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The formation of pathological bone deposits within soft tissues, termed heterotopic ossification (HO), is common after trauma. However, the severity of HO formation varies substantially between individuals, from relatively isolated small bone islands through to extensive soft tissue replacement by bone giving rise to debilitating symptoms. The aim of this study was to identify novel candidate therapeutic molecular targets for severe HO. We conducted a genome-wide scan in men and women with HO of varying severity following hip replacement for osteoarthritis. HO severity was dichotomized as mild or severe, and association analysis was performed with adjustment for age and sex. We next confirmed expression of the gene encoded by the lead signal in human bone and in primary human mesenchymal stem cells. We then examined the effect of gene knockout in a murine model of osseous trans-differentiation, and finally we explored transcription factor phosphorylation in key pathways perturbed by the gene. Ten independent signals were suggestively associated with HO severity, with

Molecular mechanisms that regulate tumor-associated macrophage (TAM) phenotype and function are incompletely understood. The pseudokinase TRIB1 has been reported as a regulator of macrophage phenotypes, both in mouse and human systems.

Simple Summary Tribbles proteins play various roles in cancer initiation and progression. However, still little is known about their molecular actions. Here we developed a mass spectrometry-based approach to study the Tribbles interactomes, allowing us to discover new interactors and functions that might help to understand their behavior better. Our proteomics data highlight the ability of TRIB3 to interact with transcription regulatory proteins and point to a new role in gene repression. Systematic analyses like these will help to evaluate the potential of Tribbles proteins as biomarkers for disease diagnosis and prognosis. Abstract The three human Tribbles (TRIB) pseudokinases have been implicated in a plethora of signaling and metabolic processes linked to cancer initiation and progression and can potentially be used as biomarkers of disease and prognosis. While their modes of action reported so far center around protein–protein interactions, the comprehensive profiling of TRIB interactomes has not been reported yet. Here, we have developed a robust mass spectrometry (MS)-based proteomics approach to characterize Tribbles’ interactomes and report a comprehensive assessment and comparison of the TRIB1, -2 and -3 interactomes, as well as domain-specific interactions for TRIB3. Interestingly, TRIB3, which is predominantly localized in the nucleus, interacts with multiple transcriptional regulators, including proteins involved in gene repression. Indeed, we found that TRIB3 repressed gene transcription when tethered to DNA in breast cancer cells. Taken together, our comprehensive proteomic assessment reveals previously unknown interacting partners and functions of Tribbles proteins that expand our understanding of this family of proteins. In addition, our findings show that MS-based proteomics provides a powerful tool to unravel novel pseudokinase biology.

SummaryMolecular mechanisms that regulate tumour-associated macrophage (TAM) phenotype and function are incompletely understood. Here, we show that the pseudokinase TRIB1 is highly expressed by TAMs in breast cancer and that its expression correlates with response to chemotherapy and patient survival. We used immune-competent murine models of breast cancer to characterise the consequences of altered (reduced or elevated) myeloid Trib1 expression on tumour growth and composition of stromal immune cells. We found that both overexpression and knockout of myeloid Trib1 promote tumour growth, albeit through distinct molecular mechanisms. Myeloid Trib1 deficiency resulted in an early accelearation of tumour growth, paired with a selective reduction in perivascular macrophage numbers in vivo and enhanced oncogenic cytokine expression in vitro. In contrast, elevated levels of Trib1 in myeloid cells led to an increase in mammary tumour volume at late stages, together with a reduction of NOS2 expressing macrophages and an overall reduction of these cells in hypoxic tumour regions. In addition, we show that myeloid Trib1 is a previously unknown, negative regulator of the anti-tumour cytokine IL-15 and that increased expression of myeloid Trib1 leads to reduced IL-15 levels in mammary tumours, with a consequent reduction in the number of T-cells, that are key to anti-tumour immune responses.Together, these results define the different roles of TRIB1 in human breast cancer and provide a mechanistic understanding for the importance of myeloid TRIB1 expression levels in the development of this disease.SignificanceTRIB1 expression is strongly associated with response to chemotherapy in breast cancer patients with aggressive tumours. This protein is also highly expressed by tumour-associated macrophages. Thus, we used myeloid-specific alterations of Trib1 expression in mice (Trib1mKO and Trib1mTg), and characterised consequent changes in the growth rate and tumour microenvironment of mammary tumours. Both Trib1mKO and Trib1mTg enhanced tumour growth, but at different stages of tumour growth and via distinct mechanisms. Trib1mKO significantly increased the expression of oncogenic cytokines, such as IL6, IL10, CCL20, PD-L1, and VEGF. In contrast, Trib1mTg accelerated at the later stage of tumour growth via inhibition of hypoxic TAMs in the TME, as well as by reduced IL-15 expression thus leading to impaired naïve and cytotoxic T cell infiltration. These data define TRIB1 as a potential novel marker of therapeutic responses in breast cancer, as well as a key mechanistic regulator of the anti-tumour cytokine, IL-15 in myeloid cells.

