Jessica Johnston

Tribbles in inflammation

Inflammation is part of the physiological innate immune response to invading pathogens and tissue injury. However, unresolved inflammation leads to human disease. The tribbles (TRIB) family of pseudokinase proteins has been shown to modulate key inflammatory signalling pathways, including the MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase) networks. This review summarizes our current knowledge on TRIBs in the context of inflammation, both at the level of molecular mechanisms and in disease development.

Neutrophil microvesicles drive atherosclerosis by delivering miR-155 to atheroprone endothelium.

Neutrophils have been implicated in the pathogenesis of atherosclerosis, a lipid-driven disease of arteries, but they are seldom found in atherosclerotic plaques. To resolve this longstanding paradox, we investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Clinical and pre-clinical studies revealed that levels of circulating neutrophil microvesicles were enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulated at disease-prone regions of arteries that are exposed to complex flow patterns, and they promoted vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, it was demonstrated that neutrophil microvesicles promoted inflammatory gene expression by delivering a microRNA (miR-155) that enhanced NF-κB activation. Similary, neutrophil microvesicles increased miR-155 and enhanced NF-κB at disease-prone sites of disturbed flow in arteries of mice. We conclude that delivery of microvesicles carrying miR-155 to disease-prone regions of arteries provides a novel mechanism by which neutrophils contribute to vascular inflammation and atherogenesis.

Evidence for a role of TRIB3 in the regulation of megakaryocytopoiesis

Megakaryocytopoiesis is a complex differentiation process driven by the hormone thrombopoietin by which haematopoietic progenitor cells give rise to megakaryocytes, the giant bone marrow cells that in turn break down to form blood platelets. The Tribbles Pseudokinase 3 gene (TRIB3) encodes a pleiotropic protein increasingly implicated in the regulation of cellular differentiation programmes. Previous studies have hinted that TRIB3 could be also involved in megakaryocytopoiesis but its role in this process has so far not been investigated. Using cellular model systems of haematopoietic lineage differentiation here we demonstrate that TRIB3 is a negative modulator of megakaryocytopoiesis. We found that in primary cultures derived from human haematopoietic progenitor cells, thrombopoietin-induced megakaryocytic differentiation led to a time and dose-dependent decrease in TRIB3 mRNA levels. In the haematopoietic cell line UT7/mpl, silencing of TRIB3 increased basal and thrombopoietin-stimulated megakaryocyte antigen expression, as well as basal levels of ERK1/2 phosphorylation. In primary haematopoietic cell cultures, silencing of TRIB3 facilitated megakaryocyte differentiation. In contrast, over-expression of TRIB3 in these cells inhibited the differentiation process. The in-vitro identification of TRIB3 as a negative regulator of megakaryocytopoiesis suggests that in-vivo this gene could be important for the regulation of platelet production.

157 Myeloid expression of trib1 regulates the polarisation state of tissue resident macrophages that has consequences on plasma lipid and metabolic homeostasis

Introduction Genome wide association studies have identified Tribbles-1 (TRIB1) to be significantly associated with all major plasma lipid traits and as a risk factor for ischaemic heart disease and myocardial infarction. Studies in mice using Trib1 full body KO and liver-specific over-expression and KO models have shown that hepatic expression of TRIB1 reduces circulating lipids. Additionally, Trib1 has been implicated as a regulator of alternatively activated macrophages. However the potential interplay between hepatocytes, macrophages and Trib1 remain unexplored. This study aimed to assess whether myeloid Trib1 regulates tissue macrophage polarisation and investigate its consequences on plasma lipid homeostasis. Methods We developed myeloid specific Trib1 conditional knockout (Trib1 fl/fl x Lyz2Cre; Trib1KO) and over-expressor mice (ROSA26Trib1.Tg x Lyz2Cre; Trib1Tg), thereby deleting or over-expressing Trib1 in myeloid cells. Plasma lipid levels were directly measured by ion mobility. Macrophage phenotype was characterised in the liver (Kupffer cells, KCs), adipose (ATMs) and BMDMs by qPCR and semi-quantitative immunofluorescence analysis. Western blotting was used to assess regulators of macrophage polarisation. Furthermore, microarray analysis of human monocyte derived macrophages (MDMs) was employed to identify potential TRIB1-regulated cytokines. Results Loss of myeloid Trib1 increased levels of plasma triglyceride, VLDL-C (p<0.05) and promoted pro-inflammatory polarisation in KCs (p<0.01), ATMs (p<0.01) and BMDMs (p<0.05), while Trib1Tg mice revealed opposing changes in all parameters assessed. Western blotting showed TRIB1 modulates protein levels of C/EBP-β2 and –β² (p<0.05), both key regulators of macrophage polarisation, via the control of COP1 activity and miR-155 expression. Microarray analysis of MDMs indicated TRIB1 may regulate production of a number of pro-inflammatory cytokines that are implicated in fatty liver disease and adipocyte lipolysis. Reduced expression of these was confirmed in in Trib1Tg BMDMs (p<0.05). Conclusions Myeloid Trib1 is a potent regulator of lipid homeostasis, the loss of which promotes inflammation in metabolic tissues. Our observations uncover a novel mechanism of KC-hepatocyte cross talk mediated through Trib1.

