1 4640 172 NEURONAL NUCLEAR CALCIUM SIGNALING SUPPRESSION OF MICROGLIAL REACTIVITY IS MEDIATED BY OSTEOPROTEGERIN AFTER TRAUMATIC BRAIN INJURY. BACKGROUND: TRAUMATIC BRAIN INJURY (TBI) IS CHARACTERIZED BY MASSIVE CHANGES IN NEURONAL EXCITATION, FROM ACUTE EXCITOTOXICITY TO CHRONIC HYPER- OR HYPOEXCITABILITY. NUCLEAR CALCIUM SIGNALING PATHWAYS ARE INVOLVED IN TRANSLATING CHANGES IN SYNAPTIC INPUTS AND NEURONAL ACTIVITY INTO DISCRETE TRANSCRIPTIONAL PROGRAMS WHICH NOT ONLY AFFECT NEURONAL SURVIVAL AND SYNAPTIC INTEGRITY, BUT ALSO THE CROSSTALK BETWEEN NEURONS AND GLIAL CELLS. HERE, WE REPORT THE EFFECTS OF BLUNTING NEURONAL NUCLEAR CALCIUM SIGNALS IN THE CONTEXT OF TBI. METHODS: WE USED AAV VECTORS TO EXPRESS THE GENETICALLY ENCODED AND NUCLEAR-TARGETED CALCIUM BUFFER PARVALBUMIN (PV.NLS.MCHERRY) OR THE CALCIUM/CALMODULIN BUFFER CAMBP4.MCHERRY IN NEURONS ONLY. UPON TBI, THE EXTENT OF NEUROINFLAMMATION, NEURONAL DEATH AND SYNAPTIC LOSS WERE ASSESSED BY IMMUNOHISTOCHEMISTRY AND TARGETED TRANSCRIPTOME ANALYSIS. MODULATION OF THE OVERALL LEVEL OF NEURONAL ACTIVITY WAS ACHIEVED BY PSAM/PSEM CHEMOGENETICS TARGETED TO PARVALBUMIN INTERNEURONS. THE FUNCTIONAL IMPACT OF NEURONAL NUCLEAR CALCIUM BUFFERING IN TBI WAS ASSESSED BY QUANTIFICATION OF SPONTANEOUS WHISKING. RESULTS: BUFFERING NEURONAL NUCLEAR CALCIUM UNEXPECTEDLY RESULTED IN A MASSIVE AND LONG-LASTING INCREASE IN THE RECRUITMENT OF REACTIVE MICROGLIA TO THE INJURY SITE, WHICH WAS CHARACTERIZED BY A DISEASE-ASSOCIATED AND PHAGOCYTIC PHENOTYPE. THIS EFFECT WAS ACCOMPANIED BY A SUBSTANTIAL SURGE IN SYNAPTIC LOSS AND SIGNIFICANTLY REDUCED WHISKING ACTIVITY. TRANSCRIPTOME ANALYSIS REVEALED A COMPLEX EFFECT OF TBI IN THE CONTEXT OF NEURONAL NUCLEAR CALCIUM BUFFERING, WITH UPREGULATION OF COMPLEMENT FACTORS, CHEMOKINES AND INTERFERON-RESPONSE GENES, AS WELL AS THE DOWNREGULATION OF SYNAPTIC GENES AND EPIGENETIC REGULATORS COMPARED TO CONTROL CONDITIONS. NOTABLY, NUCLEAR CALCIUM BUFFERING LED TO A SUBSTANTIAL LOSS IN NEURONAL OSTEOPROTEGERIN (OPG), WHEREAS STIMULATION OF NEURONAL FIRING INDUCED OPG EXPRESSION. VIRAL RE-EXPRESSION OF OPG RESULTED IN DECREASED MICROGLIAL RECRUITMENT AND SYNAPTIC LOSS. OPG UPREGULATION WAS ALSO OBSERVED IN THE CSF OF HUMAN TBI PATIENTS, UNDERSCORING ITS TRANSLATIONAL VALUE. CONCLUSION: NEURONAL NUCLEAR CALCIUM SIGNALS REGULATE THE DEGREE OF MICROGLIAL RECRUITMENT AND REACTIVITY UPON TBI VIA, AMONG OTHERS, OSTEOPROTEGERIN SIGNALS. OUR FINDINGS SUPPORT A MODEL WHEREBY NEURONAL ACTIVITY ALTERED AFTER TBI EXERTS A POWERFUL IMPACT ON THE NEUROINFLAMMATORY CASCADE, WHICH IN TURN CONTRIBUTES TO THE OVERALL LOSS OF SYNAPSES AND FUNCTIONAL IMPAIRMENT. 2022 2 4298 26 MICRORNA-146A GOVERNS FIBROBLAST ACTIVATION AND JOINT PATHOLOGY IN ARTHRITIS. SYNOVIAL FIBROBLASTS ARE KEY CELLS ORCHESTRATING THE INFLAMMATORY RESPONSE IN ARTHRITIS. HERE WE DEMONSTRATE THAT LOSS OF MIR-146A, A KEY EPIGENETIC REGULATOR OF THE INNATE IMMUNE RESPONSE, LEADS TO INCREASED JOINT DESTRUCTION IN A TNF-DRIVEN MODEL OF ARTHRITIS BY SPECIFICALLY REGULATING THE BEHAVIOR OF SYNOVIAL FIBROBLASTS. ABSENCE OF MIR-146A IN SYNOVIAL FIBROBLASTS DISPLAY A HIGHLY DEREGULATED GENE EXPRESSION PATTERN AND ENHANCED PROLIFERATION IN VITRO AND IN VIVO. DEFICIENCY OF MIR-146A INDUCES DEREGULATION OF TUMOR NECROSIS FACTOR (TNF) RECEPTOR ASSOCIATED FACTOR 6 (TRAF6) IN SYNOVIAL FIBROBLASTS, LEADING TO INCREASED PROLIFERATION. IN ADDITION, LOSS OF MIR-146A SHIFTS THE METABOLIC STATE OF FIBROBLASTS TOWARDS GLYCOLYSIS AND AUGMENTS THE ABILITY OF SYNOVIAL FIBROBLASTS TO SUPPORT THE GENERATION OF OSTEOCLASTS BY CONTROLLING THE BALANCE OF OSTEOCLASTOGENIC REGULATORY FACTORS RECEPTOR ACTIVATOR OF NF-KAPPAB LIGAND (RANKL) AND OSTEOPROTEGERIN (OPG). BONE MARROW TRANSPLANTATION EXPERIMENTS CONFIRMED THE IMPORTANCE OF MIR-146A IN THE RADIORESISTANT MESENCHYMAL COMPARTMENT FOR THE CONTROL OF ARTHRITIS SEVERITY, IN PARTICULAR FOR INFLAMMATORY JOINT DESTRUCTION. THIS STUDY THEREFORE IDENTIFIES MICRORNA-146A AS AN IMPORTANT LOCAL EPIGENETIC REGULATOR OF THE INFLAMMATORY RESPONSE IN ARTHRITIS. IT IS A CENTRAL ELEMENT OF AN ANTI-INFLAMMATORY FEEDBACK LOOP IN RESIDENT SYNOVIAL FIBROBLASTS, WHO ARE ORCHESTRATING THE INFLAMMATORY RESPONSE IN CHRONIC ARTHRITIS. MIR-146A RESTRICTS THEIR ACTIVATION, THEREBY PREVENTING EXCESSIVE TISSUE DAMAGE DURING ARTHRITIS. 