Introduction Ischemic stroke is commonly caused by large artery atherosclerosis. Patients with a high atherosclerotic burden (stenosis) and inflamed or ulcerated plaque are at increased risk of early recurrent ischaemic events. Treatment with anti-inflammatory agents may therefore reduce stroke incidence and recurrence in patients with this condition, but in vitro human studies of the possible mechanisms are lacking. We hypothesised that an anti-inflammatory compound (denoted drug X*) alters the expression of macrophage specific genes, including scavenger receptors in in vitro models of human carotid plaque macrophages. Methods CD14+ monocytes were collected from whole blood donations (volunteers) and differentiated to monocyte derived macrophages (MDMs). Macrophage polarisation with M1 (100 ng/ml LPS + 20 ng/ml INF-γ) and M2a (20 ng/ml IL-4) markers was performed in complete media supplemented with 3 different clinically relevant concentrations of drug X. Gene expression (RT-qPCR) analysis (n=10) was carried out in the differentiated hMDMs to test for macrophage specific genes and scavenger receptors. Western Blot analysis (n=7) and immunofluorescence stain (IF) (n=5) was performed on drug X- treated MDMs to validate changes in the expression of Oxidized Low Density Lipoprotein Receptor 1 (OLR1). The physiological effect of drug X was also tested on oxLDL (25 μg/ml) uptake by macrophages (n=5). Results Our data suggest that drug X may re-program macrophages to a less inflammatory state (an M2 state) by significantly altering Mannose Receptor C-type 1 (MRC1) gene expression (p:0.0183). Drug X also alters the expression level of Macrophage Scavenger Receptor 1 (MSR1) gene, provoking the pacification of the M1 state and resulting in a beneficial effect on LDL uptake (p: 0.0845). Using OLR1 gene expression as a readout, in both M1 and M2a macrophages, the average OLR1 expression was reduced by treatment with drug X (p: 0.0003). This implies that M1 macrophages can bind less oxLDL which could reduce the formation of atherosclerotic plaque in the vessel wall. OLR1 protein expression was also reduced (p:0.0003) following 24-hour exposure of the macrophages to drug X. In functional tests, oxLDL uptake by drug X treated hMDMs was reduced by 44% (p:0.003). Conclusions The gene expression of macrophage specific genes and scavenger receptors in human M1 and M2a macrophages was significantly altered following 24-hour exposure to clinically relevant concentrations of drug X in vitro. Drug X reduced ‘active’ and pathogenic cell behaviours e.g., lipid uptake and promoted the polarisation to the M2 state. These data demonstrate that human macrophages can be reprogrammed to a less pathogenic state in vitro and they provide a potential mechanism for the effectiveness of drug X in the stabilisation of carotid atherosclerotic plaques in humans. * Drug X is used for ongoing IP/patent assessment Conflict of Interest No

The pseudokinase TRIB1 controls cell function in a range of contexts, by regulating MAP kinase activation and mediating protein degradation via the COP1 ubiquitin ligase. TRIB1 regulates polarization of macrophages and dysregulated Trib1 expression in murine models has been shown to alter atherosclerosis burden and adipose homeostasis. Recently, TRIB1 has also been implicated in the pathogenesis of prostate cancer, where it is often overexpressed, even in the absence of genetic amplification. Well described TRIB1 effectors include MAP kinases and C/EBP transcription factors, both in immune cells and in carcinogenesis. However, the mechanisms that regulate TRIB1 itself remain elusive. Here, we show that the long and conserved 3’untranslated region (3’UTR) of TRIB1 is targeted by miRNAs in macrophage and prostate cancer models. By using a systematic in silico analysis, we identified multiple “high confidence” miRNAs potentially binding to the 3’UTR of TRIB1 and report that miR-101-3p and miR-132-3p are direct regulators of TRIB1 expression and function. Binding of miR-101-3p and miR-132-3p to the 3’UTR of TRIB1 mRNA leads to an increased transcription and secretion of interleukin-8. Our data demonstrate that modulation of TRIB1 by miRNAs alters the inflammatory profile of both human macrophages and prostate cancer cells.

