Oltin Tiberiu Pop

Role of expression of the tumour-associated macrophage receptor, MERTK, in hepatocellular carcinoma: Spring Meeting for Clinician Scientists in Training 2017

BACKGROUND: Venous thromboembolism is a common, potentially avoidable cause of death and morbidity in patients in hospital, including those with stroke. In surgical patients, intermittent pneumatic compression (IPC) reduces the risk of deep vein thrombosis (DVT), but no reliable evidence exists about its effectiveness in patients who have had a stroke. We assessed the effectiveness of IPC to reduce the risk of DVT in patients who have had a stroke. METHODS: The CLOTS 3 trial is a multicentre parallel group randomised trial assessing IPC in immobile patients (ie, who cannot walk to the toilet without the help of another person) with acute stroke. We enrolled patients from day 0 to day 3 of admission and allocated them via a central randomisation system (ratio 1:1) to receive either IPC or no IPC. A technician who was masked to treatment allocation did a compression duplex ultrasound (CDU) of both legs at 7-10 days and, wherever practical, at 25-30 days after enrolment. Caregivers and patients were not masked to treatment assignment. Patients were followed up for 6 months to determine survival and later symptomatic venous thromboembolism. The primary outcome was a DVT in the proximal veins detected on a screening CDU or any symptomatic DVT in the proximal veins, confirmed on imaging, within 30 days of randomisation. Patients were analysed according to their treatment allocation. TRIAL REGISTRATION: ISRCTN93529999. FINDINGS: Between Dec 8, 2008, and Sept 6, 2012, 2876 patients were enrolled in 94 centres in the UK. The included patients were broadly representative of immobile stroke patients admitted to hospital and had a median age of 76 years (IQR 67-84). The primary outcome occurred in 122 (8·5%) of 1438 patients allocated IPC and 174 (12·1%) of 1438 patients allocated no IPC; an absolute reduction in risk of 3·6% (95% CI 1·4-5·8). Excluding the 323 patients who died before any primary outcome and 41 without any screening CDU, the adjusted OR for the comparison of 122 of 1267 patients vs 174 of 1245 patients was 0·65 (95% CI 0·51-0·84; p=0·001). Deaths in the treatment period occurred in 156 (11%) patients allocated IPC and 189 (13%) patients allocated no IPC died within the 30 days of treatment period (p=0·057); skin breaks on the legs were reported in 44 (3%) patients allocated IPC and in 20 (1%) patients allocated no IPC (p=0·002); falls with injury were reported in 33 (2%) patients in the IPC group and in 24 (2%) patients in the no-IPC group (p=0·221). INTERPRETATION: IPC is an effective method of reducing the risk of DVT and possibly improving survival in a wide variety of patients who are immobile after stroke. FUNDING: National Institute of Health Research (NIHR) Health Technology Assessment (HTA) programme, UK; Chief Scientist Office of Scottish Government; Covidien (MA, USA).

Repeated PTZ Treatment at 25-Day Intervals Leads to a Highly Efficient Accumulation of Doublecortin in the Dorsal Hippocampus of Rats

Background Neurogenesis persists throughout life in the adult mammalian brain. Because neurogenesis can only be assessed in postmortem tissue, its functional significance remains undetermined, and identifying an in vivo correlate of neurogenesis has become an important goal. By studying pentylenetetrazole-induced brain stimulation in a rat model of kindling we accidentally discovered that 25±1 days periodic stimulation of Sprague-Dawley rats led to a highly efficient increase in seizure susceptibility. Methodology/Principal Findings By EEG, RT-PCR, western blotting and immunohistochemistry, we show that repeated convulsive seizures with a periodicity of 25±1 days led to an enrichment of newly generated neurons, that were BrdU-positive in the dentate gyrus at day 25±1 post-seizure. At the same time, there was a massive increase in the number of neurons expressing the migratory marker, doublecortin, at the boundary between the granule cell layer and the polymorphic layer in the dorsal hippocampus. Some of these migrating neurons were also positive for NeuN, a marker for adult neurons. Conclusion/Significance Our results suggest that the increased susceptibility to seizure at day 25±1 post-treatment is coincident with a critical time required for newborn neurons to differentiate and integrate into the existing hippocampal network, and outlines the importance of the dorsal hippocampus for seizure-related neurogenesis. This model can be used as an in vivo correlate of neurogenesis to study basic questions related to neurogenesis and to the neurogenic mechanisms that contribute to the development of epilepsy.

