1 gene in fibrosarcoma
cells and derivative mutant cells lacking components required for signaling by type I IFNs. β-R1 was readily induced by IFN-β
in the parental 2fTGH cell line, but not by recombinant IFN-α2, IFN-α Con1, or a mixture of IFN-α subtypes. IFN-α8 induced
β-R1 weakly. β-R1 was not induced by IFN-β in mutant cell lines U2A, U3A, U4A, and U6A, which lack, respectively, p48, STAT1,
JAK1, and STAT2. U5A cells, which lack the Ifnar 2.2 component of the IFN-α and -β receptor, also failed to express β-R1.
U1A cells are partially responsive to IFN-β and IFN-α8 but lacked β-R1 expression, indicating that TYK2 protein is essential
for induction of this gene. Taken together, these results suggest that the expression of β-R1 in response to type I IFN requires
IFN-stimulated gene factor 3 plus an additional component, which is more efficiently formed on induction by IFN-β compared
with IFN-α.
Herein, we demonstrate that purified Stat1 binds to its tyrosine-phosphorylated docking site on the IFNγ receptor α chain in a direct, specific, and reversible manner. Using surface plasmon resonance, we determine the affinity (KD = 137 nM) and specificity of the interaction and define the minimum affinity needed for receptor-mediated Stat1 activation. In addition, we quantitate the relative ability of purified Stat1 to interact with tyrosine-phosphorylated binding sites on other Stat proteins. Finally, we describe experiments that imply that the unidirectional release of activated Stat1 from the IFNγ receptor reflects the preference of free tyrosine-phosphorylated Stat1 monomers to form high avidity reciprocal homodimere rather than reassociating with the receptor binding site. Our results demonstrate that IFNγ-induced Stat1 activation is an ordered and affinity-driven process and we propose that this process may serve as a paradigm for Stat activation by other cytokine receptors.
We have characterized the functional properties of the short form of the human interferon α/β receptor chain 2 (IFNAR2), denoted IFNAR2.1. IFNAR2.1 contains a shortened cytoplasmic domain when compared with the recently cloned full-length IFNAR2 chain (IFNAR2.2). We show that IFNα8 and IFNβ1b induce antiviral and antiproliferative activity in mouse cell transfectants expressing the human IFNAR1 chain of the receptor and induce the formation of STAT1/STAT2 dimers in IFN-stimulated response element (ISRE)-dependent gel shift assays. In contrast, coexpression of IFNAR2.1 with IFNAR1 reduces the IFN-induced antiviral, antiproliferative and ISRE-dependent gel shift binding activity conferred by IFNAR1 alone. No antiviral or antiproliferative response to IFN, nor IFN-induced ISRE-dependent gel shift binding activity, was observed when IFNAR2.1 was expressed alone in murine cells. Therefore, IFNAR2.1 acts as a dominant negative for these IFN-induced activities. Our results suggest that IFNAR2.1 represents a nonfunctional version of the full-length chain (IFNAR2.2).
Ribonuclease L (RNase L), the 2′,5′-oligoadenylate-dependent ribonuclease, is one of the cellular antiviral systems with enhanced
activity in the presence of interferon. A reaction scheme has been developed to model the sequence of steps necessary for
the activation of RNase L (Cole, J. L., Carroll, S. S., Blue, E. S., Viscount, T., and Kuo, L. C. (1997)J. Biol. Chem. 272, 19187–19192). The model comprises three sequential binding steps: the binding of activator to enzyme monomer, the subsequent
dimerization of the activated monomer to form the active enzyme dimer, followed by the binding of substrate prior to catalysis.
The model is used to evaluate the activation of RNase L by several synthetic analogs of the native activator. The 5′-phosphate
of the activator has been determined to be an important structural determinant for the efficient activation of RNase L, and
its loss caused a loss of activator affinity of 2–3 orders of magnitude. The length of activator is not an important determinant
of activator potency for the activator analogs examined. The specific activity of the enzyme under conditions of saturation
of activator binding and complete dimerization of the activated monomers varies only by about a factor of 3 for the activators
examined, indicating that once dimerized in the presence of any of these activators, the enzyme exhibits a similar catalytic
activity.
tyk2 belongs to the JAK family of nonreceptor protein tyrosine kinases recently found implicated in signaling through a large number of cytokine receptors. These proteins are characterized by a large amino-terminal region and two tandemly arranged kinase domains, a kinase-like and a tyrosine kinase domain. Genetic and biochemical evidence supports the requirement for tyk2 in interferon-α/β binding and signaling. To study the role of the distinct domains of tyk2, constructs lacking one or both kinase domains were stably transfected in recipient cells lacking the endogenous protein. Removal of either or both kinase domains resulted in loss of the in vitro kinase activity. The mutant form truncated of the tyrosine kinase domain was found to reconstitute binding of interferon-α8 and partial signaling. While no contribution of this protein toward interferon-β binding was evident, increased signaling could be measured. The mutant form lacking both kinase domains did not exhibit any detectable activity. Altogether, these results show that a sequential deletion of domains engenders a sequential loss of function and that the different domains of tyk2 have distinct functions, all essential for full interferon-α and -β binding and signaling.
All Type I interferons (IFNα, IFNβ, IFNω) bind to the Type I IFN receptor (IFNR) and elicit a common set of signaling events,
including activation of the Jak/Stat and IRS pathways. However, IFNβ selectively induces the association of the α subunit
of the Type I IFNR with p100, a tyrosyl phosphoprotein, to transduce IFNβ-specific signals. Using antibodies raised against
the different components of the Type I IFNR, we identified p100 as the long form of the β subunit (βL subunit) of the Type I IFNR. This was also confirmed in experiments with mouse L-929 cells transfected with truncated forms
of βL. Thus, IFNβ stimulation of human cells or mouse L-929 transfectants expressing the human α and βL subunits, selectively induces the formation of a signaling complex containing the α and βL subunits of the receptor. The IFNβ-regulated interaction of the α and βL chains is rapid and transient and follows a similar time course with the tyrosine phosphorylation of these receptor components.
