Interferon-ε is a tumour suppressor and restricts ovarian cancer.

High-grade serous ovarian cancers have low survival rates because of their late presentation with extensive peritoneal metastases and frequent chemoresistance1, and require new treatments guided by novel insights into pathogenesis. Here we describe the intrinsic tumour-suppressive activities of interferon-ε (IFNε). IFNε is constitutively expressed in epithelial cells of the fallopian tube, the cell of origin of high-grade serous ovarian cancers, and is then lost during development of these tumours. We characterize its anti-tumour activity in several preclinical models: ovarian cancer patient-derived xenografts, orthotopic and disseminated syngeneic models, and tumour cell lines with or without mutations in Trp53 and Brca genes. We use manipulation of the IFNε receptor IFNAR1 in different cell compartments, differential exposure status to IFNε and global measures of IFN signalling to show that the mechanism of the anti-tumour activity of IFNε involves direct action on tumour cells and, crucially, activation of anti-tumour immunity. IFNε activated anti-tumour T and natural killer cells and prevented the accumulation and activation of myeloid-derived suppressor cells and regulatory T cells. Thus, we demonstrate that IFNε is an intrinsic tumour suppressor in the female reproductive tract whose activities in models of established and advanced ovarian cancer, distinct from other type I IFNs, are compelling indications of potential new therapeutic approaches for ovarian cancer.

Bacterial clade-specific analysis identifies distinct epithelial responses in inflammatory bowel disease.

Abnormal immune responses to the resident gut microbiome can drive inflammatory bowel disease (IBD). Here, we combine high-resolution, culture-based shotgun metagenomic sequencing and analysis with matched host transcriptomics across three intestinal sites (terminal ileum, cecum, rectum) from pediatric IBD (PIBD) patients (n = 58) and matched controls (n = 42) to investigate this relationship. Combining our site-specific approach with bacterial culturing, we establish a cohort-specific bacterial culture collection, comprising 6,620 isolates (170 distinct species, 32 putative novel), cultured from 286 mucosal biopsies. Phylogeny-based, clade-specific metagenomic analysis identifies key, functionally distinct Enterococcus clades associated with either IBD or health. Strain-specific in vitro validation demonstrates differences in cell cytotoxicity and inflammatory signaling in intestinal epithelial cells, consistent with the colonic mucosa-specific response measured in patients with IBD. This demonstrates the importance of strain-specific phenotypes and consideration of anatomical sites in exploring the dysregulated host-bacterial interactions in IBD.

Extensive genome analysis identifies novel plasmid families in Clostridium perfringens.

Globally, the anaerobic bacterium Clostridium perfringens causes severe disease in a wide array of hosts; however, C. perfringens strains are also carried asymptomatically. Accessory genes are responsible for much of the observed phenotypic variation and virulence within this species, with toxins frequently encoded on conjugative plasmids and many isolates carrying up to 10 plasmids. Despite this unusual biology, current genomic analyses have largely excluded isolates from healthy hosts or environmental sources. Accessory genomes, including plasmids, also have often been excluded from broader scale phylogenetic investigations. Here we interrogate a comprehensive collection of 464 C. perfringens genomes and identify the first putative non-conjugative enterotoxin (CPE)-encoding plasmids and a putative novel conjugative locus (Bcp) with sequence similarity to a locus reported from Clostridium botulinum. We sequenced and archived 102 new C. perfringens genomes, including those from rarely sequenced toxinotype B, C, D and E isolates. Long-read sequencing of 11 C. perfringens strains representing all toxinotypes (A-G) identified 55 plasmids from nine distinct plasmid groups. Interrogation of the 464 genomes in this collection identified 1045 plasmid-like contigs from the nine plasmid families, with a wide distribution across the C. perfringens isolates. Plasmids and plasmid diversity play an essential role in C. perfringens pathogenicity and broader biology. We have expanded the C. perfringens genome collection to include temporal, spatial and phenotypically diverse isolates including those carried asymptomatically in the gastrointestinal microbiome. This analysis has resulted in the identification of novel C. perfringens plasmids whilst providing a comprehensive understanding of species diversity.