Multivariate metabotyping of plasma predicts survival in patients with decompensated cirrhosis.

Graphical abstract

MerTK expressing hepatic macrophages promote the resolution of inflammation in acute liver failure

Objective Acute liver failure (ALF) is characterised by overwhelming hepatocyte death and liver inflammation with massive infiltration of myeloid cells in necrotic areas. The mechanisms underlying resolution of acute hepatic inflammation are largely unknown. Here, we aimed to investigate the impact of Mer tyrosine kinase (MerTK) during ALF and also examine how the microenvironmental mediator, secretory leucocyte protease inhibitor (SLPI), governs this response. Design Flow cytometry, immunohistochemistry, confocal imaging and gene expression analyses determined the phenotype, functional/transcriptomic profile and tissue topography of MerTK+ monocytes/macrophages in ALF, healthy and disease controls. The temporal evolution of macrophage MerTK expression and its impact on resolution was examined in APAP-induced acute liver injury using wild-type (WT) and Mer-deficient (Mer−/−) mice. SLPI effects on hepatic myeloid cells were determined in vitro and in vivo using APAP-treated WT mice. Results We demonstrate a significant expansion of resolution-like MerTK+HLA-DRhigh cells in circulatory and tissue compartments of patients with ALF. Compared with WT mice which show an increase of MerTK+MHCIIhigh macrophages during the resolution phase in ALF, APAP-treated Mer−/− mice exhibit persistent liver injury and inflammation, characterised by a decreased proportion of resident Kupffer cells and increased number of neutrophils. Both in vitro and in APAP-treated mice, SLPI reprogrammes myeloid cells towards resolution responses through induction of a MerTK+HLA-DRhigh phenotype which promotes neutrophil apoptosis and their subsequent clearance. Conclusions We identify a hepatoprotective, MerTK+, macrophage phenotype that evolves during the resolution phase following ALF and represents a novel immunotherapeutic target to promote resolution responses following acute liver injury.

CD14+CD15-HLA-DR- myeloid-derived suppressor cells impair antimicrobial responses in patients with acute-on-chronic liver failure

Objective Immune paresis in patients with acute-on-chronic liver failure (ACLF) accounts for infection susceptibility and increased mortality. Immunosuppressive mononuclear CD14+HLA-DR− myeloid-derived suppressor cells (M-MDSCs) have recently been identified to quell antimicrobial responses in immune-mediated diseases. We sought to delineate the function and derivation of M-MDSC in patients with ACLF, and explore potential targets to augment antimicrobial responses. Design Patients with ACLF (n=41) were compared with healthy subjects (n=25) and patients with cirrhosis (n=22) or acute liver failure (n=30). CD14+CD15−CD11b+HLA-DR− cells were identified as per definition of M-MDSC and detailed immunophenotypic analyses were performed. Suppression of T cell activation was assessed by mixed lymphocyte reaction. Assessment of innate immune function included cytokine expression in response to Toll-like receptor (TLR-2, TLR-4 and TLR-9) stimulation and phagocytosis assays using flow cytometry and live cell imaging-based techniques. Results Circulating CD14+CD15−CD11b+HLA-DR− M-MDSCs were markedly expanded in patients with ACLF (55% of CD14+ cells). M-MDSC displayed immunosuppressive properties, significantly decreasing T cell proliferation (p=0.01), producing less tumour necrosis factor-alpha/interleukin-6 in response to TLR stimulation (all p<0.01), and reduced bacterial uptake of Escherichia coli (p<0.001). Persistently low expression of HLA-DR during disease evolution was linked to secondary infection and 28-day mortality. Recurrent TLR-2 and TLR-4 stimulation expanded M-MDSC in vitro. By contrast, TLR-3 agonism reconstituted HLA-DR expression and innate immune function ex vivo. Conclusion Immunosuppressive CD14+HLA-DR− M-MDSCs are expanded in patients with ACLF. They were depicted by suppressing T cell function, attenuated antimicrobial innate immune responses, linked to secondary infection, disease severity and prognosis. TLR-3 agonism reversed M-MDSC expansion and innate immune function and merits further evaluation as potential immunotherapeutic agent.

