Nigel Stevenson - Academia.edu (original) (raw)

Papers by Nigel Stevenson

Viruses, 2021

The current COVID-19 pandemic has highlighted the need for the research community to develop a be... more The current COVID-19 pandemic has highlighted the need for the research community to develop a better understanding of viruses, in particular their modes of infection and replicative lifecycles, to aid in the development of novel vaccines and much needed anti-viral therapeutics. Several viruses express proteins capable of forming pores in host cellular membranes, termed “Viroporins”. They are a family of small hydrophobic proteins, with at least one amphipathic domain, which characteristically form oligomeric structures with central hydrophilic domains. Consequently, they can facilitate the transport of ions through the hydrophilic core. Viroporins localise to host membranes such as the endoplasmic reticulum and regulate ion homeostasis creating a favourable environment for viral infection. Viroporins also contribute to viral immune evasion via several mechanisms. Given that viroporins are often essential for virion assembly and egress, and as their structural features tend to be ev...

Viruses, 2021

Coronaviruses are a large family of zoonotic RNA viruses, whose infection can lead to mild or let... more Coronaviruses are a large family of zoonotic RNA viruses, whose infection can lead to mild or lethal respiratory tract disease. Severe Acute Respiratory Syndrome-Coronavirus-1 (SARS-CoV-1) first emerged in Guangdong, China in 2002 and spread to 29 countries, infecting 8089 individuals and causing 774 deaths. In 2012, Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) emerged in Saudi Arabia and has spread to 27 countries, with a mortality rate of ~34%. In 2019, SARS-CoV-2 emerged and has spread to 220 countries, infecting over 100,000,000 people and causing more than 2,000,000 deaths to date. These three human coronaviruses cause diseases of varying severity. Most people develop mild, common cold-like symptoms, while some develop acute respiratory distress syndrome (ARDS). The success of all viruses, including coronaviruses, relies on their evolved abilities to evade and modulate the host anti-viral and pro-inflammatory immune responses. However, we still do not fully understan...

Trials, 2021

Ever since the emergence of the coronavirus disease 2019 (COVID-19), global public health infrast... more Ever since the emergence of the coronavirus disease 2019 (COVID-19), global public health infrastructures and systems, along with community-wide collaboration and service, have risen to an unprecedented challenge. Vaccine development was immediately propelled to the centre of all our scientific, public health and community efforts. Despite the development of SARS-CoV-2 vaccines arguably being the greatest and most palpable achievements of the past 12 months, they have also been one of the most contentious and debated issues during the pandemic. However, what uniquely differentiates vaccine development is its intimate relationship with the community it seeks to serve; both in its clinical trial testing as an efficacious and safe prophylactic, and its post-developmental ‘roll-out’ success, as an effective public health tool. These relationships have birthed a myriad of complexities, from community-based mistrust, to academically contended ethical dilemmas. Indeed, the accelerated adva...

Cellular and Molecular Life Sciences, 2020

Every year there are > 33 million cases of Respiratory Syncytial Virus (RSV)-related respiratory ... more Every year there are > 33 million cases of Respiratory Syncytial Virus (RSV)-related respiratory infection in children under the age of five, making RSV the leading cause of lower respiratory tract infection (LRTI) in infants. RSV is a global infection, but 99% of related mortality is in low/middle-income countries. Unbelievably, 62 years after its identification, there remains no effective treatment nor vaccine for this deadly virus, leaving infants, elderly and immunocompromised patients at high risk. The success of all pathogens depends on their ability to evade and modulate the host immune response. RSV has a complex and intricate relationship with our immune systems, but a clearer understanding of these interactions is essential in the development of effective medicines. Therefore, in a bid to update and focus our research community's understanding of RSV's interaction with immune defences, this review aims to discuss how our current knowledgebase could be used to combat this global viral threat.

The FASEB Journal, 2019

Viruses use a spectrum of immune evasion strategies that enable infection and replication. The ac... more Viruses use a spectrum of immune evasion strategies that enable infection and replication. The acute phase of hepatitis C virus (HCV) infection is characterized by nonspecific and often mild clinical symptoms, suggesting an immunosuppressive mechanism that, unless symptomatic liver disease presents, allows the virus to remain largely undetected. We previously reported that HCV induced the regulatory protein suppressor of cytokine signaling (SOCS)3, which inhibited TNF-a-mediated inflammatory responses. However, the mechanism by which HCV up-regulates SOCS3 remains unknown. Here we show that the HCV protein, p7, enhances both SOCS3 mRNA and protein expression. A p7 inhibitor reduced SOCS3 induction, indicating that p7's ion channel activity was required for optimal up-regulation of SOCS3. Short hairpin RNA and chemical inhibition revealed that both the Janus kinase-signal transducer and activator of transcription (JAK-STAT) and MAPK pathways were required for p7-mediated induction of SOCS3. HCV-p7 expression suppressed TNF-a-mediated IkB-a degradation and subsequent NF-kB promoter activity, revealing a new and functional, anti-inflammatory effect of p7. Together, these findings identify a molecular mechanism by which HCV-p7 induces SOCS3 through STAT3 and ERK activation and demonstrate that p7 suppresses proinflammatory responses to TNF-a, possibly explaining the lack of inflammatory symptoms observed during early HCV infection.

