Invited Speakers

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Demographic development in many countries reveals that the number of elderly has increased and will increase more and more within the next decades. However, while average life expectancy has increased markedly throughout the last century, this has not been accompanied by an equivalent increase in healthy life expectancy. Indeed, chronic, low-grade inflammation also frequently referred to as inflammaging has been acknowledged as a major characteristic of aging also being suggested to be critically involved in aging-associated decline of many organs including adipose tissue and skeletal muscle but also the liver and the gut. Despite intense research efforts molecular mechanisms underlying inflammaging have not yet been fully understood. Results of several epidemiological studies also suggest that age and herein especially older age increases the odds to develop diseases among them many metabolic diseases like non-alcoholic fatty liver disease. Furthermore, it has also been discussed that nutrition may be critical for the extension of a healthy life span. Results obtained from epidemiological studies and studies in model organisms have repeatedly shown that caloric or protein restriction but maybe also an enrichment of diet with certain macronutrients or probiotics may alter aging-associated decline and inflammaging. Here, an overview summarizing recent findings and highlighting emerging trends in nutrition, aging and liver disease will be given.
We define reperfusion injury (RI) as cell death occurring after the termination of myocardial ischemia, and preventable by interventions applied at that time. The evidence accumulated over the last three decades on the existence and importance of RI in experimental models is solid and undisputed. The mechanisms of RI have been partially elucidated. Despite this, no treatment to limit RI in patients with ST elevation myocardial infarction (STEMI) has gained yet a place in standard clinical practice. Difficulties in translation have been explained by the use of inadequate preclinical models (very young, healthy animals receiving no co-medications), and to limitations in the design and methodologies of clinical trials. We will critically analyze the contribution of these limitations in pre-clinical and clinical studies to the delayed translation of treatments against RI to patients with STEMI, and will propose that selection of wrong targets and treatments in clinical trials is instead the main responsible of this delay. Moreover, when interventions with solid preclinical results are translated to patients, as is the case with remote ischemic conditioning, the results are consistently promising. Finally, we propose a pathway to accelerated translation of RI limitation to patients based on: (1) development of drugs against well established targets never studied in patients, (2) testing of these treatments in multicenter preclinical platforms, (3) the inclusion of selected co-treatments in the models, (4) combination of anti RI treatments to increase their efficacy and safety, and (5) adequately powered clinical trials with optimal end-points.
PL-5 | Telomerase activation as a therapeutic strategy for treatment of age-related diseases Cell migration in vivo mostly takes place in 3D environments. In addition to the amoeboid migration used by all leukocytes, we showed in vitro that macrophages, and more recently dendritic cells, use the mesenchymal migration in dense environments that involves proteolysis of the extracellular matrix to create paths. In most cancers, the density and stiffness of the tissue stroma are enhanced. We found in mouse fibrosarcoma in vivo and in human breast cancer ex vivo, that tumor-associated macrophages (TAM) perform the mesenchymal migration using their own matrix metalloproteases (MMPs). In tissue at the tumor periphery and in inflamed ear derma, macrophages use the amoeboid migration. As TAM favor cancer progression, controlling their infiltration and migration in tumors appears as a novel therapeutic strategy. As a proof of concept, we report that MMP inhibition correlates with decreased TAM recruitment and tumor growth. To target more specifically the TAM mesenchymal migration, we are involved in the study of podosomes, cell structures that are constitutively formed in a few cell types including macrophages. Podosomes are instrumental in that migration mode since their disruption or stabilization impacts the mesenchymal migration but not the amoeboid motility. These unique cell structures have adhesion, mechanosensing, force generation and proteolytic properties. Deciphering the architecture and mechanics of podosomes should provide new pharmacological targets.

B. Yipp
University of Calgary, Calgary, Canada Pulmonary immunity requires tight regulation, as interstitial inflammation can compromise gas exchange and lead to respiratory failure. Here we found a greater number of aged CD11b hi L-selectin lo CXCR4+ polymorphonuclear leukocytes (PMNs) in lung vasculature than in the peripheral circulation. Using pulmonary intravital microscopy, we observed lung PMNs physically interacting with B cells via b 2 integrins; this initiated neutrophil apoptosis, which led to macrophage-mediated clearance. Genetic deletion of B cells led to the accumulation of aged PMNs in the lungs without systemic inflammation, which caused pathological fibrotic interstitial lung disease that was attenuated by the adoptive transfer of B cells or depletion of PMNs. Thus, the lungs are an intermediary niche in the PMN lifecycle wherein aged PMNs are regulated by B cells, which restrains their potential to cause pulmonary pathology.
W1-L3 | How do the mechanical properties of the nuclear lamina affect granulocyte transendothelial migration and crossing through collagen barriers?

R. Alon
The Weizmann Institute Of Science, Rehovot, Israel Transendothelial migration (TEM) of leukocytes is associated with the ability of their deformable nuclei to displace endothelial cytoskeletal barriers. Lamin A is a key intermediate filament component of the nuclear lamina which is downregulated during granulopoiesis. When elevated, lamin A restricts nuclear squeezing through rigid confinements. To determine if the low lamin A expression by leukocyte nuclei is critical for their exceptional squeezing ability through endothelial barriers, we overexpressed this protein in granulocyte-like differentiated HL-60 cells. A 10-fold higher lamin A expression did not interfere with chemokinetic motility of these granulocytes. Furthermore, these lamin A high leukocytes exhibited normal chemotaxis determined in large pore transwell barriers, but poorly squeezed through 3-μm pores. Strikingly, however, these leukocytes successfully completed paracellular TEM across inflamed endothelial monolayers under shear flow, albeit with a small delay in nuclear squeezing into their subendothelial pseudopodia. In contrast, granulocyte motility through collagen I barriers was dramatically delayed by lamin A overexpression due to a failure of lamin A high nuclei to translocate into the pseudopodia of the granulocytes. Collectively our data predict that leukocytes maintain a low lamin A content in their nuclear lamina in order to optimize squeezing through extracellular collagen barriers rather than crossing the highly adaptable barriers presented by the endothelial cytoskeleton. Our ongoing studies suggest that dynamic microtubule cytoskeleton is critical for granulocyte squeezing through collagen barriers but not for crossing endothelial barriers. Collectively our results suggest that both the deformable lamin A low nuclei and the dynamic microtubule cytoskeleton play critical roles in the exceptional ability of leukocytes to rapidly squeeze through dense extracellular matrices.

A. Hidalgo
Fundaci on CNIC, Madrid, Spain Neutrophils are the most abundant innate immune cells and are generally believed to exclusively participate in antimicrobial defense, and to precipitate acute inflammatory processes when activation is deranged. Beyond these acute processes, however, little is known of many aspects of their biology and diversity. I will present our published and ongoing work illustrating other unexpected homeostatic functions of neutrophils, and argue that many of these originate from their capacity to infiltrate multiple tissues and to adopt multiple phenotypes, even in the absence of danger. I will also discuss that the phenotype, dynamics and biological functions of neutrophils are intimately linked to day-night cycles.
W1-L5 | The Janus faced gladiator: neutrophils in sterile inflammation and autoimmunity M. Hoffmann immunoregulatory functions. Neutrophil extracellular traps (NETs), aggregation, and degradation of inflammatory mediators by serine proteases are important neutrophil tools to resolve gouty arthritis and other forms of localized inflammatory conditions. On the downside, NETs come at the price of collateral tissue damage and may cause occlusion of ductal structures. Thus, rather than being brainless foot soldiers, neutrophils are actively involved in regulation of inflammatory processes and autoimmune diseases. Background: Timely resolution of infections critically depends on clearance of invading pathogens by polymorphonuclear leukocytes (PMNL). Typically, phagocytosis of bacteria accelerates PMN apoptosis and removal of apoptotic PMN by efferocytosis. Impaired PMN responses, resulting in inefficient clearance of invading bacteria and delayed apoptosis, is a characteristic feature of many pathologies. We investigated whether bacterial constituents can impair bacterial clearance and phagocytosis-induced PMN apoptosis and whether these can be countered by the specialized pro-resolving lipid mediators resolvin E1 (RvE1) and15-epi-lipoxin A4 (15-epi-LXA4). Materials and methods: Phagocytosis-induced apoptosis, expression of surface receptors and intracellular signaling pathways were assessed in human PMN following phagocytosis of opsonized E. coli. Acute lung inflammation was produced by intratracheal instillation of live E. coli AE bacterial DNA in mice and the animals were treated with RvE1 or 15-epi-LXA4 at the peak of inflammation. Results: Bacterial DNA decreased phagocytosis and killing of E. coli through TLR9-mediated downregulation of C5aR (CD88) expression, thereby reducing phagocytosis-induced apoptosis in human PMN. RvE1 through the leukotriene B4 receptor BLT1 mitigated ERK and Akt-mediated survival signals generated by ligation of Mac-1 and TLR9 through acceleration of Mcl-1 degradation and induction of mitochondrial dysfunction. 15-epi-LXA4 through the FPR2/ALX antagonized survival cues from bacterial DNA, prevented C5aR shedding, restored phagocytosis and enhanced phagocytosis-induced PMN apoptosis. In mice, bacterial DNA impaired pulmonary clearance of E. coli, suppressed PMN apoptosis and hindered resolution of lung injury evoked by E. coli. Treatment with either RvE1 or 15-epi-LXA4 efficiently countered these actions and promoted resolution. Conclusions: These data shed new light how bacterial DNA contributes to altered PMN responses and prolongation of inflammation. Our findings also identify a common effector mechanism, mitigation of anti-apoptosis cues and enhancing phagocytosis-induced PMN apoptosis, by which pro-resolving mediators acting via distinct receptors could contribute to timely resolution of infections. (Grant support: CIHR MOP-97742).

