Publications by authors named "Claire E McCoy"

29 Publications

  • Page 1 of 1

Mitochondrial arginase-2 is essential for IL-10 metabolic reprogramming of inflammatory macrophages.

Nat Commun 2021 03 5;12(1):1460. Epub 2021 Mar 5.

School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin 2, Ireland.

Mitochondria are important regulators of macrophage polarisation. Here, we show that arginase-2 (Arg2) is a microRNA-155 (miR-155) and interleukin-10 (IL-10) regulated protein localized at the mitochondria in inflammatory macrophages, and is critical for IL-10-induced modulation of mitochondrial dynamics and oxidative respiration. Mechanistically, the catalytic activity and presence of Arg2 at the mitochondria is crucial for oxidative phosphorylation. We further show that Arg2 mediates this process by increasing the activity of complex II (succinate dehydrogenase). Moreover, Arg2 is essential for IL-10-mediated downregulation of the inflammatory mediators succinate, hypoxia inducible factor 1α (HIF-1α) and IL-1β in vitro. Accordingly, HIF-1α and IL-1β are highly expressed in an LPS-induced in vivo model of acute inflammation using Arg2 mice. These findings shed light on a new arm of IL-10-mediated metabolic regulation, working to resolve the inflammatory status of the cell.
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http://dx.doi.org/10.1038/s41467-021-21617-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7936006PMC
March 2021

The Key Regulator of Necroptosis, RIP1 Kinase, Contributes to the Formation of Astrogliosis and Glial Scar in Ischemic Stroke.

Transl Stroke Res 2021 Feb 24. Epub 2021 Feb 24.

Jiangsu Key Laboratory of Neuropsychiatric Diseases and College of Pharmaceutical Sciences, Laboratory of Cerebrovascular Pharmacology, College of Pharmaceutical Science, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, School of Public Health, Soochow University, 199 Ren-Ai Road, Suzhou, 215123, Jiangsu, China.

Necroptosis initiation relies on the receptor-interacting protein 1 kinase (RIP1K). We recently reported that genetic and pharmacological inhibition of RIP1K produces protection against ischemic stroke-induced astrocytic injury. However, the role of RIP1K in ischemic stroke-induced formation of astrogliosis and glial scar remains unknown. Here, in a transient middle cerebral artery occlusion (tMCAO) rat model and an oxygen and glucose deprivation and reoxygenation (OGD/Re)-induced astrocytic injury model, we show that RIP1K was significantly elevated in the reactive astrocytes. Knockdown of RIP1K or delayed administration of RIP1K inhibitor Nec-1 down-regulated the glial scar markers, improved ischemic stroke-induced necrotic morphology and neurologic deficits, and reduced the volume of brain atrophy. Moreover, knockdown of RIP1K attenuated astrocytic cell death and proliferation and promoted neuronal axonal generation in a neuron and astrocyte co-culture system. Both vascular endothelial growth factor D (VEGF-D) and its receptor VEGFR-3 were elevated in the reactive astrocytes; simultaneously, VEGF-D was increased in the medium of astrocytes exposed to OGD/Re. Knockdown of RIP1K down-regulated VEGF-D gene and protein levels in the reactive astrocytes. Treatment with 400 ng/ml recombinant VEGF-D induced the formation of glial scar; conversely, the inhibitor of VEGFR-3 suppressed OGD/Re-induced glial scar formation. RIP3K and MLKL may be involved in glial scar formation. Taken together, these results suggest that RIP1K participates in the formation of astrogliosis and glial scar via impairment of normal astrocyte responses and enhancing the astrocytic VEGF-D/VEGFR-3 signaling pathways. Inhibition of RIP1K promotes the brain functional recovery partially via suppressing the formation of astrogliosis and glial scar.
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http://dx.doi.org/10.1007/s12975-021-00888-3DOI Listing
February 2021

Impact of Exercise on Immunometabolism in Multiple Sclerosis.

J Clin Med 2020 Sep 21;9(9). Epub 2020 Sep 21.

School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, 123 St. Stephen's Green, D02 YN77 Dublin, Ireland.