Cellular and molecular life sciences : CMLS, May 1, 2017

The cytokine, Interferon (IFN)-α, induces a wide spectrum of anti-viral mediators, via the Janus ... more The cytokine, Interferon (IFN)-α, induces a wide spectrum of anti-viral mediators, via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. STAT1 and STAT2 are well characterised to upregulate IFN-stimulated gene (ISG) expression; but even though STAT3 is also activated by IFN-α, its role in anti-viral ISG induction is unclear. Several viruses, including Hepatitis C and Mumps, reduce cellular STAT3 protein levels, via the promotion of ubiquitin-mediated proteasomal degradation. This viral immune evasion mechanism suggests an undiscovered anti-viral role for STAT3 in IFN-α signalling. To investigate STAT3's functional involvement in this Type I IFN pathway, we first analysed its effect upon the replication of two viruses, Influenza and Vaccinia. Viral plaque assays, using Wild Type (WT) and STAT3-/- Murine Embryonic Fibroblasts (MEFs), revealed that STAT3 is required for the inhibition of Influenza and Vaccinia replication. Furthermore, STAT3 shRN...

Cellular and Molecular Life Sciences, 2015

Signaling pathways are essential intracellular networks that coordinate 14 molecular outcomes to ... more Signaling pathways are essential intracellular networks that coordinate 14 molecular outcomes to external stimuli. Tight regulation of these pathways is 15 essential to ensure an appropriate response. microRNA (miRNA) is a class of 16 small, non-coding RNA that regulates gene expression at a post-transcriptional 17 level by binding to the complementary sequence on target mRNA, thus limiting 18 protein translation. Intracellular pathways are controlled by protein regulators,

Brain, Behavior, and Immunity, 2015

Journal of Translational Medicine, 2014

Background: Treatment with interferon-alpha (IFN-α) and ribavirin successfully clears hepatitis C... more Background: Treatment with interferon-alpha (IFN-α) and ribavirin successfully clears hepatitis C virus (HCV) infection in 50% of patients infected with genotype 1. Addition of NS3-4A protease inhibitors (PIs) increases response rates but results in additional side effects and significant economic costs. Here, we hypothesised that in vitro responsiveness of peripheral blood mononuclear cells (PBMCs) to IFN-α stimulation would identify patients who achieved sustained virological response (SVR) on dual therapy alone and thus not require addition of PIs. Methods: PBMCs were isolated from HCV infected patients (n = 42), infected with either HCV genotype 1 or genotype 3, before commencing therapy and stimulated in vitro with IFN-α. Expression of the IFN stimulated genes (ISGs) PKR, OAS and MxA was measured and correlated with subsequent treatment response and IL28B genotype. Results: Genotype 1 infected patients who achieved SVR had significantly higher pre-treatment expression of PKR (p = 0.0148), OAS (p = 0.0019) and MxA (p = 0.0019) in IFN-α stimulated PBMCs, compared to genotype 1 infected patients who did not achieve SVR or patients infected with genotype 3, whose in vitro ISG expression did not correlate with clinical responsiveness. IL28B genotype (rs12979860) did not correlate with endogenous or IFN-α stimulated ISG responsiveness. Conclusions: In vitro responsiveness of PBMCs to IFN-α from genotype 1 infected patients predicts clinical responsiveness to dual therapy, independently of IL28B genotype. These results indicate that this subgroup of HCV infected patients could be identified pre-treatment and successfully treated without PIs, thus reducing adverse side effects and emergence of PI resistant virus while making significant economic savings.

Nature Communications, 2011

Deubiquitinating enzymes are now emerging as potential therapeutic targets that control many cell... more Deubiquitinating enzymes are now emerging as potential therapeutic targets that control many cellular processes, but few have been demonstrated to control cell motility. Here, we show that ubiquitin-specific protease 17 (usP17) is rapidly and transiently induced in response to chemokines sDF-1/CXCL12 and IL-8/CXCL8 in both primary cells and cell lines, and that its depletion completely blocks chemokine-induced cell migration and cytoskeletal rearrangements. using live cell imaging, we demonstrate that usP17 is required for both elongated and amoeboid motility, in addition to chemotaxis. usP17 has previously been reported to disrupt Ras localization and we now find that usP17 depletion blocks chemokine-induced subcellular relocalization of GTPases Cdc42, Rac and RhoA, which are GTPases essential for cell motility. Collectively, these results demonstrate that usP17 has a critical role in cell migration and may be a useful drug target for both inflammatory and metastatic disease.