Impact of Donation Mode on the Proportion and Function of T Lymphocytes in the Liver

Background Liver T-cells respond to the inflammatory insult generated during organ procurement and contribute to the injury following reperfusion. The mode of liver donation alters various metabolic and inflammatory pathways but the way it affects intrahepatic T-cells is still unclear. Methods We investigated the modifications occurring in the proportion and function of T-cells during liver procurement for transplantation. We isolated hepatic mononuclear cells (HMC) from liver perfusate of living donors (LD) and donors after brain death (DBD) or cardiac death (DCD) and assessed the frequency of T-cell subsets, their cytokine secretion profile and CD8 T-cell cytotoxicity function, responsiveness to a danger associated molecular pattern (High Mobility Group Box1, HMGB1) and association with donor and recipient clinical parameters and immediate graft outcome. Results We found that T-cells in healthy human livers were enriched in memory CD8 T-cells exhibiting a phenotype of non-circulating tissue-associated lymphocytes, functionally dominated by more cytotoxicity and IFN-γ-production in DBD donors, including upon activation by HMGB1 and correlating with peak of post-transplant AST. This liver-specific pattern of CD8 T-cell was prominent in DBD livers compared to DCD and LD livers suggesting that it was influenced by events surrounding brain death, prior to retrieval. Conclusion Mode of liver donation can affect liver T-cells with increased liver damage in DBD donors. These findings may be relevant in designing therapeutic strategies aimed at organ optimization prior to transplantation.

Role of expression of the tumour-associated macrophage receptor, MERTK, in hepatocellular carcinoma

Abstract Background Tumour-associated macrophages are implicated in progression of hepatocellular carcinoma by dampening immune responses. Experimental models show that activation of the tumour-associated macrophage receptor, tyrosine-protein kinase mer (MERTK), in myeloid cells promotes tumour progression by suppressing innate and adaptive responses. We sought to investigate the role of MERTK and its sister molecule, AXL, in patients with hepatocellular carcinoma. Methods Immunohistochemistry and multispectral imaging were performed for MERTK, AXL, and various macrophage activation markers on explanted hepatocellular carcinoma tissue (n=10), regenerative nodules (5), dysplastic nodules (5), and cirrhotic liver (controls, 5). Immunophenotyping of circulating monocytes was done. In-vitro co-culture systems were developed to modulate tumour-associated macrophage MERTK expression and determine the effects on CD14+, CD4+, and CD8+ leucocyte function. Findings Peripheral leucocytes in hepatocellular carcinoma had higher MERTK expression (22·4% vs 10·7%, p=0·01) and a higher ratio of MERTK to AXL expression (39·1:1 vs 4·6:1, p=0·03) than cirrhotic controls. Immunohistochemical analysis confirmed increased macrophage MERTK expression in hepatocellular carcinoma compared with cirrhotic and healthy controls (p=0·01 and p=0·02, respectively). In vitro, induction of MERTK expression in CD14+ antigen-presenting cells reduced CD8+ (2·9% vs 10·7%, p=0·02) and CD4+ (3·6% vs 1·0%, p=0·04) T-cell proliferative responses to class I and II restricted antigens, respectively, with concomitant reduction (1·18% vs 5·59%, p=0·03) in the early activation marker CD69 in CD8+ T cells at 24 h. Antibody-mediated blockade of MERTK restored mean fluorescence intensity of the early activation marker CD28 in the same cells (p=0·037). Interpretation We have identified an expanded population of MERTK-expressing immunosuppressive tumour macrophages in hepatocellular carcinoma. These cells can suppress innate and adaptive immune responses. We have shown that AXL is not similarly upregulated. Inhibition of MERTK signalling restores macrophage activation, thereby potentially identifying a novel immunotherapeutic target in hepatocellular carcinoma. Funding National Institute for Health Research academic clinical fellowship.

P138 SERONEGATIVE ACUTE LIVER FAILURE REPRESENTS A MACROPHAGE–T CELL ACTIVATION SYNDROME

P136 DEFICIENCY OF IL-33 SENSITIZES TO SEVERE LIVER INJURY INDUCED BY ConA BUT NOT BY CCl4 IN MICE M.I. Arshad, A. Filliol, V. Genet, C. Lucas-Clerc, J.-P. Girard, C. Piquet-Pellorce, M. Samson. Inserm U 1085, Institut de Recherche Sante Environnement & Travail (IRSET), Rennes, France; Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan; Service de Biochimie CHU Rennes, Universite de Rennes 1, Rennes, Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique (IPBS-CNRS), Universite de Toulouse, Toulouse, Structure Federative BioSit UMS 3480 CNRS-US 18 Inserm, Rennes, France E-mail: michel.samson@univ-rennes1.fr