These data demonstrate that the signaling specificity for different Type I IFNs is established early in the signaling cascade,
at the receptor level, and results from distinct interactions between components of the Type I IFNR.
The interferon-inducible, double-stranded RNA (dsRNA)-dependent eukaryotic initiation factor-2α kinase PKR has primarily been characterized as a component of the interferon-mediated cellular antiviral response. Several lines of evidence now exist that suggest that PKR plays a role in the regulation of growth in uninfected cells. The most direct examples are the finding of an oncogenic variant of PKR and the effects of activators and inhibitors of PKR phosphorylation on the expression of platelet-derived growth factor (PDGF)-inducible genes. Previous reports have shown that 1) dsRNA, a direct activator of PKR, induces the genes c-myc, c-fos, and JE; 2) 2-aminopurine, a chemical inhibitor of PKR, blocks the induction of these genes by serum; and 3) activated p21 induces a cellular inhibitor of PKR. We report here that activation of PKR was correlated with the induction of the immediate early genes c-fos, c-myc, and JE by PDGF in the following situations: 1) PDGF induction of these genes, also inducible by dsRNA, was blocked by two inhibitors of PKR activation: 2-aminopurine and v-ras; 2) PDGF induction of another immediate early gene, egr-1, which could not be induced by dsRNA, was not blocked by 2-aminopurine or v-ras; 3) agents that reverse v-ras inhibition of PKR activation also reversed the v-ras block of PDGF induction of c-myc, c-fos, and JE; 4) down-regulation of PKR protein levels by antisense inhibition of translation blocked the induction of c-myc, c-fos, and JE by PDGF, but had no effect on egr-1 induction; and finally, 5) PKR was autophosphorylated in vivo in response to PDGF. These results provide direct evidence that PKR activation functions as a second messenger in a growth factor signal transduction pathway. Thus, PKR may serve as a common mediator of growth-promoting and growth inhibitory signals.
Interaction of certain cytokines with their corresponding cell-surface receptors induces programed cell death. Interferon-γ
induces in HeLa cells a type of cell death with features characteristic of programed cell death. Here, we report the isolation
of a novel gene, DAP3 (death-associated protein-3), involved in mediating interferon-γ-induced cell death. The rescue of this gene was performed
by a functional selection approach of gene cloning that is based on transfection with an antisense cDNA expression library.
The antisense RNA-mediated inactivation of the DAP3 gene protected the cells from interferon-γ-induced cell death. This property endowed the cells expressing it with a growth
advantage in an environment restrictive due to the continuous presence of interferon-γ and thus provided the basis of its
selection. The gene is transcribed into a single 1.7-kilobase mRNA, which is ubiquitously expressed in different tissues and
codes for a 46-kDa protein carrying a potential P-loop motif. Ectopic expression of DAP3 in HeLa cells was not compatible with cell growth, resulting in a 16-fold reduction in the number of drug-resistant stable
clones. The data presented suggest that DAP3 is a positive mediator of cell death induced by interferon-γ.
We have shown previously that a four-amino acid block residing at positions 266-269 (LPKS) in the intracellular domain of
the human interferon- (IFN-) receptor α chain is critical for IFN--dependent tyrosine kinase activation and biologic response induction. Herein we show that this sequence is required for the
constitutive attachment of the tyrosine kinase JAK-1. Using a vaccinia expression system, a receptor α chain-specific monoclonal
antibody coprecipitated JAK-1 from cells coexpressing JAK-1 and either (a) wild type IFN- receptor α chain, (b) a receptor α chain truncation mutant containing only the first 59 intracellular domain amino acids, or (c) a receptor mutant containing alanine substitutions for the functionally irrelevant residues 272-275. In contrast, JAK-1
was not coprecipitated when coexpressed with a receptor α chain mutant containing alanine substitutions for the functionally
critical residues 266-269 (LPKS). Mutagenesis of the LPKS sequence revealed that Pro-267 is the only residue obligatorily
required for receptor function. In addition, Pro-267 is required for JAK-1 binding. These results thus identify a site in
the IFN- receptor α chain required for constitutive JAK-1 association and establish that this association is critical for IFN- signal transduction.
We previously found that the level of Fas, a cell surface receptor for an apoptosis signal, increases at the mRNA level in
influenza virus-infected HeLa cells prior to their death by apoptosis. Here we investigated the mechanism of activation of
the Fas-encoding gene expression upon influenza virus infection. Nucleotide sequences for the binding of nuclear factor for
interleukin-6 expression (NF-IL6), also known as CCAAT/enhancer-binding protein β, were repeated 8 times in the 5′-end region
of the human FAS gene, spanning from −1360 to +320. This region directed the expression of a downstream marker gene when introduced into HeLa
cells and the activity of the FAS gene promoter was stimulated about 2-fold upon influenza virus infection. Gene expression driven by the FAS promoter was activated when human NF-IL6 was overproduced in a DNA co-transfection study. Moreover, the DNA-binding activity
of NF-IL6 increased after infection with the virus, whereas the amount of NF-IL6 seemed unchanged. The results suggest that
NF-IL6 is activated upon influenza virus infection through post-translational modification and that the modified factor stimulates
the transcription of the human FAS gene.
