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.

Viperin is anti-viral in vitro but is dispensable for restricting dengue virus replication or induction of innate and inflammatory responses in vivo.

Viperin has antiviral function against many viruses, including dengue virus (DENV), when studied in cells in culture. Here, the antiviral actions of viperin were defined both in vitro and in a mouse in vivo model of DENV infection. Murine embryonic fibroblasts (MEFs) derived from mice lacking viperin (vip-/-) showed enhanced DENV infection, accompanied by increased IFN-ß and induction of ISGs; IFIT1 and CXCL-10 but not IRF7, when compared to wild-type (WT) MEFs. In contrast, subcutaneous challenge of immunocompetent WT and vip-/- mice with DENV did not result in enhanced infection. Intracranial infection with DENV resulted in body weight loss and neurological disease with a moderate increase in mortality in vip-/- compared with WT mice, although this was not accompanied by altered brain morphology, immune cell infiltration or DENV RNA level in the brain. Similarly, DENV induction of IFN-ß, IFIT1, CXCL-10, IRF7 and TNF-α was not significantly different in WT and vip-/- mouse brain, although there was a modest but significant increase in DENV induction of IL-6 and IfI27la in the absence of viperin. NanoString nCounter analysis confirmed no significant difference in induction of a panel of inflammatory genes in WT compared to vip-/- DENV-infected mouse brains. Further, polyI:C stimulation of bone marrow-derived macrophages (BMDMs) induced TNF-α, IFN-ß, IL-6 and Nos-2, but responses were not different in BMDMs generated from WT or vip-/- mice. Thus, while there is significant evidence of anti-DENV actions of viperin in some cell types in vitro, for DENV infection in vivo a lack of viperin does not affect systemic or brain susceptibility to DENV or induction of innate and inflammatory responses.

Viperin binds STING and enhances the type-I interferon response following dsDNA detection.

Viperin is an interferon-inducible protein that is pivotal for eliciting an effective immune response against an array of diverse viral pathogens. Here we describe a mechanism of viperin's broad antiviral activity by demonstrating the protein's ability to synergistically enhance the innate immune dsDNA signaling pathway to limit viral infection. Viperin co-localized with the key signaling molecules of the innate immune dsDNA sensing pathway, STING and TBK1; binding directly to STING and inducing enhanced K63-linked polyubiquitination of TBK1. Subsequent analysis identified viperin's necessity to bind the cytosolic iron-sulfur assembly component 2A, to prolong its enhancement of the type-I interferon response to aberrant dsDNA. Here we show that viperin facilitates the formation of a signaling enhanceosome, to coordinate efficient signal transduction following activation of the dsDNA signaling pathway, which results in an enhanced antiviral state. We also provide evidence for viperin's radical SAM enzymatic activity to self-limit its immunomodulatory functions. These data further define viperin's role as a positive regulator of innate immune signaling, offering a mechanism of viperin's broad antiviral capacity.

Viperin interacts with PEX19 to mediate peroxisomal augmentation of the innate antiviral response.

Peroxisomes are recognized as significant platforms for the activation of antiviral innate immunity where stimulation of the key adapter molecule mitochondrial antiviral signaling protein (MAVS) within the RIG-I like receptor (RLR) pathway culminates in the up-regulation of hundreds of ISGs, some of which drive augmentation of multiple innate sensing pathways. However, whether ISGs can augment peroxisome-driven RLR signaling is currently unknown. Using a proteomics-based screening approach, we identified Pex19 as a binding partner of the ISG viperin. Viperin colocalized with numerous peroxisomal proteins and its interaction with Pex19 was in close association with lipid droplets, another emerging innate signaling platform. Augmentation of the RLR pathway by viperin was lost when Pex19 expression was reduced. Expression of organelle-specific MAVS demonstrated that viperin requires both mitochondria and peroxisome MAVS for optimal induction of IFN-ß. These results suggest that viperin is required to enhance the antiviral cellular response with a possible role to position the peroxisome at the mitochondrial/MAM MAVS signaling synapse, furthering our understanding of the importance of multiple organelles driving the innate immune response against viral infection.

MicroRNA-regulated pathways associated with endometriosis.

