A dataset of dual calcium and voltage optical mapping in healthy and hypertrophied murine hearts

Pathological hypertrophy underlies sudden cardiac death due to its high incidence of occurrence of ventricular arrhythmias. The alteration of transmural electrophysiological properties in hypertrophic cardiac murine tissue has never been explored previously. In this dataset, we have for the first time conducted high-throughput simultaneous optical imaging of transmembrane potential and calcium transients (CaT) throughout the entire hypertrophic murine hearts at high temporal and spatial resolution. Using ElectroMap, we have conducted multiple parameters analysis including action potential duration/calcium transient duration, conduction velocity, alternans and diastolic interval. Voltage-calcium latency was measured as time difference between action potential and CaT peak. The dataset therefore provides the first high spatial resolution transmural electrophysiological profiling of the murine heart, allowing interrogation of mechanisms driving ventricular arrhythmias associated with pathological hypertrophy. The dataset allows for further reuse and detailed analyses of geometrical, topological and functional analyses and reconstruction of 2-dimensional and 3-dimentional models.

There Is Strength in Numbers: Quantitation of Fc Gamma Receptors on Murine Tissue-Resident Macrophages

Many of the effector functions of antibodies rely on the binding of antibodies/immune complexes to cellular Fcγ receptors (FcγRs). Since the majority of innate immune effector cells express both activating and inhibitory Fc receptors, the outcome of the binding of immune complexes to cells of a given population is influenced by the relative affinities of the respective IgG subclasses to these receptors, as well as by the numbers of activating and inhibitory FcγRs on the cell surface. A group of immune cells that has come into focus more recently is the various subsets of tissue-resident macrophages. The central functions of FcγRs on tissue macrophages include the clearance of opsonized pathogens, the removal of small immune complexes from the circulation and the depletion of antibody-opsonized cells in the therapy of autoimmunity and cancer. Despite these essential functions of FcγRs on tissue-resident macrophages, an in-depth quantification of FcγRs is lacking. Thus, the aim of our current study was to quantify the various Fcγ receptors on macrophages in murine liver, lung, kidney, brain, skin and spleen. Our study identified a pronounced heterogeneity between FcγR expression patterns of the different tissue macrophages, which may reflect their specialized functions within their unique niches in different organ environments.

Mitoxantrone-Loaded Nanoferritin Slows Tumor Growth and Improves the Overall Survival Rate in a Subcutaneous Pancreatic Cancer Mouse Model

Pancreatic cancer (PC) represents an intriguing topic for researchers. To date, the prognosis of metastasized PC is poor with just 7% of patients exceeding a five-year survival period. Thus, molecular modifications of existing drugs should be developed to change the course of the disease. Our previously generated nanocages of Mitoxantrone (MIT) encapsulated in human H-chain Ferritin (HFt), designated as HFt-MP-PASE-MIT, has shown excellent tumor distribution and extended serum half-life meriting further investigation for PC treatment. Thus, in this study, we used the same nano-formulation to test its cytotoxicity using both in vitro and in vivo assays. Interestingly, both encapsulated and free-MIT drugs demonstrated similar killing capabilities on PaCa44 cell line. Conversely, in vivo assessment in a subcutaneous PaCa44 tumor model of PC demonstrated a remarkable capability for encapsulated MIT to control tumor growth and improve mouse survival with a median survival rate of 65 vs. 33 days for loaded and free-MIT, respectively. Interestingly, throughout the course of mice treatment, MIT encapsulation did not present any adverse side effects as confirmed by histological analysis of various murine tissue organs and body mass weights. Our results are promising and pave the way to effective PC targeted chemotherapy using our HFt nanodelivery platforms.

Elevated murine HB-EGF confers sensitivity to diphtheria toxin in EGFR-mutant lung adenocarcinoma

Conditional ablation of defined cell populations in vivo can be achieved using genetically engineered mice in which the human diphtheria toxin (DT) receptor (DTR) is placed under control of a murine tissue-specific promotor, such that delivery of diphtheria toxin selectively ablates cells expressing the high-affinity human DTR. Cells expressing only the endogenous low-affinity mouse DTR are assumed to be unaffected. Surprisingly, we found that systemic DT administration induced rapid regression of murine EGFR-mutant lung adenocarcinomas in the absence of a transgenic allele containing human DTR. DT enzymatic activity was required for tumor regression, and EGFR-mutant tumor cells were the primary targets of DT toxicity. In FVB mice, EGFR-mutant tumors upregulated expression of HB-EGF, which is the DTR in mice and humans. HB-EGF blockade with CRM197, an enzymatically inactive DT mutant, partially abrogated DT-induced tumor regression. These results suggest that elevated expression of murine HB-EGF (low-affinity DTR) confers sensitivity to DT in EGFR-mutant tumors, demonstrating a biological effect of DT in mice lacking transgenic DTR alleles and highlighting a unique vulnerability of EGFR-mutant lung cancers.

