1 2185 134 EPIGENETIC MECHANISMS UNDERLYING HIV-INFECTION INDUCED SUSCEPTIBILITY OF CD4+ T CELLS TO ENHANCED ACTIVATION-INDUCED FASL EXPRESSION AND CELL DEATH. BACKGROUND: CHRONIC IMMUNE ACTIVATION AND CD4 T CELL DEPLETION ARE SIGNIFICANT PATHOGENIC FEATURES OF HIV INFECTION. EXPRESSION OF FAS LIGAND (FASL), A KEY MEDIATOR OF ACTIVATION-INDUCED CELL DEATH IN T CELLS, IS ELEVATED IN PEOPLE LIVING WITH HIV-1 INFECTION (PLWH). HOWEVER, THE EPIGENETIC MECHANISMS UNDERLYING THE ENHANCED INDUCTION OF FASL EXPRESSION IN CD4 T LYMPHOCYTES IN PLWH ARE NOT COMPLETELY ELUCIDATED. HENCE, THE CURRENT WORK EXAMINED THE EFFECT OF HIV INFECTION ON FASL PROMOTER-ASSOCIATED HISTONE MODIFICATIONS AND TRANSCRIPTIONAL REGULATION IN CD4 T LYMPHOCYTES IN PLWH. METHOD: FLOW CYTOMETRIC ANALYSIS WAS PERFORMED TO EXAMINE THE FAS-FASL EXPRESSION ON TOTAL CD4 T CELLS AND NAIVE/MEMORY CD4 T CELL SUBSETS. EPIGENETIC FASL PROMOTER HISTONE MODIFICATIONS WERE INVESTIGATED BY CHROMATIN IMMUNOPRECIPITATION-QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION ANALYSIS USING FRESHLY ISOLATED TOTAL CD4 T LYMPHOCYTES FROM HIV-1 INFECTED AND NONINFECTED INDIVIDUALS. RESULTS: ALL NAIVE/MEMORY CD4 T CELL SUBSETS FROM PLWH SHOWED MARKEDLY GREATER FREQUENCY OF FASL EXPRESSION. NOTABLY, EXAMINATION OF FUNCTIONAL OUTCOME OF FASL/FAS CO-EXPRESSION DEMONSTRATED THE PREFERENTIAL SUSCEPTIBILITY OF TCM AND TEM SUBSETS TO ACTIVATION-INDUCED APOPTOSIS. IMPORTANTLY, THESE CD4 T CELLS COLLECTIVELY DEMONSTRATED A DISTINCT FASL PROMOTER HISTONE PROFILE INVOLVING A COORDINATED CROSS-TALK BETWEEN HISTONE H3 MODIFICATIONS LEADING TO ENHANCED FASL GENE EXPRESSION. SPECIFICALLY, LEVELS OF TRANSCRIPTIONALLY PERMISSIVE HISTONE H3K4-TRIMETHYLATION (H3K4ME3) AND HISTONE H3K9-ACETYLATION (H3K9AC) WERE INCREASED, WITH A CONCOMITANT DECREASE IN THE REPRESSIVE H3K9-TRIMETHYLATION (H3K9ME3). CONCLUSION: THE PRESENT WORK DEMONSTRATES THAT EPIGENETIC MECHANISMS INVOLVING PROMOTER-HISTONE MODIFICATIONS REGULATE TRANSCRIPTIONAL COMPETENCE AND FASL EXPRESSION IN CD4 T CELLS FROM PLWH AND RENDER THEM SUSCEPTIBLE TO ACTIVATION-INDUCED CELL DEATH. 