T. Lapidot
Weizmann Institute of Science, Rehovot, Israel Background: Murine bone marrow (BM) leukocytes predominantly differentiate during the light hours and therefore are more responsive to inflammatory challenges. The first lines of defense upon inflammation are neutrophils. Metabolic pathways and lactate levels are the best clinical markers for sepsis diagnosis. However, the mechanisms underlying lactate production and function during inflammation remain poorly understood. Material and methods: Mice were challenged with high (250 lg/mouse) or low (50 lg/mouse) doses of LPS at different time points. NAC was administered to scavenge reactive oxygen species (ROS) particles. BM and blood cells were harvested and analyzed by flow cytometry or ImageStream. BM vascular endothelial barrier function was assessed by Evans Blue Dye (EBD) assay. Results: We found that high doses of LPS administration in the afternoon (following increased BM leukocyte production) resulted in a dramatic elevation of neutrophils and monocytes recruitment from the BM, which is lethal, in contrast to LPS injection at midnight with no immune activation. Reducing differentiation in the BM by ROS inhibition decreased the levels of neutrophils and monocytes recruitment following LPS challenge in the afternoon. To further understand how inflammation affects neutrophils recruitment during the day, we administered low doses of LPS that activated BM neutrophils with an increase in BM neutrophil metabolism, reflected by elevated ROS and HIF-1a signaling. Importantly, we identified that LPS up-regulated the expression of lactate dehydrogenase A and lactate efflux transporter MCT4 on BM neutrophils. Unexpectedly, in vivo lactate treatment specifically induced neutrophil mobilization from the BM to the circulation. Moreover, lactate increased blood-BM vascular permeability and neutrophil mobilization from the BM in an endothelial lactate receptor GPR81-dependent manner. Conclusions: These results reveal that during LPS-induced inflammation, BM neutrophils produce high levels of lactate. Lactate is a potential target for attenuating blood neutrophilia associated with sepsis and other infectious conditions. W1-L8 | Signaling in phagocytes during apoptotic cell engulfment

K. Ravichandran
University of Virginia, Charlottesville, USA Every day we turnover billions of cells in the body as part of normal healthy living. Majority of these cells die via the process of apoptosis and these cells are cleared by professional phagocytes (such as macrophages and immature dendritic cells) and non-professional phagocytes (such as epithelial cells and fibroblasts). The specific recognition of apoptotic cells among the sea of living cells is achieved through specific receptors on phagocytes engaging specific molecules on apoptotic cells, and the subsequent intracellular signaling within phagocytes that mediate the corpse internalization. One of the fascinating, yet understudied, aspects of phagocyte biology is how the phagocyte manages to internalize a corpse almost the same size as itself, and how the phagocyte manages the contents of the ingested cargo, yet continues to maintain homeostasis and eat additional corpses. Further, the process of eating apoptotic cells is actively anti-inflammatory. These effects are achieved through the intracellular signaling that occurs within phagocytes during recognition, ingestion, and subsequent processing of ingested corpses. Our recent work addressing the signaling in phagocytes ingesting corpses, both at the transcriptomics and proteomics levels, suggest new insights into signatures unique to apoptotic cell clearance and anti-inflammatory signaling.

C. Rothlin
Yale University, New Haven, USA Inflammationthe physiological response elicited by injury and the recognition of foreign antigensinvolves a complex interplay of biochemical pathways that trigger and shape both the innate and adaptive immune responses.
These culminate in a coordinated response that is essential for protection against invading pathogens. Its fundamental role notwithstanding, inflammation, if unchecked, can have deleterious consequences that can favor the development of chronic inflammatory diseases and autoimmunity. Thus, endogenous mechanisms that regulate inflammation, limiting its duration and intensity as well as inducing the resolution phase of this response, are fundamental to immune homeostasis. Here I will discuss recent findings on the central function of the TAM receptor tyrosine kinases AXL and MERTK in the coordinated inhibition of inflammation, clearance of apoptotic debris and induction of wound healing responses.