TRIB1 (tribbles pseudokinase 1)

Review on TRIB1, with data on DNA, on the protein encoded, and where the gene is implicated.

165 In situ Examination of Plaque Macrophage Populations Using Multicolour Florescence Microscopy Reveals Critical Differences between Murine Models of Experimental Atherosclerosis

Macrophages play a central role in the development of atherosclerosis. They are highly plastic and heterogeneous cells that exhibit a spectrum of phenotypes, from pro-inflammatory or ‘classically activated’ to anti-inflammatory or ‘alternatively activated’. Plaque macrophages in particular have roles in the pathogenesis of lesion stability and rupture, the most common cause of cardiac associated mortality. Despite considerable work aiming to characterise these phagocytic cells in disease, our knowledge of their complexity in vivo is only partial. Here we wished to characterise plaque macrophages from two of the most common murine models; ApoE-/- (Apolipoprotein E null) and LDLR-/- (low density lipoprotein receptor null) and a relatively new and less characterised model; PCKS9-AAV8 (Adeno-associated virus serotype 8-proprotein convertase subtilisin kexin type 9) induced hyperlipidaemia. Using immunohistochemistry, plaques from high-fat diet fed ApoE-/-, LDLR-/- and PCSK9-AAV8 mice were simultaneously stained for the pan macrophage marker Mac-3 and the pro-/ anti- inflammatory markers inducible nitric oxide synthase (iNOS) and Arginase I (ArgI). Plaques were imaged using fluorescence microscopy and analysed by ImageJ. Individual Mac-3+ cells were selected as the region of interest and corresponding iNOS and ArgI staining was quantified. The analysis allowed consideration for the spectrum of marker co-expression and characterisation of individual cells based on staining intensity. Using this approach, complex populations of plaque macrophagesincluding single+ ArgI, double positive, double negative and single+ iNOS were quantified. To understand the roles of these populations in atherosclerosis further, we correlated macrophage quality and quantities with lesion size and collagen content. We show that ApoE-/- plaque macrophages are significantly more pro-inflammatory than LDLR-/- and PCSK9 plaque macrophages (p < 0.05 and p < 0.0001). The population responsible for the pro-inflammatory phenotype of ApoE-/- macrophages were single+ iNOS cells (p < 0.0001). We also show that the abundance of these cells significantly correlates (R2=0.4791, p = 0.0183) with lesion size in the aortic sinus. In addition, the frequency of double negative macrophages correlated with lesion collagen content (R2=0.4451, p = 0.0178). For the first time, plaque macrophages from three murine atherosclerosis models have been comprehensively characterised using a multi-colour image analysis strategy and suggest that plaque macrophages from ApoE-/- mice are significantly more pro-inflammatory than LDLR-/- and PCSK9 macrophages. We show that single iNOS+ cells may have a role in promoting lesion formation and double negative cells may also have a role in lesion stability. We envisage our platform provides a novel tool to gain a further, in-depth understanding of macrophage phenotype in atherosclerosis and will use it to elucidate the action of modulators of macrophage polarisation in vivo.