Endometriosis is a prevalent gynecological disease characterized by growth of endometriotic tissue outside the uterine cavity. MicroRNAs (miRNAs) are naturally occurring posttranscriptional regulatory molecules that potentially play a role in endometriotic lesion development. We assessed miRNA expression by microarray analysis in paired ectopic and eutopic endometrial tissues and identified 14 up-regulated (miR-145, miR-143, miR-99a, miR-99b, miR-126, miR-100, miR-125b, miR-150, miR-125a, miR-223, miR-194, miR-365, miR-29c and miR-1) and eight down-regulated (miR-200a, miR-141, miR-200b, miR-142-3p, miR-424, miR-34c, miR-20a and miR-196b) miRNAs. The differential expression of six miRNAs was confirmed by quantitative RT-PCR. An in silico analysis identified 3851 mRNA transcripts as putative targets of the 22 miRNAs. Of these predicted targets, 673 were also differentially expressed in ectopic vs. eutopic endometrial tissue, as determined by microarray. Functional analysis suggested that the 673 miRNA targets constitute molecular pathways previously associated with endometriosis, including c-Jun, CREB-binding protein, protein kinase B (Akt), and cyclin D1 (CCND1) signaling. These pathways appeared to be regulated both transcriptionally as well as by miRNAs at posttranscriptional level. These data are a rich and novel resource for endometriosis and miRNA research and suggest that the 22 miRNAs and their cognate mRNA target sequences constitute pathways that promote endometriosis. Accordingly, miRNAs are potential therapeutic targets for treating this disease.

Viperin is an important host restriction factor in control of Zika virus infection.

Zika virus (ZIKV) infection has emerged as a global health threat and infection of pregnant women causes intrauterine growth restriction, spontaneous abortion and microcephaly in newborns. Here we show using biologically relevant cells of neural and placental origin that following ZIKV infection, there is attenuation of the cellular innate response characterised by reduced expression of IFN-ß and associated interferon stimulated genes (ISGs). One such ISG is viperin that has well documented antiviral activity against a wide range of viruses. Expression of viperin in cultured cells resulted in significant impairment of ZIKV replication, while MEFs derived from CRISPR/Cas9 derived viperin-/- mice replicated ZIKV to higher titers compared to their WT counterparts. These results suggest that ZIKV can attenuate ISG expression to avoid the cellular antiviral innate response, thus allowing the virus to replicate unchecked. Moreover, we have identified that the ISG viperin has significant anti-ZIKV activity. Further understanding of how ZIKV perturbs the ISG response and the molecular mechanisms utilised by viperin to suppress ZIKV replication will aid in our understanding of ZIKV biology, pathogenesis and possible design of novel antiviral strategies.

HCV RNA traffic and association with NS5A in living cells.

The spatiotemporal dynamics of Hepatitis C Virus (HCV) RNA localisation are poorly understood. To address this we engineered HCV genomes harbouring MS2 bacteriophage RNA stem-loops within the 3'-untranslated region to allow tracking of HCV RNA via specific interaction with a MS2-Coat-mCherry fusion protein. Despite the impact of these insertions on viral fitness, live imaging revealed that replication of tagged-HCV genomes induced specific redistribution of the mCherry-tagged-MS2-Coat protein to motile and static foci. Further analysis showed that HCV RNA was associated with NS5A in both static and motile structures while a subset of motile NS5A structures was devoid of HCV RNA. Further investigation of viral RNA traffic with respect to lipid droplets (LDs) revealed HCV RNA-positive structures in close association with LDs. These studies provide new insights into the dynamics of HCV RNA traffic with NS5A and LDs and provide a platform for future investigations of HCV replication and assembly.

Fatty Acids Induce a Pro-Inflammatory Gene Expression Profile in Huh-7 Cells That Attenuates the Anti-HCV Action of Interferon.