J.M. Blander
Weill Cornell Medicine, New York, USA Apoptosis is an important component of normal tissue physiology, and the prompt removal of apoptotic cells is equally essential to avoid the undesirable consequences of their accumulation and disintegration. Professional phagocytes are highly specialized for engulfing apoptotic cells. Tracking apoptotic cells in situ revealed a division of labor among the tissue resident phagocytes that sample them. Macrophages are uniquely programmed to process internalized apoptotic cell-derived fatty acids, cholesterol and nucleotides, as a reflection of their dominant role in apoptotic cell clearance. Dendritic cells carry apoptotic cells to lymph nodes where they signal expansion of highly suppressive regulatory CD4 T cells. A broad suppression of inflammation is executed through distinct phagocyte-specific mechanisms. A DC-specific induction of negative regulatory nodes is notable serving to simultaneously shut down multiple pathways of inflammation. Several of the genes and pathways modulated in phagocytes in response to apoptotic cells have been linked to chronic inflammatory and autoimmune diseases such as atherosclerosis, inflammatory bowel disease and systemic lupus erythematosus. and foreign material, and orchestrate inflammation and immune defense. Most tissue-resident macrophage compartments are established prenatally and develop locally, alongside their host tissue, separated from each other. Maintenance of these populations, residing at sites like liver and brain, relies on longevity and self-renewal and seems independent from ongoing hematopoiesis. Selected barrier tissues, such as the gut and skin, but also the heart, display however substantial postnatal replacement of the embryonic populations by adult monocyte-derived cellsprobably linked to unique homeostatic challenges of these organs. Microglia and intestinal macrophages are found at the extremes of this spectrum, given their respective seclusion behind the blood brain barrier and exposure to products of the commensal microbiota, their origins, half-lives, as well as their transcriptomes and enhancer landscapes. Here I will discuss recent efforts of my laboratory to gain insights into macrophage biology through the comparative analysis of tissue macrophage populations. Specifically, we use conditional mutagenesis combined with advanced genomics to probe how these intriguing cells maintain health and cope with challenges. Neutrophils play a major role in the host resistance to infection. Neutrophils are the first to arrive at a site of infection, and they stay for only a short time (the first 24 hours). Of note, monocytes arrive somewhat later but stay for days. Patients with alcoholic cirrhosis are susceptible to develop bacterial infections. Recent study addressed the question of whether neutrophils in these patients may have defects resulting in anti-microbial activity. Bacterial products such as fMet-Leu-Phe (fMLP) stimulate neutrophils to produce reactive oxygen species (ROS). The production of ROS by neutrophils which is termed respiratory burst (RB) or oxidative stress (OS), plays a key role in antimicrobial host-defense systems (1). The enzyme responsible for the neutrophil RB, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2), is a membrane multiprotein complex whose activation requires the phosphorylation and membrane translocation of cytosolic components, among which p47phox (phox: phagocyte oxidase) plays an important role. In pathological situations, ROS production becomes inappropriately regulated. Neutrophils from patients with decompensated alcoholic cirrhosis exhibit a marked defect in RB, microbicidal activity, and phagocytosis (2). Neutrophil dysfunctions were found to be reversible after endotoxin removal from patient plasma (3). In other studies, persistent cellular defects were also observed (4). The mechanisms that explain defective RB are poorly understood. Stimulation of "healthy" neutrophils by fMLP has been shown to induce a rapid activation of mammalian target of rapamycin (mTOR) on Ser2448 through the AKT pathway (2). Activated mTOR was found to activate p38-MAPK, which, in turn, phosphorylated p47phox on its Ser345 at the membrane and thus contributed to superoxide production by NOX2. In patients with cirrhosis, NOX2 activity was decreased as a result of a deficient activation of p38-MAPK and p47phox (2). The formation of a multiprotein complex at the plasma membrane, which is required for NOX2 activation, includes the catalytic core flavocytochrome b558 heterodimer consisting in two associated transmembrane proteins, gp91 phox (NOX2) and p22 phox , and cytosolic components, p67 phox , p47 phox , p40 phox and Rac1/2. Neutrophils from patients with decompensated alcoholic cirrhosis exhibit a marked under-expression of gp91 phox , p22 phox , p47 phox and mTOR. Gp91 phox turned out to be depleted by degradation mediated by elastase in patients' plasma, and by a deficient mTOR-dependent translational machinery (5). Interestingly, the deficient ROS production was reversed through activation of intracellular TLR7/8 involving de novo gp91 phox synthesis via a mTOR-dependent process (5). Neutrophils contribute to microbial elimination via myeloperoxidase exocytosis. Neutrophils from patients with decompensated alcoholic cirrhosis have deficient AKT/p38-MAP-Kinase signaling, myeloperoxidase release and antibacterial activity, which can be reversed via TLR7/8 activation (6). These defects, together with the severe defect in superoxide production, can increase cirrhotic patients' susceptibility to bacterial infections. In addition, there are novel approaches using TLR7/8 agonists that can, at least in part, restore neutrophil responses. Alcoholic hepatitis (AH) is characterized by activation of the inflammatory cascade and liver damage. Binge drinking can be a precipitant of an acute alcoholic hepatitis event.
In AH, mortality correlates with liver neutrophil infiltration and sepsis. Neutrophil extracellular traps (NETs) can immobilize and kill invading microorganisms. We hypothesized that NET formation and its clearance by macrophages (MØ) (efferocytosis) is altered by alcohol binge drinking and in acute sepsis following a binge drinking episode. In healthy volunteers who consumed 2 mL of vodka/kg body weight and serum endotoxin and bacterial 16s rDNA, both inducers of NETs, were significantly increased after binge alcohol drinking. Ex vivo in normal peripheral neutrophils alcohol (50 mM) alone increased NET formation but attenuated NET formation upon phorbol 12-myristate 13-acetate (PMA) stimulation. Binge alcohol administration in mice in vivo resulted in a biphasic response to LPS. Initially, binge alcohol reduced NETs and resulted in a diffuse distribution of neutrophils in the liver compared to alcohol-na€ ıve mice. Moreover, indicators of NET formation including citrullinated histone H3, neutrophil elastase, and neutrophil myeloperoxidase were decreased at an early time point after an LPS challenge in mice with alcohol binge suggesting decreased NET formation. However, 15 hours after LPS in alcohol binge, citrullinated histone-H3 was increased in the liver suggesting decreased clearance of NETs. We found that in vitro alcohol treatment reduced efferocytosis and phagocytosis of NETosing neutrophils and promoted expression of CD206 on macrophages. Finally, in vivo, depletion of neutrophils with anti-Ly6G antibody (IA8) administration prior to alcohol binge ameliorated LPS-induced systemic inflammation and liver injury in mice. Our results suggest that alcohol binge alters NETosis and efferocytosis and dysfunctional neutrophil NETosis and efferocytosis after binge drinking exacerbate liver injury associated with sepsis. atherosclerosis. The identification of endogenously generated molecules including lipoxins, [LXs], which promote the physiologic resolution of inflammation suggests that these bioactions may have therapeutic potential in the context of chronic inflammation. We investigated the potential of LXA4 to modulate diabetic complications in the streptozotocin-induced diabetic ApoE-/-mice and in human carotid plaque tissue ex vivo. In order to exploit the therapeutic potential of LXs synthetic mimetics have been generated and characterised The development of diabetes-induced kidney disease as evidenced by albuminuria, mesangial expansion and collagen deposition was attenuated by LXA4. It is noteworthy that LXA4 also attenuated established kidney disease in diabetic mice. Kidney transcriptome profiling defined a diabetic signature [725 genes; FDR P ≤ 0.05]. Comparison of this murine gene signature with human DKD identified shared renal pro-inflammatory/pro-fibrotic signals Pathway analysis identified established and novel networks activated in diabetes and regulated by LXA4. The development of diabetes-induced aortic plaques and inflammatory responses of aortic tissue was significantly attenuated by LXA4 in diabetic ApoE-/-mice. In mice with established atherosclerosis LXA4 treatment led to a significant reduction in aortic arch plaque development. Treatment of human carotid plaques explants with LXA4 ex vivo attenuated secretion of proinflammatory cytokines. LXA4 inhibited PDGF-stimulated vascular smooth muscle cells proliferation and transmigration and endothelial cell inflammation. To exploit the therapeutic potential of LXA4 a panel of synthetic mimetics have been generated and characterized. We have identified an FPR2 agonist with proresolving bioactions including cardioprotection in murine sepsis. Future studies will investigate its therapeutic potential in vascular complications of diabetes. Background and Aim: Primary Biliary Cholangitis (PBC) is a chronic autoimmune liver disease characterized by the presence of anti-mitochondrial antibodies (AMA) and hepatic antigen specific CD8+ T cells infiltrate. Monocytes are innate immune cells shown to have pro-inflammatory phenotype in PBC patients. CCR2 and CCR5 are chemokine receptors, expressed by various immune cells, shown to be important for the recruitment of monocytes and T cells to inflammatory sites. Cenicriviroc (CVC) is a dual CCR2/ CCR5 inhibitor. Our aim was to assess the effectiveness of CVC treatment in a murine model of PBC. Methods: We utilized an inducible murine model of PBC by immunization of mice with the xenobiotic 2-octynoic acid conjugated to bovine serum albumin (2-OA BSA). 20 C57BL/6 mice were divided into 2 groups of 10 mice (group of CVC treatment and a control group that received vehicle only). Mice were treated with CVC 20 mg/kg/day or vehicle administered I.P once daily. Results: Histopathology evaluation revealed hepatic infiltration of mononuclear cells in mice treated with vehicle as compared to CVC treated animals that exhibited only minor liver abnormalities. Analysis for alkaline phosphatase and total bile acid in the sera revealed significantly reduced levels in CVC treated mice. Immunofluorescent microscopy revealed massive accumulation of peri-portal macrophages in control mice but not in the CVC group. Flow cytometry analysis showed reduced numbers of hepatic monocytederived cells in CVC treated mice. Sirius red staining of liver sections exhibit peri-portal collagen deposition only in the control group, a finding that was supported by a profibrotic hepatic gene-expression signature in this group. Conclusions: Our results indicate a major role for CCR2/ CCR5 in PBC pathogenesis and proved that inhibition of these axes by CVC significantly ameliorated both liver inflammation and fibrosis. These results reinforce further investigation of this therapeutic option in clinical trials. Background: Macrophages are known for their versatile role in patho-physiology. During innate immunity and the onset of inflammation an inherent destructive behavior is directed against foreign or 'altered self' structures. However, macrophages can also be turned into a tissue regenerative, proresolving, or healing phenotype. Their functional responses represent a continuum between classical and alternative activation. Material and methods: Diverse chemical signals, originating from their environment, contribute to the phenotype shift. Among these signals, molecules released by apoptotic cells and/or macrophage activation via the apoptotic synapse followed by the concomitant phagocytic process add to the rich educational diversity of macrophage phenotypes. Results: We present evidence that lipids, particularly sphingosine-1-phosphate (S1P) generated by apoptotic cells, shift the macrophage phenotype to an angiogenic, lymphangiogenic, immunosuppressive, tumor supportive, and survival-promoting one. Pathways involved comprise activation of the S1P-receptor 1 in close association with upregulating hypoxia-inducible factor 1 (HIF-1) target genes, induction of cyclooxygenase-2 (COX-2) as well as microsomal prostaglandin E-synthase-1 (mPGES1) with the concomitant production of prostaglandin E2 (PGE2), and expression regulation of the iron chelating and transporting protein lipocalin-2 (LCN-2) via a STAT3 transcriptional regulatory program. Conclusions: Activation of these pathways drastically changes the macrophage output system and thus, ways how these cells communicate with and shape their environment. This also adds to our understanding how blood/lymph vessel growth and tumor cell proliferation/migration is affected by macrophages in the tumor microenvironment. Maintenance of homeostasis requires macrophages to display both pro-and anti-inflammatory functions, and deregulation of this dynamic balance results in chronic inflammatory pathologies. The main factors promoting macrophage differentiation (GM-CSF and M-CSF) lead to generation of macrophages with opposite effector functions. M-CSF is constitutively present in serum and controls monocyte/macrophage recruitment and polarization under physiological and pathological settings. M-CSF primes macrophages to acquire trophic and anti-inflammatory functions in homeostasis, but drives the generation of IL-10-producing pro-tumoral macrophages within the tumor environment. Given the pathophysiological relevance of IL-10-producing M-CSF-dependent macrophages, we have determined the transcriptional signature of macrophages generated in the presence of GM-CSF (GM-MØ) or M-CSF (M-MØ) in response to pathogenic stimulation, and identified a panel of genes ("activated M-MØ gene set") whose modulation by TLR ligands only occurs in M-MØ, correlates with IL-10 expression and primarily depends on MAPK and GSK3. The "activated M-MØ gene set" includes growth factors, signaling molecules and chemokines that might contribute to the trophic and immunosuppressive action of macrophages, and whose coexpression is observed in macrophages within tissues undergoing senescence-induced inflammation like naevi. The identification of the "activated M-MØ gene set" provides a novel set of biomarkers to assess the functional state of macrophages in the increasing number of diseases where deregulated macrophage polarization is pathologically relevant.
W2-L1 | Back to the basics: evolution of mitochondrial respirometry and basic concepts P. Oliveira CNC -Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal From the basic mechanisms of energy production, stored as adenosine triphosphate (ATP), through means of oxidative phosphorylation, to control of cytosolic calcium, regulation of cell death, intermediate metabolism, and production of reactive oxygen species, to name just a few of the cellular roles of mitochondria, it is clear why they are thus considered the "powerhouses" of cells. Still, more than being simply "powerhouses", mitochondria act as factories and processors of cellular dynamics. In fact, even regulation of gene expression in the nucleus is regulated by mitochondria, which emphasizes the importance of this organelle in the cell. Mitochondrial respiration is a marker of mitochondrial viability. Different substrates and inhibitors can be added to isolated mitochondrial preparations or permeabilized/intact fibers and cells so that a complete profile of mitochondrial respiration can be obtained. The use of modern technology to assess mitochondrial respiration leads to a fast and easy way to obtain data. But the analysis of the resulting data is often not seen in the light of basic concepts of mitochondrial bioenergetics, which is often ignored. We review here some basic mitochondrial bioenergetic concepts and present some of the classic methodology that was and is used to measure oxygen consumption, providing some of the most extraordinary contributions to the field of cell energy metabolism. Mitochondria (mt) are the powerhouses of the cell and the main source and scavenger of reactive oxygen and nitrogen species. In recent years, evaluation of mitochondrial function received a broadening attention in studies of many physio-pathological conditions, with increasing relevance in clinical applications. High-Resolution Fluorespirometry (HRFR) provides the state-of-the-art platform for simultaneous measurement of mitochondrial and cell respiration and additional bioenergetic parameters (''i.e.'', hydrogen peroxide production, mitochondrial membrane potential, ADP-ATP exchange, Ca²+ dynamics). Any mitochondrial preparation (mtprep) can be applied, from isolated mitochondria, tissue homogenate, permeabilized muscle fibers, permeabilized cells, to intact cells, to optimally address the variety of research questions. Substrate-uncoupler-inhibitor titration (SUIT) protocols have been evaluated and standardized, since the appropriated design of SUIT protocols is decisive for an accurate evaluation of mitochondrial function and dysfunction. Therefore, we developed the SUIT reference protocol (RP) with the objective to provide a common reference for establishing a comprehensive mtphysiology database. The SUIT-RP consists of two complementary SUIT protocols (RP1 and RP2) which interrogate several mt-pathways converging at the Q-junction, allowing the identification of specific mt-related injuries. Application examples are shown with the SUIT-RP applied in different mtprep including cardiac isolated mitochondria, brain tissue homogenate, cryopreserved cells, permeabilized fibers and human blood cells. In summary, standardized respiratory SUIT protocols combined with additional bioenergetic parameters offer a powerful tool in biomedical research and clinical diagnostics, providing important information about mitochondrial function in health and disease. Mitochondrial function and glycolysis play critical roles in a variety of vital cellular processes, including cellular activation, proliferation, differentiation, cell death, and disease progression. Seahorse Bioscience has developed a technology that enables the measurement of various metabolic parameters and functions using live cells, in real-time, in a microplate. Seahorse Analyzers profile cellular metabolic functions, using label-free, solid-state disposable optical sensors. The Seahorse Analyzers simultaneously measure mitochondrial respiration (oxidative phosphorylation; OXPHOS) via the oxygen consumption rate (OCR), and glycolysis via the extracellular acidification rate (ECAR). Integrated drug injection ports allow for up to 4 reagent additions (e.g. drug or substrate) that can be programmed for automated delivery into the independent cell culture wells. Assay kits and reagents provide standard methods for quantifying mitochondrial respiration, glycolytic activity, endogenous and exogenous fatty acid oxidation, substrate oxidation, and metabolic phenotype. Seahorse XF technology has been applied to multiple research areas, including cancer, obesity, diabetes, metabolic disorders, immunology, cardiovascular function, neurodegeneration, virology, and aging. Knowledge of cellular oxygenation is central to the development physiologically relevant in vitro cell models. It insures a more accurate reflection of the in vivo condition, and facilitates in vitro modeling of the hypoxic events central to many disease states including diabetes, ischemia/ reperfusion injury, and cancer. This is particularly important in tumor metabolism, where oncogene-driven proliferation and aberrant angiogenesis can cause areas of localized hypoxia, activating O2-sensitive pathways, and driving metabolic reprogramming that confers specific survival advantages. Enabling researchers to measure and perturb cellular oxygenation and relate this to other key metabolic and signaling end-points is of critical importance in advancing our understanding of these diseases. Despite its importance, detailed analysis of the interplay between cellular oxygenation, signaling, and metabolic reprogramming is still limited. Indeed, most in vitro assays investigate these relationships without specific knowledge of the actual cellular oxygenation levels driving the signaling events under study. This is in large part due to the absence of methodologies capable of controlling and monitoring oxygenation, while in parallel probing other key metabolic parameters.
Here we address this deficit and describe how, using a nanoparticulate intracellular oxygen probe (MitoXpress®-Intra), cellular oxygenation can be monitored in real-time in conventional microtiter plates on a commercially available fluorescence plate reader. To demonstrate the utility of such an approach, tumor hypoxia and ischemia/reperfusion are modeled in vitro in 2D using a fluorescence plate reader equipped with dynamic atmospheric control. Mitochondria play a central role in cellular (patho)physiology and display a highly variable morphology that is probably coupled to their functional state (Willems, Cell Metab., 2015). Our research aims to quantitatively understand the molecular connection between mitochondrial metabolism and (ultra)structure with particular attention to redox and energy homeostasis (Liemburg-Apers, J. Cell Sci., 2016; Teixeira, Redox Biol., 2018). In primary fibroblasts from Leigh Syndrome (LS) patients, isolated CI deficiency is associated with mitochondrial morphological and functional changes and increased reactive oxygen species (ROS) levels. We developed a high-content ("cellomics") strategy (Iannetti, Nature Protocols, 2016) allowing unbiased and automated quantification of mitochondrial "morphofunction" (i.e. morphology and membrane potential), cellular parameters (size, confluence) and nuclear parameters (number, morphology) in intact living Primary Human Skin Fibroblasts. Cells are cultured in 96well plates and stained with tetramethyl rhodamine methyl ester (TMRM), Calcein-AM and Hoechst 33258. Next, multispectral fluorescence images are acquired using automated microscopy and processed to extract 44 descriptors. Subsequently, the descriptor data is subjected to a quality control (QC) algorithm based upon Principal Component Analysis (PCA) and interpreted using univariate, bivariate and multivariate analysis. Using a machine learning strategy (Blanchet, Sci. Rep., 2015), we discriminated between fibroblasts of a healthy individual and an LS patient based upon their mitochondrial morpho-functional phenotype. This allowed evaluating the therapeutic potential of newly developed small-molecules (Koopman, EMBO Mol. Med., 2016). Substrate oxidation by the Krebs cycle is the major pathway for generation of ATP and reducing equivalents in most cell types of the body. The selection of substrates for oxidation can be influenced by both substrate and oxygen availability as well as through regulation of strategic metabolic enzymes such as pyruvate dehydrogenase or transport into mitochondria such as that of long-chain fatty acids mediated by CPT1A. Meanwhile, the yield of reducing equivalents and/or ATP per mol of substrate oxidized is dependent on functional electron transport and oxidative phosphorylation by the mitochondria. Since the pathophysiology of many diseases may involve alterations in substrate and oxygen availability and/or mitochondrial dysfunction, a holistic analysis of substrate oxidation fluxes and mitochondrial function should provide powerful insights into disease progression and management. The objective of this presentation is to describe 13C-NMR analyses of 13C-enriched substrates metabolism for evaluating Krebs cycle substrate selection in various tissues and propose a framework for integrating this information with measurements of mitochondrial oxygen consumption and ATP generation. Diabetes can lead to diabetic complications affecting all the major systems in the body, and increases the risk of Alzheimer's disease (AD). We have previously shown that diabetes leads to changes in mitochondrial DNA (MtDNA),¹ proposed the use of MtDNA as a biomarker of mitochondrial dysfunction ₂ and demonstrated that changes in MtDNA content are indicative of mitochondrial dysfunction ₃ Since mitochondrial dysfunction has been implicated in AD ₄ , and diabetes increases the risk of AD by 2-fold, we hypothesised that there would be detectable and similar changes in MtDNA in the brain of subjects with both diabetes and AD. ABSTRACTS Therefore, the aim of this work was to determine the combined and individual effect of AD and diabetes on MtDNA content in the human brain. Post-mortem human brain samples, comprising of the frontal cortex, parietal cortex, and cerebellum, were obtained from a total of 74 cases, including healthy controls (HC, n = 30), those with mild cognitive impairment (MCI, n = 10), and those with AD (n = 34). A proportion of cases (4-14) within each group were also diabetic. Absolute MtDNA copy number per cell (MtDNA content) was measured as mitochondrial to nuclear genome ratio, and alterations in MAP2/HuCD-positive neurons, IBA1/AIF1-positive microglia and GFAPpositive astrocyte densities were assessed using mRNA expression and immunohistochemistry.
We found that MtDNA content shows regional variation in the human brain, potentially indicative of differences in bioenergetic need. Both AD and diabetes were found to significantly affect MtDNA content but our data suggests that rather than exacerbate the changes seen in AD, diabetes has the opposite impact on brain MtDNA. The data and the potential implications of these findings will be dis- The term "mitochondrial permeability transition" (MPT) refers to an abrupt increase in the permeability of the inner mitochondrial membrane. MPT drives cell death and provides an etiological to several human disorders characterized by the acute loss of post-mitotic cells. These conditions include ischemia/reperfusion injury, cancer and neurodegenerative disorders. Classically, MPT appears to be mediated by the opening of the so-called "permeability transition pore complex" (PTPC), a poorly characterized and versatile supramolecular entity assembled at the junctions between the inner and outer mitochondrial membranes. The recent key discoveries surrounding the composition of the PTPC, particularly the F1FO ATP synthase, have opened new perspectives into the molecular definition of its role in pathophysiology. During the lecture it will be demonstrated current evidences about molecular structure and regulatory components of PTPC. In particular an attention on new two proteins which recently were added to the list of PTPC components: the mitochondrial F1FO ATP synthase, particularly and the SPG7 paraplegin matrix AAA peptidase subunit will be paid. Detailed overview of MPT contribution to pathological condition will be provided, focusing on the idea that to develop therapeutic drugs, it will be fundamental to understand the molecular composition of the PTPC. Background: Cancer cells without mitochondrial DNA (mtDNA) need to reconstitute oxidative phosphorylation (OXPHOS) by acquisition of host mitochondria to form tumors (1,2), but the reasons why functional respiration is crucial for tumorigenesis remain unclear. Materials and Methods: Using time-resolved analysis of the initial stages of tumor formation by mtDNA-devoid cells and genetic manipulations of OXPHOS components. Results: We show that pyrimidine biosynthesis, supported by the respiration-linked dihydroorotate dehydrogenase (DHODH), is strictly required to overcome cell cycle arrest, while mitochondrial ATP generation is dispensable for tumorigenesis. Primed DHODH is present in mtDNAdevoid cells and becomes fully active by complex III/IV respiration after mitochondrial transfer, or by the introduction of an alternative oxidase. Conversely, DHODH deletion interferes with tumor formation even in cells with functional OXPHOS, whereas disruption of mitochondrial ATP synthase has little or no effect. Conclusions: Collectively, our results show that pyrimidine biosynthesis via DHODH is the essential pathway that links respiration to tumorigenesis. 1. Tan Mitochondria play a pivotal role in neuronal cell survival since they are involved in energy production and function as the metabolic hub of cells. A great body of evidence link mitochondrial dysfunction to Parkinson´s disease etiopathogenesis, where mitochondria abnormalities have been described in the brain and peripheral tissues. Indeed, mitochondrial dysfunction, protein aggregation and neuroinflammation are key features of Parkinson's disease. The endosymbiotic theory argues a bacterial origin for mitochondria, which suggests that they may also signal cells to activate innate immunity. Recent data highlight the role mitochondrial damage-associated molecular patterns in the intracellular signaling that activate innate immunity and inflammation. In fact, it has been demonstrated that neurons are also able to orchestrate innate immune responses against infection and/or injury. We will discuss the effect of bacterial toxins on mitochondrial function, keeping in mind mitochondria's ability to activate neuronal innate immunity to elicit a sterile inflammation. We hypothesize that a dysbiotic gut microbiota will signal the brain through the gut-brain axis targeting the mitochondria and in this way inducing a neurodegenerative process that leads to Parkinson's disease.
W2-L11 | Cross-regulatory mechanisms between mitochondria and lysosomes N. Raimundo; L. Fernandez-Mosquera; K.F. Yambire Universitatsmedizin Goettingen, Goettingen, Germany Mitochondria and lysosomes are interdependent, but the mechanisms underlying the mitochondria-lysosome crosstalk remain unclear. We show here that chronic mitochondrial stress (respiratory chain disruption by stable silencing of a complex III subunit) impairs lysosomal Ca2+ homeostasis, resulting in lysosomal Ca2+ accumulation and swelling, impaired lysosomal acidification and catabolic activity and stalled autophagy. These effects on lysosomal function are rescued by reactivation of the MCOLN1/TRPML1 lysosomal Ca2+ channel. The decrease in MCOLN1 activity under chronic mitochondrial defects is due to a decrease in AMPK activity. Accordingly, reactivation of AMPK or of the MCOLN1 channel rescues lysosomal function, and AMP-Ka1a2 DKO fibroblasts present defects in lysosomal function similar to chronic mitochondrial malfunction. Importantly, the impairment of lysosomal function in the presence of chronic mitochondrial stress is beneficial, as reactivation of lysosomes results in increased cell death. These results show that chronic mitochondrial deficiency exerts a regulatory inhibition on lysosomes, which can be interpreted as a protective mechanism against continuous mitophagy, suggesting that the cells prioritize maintenance of mitochondria, even if defective, over complete clearance of the mitochondrial network. Reciprocally, most lysosomal diseases also present defects in mitochondrial function. We have observed that in Niemann-Pick, a storage disease with lysosomal cholesterol and sphingomyelin accumulation, mitochondrial respiratory capacity is impaired, and, most notably, the transcriptional program of mitochondrial biogenesis is strongly repressed. This repression is mediated by two novel transcription factors, whose silencing rescues both mitochondrial biogenesis and mitochondrial function. These results suggest that under lysosomal dysfunction, cells/tissues actively repress mitochondrial biogenesis, possibly to decrease the need for mitophagy. Our data mechanistically underpins the role of cross-regulation between mitochondria and lysosomes in metabolic diseases as well as other pathologies. Obesity-related type 2 diabetes is a multi-organic disease. Hence, our work ambitions to develop broad, systems-biology approaches to supply new insights and a better understanding of the integrated mechanisms that regulate this predominant disease. For this purpose, a diet-induced obese animal model of T2DM is used. A phenotypical and functional study is being performed both, at systemic level and in pancreas, white adipose tissue, liver, oxidative and glycolytic skeletal muscle, and hypothalamus. Three experimental groups are defined representing the metabolic stages of interest: control group (Ctrl); pathologic group (high-fat diet, HFD, that mimic diet-induced T2DM after 16 weeks on HFD); and a third group (Int) that follows a lifestyle intervention afterwards. The diet-induced obese experimental group (HFD group) reported the typical physiological features of the pathological state: overweight, fasting hyperglycaemia, hyperinsulinaemia and hyperleptinemia, increase in fat mass and volume, increase of white adipose tissue, liver and pancreas weight, increase of liver and oxidative skeletal muscle triglyceride levels, glucose intolerance, insulin resistance, increase in beta-cell mass along with hypertrophic enlarged islets and dysfunction in glucose-stimulated insulin secretion in vivo and in vitro, and a diminishment in oxygen consumption, heat production and scapular temperature. Lifestyle intervention was enough to revert most of the disruptions reported in the pathological group. However, certain irreversibility degree was still observed in some specific parameters: (1) alteration in fasting glucose and (2) white adipose tissue mitochondrial dysfunction, already observed in the pathological state. In summary, our pathological state resembled obesityrelated T2DM phenotype. Lifestyle intervention significantly reverted the pathological state. Furthermore, data integration highlights an important degree of metabolic irreversibility in white adipose tissue, and tissue-specific analysis has enabled us to identify mitochondria-related metabolic derangements as a key biological process implicated in this loss of metabolic plasticity. Ischemia/reperfusion (I/R) injury in liver transplantation can disrupt the normal activity of mitochondria in the hepatic parenchyma. This potential dysfunction of mitochondria bioenergetic after I/R injury could be responsible for the initial poor graft function or primary nonfunction observed after liver transplantation. Thus, determining the mechanisms that lead to human hepatic mitochondrial dysfunction might contribute to improving the outcome of liver transplantation. The availability in the market of several preservation solutions allowed several clinical trials and experimental works comparing all the solutions, however the comparative appraisals are poor; especially when they deal with the underlying protection mechanisms of the fatty liver graft during cold storage. Additionally, various pharmacological strategies have been applied to preserve/improve mitochondrial function, during cold preservation and consequent reperfusion, some of them being very promising in the future. All these beneficial effects would contribute to limit the subsequent extension of reperfusion injury after graft revascularization in liver transplantation procedures. Background: Mitochondrial disorders represent a common cause of morbidity and are more prevalent than previously thought since they are found in approximately 1:4300 of the population; up to date no effective treatments exist. Our aim is to develop a gene therapy with a large spectrum of action to prevent neuronal cell degeneration which is one of the most devastating symptoms of mitochondrial diseases. Therefore, we choose the mouse model of mitochondrial disease due to the depletion of the Apoptosis Inducing Factor (AIF) a mitochondrial protein essential for the organelle biogenesis. These mice known as Harlequin mice exhibit optic neuropathy; to prevent optic neuropathy we use the neuroglobin. This mitochondrial protein is abundant in neurons and plays an important role in their protection probably by its involvement in mitochondrial homeostasis. Material and methods: Adeno-associated viral vector serotype 2 encompassing the neuroglobin gene (AAV2/2-NGB) was administrated in the vitreous body of Harlequin mice aged 4-5 months. At this age the disappearance of retinal ganglion cells, which axons composed the optic nerve, was already started. The visual function of treated mice was evaluated using the OptoMotry devise. Before euthanasia, Flash-Visual Evoked Potentials were performed to monitor the communication from the RGC soma, through the axon, to the visual cortex. Histochemistry and respiratory chain assessments were performed in retinas and optic nerves. Results: The increment of NGB expression prevented respiratory chain complex I failure in optic nerves from treated mice. Moreover the surviving neurons were able to enhance visual cortical function via morphologic changes within the retina. These changes were sufficient to durably protect visual function. Conclusions: The beneficial impact of neuroglobin for neuronal survival and its implication in mitochondrial homeostasis allow envisioning that therapeutic strategies leading to its up-regulation can become a powerful tool for managing neurological disorders.
W2-L15 | The role of mitochondria to explain gender associated differences in aging and ageassociated diseases Mitochondria are the major source of radicals in the aging cell and they are also the major target for damage of these radicals. Females live longer than males in many species including humans. Moreover, most of the age-associated diseases increase their incidence after menopause in women. We found that gender differences in longevity have a biological basis: mitochondria from females produce less free radicals than those from males, because estrogens induce the expression of mitochondrial antioxidant enzymes, and this protects them against oxidative stress, therefore conferring them greater longevity. Then, we tested gender differences in age-associated diseases where mitochondria have been shown to play a role, i.e. in type 2 diabetes and Alzheimer's. Again, we found gender differences in mitochondrial oxidative stress parameters, and a protective role of estrogens, which could explain, at least in part, the lower incidence of these ageassociated diseases in pre-menopausal women compared to men. However, estrogens could have undesirable effects and are feminising. Therefore, we tested the effect of phytoestrogens in our models, and found that they can also activate mitochondrial antioxidant genes at nutritionally relevant concentrations, becoming to possible candidates for treating age-associated diseases such as type 2 diabetes or Alzheimer disease. In conclusion, mitochondria play a role in gender differences in longevity and in age-associated diseases such as type 2 diabetes and Alzheimer's disease.