223 MIRNA202 is a Novel Regulator of Tribbles-1 Expression

Introduction Tribbles-1 (trib-1) pseudokinase is a regulatory protein that has been shown to be a protective gene in a number of cell types and processes, relevant to the development of atherosclerosis. These include inhibition of vascular smooth muscle cell proliferation, polarisation of macrophages towards an alternatively activated phenotype and lowering the production and release of LDL in hepatic tissues. Therefore, understanding the molecular mechanisms, which regulate trib-1 expression are of significant interest. Our group have identified miRNA202 as a controller of trib-1 levels. Rationale Trib-1 mRNA is highly unstable with a half-life of less than 1 h; the 3’ UTR encodes for a number of putative binding sites for miRNAs, including miRNA202. This study aimed to experimentally validate the importance of miRNA202 in the control of trib-1 expression. Methods 1. We have used a luciferase reporter to demonstrate the role of trib-1 3’UTR in mRNA stability and to characterise the impact of miR202 on trib-1 3’UTR in hepatic cells (HepG2). 2. qRT-PCR was used to quantify endogenous trib-1 and miRNA202 levels upon stimulation of IL-1 and in high glucose media. 3. Western blotting was used to elucidate the effect of miRNA202 on trib-1 protein levels. Results 1. Overexpression of miR202 reduced luciferase – trib1-3’UTR reporter expression. 2. The endogenous trb-1 mRNA was also modulated by miR202. Furthermore, stimulation of IL-1 in HepG2 cells has shown trb-1 levels to reduce by >70% and miR202 levels to increase by 2 fold. 3. Trib-1 protein levels have shown a reduction upon overexpression of miRNA202. Conversely, miRNA202 inhibitor increased trib-1 protein levels. Discussion miR202 is a novel regulator of trib-1 expression and may represent a target by which trib-1 levels could be raised in vivo, thereby providing a mechanism to augment the anti-atherosclerotic effects of this protein.

186 Assessment of Plaque Macrophage Phenotype in situ by Multicolour Fluorescence Microscopy

Background Macrophages are a heterogeneous and diverse population of cells with an important role in immunity and also in the development of diseases including atherosclerosis. Macrophages exhibit a spectrum of phenotypes, from pro-inflammatory or M1/ ‘classically activated’ to anti-inflammatory or M2/ ‘alternatively activated’ cells with wound healing properties. These cells are highly plastic and can switch from one phenotype to another depending on environmental cues. Despite considerable work aiming to characterise the polarisation of macrophages in disease, there have been few studies that have elucidated macrophage phenotype in situ and our knowledge of their complexity in vivo is only partial. Here we wished to establish a robust analysis pipeline to assess macrophage phenotype in aortic root plaques of high-fat diet fed mice. This approach will provide a unique insight into macrophage phenotype within the plaque and will advance our understanding of plaque development and phenotypic switching of macrophages in vivo. Methods Using multicolour immunohistochemistry, formalin-fixed paraffin embedded (FFPE) aortic root sections from high-fat diet fed (ApoE–/– and LDLR–/–) mice were simultaneously stained for the pan macrophage marker Mac-3 (CD107b), iNOS (M1 marker) and Arginase I (M2 marker) in situ, respectively. Plaques from aortic roots were imaged using multi-colour fluorescence microscopy (Leica AF6000). The resulting images were analysed by Image J. Briefly, individual Mac-3 positive cells were selected as the region of interest (ROI) and corresponding iNOS and ArgI positive staining was evaluated. The analysis allowed consideration for the spectrum of M1/M2 associated marker expression and enabled characterisation of individual cells based on staining intensity. Using this approach it is possible to reveal and quantify complex populations of plaque macrophages in situ. Results We have successfully optimised a simultaneous staining protocol for a pan macrophage marker and M1/M2 associated markers and quantified differential expression of these markers in individual macrophages. We have created a robust, semi-automated pipeline to analyse plaque macrophage phenotype andvalidated this approach by comparing macrophage phenotypes in LDLR-/- vs. ApoE-/- models of experimental atherosclerosis. Conclusions/implications We envisage that our platform provides a novel tool to gain an in-depth understanding of macrophage phenotype in atherosclerosis and we will use this to elucidate the action of modulators of macrophage polarisation in vivo.