PLoS ONE, 2011

The nucleoside analogue Ribavirin significantly increases patient response to IFN-a treatment of ... more The nucleoside analogue Ribavirin significantly increases patient response to IFN-a treatment of HCV, by directly inhibiting viral replication. Recent studies indicate that Ribavirin also regulates immunity and we propose that Ribavirin enhances specific interferon sensitive gene (ISG) expression by amplifying the IFN-a-JAK/STAT pathway. We found that IFN-a-induced STAT1 and STAT3 phosphorylation was increased in hepatocytes co-treated with Ribavirin and IFN-a, compared to IFN-a alone. Ribavirin specifically enhanced IFN-a induced mRNA and protein of the anti-viral mediator MxA, which co-localised with HCV core protein. These novel findings indicate for the first time that Ribavirin, in addition to its viral incorporation, also enhances IFN-a-JAK/STAT signalling, leading to a novel MxA-mediated immuno-modulatory mechanism that may enhance IFN-a anti-viral activity against HCV.

PLoS ONE, 2013

Suppressors of cytokine signalling (SOCS) proteins are classic inhibitors of the Janus kinase-sig... more Suppressors of cytokine signalling (SOCS) proteins are classic inhibitors of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Many cytokines and pathogenic mediators induce expression of SOCS, which act in a negative feedback loop to inhibit further signal transduction. SOCS mRNA expression is regulated by DNA binding of STAT proteins, however, their post-transcriptional regulation is poorly understood. microRNAs (miRNAs) are small non-coding RNAs that bind to complementary sequences on target mRNAs, often silencing gene expression. miR-19a has been shown to regulate SOCS1 expression during mutiple myeloma and be induced by the anti-viral cytokine interferon-(IFN)-α, suggesting a role in the regulation of the JAK-STAT pathway. This study aimed to identify targets of miR-19a in the JAK-STAT pathway and elucidate the functional consequences. Bioinformatic analysis identified highly conserved 3'UTR miR-19a target sequences in several JAK-STAT associated genes, including SOCS1, SOCS3, SOCS5 and Cullin (Cul) 5. Functional studies revealed that miR-19a significantly decreased SOCS3 mRNA and protein, while a miR-19a antagomir specifically reversed its inhibitory effect. Furthermore, miR-19a-mediated reduction of SOCS3 enhanced IFN-α and interleukin (IL)-6 signal transduction through STAT3. These results reveal a novel mechanism by which miR-19a may augment JAK-STAT signal transduction via control of SOCS3 expression and are fundamental to the understanding of inflammatory regulation.

Journal of Viral Hepatitis, 2011

Dendritic cells (DCs) are likely to play a key role in the compromised T-cell function associated... more Dendritic cells (DCs) are likely to play a key role in the compromised T-cell function associated with hepatitis C Virus (HCV) infection. However, studies of DC function in HCV-infected patients to date have yielded conflicting findings possibly because of patient and virus heterogeneity. Here, we report the characterization of monocyte-derived DCs obtained from a homogenous cohort of women who were infected with HCV genotype 1b following exposure to contaminated anti-D immunoglobulin from a single donor source. Patients included in the study had not received antiviral therapy and all had mild liver disease. We show that phenotypically normal monocyte-derived dendritic cells (MDDCs) (CD11c + HLA) DR + CD1a + CD14 lo) can be obtained from these patients. These cells respond to both Poly(I:C) and LPS, by up-regulating expression of CD86. They secrete high levels of IL-8 and CCL5 in response to LPS, an indication that the MyD88-dependent and MyD88-independent signalling pathways downstream of TLR4 ligation are functioning normally. However, these cells are poor stimulators of T-cell proliferation in allogeneic mixed lymphocyte reactions. Furthermore, patient MDDCs fail to secrete IFN-a in response to poly(I:C) or IFN-b stimulation. Altered DC function may contribute to impaired cellular immune responses and chronicity of disease following HCV infection in this cohort. An effective therapeutic vaccine for chronic HCV infection will most likely need to target DCs to elicit an appropriate cellular response; therefore, it is important to resolve how the DCs of different patient cohorts respond to stimulation via TLRs.

Journal of Leukocyte Biology, 2014

TNF-␣ is a proinflammatory cytokine, dramatically elevated during pathogenic infection and often ... more TNF-␣ is a proinflammatory cytokine, dramatically elevated during pathogenic infection and often responsible for inflammation-induced disease pathology. SOCS proteins are inhibitors of cytokine signaling and regulators of inflammation. In this study, we found that both SOCS1 and SOCS3 were transiently induced by TNF-␣ and negatively regulate its NF-B-mediated signal transduction. We discovered that PBMCs from HCV-infected patients have elevated endogenous SOCS3 expression but less TNF-␣-mediated IB degradation and proinflammatory cytokine production than healthy controls. HCV protein expression in Huh7 hepatocytes also induced SOCS3 and directly inhibited TNF-␣-mediated IL-8 production. Furthermore, we found that SOCS3 associates with TRAF2 and inhibits TRAF2-mediated NF-B promoter activity, suggesting a mechanism by which SOCS3 inhibits TNF-␣-mediated signaling. These results demonstrate a role for SOCS3 in regulating proinflammatory TNF-␣ signal transduction and reveal a novel immunemodulatory mechanism by which HCV suppresses inflammatory responses in primary immune cells and hepatocytes, perhaps explaining mild pathology often associated with acute HCV infection.