© 2020 by the authors., Prostate cancer is the most frequent malignancy in European men and the second worldwide. One of the major oncogenic events in this disease includes amplification of the transcription factor cMYC. Amplification of this oncogene in chromosome 8q24 occurs concomitantly with the copy number increase in a subset of neighboring genes and regulatory elements, but their contribution to disease pathogenesis is poorly understood. Here we show that TRIB1 is among the most robustly upregulated coding genes within the 8q24 amplicon in prostate cancer. Moreover, we demonstrate that TRIB1 amplification and overexpression are frequent in this tumor type. Importantly, we find that, parallel to its amplification, TRIB1 transcription is controlled by cMYC. Mouse modeling and functional analysis revealed that aberrant TRIB1 expression is causal to prostate cancer pathogenesis. In sum, we provide unprecedented evidence for the regulation and function of TRIB1 in prostate cancer., We are grateful to the Carracedo lab for valuable input, to Ana M. Aransay and F. Elortza for technical advice. P.S. was funded by the European Training Networks Project (H2020-MSCA-ITN-308 2016 721532). V.T. is funded by Fundación Vasca de Innovación e Investigación Sanitarias, BIOEF (BIO15/CA/052), the AECC J.P. Bizkaia, the Basque Department of Health (2016111109) and the MICINN RTI2018-097267-B-I00. A. Macchia was funded by a FPI predoctoral fellowship from MICINN (PRE2018-083607). EB is funded by the MICINN (BFU2016-76872-R (FEDER/EU), PID2019-108112RB-I00, Severo Ochoa Excellence Accreditation SEV-2016-0644 and Excellence Networks SAF2017-90794-REDT). The work of A. Carracedo is supported by the Basque Department of Industry, Tourism and Trade (Elkartek), the department of education (IKERTALDE IT1106-16) and health (RIS3), the BBVA foundation and the MICINN (SAF2016-79381-R (FEDER/EU; PID2019-108787RB-I00) Severo Ochoa Excellence Accreditation SEV-2016-0644 and Excellence Networks SAF2016-81975-REDT and RED2018-102769-T), European Training Networks Project (H2020-MSCA-ITN-308 2016 721532), the AECC (IDEAS175CARR, GCTRA18006CARR), La Caixa Foundation (ID 100010434), under the agreement LCF/PR/HR17/ and the European Research Council (Starting Grant 336343, Consolidator Grant 819242). CIBERONC was co-funded with FEDER funds and funded by ISCIII.

Mesenchymal stem cells (MSC) are emerging as an effective therapeutic tool in treating canine osteoarthritis (OA). In this report, we focused on the questions of whether MSC transplantation has long-term beneficial effects for the improvement in motion and also evaluated the safety of MSC injection. Visceral adipose tissue, a surgical waste obtained during routine ovariectomy served as a source of allogeneic MSCs and used to treat OA. Altogether, fifty-eight dogs were transplanted in the study suffering from OA in the elbow (42 animals), hip (5), knee (8), ankle (2), and hock (1). The effect of MSC transplantation was evaluated by the degree of lameness at a 4-5-years follow-up period based on the owners' subjective observations. The results showed that 83% of the OA patients improved or retained improvement in lameness. Clinical safety of the treatment was assessed by evaluating the coincidence of tumors or other diseases and other adverse reactions (such as local inflammation) after MSC cell therapy. Two incidences of local inflammation for

Background and aims\ud \ud Lipid-rich foam cell macrophages drive atherosclerosis via several mechanisms, including inflammation, lipid uptake, lipid deposition and plaque vulnerability. The atheroma environment shapes macrophage function and phenotype; anti-inflammatory macrophages improve plaque stability while pro-inflammatory macrophages promote rupture. Current evidence suggests a variety of macrophage phenotypes occur in atherosclerotic plaques with local lipids, cytokines, oxidised phospholipids and pathogenic stimuli altering their phenotype. In this study, we addressed differential functioning of macrophage phenotypes via a systematic analysis of in vitro polarised, human monocyte-derived macrophage phenotypes, focussing on molecular events that regulate foam-cell formation.\ud \ud \ud \ud Methods\ud \ud We examined transcriptomes, protein levels and functionally determined lipid handling and foam cell formation capacity in macrophages polarised with IFNγ+LPS, IL–4, IL–10, oxPAPC and CXCL4.\ud \ud \ud \ud Results\ud \ud RNA sequencing of differentially polarised macrophages revealed distinct gene expression changes, with enrichment in atherosclerosis and lipid-associated pathways. Analysis of lipid processing activity showed IL–4 and IL–10 macrophages have higher lipid uptake and foam cell formation activities, while inflammatory and oxPAPC macrophages displayed lower foam cell formation. Inflammatory macrophages showed low lipid uptake, while higher lipid uptake in oxPAPC macrophages was matched by increased lipid efflux capacity.\ud \ud \ud \ud Conclusions\ud \ud Atherosclerosis-associated macrophage polarisation dramatically affects lipid handling capacity underpinned by major transcriptomic changes and altered protein levels in lipid-handling gene expression. This leads to phenotype-specific differences in LDL uptake, cellular cholesterol levels and cholesterol efflux, informing how the plaque environment influences atherosclerosis progression by influencing macrophage phenotypes.