2017 3 6111 24 THE EPIGENETIC ARCHITECTURE AT GENE PROMOTERS DETERMINES CELL TYPE-SPECIFIC LPS TOLERANCE. SYNOVIAL FIBROBLASTS (SF) DRIVE INFLAMMATION AND JOINT DESTRUCTION IN CHRONIC ARTHRITIS. HERE WE SHOW THAT SF POSSESS A DISTINCT TYPE OF LPS TOLERANCE COMPARED TO MACROPHAGES AND OTHER TYPES OF FIBROBLASTS. IN SF AND DERMAL FIBROBLASTS, GENES THAT WERE NON-TOLERIZABLE AFTER REPEATED LPS STIMULATION INCLUDED PRO-INFLAMMATORY CYTOKINES, CHEMOKINES AND MATRIX METALLOPROTEINASES, WHEREAS ANTI-VIRAL GENES WERE TOLERIZABLE. IN MACROPHAGES, ALL MEASURED GENES WERE TOLERIZABLE, WHEREAS IN GINGIVAL AND FORESKIN FIBROBLASTS THESE GENES WERE NON-TOLERIZABLE. REPEATED STIMULATION OF SF WITH LPS RESULTED IN LOSS OF ACTIVATING HISTONE MARKS ONLY IN PROMOTERS OF TOLERIZABLE GENES. THE EPIGENETIC LANDSCAPE AT PROMOTERS OF TOLERIZABLE GENES WAS SIMILAR IN UNSTIMULATED SF AND MONOCYTES, WHEREAS THE BASAL CONFIGURATION OF HISTONE MARKS PROFOUNDLY DIFFERED IN GENES THAT WERE NON-TOLERIZABLE IN SF ONLY. OUR DATA SUGGEST THAT THE EPIGENETIC CONFIGURATION AT GENE PROMOTERS REGULATES CELL-SPECIFIC LPS-INDUCED RESPONSES AND PRIMES SF TO SUSTAIN THEIR INFLAMMATORY RESPONSE IN CHRONIC ARTHRITIS. 2017 4 5227 34 PRMT6 MEDIATES INFLAMMATION VIA ACTIVATION OF THE NF-KAPPAB/P65 PATHWAY ON A CIGARETTE SMOKE EXTRACT-INDUCED MURINE EMPHYSEMA MODEL. INTRODUCTION: SMOKE-DRIVEN LUNG INFLAMMATION IS CONSIDERED TO BE THE MAJOR PATHOPHYSIOLOGY MECHANISM OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD)/EMPHYSEMA. PROTEIN ARGININE METHYLTRANSFERASE 6 (PRMT6) IS A KEY EPIGENETIC ENZYME, WHICH IS RELATED TO PROTECTING THE TRI-METHYLATION OF H3K4 (H3K4ME3). WE HYPOTHESIZED THAT PTMT6 PROTECTS LUNG INFLAMMATION THROUGH THE NUCLEAR FACTOR KAPPA B (NF-KAPPAB) PATHWAY. METHODS: MICE WERE INJECTED WITH CIGARETTE SMOKE EXTRACT (CSE) OR PBS TO ESTABLISH A MICE MODEL, INTRATRACHEALLY INSTILLED WITH OVEREXPRESSED PRMT6 OR NEGATIVE CONTROL VECTOR. MORPHOMETRY OF LUNG SLIDES AND LUNG FUNCTION WERE MEASURED. WE DETERMINED THE PROTEIN EXPRESSION OF PRMT6 AND ITS RELATED HISTONE TARGETS, THE ACTIVATION OF NF-KAPPAB PATHWAY, THE LEVEL OF TUMOR NECROSIS FACTOR ALPHA (TNFALPHA) AND INTERLEUKIN-1BETA (IL-1BETA). RESULTS: AFTER PRMT6 OVEREXPRESSION, THE MORPHOMETRY INDEXES AND LUNG FUNCTION WERE IMPROVED. ALSO, THE EXPRESSION OF H3K4ME3 WAS DECREASED. OVEREXPRESSED PRMT6 COULD SUPPRESS CSE-INDUCED NF-KAPPAB ACTIVATION AND PRO-INFLAMMATION GENES EXPRESSION. CONCLUSIONS: THE OVEREXPRESSED PRMT6 COULD SERVE AS AN INFLAMMATION INHIBITOR, POTENTIALLY THROUGH BLOCKING THE NF-KAPPAB/P65 PATHWAY IN THE MURINE EMPHYSEMA MODEL. 2020 5 2065 30 EPIGENETIC CONTROL OF INTESTINAL BARRIER FUNCTION AND INFLAMMATION IN ZEBRAFISH. THE INTESTINAL EPITHELIUM FORMS A BARRIER PROTECTING THE ORGANISM FROM MICROBES AND OTHER PROINFLAMMATORY STIMULI. THE INTEGRITY OF THIS BARRIER AND THE PROPER RESPONSE TO INFECTION REQUIRES PRECISE REGULATION OF POWERFUL IMMUNE HOMING SIGNALS SUCH AS TUMOR NECROSIS FACTOR (TNF). DYSREGULATION OF TNF LEADS TO INFLAMMATORY BOWEL DISEASES (IBD), BUT THE MECHANISM CONTROLLING THE EXPRESSION OF THIS POTENT CYTOKINE AND THE EVENTS THAT TRIGGER THE ONSET OF CHRONIC INFLAMMATION ARE UNKNOWN. HERE, WE SHOW THAT LOSS OF FUNCTION OF THE EPIGENETIC REGULATOR UBIQUITIN-LIKE PROTEIN CONTAINING PHD AND RING FINGER DOMAINS 1 (UHRF1) IN ZEBRAFISH LEADS TO A REDUCTION IN TNFA PROMOTER METHYLATION AND THE INDUCTION OF TNFA EXPRESSION IN INTESTINAL EPITHELIAL CELLS (IECS). THE INCREASE IN IEC TNFA LEVELS IS MICROBE-DEPENDENT AND RESULTS IN IEC SHEDDING AND APOPTOSIS, IMMUNE CELL RECRUITMENT, AND BARRIER DYSFUNCTION, CONSISTENT WITH CHRONIC INFLAMMATION. IMPORTANTLY, TNFA KNOCKDOWN IN UHRF1 MUTANTS RESTORES IEC MORPHOLOGY, REDUCES CELL SHEDDING, AND IMPROVES BARRIER FUNCTION. WE PROPOSE THAT LOSS OF EPIGENETIC REPRESSION AND TNF INDUCTION IN THE INTESTINAL EPITHELIUM CAN LEAD TO IBD ONSET. 2015 6 5868 37 SUPPRESSIVE EFFECTS OF METFORMIN ON T-HELPER 1-RELATED CHEMOKINES EXPRESSION IN THE HUMAN MONOCYTIC LEUKEMIA CELL LINE THP-1. PURPOSE OF THE STUDY: TYPE 1 AND TYPE 2 DIABETES MELLITUS (DM) ARE CHRONIC T-CELL-MEDIATED INFLAMMATORY DISEASES. METFORMIN IS A WIDELY USED DRUG FOR TYPE 2 DM THAT REDUCES THE NEED FOR INSULIN IN TYPE 1 DM. HOWEVER, WHETHER METFORMIN HAS AN ANTI-INFLAMMATORY EFFECT FOR TREATING DM IS UNKNOWN. WE INVESTIGATED THE ANTI-INFLAMMATORY MECHANISM OF METFORMIN IN THE HUMAN MONOCYTIC LEUKEMIA CELL LINE THP-1. MATERIALS AND METHODS: THE HUMAN MONOCYTIC LEUKEMIA CELL LINE THP-1 WAS PRETREATED WITH METFORMIN AND STIMULATED WITH LIPOPOLYSACCHARIDE (LPS). THE PRODUCTION OF T-HELPER (TH)-1-RELATED CHEMOKINES INCLUDING INTERFERON-GAMMA-INDUCED PROTEIN-10 (IP-10) AND MONOCYTE CHEMOATTRACTANT PROTEIN-1 (MCP-1), TH2-RELATED CHEMOKINE MACROPHAGE-DERIVED CHEMOKINE, AND THE PROINFLAMMATORY CHEMOKINE TUMOR NECROSIS FACTOR-ALPHA WAS MEASURED USING ENZYME-LINKED IMMUNOSORBENT ASSAY. INTRACELLULAR SIGNALING PATHWAYS WERE INVESTIGATED USING WESTERN BLOT ANALYSIS AND CHROMATIN IMMUNOPRECIPITATION ASSAY. RESULTS: METFORMIN SUPPRESSED