Journal of Leukocyte Biology, 2008

The chemokine eotaxin/CCL11 is an important mediator of leukocyte migration, but its effect on in... more The chemokine eotaxin/CCL11 is an important mediator of leukocyte migration, but its effect on inflammatory cytokine signaling has not been explored. In this study, we find that CCL11 induces suppressor of cytokine signaling (SOCS)1 and SOCS3 expression in murine macrophages, human monocytes, and dendritic cells (DCs). We also discover that CCL11 inhibits GM-CSF-mediated STAT5 activation and IL-4-induced STAT6 activation in a range of hematopoietic cells. This blockade of cytokine signaling by CCL11 results in reduced differentiation and endocytic ability of DCs, implicating CCL11-induced SOCS as mediators of chemotactic inflammatory control. These findings demonstrate cross-talk between chemokine and cytokine responses, suggesting that myeloid cells tracking to the inflammatory site do not differentiate in the presence of this chemokine, revealing another role for SOCS in inflammatory regulation.

The Journal of Immunology, 2004

Journal of Biological Chemistry, 2005

The suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine signal ... more The suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine signal transduction. SOCS3 is a key negative regulator of interleuking-6 (IL-6) signal transduction. Furthermore, SOCS3 was shown to be phosphorylated upon treatment of cells with IL-2, and this has been reported to regulate its function and half-life. We set out to investigate whether SOCS3 phosphorylation may play a role in IL-6 signaling. Tyrosine-phosphorylated SOCS3 was detected upon treatment of mouse embryonic fibroblasts with IL-6. Interestingly, the observed SOCS3 phosphorylation does not require SOCS3 recruitment to phosphotyrosine (Tyr(P)) 759 of gp130, and the kinetics of SOCS3 phosphorylation do not match the activation kinetics of the Janus kinases. This suggests that other kinases may be involved in SOCS3 phosphorylation. Using Src and Janus kinase inhibitors as well as Src kinase-deficient mouse embryonic fibroblasts, we provide evidence that Src kinases, which we found to be constitutively active in these cells, are involved in the phosphorylation of IL-6-induced SOCS3. In addition, we found that receptor-tyrosine kinases such as platelet-derived growth factor receptor or epidermal growth factor receptor can very potently phosphorylate IL-6-induced SOCS3. Taken together, these results suggest that SOCS3 phosphorylation is not a JAK-mediated phenomenon but is dependent on the activity of other kinases such as Src kinases or receptor-tyrosine kinases, which can either be constitutively active or activated by an additional stimulus.

Human Immunology, 2010

CD209, a c-type lectin expressed by dendritic cells (DCs) acts as a pathogen recognition receptor... more CD209, a c-type lectin expressed by dendritic cells (DCs) acts as a pathogen recognition receptor. A single nucleotide polymorphism (SNP) in the promoter region of CD209 (-336 A/G; rs4804803) affects transcription and is associated with the severity of Tuberculosis and Dengue fever. As CD209 binds Hepatitis C Virus (HCV) glycoprotein-E2, we investigated this SNP in the context of chronic HCV infection. 131 Irish women who had received HCV contaminated Anti-D immunoglobulin and 79 healthy controls were genotyped. We found no association between rs4804803 and the risk of HCV chronicity. However, of those with chronic infection, possession of at least one g-allele was associated with more advanced liver disease, with significantly higher liver fibrosis scores and levels of alanine transaminase (ALT) observed. We conclude that rs4804803, a SNP in the CD209 promoter contributes to the severity of liver disease in chronic HCV infection.

FEBS Letters, 2013

JAK/STAT signalling is essential for anti-viral immunity, making IFN-a an obvious anti-viral ther... more JAK/STAT signalling is essential for anti-viral immunity, making IFN-a an obvious anti-viral therapeutic. However, many HCV+ patients fail treatment, indicating that the virus blocks successful IFN-a signalling. We found that STAT1 and STAT3 proteins, key components of the IFN-a signalling pathway were reduced in immune cells and hepatocytes from HCV infected patients, and upon HCV expression in Huh7 hepatocytes. However, STAT1 and STAT3 mRNA levels were normal. Mechanistic analysis revealed that in the presence of HCV, STAT3 protein was preferentially ubiquitinated, and degradation was blocked by the proteasomal inhibitor MG132. These findings show that HCV inhibits IFN-a responses in a broad spectrum of cells via proteasomal degradation of JAK/STAT pathway components.

FEBS Letters, 2010

Suppressors of cytokine signalling (SOCS) proteins regulate signal transduction, but their role i... more Suppressors of cytokine signalling (SOCS) proteins regulate signal transduction, but their role in 28 responses to chemokines remains poorly understood. We report that cells expressing SOCS1 and 3 29 exhibit enhanced adhesion and reduced migration towards the chemokine CCL11. Focal adhesion 30 kinase (FAK) and the GTPase RhoA, control cell adhesion and migration and we show the presence 31 of SOCS1 or 3 regulates expression and tyrosine phosphorylation of FAK, while also enhancing acti-32 vation of RhoA. Our novel findings suggest that SOCS1 and 3 may control chemotaxis and adhesion 33 by significantly enhancing both FAK and RhoA activity, thus localizing immune cells to the site of 34 allergic inflammation.