We report that interferon γ (IFN-γ ) inhibits transcription
of the macrophage scavenger receptor gene by antagonizing the
Ras-dependent activities of AP-1 and cooperating ets domain
transcription factors, apparently as a result of competition between
AP-1/ets factors and activated STAT1 for limiting amounts of CBP and
p300. Consistent with this model, STAT1α interacts directly with
CBP in cells, and microinjection of anti-CBP and anti-p300 antibodies
blocks transcriptional responses to IFN-γ . Cells lacking STAT1
fail to inhibit AP-1/ets activity, and overexpression of CBP both
potentiates IFN-γ -dependent transcription and relieves AP-1/ets
repression. Thus, CBP and p300 integrate both positive and negative
effects of IFN-γ on gene expression by serving as essential
coactivators of STAT1α , modulating gene-specific responses to
simultaneous activation of two or more signal transduction pathways.
The interferon (IFN)-induced double-stranded RNA (dsRNA)-activated Ser/Thr protein kinase (PKR) plays a role in the antiviral and antiproliferative effects of IFN. PKR phosphorylates initiation factor eIF2, thereby inhibiting protein synthesis, and also activates the transcription factor, nuclear factor-B (NF-B), by phosphorylating the inhibitor of NF-B, IB. Mice devoid of functional PKR (Pkr°/°) derived by targeted gene disruption exhibit a diminished response to IFN- and poly(rI:rC) (pIC). In embryo fibroblasts derived from Pkr°/° mice, interferon regulatory factor 1 (IRF-1) or guanylate binding protein (Gbp) promoter–reporter constructs were unresponsive to IFN- or pIC but response could be restored by co-transfection with PKR. The lack of responsiveness could be attributed to a diminished activation of IRF-1 and/or NF-B in response to IFN- or pIC. Thus, PKR acts as a signal transducer for IFN-stimulated genes dependent on the transcription factors IRF-1 and NF-B.
Ribonuclease L is an endoribonuclease that is activated by binding of 2′,5′-linked oligoadenylates. Activation of ribonuclease
L also induces dimerization. Here, we demonstrate using equilibrium sedimentation that dimerization requires the binding of
one 5′-monophosphate 2′,5′-(adenosine) molecule per ribonuclease L monomer. No dimerization was observed in the absence of activator up to a protein concentration
of 18 μM, indicating that unliganded enzyme is unable to dimerize or the association is very weak. In parallel with dimerization,
enzymatic activity is also maximized at a 1:1 activator: ribonuclease L stoichiometry. The same stoichiometry for dimerization
is observed using a nonphosphorylated activator 2′-5′-(adenosine). Adenosine triphosphate or RNA oligonucleotide substrates do not induce dimerization. The observed stoichiometry supports
a model for ribonuclease L dimerization in which activator binds to monomer, which subsequently dimerizes.
RNase L, the 2',5' oligoadenylate-dependent ribonuclease, is one of the enzyme systems important in the cellular response to interferon, When activated in the presence of 2',5'-linked oligoadenylates, RNase L can catalyze the cleavage of synthetic oligoribonucleotides that contain dyad sequences of the forms UU, UA, AU, AA, and UG, but it cannot catalyze the cleavage of an oligoribonucleotide containing only cytosines, The primary site of the cleavage reaction with the substrate C11UUC7 has been defined to be 3' of the UU dyad by labeling either the 5' or the 3' end of the oligoribonucleotide and by examining the reaction products on polyacrylamide sequencing gels, Reaction time courses have been used to determine the kinetic parameters of the cleavage reactions, The effect of the overall length of the oligomeric substrate as well as the sequence of the bases around the position of the cleavage site on the kinetics of the cleavage reaction has been examined. The efficiency with which activated RNase L catalyzes the cleavage of the substrate C11UUC7 is 1.9 x 10(7) M(-1) s(-1). Because the cleavage of the synthetic oligoribonucleotide can be used to monitor the steady-state kinetics of catalysis by activated RNase L, this method offers an advantage over previous methods of assay for RNase L activity.
The tyrosine kinase JAK1 and the transcription factors STAT1 and STAT3 are phosphorylated in response to epidermal growth factor (EGF) and other growth factors. We have used EGF receptor-transfected cell lines defective in individual JAKs to assess the roles of these kinases in STAT activation and signal transduction in response to EGF. Although JAK1 is phosphorylated in response to EGF, it is not required for STAT activation or for induction of the c-fos gene. STAT activation in JAK2- and TYK2-defective cells is also normal, and the tyrosine phosphorylation of these two kinases does not increase upon EGF stimulation in wild-type or JAK1-negative cells. In cells transfected with a kinase-negative mutant EGF receptor, there is no STAT activation in response to EGF and c-fos is not induced, showing that the kinase activity of the receptor is required, directly or indirectly, for these two responses. The data do not support a role for any of the three JAK family members tested in STAT activation and are consistent with a JAK-independent pathway in which the intrinsic kinase domain of the EGF receptor is crucial. Furthermore, data from transient transfection experiments in HeLa cells, using c-fos promoters lacking the STAT regulatory element c-sis-inducible element, indicate that this element may play only a minor role in the induction of c-fos by EGF in these cells.
Interferons (IFNs) induce early-response genes by stimulating Janus family (Jak) tyrosine kinases, leading to tyrosine phosphorylation
of Stat transcription factors. Previous studies implicated protein-tyrosine phosphatase (PTP) activity in the control of IFN-regulated
Jak/Stat signaling, but the specific PTPs responsible remained unidentified. We have found that SH2 domain-containing PTP1
(SHPTP1; also called PTP1C, HCP, or SHP) reversibly associates with the IFN-alpha receptor complex upon IFN addition. Compared
with macrophages from normal littermate controls, macrophages from motheaten mice, which lack SHPTP1, show dramatically increased
Jak1 and Stat1 alpha tyrosine phosphorylation, whereas Tyk2 and Stat2 activation is largely unaffected. These findings correlate
with selectively increased complex formation on a gamma response element, but not an IFN-stimulated response element, in motheaten
macrophages. Our results establish that SHPTP1 selectively regulates distinct components of Jak/Stat signal transduction pathways
in vivo.