Neutrophils are implicated in the pathogenesis of atherosclerosis but are seldom detected in atherosclerotic plaques. We investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Here we report that levels of circulating neutrophil microvesicles are enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulate at disease-prone regions of arteries exposed to disturbed flow patterns, and promote vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, we demonstrate that neutrophil microvesicles promote inflammatory gene expression by delivering miR-155, enhancing NF-κB activation. Similarly, neutrophil microvesicles increase miR-155 and enhance NF-κB at disease-prone sites of disturbed flow in vivo. Enhancement of atherosclerotic plaque formation and increase in macrophage content by neutrophil microvesicles is dependent on miR-155. We conclude that neutrophils contribute to vascular inflammation and atherogenesis through delivery of microvesicles carrying miR-155 to disease-prone regions., The role of neutrophils in the development of atherosclerosis has long been an enigma, with few neutrophils detected within the plaque. Here, the authors show that microvesicles released from neutrophils increase vascular inflammation and enhance atherosclerotic plaque formation through delivery of miR-155.

The presence of cytosolic double-stranded DNA molecules can trigger multiple innate immune signalling pathways which converge on the activation of an ER-resident innate immune adaptor named “STimulator of INterferon Genes (STING)”. STING has been found to mediate type I interferon response downstream of cyclic dinucleotides and a number of DNA and RNA inducing signalling pathway. In addition to its physiological function, a rapidly increasing body of literature highlights the role for STING in human disease where variants of the STING proteins, as well as dysregulated STING signalling, have been implicated in a number of inflammatory diseases. This review will summarise the recent structural and functional findings of STING, and discuss how STING research has promoted the development of novel therapeutic approaches and experimental tools to improve treatment of tumour and autoimmune diseases.

Heterotopic ossification (HO) is bone formation that occurs after trauma within tissues that do not normally have the property of ossification, resulting in pain and disability. The genetic architecture of HO remains unclear. In the first genome-wide association studies of this disease, we identify the human-only long non-coding RNA-encoding gene CASC20 as a robust, replicating susceptibility locus for HO and KIF26B as a potential severity locus. We find that both CASC20 and KIF26B are expressed in human bone. Both CASC20 and KIF26B expression is upregulated upon BMP2 induced osteogenic differentiation in primary human mesenchymal stem cells, followed by RUNX2 and OSTERIX upregulation and mineralised nodule formation. A CRISPR-Cas9 mediated knockout of Kif26b inhibits BMP2-induced Runx2, Sp7/Osterix, Col1A1, Alp, and Bglap/Osteocalcin expression in a murine myocyte model of osteogenic trans-differentiation, and prevents mineralised nodule formation. These studies provide the first insights into the heritable biology of common, complex HO.

Trib1 controls atherosclerotic plaque macrophage function by up-regulating OLR1, promoting foam cell formation and atherosclerosis., Macrophages drive atherosclerotic plaque progression and rupture; hence, attenuating their atherosclerosis-inducing properties holds promise for reducing coronary heart disease (CHD). Recent studies in mouse models have demonstrated that Tribbles 1 (Trib1) regulates macrophage phenotype and shows that Trib1 deficiency increases plasma cholesterol and triglyceride levels, suggesting that reduced TRIB1 expression mediates the strong genetic association between the TRIB1 locus and increased CHD risk in man. However, we report here that myeloid-specific Trib1 (mTrib1) deficiency reduces early atheroma formation and that mTrib1 transgene expression increases atherogenesis. Mechanistically, mTrib1 increased macrophage lipid accumulation and the expression of a critical receptor (OLR1), promoting oxidized low-density lipoprotein uptake and the formation of lipid-laden foam cells. As TRIB1 and OLR1 RNA levels were also strongly correlated in human macrophages, we suggest that a conserved, TRIB1-mediated mechanism drives foam cell formation in atherosclerotic plaque and that inhibiting mTRIB1 could be used therapeutically to reduce CHD.

Introduction MiR-101-3p has been implicated in the regulation of macrophage cholesterol efflux and pro-inflammatory genes by directly targeting ABCA1 and MKP-1, which are both downregulated in atherosclerosis. By using bioinformatic analysis we identified TRIB1 as a direct target of miR-101. The TRIB1 gene plays major roles in myeloid cells, regulating inflammation and lipid metabolism. Its deficiency is associated with a severe reduction of anti-inflammatory tissue macrophages (M2-like cells) and an increase in plasma triglycerides. Here we aimed to uncover the role of miR-101-3p/TRIB1 interaction and its consequences on human macrophage immunometabolism. Methods MiRanda target prediction algorithm was used to identify macrophage-specific miRNAs targeting TRIB1, along with additional web-based tools. MiR-101-3p was selected based on its conservation among species as well as its high predictive score and free energy. A luciferase reporter assay was used to validate the miR-101-3p/TRIB1 interaction, employing a miR-101 mimic and inhibitor. Gene expression (RT-qPCR) and protein analysis (Western Blot) was carried out in human monocyte-derived macrophages (hMDMs) following transient manipulation of TRIB1 and miR-101 levels. Results miR-101-3p has a functional binding site for the 3’UTR of TRIB1 and when overexpressed in hMDMs is able to reduce TRIB1 expression at both mRNA and protein levels. Over-expression of TRIB1 in hMDMs alters the levels of pro-inflammatory genes and scavenger receptors, inducing an M2-like phenotype. Conversely, overexpression of miR-101 is associated with a pro-inflammatory phenotype (M1-like). Conclusion miR-101-3p offers a potential target to simultaneously enhance the expression of ABCA1, MKP-1 and TRIB1, thus improving macrophage lipid metabolism and opposing inflammation, thereby attenuating atherosclerosis development. Conflict of interest No