The pathogenesis of nonalcoholic steatohepatitis is primarily an immune-driven disease and a known factor associated with treatment failure of chronic hepatitis C with interferon (IFN) and ribavirin. We studied the hepatocyte response in a model of steatosis at the transcriptome level and the antiviral action of IFN against hepatitis C virus (HCV) in this setting. In this study, we have shown that lipid loading (oleic acid and palmitic acid, OA:PA) of Huh-7 cells leads to increased expression of classical interferon-stimulated genes (ISGs) and NF-κß-dependent pro-inflammatory genes. A selective blocker of Toll-like receptor (TLR)2 signaling suppressed NF-κß promoter activity by OA:PA, suggesting that free fatty acids (FFAs) act as a TLR2 pathogen-associated molecular pattern. Furthermore, in the presence of OA:PA, IFN stimulation and HCV infection (Jc1) increased ISG expression. Somewhat counterintuitive to the increase in ISGs, the anti-HCV activity of IFN was attenuated in the presence of OA:PA. Interestingly, the combination of OA:PA, HCV, and IFN-α stimulation resulted in a significant increase in CXCL8 protein production, a cytokine known to have anti-IFN modulating activity. Thus, in an in vitro model of steatosis, the FFAs OA and PA drive an NF-κß-dependent inflammatory and ISG gene expression profile via TLR2 activation. Furthermore, FFA synergistically increases IFN-driven gene expression that may account for HCV treatment failure in vivo.

Leptin and leptin receptor expression in the rat ovary.

Leptin is an important satiety hormone and reproductive regulator and is found, along with its receptors, throughout the ovary. To date, the changes in ovarian expression of both of these proteins throughout the estrous cycle has not been studied, and the examination of protein expression has not distinguished between different forms of the receptor. In this study leptin mRNA expression in the immature gonadotropin-primed rat ovary increased 3-fold after human chorionic gonadotropin administration, followed by a dramatic increase in mRNA for both the short form (Ob-Ra) and the long form (Ob-Rb) of the leptin receptor (approximately 8- and 7-fold, respectively). A corresponding increase in mRNA expression of the receptor was not observed in isolated preovulatory follicles. Using immunohistochemistry, we observed protein expression of the long form of the leptin receptor (Ob-Rb) in the ovary, with high intensities observed in oocytes and endothelial cells as well as thecal cells and corpora lutea. These results suggest that ovarian expression of leptin and its receptor is regulated across the cycle by gonadotropins, with peak expression at ovulation, indicating a possible involvement in oocyte maturation, angiogenesis, follicle rupture, or subsequent corpus luteum formation.

Osteopontin increases hepatocellular carcinoma cell growth in a CD44 dependant manner.

AIM: To investigate the role of osteopontin (OPN) and its splice variants in the proliferation of hepatocellular carcinoma (HCC). METHODS: The expression of OPN variants in HCC cell lines as well as HCC tissue samples and non-tumour tissue was studied using polymerase chain reaction. OPN variant cDNAs were cloned into a mammalian expression vector allowing both transient expression and the production of stable OPN expressing cell lines. OPN expression was studied in these cells using Western blotting, immunofluoresnce and enzyme linked immunosorbent assay. A CD44 blocking antibody and siRNA targeting of CD44 were used to examine the role of this receptor in the OPN stimulated cell growth observed in culture. Huh-7 cells stably expressing either OPN-A, -B or -C were injected subcutaneously into the flanks of nude mice to observe in vivo tumour growth. Expression of OPN mRNA and protein in these tumours was examined using reverse transcription-polymerase chain reaction and immunohistochemistry. RESULTS: OPN is expressed in HCC in 3 forms, the full length OPN-A and 2 splice variants OPN-B and -C. OPN variant expression was noted in HCC tissue as well as cognate surrounding cirrhotic liver tissue. Expression of these OPN variants in the HCC derived cell line Huh-7 resulted in secretion of OPN into the culture medium. Transfer of OPN conditioned media to naïve Huh-7 and HepG2 cells resulted in significant cell growth suggesting that all OPN variants can modulate cell proliferation in a paracrine manner. Furthermore the OPN mediated increase in cellular proliferation was dependent on CD44 as only CD44 positive cell lines responded to OPN conditioned media while siRNA knockdown of CD44 blocked the proliferative effect. OPN expression also increased the proliferation of Huh-7 cells in a subcutaneous nude mouse tumour model, with Huh-7 cells expressing OPN-A showing the greatest proliferative effect. CONCLUSION: This study demonstrates that OPN plays a significant role in the proliferation of HCC through interaction with the cell surface receptor CD44. Modulation of this interaction could represent a novel strategy for the control of HCC.