Multiple Sclerosis (MS) is a chronic, autoimmune condition characterized by demyelinating lesions and axonal degradation. Even though the cause of MS is heterogeneous, it is known that peripheral immune invasion in the central nervous system (CNS) drives pathology at least in the most common form of MS, relapse-remitting MS (RRMS). The more progressive forms' mechanisms of action remain more elusive yet an innate immune dysfunction combined with neurodegeneration are likely drivers. Recently, increasing studies have focused on the influence of metabolism in regulating immune cell function. In this regard, exercise has long been known to regulate metabolism, and has emerged as a promising therapy for management of autoimmune disorders. Hence, in this review, we inspect the role of key immunometabolic pathways specifically dysregulated in MS and highlight potential therapeutic benefits of exercise in modulating those pathways to harness an anti-inflammatory state. Finally, we touch upon current challenges and future directions for the field of exercise and immunometabolism in MS.
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http://dx.doi.org/10.3390/jcm9093038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564219PMC
September 2020

The Role of MicroRNAs in Repair Processes in Multiple Sclerosis.

Cells 2020 07 16;9(7). Epub 2020 Jul 16.

School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, 123 St Stephen's Green, Dublin 2 D02 YN77, Ireland.

Multiple sclerosis (MS) is an autoimmune disorder characterised by demyelination of central nervous system neurons with subsequent damage, cell death and disability. While mechanisms exist in the CNS to repair this damage, they are disrupted in MS and currently there are no treatments to address this deficit. In recent years, increasing attention has been paid to the influence of the small, non-coding RNA molecules, microRNAs (miRNAs), in autoimmune disorders, including MS. In this review, we examine the role of miRNAs in remyelination in the different cell types that contribute to MS. We focus on key miRNAs that have a central role in mediating the repair process, along with several more that play either secondary or inhibitory roles in one or more aspects. Finally, we consider the current state of miRNAs as therapeutic targets in MS, acknowledging current challenges and potential strategies to overcome them in developing effective novel therapeutics to enhance repair mechanisms in MS.
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http://dx.doi.org/10.3390/cells9071711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408558PMC
July 2020

Nanomodulation of Macrophages in Multiple Sclerosis.

Cells 2019 06 5;8(6). Epub 2019 Jun 5.

Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, 123 St Stephen's Green, 2 D02 YN77 Dublin, Ireland.

Multiple Sclerosis (MS) is a chronic demyelinating autoimmune disease primarily affecting young adults. Despite an unclear causal factor, symptoms and pathology arise from the infiltration of peripheral immune cells across the blood brain barrier. Accounting for the largest fraction of this infiltrate, macrophages are functionally heterogeneous innate immune cells capable of adopting either a pro or an anti-inflammatory phenotype, a phenomenon dependent upon cytokine milieu in the CNS. This functional plasticity is of key relevance in MS, where the pro-inflammatory state dominates the early stage, instructing demyelination and axonal loss while the later anti-inflammatory state holds a key role in promoting tissue repair and regeneration in later remission. This review highlights a potential therapeutic benefit of modulating macrophage polarisation to harness the anti-inflammatory and reparative state in MS. Here, we outline the role of macrophages in MS and look at the role of current FDA approved therapeutics in macrophage polarisation. Moreover, we explore the potential of particulate carriers as a novel strategy to manipulate polarisation states in macrophages, whilst examining how optimising macrophage uptake via nanoparticle size and functionalisation could offer a novel therapeutic approach for MS.
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http://dx.doi.org/10.3390/cells8060543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628349PMC
June 2019

Optimization Techniques for miRNA Expression in Low Frequency Immune Cell Populations.

Methods Mol Biol 2018 ;1725:237-256

Hudson Institute of Medical Research, Department of Molecular and Translational Science, Monash University Faculty of Medicine, Nursing and Health Sciences, Clayton, VIC, Australia.

In this chapter we outline a RNA extraction method for very low immune cell populations isolated from the central nervous system of mice undergoing experimental autoimmune encephalomyelitis. We compare various normalization and quantification techniques to examine miRNA expression. Our data highlight that employing a mean normalization procedure using a number of well-selected housekeeping miRNA genes, followed by absolute quantification with a standard curve generated from a commercial miRNA oligo, gave the most robust and reproducible miRNA expression results.
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http://dx.doi.org/10.1007/978-1-4939-7568-6_19DOI Listing
January 2019

miR-222 isoforms are differentially regulated by type-I interferon.

RNA 2018 03 20;24(3):332-341. Epub 2017 Dec 20.

Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Clayton, Victoria 3168, Australia.