Constitutive expression and distinct properties of IFN-epsilon protect the female reproductive tract from Zika virus infection.

The immunological surveillance factors controlling vulnerability of the female reproductive tract (FRT) to sexually transmitted viral infections are not well understood. Interferon-epsilon (IFNɛ) is a distinct, immunoregulatory type-I IFN that is constitutively expressed by FRT epithelium and is not induced by pathogens like other antiviral IFNs α, ß and λ. We show the necessity of IFNɛ for Zika Virus (ZIKV) protection by: increased susceptibility of IFNɛ-/- mice; their "rescue" by intravaginal recombinant IFNɛ treatment and blockade of protective endogenous IFNɛ by neutralising antibody. Complementary studies in human FRT cell lines showed IFNɛ had potent anti-ZIKV activity, associated with transcriptome responses similar to IFNλ but lacking the proinflammatory gene signature of IFNα. IFNɛ activated STAT1/2 pathways similar to IFNα and λ that were inhibited by ZIKV-encoded non-structural (NS) proteins, but not if IFNε exposure preceded infection. This scenario is provided by the constitutive expression of endogenous IFNε. However, the IFNɛ expression was not inhibited by ZIKV NS proteins despite their ability to antagonise the expression of IFNß or λ. Thus, the constitutive expression of IFNɛ provides cellular resistance to viral strategies of antagonism and maximises the antiviral activity of the FRT. These results show that the unique spatiotemporal properties of IFNε provides an innate immune surveillance network in the FRT that is a significant barrier to viral infection with important implications for prevention and therapy.

Strain-level characterization of broad host range mobile genetic elements transferring antibiotic resistance from the human microbiome.

Mobile genetic elements (MGEs) carrying antibiotic resistance genes (ARGs) disseminate ARGs when they mobilise into new bacterial hosts. The nature of such horizontal gene transfer (HGT) events between human gut commensals and pathogens remain poorly characterised. Here, we compare 1354 cultured commensal strains (540 species) to 45,403 pathogen strains (12 species) and find 64,188 MGE-mediated ARG transfer events between the two groups using established methods. Among the 5931 MGEs, we find 15 broad host range elements predicted to have crossed different bacterial phyla while also occurring in animal and environmental microbiomes. We experimentally demonstrate that predicted broad host range MGEs can mobilise from commensals Dorea longicatena and Hungatella hathewayi to pathogen Klebsiella oxytoca, crossing phyla simultaneously. Our work establishes the MGE-mediated ARG dissemination network between human gut commensals and pathogens and highlights broad host range MGEs as targets for future ARG dissemination management.

CiiiDER: A tool for predicting and analysing transcription factor binding sites.

The availability of large amounts of high-throughput genomic, transcriptomic and epigenomic data has provided opportunity to understand regulation of the cellular transcriptome with an unprecedented level of detail. As a result, research has advanced from identifying gene expression patterns associated with particular conditions to elucidating signalling pathways that regulate expression. There are over 1,000 transcription factors (TFs) in vertebrates that play a role in this regulation. Determining which of these are likely to be controlling a set of genes can be assisted by computational prediction, utilising experimentally verified binding site motifs. Here we present CiiiDER, an integrated computational toolkit for transcription factor binding analysis, written in the Java programming language, to make it independent of computer operating system. It is operated through an intuitive graphical user interface with interactive, high-quality visual outputs, making it accessible to all researchers. CiiiDER predicts transcription factor binding sites (TFBSs) across regulatory regions of interest, such as promoters and enhancers derived from any species. It can perform an enrichment analysis to identify TFs that are significantly over- or under-represented in comparison to a bespoke background set and thereby elucidate pathways regulating sets of genes of pathophysiological importance.