Troglitazone regulates peroxisome proliferator-activated receptors and inducible nitric oxide synthase in murine ovarian macrophages.

Peroxisome proliferator-activated receptor-gamma (PPARG) and PPAR-alpha (PPARA) control metabolic processes in many cell types and act as anti-inflammatory regulators in macrophages. PPAR-activating ligands include thiazolidinediones (TZDs), such as troglitazone, once frequently used to treat insulin resistance as well as symptoms of polycystic ovary syndrome (PCOS). Since macrophages within the ovary mediate optimal follicle development, TZD actions to improve PCOS symptoms are likely to be partly mediated through these specifically localized immune cells. In mouse ovary, PPARG protein was expressed in granulosa cells and in isolated cells localized to theca, stroma, and corpora lutea, consistent with EMR1+ macrophages. Isolation of immune cells (EMR1+ or H2+) showed that Pparg and Ppara were expressed in ovarian macrophages at much higher levels than in peritoneal macrophages. Ovulatory human chorionic gonadotropin downregulated expression of Pparg and Ppara in EMR1+ ovarian macrophages, but no hormonal responsiveness was observed in H2+ cells. Downstream anti-inflammatory effects of PPARG activation were analyzed by in vitro treatment of isolated macrophages with troglitazone. Interleukin-1 beta (Il1b) expression was not altered, and tumor necrosis factor-alpha (Tnf) expression was affected in peritoneal macrophages only. In ovarian macrophages, inducible nitric oxide synthase (Nos2), an important proinflammatory enzyme that regulates ovulation, was significantly reduced by troglitazone treatment, an effect that was restricted to cells from the preovulatory ovary. Thus, expression of PPARs within ovarian macrophages is hormonally regulated, reflecting the changing roles of these cells during the ovulatory cycle. Additionally, ovarian macrophages respond directly to troglitazone to downregulate expression of proinflammatory Nos2, providing mechanistic information about ovarian effects of TZD treatment.

Macrophage contributions to ovarian function.

Macrophages are multifunctional cells that play key roles in the immune response and are abundant throughout female reproductive tissues. Macrophages are identified in tissues by their expression of cell surface receptors and can execute diverse functional activities, including phagocytosis and degradation of foreign antigens, matrix dissolution and tissue remodelling, and production and secretion of cytokines, chemokines and growth factors. Their specific localization and variations in distribution in the ovary during different stages of the cycle, as well as their presence in peri-ovulatory human follicular fluid, suggest that macrophages play diverse roles in intra-ovarian events including folliculogenesis, tissue restructuring at ovulation and corpus luteum formation and regression. This review presents the existing evidence for the regulation of ovarian function by macrophages and macrophage-derived products, highlighting the implications of these cells in ovarian diseases, particularly polycystic ovary syndrome, endometriosis and premature ovarian failure.

Expression of leptin and its receptor in the murine ovary: possible role in the regulation of oocyte maturation.

Leptin is a product of the ob gene that is produced primarily by adipose tissue. Leptin and its receptors are found within the ovary, but it is unclear what function this hormone has in the ovary. Using immunohistochemistry, we determined that leptin is found in most cell types in the murine ovary, with the highest staining levels observed in the oocyte. Leptin receptor was also expressed in all of the main ovarian cell types, with the thecal cell layer exhibiting the highest staining levels. Leptin administration did not affect spontaneous or induced maturation of either isolated denuded oocytes or cumulus-oocyte complexes, but it did significantly increase the rate of meiotic resumption in preovulatory follicle-enclosed oocytes (P < 0.01). Measurements of cAMP within oocytes cultured with leptin showed that this enhanced ability to resume meiosis does not occur via activation of phosphodiesterase 3B and subsequent cAMP reduction. These results provide evidence that leptin affects oocyte maturation when the oocyte is cultured within its normal follicular environment. It is suggested that leptin may induce the production of another factor, possibly from thecal cells, that directly or indirectly acts on the oocyte to initiate germinal vesicle breakdown in this species.