LPS-INDUCED IP-10 AND MCP-1 PRODUCTION AS WELL AS LPS-INDUCED PHOSPHORYLATION OF C-JUN N-TERMINAL KINASE (JNK), P38, EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK), AND NUCLEAR FACTOR-KAPPA B (NF-KAPPAB). MOREOVER, METFORMIN SUPPRESSED LPS-INDUCED ACETYLATION OF HISTONES H3 AND H4 AT THE IP-10 PROMOTER. CONCLUSIONS: METFORMIN SUPPRESSED THE PRODUCTION OF TH1-RELATED CHEMOKINES IP-10 AND MCP-1 IN THP-1 CELLS. SUPPRESSIVE EFFECTS OF METFORMIN ON IP-10 PRODUCTION MIGHT BE ATTRIBUTED AT LEAST PARTIALLY TO THE JNK, P38, ERK, AND NF-KAPPAB PATHWAYS AS WELL AS TO EPIGENETIC REGULATION THROUGH THE ACETYLATION OF HISTONES H3 AND H4. THESE RESULTS INDICATED THE THERAPEUTIC ANTI-INFLAMMATORY POTENTIAL OF METFORMIN. 2018 7 5479 33 RESVERATROL ATTENUATES CIGARETTE SMOKE EXTRACT INDUCED CELLULAR SENESCENCE IN HUMAN AIRWAY EPITHELIAL CELLS BY REGULATING THE MIR-34A/SIRT1/NF-KAPPAB PATHWAY. CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) IS CHARACTERIZED BY ACCELERATED LUNG AGING. SMOKING IS THE CRITICAL RISK FACTOR FOR COPD. CELLULAR SENESCENCE OF AIRWAY EPITHELIAL CELLS IS THE CYTOLOGICAL BASIS OF ACCELERATED LUNG AGING IN COPD, AND THE REGULATION OF MICRORNAS (MIRNAS) IS THE CENTRAL EPIGENETIC MECHANISM OF CELLULAR SENESCENCE. RESVERATROL (RES) IS A POLYPHENOL WITH ANTI-AGING PROPERTIES. THIS STUDY INVESTIGATED WHETHER RES ATTENUATES CIGARETTE SMOKE EXTRACT (CSE)-INDUCED CELLULAR SENESCENCE IN HUMAN AIRWAY EPITHELIAL CELLS (BEAS-2B) THROUGH THE MIR-34A/SIRT1/NUCLEAR FACTOR-KAPPAB (NF-KAPPAB) PATHWAY. BEAS-2B CELLS WERE TREATED WITH RES, CSE AND TRANSFECTED WITH MIR-34A-5P MIMICS. CELLULAR SENESCENCE WAS EVALUATED BY SENESCENCE -RELATED BETA-GALACTOSIDASE (SA-BETA-GAL) STAINING AND EXPRESSION OF SENESCENCE-RELATED GENES (P16, P21, AND P53). THE EXPRESSIONS OF MIR-34A-5P, SIRT1, AND NF-KAPPAB P65 WERE EXAMINED USING QUANTITATIVE REAL TIME POLYMERASE CHAIN REACTION AND WESTERN BLOTTING. THE SENESCENCE-ASSOCIATED SECRETORY PHENOTYPE (SASP) CYTOKINES (IL-1BETA, IL-6, IL-8, TNF-ALPHA) WERE ASSESSED BY ENZYME-LINKED IMMUNOSORBENT ASSAY. THE BINDING BETWEEN MIR-34A-5P AND SIRT1 WAS CONFIRMED BY DUAL-LUCIFERASE REPORTER ASSAY. THE RESULTS SHOWED THAT CSE DOSE-DEPENDENTLY DECREASED CELL VIABILITY AND ELEVATED CELLULAR SENESCENCE, CHARACTERIZED BY INCREASED SA-BETA-GAL STAINING AND SENESCENCE-RELATED GENE EXPRESSIONS (P16, P21, AND P53). FURTHER, CSE DOSE-DEPENDENTLY INCREASED THE EXPRESSION OF MIR-34A-5P AND SASP CYTOKINES (IL-1BETA, IL-6, IL-8, TNF-ALPHA) IN BEAS-2B CELLS. PRETREATMENT WITH RES INHIBITED CSE-INDUCED CELLULAR SENESCENCE AND SECRETION OF SASP CYTOKINES (IL-1BETA, IL-6, IL-8, TNF-ALPHA) IN A DOSE-DEPENDENT MANNER. MOREOVER, RES REVERSED THE CSE-INDUCED DOWN-REGULATION OF SIRT1 AND UP-REGULATION OF MIR-34A-5P AND NF-KAPPAB P65. SIRT1 IS A TARGET OF MIR-34A-5P. OVEREXPRESSION OF MIR-34A-5P VIA TRANSFECTION WITH MIR-34A-5P MIMIC IN BEAS-2B CELLS ATTENUATED THE INHIBITORY EFFECT OF RES ON CELLULAR SENESCENCE, ACCOMPANIED BY REVERSING THE EXPRESSION OF SIRT1 AND NF-KAPPAB P65. IN CONCLUSION, RES ATTENUATED CSE-INDUCED CELLULAR SENESCENCE IN BEAS-2B CELLS BY REGULATING THE MIR-34A/SIRT1/NF-KAPPAB PATHWAY, WHICH MAY PROVIDE A NEW APPROACH FOR COPD TREATMENT. 2022 8 4303 39 MICRORNA-223 INHIBITS TISSUE FACTOR EXPRESSION IN VASCULAR ENDOTHELIAL CELLS. OBJECTIVE: ATHEROSCLEROSIS IS A CHRONIC INFLAMMATORY PROCESS, IN WHICH VASCULAR ENDOTHELIAL CELLS (ECS) BECOME DYSFUNCTIONAL OWING TO THE EFFECTS OF CHEMICAL SUBSTANCES, SUCH AS INFLAMMATORY FACTOR AND GROWTH FACTORS. TISSUE FACTOR (TF) EXPRESSION IS INDUCED BY THE ABOVE CHEMICAL SUBSTANCES IN ACTIVATED ECS. TF INITIATES THROMBOSIS ON DISRUPTED ATHEROSCLEROTIC PLAQUES WHICH PLAYS AN ESSENTIAL ROLE DURING THE ONSET OF ACUTE CORONARY SYNDROMES (ACS). INCREASING EVIDENCES SUGGEST THE IMPORTANT ROLE OF MICRORNAS AS EPIGENETIC REGULATORS OF ATHEROSCLEROTIC DISEASE. THE AIM OF OUR STUDY IS TO IDENTIFY IF MICRORNA-223 (MIR-223) TARGETS TF IN ECS. METHODS AND RESULTS: BIOINFORMATIC ANALYSIS SHOWED THAT TF IS A TARGET CANDIDATE OF MIR-223. WESTERN BLOTTING ANALYSIS REVEALED THAT TUMOR NECROSIS FACTOR ALPHA (TNF-ALPHA) INCREASED TF EXPRESSION IN AORTA OF C57BL/6J MICE AND CULTURED ECS (EA.HY926 CELLS AND HUVEC) AFTER 4 H TREATMENT. IN TNF-ALPHA TREATED ECS, TF MRNA WAS ALSO INCREASED MEASURED BY REAL-TIME PCR. REAL-TIME PCR RESULTS SHOWED THAT MIR-223 LEVELS WERE DOWNREGULATED IN TNF-ALPHA-TREATED AORTA OF C57BL/6J MICE AND CULTURED ECS. TRANSFECTION OF ECS WITH MIR-223 MIMIC OR MIR-223 INHIBITOR MODIFIED TF EXPRESSION BOTH IN MRNA AND PROTEIN LEVELS. LUCIFERASE ASSAYS CONFIRMED THAT MIR-223 SUPPRESSED TF EXPRESSION BY BINDING TO THE SEQUENCE OF TF 3'-UNTRANSLATED REGIONS (3'UTR). TF PROCOAGULANT ACTIVITY WAS INHIBITED BY OVEREXPRESSING MIR-223 WITH OR WITHOUT TNF-ALPHA STIMULATION. CONCLUSIONS: MIR-223-MEDIATED SUPPRESSION OF TF EXPRESSION PROVIDES A NOVEL MOLECULAR MECHANISM FOR THE REGULATION OF COAGULATION CASCADE, AND SUGGESTS A CLUE AGAINST THROMBOGENESIS DURING THE PROCESS OF ATHEROSCLEROTIC PLAQUE RUPTURE. 