Concurrent Host-Pathogen Transcriptional Responses in a Clostridium perfringens Murine Myonecrosis Infection.

To obtain an insight into host-pathogen interactions in clostridial myonecrosis, we carried out comparative transcriptome analysis of both the bacterium and the host in a murine Clostridium perfringens infection model, which is the first time that such an investigation has been conducted. Analysis of the host transcriptome from infected muscle tissues indicated that many genes were upregulated compared to the results seen with mock-infected mice. These genes were enriched for host defense pathways, including Toll-like receptor (TLR) and Nod-like receptor (NLR) signaling components. Real-time PCR confirmed that host TLR2 and NLRP3 inflammasome genes were induced in response to C. perfringens infection. Comparison of the transcriptome of C. perfringens cells from the infected tissues with that from broth cultures showed that host selective pressure induced a global change in C. perfringens gene expression. A total of 33% (923) of C. perfringens genes were differentially regulated, including 10 potential virulence genes that were upregulated relative to their expression in vitro These genes encoded putative proteins that may be involved in the synthesis of cell wall-associated macromolecules, in adhesion to host cells, or in protection from host cationic antimicrobial peptides. This report presents the first successful expression profiling of coregulated transcriptomes of bacterial and host genes during a clostridial myonecrosis infection and provides new insights into disease pathogenesis and host-pathogen interactions.IMPORTANCEClostridium perfringens is the causative agent of traumatic clostridial myonecrosis, or gas gangrene. In this study, we carried out transcriptional analysis of both the host and the bacterial pathogen in a mouse myonecrosis infection. The results showed that in comparison to mock-infected control tissues, muscle tissues from C. perfringens-infected mice had a significantly altered gene expression profile. In particular, the expression of many genes involved in the innate immune system was upregulated. Comparison of the expression profiles of C. perfringens cells isolated from the infected tissues with those from equivalent broth cultures identified many potential virulence genes that were significantly upregulated in vivo These studies have provided a new understanding of the range of factors involved in host-pathogen interactions in a myonecrosis infection.

Obtaining high quality transcriptome data from formalin-fixed, paraffin-embedded diagnostic prostate tumor specimens.

Prognostic genomic biomarkers that can be measured at diagnosis to aid choice of treatment options are unavailable for most common cancers. This is due in part to the poor quality and quantity of available diagnostic specimens for discovery research and to limitations in genomic technologies. Recent technical advances now enable high-density molecular analyses using suboptimal biological specimens. Here we describe the optimization of a transcriptome-specific protocol for use with formalin-fixed, paraffin-embedded (FFPE) diagnostic prostate cancer (PrCa) specimens. We applied the Ion AmpliSeq Transcriptome Human Gene Expression Kit (AmpliSeq Kit) to RNA samples extracted from 36 tumor-enriched and 16 adjacent normal tissues (ADJNT) from 37 FFPE PrCa specimens over a series of eight pilot studies, incorporating protocol modifications from Pilots 2 to 5. Data quality were measured by (1) the total number of mapped reads; (2) the percentage of reads that mapped to AmpliSeq target regions (OnTarget%); (3) the percentage of genes on the AmpliSeq panel with a read count ≥10 (TargetsDetected%); and (4) comparing the gene read-count distribution of the prostate tissue samples with the median gene read-count distribution of cell line-derived RNA samples. Modifications incorporated into Pilot study 5 provided gene expression data equivalent to cell line-derived RNA samples. These modifications included the use of freshly cut slides for macrodissection; increased tissue section thickness (8 µm); RNA extraction using the RecoverAll Total Nucleic Acid Isolation Kit for FFPE (ThermoFisher); 18 target amplification cycles; and processing six samples per Ion PI chip. This protocol will facilitate the discovery of prognostic biomarkers for cancer by allowing researchers to exploit previously underutilized diagnostic FFPE specimens.