IRB Barcelona, UB & Ciberdem, Barcelona, Spain
Mitochondrial fusion and fission are key processes that regulate mitochondrial morphology. Mitochondrial fusion is catalyzed by MFN1, MFN2 (Mitofusins) and OPA1 proteins in human cells. MFN2 protein plays a complex set of functions. It regulates mitochondrial morphology, and, in addition, also controls the morphology and function of the endoplasmic reticulum. Expression of MFN2 is exquisitely regulated in tissues. Thus, it is induced in skeletal muscle in response to chronic exercise and after exposure to cold. In contrast, MFN2 is repressed in muscle or in the hypothalamus of mice fed a high fat diet. On the other hand, MFN2 is repressed in type 2 diabetic patients or in obese subjects. In turn, changes in MFN2 expression have a marked impact on mitochondrial metabolism. Skeletal muscles obtained from MFN2 knockout mice show a reduction in respiratory control, glucose oxidation, and expression of some subunits of oxidative phosphorylation. MFN2 deficiency causes major alterations in muscle biology. Thus, skeletal muscle-ablated MFN2 KO mice show susceptibility to develop glucose intolerance and insulin resistance in response to a high-fat diet or to aging. In ABSTRACTS addition, Mfn2-deficient muscle show atrophy and a gene signature linked to aging. In summary, available data indicate that the MFN2 protein regulates metabolic homeostasis, insulin signaling, and maintenance of muscle mass.
W2-L17 | Transcriptomic analysis reveals a distinct control of apoptosis in exceptional aging: role of mitochondria J. Viña