P030 SUCNR1 mediates inflammasome activation: Relevance in Ulcerative Colitis

Background Ulcerative colitis (UC) is characterized by a diffuse, continuous, and chronic inflammation of mucosa and submucosa layers in the colon1. Inflammasome complex is involved in the intestinal homeostasis regulation, but its role in UC has not been established yet. We have recently reported that SUCNR1 mediates intestinal inflammation and fibrosis2. We aim to analyze the role of SUCNR1 in inflammasome activation and UC. Methods Intestinal resections from UC and non-IBD patients were obtained. HT29 cells were treated with succinate 1mM and an inflammasome activator cocktail (TNF-α 25ng/ml, IFN-γ 20 ng/ml and LPS 1µg/ml) for 24 hours and transfected with SUCNR1 siRNA. Chronic DSS-colitis was induced in wild-type (WT) and SUNCR1-/- (KO) mice with 4 cycles of DSS. Gene expression and protein levels of SUCNR1 and inflammasome markers were analyzed by qPCR, WesternBlot and ELISA. Histology of murine tissue was analyzed by Hematoxylin-Eosin staining. Results were expressed as fold induction (mean±SEM, n≥5). Statistical analysis was performed with one-way ANOVA followed by Newman-Keuls test. Correlations were analyzed with the Spearman coefficient. Results In UC patients, gene expression of SUCNR1 (4.47±1.70), Nlrp3 (1.97±0.38), Caspase-1 (2.08±0.33) and IL-1β (6.90±2.02) were significantly increased vs non-IBD. SUCNR1 positively correlates with the expression of Caspase-1 (r=0.46) and ASC (r=0.45). Protein levels of SUCNR1 (151.00±8.29), Nlrp3 (215.20±53.35) and Caspase-1 (518.30±231.50) were increased in UC vs non-IBD. HT29 cells treated with succinate and inflammasome cocktail showed an increased gene expression of SUCNR1 (4.45±1.17), Nlrp3 (3.13±0.29), Caspase-1 (70.40±16.14), ASC (2.38±0.37), IL-18 (2.52±0.31) and IL-1β (2.32±0.18). Of interest, siSUCNR1 cells treated with the cocktail and succinate showed a significant reduction in the expression of Nlrp3 (1.00±0.15), IL-1β (0.98±0.21), and ASC (1.19±0.14). In parallel, protein levels of Caspase-1 in siSUCNR1 cells treated with cocktail and succinate were significantly reduced (609.00±116.20) vs non-transfected cells (1095.00±148.30). IL-1β levels were also significantly reduced in siSUCNR1 cells (148.60±16.13) vs non-transfected cells (744.70±94.06). Finally, WT-DSS mice exhibited a worse colon histology and an increased protein expression of Caspase-1 (170.60±32.20), compared with KO-DSS (61.39±4.59). Conclusion SUCNR1 is increased and positively correlates with the expression of inflammasome components in UC patients. Moreover, SUCNR1 mediates inflammasome activation and its absence ameliorates chronic DSS-colitis. Hence, SUCNR1 might be a potential pharmacological target for UC treatment. Reference

Perfluoroalkyl substances are increased in patients with late-onset ulcerative colitis and induce intestinal barrier defects ex vivo in murine intestinal tissue