Endogenous microRNAs (miRNAs) often exist as multiple isoforms (known as "isomiRs") with predominant variation around their 3'-end. Increasing evidence suggests that different isomiRs of the same family can have diverse functional roles, as recently demonstrated with the example of miR-222-3p 3'-end variants. While isomiR levels from a same miRNA family can vary between tissues and cell types, change of templated isomiR stoichiometry to stimulation has not been reported to date. Relying on small RNA-sequencing analyses, we demonstrate here that miR-222-3p 3'-end variants >23 nt are specifically decreased upon interferon (IFN) β stimulation of human fibroblasts, while shorter isoforms are spared. This length-dependent dynamic regulation of long miR-222-3p 3'-isoforms and >40 other miRNA families was confirmed in human monocyte-derived dendritic cells following infection with Typhimurium, underlining the breadth of 3'-length regulation by infection, beyond the example of miR-222-3p. We further show that stem-loop miRNA Taqman RT-qPCR exhibits selectivity between 3'-isoforms, according to their length, and that this can lead to misinterpretation of results when these isoforms are differentially regulated. Collectively, and to our knowledge, this work constitutes the first demonstration that the stoichiometry of highly abundant templated 3'-isoforms of a same miRNA family can be dynamically regulated by a stimulus. Given that such 3'-isomiRs can have different functions, our study underlines the need to consider isomiRs when investigating miRNA-based regulation.
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http://dx.doi.org/10.1261/rna.064550.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5824353PMC
March 2018

miR-155 Dysregulation and Therapeutic Intervention in Multiple Sclerosis.

Authors:
Claire E McCoy

Adv Exp Med Biol 2017 ;1024:111-131

Hudson Institute of Medical Research, Department of Molecular and Translational Science, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, 3168, Australia.

microRNAs play a fundamental role in the immune system. One particular microRNA, miR-155 plays a critical role in hematopoietic cell development and tightly regulates innate and adaptive immune responses in response to infection. However, its dysregulation, more specifically its overexpression, is closely associated with various inflammatory disorders. The purpose of this review is to consolidate how miR-155 underpins a variety of processes that contribute to the pathology of multiple sclerosis (MS). In particular, the impact of miR-155 is discussed with respect to human pathology and animal models. How miR-155 contributes to the activation of pathogenic immune cells, the permeability of the blood-brain barrier, and neurodegeneration in relation to MS is described. Many environmental risk factors associated with MS susceptibility can cause upregulation of miR-155, while many of the current disease-modifying treatments may work by inhibiting miR-155. From this review, it is clear that miR-155 is a realistic and feasible diagnostic, prognostic, and therapeutic target for the treatment of MS.
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http://dx.doi.org/10.1007/978-981-10-5987-2_5DOI Listing
April 2018

Preface.

Authors:
Claire E McCoy

Methods Mol Biol 2016 ;1390

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http://dx.doi.org/10.1007/978-1-4939-3335-8DOI Listing
October 2016

Simple Methods to Investigate MicroRNA Induction in Response to Toll-Like Receptors.

Methods Mol Biol 2016 ;1390:159-82

School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland.

In this chapter, we describe simple methods to investigate microRNA (miRNA) induction in response to lipopolysaccharide, the ligand for Toll-Like Receptor-4 activation. In brief, we demonstrate how to investigate global miRNA induction and/or repression in bone marrow-derived macrophages using TaqMan MicroRNA Arrays, followed by methods to measure individual miRNAs and target mRNA expression. Moreover, we explain step-by-step instructions on how to modulate endogenous miRNA expression through the use of miRNA inhibitors and mimics as well as highlight how miRNA modulation can be used to confirm mRNA targeting via Luciferase reporter assay. Moreover, these methods can be applied to whichever cell type and cellular function under investigation.
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http://dx.doi.org/10.1007/978-1-4939-3335-8_11DOI Listing
October 2016

Sequence-dependent off-target inhibition of TLR7/8 sensing by synthetic microRNA inhibitors.

Nucleic Acids Res 2015 Jan 24;43(2):1177-88. Epub 2014 Dec 24.

Centre for Cancer Research, MIMR-PHI Institute of Medical Research, Clayton, Victoria 3168, Australia Department of Molecular and Translational Science, Monash University, Clayton, Victoria 3168, Australia