Universidad De Valencia, Valencia, Spain
Centenarians not only reach exceptional longevity, they also evade age-related morbidities and exhibit "successful aging." Using functional transcriptomic analysis (microarrays) of peripheral blood mononuclear cells, we identified 1721 mRNAs expressed by centenarians but not by septuagenarians and young people. A sub-network analysis on these 1721 mRNAs showed six common genes: interferon (IFN)-c (IFNG), T-cell receptor (TCR),tumor necrosis factor (TNF), SP1 transcription factor (SP1), transforming growth factor (TGF)-b1 (TGFb1) and IL-32. These six centenarian-specific genes are related to Bcl-xL, Fas, and Fas ligand all of them involved in the control of apoptosis. Further analysis confirmed that centenarians up-regulate Bcl-xL, have lower plasma cytochrome C levels and higher chemotaxis and phagocytic capacity than septuagenarians and similar to young people. In vitro experiments in mouse embryo fibroblasts (MEFs) demonstrated that cells overexpressing Bcl-xL display increased proliferation, decreased expression of p16Ink4a, p19Arf, and p21cip cell cycle regulators and diminished activity of senescence markers bgalactosidase and Dcr2 compared with control MEFs. Moreover, expression of Bcl-xL, significantly, bestowed cytoprotective effects against oxidative damage. These experiments underpin the role of mitochondrial proteins like Bcl-xL in exceptional aging. The implication of mitochondria in the core mechanisms responsible for aging is a constant theme. Thus, mitochondrial DNA (mtDNA), mitochondrial ROS production and/ or mitochondrial metabolisms in aging have been proposed individually or in combination as the driving force causing aging. A number of animal models targeting different aspects of mitochondrial biology causing progeria syndromes has been develop that support or contradict their role in aging. However, they did not provide sufficient mechanistic inside to reach conclusive answers. Here we have investigated in mice the impact of natural genetic variability and disturbance modulation of mtDNA in physiological aging. By a combination of transcriptomic, proteomic, metabolic and physiological analysis we exhaustively investigated the impact on different tissues and the integrated response at organismal level. We find that the tree levels: mtDNA genetics, mitochondrial ROS production and the impact of mitochondria in the cellular, tissue and organismal metabolism are fully integrated to modulate frailty and healthy aging. Background: Fatty liver disease is one of the most prevalent chronic liver diseases in the world and encompasses both alcoholic and nonalcoholic liver disease. The progression from the initial stage of hepatic steatosis to advanced states, such as steatohepatitis, which is characterized by liver injury and inflammation, is not fully understood. In this regard, the mitochondrial cholesterol pool has emerged as a key factor in this progression. Although StARD1 regulates mitochondrial cholesterol trafficking its role in alcoholic steatohepatitis (ASH) has not been investigated. Material and methods: We floxed StARD1 gene in exons 2-5 to produce mice with StARD1 deletion in hepatocytes (StARD1DHep) or macrophages (StARD1DMac) to examine the contribution of cell-specific StARD1 to cholesterolmediated switch from alcoholic steatosis to ASH. Results: StARD1 floxed mice mice exhibited chronic and acute-onchronic ASH, with severe steatosis, liver injury, fibrosis and inflammation caused by synergism between cholesterol and ethanol. StARD1DHep mice but not StARD1DMac mice were resistant to cholesterol plus ethanol-mediated ASH. StARD1DHep mice were refractory to early mitochondrial cholesterol accumulation and mitochondrial GSH depletion caused by cholesterol plus ethanol. Sab (SH3BP5) expression and mitochondrial translocation of phospho-JNK (p-JNK) was similar in StARD1DHep and StARD1 floxed mice. Mitochondrial GSH recovery by GSH ethyl ester protected StARD1 floxed mice against cholesterol plus ethanol induced ASH. Moreover, mitochondria isolated from liver explants of patients with alcoholic cirrhosis exhibited increased StARD1 expression, mitochondrial cholesterol accumulation and GSH depletion as well as enhanced mitochondrial Sab levels and p-JNK translocation. Conclusions: These findings underscore a key role for hepatocyte StARD1 in mitochondrial cholesterol trafficking and indicate that its targeting may be of potential relevance in ASH. Background: Western lifestyle-associated high-fat and high-sugar malnutrition ("Western diet" (WD)) causes steatosis that may progress to liver inflammation. Mitochondrial dysfunction has been suggested as a key factor in promoting this disease. Methods: We have molecularly, biochemically and biophysically analyzed mitochondria from WD-fed steatotic mice.
Results: WD-mitochondria demonstrated lipidomic changes, a decreased mitochondrial ATP production capacity and a significant sensitivity to calcium in comparison to mitochondria isolated from mice fed a normal diet. These changes preceded hepatocyte damage and were not associated with enhanced mitochondrial ROS production. However, upon further mechanistically different pathological impacts on WD-mitochondria severe liver damage occurred. Conclusions: WD-mitochondria do not promote steatohepatitis per se, but demonstrate bioenergetic deficits and increased sensitivity to stress signals. These findings may explain why steatosis is mostly reversible in the majority of patients, but also suggests that second pathological hits on mitochondria may severely aggravate liver damage in affected patients. Altered lipid homeostasis in liver is associated with the promotion of hepatic damage, ranging from nonalcoholic steatohepatitis to hepatocarcinoma. Molecular and cellular mechanisms underlying liver damage, malignant transformation of hepatocytes and hepatocarcinoma development in response to lipotoxicity remain poorly known. Mitochondrial function is involved in lipid homeostasis management in liver. ARMCX3 is a member of the ARMC/ARMCX family of proteins, known to be involved in mitochondrial biology regulation. ARMCX3 expression in liver is strongly regulated in response to metabolic challenges, being strongly up-regulated after experimental high-fat diet treatment or pharmacologically-induced hepatic steatosis, but repressed after starvation. Targeted deletion of ARMCX3 protects mice from the development of DEN-induced hepatocarcinoma, and the protective effects are specially pronounced for the capacity of a high-fat diet to enhance DEN-induced hepatocarcinogenesis. In fact, ARMCX3-null mice show protection against the hepatic steatosis and metabolic derangements elicited by a high fat diet. A relevant component of the systemic metabolic protection caused by ARMCX3 invalidation appears to be the induction of the browning of adipose tissue and the subsequent enhancement of an extra-hepatic site of lipid oxidation. This is consistent with the reciprocal relationship between ARMCX3 expression and the extent of thermogenic activation in adipose tissues under distinct adaptive experimental conditions. ARMCX3 is emerging as a novel mitochondrial actor in the control of lipid metabolism, with consequences in the lipid management in liver and the propensity of hepatic damage in response to metabolic insults.
Background: Electrical isolation of fastest-activated atrial fibrillation (AF) drivers has been proven as an effective therapy for drug refractory AF patients. High-frequency atrial regions can be identified by different techniques as intracardiac basket mapping or non-invasive recordings (electrocardiographic imaging, ECGi), yet they have not been compared in the same patients. This study compares endocardial vs non-invasive frequential maps in simultaneous recordings on a large cohort of AF patients. Material and methods: Intracardiac electrograms of 29 AF patients (45% persistent AF) were acquired with one 64-pole basket catheter in each atrium simultaneously to 57-lead body surface recordings. Atrial and torso anatomy were reconstructed by using segmented magnetic resonance images. ECGi signals were obtained from surface signals by using zero-order Tikhonov regularization after software QRST subtraction. We used Dominant Frequency (DF) analysis to identify high activation frequency regions in both endocardial and non-invasive signals. Results: Non-invasive measures of DF agreed with endocardial measurements: they presented an absolute deviation of 1.7 Hz [0.5-3.4] vs invasive recordings, consisting on a relative deviation of 42% . ECGi high-frequency regions (defined as <1 Hz from fastest recording) presented a higher agreement with endocardial measurements than slower-activated regions both in absolute ( Biomarkers have emerged as novel tools for diagnosis and treatment in heart failure (HF) over the past 15 years. Some of them are already included in international guidelines. Indeed, natriuretic peptides -NP-(BNP and NT-proBNP) are clearly indicated for HF diagnosis; and NP and high-sensitivity troponins also provide value in prognostication. Their value to tailor therapeutic strategies is far more controversial. The use of NPs to guide therapy has been tested in numerous clinical trials in the acute and chronic setting with controversial results. The most recent GUIDE-IT and PRIMA-2 trials, in chronic and acute HF respectively, demonstrated negligible value of the NPguided therapy. Other biomarkers have emerged in recent years, of which ST2 is the most promising. On one hand, it reflects three pathobiological pathways related to HF, namely inflammation, stretch and extracellular remodelling. Further, its circulating concentrations are not significantly influenced by age, renal dysfunction or BMI, all of them impactful in NPs concentrations. Thus, ST2 is a robust biomarker for HF prognostication, in both acute and chronic HF, and it has been acknowledged as the new gold standard. Preliminary evidence also suggests that ST2 concentrations may be modified by targeting specific treatment, including beta-blockers and MRA. The advent of canakinumab, an IL-1b antagonist, as a powerful new therapeutic tool in the cardiovascular arena, increases even further the potential of ST2, which is the soluble part of IL-1 receptor-1. In sum, the HF field is facing the move from one-fits-it-all therapies to tailored treatment according to biological phenotyping. In this process biomarkers will become main actors.
W3-L9 | Towards a personalized therapy of myocardial fibrosis in heart failure J. Diez