Mitochondrial DNA haplotypes induce differential patterns of DNA methylation that result in differential chromosomal gene expression patterns.

Mitochondrial DNA copy number is strictly regulated during development as naive cells differentiate into mature cells to ensure that specific cell types have sufficient copies of mitochondrial DNA to perform their specialised functions. Mitochondrial DNA haplotypes are defined as specific regions of mitochondrial DNA that cluster with other mitochondrial sequences to show the phylogenetic origins of maternal lineages. Mitochondrial DNA haplotypes are associated with a range of phenotypes and disease. To understand how mitochondrial DNA haplotypes induce these characteristics, we used four embryonic stem cell lines that have the same set of chromosomes but possess different mitochondrial DNA haplotypes. We show that mitochondrial DNA haplotypes influence changes in chromosomal gene expression and affinity for nuclear-encoded mitochondrial DNA replication factors to modulate mitochondrial DNA copy number, two events that act synchronously during differentiation. Global DNA methylation analysis showed that each haplotype induces distinct DNA methylation patterns, which, when modulated by DNA demethylation agents, resulted in skewed gene expression patterns that highlight the effectiveness of the new DNA methylation patterns established by each haplotype. The haplotypes differentially regulate α-ketoglutarate, a metabolite from the TCA cycle that modulates the TET family of proteins, which catalyse the transition from 5-methylcytosine, indicative of DNA methylation, to 5-hydroxymethylcytosine, indicative of DNA demethylation. Our outcomes show that mitochondrial DNA haplotypes differentially modulate chromosomal gene expression patterns of naive and differentiating cells by establishing mitochondrial DNA haplotype-specific DNA methylation patterns.

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

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

Restoration of normal embryogenesis by mitochondrial supplementation in pig oocytes exhibiting mitochondrial DNA deficiency.

An increasing number of women fail to achieve pregnancy due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Indeed, reduced mitochondrial DNA copy number (mitochondrial DNA deficiency) may disrupt oocyte quality in some women. To overcome mitochondrial DNA deficiency, whilst maintaining genetic identity, we supplemented pig oocytes selected for mitochondrial DNA deficiency, reduced cytoplasmic maturation and lower developmental competence, with autologous populations of mitochondrial isolate at fertilization. Supplementation increased development to blastocyst, the final stage of preimplantation development, and promoted mitochondrial DNA replication prior to embryonic genome activation in mitochondrial DNA deficient oocytes but not in oocytes with normal levels of mitochondrial DNA. Blastocysts exhibited transcriptome profiles more closely resembling those of blastocysts from developmentally competent oocytes. Furthermore, mitochondrial supplementation reduced gene expression patterns associated with metabolic disorders that were identified in blastocysts from mitochondrial DNA deficient oocytes. These results demonstrate the importance of the oocyte's mitochondrial DNA investment in fertilization outcome and subsequent embryo development to mitochondrial DNA deficient oocytes.

Transcriptomic analysis of stage 1 versus advanced adult granulosa cell tumors.

Ovarian granulosa cell tumors (GCT) are hormonally-active neoplasms characterized, in the adult-subtype, by a mutation in the FOXL2 gene (C134W). They exhibit an indolent course with an unexplained propensity for late recurrence; ~80% of patients with aggressive, advanced stage tumors die from their disease; aside from surgery, therapeutic options are limited. To identify the molecular basis of advanced stage disease we have used whole transcriptome analysis of FOXL2 C134W mutation positive adult (a)GCT to identify genes that are differentially expressed between early (stage 1) and advanced (stage 3) aGCT. Transcriptome profiles for early (n = 6) and stage 3 (n = 6) aGCT, and for the aGCT-derived KGN, cell line identified 24 genes whose expression significantly differs between the early and stage 3 aGCT. Of these, 16 were more abundantly expressed in the stage 3 aGCT and 8 were higher in the stage 1 tumors. These changes were further examined for the genes which showed the greatest fold change: the cytokine CXCL14, microfibrillar-associated protein 5, insulin-like 3 and desmin. Gene Set Enrichment Analysis identified overexpression of genes on chromosome 7p15 which includes the homeobox A gene locus. The analysis therefore identifies a small number of genes with clearly discriminate patterns of expression arguing that the clinicopathological-derived distinction of the tumor stage is robust, whilst confirming the relative homogeneity of expression for many genes across the cohort and hence of aGCT. The expression profiles do however identify several overexpressed genes in both stage 1 and/or stage 3 aGCT which warrant further study as possible therapeutic targets.