2014 9 1158 44 CONTEXT-DEPENDENT EPIGENETIC REGULATION OF NUCLEAR FACTOR OF ACTIVATED T CELLS 1 IN PANCREATIC PLASTICITY. BACKGROUND & AIMS: THE ABILITY OF EXOCRINE PANCREATIC CELLS TO CHANGE THE CELLULAR PHENOTYPE IS REQUIRED FOR TISSUE REGENERATION UPON INJURY, BUT ALSO CONTRIBUTES TO THEIR MALIGNANT TRANSFORMATION AND TUMOR PROGRESSION. WE INVESTIGATED CONTEXT-DEPENDENT SIGNALING AND TRANSCRIPTION MECHANISMS THAT DETERMINE PANCREATIC CELL FATE DECISIONS TOWARD REGENERATION AND MALIGNANCY. IN PARTICULAR, WE STUDIED THE FUNCTION AND REGULATION OF THE INFLAMMATORY TRANSCRIPTION FACTOR NUCLEAR FACTOR OF ACTIVATED T CELLS 1 (NFATC1) IN PANCREATIC CELL PLASTICITY AND TISSUE ADAPTATION. METHODS: WE ANALYZED CELL PLASTICITY DURING PANCREATIC REGENERATION AND TRANSFORMATION IN MICE WITH PANCREAS-SPECIFIC EXPRESSION OF A CONSTITUTIVELY ACTIVE FORM OF NFATC1, OR DEPLETION OF ENHANCER OF ZESTE 2 HOMOLOGUE 2 (EZH2), IN THE CONTEXT OF WILD-TYPE OR CONSTITUTIVELY ACTIVATE KRAS, RESPECTIVELY. ACUTE AND CHRONIC PANCREATITIS WERE INDUCED BY INTRAPERITONEAL INJECTION OF CAERULEIN. EZH2-DEPENDENT REGULATION OF NFATC1 EXPRESSION WAS STUDIED IN MOUSE IN HUMAN PANCREATIC TISSUE AND CELLS BY IMMUNOHISTOCHEMISTRY, IMMUNOBLOTTING, AND QUANTITATIVE REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION. WE USED GENETIC AND PHARMACOLOGIC APPROACHES OF EZH2 AND NFATC1 INHIBITION TO STUDY THE CONSEQUENCES OF PATHWAY DISRUPTION ON PANCREATIC MORPHOLOGY AND FUNCTION. EPIGENETIC MODIFICATIONS ON THE NFATC1 GENE WERE INVESTIGATED BY CHROMATIN IMMUNOPRECIPITATION ASSAYS. RESULTS: NFATC1 WAS RAPIDLY AND TRANSIENTLY INDUCED IN EARLY ADAPTATION TO ACINAR CELL INJURY IN HUMAN SAMPLES AND IN MICE, WHERE IT PROMOTED ACINAR CELL TRANSDIFFERENTIATION AND BLOCKED PROLIFERATION OF METAPLASTIC PANCREATIC CELLS. HOWEVER, IN LATE STAGES OF REGENERATION, NFATC1 WAS EPIGENETICALLY SILENCED BY EZH2-DEPENDENT HISTONE METHYLATION, TO ENABLE ACINAR CELL REDIFFERENTIATION AND PREVENT ORGAN ATROPHY AND EXOCRINE INSUFFICIENCY. IN CONTRAST, ONCOGENIC ACTIVATION OF KRAS SIGNALING IN PANCREATIC DUCTAL ADENOCARCINOMA CELLS REVERSED THE EZH2-DEPENDENT EFFECTS ON THE NFATC1 GENE AND WAS REQUIRED FOR EZH2-MEDIATED TRANSCRIPTIONAL ACTIVATION OF NFATC1. CONCLUSIONS: IN STUDIES OF HUMAN AND MOUSE PANCREATIC CELLS AND TISSUE, WE IDENTIFIED CONTEXT-SPECIFIC EPIGENETIC REGULATION OF NFATC1 ACTIVITY AS AN IMPORTANT MECHANISM OF PANCREATIC CELL PLASTICITY. INHIBITORS OF EZH2 MIGHT THEREFORE INTERFERE WITH ONCOGENIC ACTIVITY OF NFATC1 AND BE USED IN TREATMENT OF PANCREATIC DUCTAL ADENOCARCINOMA. 2017 10 4041 34 MACROPHAGE PLASTICITY IN DUCHENNE MUSCULAR DYSTROPHY: A NEXUS OF PATHOLOGICAL REMODELLING WITH THERAPEUTIC IMPLICATIONS. DUCHENNE MUSCULAR DYSTROPHY (DMD) IS CHARACTERIZED BY CHRONIC SKELETAL MUSCLE NECROSIS, LEADING TO MUSCLE REGENERATION FAILURE AND FIBROSIS. ALTHOUGH MACROPHAGES (MPS) ARE NORMALLY ESSENTIAL FOR MUSCLE REGENERATION, DYSREGULATED MP FUNCTION PROMOTES PATHOLOGICAL MUSCLE REMODELLING. INFILTRATING MPS CAN BE PREDOMINANTLY PRO-INFLAMMATORY (M1 BIASED), ANTI-INFLAMMATORY (M2 BIASED) OR OF A MIXED PHENOTYPE AND CAN ORIGINATE FROM THE ADULT BONE MARROW (MONOCYTE DEPENDENT) OR EMBRYONIC PRECURSORS (MONOCYTE INDEPENDENT). IN MDX MICE (GENETIC MODEL OF DMD) LACKING EITHER TOLL-LIKE RECEPTOR (TLR) 2 OR TLR4, IT IS FOUND THAT MP INFILTRATION OF DYSTROPHIC MUSCLE IS SIGNIFICANTLY REDUCED AND THAT THE MP PHENOTYPE IS SHIFTED TOWARD A MORE ANTI-INFLAMMATORY PROFILE. THIS IS ACCOMPANIED BY SIGNIFICANT IMPROVEMENTS IN MUSCLE HISTOLOGY AND FORCE PRODUCTION. LACK OF THE CHEMOKINE RECEPTOR CCR2, WHICH IMPEDES MONOCYTE RELEASE FROM THE BONE MARROW, LEADS TO SIMILAR BENEFICIAL EFFECTS IN MDX MICE. EVIDENCE WAS ALSO FOUND FOR TLR4-REGULATED INDUCTION OF TRAINED INNATE IMMUNITY IN MPS CULTURED FROM THE BONE MARROW OF MDX MICE BEFORE THEIR ENTRY INTO THE MUSCLE. THESE MPS EXHIBIT EPIGENETIC AND METABOLIC ALTERATIONS, ACCOMPANIED BY NON-SPECIFIC HYPER-RESPONSIVENESS TO MULTIPLE STIMULI, WHICH IS MANIFESTED BY POTENTIATED UPREGULATION OF BOTH PRO- AND ANTI-INFLAMMATORY GENES. IN SUMMARY, EXAGGERATED RECRUITMENT OF MONOCYTE-DERIVED MPS AND SIGNS OF TRAINED INNATE IMMUNITY AT THE LEVEL OF THE BONE MARROW ARE FEATURES OF THE IMMUNOPHENOTYPE ASSOCIATED WITH DYSTROPHIC MUSCLE DISEASE. THESE PHENOMENA ARE REGULATED BY TOLL-LIKE RECEPTORS THAT BIND ENDOGENOUS DAMAGE-ASSOCIATED MOLECULAR PATTERN (DAMP) MOLECULES, SUGGESTING THAT DAMP RELEASE FROM DYSTROPHIC MUSCLES MODULATES MP PLASTICITY AT THE BONE MARROW LEVEL THROUGH TOLL-LIKE RECEPTOR-DRIVEN MECHANISMS. 