Viruses, 2021

The current COVID-19 pandemic has highlighted the need for the research community to develop a be... more The current COVID-19 pandemic has highlighted the need for the research community to develop a better understanding of viruses, in particular their modes of infection and replicative lifecycles, to aid in the development of novel vaccines and much needed anti-viral therapeutics. Several viruses express proteins capable of forming pores in host cellular membranes, termed “Viroporins”. They are a family of small hydrophobic proteins, with at least one amphipathic domain, which characteristically form oligomeric structures with central hydrophilic domains. Consequently, they can facilitate the transport of ions through the hydrophilic core. Viroporins localise to host membranes such as the endoplasmic reticulum and regulate ion homeostasis creating a favourable environment for viral infection. Viroporins also contribute to viral immune evasion via several mechanisms. Given that viroporins are often essential for virion assembly and egress, and as their structural features tend to be ev...

Viruses, 2021

Coronaviruses are a large family of zoonotic RNA viruses, whose infection can lead to mild or let... more Coronaviruses are a large family of zoonotic RNA viruses, whose infection can lead to mild or lethal respiratory tract disease. Severe Acute Respiratory Syndrome-Coronavirus-1 (SARS-CoV-1) first emerged in Guangdong, China in 2002 and spread to 29 countries, infecting 8089 individuals and causing 774 deaths. In 2012, Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) emerged in Saudi Arabia and has spread to 27 countries, with a mortality rate of ~34%. In 2019, SARS-CoV-2 emerged and has spread to 220 countries, infecting over 100,000,000 people and causing more than 2,000,000 deaths to date. These three human coronaviruses cause diseases of varying severity. Most people develop mild, common cold-like symptoms, while some develop acute respiratory distress syndrome (ARDS). The success of all viruses, including coronaviruses, relies on their evolved abilities to evade and modulate the host anti-viral and pro-inflammatory immune responses. However, we still do not fully understan...

Trials, 2021

Ever since the emergence of the coronavirus disease 2019 (COVID-19), global public health infrast... more Ever since the emergence of the coronavirus disease 2019 (COVID-19), global public health infrastructures and systems, along with community-wide collaboration and service, have risen to an unprecedented challenge. Vaccine development was immediately propelled to the centre of all our scientific, public health and community efforts. Despite the development of SARS-CoV-2 vaccines arguably being the greatest and most palpable achievements of the past 12 months, they have also been one of the most contentious and debated issues during the pandemic. However, what uniquely differentiates vaccine development is its intimate relationship with the community it seeks to serve; both in its clinical trial testing as an efficacious and safe prophylactic, and its post-developmental ‘roll-out’ success, as an effective public health tool. These relationships have birthed a myriad of complexities, from community-based mistrust, to academically contended ethical dilemmas. Indeed, the accelerated adva...

Cellular and Molecular Life Sciences, 2020

Every year there are > 33 million cases of Respiratory Syncytial Virus (RSV)-related respiratory ... more Every year there are > 33 million cases of Respiratory Syncytial Virus (RSV)-related respiratory infection in children under the age of five, making RSV the leading cause of lower respiratory tract infection (LRTI) in infants. RSV is a global infection, but 99% of related mortality is in low/middle-income countries. Unbelievably, 62 years after its identification, there remains no effective treatment nor vaccine for this deadly virus, leaving infants, elderly and immunocompromised patients at high risk. The success of all pathogens depends on their ability to evade and modulate the host immune response. RSV has a complex and intricate relationship with our immune systems, but a clearer understanding of these interactions is essential in the development of effective medicines. Therefore, in a bid to update and focus our research community's understanding of RSV's interaction with immune defences, this review aims to discuss how our current knowledgebase could be used to combat this global viral threat.

The FASEB Journal, 2019

Viruses use a spectrum of immune evasion strategies that enable infection and replication. The ac... more Viruses use a spectrum of immune evasion strategies that enable infection and replication. The acute phase of hepatitis C virus (HCV) infection is characterized by nonspecific and often mild clinical symptoms, suggesting an immunosuppressive mechanism that, unless symptomatic liver disease presents, allows the virus to remain largely undetected. We previously reported that HCV induced the regulatory protein suppressor of cytokine signaling (SOCS)3, which inhibited TNF-a-mediated inflammatory responses. However, the mechanism by which HCV up-regulates SOCS3 remains unknown. Here we show that the HCV protein, p7, enhances both SOCS3 mRNA and protein expression. A p7 inhibitor reduced SOCS3 induction, indicating that p7's ion channel activity was required for optimal up-regulation of SOCS3. Short hairpin RNA and chemical inhibition revealed that both the Janus kinase-signal transducer and activator of transcription (JAK-STAT) and MAPK pathways were required for p7-mediated induction of SOCS3. HCV-p7 expression suppressed TNF-a-mediated IkB-a degradation and subsequent NF-kB promoter activity, revealing a new and functional, anti-inflammatory effect of p7. Together, these findings identify a molecular mechanism by which HCV-p7 induces SOCS3 through STAT3 and ERK activation and demonstrate that p7 suppresses proinflammatory responses to TNF-a, possibly explaining the lack of inflammatory symptoms observed during early HCV infection.