2021 2 2392 35 EPIGENETIC REPRESSION OF INTERLEUKIN 2 EXPRESSION IN SENESCENT CD4+ T CELLS DURING CHRONIC HIV TYPE 1 INFECTION. THE MOLECULAR MECHANISMS FOR IL2 GENE-SPECIFIC DYSREGULATION DURING CHRONIC HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1) INFECTION ARE UNKNOWN. HERE, WE INVESTIGATED THE ROLE OF DNA METHYLATION IN SUPPRESSING INTERLEUKIN 2 (IL-2) EXPRESSION IN MEMORY CD4(+) T CELLS DURING CHRONIC HIV-1 INFECTION. WE OBSERVED THAT CPG SITES IN THE IL2 PROMOTER OF CD4(+) T CELLS WERE FULLY METHYLATED IN NAIVE CD4(+) T CELLS AND SIGNIFICANTLY DEMETHYLATED IN THE MEMORY POPULATIONS. INTERESTINGLY, WE FOUND THAT THE MEMORY CELLS THAT HAD A TERMINALLY DIFFERENTIATED PHENOTYPE AND EXPRESSED CD57 HAD INCREASED IL2 PROMOTER METHYLATION RELATIVE TO LESS DIFFERENTIATED MEMORY CELLS IN HEALTHY INDIVIDUALS. IMPORTANTLY, EARLY EFFECTOR MEMORY SUBSETS FROM HIV-1-INFECTED SUBJECTS EXPRESSED HIGH LEVELS OF CD57 AND WERE HIGHLY METHYLATED AT THE IL2 LOCUS. FURTHERMORE, THE INCREASED CD57 EXPRESSION ON MEMORY CD4(+) T CELLS WAS INVERSELY CORRELATED WITH IL-2 PRODUCTION. THESE DATA SUGGEST THAT DNA METHYLATION AT THE IL2 LOCUS IN CD4(+) T CELLS IS COUPLED TO IMMUNOSENESCENCE AND PLAYS A CRITICAL ROLE IN THE BROAD DYSFUNCTION THAT OCCURS IN POLYCLONAL T CELLS DURING HIV-1 INFECTION. 2015 3 1500 39 DNA METHYLATION ANALYSIS OF CD4+ T CELLS IN PATIENTS WITH PSORIASIS. PSORIASIS IS A CHRONIC INFLAMMATORY SKIN DISEASE THAT IS CHARACTERIZED BY ABERRANT CROSS-TALK BETWEEN KERATINOCYTES AND IMMUNE CELLS SUCH AS CD4+ T CELLS, RESULTING IN KERATINOCYTE HYPERPROLIFERATION IN THE EPIDERMIS. DNA METHYLATION, ONE OF SEVERAL EPIGENETIC MECHANISMS, PLAYS AN IMPORTANT ROLE IN GENE EXPRESSION WITHOUT CHANGING THE DNA SEQUENCE. SEVERAL STUDIES HAVE SUGGESTED THE INVOLVEMENT OF EPIGENETIC REGULATION IN SKIN LESIONS FROM PATIENTS WITH PSORIASIS. IN THIS STUDY, WE INVESTIGATED THE GENOME-WIDE DNA METHYLATION STATUS OF CD4+ T CELLS IN PATIENTS WITH PSORIASIS COMPARED WITH HEALTHY SUBJECTS USING METHYLATED DNA IMMUNOPRECIPITATION SEQUENCING (MEDIP-SEQ). THE RESULTS OF MEDIP-SEQ SHOWED THAT THE GLOBAL METHYLATION VALUES OF CD4+ T CELLS ARE HIGHER IN PATIENTS WITH PSORIASIS THAN IN HEALTHY CONTROLS, PARTICULARLY IN THE PROMOTER REGIONS. AMONG THE MOST HYPERMETHYLATED GENES IN THE PROMOTER REGIONS, WE SELECTED THE GENES WHOSE EXPRESSION IS SIGNIFICANTLY REDUCED IN THE CD4+ T CELLS OF PSORIASIS PATIENTS. STUDIES USING THE METHYLATION INHIBITOR 5-AZACYTIDINE IN VITRO METHYLATION ASSAYS HAVE SHOWN THAT THE DIFFERENTIAL EXPRESSION LEVELS WERE ASSOCIATED WITH THE METHYLATION STATUS OF EACH GENE. BISULFITE SEQUENCING OF THE TRANSCRIPTION START REGION OF PHOSPHATIDIC ACID PHOSPHATASE TYPE 2 DOMAIN CONTAINING 3 (PPAPDC3), ONE OF THE SELECTED GENES, SHOWED HYPERMETHYLATION IN THE CD4+ T CELLS OF PSORIASIS PATIENTS. THESE RESULTS SUGGESTED THAT THE METHYLATION STATUS, WHICH IS IDENTIFIED BY MEDIP-SEQ OF THE GENES, WAS CORRELATED WITH THE MRNA EXPRESSION LEVEL OF THE GENES. COLLECTIVELY, THE DNA METHYLATION STATUS IN CD4+ T CELLS MIGHT BE ASSOCIATED WITH THE PATHOGENESIS OF PSORIASIS. 