Introduction Atherosclerosis is a complex inflammatory disease in which major arteries narrow, and an atherosclerotic plaque develops, modulated by genetic and environmental factors. Macrophages participate in all stages of the plaque formation and progression. Different macrophage subtypes within carotid plaques have been shown to be altered in unstable vs. stable plaques. M1 proinflammatory macrophages are mostly abundant in symptomatic and unstable plaques. In contrast, M2 macrophages are associated with regulatory and wound-healing properties and generally found in stable lesions. In the future, the characterisation of these changes in situ in the carotid plaques, associated with in vitro macrophage studies, and blood composition may lead to the identification of diagnostic markers to direct the best treatment for stroke patients. Methods Carotid plaques were obtained from patients with a recent stroke or TIA and with a greater than 50% stenosis of the internal carotid artery. Plaques were removed during carotid endarterectomy and fixed in 10% (v/v) neutral buffered formalin for maximum 36h. Macrophage phenotypes within the shoulder regions were characterised with immunofluorescence microscopy. A novel immunofluorescence staining, image acquisition and analysis technique was developed to identify single M1 (CD68+CD86+MRC1-), single M2 (CD68+CD86-MRC1+), double positive (CD68+CD86+MRC1+) and double negative (CD68+CD86-MRC1-) macrophages in the human carotid atherosclerotic plaques. Results 20 carotid plaques were collected from patients with recent stroke or TIA (73% male, 70% smokers, 87% hypertensive). In 17 plaques the shoulder region (either side of a thinned fibrous cap) was sufficiently intact for histological analysis. Dense infiltration with macrophages was seen in all 17 specimens. The population comprised double positive (CD86+MRC1+) (71%), M1 single positive (CD86+MRC1-) (19%), and M2 single positive (CD86-MRC1+) (4%). Double negative (CD86-MRC1-) macrophages represented 6% of all macrophages. Amongst 10 unstable plaques (defined as AHA Grade 6 or above) the majority (80%) of shoulder macrophages were M1 single positive. In contrast, amongst 7 stable plaques, the majority of plaque shoulder macrophages (65%) were M2 single positive. Conclusions Macrophages within symptomatic carotid plaques can express both M1 (CD86) and M2 (MRC1) markers, or can express neither. Pro-inflammatory macrophage phenotypes cluster in areas of unstable carotid plaque. Larger studies are needed to confirm the role of dual staining macrophages and double negative macrophages. If such in situ macrophages can be manipulated towards one phenotype with pharmaceutical interventions, this could offer a potential new approach to plaque stabilising therapies. Conflict of interest No