Microarray profiling to analyse adult cardiac fibroblast identity.

Heart failure is one of the leading causes of death worldwide [1-4]. Current therapeutic strategies are inefficient and cannot cure this chronic and debilitating condition [5]. Ultimately, heart transplants are required for patient survival, but donor organs are scarce in availability and only prolong the life-span of patients for a limited time. Fibrosis is one of the main pathological features of heart failure [6,7], caused by inappropriate stimulation of fibroblasts and excessive extracellular matrix production. Therefore, an in-depth understanding of the cardiac fibroblast is essential to underpin effective therapeutic treatments for heart failure [5]. Fibroblasts in general have been an underappreciated cell type, regarded as relatively inert and providing only basic functionality; they are usually referred to as the  'biological glue' of all tissues in the body. However, more recent literature suggests that they actively participate in organ homeostasis and disease [7,8]. We have recently uncovered a unique molecular identity for fibroblasts isolated from the heart [9], expressing a set of cardiogenic transcription factors that have been previously associated with cardiomyocyte ontogenesis. This signature suggests that cardiac fibroblasts may be ideal for use in stem cell replacement therapies, as they may retain the memory of where they derive from embryologically. Our data also revealed that about 90% of fibroblasts from both tail and heart origins share a cell surface signature that has previously been described for mesenchymal stem cells (MSCs), raising the possibility that fibroblasts and MSCs may in fact be the same cell type. Thus, our findings carry profound implications for the field of regenerative medicine. Here, we describe detailed methodology and quality controls related to the gene expression profiling of cardiac fibroblasts, deposited at the Gene Expression Omnibus (GEO) under the accession number GSE50531. We also provide the R code to easily reproduce the data quantification and analysis processes.

Purification and biological characterization of soluble, recombinant mouse IFNß expressed in insect cells.

Interferon ß (IFNß) is a member of the type I interferon family of cytokines widely recognised for their anti-viral, anti-proliferative and immunomodulatory properties. Recombinant, biologically active forms of this cytokine are used clinically for the treatment of multiple sclerosis and in laboratories to study the role of this cytokine in health and disease. Established methods for expression of IFNß utilise either bacterial systems from which the insoluble recombinant proteins must be refolded, or mammalian expression systems in which large volumes of cell culture are required for recovery of acceptable yields. Utilising the baculovirus expression system and Trichoplusia ni (Cabbage Looper) BTI-TN-5B1-4 cell line, we report a reproducible method for production and purification of milligram/litre quantities of biologically active murine IFNß. Due to the design of our construct and the eukaryotic nature of insect cells, the resulting soluble protein is secreted allowing purification of the Histidine-tagged natively-folded protein from the culture supernatant. The IFNß purification method described is a two-step process employing immobilised metal-ion affinity chromatography (IMAC) and reverse-phase high performance liquid chromatography (RP-HPLC) that results in production of significantly more purified IFNß than any other reported eukaryotic-based expression system. Recombinant murine IFNß produced by this method was natively folded and demonstrated hallmark type I interferon biological effects including antiviral and anti-proliferative activities, and induced genes characteristic of IFNß activity in vivo. Recombinant IFNß also had specific activity levels exceeding that of the commercially available equivalent. Together, our findings provide a method for production of highly pure, biologically active murine IFNß.

Structural basis of a unique interferon-ß signaling axis mediated via the receptor IFNAR1.