Anti-microRNA (miRNA) oligonucleotides (AMOs) with 2'-O-Methyl (2'OMe) residues are commonly used to study miRNA function and can achieve high potency, with low cytotoxicity. Not withstanding this, we demonstrate the sequence-dependent capacity of 2'OMe AMOs to inhibit Toll-like receptor (TLR) 7 and 8 sensing of immunostimulatory RNA, independent of their miRNA-targeting function. Through a screen of 29 AMOs targeting common miRNAs, we found a subset of sequences highly inhibitory to TLR7 sensing in mouse macrophages. Interspecies conservation of this inhibitory activity was confirmed on TLR7/8 activity in human peripheral blood mononuclear cells. Significantly, we identified a core motif governing the inhibitory activity of these AMOs, which is present in more than 50 AMOs targeted to human miRNAs in miRBaseV20. DNA/locked nucleic acids (LNA) AMOs synthesized with a phosphorothioate backbone also inhibited TLR7 sensing in a sequence-dependent manner, demonstrating that the off-target effects of AMOs are not restricted to 2'OMe modification. Taken together, our work establishes the potential for off-target effects of AMOs on TLR7/8 function, which should be taken into account in their therapeutic development and in vivo application.
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http://dx.doi.org/10.1093/nar/gku1343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4333393PMC
January 2015

IL-10 regulates Aicda expression through miR-155.

J Leukoc Biol 2015 Jan 7;97(1):71-8. Epub 2014 Nov 7.

*Faculty of Medicine, Department of Immunology, Monash University, Prahran, and Department of Molecular and Translational Science, Monash University, Clayton, Victoria, Australia; The Department of Experimental Medicine, University of Melbourne, Parkville, Victoria, Australia; Division of Molecular Medicine, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; Centre for Cancer Research, Monash Institute of Medical Research-Prince Henry's Institute of Medical Research, Clayton, Victoria, Australia; and School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland

Aicda is a critical component of antibody class-switching in B cells. In this work, we study the impact of TLR4 activation and IL-10 stimulation on Aicda expression in B cells. Through the global analysis of miRNAs in response to TLR4 activation, in combination with IL-10 stimulation, we identified that IL-10 can suppress TLR4-induced miR-155 expression, an effect that resulted in enhanced Aicda expression. Furthermore, when preventing miR-155 control of Aicda expression, by genetic mutation of its target site in the Aicda mRNA, IL-10 could further potentiate Aicda expression. Given that miR-155 expression is lost, and expression levels of both Aicda and IL-10 are high in diseases, such as Burkitt's lymphoma, our results suggest a stringent and sophisticated control of Aicda by a novel IL-10/miR-155 axis, where the imbalance of IL-10 and/or miR-155 may contribute to disease pathogenesis.
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http://dx.doi.org/10.1189/jlb.2A0314-178RDOI Listing
January 2015

The role of Ets2 transcription factor in the induction of microRNA-155 (miR-155) by lipopolysaccharide and its targeting by interleukin-10.

J Biol Chem 2014 Feb 20;289(7):4316-25. Epub 2013 Dec 20.

From the School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland.

MicroRNA-155 (miR-155) is highly expressed in many cancers such as B cell lymphomas and myeloid leukemia and inflammatory disorders such as rheumatoid arthritis, atopic dermatitis, and multiple sclerosis. The role of miR-155 as both a promoter of inflammation and an oncogenic agent provides a clear need for miR-155 itself to be stringently regulated. We therefore investigated the transcriptional regulation of miR-155 in response to the respective pro- and anti-inflammatory mediators LPS and IL-10. Bioinformatic analysis revealed Ets binding sites on the miR-155 promoter, and we found that Ets2 is critical for miR-155 induction by LPS. Truncation and mutational analysis of the miR-155 promoter confirmed the role of the Ets2 binding site proximal to the transcription start site for LPS responsiveness. We observed increased binding of Ets2 to the miR-155 promoter and Ets2 deficient mice displayed decreased induction of miR-155 in response to LPS. IL-10 inhibited the induction of Ets2 mRNA and protein by LPS, thereby decreasing Ets2 function on the pri-155 promoter. We have thus identified Ets2 as a key novel regulator in both the positive and negative control of miR-155 in the inflammatory response.
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http://dx.doi.org/10.1074/jbc.M113.522730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3924294PMC
February 2014

Inosine-mediated modulation of RNA sensing by Toll-like receptor 7 (TLR7) and TLR8.

J Virol 2014 Jan 13;88(2):799-810. Epub 2013 Nov 13.

Centre for Cancer Research, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia.