2014 4 5223 34 PRIMARY MURINE CD4+ T CELLS FAIL TO ACQUIRE THE ABILITY TO PRODUCE EFFECTOR CYTOKINES WHEN ACTIVE RAS IS PRESENT DURING TH1/TH2 DIFFERENTIATION. CONSTITUTIVE RAS SIGNALING HAS BEEN SHOWN TO AUGMENT IL-2 PRODUCTION, REVERSE ANERGY, AND FUNCTIONALLY REPLACE MANY ASPECTS OF CD28 CO-STIMULATION IN CD4+ T CELLS. THESE DATA RAISE THE POSSIBILITY THAT INTRODUCTION OF ACTIVE RAS INTO PRIMARY T CELLS MIGHT RESULT IN IMPROVED FUNCTIONALITY IN PATHOLOGIC SITUATIONS OF T CELL DYSFUNCTION, SUCH AS CANCER OR CHRONIC VIRAL INFECTION. TO TEST THE BIOLOGIC EFFECTS OF ACTIVE RAS IN PRIMARY T CELLS, CD4+ T CELLS FROM COXSACKIE-ADENOVIRUS RECEPTOR TRANSGENIC MICE WERE TRANSDUCED WITH AN ADENOVIRUS ENCODING ACTIVE RAS. AS EXPECTED, ACTIVE RAS AUGMENTED IL-2 PRODUCTION IN NAIVE CD4+ T CELLS. HOWEVER, WHEN CELLS WERE CULTURED FOR 4 DAYS UNDER CONDITIONS TO PROMOTE EFFECTOR CELL DIFFERENTIATION, ACTIVE RAS INHIBITED THE ABILITY OF CD4+ T CELLS TO ACQUIRE A TH1 OR TH2 EFFECTOR CYTOKINE PROFILE. THIS DIFFERENTIATION DEFECT WAS NOT DUE TO DEFICIENT STAT4 OR STAT6 ACTIVATION BY IL-12 OR IL-4, RESPECTIVELY, NOR WAS IT ASSOCIATED WITH DEFICIENT INDUCTION OF T-BET AND GATA-3 EXPRESSION. IMPAIRED EFFECTOR CYTOKINE PRODUCTION IN ACTIVE RAS-TRANSDUCED CELLS WAS ASSOCIATED WITH DEFICIENT DEMETHYLATION OF THE IL-4 GENE LOCUS. OUR RESULTS INDICATE THAT, DESPITE AUGMENTING ACUTE ACTIVATION OF NAIVE T CELLS, CONSTITUTIVE RAS SIGNALING INHIBITS THE ABILITY OF CD4+ T CELLS TO PROPERLY DIFFERENTIATE INTO TH1/TH2 EFFECTOR CYTOKINE-PRODUCING CELLS, IN PART BY INTERFERING WITH EPIGENETIC MODIFICATION OF EFFECTOR GENE LOCI. ALTERNATIVE STRATEGIES TO POTENTIATE RAS PATHWAY SIGNALING IN T CELLS IN A MORE REGULATED FASHION SHOULD BE CONSIDERED AS A THERAPEUTIC APPROACH TO IMPROVE IMMUNE RESPONSES IN VIVO. 2014 5 2143 38 EPIGENETIC LANDSCAPE OF HIV-1 INFECTION IN PRIMARY HUMAN MACROPHAGE. HUMAN IMMUNODEFICIENCY VIRUS (HIV)-INFECTED MACROPHAGES ARE LONG-LIVED CELLS THAT SUSTAIN PERSISTENT VIRUS EXPRESSION, WHICH IS BOTH A BARRIER TO VIRAL ERADICATION AND CONTRIBUTOR TO NEUROLOGICAL COMPLICATIONS IN PATIENTS DESPITE