University of Navarra, Pamplona, Spain
One hallmark myocardial lesion in heart failure (HF) is the diffuse accumulation of collagen type I fibers (ie, myocardial fibrosis [MF]). MF is mainly the result of the predominance of collagen type I synthesis (mediated by enzymes such as procollagen type I carboxy-terminal proteinase [PCP]) and cross-linking (mediated by enzymes such as lysyl oxidase [LOX]) over its degradation (mediated by enzymes such as matrix metalloproteinases) as a result of the actions of myofibroblasts. MF leads to left ventricular dysfunction, arrhythmias, and poor outcomes, underscoring the need to integrate it into diagnosis and management of patients. The question is how to identify those HF patients with increased myocardial PCP/LOX-mediated collagen type I synthesis and cross-linking and thus who would be susceptible to a personalized treatment aimed to inhibit the PCP/ LOX axis. Recently, we have demonstrated that a combination of 2 circulating biomarkers (ie, high serum carboxyterminal propeptide of procollagen type I [PICP], and low serum collagen type I telopeptide to serum matrix metalloproteinase-1 (CITP: MMP-1) ratio) identifies with good sensitivity and specificity HF patients with histologically proven excessive myocardial collagen type I synthesis and cross-linking, which account for approximately one third of all HF patients. Furthermore, HF patients presenting with the combination of the 2 biomarkers exhibited the worst outcome (ie, high risk of HF hospitalization or mortality). The use of panels combining circulating (and/or imaging) biomarkers of MF may contribute to personalize HF therapy. From this perspective and as an example, it can be hypothesized that therapies able to regress MF as a result of reductions in both synthesis and cross-linking of collagen type I fibers (e.g. torasemide) may be specifically beneficial for HF patients presenting with the combination of ABSTRACTS high serum PICP and low serum CITP: MMP-1 ratio. This hypothesis remains to be tested in an adequately designed trial.

W3-L10 | Innovation in electrophysiology
The objective of this presentation is to review the most recent advances in the clinical recognition and treatment of cardiac arrhythmias. As the most prevalent arrhythmia, atrial fibrillation keeps generating a huge number of clinical and basic research studies. Radiofrequency ablation is widely used in patients with AF although it has an heterogeneous rate of success and some uncertainty predicting arrhythmia recurrences. These limitations have prompted to develop innovative diagnostic and therapeutic therapies in a short period of time. Among the most relevant achievements we shall consider the development of very high density mapping systems implemented in sophisticated electromagnetic cardiac navigators such as Carto3, Rhythmia or Ensite Navex. These systems have permitted to explore and identify new anatomical structures and regions that may potentially be the origin of arrhythmogenic foci. Examples of these new target regions for RF ablation are the sinoatrial nodal region, the Marshall's ligament, or the pulmonary sinus cusp in patients with right ventricular outflow tract arrhythmias. The ultra-high-resolution mapping of the superior vena cava spiral activation may also help in the caval isolation in patients with atrial fibrillation. The 3-dimensional noncontact left atrial mapping during AF allows examining the prevalence and location of rotor activation and a successful ablation in a stepwise fashion. Moreover, mapping of the activity of papillary muscle is being attempted in patients with ventricular arrhythmias. The neural interaction with cardiac arrhythmias has been the subject of relevant technical advances that permit recording noninvasively the neural activity of the autonomic nervous system and that of the cardiac intrinsic plexuses during arrhythmia episodes. Cardiac denervation techniques have been also developed to treat patients with recurrent ventricular or atrial arrhythmias. The use of antiarrhythmic drugs and the effects of atrial fibrillation on the structure and cognitive function of the brain are also reviewed. Background: Several inflammatory molecules have been recently identified to play a promising role as prognostic biomarkers in patients with severe carotid plaque stenosis. Material and methods: This scientific overview was based on the works detected on PubMed and MEDLINE up to January 2018. Results: Among systemic mediators, serum levels of osteopontin, adiponectin, lipoprotein(a) [Lp(a)] and the anti-apolipoprotein A-1 auto-antibodies were found associated with major adverse cardiovascular events after carotid endarterectomy (CEA). Considering intraplaque mediators, the expression levels of some receptors (i.e. the cannabinoid type 2 receptor), or inflammatory molecules (i.e. C-reactive protein) were strongly associated with plaque vulnerability. Investigations targeting both intraplaque and systemic cytokines and chemokines have been recently performed in animal models of carotid stenosis and might be promising for being tested in human pathophysiology. Conclusion: Although still preliminary evidence exists, the assessment of inflammatory molecule expression both in the systemic circulation and within carotid plaques might represent a promising prognostic tool in patients with severe carotid atherogenesis after CEA. Additional larger clinical trials are needed to support evidence from observational studies.