2022 11 2702 27 EXCITOTOXICITY AND OVERNUTRITION ADDITIVELY IMPAIR METABOLIC FUNCTION AND IDENTITY OF PANCREATIC BETA-CELLS. A SUSTAINED INCREASE IN INTRACELLULAR CA(2+) CONCENTRATION (REFERRED TO HEREAFTER AS EXCITOTOXICITY), BROUGHT ON BY CHRONIC METABOLIC STRESS, MAY CONTRIBUTE TO PANCREATIC BETA-CELL FAILURE. TO DETERMINE THE ADDITIVE EFFECTS OF EXCITOTOXICITY AND OVERNUTRITION ON BETA-CELL FUNCTION AND GENE EXPRESSION, WE ANALYZED THE IMPACT OF A HIGH-FAT DIET (HFD) ON ABCC8 KNOCKOUT MICE. EXCITOTOXICITY CAUSED BETA-CELLS TO BE MORE SUSCEPTIBLE TO HFD-INDUCED IMPAIRMENT OF GLUCOSE HOMEOSTASIS, AND THESE EFFECTS WERE MITIGATED BY VERAPAMIL, A CA(2+) CHANNEL BLOCKER. EXCITOTOXICITY, OVERNUTRITION, AND THE COMBINATION OF BOTH STRESSES CAUSED SIMILAR BUT DISTINCT ALTERATIONS IN THE BETA-CELL TRANSCRIPTOME, INCLUDING ADDITIVE INCREASES IN GENES ASSOCIATED WITH MITOCHONDRIAL ENERGY METABOLISM, FATTY ACID BETA-OXIDATION, AND MITOCHONDRIAL BIOGENESIS AND THEIR KEY REGULATOR PPARGC1A OVERNUTRITION WORSENED EXCITOTOXICITY-INDUCED MITOCHONDRIAL DYSFUNCTION, INCREASING METABOLIC INFLEXIBILITY AND MITOCHONDRIAL DAMAGE. IN ADDITION, EXCITOTOXICITY AND OVERNUTRITION, INDIVIDUALLY AND TOGETHER, IMPAIRED BOTH BETA-CELL FUNCTION AND IDENTITY BY REDUCING EXPRESSION OF GENES IMPORTANT FOR INSULIN SECRETION, CELL POLARITY, CELL JUNCTION, CILIA, CYTOSKELETON, VESICULAR TRAFFICKING, AND REGULATION OF BETA-CELL EPIGENETIC AND TRANSCRIPTIONAL PROGRAM. SEX HAD AN IMPACT ON ALL BETA-CELL RESPONSES, WITH MALE ANIMALS EXHIBITING GREATER METABOLIC STRESS-INDUCED IMPAIRMENTS THAN FEMALES. TOGETHER, THESE FINDINGS INDICATE THAT A SUSTAINED INCREASE IN INTRACELLULAR CA(2+), BY ALTERING MITOCHONDRIAL FUNCTION AND IMPAIRING BETA-CELL IDENTITY, AUGMENTS OVERNUTRITION-INDUCED BETA-CELL FAILURE. 2020 12 1357 38 DEVELOPMENT OF INNATE IMMUNE MEMORY BY NON-IMMUNE CELLS DURING STAPHYLOCOCCUS AUREUS INFECTION DEPENDS ON REACTIVE OXYGEN SPECIES. INTRODUCTION: THE MECHANISMS UNDERLYING INNATE IMMUNE MEMORY (TRAINED IMMUNITY) COMPRISE EPIGENETIC REPROGRAMMING OF TRANSCRIPTIONAL PATHWAYS ASSOCIATED WITH ALTERATIONS OF INTRACELLULAR METABOLISM. WHILE THE MECHANISMS OF INNATE IMMUNE MEMORY CARRIED OUT BY IMMUNE CELLS ARE WELL CHARACTERIZED, SUCH PROCESSES IN NON-IMMUNE CELLS, ARE POORLY UNDERSTOOD. THE OPPORTUNISTIC PATHOGEN, STAPHYLOCOCCUS AUREUS, IS RESPONSIBLE FOR A MULTITUDE OF HUMAN DISEASES, INCLUDING PNEUMONIA, ENDOCARDITIS AND OSTEOMYELITIS, AS WELL AS ANIMAL INFECTIONS, INCLUDING CHRONIC CATTLE MASTITIS THAT ARE EXTREMELY DIFFICULT TO TREAT. AN INDUCTION OF INNATE IMMUNE MEMORY MAY BE CONSIDERED AS A THERAPEUTIC ALTERNATIVE TO FIGHT S. AUREUS INFECTION. METHODS: IN THE CURRENT WORK, WE DEMONSTRATED THE DEVELOPMENT OF INNATE IMMUNE MEMORY IN NON-IMMUNE CELLS DURING S. AUREUS INFECTION EMPLOYING A COMBINATION OF TECHNIQUES INCLUDING ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA), MICROSCOPIC ANALYSIS, AND CYTOMETRY. RESULTS: WE OBSERVED THAT TRAINING OF HUMAN OSTEOBLAST-LIKE MG-63 CELLS AND LUNG EPITHELIAL A549 CELLS WITH BETA-GLUCAN INCREASED IL-6 AND IL-8 PRODUCTION UPON A STIMULATION WITH S. AUREUS, CONCOMITANT WITH HISTONES MODIFICATIONS. IL-6 AND IL-8 PRODUCTION WAS POSITIVELY CORRELATED WITH AN ACETYLATION OF HISTONE 3 AT LYSINE 27 (H3K27), THUS SUGGESTING EPIGENETIC REPROGRAMMING IN THESE CELLS. AN ADDITION OF THE ROS SCAVENGER N-ACETYLCYSTEINE, NAC, PRIOR TO BETA-GLUCAN PRETREATMENT FOLLOWED BY AN EXPOSURE TO S. AUREUS, RESULTED IN DECREASED IL-6 AND IL-8 PRODUCTION, THEREBY SUPPORTING THE INVOLVEMENT OF ROS IN THE INDUCTION OF INNATE IMMUNE MEMORY. EXPOSURE OF CELLS TO LACTOCOCCUS LACTIS RESULTED IN INCREASED IL-6 AND IL-8 PRODUCTION BY MG-63 AND A549 CELLS UPON A STIMULATION WITH S. AUREUS THAT WAS CORRELATED WITH H3K27 ACETYLATION, SUGGESTING THE ABILITY OF THIS BENEFICIAL BACTERIUM TO INDUCE INNATE IMMUNE MEMORY. DISCUSSION: THIS WORK IMPROVES OUR UNDERSTANDING OF INNATE IMMUNE MEMORY IN NON-IMMUNE CELLS IN THE CONTEXT OF S. AUREUS INFECTION. IN ADDITION TO KNOWN INDUCERS, PROBIOTICS MAY REPRESENT GOOD CANDIDATES FOR THE INDUCTION OF INNATE IMMUNE MEMORY. OUR FINDINGS MAY HELP THE DEVELOPMENT OF ALTERNATIVE THERAPEUTIC APPROACHES FOR THE PREVENTION OF S. AUREUS INFECTION. 