The data presented in this manuscript extend our previous observations that recombinant interferon-gamma (reIFN-gamma) can suppress anti-immunoglobulin (anti-Ig)-stimulated B cell proliferation, and demonstrate that reIFN-gamma can also suppress B cell stimulation factor type 1 (BSF-1)-stimulated increases in expression of MHC class II molecules (Ia) on B cells. This suppression is most effective when relatively low concentrations of BSF-1 are employed, but is still very substantial even when optimal concentrations of BSF-1 were used. This suppression is also observed when size-separated small B cells which are devoid of detectable macrophages or NK cells are cultured with BSF-1 and reIFN-gamma, thus suggesting that IFN-gamma-mediated inhibition is a consequence of a direct effect on the B cells. Incubation of B cells with reIFN-gamma for 24 hr before their culture with BSF-1 did not prevent BSF-1-stimulated increases in sIa. This finding supports the contention that the effect of IFN-gamma is not mediated via the stimulation of "suppressor" influences in these cell cultures. The inhibition of B cell activation by IFN-gamma occurs within the first 3 hr after the onset of culture, as demonstrated by the inability of antibody to IFN-gamma to totally reverse the IFN-gamma-mediated suppressive effects on B cell proliferation if it is added later than 3 hr after the onset of culture. These results suggest a role for IFN-gamma in down-regulating the ability of B cells to function as antigen-presenting cells in non-cognate T cell-dependent responses.
2‐5A‐dependent RNase is the terminal factor in the interferon‐regulated 2‐5A system thought to function in both the molecular mechanism of interferon action and in the general control of RNA stability. However, direct evidence for specific functions of 2‐5A‐dependent RNase has been generally lacking. Therefore, we developed a strategy to block the 2‐5A system using a truncated form of 2‐5A‐dependent RNase which retains 2‐5A binding activity while lacking RNase activity. When the truncated RNase was stably expressed to high levels in murine cells, it prevented specific rRNA cleavage in response to 2‐5A transfection and the cells were unresponsive to the antiviral activity of interferon alpha/beta for encephalomyocarditis virus. Remarkably, cells expressing the truncated RNase were also resistant to the antiproliferative activity of interferon. The truncated RNase is a dominant negative mutant that binds 2‐5A and that may interfere with normal protein‐protein interactions through nine ankyrin‐like repeats.
The malignant, CD5+ B lymphocytes of B cell chronic lymphocytic leukemia (B-CLL) die by apoptosis in vitro. This is in contrast to the prolonged life span of the leukemic cells in vivo and likely reflects the lack of essential growth factors in the tissue culture medium. We found that interferon gamma (IFN-gamma) inhibits programmed cell death and promotes survival of B-CLL cells in culture. This effect may also be important in vivo: increased serum levels of IFN-gamma, ranging from 60 to > 2,200 pg/ml, were found in 7 of 10 B-CLL samples tested, whereas the sera of 10 healthy individuals did not contain detectable levels of this cytokine (< 20 pg/ml). High levels of IFN-gamma message were detected in RNA from T cell-depleted B-CLL peripheral blood samples by Northern blot analysis. Synthesis of IFN-gamma by B-CLL lymphocytes was confirmed by in situ hybridization and flow cytometry. The majority of B-CLL cells (74-82%) expressed detectable levels of IFN-gamma mRNA, and CD19+ B-CLL cells were labeled with anti-IFN-gamma monoclonal antibodies. These results show that IFN-gamma inhibits programmed cell death in B-CLL cells and suggest that the malignant cells are able to synthesize this cytokine. By delaying apoptosis, IFN-gamma may extend the life span of the malignant cells and thereby contribute to their clonal accumulation.
Oncostatin M (OSM) is a 28-kD glycoprotein recently identified as a growth factor for human multiple myeloma cells. It belongs to a family of distantly related cytokines that includes interleukin 6, ciliary neurotrophic factor, leukemia-inhibitory factor, and interleukin 11. These cytokines initiate signaling by inducing either homodimerization of gp130 or heterodimerization of gp130 with leukemia-inhibitory factor receptor beta components. Such dimerization in turn activates receptor-associated tyrosine kinases. In the present study using U266B1 human multiple myeloma cells, we show that OSM induces tyrosine phosphorylation and activation of JAK2, but not JAK1 or Tyk2, kinases. The results also demonstrate that OSM induces direct interaction of JAK2 kinase with Grb2, an SH2/SH3 domain containing adaptor protein. The SH2 domain of Grb2 is directly associated with tyrosine-phosphorylated JAK2. Furthermore, the presence of Sos in the JAK2-Grb2 complex suggests a role for Ras in OSM-transduced signaling.
Signal transducers and activators of transcription (STATs) enhance transcription of specific genes in response to cytokines and growth factors. STAT1 is also required for efficient constitutive expression of the caspases Ice, Cpp32, and Ich-1 in human fibroblasts. As a consequence, STAT1-null cells are resistant to apoptosis by tumor necrosis factor α (TNF-α). Reintroduction of STAT1α restored both TNF-α–induced apoptosis and the expression of Ice, Cpp32, and Ich-1. Variant STAT1 proteins carrying point mutations that inactivate domains required for STAT dimer formation nevertheless restored protease expression and sensitivity to apoptosis, indicating that the functions of STAT1 required for these activities are different from those that mediate induced gene expression.