The Tribbles (TRIB) family of pseudokinase proteins has been shown to play key roles in cell cycle, metabolic diseases, chronic inflammatory disease, and cancer development. A better understanding of the mechanisms of TRIB pseudokinases could provide new insights for disease development and help promote TRIB proteins as novel therapeutic targets for drug discovery. At the 2nd International Symposium on Tribbles and Diseases held on May 7-9, 2018 in Beijing, China, a group of leading Tribbles scientists reported their findings and ongoing studies about the effects of the different TRIB proteins in the areas of immunity, metabolism, fundamental cell biology and cancer. Here, we summarize important and insightful overviews from 4 keynote lectures, 13 plenary lectures and 8 short talks that took place during this meeting. These findings may offer new insights for the understanding of the roles of TRIB pseudokinases in the development of various diseases. (C) 2019 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V. Dr. Zhuowei Hu' s work was supported by National Key R&D Program of China (Grant No. 2017YFA0205400, China), the National Natural Science Foundation of China (Grant Nos. 81530093 and 81773781, China), Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (Grant No. 2016-12M-1-007, China) and CAMS Central Public-interest Scientific Institution Basic Research Fund (Grant No. 2017PT3104, China); Dr. Bing Cui' s work was supported by grants of the National Natural Science Foundation of China (Grant No. 81874316, China), the CAMS Innovation Fund for Medical Sciences (Grant No. 2016-I2M-3-008, China); Dr. Patrick A. Eyers' s work was supported by grants of from the BBSRC and NWCR (Grant Nos. 1088 and 1097, UK); Dr. Leonard L. Dobens' s work was supported by grants of NSF (Grant No. IOS-1456023, USA), NIH (Grant No. NIH R21 CA197317, USA); Dr. Nguan Soon Tan' s work was supported by grants of Ministry of Education, Singapore (Grant Nos. MOE2014-T2-1-012 and 2012-T1-001-036, Singapore); Dr. Peter D. Mace' s work was supported by grants from the Health Research Council of New Zealand, and additionally supported by a Rutherford Discovery Fellowship from the New Zealand government administered by the Royal Society of New Zealand; Dr. Wolfgang Link' s work was supported by Fundacao para a Ciencia e a Tecnologia (FCT) Research Center Grant UID/KM/04773/2013 Centre for Biomedical Research 1334, by a research grant from Liga Portuguesa Contra o Cancro NUcleo Regional do Sul (LPCC/NRS, Portugal), Terry Fox, by a FCT 2014 research grant SFRH/BPD/100434/2014 and a ProRegem grant PD/BD/114258/2016 (Portugal); Dr. Endre Kiss-Toth' s work was supported by European Marie Sklodowska Curie ITN Project TRAIN-TRIBBLES Research and Innovation Network (Grant No. 721532, EU)and Innovation Network and the British Heart Foundation (PG/16/44/32146, UK); Dr. Karen Keeshan' s work was supported by grants from The Howat Foundation Ltd. (UK), Children with Cancer UK, Bloodwise and the Friends of Paul O' Gorman (UK); Dr. Takuro Nakamura' s work was supported by grants of P-CREATE from Japan Agency for Medical Research and Development; Dr. Warren S. Pear' s work was supported by grants from the NIH (NIAID, USA), Alex' s Lemonade Stand Foundation (USA) and the Samuel Waxman Cancer Research Foundation (USA); Yodit Feseha is supported by European Marie Sklodowska Curie ITN Project TRAIN-TRIBBLES Research and Innovation Network (Grant No. 721532, EU). Yodit Feseha' s work is conducted in the context of the "Fondation Centaure" (RTRS), which supports a French transplantation research network, the 1HU-Cesti project, the DHU Oncogreffe and the LabEX IGO thanks to French government financial support managed by the National Research Agency via the "Investment into the Future" program (Grant Nos. ANR-10-IBHU-005 and ANR-11-LABX-0016-01, France). The IHU-Cesti project is also supported by Nantes Metropole and Region Pays de la Loire (France); Dr. Jessica Johnston's work was supported by grants of the British Heart Foundation (PG/16/44/32146, UK); Dr. Arkatiz Carracedo' s work was supported by European Marie Sklodowska Curie ITN Project TRAIN-TRIBBLES Research and Innovation Network (Grant No. 721532, EU); Dr. Marcel Scheideler' s work was supported by European Marie Sklodowska Curie ITN Project TRAIN-TRIBBLES Research and Innovation Network (Grant No. 721532, EU); Dr. Zabran llyas' s work was supported by a joint Ph.D studentship beween the A* Star Institute and the University of Sheffield (UK); Dr. Robert C. Bauer s work was supported by funding from the National Institutes of Health National Heart, Lung, and Blood Institute (R01HL141745, USA); Dominika Grzesik' s work was supported by European Marie Sklodowska Curie ITN Project TRAIN-TRIBBLES Research and Innovation Network (Grant No. 721532, EU); Juan Salamanca-Viloria's work was supported by European Marie Sklodowska Curie ITN Project TRAIN-TRIBBLES Research and Innovation Network (Grant No. 721532, EU); Dr. Xiaoxi Lv' s work was supported by the National Natural Science Foundation of China (Grant No. 81503128, China) and CAMS Innovation Fund for Medical Sciences (Grant No. 201642M-1-008, China); Dr. Yishi Jin' s work was supported by National Institute of Health (NS R01-035546, USA); Dr. Ke Li' s work was supported by the National Natural Science Foundation of China (Grant No. 81400140, China), and CAMS Innovation Fund for Medical Sciences (Grant No. 201642M-1-011, China); Dr. Guillermo Velasco' s work was supported by European Marie Sklodowska Curie ITN Project TRAIN-TRIBBLES Research and Innovation Network (Grant No. 721532, EU); Dr. Jose M. Lizcano's work was supported by Spanish Ministry of Economy and Competitiveness (MINECO) and Fondo Europeo de desarrollo Regional (FEDER) (Grant No. INNPACTO/IPT-2012-0614-010000, Spain); Dr. Xiaowei Zhang' s work was supported by the National Natural Science Foundation of China (Grant Nos. 81400286 and 81530093, China), and the CAMS Innovation Fund for Medical Sciences (Grant No. 201642M-1-010, China); Dr. Fang Hua' s work was supported by the National Natural Science Foundation of China (Grant Nos. 81472717 and 81673474, China), Beijing Natural Science Foundation (Grant No. 7162133, China), and the CAMS Innovation Fund for Medical Sciences (Grant No. 201642M-1-007, China); Dr. Jiaojiao Yu' s work was supported by the National Natural Science Foundation of China (Grant No. 81703564, China); Dr. Jinmei Yu' s work was supported by the National Natural Science Foundation of China (Grant No. 81603129, China). In addition, the Meeting Organizing Committee was greatly thankful to PerkinElmer and Innovent Biologics for their generous financial supports, as well as Dr. Zhuowei Hu' s laboratory members for their assistance in preparing and organizing the conference.