F. Mach
Geneva, Switzerland W3-L16 | Conditioning the heart G. Vilahur Barcelona, Spain Ischemic heart disease, mainly presented as ST-segment elevation myocardial infarction (STEMI) due to complete occlusion of an epicardial coronary artery, remains the leading cause of death and disability worldwide. Despite timely reperfusion, the number of surviving patients going on to develop heart failure is increasing. There is a need to find therapeutic and/or pharmacological interventions targeted to attenuate the detrimental effects of acute myocardial ischemia/reperfusion injury and to prevent the development of adverse left ventricular overall reducing the burden of heart failure. Intentional exposure to brief periods of ischemia and reperfusion including local and remote ischemic pre-, perand post-conditioning, have demonstrated consistent cardioprotective potential in experimental models of acute ischemia/reperfusion injury. Their translation to the clinical scenario, however, has been challenging. On the other hand, numerous evidences from experimental studies have suggested that several ischemic-post-conditioning mimicking drugs exert cardioprotective effects limiting the size of infarction and improving coronary flow. Yet, so far, none has shown to be successful in Phase-III clinical trials. Besides optimizing the design of both experimental studies and clinical trials, there is an urgent need to find new cardioprotective targets. In this regard, implementation of systems biology approaches and "omic" technologies not only have further enhanced our understanding of the molecular events triggered during ischemia and further reperfusion but have opened new venues for the identification of novel molecules and signalling pathways with cardioprotective potential. Advanced age increases the extent of myocardial infarcts. The increased vulnerability of the senescent heart to cell damage has been reproduced in isolated cardiomyocytes, in which the contribution of cell-to-cell interaction, extracellular components, vascular factors and comorbidities is absent. Experimental studies indicate that mitochondria play a prominent role in cell dysfunction during aging. Recently, mitochondrial ATP synthase has been proposed to conform the molecular entity of mitochondrial permeability transition (mPTP). We investigated the contribution of aging on the accumulation of advanced glycation end-products in the senescent heart and their impact on mitochondrial damage and ATP synthase. Our results disclosed a significant increase in AGEs content both in human and murine myocardium of aged individuals secondary to deficient glyoxalasedependent detoxification system. Proteomic analysis identified mitochondrial ATP synthase as one of the target proteins of glycative damage. Mitochondria from human and murine aged hearts exhibit a reduction in ADP-dependent O2 consumption. In mouse cardiomyocytes submitted to transient ischemia-reperfusion, glycative damage of mitochondrial ATP synthase is associated with more pronounced failure in mitochondrial energy recovery upon reperfusion and higher susceptibility to undergo mPTP. These effects were paralleled by an increased cell death. These results identify a new pathophysiological mechanism that may underlie the increased vulnerability of senescent heart to ischemia-reperfusion damage. The translation from numerous successful animal experiments on adjunct cardioprotection beyond that by reperfusion to clinical practice has so far been disappointing. Animal experiments often use reductionist approaches and are mostly performed in young and healthy animals which lack the risk factors, co-morbidities and co-medications which are characteristic of patients suffering an acute myocardial infarction or undergoing cardiovascular surgery. Conceptually, it is still unclear by how much the time window for successful reperfusion is extended by preconditioning, and how long the duration of ischemia can be so that adjunct cardioprotection by postconditioning at reperfusion still protects. Experimental studies addressing long-term effects of adjunct cardioprotection beyond infarct size reduction, i.e. on repair, remodeling and mortality are lacking. Technically, reproducibility and robustness of experimental studies are often limited. Faults in design and ABSTRACTS | 35 conduct of clinical trials have also substantially contributed to the failure of translation of cardioprotection to clinical practice. Cardiovascular surgery with ischemic cardioplegic arrest is only a surrogate of acute myocardial infarction and confounded by the choice of anesthesia, hypothermia, cardioplegia and traumatic myocardial injury. Trials in patients with acute myocardial infarction have been performed on agents/interventions with no or inconsistent prior animal data and in patients who had either some reperfusion already at admission or were reperfused too late to expect any myocardial salvage. Of greatest concern is the lack of adequate phase II dosing and timing studies when rushing from promising proof-of-concept trials with surrogate endpoints such as infarct size to larger clinical outcome trials. Future trials must focus on interventions/agents with robust preclinical evidence, have solid phase II dosing and timing data and recruit patients who have truly a chance to benefit from adjunct cardioprotection. Discovery of the hepatokine FGF21 (Fibroblast Growth factor 21) and its effect on glucose homoeostasis has highlighted the importance of the liver in development of novel diabetic therapies. While the physiological benefits of FGF21 have been documented, little information is available on the regulation of FGF21 in the context of glucose overload. New developments have reported the importance of the glucose sensitive transcription factor ChREBP (Carbohydrate Responsive Element Binding Protein) for the regulation of FGF21 in response to glucose and fructose. Microarray analysis in liver confirms that few genes respond in the same unique fashion as FGF21 to fasting and glucose feeding. Here we report that PPARa, a key nuclear receptor for the adaptive fasting response, is required together with ChREBP, to balance the glucosemediated FGF21 response. In vitro and in vivo analyses reported lack of FGF21 in ChREBP-/-mice. Rescue of ChREBP activity in liver of ChREBP-/-mice through adenoviral gene transfer fully restored FGF21 expression and secretion in these mice. Unexpectedly, carbohydrate challenge of liver specific PPARa knockout mice, demonstrates a PPARa-dependent glucose-mediated FGF21 response. In vivo ChIP experiments reveal that in the absence of PPARa, ChREBP binding to the Fgf21 ChoRE binding site is decreased demonstrating, for the first time, a PPARa-ChREBP crosstalk in response to glucose. Stimulation of mouse and human primary hepatocytes with a combination of glucose and Wy-14643, a PPARa agonist, resulted in a synergetic effect on Fgf21 mRNA. Physiologically, decreased circulating FGF21 levels in liver specific PPARa knockout mice led to increased sucrose preference. In conclusion, our study reports that ChREBP and PPARa codependently regulate the glucose-mediated induction of FGF21. Future studies will identify the molecular link between ChREBP and PPARa and will determine whether other key hepatokines are dependent on the synergistic action of ChREBP and PPARa. Health Related Quality of Life (HRQoL) is defined as functional effect of medical condition/therapy upon a patient. It is subjective and multidimensional. In liver conditions it has been best studied in viral hepatitis and endstage liver disease. The data are less impressive in terms of autoimmune liver diseases (AILD), autoimmune hepatitis in particular. Primary biliary cholangitis (PBC). Unlike in other AILD, there is an internationally validated HRQoL questionnaire called PBC-40. It contains six domains including fatigue and pruritus. Large studies showed that HRQoL does not depend on disease severity (i.e. whether patient is cirrhotic or not) and response to ursodeoxycholic acid. Younger patients seems to have worse quality of life and social dysfunction is the greatest contributor to HRQoL. Patients who are AMA negative score worse in social and emotional domains of PBC-40 and experience more itching. Primary sclerosing cholangitis (PSC). Pruritus may be the greatest negative contributor to HRQoL. Unlike in PBC, cirrhotic patients have worse quality of life. Also females suffer from worse HRQoL but inflammatory bowel disease does not seem to affect significantly their well being. PBC-40 questionnaire, designed for PBC may also of use in PSC as fatigue/pruritus are crucial factors affecting life`s quality in both conditions. A dedicated questionnaire for PSC, a 42-item PSC-PRO form has been recently proposed but it needs further validation.
Autoimmune hepatitis (AIH). The data are very scanty. Patients with AIH have significantly worse HRQoL than matched controls. Depression and anxiety occur significantly more common than in general population. These patients are also more fatigued, this symptom may be more pronounced in females. Severe depression seems to affect more commonly female patients. When patients with PBC, PSC and AIH are compared by far the worse quality of life is reported in PBC.
W4/W7-L3 | The burden of fatty liver disease in an aging population

E. Bugianesi
Department of Medical Sciences, University of Torino, Torino, Italy NAFLD is one of the most important causes of liver disease worldwide and will probably emerge as the leading cause of end-stage liver disease in the coming decades, with the disease affecting both adults and children. The epidemiology and demographic characteristics of NAFLD vary worldwide, usually parallel to the prevalence of obesity, although a substantial proportion of patients are lean. The total NAFLD population in USA in 2015 was estimated at 83.1 million with a prevalence rate of 30.0% among the population aged ≥15 years and 25.8% among all ages. The reported ratio of male to female prevalence varied by age group, with the lowest male/female ratio among individuals <30 years and the highest among individuals aged 40-49 years. Prevalence of NAFLD in 2030 is estimated at 33.5% (aged ≥15 years) and 28.4% (all ages). The median age of the NAFLD population was estimated at 50 years of age (2015), increasing to 55 years (2030). The large number of patients with NAFLD with potential for progressive liver disease creates challenges for screening, as the diagnosis of NASH necessitates invasive liver biopsy. While awaiting the development effective therapies, this disease warrants the attention of primary care physicians, specialists and health policy makers. Background: Acute and chronic liver diseases are accompanied by cholestasis. Intrahepatic accumulation of bile acids (BAs) causes hepatocytes and cholangiocytes death. Liver injury elicits a potent protective/regenerative response. However, when this response chronifies fibrosis and tumorigenesis may ensue. Understanding this reaction is required to devise hepatoprotective, antifibrogenic and antineoplastic therapies. The epidermal growth factor receptor (EGFR) signaling system is essential for regeneration after liver injury, including cholestatic injury. EGFR is activated by various ligands, among which amphiregulin (AR) is a key mediator of liver regeneration. We studied AR's role during cholestatic liver injury and the mutual regulation of AR expression and BA synthesis. Method: We used two models of cholestatic liver injury: bile duct ligation (BDL) and oral alpha-naphtyl-isothiocyanate (ANIT) administration in wild type (AR-WT) and AR knockout (AR-KO) mice. AR expression was examined in: (i) livers from patients with primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC); (ii) mice and cultured liver cells treated with BAs; and (iii) farnesoid X receptor knockout mice (FXR-KO) after BDL. In vitro and in vivo AR-mediated cytoprotection was evaluated. Results: Hepatic AR expression was up-regulated in PBC and PSC patients. Oral BA administration to mice induces ileal and hepatic AR expression, and cholestyramine feeding reduces postprandial ileal and liver AR upregulation. AR-KO display higher Cyp7a1 expression and intrahepatic BA concentrations than AR-WT. Liver AR expression was markedly enhanced in BDL and ANIT groups of AR-WT. Liver damage was higher in AR-KOs. BAs induced AR expression in cultured liver cells partially through FXR. Consistently, after BDL FXR-KO show reduced liver AR expression than FXR-WT. AR protected from BDLinduced liver injury and from BAs toxicity in cultured liver cells. ABSTRACTS | 37 Conclusion: AR participates in physiological BA homeostasis. Liver AR expression increases during cholestasis partially through FXR. AR protects the liver from BAinduced toxicity W4/W7-L5 | More metabolic syndrome after cholecystectomy? A lesson from bile acid (BA) function