ANTIRETROVIRAL THERAPY (ART). TO BETTER UNDERSTAND THE REGULATION OF HIV-1 IN MACROPHAGES, WE COMPARED HIV-INFECTED PRIMARY HUMAN MONOCYTE-DERIVED MACROPHAGES (MDM) TO ACUTELY INFECTED PRIMARY CD4 T CELLS AND JURKAT CELLS LATENTLY INFECTED WITH HIV (JLAT 8.4). HIV GENOMES IN MDM WERE ACTIVELY TRANSCRIBED DESPITE ENRICHMENT WITH HETEROCHROMATIN-ASSOCIATED H3K9ME3 ACROSS THE COMPLETE HIV GENOME IN COMBINATION WITH ELEVATED ACTIVATION MARKS OF H3K9AC AND H3K27AC AT THE LONG TERMINAL REPEAT (LTR). MACROPHAGE PATTERNS CONTRASTED WITH JLAT CELLS, WHICH SHOWED CONVENTIONAL BIVALENT H3K4ME3/H3K27ME3, AND ACUTELY INFECTED CD4 T CELLS, WHICH SHOWED AN INTERMEDIATE EPIGENOTYPE. 5'-METHYLCYTOSINE (5MC) WAS ENRICHED ACROSS THE HIV GENOME IN LATENTLY INFECTED JLAT CELLS, WHILE 5'-HYDROXYMETHYLCYTOSINE (5HMC) WAS ENRICHED IN CD4 CELLS AND MDMS. HIV INFECTION INDUCED MULTINUCLEATION OF MDMS ALONG WITH DNA DAMAGE-ASSOCIATED P53 PHOSPHORYLATION, AS WELL AS LOSS OF TET2 AND THE NUCLEAR REDISTRIBUTION OF 5-HYDOXYMETHYLATION. TAKEN TOGETHER, OUR FINDINGS SUGGEST THAT HIV INDUCES A UNIQUE MACROPHAGE NUCLEAR AND TRANSCRIPTIONAL PROFILE, AND VIRAL GENOMES ARE MAINTAINED IN A NONCANONICAL BIVALENT EPIGENETIC STATE. IMPORTANCE MACROPHAGES SERVE AS A RESERVOIR FOR LONG-TERM PERSISTENCE AND CHRONIC PRODUCTION OF HIV. WE FOUND AN ATYPICAL EPIGENETIC CONTROL OF HIV IN MACROPHAGES MARKED BY HETEROCHROMATIC H3K9ME3 DESPITE ACTIVE VIRAL TRANSCRIPTION. HIV INFECTION INDUCED CHANGES IN MACROPHAGE NUCLEAR MORPHOLOGY AND EPIGENETIC REGULATORY FACTORS. THESE FINDINGS MAY IDENTIFY NEW MECHANISMS TO CONTROL CHRONIC HIV EXPRESSION IN INFECTED MACROPHAGES. 2022 6 5902 40 T-HELPER 17 CELL POLARIZATION IN PULMONARY ARTERIAL HYPERTENSION. BACKGROUND: INFLAMMATION MAY CONTRIBUTE TO THE PATHOBIOLOGY OF PULMONARY ARTERIAL HYPERTENSION (PAH). DECIPHERING THE PAH FINGERPRINT ON THE INFLAMMATION ORCHESTRATED BY DENDRITIC CELLS (DCS) AND T CELLS, KEY DRIVER AND EFFECTOR CELLS, RESPECTIVELY, OF THE IMMUNE SYSTEM, MAY ALLOW THE IDENTIFICATION OF IMMUNOPATHOLOGIC APPROACHES TO PAH MANAGEMENT. METHODS: USING FLOW CYTOMETRY, WE PERFORMED IMMUNOPHENOTYPING OF MONOCYTE-DERIVED DCS (MODCS) AND CIRCULATING LYMPHOCYTES FROM PATIENTS WITH IDIOPATHIC PAH AND CONTROL SUBJECTS. WITH THE SAME TECHNIQUE, WE PERFORMED CYTOKINE PROFILING OF BOTH POPULATIONS FOLLOWING STIMULATION, COCULTURE, OR BOTH. WE TESTED THE IMMUNOMODULATORY EFFECTS OF A GLUCOCORTICOID (DEXAMETHASONE [DEX]) ON