2023 13 3373 28 HISTONE MODULATION BLOCKS TREG-INDUCED FOXP3 BINDING TO THE IL-2 PROMOTER OF VIRUS-SPECIFIC CD8(+) T CELLS FROM FELINE IMMUNODEFICIENCY VIRUS-INFECTED CATS. CD8(+) T CELLS ARE CRITICAL FOR CONTROLLING HIV INFECTION. DURING THE CHRONIC PHASE OF LENTIVIRAL INFECTION, CD8(+) T CELLS LOSE THEIR PROLIFERATIVE CAPACITY AND EXHIBIT IMPAIRED ANTIVIRAL FUNCTION. THIS LOSS OF CD8(+) T CELL FUNCTION IS DUE, IN PART, TO CD4(+)CD25(+) T REGULATORY (TREG) CELL-MEDIATED SUPPRESSION. OUR RESEARCH GROUP HAS DEMONSTRATED THAT LENTIVIRUS-ACTIVATED CD4(+)CD25(+) TREG CELLS INDUCE THE REPRESSIVE TRANSCRIPTION FACTOR FORKHEAD BOX P3 (FOXP3) IN AUTOLOGOUS CD8(+) T CELLS FOLLOWING CO-CULTURE. WE HAVE RECENTLY REPORTED THAT TREG-INDUCED FOXP3 BINDS THE INTERLEUKIN-2 (IL-2), INTERFERON-GAMMA (IFN- GAMMA), AND TUMOR NECROSIS FACTOR-ALPHA (TNF-ALPHA) PROMOTERS IN VIRUS-SPECIFIC CD8(+) T CELLS. THESE DATA SUGGEST AN IMPORTANT ROLE OF FOXP3-MEDIATED CD8(+) T CELL DYSFUNCTION IN LENTIVIRAL INFECTION. TO ELUCIDATE THE MECHANISM OF THIS SUPPRESSION, WE PREVIOUSLY REPORTED THAT DECREASED METHYLATION FACILITATES FOXP3 BINDING IN MITOGEN-ACTIVATED CD8(+) T CELLS FROM FELINE IMMUNODEFICIENCY VIRUS (FIV)-INFECTED CATS. WE DEMONSTRATED THE REDUCED BINDING OF FOXP3 TO THE IL-2 PROMOTER BY INCREASING METHYLATION OF CD8(+) T CELLS. IN THE STUDIES PRESENTED HERE, WE ASK IF ANOTHER FORM OF EPIGENETIC MODULATION MIGHT ALLEVIATE FOXP3-MEDIATED SUPPRESSION IN CD8(+) T CELLS. WE HYPOTHESIZED THAT DECREASING HISTONE ACETYLATION IN VIRUS-SPECIFIC CD8(+) T CELLS WOULD DECREASE TREG-INDUCED FOXP3 BINDING TO THE IL-2 PROMOTER. INDEED, USING ANACARDIC ACID (AA), A KNOWN HISTONE ACETYL TRANSFERASE (HAT) INHIBITOR, WE DEMONSTRATE A REDUCTION IN FOXP3 BINDING TO THE IL-2 PROMOTER IN VIRUS-SPECIFIC CD8(+) T CELLS CO-CULTURED WITH AUTOLOGOUS TREG CELLS. THESE DATA IDENTIFY A NOVEL MECHANISM OF FOXP3-MEDIATED CD8(+) T CELL DYSFUNCTION DURING LENTIVIRAL INFECTION. 2018 14 1667 38 DOWNREGULATION OF PCAF BY MIR-181A/B PROVIDES FEEDBACK REGULATION TO TNF-ALPHA-INDUCED TRANSCRIPTION OF PROINFLAMMATORY GENES IN LIVER EPITHELIAL CELLS. ABERRANT CELLULAR RESPONSES TO PROINFLAMMATORY CYTOKINES, SUCH AS TNF-ALPHA, ARE PATHOGENIC FEATURES IN MOST CHRONIC INFLAMMATORY DISEASES. A VARIETY OF EXTRACELLULAR AND INTRACELLULAR FEEDBACK PATHWAYS HAS EVOLVED TO PREVENT AN INAPPROPRIATE CELLULAR REACTION TO THESE PROINFLAMMATORY CYTOKINES. IN THIS STUDY, WE REPORT THAT TNF-ALPHA TREATMENT OF HUMAN AND MOUSE CHOLANGIOCYTES AND HEPATOCYTES DOWNREGULATED EXPRESSION OF P300/CBP-ASSOCIATED FACTOR (PCAF), A COACTIVATOR AND AN ACETYLTRANSFERASE THAT PROMOTES HISTONE ACETYLATION AND GENE TRANSCRIPTION. OF THESE UPREGULATED MICRORNAS IN TNF-ALPHA-TREATED CELLS, MIR-181A/B (MIR-181A AND MIR-181B) SUPPRESSED TRANSLATION OF PCAF MRNA. FUNCTIONAL MANIPULATION OF MIR-181A/B CAUSED RECIPROCAL ALTERATIONS IN PCAF PROTEIN EXPRESSION IN CULTURED CHOLANGIOCYTES AND HEPATOCYTES. INHIBITION OF MIR-181A/B FUNCTION WITH ANTI-MIRS BLOCKED TNF-ALPHA-INDUCED SUPPRESSION OF PCAF EXPRESSION. PROMOTER RECRUITMENT OF PCAF WAS SHOWN TO BE ASSOCIATED WITH TNF-ALPHA-INDUCED TRANSCRIPTION OF INFLAMMATORY GENES. INTRIGUINGLY, PRETREATMENT OF CELLS WITH TNF-ALPHA INHIBITED TRANSCRIPTION OF INFLAMMATORY GENES IN RESPONSE TO SUBSEQUENT TNF-ALPHA STIMULATION. OVEREXPRESSION OF PCAF OR INHIBITION OF MIR-181A/B FUNCTION WITH ANTI-MIRS ATTENUATED THE INHIBITORY EFFECTS OF TNF-ALPHA PRETREATMENT ON EPITHELIAL INFLAMMATORY RESPONSE TO SUBSEQUENT TNF-ALPHA STIMULATION. DOWNREGULATION OF PCAF AND THE INHIBITORY EFFECTS OF TNF-ALPHA PRETREATMENT ON LIVER EPITHELIAL INFLAMMATORY RESPONSE WERE FURTHER CONFIRMED IN A MOUSE MODEL OF TNF-ALPHA I.P. INJECTION. THESE DATA SUGGEST THAT PCAF IS A TARGET FOR MIR-181A/B, AND DOWNREGULATION OF PCAF BY TNF-ALPHA PROVIDES NEGATIVE FEEDBACK REGULATION TO INFLAMMATORY REACTIONS IN LIVER EPITHELIAL CELLS, A PROCESS THAT MAY BE RELEVANT TO THE EPIGENETIC FINE-TUNING OF EPITHELIAL INFLAMMATORY PROCESSES IN GENERAL. 2012 15 2748 33 EXPRESSION AND FUNCTION OF EZH2 IN SYNOVIAL FIBROBLASTS: EPIGENETIC REPRESSION OF THE WNT INHIBITOR SFRP1 IN RHEUMATOID ARTHRITIS. OBJECTIVES: TO STUDY THE EXPRESSION, REGULATION AND FUNCTION OF THE HISTONE METHYLTRANSFERASE ENHANCER OF ZESTE HOMOLOGUE 2 (EZH2) IN SYNOVIAL FIBROBLASTS (SF) FROM PATIENTS WITH RHEUMATOID ARTHRITIS (RA) AND OSTEOARTHRITIS (OA). METHODS: SF WERE OBTAINED FROM RA AND OA PATIENTS UNDERGOING JOINT SURGERY. EXPRESSION LEVELS WERE ASSESSED BY QUANTITATIVE REAL-TIME PCR AND WESTERN BLOT. KINASE INHIBITORS AND REPORTER GENE ASSAYS WERE EMPLOYED TO STUDY SIGNALLING PATHWAYS. FUNCTIONAL ANALYSES INCLUDED EZH2 OVEREXPRESSION BY PLASMID TRANSFECTION AND GENE SILENCING BY SMALL INTERFERING RNA. CHROMATIN IMMUNOPRECIPITATION ASSAY WAS USED TO ANALYSE HISTONE METHYLATION WITHIN DISTINCT PROMOTER REGIONS. RESULTS: BY STUDYING THE EXPRESSION AND FUNCTION OF EZH2 IN SF THE AUTHORS