Cellular and molecular life sciences : CMLS, May 1, 2017

The cytokine, Interferon (IFN)-α, induces a wide spectrum of anti-viral mediators, via the Janus ... more The cytokine, Interferon (IFN)-α, induces a wide spectrum of anti-viral mediators, via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. STAT1 and STAT2 are well characterised to upregulate IFN-stimulated gene (ISG) expression; but even though STAT3 is also activated by IFN-α, its role in anti-viral ISG induction is unclear. Several viruses, including Hepatitis C and Mumps, reduce cellular STAT3 protein levels, via the promotion of ubiquitin-mediated proteasomal degradation. This viral immune evasion mechanism suggests an undiscovered anti-viral role for STAT3 in IFN-α signalling. To investigate STAT3's functional involvement in this Type I IFN pathway, we first analysed its effect upon the replication of two viruses, Influenza and Vaccinia. Viral plaque assays, using Wild Type (WT) and STAT3-/- Murine Embryonic Fibroblasts (MEFs), revealed that STAT3 is required for the inhibition of Influenza and Vaccinia replication. Furthermore, STAT3 shRN...

Cellular and Molecular Life Sciences, 2015

Signaling pathways are essential intracellular networks that coordinate 14 molecular outcomes to ... more Signaling pathways are essential intracellular networks that coordinate 14 molecular outcomes to external stimuli. Tight regulation of these pathways is 15 essential to ensure an appropriate response. microRNA (miRNA) is a class of 16 small, non-coding RNA that regulates gene expression at a post-transcriptional 17 level by binding to the complementary sequence on target mRNA, thus limiting 18 protein translation. Intracellular pathways are controlled by protein regulators,

Brain, Behavior, and Immunity, 2015

Journal of Translational Medicine, 2014

Background: Treatment with interferon-alpha (IFN-α) and ribavirin successfully clears hepatitis C... more Background: Treatment with interferon-alpha (IFN-α) and ribavirin successfully clears hepatitis C virus (HCV) infection in 50% of patients infected with genotype 1. Addition of NS3-4A protease inhibitors (PIs) increases response rates but results in additional side effects and significant economic costs. Here, we hypothesised that in vitro responsiveness of peripheral blood mononuclear cells (PBMCs) to IFN-α stimulation would identify patients who achieved sustained virological response (SVR) on dual therapy alone and thus not require addition of PIs. Methods: PBMCs were isolated from HCV infected patients (n = 42), infected with either HCV genotype 1 or genotype 3, before commencing therapy and stimulated in vitro with IFN-α. Expression of the IFN stimulated genes (ISGs) PKR, OAS and MxA was measured and correlated with subsequent treatment response and IL28B genotype. Results: Genotype 1 infected patients who achieved SVR had significantly higher pre-treatment expression of PKR (p = 0.0148), OAS (p = 0.0019) and MxA (p = 0.0019) in IFN-α stimulated PBMCs, compared to genotype 1 infected patients who did not achieve SVR or patients infected with genotype 3, whose in vitro ISG expression did not correlate with clinical responsiveness. IL28B genotype (rs12979860) did not correlate with endogenous or IFN-α stimulated ISG responsiveness. Conclusions: In vitro responsiveness of PBMCs to IFN-α from genotype 1 infected patients predicts clinical responsiveness to dual therapy, independently of IL28B genotype. These results indicate that this subgroup of HCV infected patients could be identified pre-treatment and successfully treated without PIs, thus reducing adverse side effects and emergence of PI resistant virus while making significant economic savings.

Nature Communications, 2011

Deubiquitinating enzymes are now emerging as potential therapeutic targets that control many cell... more Deubiquitinating enzymes are now emerging as potential therapeutic targets that control many cellular processes, but few have been demonstrated to control cell motility. Here, we show that ubiquitin-specific protease 17 (usP17) is rapidly and transiently induced in response to chemokines sDF-1/CXCL12 and IL-8/CXCL8 in both primary cells and cell lines, and that its depletion completely blocks chemokine-induced cell migration and cytoskeletal rearrangements. using live cell imaging, we demonstrate that usP17 is required for both elongated and amoeboid motility, in addition to chemotaxis. usP17 has previously been reported to disrupt Ras localization and we now find that usP17 depletion blocks chemokine-induced subcellular relocalization of GTPases Cdc42, Rac and RhoA, which are GTPases essential for cell motility. Collectively, these results demonstrate that usP17 has a critical role in cell migration and may be a useful drug target for both inflammatory and metastatic disease.