RNA-specific adenosine deaminase (ADAR)-mediated adenosine-to-inosine (A-to-I) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of nonself RNA by innate immune sensors and accordingly investigated the impact of inosine-modified RNA on Toll-like receptor 7 and 8 (TLR7/8) sensing. Inosine incorporation into synthetic single-stranded RNA (ssRNA) potentiated tumor necrosis factor alpha (TNF-α) or alpha interferon (IFN-α) production in human peripheral blood mononuclear cells (PBMCs) in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs and was not seen with inosine-containing short double-stranded RNAs or with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated IFN-α production in human PBMCs through TLR7 recruitment, as established through the use of a TLR7 antagonist and Tlr7-deficient cells. There was a correlation between hyperediting of influenza A viral ssRNA and its ability to stimulate TNF-α, independent of 5'-triphosphate residues, and involving Adar-1. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus, we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate TLR7/8 sensing of phagocytosed viral RNA.
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http://dx.doi.org/10.1128/JVI.01571-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911656PMC
January 2014

miR-19a: an effective regulator of SOCS3 and enhancer of JAK-STAT signalling.

PLoS One 2013 22;8(7):e69090. Epub 2013 Jul 22.

School of Biochemistry and Immunology, Trinity College, University of Dublin, Dublin, Ireland.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069090PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3718810PMC
February 2014

Conjugated linoleic acid suppresses IRF3 activation via modulation of CD14.

J Nutr Biochem 2013 May 15;24(5):920-8. Epub 2012 Aug 15.

Immunomodulation Group, School of Biotechnology, Dublin City University, Dublin 9, Ireland.

Polyunsaturated fatty acids (PUFA) can modulate the immune response, however the mechanism by which they exert this effect remains unclear. Previous studies have clearly demonstrated that the cis-9, trans-11 isomer of conjugated linoleic acid (c9,t11-CLA), found predominantly in beef and dairy products, can modulate the response of immune cells to the toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). This study aimed to investigate further the mechanism by which these effects are mediated. Treatment of macrophages with c9,t11-CLA significantly decreased CD14 expression and partially blocked its association with lipid rafts following stimulation with LPS. Furthermore the c9,t11-CLA isomer inhibited both nuclear factor-κB (NF-κB) and IRF3 activation following TLR4 ligation while eicosapentaenoic acid (EPA) only suppressed NF-κB activation. Given that the ability of LPS to activate IRF3 downstream of TLR4 depends on internalisation of the TLR4 complex and involves CD14, we examined TLR4 endocytosis. Indeed the internalisation of TLR4 to early endosomes following activation with LPS was markedly inhibited in c9,t11-CLA treated cells. These effects were not seen with the n-3 fatty acid, EPA, which was used as a comparison. Our data demonstrates that c9,t11-CLA inhibits IRF3 activation via its effects on CD14 expression and localisation. This results in a decrease in the endocytosis of TLR4 which is necessary for IRF3 activation, revealing a novel mechanism by which this PUFA exerts its anti-inflammatory effects.
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http://dx.doi.org/10.1016/j.jnutbio.2012.06.010DOI Listing
May 2013

A miR-19 regulon that controls NF-κB signaling.

Nucleic Acids Res 2012 Sep 7;40(16):8048-58. Epub 2012 Jun 7.

Centre for Cancer Research, Monash Institute of Medical Research, Clayton, Victoria 3168, Australia.

Fine-tuning of inflammatory responses by microRNAs (miRNAs) is complex, as they can both enhance and repress expression of pro-inflammatory mediators. In this study, we investigate inflammatory responses following global miRNA depletion, to better define the overall contribution of miRNAs to inflammation. We demonstrate that miRNAs positively regulate Toll-like receptor signaling using inducible Dicer1 deletion and global miRNA depletion. We establish an important contribution of miR-19b in this effect, which potentiates nuclear factor-κB (NF-κB) activity in human and mouse cells. Positive regulation of NF-κB signaling by miR-19b involves the coordinated suppression of a regulon of negative regulators of NF-κB signaling (including A20/Tnfaip3, Rnf11, Fbxl11/Kdm2a and Zbtb16). Transfection of miR-19b mimics exacerbated the inflammatory activation of rheumatoid arthritis primary fibroblast-like synoviocytes, demonstrating its physiological importance in the pathology of this disease. This study constitutes, to our knowledge, the first description of a miR-19 regulon that controls NF-κB signaling, and suggests that targeting this miRNA and linked family members could regulate the activity of NF-κB signaling in inflammation.
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http://dx.doi.org/10.1093/nar/gks521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3439911PMC
September 2012

Characterization of the cellular action of the MSK inhibitor SB-747651A.

Biochem J 2012 Jan;441(1):347-57

MRC Protein Phosphorylation Unit, Sir James Black Complex, University of Dundee, Dundee DD1 5EH, Scotland, U.K.