Antigens and infectious agents that stimulate interferon alpha(IFN-alpha) production in mice induce antibody responses that are predominantly of the immunoglobulin (Ig)G2a isotype and contain little or no IgE. This suggested the possibility that IFN-alpha might have a role in directing Ig isotype selection. Consistent with this possibility, we have found that injection of mice with recombinant mouse IFN-alpha suppresses IgE secretion, enhances IgG2a secretion, and has no independent effect on IgG1 secretion in mice stimulated with a foreign anti-IgD antibody. Injection of mice with polyinosinic acid.polycytidylic acid (poly I.C), an inducer of macrophage IFN-alpha production, also suppresses the anti-IgD antibody-induced IgE response and stimulates the IgG2a response; these effects are blocked by a sheep antibody that neutralizes mouse IFN-alpha/beta. Both recombinant IFN-alpha and poly I.C have maximum IgE suppressive and IgG2a stimulatory effects when injected early in the anti-IgD antibody-induced immune response. Addition of IFN-alpha to mouse B cells cultured with lipopolysaccharide (LPS) + interleukin 4 (IL-4) suppresses both IgG1 and IgE production, but much less potently than IFN-gamma. IFN-alpha suppresses anti-IgD antibody-induced increases in the level of splenic IL-4 mRNA, but enhances the anti-IgD antibody-induced increase in the splenic level of IFN-gamma mRNA. These results are consistent with the effect of IFN-alpha on Ig isotype expression in mice, as IL-4 stimulates IgE and suppresses IgG2a secretion while IFN-gamma exerts opposite effects. These observations suggest that antigen presenting cells, by secreting IFN-alpha early in the course of an immune response, can influence the nature of that response both through direct effects on B cells and by influencing the differentiation of T cells.
Background: Efficient production of interferons (IFNs) in virally infected cells is an essential aspect of the host defence. The transcription factor complex ISGF3 (IFN-stimulated gene factor 3) was originally identified as a critical mediator of the IFN signal; it is formed upon IFN receptor (IFNR) stimulation and binds to ISREs (IFN-stimulated response elements) to activate IFN-inducible genes. It has recently been shown that the DNA binding component of ISGF3, p48 (ISGF3γ) also binds to virus-inducible elements in the IFN-α/β genes, suggesting a potential new role of p48 in IFN production.
Results: Primary cells from mice with a targeted disruption of the p48 gene show severe defects in virus-induced IFN-α/β gene expression. A similar defect was also observed in cells lacking type I IFNR or Stat1, further demonstrating the role of IFN signalling in the induction of these IFN genes. ISGF3 in fact binds to the virus-inducible elements within the IFN-α/β promoters. We also provide evidence showing that these elements are additionally controlled by an unidentified factor(s) which presumably triggers the primary phase of IFN gene induction.
Conclusions: Our results demonstrate that the IFN signal transducing complex ISGF3 plays a crucial role in IFN production and suggest that ISGF3 may participate directly in the activation of IFN-α/β promoters. This dual function of ISGF3 may insure the efficient operation of this cytokine system in the host defence.
Major histocompatibility complex (MHC) class I molecules bind peptides derived from cellular proteins and display them for surveillance by the immune system. These peptide-binding molecules are composed of a heavy chain, containing an antigen-binding groove, which is tightly associated with a light chain (beta 2-microglobulin). The majority of presented peptides are generated by degradation of proteins in the cytoplasm, in many cases by a large multicatalytic proteolytic particle, the proteasome. Two beta-subunits of the proteasome, LMP2 and LMP7, are inducible by interferon-gamma and alter the catalytic activities of this particle, enhancing the presentation of at least some antigens. After production of the peptide in the cytosol, it is transported across the endoplasmic reticulum (ER) membrane in an ATP-dependent manner by TAP (transporter associated with antigen presentation), a member of the ATP-binding cassette family of transport proteins. There are minor pathways for generating presented peptides directly in the ER, and some evidence suggests that peptides may be further trimmed in this location. The class I heavy chain and beta 2-microglobulin are cotranslationally translocated into the endoplasmic reticulum where their assembly may be facilitated by the sequential association of the heavy chain with chaperone proteins BiP and calnexin. The class I molecule then associates with the lumenal face of TAP where it is retained, presumably awaiting a peptide. After the class I molecule binds a peptide, it is released for exocytosis to the cell surface where cytotoxic T lymphocytes examine it for peptides derived from foreign proteins.
IFNα and IFNγ induce rapid activation of gene expression following binding to cell surface receptors on target cells. Gene transcription depends on latent transcription factors that become activated in response to IFN treatment. These proteins, termed Stats for signal transducers and activators of transcription, serve as intrinsic elements of the signaling pathway. In untreated cells, they are sequestered in the cytoplasm. Upon treatment of cells with IFN, they become tyrosine phophorylated by receptor-bound protein tyrosine kinases of the Jak family, assemble into multimeric complexes, and localize to the cell nucleus. Differential activation and combinatorial association of these transcription factors produce the biological responses characteristic of each IFN.
We describe a universal ligand-binding receptor for human interferons α and interferon β (type I IFNs). A soluble 40 kDa receptor (p40) that blocks the activity of type I IFNs was purified from urine and sequenced. Antibodies raised against p40 completely block the activity of several type I IFNs and immunoprecipltate both a cellular 102 kDa receptor and its cross-linked complexes with IFN-α2. The receptor is a disulfide-linked dimer, consisting of 51 kDa subunits. We isolated and expressed a 1.5 kb cDNA, coding for the receptor. Its 331 amino acid sequence includes a leader and a transmembrane region, while its ectodomain corresponds to p40. receptor is physically associated with the cytoplasmic Tyr kinase JAK1, hence, in addition to ligand binding, it is directly involved in signal transduction.