Background: Proinflammatory signaling by toll-like receptors (TLRs) likely contributes to biologic responses to wear particles causing aseptic loosening. We recently reported associations with aseptic loosening in patients with polymorphisms in the locus encoding an adapter protein specific for TLR-2 and TLR-4 known as toll/interleukin-1 receptor domain-containing adapter protein/MyD88 adapter-like (TIRAP/Mal). To directly examine the contribution of TIRAP/Mal, we tested the hypothesis that TIRAP/Mal deficiency reduces the activity of wear particles. Signaling by TLR-2 and TLR-4 through TIRAP/Mal can be activated by bacterial pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide or endogenous alarmins. To distinguish between those possibilities, we tested the hypothesis that the effects of TIRAP/Mal depend on the adherence of bacterial PAMPs to the particles. Methods: In vitro mRNA levels and secretion of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 were measured after incubating wild-type and TIRAP/Mal−/− macrophages in the presence or absence of titanium particles with adherent bacterial debris, so-called endotoxin-free particles, or particles with adherent lipopolysaccharide. In vivo osteolysis was measured after implanting titanium particles on the calvaria of wild-type and TIRAP/Mal−/− mice. Results: TIRAP/Mal deficiency significantly inhibited the activity of titanium particles with adherent bacterial debris to stimulate in vivo osteolysis and in vitro cytokine mRNAs and secretion. Those effects are dependent on adherent PAMPs because removal of >99% of the adherent bacterial debris from the particles significantly reduced their activity and the remaining activity was not dependent on TIRAP/Mal. Moreover, adherence of highly purified lipopolysaccharide to the endotoxin-free particles reconstituted the activity and the dependence on TIRAP/Mal. Conclusions: TIRAP/Mal deficiency reduces inflammatory responses and osteolysis induced by particles with adherent PAMPs. Clinical Relevance: Our results, coupled with the genetic associations between aseptic loosening and polymorphisms within the TIRAP/Mal locus, support TLR signaling through TIRAP/Mal as one of the factors that enhances the activity of wear particles and further support the hypothesis that bacterial PAMPs likely contribute to aseptic loosening in a subset of patients.

Introduction Genome Wide Association Studies have identified Tribbles-1 (TRIB1) as a regulator of plasma lipid levels and a risk factor for MI. Studies using Trib1 full body- and liver specific knockout mice have shown that hepatic expression of Trib1 reduces circulating lipids1. Additionally, Trib1 has been shown to be a regulator of alternatively activated macrophage polarisation2. However, there has been no study to directly evaluate the role of myeloid Trib1 (mTrib1) in atherogenesis. Methods To determine the role of mTrib1 in atherosclerosis, we developed myeloid-Trib1 deficient (mTrib1KO) and overexpression (mTrib1Tg) mouse strains. To distinguish between metabolic and inflammatory drivers of atherosclerosis, bone marrow from these strains were transplanted into lethally irradiated ApoE-/- mice and fed on a western diet for 12 weeks. Additionally, we also induced atherosclerosis in mTrib1Tg and mTrib1WT strains by injecting rAAV8/mPCSK9 and fed western diet for 12 weeks. Results mTrib1KO→ApoE-/- mice were protected while mTrib1Tg→ApoE-/- mice presented with increased atherosclerotic burden in both the aorta (p Conclusion We conclude that Trib1 is a potent regulator of atherosclerosis, the over-expression of which promotes atherogenesis through elevated oxLDL uptake and subsequent foam cell formation in plaque macrophages. References . Burkhardt et al. JCI (2010);120:4410–4414. . Satoh et al. Nature (2013);7442:524–8.