P. Portincasa
Department of Biomedical Sciences & Human Oncology, University "Aldo Moro" of Bari Medical School, Bari, Italy Background: The gallbladder physiologically concentrates and stores bile during fasting and provides rhythmic bile secretion both during fasting and in the postprandial phase to solubilize dietary lipids and fat-soluble vitamins. About 15% af adult population worldwide develop cholelithiasis, and a subgroup (20%) will develop symptoms i.e., biliary colic or gallstone-related complications. Whereas the pathogenesis of cholesterol gallstones depends on metabolic abnormalities (i.e., genetic background, excess secretion and aggregation/crystallization of hepatic cholesterol in bile, gallbladder stasis and increased absorption of cholesterol from the small intestine), the pathogenesis of pigment stones is due to abnormalities in the bilirubin metabolism in the gut-liver axis. BAs, together with cholesterol and phospholipids, are major lipid components of bile. BAs play a key role as signaling molecules in modulating gene expression related to cholesterol, BA, glucose and energy metabolism. Cholecystectomy remains the standard surgical procedure in patients with symptomatic gallstones of any type, and is largely performed worldwide. Cholecystectomy per se, however, might cause abnormal metabolic consequences, i.e., alterations in glucose, insulin (and insulinresistance), lipid and lipoprotein levels, liver steatosis and the metabolic syndrome. Mechanisms are likely mediated by the post-cholecystectomy abnormal transintestinal flow of BAs, producing metabolic signaling that acts without gallbladder rhythmic function and involves the BAs/farnesoid X receptor (FXR) and the BA/G protein-coupled BA receptor 1 (GPBAR-1) axes in the liver, intestine, brown adipose tissue and muscle. Conclusions: Alterations of intestinal microbiota leading to disturbed biotransformation of primary to secondary BAs, and distorted homeostatic processes are also possible. Thus, cholecystectomy, by inducing BA "hormonal" changes in the enterohepatic circulation, becomes either a risk factor, or an aggravating factor for the metabolic syndrome. Proper functioning of the endoplasmic reticulum (ER) is vital for cellular physiology since this organelle serves for the correct folding of cellular proteins. Metabolic stress caused by high-fat diet feeding stresses the ER, resulting in accumulation of unfolded proteins within the ER lumen referred to as ER stress. ER stress activates the adaptive unfolded protein response (UPR) to restore ER homeostasis by attenuating general protein synthesis, and, concomitantly, upregulating selective synthesis of key proteins that increase the capacity of the ER to fold and process these proteins. In a recent study*, we have found that the RNA-binding protein CPEB4 (cytoplasmic polyadenylation element-binding protein-4) protects against fatty liver caused by uncontrolled ingestion of fats by regulating a new adaptive branch of the UPR that helps to re-establish cell equilibrium. Thus, hepatic CPEB4 expression is upregulated in response to ER stress in a dual-mode manner. First, CPEB4 mRNA transcription is controlled by the circadian clock and then, its translation is regulated by the UPR through upstream open reading frames (uORFs) within the 5 0untranslated regions. Hence, CPEB4 protein is synthesized only upon ER-stress, but the induction amplitude is circadian. In turn, CPEB4 activates a second wave of UPRtranslation required to maintain ER homeostasis. This "clean-up" mechanism orchestrated by CPEB4 varies in function of the time of day, being more active in humans during the day (when the liver has most work) and dropping off at night. These results suggest that combined transcriptional and translational CPEB4 regulation generates a "circadian mediator", which coordinates hepatic UPR activity with periods of high ER protein-folding demand. Accordingly, in the absence of CPEB4, the liver becomes hypersensitive to steatosis induced by nutritional excess. For many years obesity has been regarded as the main risk factor for the fatty liver disease (FLD). Non-obese individuals have been, in turn, thought to only rarely suffer from FLD (Bellentani et al., Liver Int. 2017). Nevertheless, numerous studies indicate that hepatic steatosis develops also in the absence of obesity or metabolic disturbances. For example, the prevalence of NAFLD in a large population of lean North-American individuals was reported at 7.4% (Younossi et al., Medicine (Baltimore) 2012), some studies estimated that the prevalence of non-obese NAFLD worldwide might even be as high as 30%. Thus, FLD is prevalent and relevant even in non-obese individuals. The pathogenesis of fatty liver is multifactorial and involves both exogenous and inherited determinants. Our most recent analysis of non-obese and non-diabetic German patients with fatty liver demonstrated that common prosteatotic variants, especially the PNPLA3 p.I148M polymorphism, are highly prevalent in these patients (Krawczyk et al., J Huma Genet in press). Overall, as compared to healthy controls, non-obese patients with FLD seem to suffer from more insulin resistance and more hypertriglyceridemia even in the absence of obesity. Of note, alike obese patients, they have increased risk of developing nonalcoholic steatohepatitis (NASH) as well as cirrhosis and hepatocellular carcinoma. Here we will present the most recent data concerning the prevalence and pathogenesis of fatty liver in non-obese. We will also address the topic of therapy and discuss the involvement of inherited predisposition in the overall FLD risk in lean individuals. Background: In recent years, the implication of different epigenetic processes in the development of obesity is being extensively investigated. Taste and olfaction are important sensorial factors influencing food preferences, appetite, and eating behaviors. Epigenetic modifications in genes regulating taste and olfactory perceptions may account for differences in dietary intakes, and subsequently weight gain. Material and methods. A nutriepigenomic analysis was conducted in adults from the Methyl Epigenome Network Association (MENA) project (n = 474). DNA methylation was measured in circulating white blood cells by a microarray strategy (Infinium Human Methylation 450K BeadChips). Pathway mapping of genes involved in taste and olfactory transduction was performed using the KEGG reference database. Additionally, pathway enrichment analyses were further run in the pathDIP platform. Anthropometric measurements, the blood metabolic profile, and nutritional intake were analyzed. Results. Overall, 28 CpG sites at taste (n = 12) and olfactory (n = 16) genes were strongly associated with body mass index (P < 0.0001). These genes significantly contributed to the regulation of taste and olfaction transduction, affecting key processes such as taste and odor detection, and signal transmission to the nervous system. In addition, sweet taste receptor (TAS1R2) methylation correlated with total energy and carbohydrate intakes, meanwhile olfactory receptors (OR4D2 and OR2Y1) methylation levels correlated with daily intakes of total energy, carbohydrates, protein, and fat. Conclusions. Epigenetic-environmental interactions might be involved in the regulation of taste and olfaction and dietary intake. Setting an epigenetic basis for taste and olfactory function may help to understand, at least in part, relationships between sensory capacity, food consumption and body weight regulation. In turn, this knowledge may contribute to identify epigenetic biomarkers to predict the risk of developing excessive adiposity and associated comorbidities, as well as implement targeted epigenome-based strategies for prevention, prognosis and treatment of obesity within the era of precision nutrition. associated with an increased risk for many obesity-related diseases. Little is known about alterations to the cytoskeletal networks, yet remodelling of actin microfilaments, microtubules and vimentin intermediate filaments is essential for adipogenesis and for lipid accumulation during adipocyte hypertrophy. Recently, a fourth component of the cytoskeleton, septins, has been recognized (Mostowy & Cossart, 2012). These GTP-binding proteins assemble into filaments or rings and act as scaffolds for recruiting proteins and as membrane diffusion barriers, participating in cytoskeletal organization, vesicular transport, autophagy, and membrane remodelling (Menon & Gaestel, 2017). Misregulation of septins leads to human diseases such as neurodegenerative disorders and cancer. Material & Methods: Herein, we will discuss our work on septin 11 (SEPT11), which was identified by us in a proteomic screening of human adipose tissue (Moreno-Castellanos et al., 2015). Results: Our studies show that SEPT11 forms filaments and rings that associate with actin during adipocyte differentiation and to caveolae clusters (i.e., rosetta) in mature adipocytes, respectively. Indeed, SEPT11 associates with caveolin-1 in caveolae and redistributes, together with caveolin-1 and the lipid chaperone, FABP5, to the lipid droplet surface upon exposure of adipocytes to fatty acids. Moreover, SEPT11 silencing impairs insulin signalling and lipogenesis, indicating a role for the SEPT11 network in lipid traffic and accumulation in adipocytes. This role is consistent with our findings showing that SEPT11 in the adipose tissue is increased in human obesity and related to adipocyte size in omental fat and to insulin resistance markers in subcutaneous fat. The past decade has witnessed an impressive increase in our appreciation of the importance of inflammation in metabolic disease. Growing evidence implicates neuroinflammation in the pathogenesis of diet-induced obesity and cognitive dysfunction in rodent models. In humans, it has been recently shown how circulating markers of inflammation associated with brain white matter integrity and working memory/short-term verbal memory in obese and nonobese subjects. On the other hand, gut microbiota could constitute a critical modulating factor. The Shannon index (indicating biodiversity of the gut microbiota) has been found to be linked to the microstructure of the hypothalamus, caudate nucleus, and hippocampus, suggesting sparing of these brain structures with increased bacterial biodiversity. Microbiota profile also clustered with cognitive function. The relative abundance of Actinobacteria phylum was linked not only to magnetic resonance imaging diffusion tensor imaging variables in the thalamus, hypothalamus, and amygdala but also to cognitive test scores related to speed, attention, and cognitive flexibility. In addition, iron accumulates in the brain of obese subjects. A significant increase in iron load was detected at the caudate nucleus, lenticular nucleus, hypothalamus, hippocampus and liver of obese subjects, that was independently associated with worse cognitive performance. In sum, inflammation, gut microbiota composition and iron constitute novel potentially modifiable factors influencing brain microstructure and cognition. Introduction: Aging causes a progressive loss of muscle mass and strength called sarcopenia. In addition, sarcopenia maintains a chronic inflammatory state, causes chronic disease, and ultimately increases mortality. Methods and Results: Recent findings suggest that there may be an association between insulin resistance and systemic inflammation with sarcopenia and Obesity together, for example the serum hs-CRP levels were significantly increased by obesity. As regards the cognitive decline, there are studies that revealed that the Sarcopenia and Obesity conditions was significantly associated with the increased risk for cognitive decline, independent of age, gender, and education level. Recent studies provide preliminary evidence to support the hypothesis that insulin resistance is an important mediator to consider in the association between sarcopenia, obesity, and cognitive functioning. Regarding the bone mineral density, as suggested by a recent study performed in community-dwelling older men, the Sarcopenic Obesity is associated with increased fall rates over 2 years, and (SO) men have increased fracture risk over 6 years compared with non-(SO) men. In this way it has been defined Osteosarcopenic obesity (OSO) that is a multifactorial syndrome that include the following conditions: decrease of muscle mass and bone (osteopenia/osteoporosis, sarcopenia) and an increase of adiposiy (obesity) As regards the condition of adipositity, related to sarcopenia and osteoporosis, until now any study has considered the role of visceral fat in this condition. As matter of fact no studies show the effects of visceral of subcutaneous fat as adverse prognostic factors in sarcopenic patients. Insulin resistance plays an important role in muscle fiber atrophy and mitochondrial dysfunction. Also Intermuscular adipose tissue (IMAT) has been observed in the skeletal muscles of older adults with sarcopenia. Conclusion: Most mechanisms of sarcopenia are also associated with visceral obesity, which may lead to a vicious cycle of intricate interactions among risk factors. Design thinking (DT) is an innovation and creative problem-solving methodology, that has demonstrated its power in business. The most important thing that DT brings to any field of innovation is the HCDhuman-centered design. With this new vision, we are able to put people in the middle of the innovation. Through the main phases of this methodology that are: Empathy, Define, Ideate, Prototype, and Test, we will be able to create better solutions for wicked problems. Some people compare it with the scientific method (try &error). So, some authors have decided to capture the essence and main tools from this methodology, to redesign medical education and investigation, and take advantage of its characteristics. This new vision could bring new ideas, new rules and even new ways of collaborating between different areas of medicine and science. From Medical Education department in Universidad de Navarra, we are working with this methodology trying to innovate and redesign the curriculum, by using tools and essence from DT, and putting the students in the center of this new era. Learning Outcomes are: 1-Understand the potential benefits of this innovation and Creative Problem Solving methodology. 2-Identify the main steps of Design Thinking. 3-Analyze the opportunity that brings Design Thinking in healthcare/medical education and investigation. Methodology: Combining interactive lecture with practical tools and games we will create the perfect environment to learn from/with this methodology and its capabilities. Results: The attendees in this workshop will discover the key concepts of Design Thinking by playing with some of the main tools from this methodology and the end we hope the will open their mind about new ways of innovating in medical education and investigation.