Background and aimEnvironmental factors are strongly implicated in late-onset inflammatory bowel disease. By measuring perfluoroalkyl substances we investigate whether high exposure correlates with late-onset inflammatory bowel disease, and disturbances of the bile acid pool. We further explore the effect of perfluoronoctanoic acid on intestinal barrier function in murine tissue.MethodsSerum levels of perfluoroalkyl substances and bile acids were assessed in matched samples from patients with ulcerative colitis (n = 20) and Crohn’s disease (n = 20) diagnosed at the age of ≥55 years. Blood donors (n = 20), were used as healthy controls. The metabolites were assessed by ultra-performance liquid chromatography coupled to a triple-quadrupole mass spectrometer. Ex vivo exposure of perfluoronoctanoic acid in ileal and colonic murine tissue was assessed with the Ussing Chamber methodology (n = 5).ResultsThe total level of perfluoroalkyl substances was significantly increased in patients with ulcerative colitis compared to healthy controls or patients with Crohn’s disease (p< 0.05). Ex vivo exposure of 100 μM perfluoronoctanoic acid induced a significantly increased paracellular permeability across ileum (p< 0.05) and an enhanced carbachol-induced ion secretion in colon. The distribution of bile acids as well as the correlation pattern between perfluoroalkyl substances and bile acids differed between patient groups and controls.ConclusionOur results demonstrate that the levels of perfluoroalkyl substances are increased in patients with late-onset ulcerative colitis and might contribute to the disease by inducing a dysfunctional intestinal barrier or indirectly by interfering with the bile acid metabolism and thereby alter the intestinal barrier function.

THU0014 COMPARATIVE TRANSCRIPTOME ANALYSES ACROSS TISSUES AND SPECIES IDENTIFY TARGETABLE GENES FOR HUMAN SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) AND LUPUS NEPHRITIS (LN)

Background:Systemic Lupus Erythematosus (SLE) is a complex disease associated with the dysfunction of multiple tissues and cells. The causal tissue for each disease phenotype is not known a priori. Despite improvements in diagnosis and treatment, major organ involvement (such as the kidneys) contributes significantly to morbidity and mortality that still remain increased. There is an unmet need for timely targeted therapy.Objectives:RNA-sequencing was performed to investigate the patterns of transcription variation across tissues between healthy and lupus-prone mice at different stages of lupus, and how these patterns associate with human Systemic Lupus Erythematosus (SLE).Methods:NZB/W-F1 lupus prone mice were sacrificed at the pre-puberty, pre-autoimmunity and nephritic stage. Age-matched C57BL/6 were used as controls. An “effector” tissue (spleen) and “end-organs” (kidneys, brain) were collected. Total RNA was isolated, and mRNA-sequencing was performed. A time-series analysis was developed and differentially expressed genes (DEGs) were analyzed with DESeq. Hierarchical clustering and functional enrichment analysis were performed with gProfiler. Human orthologs of mouse tissue DEGs were identified in the whole-blood RNA-sequencing dataset comprised of 55 lupus-nephritis (LN), 65 non-LN SLE patients and 58 healthy individuals (HI). Human orthologs were compared to human DEGs. Using machine learning, human orthologs identified in the mouse dataset were used to predict kidney involvement in the human dataset, which was split in training and validation sets.Results:Lupus susceptibility and progression signatures at different tissues and different stages of the disease were identified. Tissue-specific signatures and a common cross-tissue signature were also described. Previously described and novel biological processes and pathways were revealed. The comparative murine-human transcriptome analysis identified human orthologs from the mouse spleen-signature (including CCL5, IFIT and HLA genes) that are involved in systemic autoimmunity. It also identified human orthologs from the kidney- and brain-signature (including FCGR2A, C1Q, JAK1 and APOA2) that are involved in major “end-organ” damage and response mechanisms. Using a neural network model, 193 human orthologs accurately predicted LN patients vs HI (accuracy=0.86, sensitivity=0.82, specificity=0.91 in the validation set). Using a support vector machine model, 30 human orthologs and age and gender were the best predictors of LN vs non-LN SLE patients (accuracy=0.71, sensitivity=0.73, specificity=0.69 in the validation set).Conclusion:Murine tissue gene signatures identified by RNA-sequencing analysis revealed biological processes and pathways that could be potentially used as biomarkers or therapeutic targets in human SLE. Comparison of the murine tissue-transcriptome with the whole-blood human-transcriptome revealed common gene signatures, demonstrating similar biological processes and pathways. Machine learning identified a murine kidney lupus signature that can accurately predict kidney involvement in human SLE. Validation in other datasets is ongoing.References:[1]Panousis NI, et al. Ann Rheum Dis 2019;78:1079Acknowledgments:This work was supported by FOREUM, SYSCID and ERC -Advanced GrantDisclosure of Interests:Eleni Frangou: None declared, Panayiotis Garantziotis: None declared, Maria Grigoriou: None declared, Aggelos Banos: None declared, Nikolaos Panousis: None declared, Emmanouil Dermitzakis: None declared, George Bertsias Grant/research support from: GSK, Consultant of: Novartis, Dimitrios Boumpas: None declared, Anastasia Filia: None declared