MSK1 (mitogen- and stress-activated kinase 1) and MSK2 are nuclear protein kinases that regulate transcription downstream of the ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38α MAPKs (mitogen-activated protein kinases) via the phosphorylation of CREB (cAMP-response-element-binding protein) and histone H3. Previous studies on the function of MSKs have used two inhibitors, H89 and Ro 31-8220, both of which have multiple off-target effects. In the present study, we report the characterization of the in vitro and cellular properties of an improved MSK1 inhibitor, SB-747651A. In vitro, SB-747651A inhibits MSK1 with an IC50 value of 11 nM. Screening of an in vitro panel of 117 protein kinases revealed that, at 1 μM, SB-747651A inhibited four other kinases, PRK2 (double-stranded-RNA-dependent protein kinase 2), RSK1 (ribosomal S6 kinase 1), p70S6K (S6K is S6 kinase) (p70RSK) and ROCK-II (Rho-associated protein kinase 2), with a similar potency to MSK1. In cells, SB-747651A fully inhibited MSK activity at 5-10 μM. SB-747651A was found to inhibit the production of the anti-inflammatory cytokine IL-10 (interleukin-10) in wild-type, but not MSK1/2-knockout, macrophages following LPS (lipopolysaccharide) stimulation. Both SB-747651A and MSK1/2 knockout resulted in elevated pro-inflammatory cytokine production by macrophages in response to LPS. Comparison of the effects of SB-747651A, both in vitro and in cells, demonstrated that SB-747651A exhibited improved selectivity over H89 and Ro 31-8220 and therefore represents a useful tool to study MSK function in cells.
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http://dx.doi.org/10.1042/BJ20110970DOI Listing
January 2012

A role for TLR4 in Clostridium difficile infection and the recognition of surface layer proteins.

PLoS Pathog 2011 Jun 30;7(6):e1002076. Epub 2011 Jun 30.

Immunomodulation Research Group, School of Biotechnology, Dublin City University, Ireland.

Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4⁻/⁻ and Myd88⁻/⁻, but not TRIF⁻/⁻ mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.
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http://dx.doi.org/10.1371/journal.ppat.1002076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3128122PMC
June 2011

The role of miRNAs in cytokine signaling.

Authors:
Claire E McCoy

Front Biosci (Landmark Ed) 2011 Jun 1;16:2161-71. Epub 2011 Jun 1.

Monash Institute of Medical Research, Monash University, Clayton, Victoria 3168, Australia.

This review explores the relationship between cytokines and microRNAs (miRNAs). In particular, the regulation of miRNAs by pro-inflammatory cytokines, anti-inflammatory cytokines, interferons and transforming growth factor beta are examined, highlighting how miRNAs can mediate some of the known functions of these cytokines, as well as identifying novel gene targets, mechanisms and cross-talk between pathways.
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http://dx.doi.org/10.2741/3845DOI Listing
June 2011

Analysis of microRNA turnover in mammalian cells following Dicer1 ablation.

Nucleic Acids Res 2011 Jul 29;39(13):5692-703. Epub 2011 Mar 29.

Centre for Cancer Research, Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Clayton, Victoria 3168, Australia.

Although microRNAs (miRNAs) are key regulators of gene expression, little is known of their overall persistence in the cell following processing. Characterization of such persistence is key to the full appreciation of their regulatory roles. Accordingly, we measured miRNA decay rates in mouse embryonic fibroblasts following loss of Dicer1 enzymatic activity. The results confirm the inherent stability of miRNAs, the intracellular levels of which were mostly affected by cell division. Using the decay rates of a panel of six miRNAs representative of the global trend of miRNA decay, we establish a mathematical model of miRNA turnover and determine an average miRNA half-life of 119 h (i.e. ∼5 days). In addition, we demonstrate that select miRNAs turnover more rapidly than others. This study constitutes, to our knowledge, the first in-depth characterization of miRNA decay in mammalian cells. Our findings indicate that miRNAs are up to 10× more stable than messenger RNA and support the existence of novel mechanism(s) controlling selective miRNA cellular concentration and function.
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http://dx.doi.org/10.1093/nar/gkr148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3141258PMC
July 2011

MicroRNAs: the fine-tuners of Toll-like receptor signalling.

Nat Rev Immunol 2011 Mar 18;11(3):163-75. Epub 2011 Feb 18.

School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland.