THIS IMMUNOPHENOTYPE AND CYTOKINE PROFILE. USING AN EPIGENETIC APPROACH, WE CONFIRMED THE IMMUNE POLARIZATION IN BLOOD DNA OF PATIENTS WITH PAH. RESULTS: THE PROFILE OF MEMBRANE COSTIMULATORY MOLECULES OF PAH MODCS WAS SIMILAR TO THAT OF CONTROL SUBJECTS. HOWEVER, PAH MODCS RETAINED HIGHER LEVELS OF THE T-CELL ACTIVATING MOLECULES CD86 AND CD40 AFTER DEX PRETREATMENT THAN DID CONTROL MODCS. THIS WAS ASSOCIATED WITH AN INCREASED EXPRESSION OF IL-12P40 AND A REDUCED MIGRATION TOWARD CHEMOKINE (C-C MOTIF) LIGAND 21. MOREOVER, BOTH WITH AND WITHOUT DEX, PAH MODCS INDUCED A HIGHER ACTIVATION AND PROLIFERATION OF CD4+ T CELLS, ASSOCIATED WITH A REDUCED EXPRESSION OF IL-4 (T HELPER 2 RESPONSE) AND A HIGHER EXPRESSION OF IL-17 (T HELPER 17 RESPONSE). PURIFIED PAH CD4+ T CELLS EXPRESSED A HIGHER LEVEL OF IL-17 AFTER ACTIVATION THAN DID THOSE OF CONTROL SUBJECTS. LASTLY, THERE WAS SIGNIFICANT HYPOMETHYLATION OF THE IL-17 PROMOTER IN THE PAH BLOOD DNA AS COMPARED WITH THE CONTROL BLOOD. CONCLUSIONS: WE HAVE HIGHLIGHTED T HELPER 17 CELL IMMUNE POLARIZATION IN PATIENTS WITH PAH, AS HAS BEEN PREVIOUSLY DEMONSTRATED IN OTHER CHRONIC INFLAMMATORY AND AUTOIMMUNE CONDITIONS. 2015 7 2200 41 EPIGENETIC MODIFICATION OF FOXP3 IN PATIENTS WITH CHRONIC HIV INFECTION. OBJECTIVES: HIV-1 MODULATES HOST CELL EPIGENETIC MACHINERY TO CONTROL ITS OWN REPLICATION AND INDUCE IMMUNE SUPPRESSION. HIV-1 INFECTION LEADS TO ACTIVATION OF T REGULATORY CELL (T(REG)), BUT THE MECHANISM UNDERLYING THIS IMMUNE MODULATION IS UNCLEAR. T(REG) PLAYS A PROMINENT ROLE IN GUT-MUCOSAL IMMUNE TOLERANCE BY RESTRAINING EXCESSIVE EFFECTOR T-CELL RESPONSES, A MECHANISM THAT IS KNOWN TO BE DISTURBED IN CHRONIC HIV-1 INFECTION. DNA METHYLATION PLAYS A MAJOR ROLE IN T(REG) LINEAGE COMMITMENT AND IMMUNE HOMEOSTASIS, WHICH MAY BE REGULATED BY HIV. TO INVESTIGATE THE MECHANISMS OF ABERRANT METHYLATION OF THE T(REG) MARKER FOXP3 IN HIV-1 INFECTION, WE EVALUATED THE EXPRESSION PATTERN OF METHYLATION-RELATED ENZYMES AND ITS CORRELATION TO FOXP3 METHYLATION. METHODS: FOXP3 PROMOTER METHYLATION IN THE COLON MUCOSA AND PERIPHERAL BLOOD FROM HIV-INFECTED PATIENTS AND CONTROL SUBJECTS WAS MEASURED USING PYROSEQUENCING. GENE EXPRESSION PATTERN OF DNA METHYLATION ENZYMES IN THE COLON MUCOSA WAS INVESTIGATED BY MICROARRAY AND QUANTITATIVE REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION ANALYSIS IN THE SAME SUBJECTS. RESULTS: FOXP3 PROMOTER WAS SIGNIFICANTLY (P