Introduction Atherosclerosis is a pathological chronic inflammatory process where lipoprotein, cholesterol, calcium and apoptotic cells build up a lipid-rich plaque within an artery. Inflammatory cells such as macrophages are a major component of the atherosclerotic plaque and participate in all stages of plaque formation and progression. The plaque can be classified as histologically stable or unstable, depending on the extent of inflammation, composition and the thickness of fibrous cap. Macrophage subtypes within carotid plaques have been shown to be altered in unstable vs. stable plaques. Broadly, M1 macrophages are proinflammatory and promote atherogenesis as well as enhancing plaque instability. In contrast, M2 macrophages have regulatory and wound-healing properties including collagen synthesis and promote tissue repair. Detailed characterisation of these macrophage subtypes and correlative analysis with in vivo carotid plaque MRI and blood composition may lead to the identification of diagnostic markers to direct the best treatment for stroke patients. Methodology Carotid plaques were obtained from patients undergoing carotid endarterectomy for recently symptomatic carotid stenosis. A validated clinical histology grading was used to define degree of overall plaque instability. The plaque ’shoulder’ regions were identified on H and E stained sections and were deemed the regions of interest (ROI). Macrophage phenotypes within the shoulder regions were characterised with immunofluorescence (IF) microscopy. An IF protocol to simultaneously stain for M1 and M2 macrophages alongside an image acquisition and analysis strategy was then developed. We used single IF with a pan macrophage polarisation marker (CD68), and dual IF with M1 (CD86) and M2 marker (MR: Mannose Receptor). Plaques were imaged using multi-colour fluorescence microscopy (Leica AF6000). After image registration, the regions of positive staining for CD68 were overlapped with the dual staining (CD86/MR) section and the prevalence of CD86 +and MR +macrophages were measured. Results Of the 16 collected plaques, an intact shoulder was identified in 13 samples and in these regions, macrophages were abundant. A median of 31.1% (4.5%–70.6%) macrophages stained for both M1 (CD86) and M2 (MR) markers. There was a higher prevalence of CD86 +MR + macrophages in shoulder regions from unstable versus stable plaques but this did not reach significance. Single positive M2 (MR +CD86-) cells were of higher prevalence in stable plaques. Hemosiderin-laden macrophages were also identified via their strong auto-fluorescence. Conclusions M1 (CD86+) and dual staining (CD86 +MR+) macrophage populations were predominant in the unstable plaques whereas single positive M2 (MR +CD86-) cells were predominant in stable plaques. Future analysis on a larger number of carotid plaques is required to better understand the prevalence and potential influence of different macrophage populations on plaque instability.

Background Chronic inflammation is one of the key processes in the development of metabolic dysfunction under obesity which represents a major risk for cardiovascular disease. The metabolic and immune responses to obesity affect many organs and particularly the adipose tissue where macrophages are key effector cells modulating inflammation through the signals received from adipocytes. The Tribbles (Trib) family of proteins has been described as major regulators of inflammation and energy metabolism. Specifically, Trib3 participates in a wide range of inflammatory and metabolic signalling pathways. We propose that Trib3 may modulate macrophage polarisation and therefore modulate the immune response to promote obesity. Methodology Trib3 full body knock out (Trib3KO) mice were used to study morphological changes in the adipose tissue. To investigate whether Trib3 regulates the polarisation state of macrophages, bone marrow derived macrophages (BMDMs) were isolated and polarised in vitro to further characterise their pro- or anti- inflammatory properties. Adipose tissue macrophages (CD11b+ATMs) were isolated from adipose tissue using magnetics beads and the expression of polarisation markers was evaluated by qPCR. Results Trib3 deficiency increased subcutaneous adipose tissue mass. Cell content showed that adipocytes were the same size compared to wild-type. However, adipocyte and macrophage numbers were increased in the Trib3KO mice. No significant differences in gene expression of classical polarisation markers were found in ATMs, however differential gene expression of proteins involved in macrophage function and phenotype was found in Trib3KO BMDMs compared to wild type results among the different polarisation states. Conclusion The results obtained in this pilot study suggest a role for Trib3 in macrophages due to expression differences measured in Trib3 deficient BMDMs, linked to a pro-inflammatory macrophage phenotype.

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.

Tribbles pseudokinase 3 (TRIB3) belongs to the tribbles family of pseudokinases. In this article, we summarize several observation obtained by our laboratories supporting that TRIB3 plays a crucial role in the anti-cancer activity of cannabinoids (a novel family of potential anti-cancer agents derived from marijuana) and that TRIB3 genetic inactivation enhances cancer generation and progression.

Tribbles (TRIB) proteins, a family of evolutionary conserved psuedokinase proteins, modulate various signalling pathways within the cell. The regulatory roles of TRIB make them an important part of a number of biological processes ranging from cell proliferation to metabolism, immunity, inflammation and carcinogenesis. Innate immune system plays a pivotal role during the regulation of reproductive processes that allows successful creation of an offspring. Its involvement initiates from fertilization of the oocyte by spermatozoon and lasts throughout early embryonic development, pregnancy and labour. Therefore, there is a close cooperation between the reproductive system and the innate immune system. Evidence from our lab has demonstrated that improper activation of the innate immune system can reduce embryo implantation, thus leading to infertility. Therefore, control mechanisms regulating the innate immune system function can be critical for successful reproductive events.

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.