FOUND THAT EZH2 IS OVEREXPRESSED IN RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS (RASF) AND FURTHER INDUCED BY TUMOUR NECROSIS FACTOR ALPHA THROUGH THE NUCLEAR FACTOR KAPPA B AND JUN KINASE PATHWAYS. AS A TARGET GENE OF EZH2 THE AUTHORS IDENTIFIED SECRETED FRIZZLED-RELATED PROTEIN 1 (SFRP1), AN INHIBITOR OF WNT SIGNALLING, WHICH IS ASSOCIATED WITH THE ACTIVATION OF RASF, AND SHOW THAT SFRP1 EXPRESSION CORRELATES WITH THE OCCUPATION OF ITS PROMOTER WITH ACTIVATING AND SILENCING HISTONE MARKS. CONCLUSIONS: THESE DATA STRONGLY SUGGEST THAT THE CHRONIC INFLAMMATORY ENVIRONMENT OF THE RA JOINT INDUCES EZH2 AND THUS MIGHT CAUSE CHANGES IN THE EPIGENETIC PROGRAMMES OF SF. 2011 16 593 43 BET PROTEIN INHIBITION REGULATES CYTOKINE PRODUCTION AND PROMOTES NEUROPROTECTION AFTER SPINAL CORD INJURY. BACKGROUND: SPINAL CORD INJURY (SCI) USUALLY CAUSES A DEVASTATING LIFELONG DISABILITY FOR PATIENTS. AFTER A TRAUMATIC LESION, DISRUPTION OF THE BLOOD-SPINAL CORD BARRIER INDUCES THE INFILTRATION OF MACROPHAGES INTO THE LESION SITE AND THE ACTIVATION OF RESIDENT GLIAL CELLS, WHICH RELEASE CYTOKINES AND CHEMOKINES. THESE EVENTS RESULT IN A PERSISTENT INFLAMMATION, WHICH HAS BOTH DETRIMENTAL AND BENEFICIAL EFFECTS, BUT EVENTUALLY LIMITS FUNCTIONAL RECOVERY AND CONTRIBUTES TO THE APPEARANCE OF NEUROPATHIC PAIN. BROMODOMAIN AND EXTRA-TERMINAL DOMAIN (BET) PROTEINS ARE EPIGENETIC READERS THAT REGULATE THE EXPRESSION OF INFLAMMATORY GENES BY INTERACTING WITH ACETYLATED LYSINE RESIDUES. WHILE BET INHIBITORS ARE A PROMISING THERAPEUTIC STRATEGY FOR CANCER, LITTLE IS KNOWN ABOUT THEIR IMPLICATION AFTER SCI. THUS, THE CURRENT STUDY WAS AIMED TO INVESTIGATE THE ANTI-INFLAMMATORY ROLE OF BET INHIBITORS IN THIS PATHOLOGIC CONDITION. METHODS: WE EVALUATED THE EFFECTIVENESS OF THE BET INHIBITOR JQ1 TO MODIFY MACROPHAGE REACTIVITY IN VITRO AND TO MODULATE INFLAMMATION IN A SCI MICE MODEL. WE ANALYZED THE EFFECTS OF BET INHIBITION IN PRO-INFLAMMATORY AND ANTI-INFLAMMATORY CYTOKINE PRODUCTION IN VITRO AND IN VIVO. WE DETERMINED THE EFFECTIVENESS OF BET INHIBITION IN TISSUE SPARING, INFLAMMATION, NEURONAL PROTECTION, AND BEHAVIORAL OUTCOME AFTER SCI. RESULTS: WE HAVE FOUND THAT THE BET INHIBITOR JQ1 REDUCED THE LEVELS OF PRO-INFLAMMATORY MEDIATORS AND INCREASED THE EXPRESSION OF ANTI-INFLAMMATORY CYTOKINES. A PROLONGED TREATMENT WITH JQ1 ALSO DECREASED REACTIVITY OF MICROGLIA/MACROPHAGES, ENHANCED NEUROPROTECTION AND FUNCTIONAL RECOVERY, AND ACUTELY REDUCED NEUROPATHIC PAIN AFTER SCI. CONCLUSIONS: BET PROTEIN INHIBITION IS AN EFFECTIVE TREATMENT TO REGULATE CYTOKINE PRODUCTION AND PROMOTE NEUROPROTECTION AFTER SCI. THESE NOVEL RESULTS DEMONSTRATE FOR THE FIRST TIME THAT TARGETING BET PROTEINS IS AN ENCOURAGING APPROACH FOR SCI REPAIR AND A POTENTIAL STRATEGY TO TREAT OTHER INFLAMMATORY PATHOLOGIES. 2019 17 5795 32 STAT3 INDUCTION OF MIR-146B FORMS A FEEDBACK LOOP TO INHIBIT THE NF-KAPPAB TO IL-6 SIGNALING AXIS AND STAT3-DRIVEN CANCER PHENOTYPES. INTERLEUKIN-6 (IL-6)-MEDIATED ACTIVATION OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 3 (STAT3) IS A MECHANISM BY WHICH CHRONIC INFLAMMATION CAN CONTRIBUTE TO CANCER AND IS A COMMON ONCOGENIC EVENT. WE DISCOVERED A PATHWAY, THE LOSS OF WHICH IS ASSOCIATED WITH PERSISTENT STAT3 ACTIVATION IN HUMAN CANCER. WE FOUND THAT THE GENE ENCODING THE TUMOR SUPPRESSOR MICRORNA MIR-146B IS A DIRECT STAT3 TARGET GENE, AND ITS EXPRESSION WAS INCREASED IN NORMAL BREAST EPITHELIAL CELLS BUT DECREASED IN TUMOR CELLS. METHYLATION OF THE MIR-146B PROMOTER, WHICH INHIBITED STAT3-MEDIATED INDUCTION OF EXPRESSION, WAS INCREASED IN PRIMARY BREAST CANCERS. MOREOVER, WE FOUND THAT MIR-146B INHIBITED NUCLEAR FACTOR KAPPAB (NF-KAPPAB)-DEPENDENT PRODUCTION OF IL-6, SUBSEQUENT STAT3 ACTIVATION, AND IL-6/STAT3-DRIVEN MIGRATION AND INVASION IN BREAST CANCER CELLS, THEREBY ESTABLISHING A NEGATIVE FEEDBACK LOOP. IN ADDITION, HIGHER EXPRESSION OF MIR-146B WAS POSITIVELY CORRELATED WITH PATIENT SURVIVAL IN BREAST CANCER SUBTYPES WITH INCREASED IL6 EXPRESSION AND STAT3 PHOSPHORYLATION. OUR RESULTS IDENTIFY AN EPIGENETIC MECHANISM OF CROSSTALK BETWEEN STAT3 AND NF-KAPPAB RELEVANT TO CONSTITUTIVE STAT3 ACTIVATION IN MALIGNANCY AND THE ROLE OF INFLAMMATION IN ONCOGENESIS. 