PLoS ONE, 2011

The nucleoside analogue Ribavirin significantly increases patient response to IFN-a treatment of ... more The nucleoside analogue Ribavirin significantly increases patient response to IFN-a treatment of HCV, by directly inhibiting viral replication. Recent studies indicate that Ribavirin also regulates immunity and we propose that Ribavirin enhances specific interferon sensitive gene (ISG) expression by amplifying the IFN-a-JAK/STAT pathway. We found that IFN-a-induced STAT1 and STAT3 phosphorylation was increased in hepatocytes co-treated with Ribavirin and IFN-a, compared to IFN-a alone. Ribavirin specifically enhanced IFN-a induced mRNA and protein of the anti-viral mediator MxA, which co-localised with HCV core protein. These novel findings indicate for the first time that Ribavirin, in addition to its viral incorporation, also enhances IFN-a-JAK/STAT signalling, leading to a novel MxA-mediated immuno-modulatory mechanism that may enhance IFN-a anti-viral activity against HCV.

PLoS ONE, 2013

Suppressors of cytokine signalling (SOCS) proteins are classic inhibitors of the Janus kinase-sig... more Suppressors of cytokine signalling (SOCS) proteins are classic inhibitors of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Many cytokines and pathogenic mediators induce expression of SOCS, which act in a negative feedback loop to inhibit further signal transduction. SOCS mRNA expression is regulated by DNA binding of STAT proteins, however, their post-transcriptional regulation is poorly understood. microRNAs (miRNAs) are small non-coding RNAs that bind to complementary sequences on target mRNAs, often silencing gene expression. miR-19a has been shown to regulate SOCS1 expression during mutiple myeloma and be induced by the anti-viral cytokine interferon-(IFN)-α, suggesting a role in the regulation of the JAK-STAT pathway. This study aimed to identify targets of miR-19a in the JAK-STAT pathway and elucidate the functional consequences. Bioinformatic analysis identified highly conserved 3'UTR miR-19a target sequences in several JAK-STAT associated genes, including SOCS1, SOCS3, SOCS5 and Cullin (Cul) 5. Functional studies revealed that miR-19a significantly decreased SOCS3 mRNA and protein, while a miR-19a antagomir specifically reversed its inhibitory effect. Furthermore, miR-19a-mediated reduction of SOCS3 enhanced IFN-α and interleukin (IL)-6 signal transduction through STAT3. These results reveal a novel mechanism by which miR-19a may augment JAK-STAT signal transduction via control of SOCS3 expression and are fundamental to the understanding of inflammatory regulation.

Journal of Viral Hepatitis, 2011

Dendritic cells (DCs) are likely to play a key role in the compromised T-cell function associated... more Dendritic cells (DCs) are likely to play a key role in the compromised T-cell function associated with hepatitis C Virus (HCV) infection. However, studies of DC function in HCV-infected patients to date have yielded conflicting findings possibly because of patient and virus heterogeneity. Here, we report the characterization of monocyte-derived DCs obtained from a homogenous cohort of women who were infected with HCV genotype 1b following exposure to contaminated anti-D immunoglobulin from a single donor source. Patients included in the study had not received antiviral therapy and all had mild liver disease. We show that phenotypically normal monocyte-derived dendritic cells (MDDCs) (CD11c + HLA) DR + CD1a + CD14 lo) can be obtained from these patients. These cells respond to both Poly(I:C) and LPS, by up-regulating expression of CD86. They secrete high levels of IL-8 and CCL5 in response to LPS, an indication that the MyD88-dependent and MyD88-independent signalling pathways downstream of TLR4 ligation are functioning normally. However, these cells are poor stimulators of T-cell proliferation in allogeneic mixed lymphocyte reactions. Furthermore, patient MDDCs fail to secrete IFN-a in response to poly(I:C) or IFN-b stimulation. Altered DC function may contribute to impaired cellular immune responses and chronicity of disease following HCV infection in this cohort. An effective therapeutic vaccine for chronic HCV infection will most likely need to target DCs to elicit an appropriate cellular response; therefore, it is important to resolve how the DCs of different patient cohorts respond to stimulation via TLRs.

Journal of Leukocyte Biology, 2014

TNF-␣ is a proinflammatory cytokine, dramatically elevated during pathogenic infection and often ... more TNF-␣ is a proinflammatory cytokine, dramatically elevated during pathogenic infection and often responsible for inflammation-induced disease pathology. SOCS proteins are inhibitors of cytokine signaling and regulators of inflammation. In this study, we found that both SOCS1 and SOCS3 were transiently induced by TNF-␣ and negatively regulate its NF-B-mediated signal transduction. We discovered that PBMCs from HCV-infected patients have elevated endogenous SOCS3 expression but less TNF-␣-mediated IB degradation and proinflammatory cytokine production than healthy controls. HCV protein expression in Huh7 hepatocytes also induced SOCS3 and directly inhibited TNF-␣-mediated IL-8 production. Furthermore, we found that SOCS3 associates with TRAF2 and inhibits TRAF2-mediated NF-B promoter activity, suggesting a mechanism by which SOCS3 inhibits TNF-␣-mediated signaling. These results demonstrate a role for SOCS3 in regulating proinflammatory TNF-␣ signal transduction and reveal a novel immunemodulatory mechanism by which HCV suppresses inflammatory responses in primary immune cells and hepatocytes, perhaps explaining mild pathology often associated with acute HCV infection.