Type I interferons are important in regulating immune responses to pathogens and tumors. All interferons are considered to signal via the heterodimeric IFNAR1-IFNAR2 complex, yet some subtypes such as interferon-ß (IFN-ß) can exhibit distinct functional properties, although the molecular basis of this is unclear. Here we demonstrate IFN-ß can uniquely and specifically ligate to IFNAR1 in an IFNAR2-independent manner, and we provide the structural basis of the IFNAR1-IFN-ß interaction. The IFNAR1-IFN-ß complex transduced signals that modulated expression of a distinct set of genes independently of Jak-STAT pathways. Lipopolysaccharide-induced sepsis was ameliorated in Ifnar1(-/-) mice but not Ifnar2(-/-) mice, suggesting that IFNAR1-IFN-ß signaling is pathologically relevant. Thus, we provide a molecular basis for understanding specific functions of IFN-ß.

Mitochondrially localised MUL1 is a novel modulator of antiviral signaling.

The innate immune response to virus must be balanced to eliminate infection yet limit damaging inflammation. A critical arm of the antiviral response is launched by the retinoic acid-inducible-gene I (RIG-I) protein. RIG-I is activated by viral RNA then associates with the mitochondrial antiviral signaling (MAVS) protein to subsequently induce potent inflammatory cytokines. Here, we demonstrate the mitochondrial E3 ubiquitin protein ligase 1 (MUL1) is a crucial moderator of RIG-I signaling. MUL1 is localized to the mitochondria where it interacts with MAVS and catalyzes RIG-I post-translational modifications that inhibit RIG-I-dependent cell signaling. Accordingly, depletion of MUL1 potentiated RIG-I mediated nuclear factor-kappa B (NF-κB) and interferon (IFN) ß reporter activity. Moreover, depletion of MUL1 boosted the antiviral response and increased proinflammatory cytokines following challenge with the RNA mimetic poly I:C and Sendai virus. We therefore submit that MUL1 is a novel regulator of the RIG-I-like receptor-dependent antiviral response, that otherwise functions to limit inflammation.

RNA-eXpress annotates novel transcript features in RNA-seq data.

Next-generation sequencing is rapidly becoming the approach of choice for transcriptional analysis experiments. Substantial advances have been achieved in computational approaches to support these technologies. These approaches typically rely on existing transcript annotations, introducing a bias towards known genes, require specific experimental design and computational resources, or focus only on identification of splice variants (ignoring other biologically relevant transcribed features contained within the data that may be important for downstream analysis). Biologically relevant transcribed features also include large and small non-coding RNA, new transcription start sites, alternative promoters, RNA editing and processing of coding transcripts. Also, many existing solutions lack accessible interfaces required for wide scale adoption. We present a user-friendly, rapid and computation-efficient feature annotation framework (RNA-eXpress) that enables identification of transcripts and other genomic and transcriptional features independently of current annotations. RNA-eXpress accepts mapped reads in the standard binary alignment (BAM) format and produces a study-specific feature annotation in GTF format, comparison statistics, sequence extraction and feature counts. The framework is designed to be easily accessible while allowing advanced users to integrate new feature-identification algorithms through simple class extension, thus facilitating expansion to novel feature types or identification of study-specific feature types.

Silencing of Irf7 pathways in breast cancer cells promotes bone metastasis through immune escape.

Breast cancer metastasis is a key determinant of long-term patient survival. By comparing the transcriptomes of primary and metastatic tumor cells in a mouse model of spontaneous bone metastasis, we found that a substantial number of genes suppressed in bone metastases are targets of the interferon regulatory factor Irf7. Restoration of Irf7 in tumor cells or administration of interferon led to reduced bone metastases and prolonged survival time. In mice deficient in the interferon (IFN) receptor or in natural killer (NK) and CD8(+) T cell responses, metastasis was accelerated, indicating that Irf7-driven suppression of metastasis was reliant on IFN signaling to host immune cells. We confirmed the clinical relevance of these findings in over 800 patients in which high expression of Irf7-regulated genes in primary tumors was associated with prolonged bone metastasis-free survival. This gene signature may identify patients that could benefit from IFN-based therapies. Thus, we have identified an innate immune pathway intrinsic to breast cancer cells, the suppression of which restricts immunosurveillance to enable metastasis.