Encephalomyocarditis virus infection of mouse Ehrlich ascites tumour or L-cells or human HeLa cells inhibits the activation of the ppp(A2′p)n A-dependent nuclease and prevents the binding of a radioactive analogue of ppp(A2′p)n A to the ppp(A2′p)n A binding protein. This loss or inactivation of the ppp(A2′p)n A-dependent nuclease in response to virus infection is prevented by interferon pretreatment of the cells.
Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon γ (Hu-IFN-γ), as measured by induction of class I human leukocyte antigen (HLA) and protection against encephalomyocarditis virus (EMCV) infection. Whereas human chromosome 6 encodes the Hu-IFN-γ receptor, human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-γ receptor. Probes from a genomic clone were used to identity cDNA clones expressing a species-specific accessory factor. These cDNA clones are able to substitute for human chromosome 21 to reconstitute the Hu-IFN-γ receptor-mediated induction of class I HLA antigens. However, the factor encoded by the cDNA does not confer full antiviral protection against EMCV, confirming that an additional factor encoded on human chromosome 21 is required for reconstitution of antiviral activity against EMCV. We conclude that this accessory factor belongs to a family of such accessory factors responsible for different actions of IFN-γ.
The interferon-induced enzyme 2–5A synthetase is shown to adenylate tRNA. Yeast tRNAPhe was incubated with the enzyme in the presence of double stranded RNA (in this case polyI-polyC) and ATP or deoxyATP. The reaction products were analyzed by ribonuclease T1 digestion of the tRNA, polyacrylamide gel electrophoresis and autoradiography. Using ATP, the 2–5A synthetase adds one, two or three AMP residues to the 3′-end of the tRNA whereas when dATP is replacing ATP, only one nucleotide unit is added.
It is concluded that one of the mechanisms of the interferon-induced antiviral effect may be an inhibition of the translation process caused by an inactivation of tRNA molecules by a 2–5A synthetase catalyzed 2′-adenylation of the 3′-end.
Activation of the transcription factor NF-κB by tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the NF-κB-inducing kinase (NIK). In a yeast two-hybrid screen for NIK-interacting proteins, we have identified a protein kinase previously known as CHUK. Overexpression of CHUK activates a NF-κB-dependent reporter gene. A catalytically inactive mutant of CHUK is a dominant-negative inhibitor of TNF-, IL-1-, TRAF-, and NIK-induced NF-κB activation. CHUK associates with the NF-κB inhibitory protein, IκB-α, in mammalian cells. CHUK specifically phosphorylates IκB-α on both serine 32 and serine 36, modifications that are required for targeted degradation of IκB-α via the ubiquitin-proteasome pathway. This phosphorylation of IκB-α is greatly enhanced by NIK costimulation. Thus, CHUK is a NIK-activated IκB-α kinase that links TNF- and IL-1-induced kinase cascades to NF-κB activation.
The double stranded RNA (dsRNA) dependent, interferon (IFN)-induced protein kinase (PKR) plays a dual role in the interferon system. As an IFN-induced enzyme its primary role is in regulating protein synthesis initiation via phosphorylation of initiation factor eIF-2. Recently, manipulation of basal levels of PKR using antisense or gene ablation technologies has identified IκB, the inhibitor of transcription factor NFκB, as a new substrate for PKR. Phosphorylation of IκB by PKR results in the transcriptional activation of NFκB-dependent genes including interferon beta (IFN-β). PKR is also implicated as playing a role at either the translational or transcriptional level in the signal transduction pathways of other cytokines including IL3, PDGF and IFN γ.
In cells derived from congenic BALB.A2G-Mx mice carrying the resistance gene Mx, but not in cells from BALB/c mice lacking Mx, mouse interferons α and β induced the synthesis of a unique cellular protein that was associated with an efficient antiviral state with selectivity for influenza viruses. In contrast, native or recombinant mouse interferon γ failed to efficiently protect Mx-bearing cells against influenza viruses and did not noticeably induce the synthesis of the Mx-associated protein, although interferon γ was as effective as interferons α and β in protecting BALB.A2G-Mx and BALB/c cells against the rhabdovirus VSV. These results demonstrate that different types of interferons differentially regulate the expression of the Mx gene and thereby induce distinct antiviral states.
The signalling pathway that comprises JAK kinases and STAT proteins (for signal transducer and activator of transcription) is important for relaying signals from various cytokines outside the cell to the inside. The feedback mechanism responsible for switching off the cytokine signal has not been elucidated. We now report the cloning and characterization of an inhibitor of STAT activation which we name SSI-1 (for STAT-induced STAT inhibitor-1). We found that SSI-1 messenger RNA was induced by the cytokines interleukins 4 and 6 (IL-4, IL-6), leukaemia-inhibitory factor (LIF), and granulocyte colony-stimulating factor (G-CSF). Stimulation by IL-6 or LIF of murine myeloid leukaemia cells (M1 cells) induced SSI-1 mRNA expression which was blocked by transfection of a dominant-negative mutant of Stat3, indicating that the SSI-1 gene is a target of Stat3. Forced overexpression of SSI-1 complementary DNA interfered with IL-6- and LIF-mediated apoptosis and macrophage differentiation of M1 cells, as well as IL-6 induced tyrosine-phosphorylation of a receptor glycoprotein component, gp130, and of Stat3. When SSI-1 is overexpressed in COS7 cells, it can associate with the kinases Jak2 and Tyk2. These findings indicate that SSI-1 is responsible for negative-feedback regulation of the JAK-STAT pathway induced by cytokine stimulation.