Toll-like receptor (TLR) signalling must be tightly regulated to avoid excessive inflammation and to allow for tissue repair and the return to homeostasis after infection and tissue injury. MicroRNAs (miRNAs) have emerged as important controllers of TLR signalling. Several miRNAs are induced by TLR activation in innate immune cells and these and other miRNAs target the 3' untranslated regions of mRNAs encoding components of the TLR signalling system. miRNAs are also proving to be an important link between the innate and adaptive immune systems, and their dysregulation might have a role in the pathogenesis of inflammatory diseases.
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http://dx.doi.org/10.1038/nri2957DOI Listing
March 2011

IL-10 inhibits miR-155 induction by toll-like receptors.

J Biol Chem 2010 Jul 30;285(27):20492-8. Epub 2010 Apr 30.

School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland.

IL-10 is a potent anti-inflammatory cytokine that is crucial for down-regulating pro-inflammatory genes, which are induced by Toll-like receptor (TLR) signaling. In this study, we have examined whether modulation of microRNAs plays a role in the inhibitory effect of IL-10 on TLR4 signaling. Analyzing microRNAs known to be induced by TLR4, we found that IL-10 could inhibit the expression of miR-155 in response to lipopolysaccharide but had no effect on miR-21 or miR-146a. IL-10 inhibited miR-155 transcription from the BIC gene in a STAT3-dependent manner. This inhibitory effect of IL-10 on miR-155 led to an increase in the expression of the miR-155 target, SHIP1. This is the first example of IL-10 playing a role in microRNA function and suggests that through its inhibitory effect on miR-155, IL-10 has the ability to promote anti-inflammatory gene expression.
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http://dx.doi.org/10.1074/jbc.M110.102111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2898307PMC
July 2010

ERK5 regulation in naïve T-cell activation and survival.

Eur J Immunol 2008 Sep;38(9):2534-47

MRC Protein Phosphorylation Unit, College of Life Sciences, Sir James Black Centre, University of Dundee, Dundee, UK.

ERK5 has been implicated in regulating the MEF2-dependent genes Klf2 and nur77 downstream of the TCR and the maintenance of expression of CD62L on peripheral T cells. Based on this data, knockout of ERK5 would be predicted to compromise T-cell development and the maintenance of T cells in the periphery. Using an ERK5 conditional knockout, driven by CD4-CRE or Vav-CRE transgenes resulting in the loss of ERK5 in T cells, we have found that ERK5 is not required for T-cell development. In addition, normal numbers of T cells were found in the spleens and lymph nodes of these mice. We also find that TCR stimulation is not a strong signal for ERK5 activation in primary murine T cells. ERK5 was found to contribute to the induction of Klf2 but not nur77 mRNA following TCR activation. Despite the reduction in Klf2 mRNA, no effect was seen in ERK5 knockouts on either the mRNA levels for the Klf2 target genes CD62L, CCR7 and S1P, or the cell surface expression of CD62L. These results suggest that while ERK5 does contribute to Klf2 regulation in T cells, it is not essential for the expression of CD62L or T-cell survival.
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http://dx.doi.org/10.1002/eji.200737867DOI Listing
September 2008

Glucocorticoids inhibit IRF3 phosphorylation in response to Toll-like receptor-3 and -4 by targeting TBK1 activation.

J Biol Chem 2008 May 20;283(21):14277-85. Epub 2008 Mar 20.

School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland.

Phosphorylation of the transcription factor interferon regulatory factor 3 (IRF3) is essential for the induction of promoters which contain the interferon-stimulated response element (ISRE). IRF3 can be activated by Toll-like receptor 3 (TLR3) in response to the double-stranded RNA mimic poly(I-C) and by TLR4 in response to lipopolysaccharide (LPS). Here we have analyzed the effect of the glucocorticoid dexamethasone on this response. Dexamethasone inhibited the induction of the ISRE-dependent gene RANTES (regulated on activation normal T cell expressed and secreted) in both U373-CD14 cells and human peripheral blood mononuclear cells and also an ISRE luciferase construct, activated by either TLR3 or TLR4. It also inhibited increased phosphorylation of IRF3 in its N terminus in response to LPS and in its C terminus on Ser-396 in response to either poly(I-C) or LPS. Several dexamethasone-induced phosphatases were tested for possible involvement in these effects; MKP1 did not appear to be involved, although MKP2 and MKP5 both partially inhibited induction of the ISRE, pointing to their possible involvement in the effect of dexamethasone. Importantly, we found that dexamethasone could inhibit TBK1 kinase activity and TBK1 phosphorylation on Ser-172, both of which are required for IRF3 phosphorylation downstream of TLR3 and TLR4 stimulation. Our study, therefore, demonstrates that TBK1 is a target for dexamethasone, common to both TLR3 and TLR4 signaling.
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http://dx.doi.org/10.1074/jbc.M709731200DOI Listing
May 2008

The role of toll-like receptors in macrophages.