LPS hypersensitivity of gp130 mutant mice is independent of elevated haemopoietic TLR4 signaling.

Among the many inflammatory mediators induced by the prototypical inflammatory stimulus lipopolysaccharide (LPS), which signals via Toll-like receptor (TLR)-4, interleukin (IL)-6 has recently been shown to feedback and augment TLR4 signaling when overproduced in LPS hypersensitive gp130(F/F) mice. This regulation by IL-6 in gp130(F/F) mice requires hyperactivation of the latent transcription factor signal transducer and activator of transcription (STAT) 3 via the IL-6 signaling receptor subunit gp130. However, the identity of LPS/TLR4-responsive inflammatory signaling pathways and gene networks, which are modulated by IL-6 (via gp130/STAT3), and the extent to which the tissue and cellular context of this regulation contributes to LPS-induced endotoxic shock in gp130(F/F) mice, are unknown. We report here that in LPS-treated macrophages from gp130(F/F) mice, gp130 hyperactivation upregulated the LPS-induced expression of inflammatory mediators downstream of Janus kinase (JAK)/STAT, nuclear factor κ-light-chain-enhancer of activated B cells, interferon regulatory factor and c-Jun N-terminal kinase/p38 mitogen-activated protein kinase pathways. Notably, however, LPS administration to bone marrow chimeras indicated that heightened LPS/TLR4 signaling in haemopoietic-derived gp130(F/F) immune cells is dispensable for the hypersensitivity of gp130(F/F) mice to LPS-induced endotoxemia. To understand the molecular consequences of gp130 hyperactivity in non-haemopoietic tissue on LPS-induced systemic inflammation, global gene expression profiling of livers from LPS-treated gp130(F/F) mice was performed and identified 264 hepatic LPS-responsive genes, which are differentially regulated by hyperactive gp130 signaling. Collectively, the substantial transcriptional reprogramming of LPS-responsive genes in gp130(F/F) mice emphasizes non-haemopoietic gp130 signaling as a key regulator of systemic inflammatory responses during LPS-induced endotoxemia.

Suppressor of cytokine signaling (SOCS) 1 inhibits type I interferon (IFN) signaling via the interferon alpha receptor (IFNAR1)-associated tyrosine kinase Tyk2.

Type I IFNs are critical players in host innate and adaptive immunity. IFN signaling is tightly controlled to ensure appropriate immune responses as imbalance could result in uncontrolled inflammation or inadequate responses to infection. It is therefore important to understand how type I IFN signaling is regulated. Here we have investigated the mechanism by which suppressor of cytokine signaling 1 (SOCS1) inhibits type I IFN signaling. We have found that SOCS1 inhibits type I IFN signaling not via a direct interaction with the IFN α receptor 1 (IFNAR1) receptor component but through an interaction with the IFNAR1-associated kinase Tyk2. We have characterized the residues/regions involved in the interaction between SOCS1 and Tyk2 and found that SOCS1 associates via its SH2 domain with conserved phosphotyrosines 1054 and 1055 of Tyk2. The kinase inhibitory region of SOCS1 is also essential for its interaction with Tyk2 and inhibition of IFN signaling. We also found that Tyk2 is preferentially Lys-63 polyubiquitinated and that this activation reaction is inhibited by SOCS1. The consequent effect of SOCS1 inhibition of Tyk2 not only results in a reduced IFN response because of inhibition of Tyk2 kinase-mediated STAT signaling but also negatively impacts IFNAR1 surface expression, which is stabilized by Tyk2.