PROTEIN SYNTHESIS in cell-free systems from interferontreated cells and rabbit reticulocyte lysates is exceptionally sensitive to inhibition by double-stranded RNA (dsRNA)1,2. Two distinct mechanisms seem to be involved: (1) inactivation of initiation factor eIF2 by dsRNA-mediated phosphorylation and (2) degradation of mRNA by one or more nucleases which are activated by sub-nanomolar levels of an unusual oligonucleotide, pppA2′p5′A2′p5′A (refs 3–10). The enzyme responsible for synthesising pppA2′p5′A2′p5′A requires dsRNA for activity, binds to poly (I)·poly(C)-Sepharose, and can be used conveniently in this insoluble form to synthesise pppA2′p5′A2′p5′A and related oligomers4,11, collectively referred to as 2-5A. The discovery of relatively high levels of 2-5A synthetase in rabbit reticulocyte lysates12 raised the possibility that the 2-5A system might be found in other cells which have not been treated with interferon and might, therefore, have a wider significance. We have developed a more convenient assay for the synthetase based on its activity when bound to poly(I)·poly(C)-paper rather than poly(I)·poly(C)-Sepharose and show here that this enzyme is distributed widely in mammalian cells. Moreover, different cells or tissues have widely differing levels of this enzyme and in the case of chick oviducts there is a substantial increase in synthetase level after withdrawal from stimulation by oestrogen.
Complex mixtures of 2′,5′-oligoadenylates are formed in cells and tissues under several different circumstances, and methods for analyzing such mixtures are reviewed. Separation is achieved by high-performance liquid chromatography and quantitation by competition-binding assays, using three different types of antibodies or a specific binding protein, or by functional assay, using preparations of an endonuclease specifically activated by some of the 2′,5′-oligoadenylates. Representative results from three different biological systems are presented. The function of 2′,5′-oligoadenylates as activators of intracellular RNA degradation is discussed, along with the possibility that these compounds may serve as signals for other intracellular regulatory processes.
Double-stranded RNA (dsRNA) induces the vascular cell adhesion molecule VCAM-1 to high levels of expression in human umbilical vein endothelial (HUVE) cells. Although VCAM-1 is also induced by the cytokine interleukin 1β (IL-1β), activation of the dsRNA-activated protein kinase (PKR) occurs only in response to incubation with dsRNA but not with IL-1β. Incubation of HUVE cells with the synthetic dsRNA, poly (I) · poly (C), activates PKR with increased autophosphorylation, increased phosphorylation of the translation factor eIF2α, and increased activation of the transcription factor NF-кB. Promoter analysis in HUVE cells using a VCAM-1 promoter linked to CAT reporter gene demonstrates that poly (I) · poly (C) responsiveness resides in the minimal VCAM-1 promoter that contains two NF-кB sites, and deletion of the NF-кB sites eliminates basal and poly (I) · poly (C)-induced CAT activity, supporting the importance of NF-кB in the poly (I) · poly (C)-mediated induction of VCAM-1. In vitro studies using purified reagents demonstrate that PKR is capable of phosphorylating IкBα (the inhibitory subunit of NF-кB) in a dsRNA-dependent manner. This suggests that phosphorylation of IкBα by PKR could be an initial step in the activation of NF-кB by dsRNA. NF-кB is also activated by IL-1β in HUVE cells, but this activation occurs without increased PKR autophosphorylation or eIF2α phosphorylation. Poly (I) · poly (C) induces VCAM-1 mRNA levels that are dramatically higher and sustained longer than levels induced by IL-1β. Although phosphorylation of eIF2α interferes with protein translation, sufficient VCAM-1 mRNA translation occurs in response to poly (I) · poly (C) to yield VCAM-1 protein levels that are similar to levels that are induced by IL-1bT. This suggests that the higher, sustained VCAM-1 mRNA levels that occur in response to incubation with poly (I) · poly (C) compensate for the partial translational block resulting from increased eIF2α phosphorylation. These studies indicate that transcrip-tional and translational regulatory events that occur in response to activation of PKR by dsRNA are important in the regulation of VCAM-1 gene expression in HUVE cells.
p21CIP11WAF1 is a CDK Inhibitor regulated by the tumor suppressor p53 and is hypothesized to mediate G1 arrest. p53 has been suggested to derive anti-oncogenic properties from this relationship. To test these notions, we created mice lacking p21CIP1/WAF1. They develop normally and (unlike p53−/− mice) have not developed spontaneous malignancies during 7 months of observation. Nonetheless, p21−/− embryonic fibroblasts are significantly deficient in their ability to arrest in G1 In response to DNA damage and nucleotide pool perturbation. p21−/− cells also exhibit a significant growth alteration in vitro, achieving a saturation density as high as that observed In p53−/− cells. In contrast, other aspects of p53 function, such as thymocytic apoptosis and the mitotic spindle checkpoint, appear normal. These results establish the role of p21CIP1/WAF1 in the G1 checkpoint, but suggest that the antiapoptotic and the anti-oncogenic effects of p53 are more complex.
Viral infections commonly induce expression of type I interferon (IFN) genes. The induction is transient and involves transcriptional activation wherein a positive factor, IRF-1, binds to upstream regulatory cis elements. In the present study we report the isolation of a cDNA encoding a novel factor, termed IRF-2, that interacts with the same nucleotide sequence elements as IRF-1. Both genes are inducible not only by virus but also by IFN. Unlike IRF-1, IRF-2 does not function as an activator; rather, it suppresses the function of IRF-1 under certain circumstances. Our results suggest that transcription of the IFN and IFN-inducible genes is regulated by two similar trans-acting factors that apparently compete for the same cis-acting recognition sequences, but which have opposite effects.