Front Biosci 2008 Jan 1;13:62-70. Epub 2008 Jan 1.

Department of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland.

Macrophages play diverse roles including but not limited to homeostatic clearance, detection of invading pathogens and induction of the adaptive immune response. Since their discovery, it has become clear that Toll-like receptors (TLRs) can play a major role in these macrophage functions. This review will describe the TLRs, their signalling pathways and their role in macrophage function.
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http://dx.doi.org/10.2741/2660DOI Listing
January 2008

Identification of novel phosphorylation sites in MSK1 by precursor ion scanning MS.

Biochem J 2007 Mar;402(3):491-501

MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.

MSK1 (mitogen- and stress-activated kinase 1) is a dual kinase domain protein that acts downstream of the ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38 MAPK (mitogen-activated protein kinase) signalling pathways in cells. MSK1, and its related isoform MSK2, phosphorylate the transcription factors CREB (cAMP-response-element-binding protein) and ATF1 (activating transcription factor 1), and the chromatin proteins histone H3 and HMGN1 (high-mobility-group nucleosomal-binding protein 1) in response to either mitogenic stimulation or cellular stress. MSK1 activity is tightly regulated in cells, and activation requires the phosphorylation of MSK1 by either ERK1/2 or p38a. This results in activation of the C-terminal kinase domain, which then phosphorylates further sites in MSK1, leading to the activation of the N-terminal kinase domain and phosphorylation of substrates. Here, we use precursor ion scanning MS to identify five previously unknown sites in MSK1: Thr630, Ser647, Ser657, Ser695 and Thr700. One of these sites, Thr700, was found to be a third site in MSK1 phosphorylated by the upstream kinases ERK1/2 and p38a. Mutation of Thr700 resulted in an increased basal activity of MSK1, but this could be further increased by stimulation with PMA or UV-C radiation. Surprisingly, however, mutation of Thr700 resulted in a dramatic loss of Thr581 phosphorylation, a site essential for activity. Mutation of Thr700 and Thr581 to an alanine residue resulted in an inactive kinase, while mutation of both sites to an aspartic acid residue resulted in a kinase with a significant basal activity that could not be further stimulated. Together these results are consistent with a mechanism by which Thr700 phosphorylation relieves the inhibition of MSK1 by a C-terminal autoinhibitory helix and helps induce a conformational shift that protects Thr581 from dephosphorylation.
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http://dx.doi.org/10.1042/BJ20061183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1863562PMC
March 2007

MSK1 activity is controlled by multiple phosphorylation sites.

Biochem J 2005 Apr;387(Pt 2):507-17

MRC Protein Phosphorylation Unit, Faculty of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.

MSK1 (mitogen- and stress-activated protein kinase) is a kinase activated in cells downstream of both the ERK1/2 (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) cascades. In the present study, we show that, in addition to being phosphorylated on Thr-581 and Ser-360 by ERK1/2 or p38, MSK1 can autophosphorylate on at least six sites: Ser-212, Ser-376, Ser-381, Ser-750, Ser-752 and Ser-758. Of these sites, the N-terminal T-loop residue Ser-212 and the 'hydrophobic motif' Ser-376 are phosphorylated by the C-terminal kinase domain of MSK1, and their phosphorylation is essential for the catalytic activity of the N-terminal kinase domain of MSK1 and therefore for the phosphorylation of MSK1 substrates in vitro. Ser-381 is also phosphorylated by the C-terminal kinase domain, and mutation of Ser-381 decreases MSK1 activity, probably through the inhibition of Ser-376 phosphorylation. Ser-750, Ser-752 and Ser-758 are phosphorylated by the N-terminal kinase domain; however, their function is not known. The activation of MSK1 in cells therefore requires the activation of the ERK1/2 or p38 MAPK cascades and does not appear to require additional signalling inputs. This is in contrast with the closely related RSK (p90 ribosomal S6 kinase) proteins, whose activity requires phosphorylation by PDK1 (3-phosphoinositide-dependent protein kinase 1) in addition to phosphorylation by ERK1/2.
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http://dx.doi.org/10.1042/BJ20041501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1134980PMC
April 2005
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