Journal of Leukocyte Biology, 2008

The chemokine eotaxin/CCL11 is an important mediator of leukocyte migration, but its effect on in... more The chemokine eotaxin/CCL11 is an important mediator of leukocyte migration, but its effect on inflammatory cytokine signaling has not been explored. In this study, we find that CCL11 induces suppressor of cytokine signaling (SOCS)1 and SOCS3 expression in murine macrophages, human monocytes, and dendritic cells (DCs). We also discover that CCL11 inhibits GM-CSF-mediated STAT5 activation and IL-4-induced STAT6 activation in a range of hematopoietic cells. This blockade of cytokine signaling by CCL11 results in reduced differentiation and endocytic ability of DCs, implicating CCL11-induced SOCS as mediators of chemotactic inflammatory control. These findings demonstrate cross-talk between chemokine and cytokine responses, suggesting that myeloid cells tracking to the inflammatory site do not differentiate in the presence of this chemokine, revealing another role for SOCS in inflammatory regulation.

The Journal of Immunology, 2004

Journal of Biological Chemistry, 2005

The suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine signal ... more The suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine signal transduction. SOCS3 is a key negative regulator of interleuking-6 (IL-6) signal transduction. Furthermore, SOCS3 was shown to be phosphorylated upon treatment of cells with IL-2, and this has been reported to regulate its function and half-life. We set out to investigate whether SOCS3 phosphorylation may play a role in IL-6 signaling. Tyrosine-phosphorylated SOCS3 was detected upon treatment of mouse embryonic fibroblasts with IL-6. Interestingly, the observed SOCS3 phosphorylation does not require SOCS3 recruitment to phosphotyrosine (Tyr(P)) 759 of gp130, and the kinetics of SOCS3 phosphorylation do not match the activation kinetics of the Janus kinases. This suggests that other kinases may be involved in SOCS3 phosphorylation. Using Src and Janus kinase inhibitors as well as Src kinase-deficient mouse embryonic fibroblasts, we provide evidence that Src kinases, which we found to be constitutively active in these cells, are involved in the phosphorylation of IL-6-induced SOCS3. In addition, we found that receptor-tyrosine kinases such as platelet-derived growth factor receptor or epidermal growth factor receptor can very potently phosphorylate IL-6-induced SOCS3. Taken together, these results suggest that SOCS3 phosphorylation is not a JAK-mediated phenomenon but is dependent on the activity of other kinases such as Src kinases or receptor-tyrosine kinases, which can either be constitutively active or activated by an additional stimulus.

Human Immunology, 2010

CD209, a c-type lectin expressed by dendritic cells (DCs) acts as a pathogen recognition receptor... more CD209, a c-type lectin expressed by dendritic cells (DCs) acts as a pathogen recognition receptor. A single nucleotide polymorphism (SNP) in the promoter region of CD209 (-336 A/G; rs4804803) affects transcription and is associated with the severity of Tuberculosis and Dengue fever. As CD209 binds Hepatitis C Virus (HCV) glycoprotein-E2, we investigated this SNP in the context of chronic HCV infection. 131 Irish women who had received HCV contaminated Anti-D immunoglobulin and 79 healthy controls were genotyped. We found no association between rs4804803 and the risk of HCV chronicity. However, of those with chronic infection, possession of at least one g-allele was associated with more advanced liver disease, with significantly higher liver fibrosis scores and levels of alanine transaminase (ALT) observed. We conclude that rs4804803, a SNP in the CD209 promoter contributes to the severity of liver disease in chronic HCV infection.

FEBS Letters, 2013

JAK/STAT signalling is essential for anti-viral immunity, making IFN-a an obvious anti-viral ther... more JAK/STAT signalling is essential for anti-viral immunity, making IFN-a an obvious anti-viral therapeutic. However, many HCV+ patients fail treatment, indicating that the virus blocks successful IFN-a signalling. We found that STAT1 and STAT3 proteins, key components of the IFN-a signalling pathway were reduced in immune cells and hepatocytes from HCV infected patients, and upon HCV expression in Huh7 hepatocytes. However, STAT1 and STAT3 mRNA levels were normal. Mechanistic analysis revealed that in the presence of HCV, STAT3 protein was preferentially ubiquitinated, and degradation was blocked by the proteasomal inhibitor MG132. These findings show that HCV inhibits IFN-a responses in a broad spectrum of cells via proteasomal degradation of JAK/STAT pathway components.

FEBS Letters, 2010

Suppressors of cytokine signalling (SOCS) proteins regulate signal transduction, but their role i... more Suppressors of cytokine signalling (SOCS) proteins regulate signal transduction, but their role in 28 responses to chemokines remains poorly understood. We report that cells expressing SOCS1 and 3 29 exhibit enhanced adhesion and reduced migration towards the chemokine CCL11. Focal adhesion 30 kinase (FAK) and the GTPase RhoA, control cell adhesion and migration and we show the presence 31 of SOCS1 or 3 regulates expression and tyrosine phosphorylation of FAK, while also enhancing acti-32 vation of RhoA. Our novel findings suggest that SOCS1 and 3 may control chemotaxis and adhesion 33 by significantly enhancing both FAK and RhoA activity, thus localizing immune cells to the site of 34 allergic inflammation.