Ten years have passed since the molecular cloning of interleukin 6 (IL-6) in 1986. IL-6 is a typical cytokine, exhibiting functional pleiotropy and redundancy. IL-6 is involved in the immune response, inflammation, and hematopoiesis. The IL-6 receptor consists of an IL-6 binding alpha chain and a signal transducer, gp130, which is shared among the receptors for the IL-6 related cytokine subfamily. The sharing of a receptor subunit is a general feature of cytokine receptors and provides the molecular basis for the functional redundancy of cytokines. JAK tyrosine kinase is a key molecule that can initiate multiple signal-transduction pathways by inducing the tyrosine-phosphorylation of the cytokine receptor, gp130 in the case of IL-6, on which several signaling molecules are recruited, including STAT, a signal transducer and activator of transcription, and SHP-2, which links to the Ras-MAP kinase pathway. JAK can also directly activate signaling molecules such as STAT and Tec. These multiple signal-transduction pathways intimately regulate the expression of several genes including c-myc, c-myb, junB, IRF1, egr-1, and bcl-2, leading to the induction of cell growth, differentiation, and survival. The deregulated expression of IL-6 and its receptor is involved in a variety of diseases.
Five human interferon-alpha (leukocyte) subtypes derived from genes cloned in Escherichia coli have been compared for their ability to induce antiviral activity against vesicular stomatitis virus infection of various mammalian cell cultures. These interferons, designated LeIF-A (IFN-alpha 2), -B, -C, -D (IFN-alpha 1) and LeIF-F, show different relative activities when assayed on human, bovine, hamster, mouse, rabbit and monkey cell lines. As with a natural human buffy-coat interferon-alpha preparation, three subtypes (LeIF-B, -C and -D) showed considerable activity on RK-13 rabbit cells, but two (LeIF-D and -F) also showed some activity on mouse L-929 cells. Of the five interferon subtypes examined, LeIF-F demonstrated the highest degree of species specificity.
Since the IFN system has been implicated in cell growth and differentiation control mechanisms, we evaluated the influence of the expression of HPV-16 E6 and E7 oncoproteins on IFN signaling by using cotransfection experiments. Both viral oncoproteins differentially interfered with the inducibility of IFN-beta promoter by Sendai virus. The activation by IFN-gamma of a GBP ISRE reporter was dramatically affected by both viral proteins suggesting a disruption of STATs/IRFs function. Further, the inducibility of 6-16 gene ISRE reporter by IFN-alpha was decreased to varying degrees by both viral oncoproteins, implying that ISGF3 function is also impaired. Taken together, these observations suggest that HPV-16 negatively interacts with cellular targets of the IFN system, and these interactions may be implicated in cellular transformation caused by HPVs and their refractory response to IFN treatment.
Apoptosis occurs in response to different cellular stresses, including viral infection, inflammatory cytokines, growth factor deprivation, and UV light, but it is unclear whether these inducers share a common mechanism of induction. The interferon-induced, double-stranded RNA-activated protein kinase (PKR) has been implicated in processes that rely on apoptosis as control mechanisms in vivo, including antiviral activities, cell growth regulation, and tumorigenesis. Here we report that mouse embryo fibroblasts from mutant mice containing homozygous deletions in the PKR gene (Pkro/o mice) were resistant to apoptotic cell death in response to double-stranded RNA, tumor necrosis factor-α, or lipopolysaccharide. The mechanism underlying the suppression of apoptosis in the Pkro/o cells could be attributed to defects in the activation of DNA-binding activity for the transcription factor interferon regulatory factor-1 and in Fas mRNA induction. Thus, these results provide genetic evidence implicating a requirement for PKR in mediating different forms of stress-related apoptosis.
A low molecular weight inhibitor of cell-free protein synthesis effective at subnanomolar concentrations is formed on incubation of cytoplasmic extracts from interferon-treated cells with double-stranded RNA and ATP. It can be conveniently synthesized by incubating a poly(I).poly(C)-Sepharose-bound enzyme fraction from such cells with [3H]- or [alpha- or gamma-32P]ATP. The radioactive inhibitor has been characterized by its behavior on DEAE-Sephadex in the presence of urea and on the basis of the products obtained on enzymic, alkaline, and sequential degradation by periodate oxidation and beta elimination. Its structure appears to be pppA2'p5'A2'p5'A. We have found no evidence for any modification or abnormality other than the 2'-5' linkage. On occasion the inhibitor preparations have included what seems to be the corresponding dimer (pppA2'p5'A), tetramer [ppp(A2'p)3A], pentamer [ppp(A2'p)4A], and higher oligomers in decreasing amounts. The trimer, tetramer, and pentamer are similar in activity, but the dimer is less potent if active at all.
Interferon-gamma (IFN-gamma) induces the transcription of the gene encoding a guanylate binding protein by activating a latent cytoplasmic factor, GAF (gamma-activated factor). GAF is translocated to the nucleus and binds a DNA element, the gamma-activated site. Through cross-linking and the use of specific antibodies GAF was found to be a 91-kilodalton DNA binding protein that was previously identified as one of four proteins in interferon-stimulated gene factor-3 (ISGF-3), a transcription complex activated by IFN-alpha. The IFN-gamma-dependent activation of the 91-kilodalton DNA binding protein required cytoplasmic phosphorylation of the protein on tyrosine. The 113-kilodalton ISGF-3 protein that is phosphorylated in response to IFN-alpha was not phosphorylated nor translocated to the nucleus in response to IFN-gamma. Thus the two different ligands result in tyrosine phosphorylation of different combinations of latent cytoplasmic transcription factors that then act at different DNA binding sites.