2014 18 5908 33 TARGETED DE-METHYLATION OF THE FOXP3-TSDR IS SUFFICIENT TO INDUCE PHYSIOLOGICAL FOXP3 EXPRESSION BUT NOT A FUNCTIONAL TREG PHENOTYPE. CD4+ REGULATORY T CELLS (TREGS) ARE KEY MEDIATORS OF IMMUNOLOGICAL TOLERANCE AND PROMISING EFFECTOR CELLS FOR IMMUNO-SUPPRESSIVE ADOPTIVE CELLULAR THERAPY TO FIGHT AUTOIMMUNITY AND CHRONIC INFLAMMATION. THEIR FUNCTIONAL STABILITY IS CRITICAL FOR THEIR CLINICAL UTILITY AND HAS BEEN CORRELATED TO THE DEMETHYLATED STATE OF THE TSDR/CNS2 ENHANCER ELEMENT IN THE TREG LINEAGE TRANSCRIPTION FACTOR FOXP3. HOWEVER, PROOF FOR A CAUSAL CONTRIBUTION OF THE TSDR DE-METHYLATION TO FOXP3 STABILITY AND TREG INDUCTION IS SO FAR LACKING. WE HERE ESTABLISHED A POWERFUL TRANSIENT-TRANSFECTION CRISPR-CAS9-BASED EPIGENETIC EDITING METHOD FOR THE SELECTIVE DE-METHYLATION OF THE TSDR WITHIN THE ENDOGENOUS CHROMATIN ENVIRONMENT OF A LIVING CELL. THE INDUCED DE-METHYLATED STATE WAS STABLE OVER WEEKS IN CLONAL T CELL PROLIFERATION CULTURES EVEN AFTER EXPRESSION OF THE EDITING COMPLEX HAD CEASED. EPIGENETIC EDITING OF THE TSDR RESULTED IN FOXP3 EXPRESSION, EVEN IN ITS PHYSIOLOGICAL ISOFORM DISTRIBUTION, PROVING A CAUSAL ROLE FOR THE DE-METHYLATED TSDR IN FOXP3 REGULATION. HOWEVER, SUCCESSFUL FOXP3 INDUCTION WAS NOT ASSOCIATED WITH A SWITCH TOWARDS A FUNCTIONAL TREG PHENOTYPE, IN CONTRAST TO WHAT HAS BEEN REPORTED FROM FOXP3 OVEREXPRESSION APPROACHES. THUS, TSDR DE-METHYLATION IS REQUIRED, BUT NOT SUFFICIENT FOR A STABLE TREG PHENOTYPE INDUCTION. THEREFORE, TARGETED DEMETHYLATION OF THE TSDR MAY BE A CRITICAL ADDITION TO PUBLISHED IN VITRO TREG INDUCTION PROTOCOLS WHICH SO FAR LACK FOXP3 STABILITY. 2020 19 674 34 BRAHMA-RELATED GENE 1 BRIDGES EPIGENETIC REGULATION OF PROINFLAMMATORY CYTOKINE PRODUCTION TO STEATOHEPATITIS IN MICE. CHRONIC INFLAMMATION, INFLICTED BY THE SPILLOVER OF PROINFLAMMATORY MEDIATORS, LINKS METABOLIC DYSFUNCTION TO NONALCOHOLIC STEATOHEPATITIS (NASH). THE EPIGENETIC MANEUVERINGS THAT UNDERSCORE ACCELERATED SYNTHESIS OF PROINFLAMMATORY MEDIATORS IN RESPONSE TO NUTRITIONAL INPUTS ARE NOT CLEARLY DEFINED. HERE WE REPORT THAT THE ATP-DEPENDENT CHROMATIN REMODELING PROTEINS BRAHMA-RELATED GENE 1 (BRG1) AND BRAHMA (BRM) WERE UP-REGULATED IN VITRO IN CULTURED HEPATOCYTES TREATED WITH FREE FATTY ACID OR GLUCOSE AND IN VIVO IN ANIMAL MODELS OF NASH. OCCUPANCY OF BRG1 AND BRM ON THE PROMOTER REGIONS OF PROINFLAMMATORY GENES WAS INCREASED IN VITRO IN CELLS AND EX VIVO IN LIVER TISSUES. ESTRADIOL SUPPRESSED THE INDUCTION AND RECRUITMENT OF BRG1/BRM BY PALMITATE. RECRUITMENT OF BRG1 AND BRM RELIED ON NUCLEAR FACTOR KAPPA B/P65; RECIPROCALLY, BRG1 AND BRM CONTRIBUTED TO THE STABILIZATION OF P65 BINDING. IMPORTANTLY, OVEREXPRESSION OF BRG1/BRM ENHANCED, WHEREAS KNOCKDOWN OF BRG1/BRM ATTENUATED, THE INDUCTION OF PROINFLAMMATORY MEDIATORS IN HEPATOCYTES CHALLENGED WITH EXCESSIVE NUTRIENT. MECHANISTICALLY, BRG1 AND BRM WERE INVOLVED IN THE MAINTENANCE OF A CHROMATIN MICROENVIRONMENT MARKED BY ACTIVE HISTONE MODIFICATIONS AND FRIENDLY TO THE ACCESS OF THE GENERAL TRANSCRIPTIONAL MACHINERY. FINALLY, DEPLETION OF BRG1/BRM BY SHORT HAIRPIN RNA ATTENUATED THE RELEASE OF PROINFLAMMATORY MEDIATORS IN THE LIVER AND SIGNIFICANTLY AMELIORATED HEPATIC PATHOLOGY IN NASH MICE. CONCLUSION: OUR DATA ILLUSTRATE A BRG1-DEPENDENT PATHWAY THAT CONNECTS THE EPIGENETIC REGULATION OF PROINFLAMMATORY GENES TO THE PATHOGENESIS OF NASH AND POINT TO A POTENTIAL DRUGGABLE TARGET IN THE THERAPEUTIC INTERVENTION OF NASH. 2013 20 4357 29 MIR-30E* IS OVEREXPRESSED IN PROSTATE CANCER AND PROMOTES NF-KAPPAB-MEDIATED PROLIFERATION AND TUMOR GROWTH. ACCORDING TO THE CDC PROSTATE CANCER (CAP) HAS THE HIGHEST INCIDENCE AND SECOND HIGHEST MORTALITY RATE AMONGST CANCERS IN AMERICAN MEN. CONSTITUTIVE NF-KAPPAB ACTIVATION IS A HALLMARK OF CAP AND THIS PATHWAY DRIVES MANY PRO-TUMORIGENIC CHARACTERISTICS OF CAP CELLS, INCLUDING CELL PROLIFERATION AND SURVIVAL. AN ACTIVATED NF-KAPPAB GENE SIGNATURE IS PREDICTIVE OF CAP PROGRESSION AND BIOCHEMICAL RECURRENCE FOLLOWING THERAPEUTIC INTERVENTION. HOWEVER, THE MECHANISMS THAT PERPETUATE NF-KAPPAB ACTIVATION ARE INCOMPLETELY UNDERSTOOD. GENES THAT CONTROL NF-KAPPAB ACTIVITY ARE RARELY MUTATED IN CAP SUGGESTING THAT EPIGENETIC MECHANISMS MAY CONTRIBUTE TO CONSTITUTIVE NF-KAPPAB ACTIVATION. MICRORNAS (MIRS) EPIGENETICALLY REGULATE MANY GENES INVOLVED WITH NF-KAPPAB ACTIVATION. IKAPPABALPHA IS A DIRECT INHIBITOR OF NF-KAPPAB; IT BINDS TO AND SEQUESTERS NF-KAPPAB IN THE CYTOPLASM RESULTING IN FUNCTIONAL INHIBITION. IKAPPABALPHA IS A TARGET GENE OF MIR-30E* YET THE EXPRESSION AND ONCOLOGICAL IMPACT OF MIR-30E* IN CAP IS UNKNOWN. WE REPORT THAT MIR-30E* EXPRESSION IS ELEVATED IN MULTIPLE MURINE MODELS OF CAP AND IS MOST PRONOUNCED IN LATE STAGE DISEASE. MIR-30E* DRIVES CAP PROLIFERATION AND TUMOR GROWTH THROUGH INHIBITION OF IKAPPABALPHA, WHICH RESULTS IN CHRONIC ACTIVATION OF NF-KAPPAB. ADDITIONALLY, WE SHOW THAT INHIBITION OF MIR-30E* IMPROVES CHEMOTHERAPEUTIC CONTROL OF CAP. THUS, MIR-30E* MAY PROVE TO BE A NOVEL CLINICAL TARGET WHOSE INHIBITION LEADS TO DECREASED CAP CELL PROLIFERATION AND SENSITIZATION OF CAP CELLS TO CHEMOTHERAPEUTICS. 2017