1 5509 127 RHPLD2 INHIBITS AIRWAY INFLAMMATION IN AN ASTHMATIC MURINE MODEL THROUGH INDUCTION OF STABLE CD25(+) FOXP3(+) TREGS. OUR PREVIOUS STUDIES HAVE SHOWN THAT RECOMBINANT HUMAN PHOSPHOLIPASE D2 (RHPLD2) PLAYS A MODULATOR ROLE ON NF-KAPPAB AND PKC SIGNALING PATHWAYS. IT ALSO INHIBITS IL-5-INDUCED INFLAMMATORY RESPONSE IN CHRONIC ASTHMATIC GUINEA PIGS. ADDITIONALLY, INCREASING EVIDENCE ALSO HAS REVEALED THAT THE ADOPTIVE TRANSFER OF INDUCED REGULATORY T CELLS (TREGS) MAY BE A THERAPEUTIC SOLUTION TO AIRWAY ALLERGIC DISEASES. TO INVESTIGATE THE EPIGENETIC, TRANSCRIPTOMIC AND PHENOTYPIC VARIABILITY OF TREG POPULATION IN AN OVALBUMIN (OVA)-INDUCED AIRWAY INFLAMMATION MODEL DERIVED FROM THE INDUCTION OF RHPLD2, OVA-INDUCED ASTHMATIC MURINE MODEL IS USED IN THIS STUDY. THE LUNG INFLAMMATION, EOSINOPHIL INFILTRATION, THE DIFFERENTIATION AND PROLIFERATION OF T HELPER CELLS AND THE AMPLIFICATION OF TREGS WERE EXAMINED IN THIS MOUSE MODEL WITH AND WITHOUT RHPLD2 INDUCTION. OUR DATA SHOWED THAT RHPLD2 ADMINISTRATION IN ASTHMATIC MICE SIGNIFICANTLY INCREASES CD4(+)CD25(+) FOXP3(+) TREG CELL NUMBERS AND ALLEVIATES LUNG INFLAMMATION. THE ADDITION OF RHPLD2 IN VITRO ENHANCED THE DEMETHYLATION OF TREG-SPECIFICDEMETHYLATED REGION (TSDR) IN ITREGS, SUGGESTING THAT RHPLD2 PROTEIN MAY BE INVOLVED IN IMPROVING THE QUALITY AND QUANTITY OF TREG CELLS THAT EVENTUALLY SIGNIFICANTLY REDUCES LUNG INFLAMMATION IN ASTHMATIC MURINE MODEL. THESE RESULTS SUGGEST THAT RHPLD2 COULD HAVE A CLINICAL IMPACT TREATING PATIENTS WITH ALLERGIC AIRWAY INFLAMMATION VIA PROMOTING AND STABILIZING ITREG DIFFERENTIATION AND FUNCTION. 2018 2 3662 40 INDUCTION OF STABLE HUMAN FOXP3(+) TREGS BY A PARASITE-DERIVED TGF-BETA MIMIC. IMMUNE HOMEOSTASIS IN THE INTESTINE IS TIGHTLY CONTROLLED BY FOXP3(+) REGULATORY T CELLS (TREGS), DEFECTS OF WHICH ARE LINKED TO THE DEVELOPMENT OF CHRONIC CONDITIONS, SUCH AS INFLAMMATORY BOWEL DISEASE (IBD). AS A MECHANISM OF IMMUNE EVASION, SEVERAL SPECIES OF INTESTINAL PARASITES BOOST TREG ACTIVITY. THE PARASITE HELIGMOSOMOIDES POLYGYRUS IS KNOWN TO SECRETE A MOLECULE (HP-TGM) THAT MIMICS THE ABILITY OF TGF-BETA TO INDUCE FOXP3 EXPRESSION IN CD4(+) T CELLS. THE STUDY AIMED TO INVESTIGATE WHETHER HP-TGM COULD INDUCE HUMAN FOXP3(+) TREGS AS A POTENTIAL THERAPEUTIC APPROACH FOR INFLAMMATORY DISEASES. CD4(+) T CELLS FROM HEALTHY VOLUNTEERS WERE EXPANDED IN THE PRESENCE OF HP-TGM OR TGF-BETA. TREG INDUCTION WAS MEASURED BY FLOW CYTOMETRIC DETECTION OF FOXP3 AND OTHER TREG MARKERS, SUCH AS CD25 AND CTLA-4. EPIGENETIC CHANGES WERE DETECTED USING CHIP-SEQ AND PYROSEQUENCING OF FOXP3. TREG PHENOTYPE STABILITY WAS ASSESSED FOLLOWING INFLAMMATORY CYTOKINE CHALLENGE AND TREG FUNCTION WAS EVALUATED BY CELLULAR CO-CULTURE SUPPRESSION ASSAYS AND CYTOMETRIC BEAD ARRAYS FOR SECRETED CYTOKINES. HP-TGM EFFICIENTLY INDUCED FOXP3 EXPRESSION (> 60%), IN ADDITION TO CD25 AND CTLA-4, AND CAUSED EPIGENETIC MODIFICATION OF THE FOXP3 LOCUS TO A GREATER EXTENT THAN TGF-BETA. HP-TGM-INDUCED TREGS HAD SUPERIOR SUPPRESSIVE FUNCTION COMPARED WITH TGF-BETA-INDUCED TREGS, AND RETAINED THEIR PHENOTYPE FOLLOWING EXPOSURE TO INFLAMMATORY CYTOKINES. FURTHERMORE, HP-TGM INDUCED A TREG-LIKE PHENOTYPE IN IN VIVO DIFFERENTIATED TH1 AND TH17 CELLS, INDICATING ITS POTENTIAL TO RE-PROGRAM MEMORY CELLS TO ENHANCE IMMUNE TOLERANCE. THESE DATA INDICATE HP-TGM HAS POTENTIAL TO BE USED TO GENERATE STABLE HUMAN FOXP3(+) TREGS TO TREAT IBD AND OTHER INFLAMMATORY DISEASES. 2021 3 272 48 AGE-DEPENDENT DECREASE IN THE INDUCTION OF REGULATORY T CELLS IS ASSOCIATED WITH DECREASED EXPRESSION OF RALDH2 IN MESENTERIC LYMPH NODE DENDRITIC CELLS. A DECLINE IN IMMUNE FUNCTION WITH AGING HAS BEEN REPORTED. REGULATORY T CELL (TREG) INDUCTION IS KNOWN TO DECREASE WITH AGE, AND ELUCIDATING THE UNDERLYING MECHANISM IS IMPORTANT FOR PREVENTING AGE-RELATED DISEASES DUE TO AGE-RELATED CHRONIC INFLAMMATION. IN THE INTESTINE, DENDRITIC CELLS (DCS) PLAY AN IMPORTANT ROLE IN INDUCING TREGS SPECIFIC TO ORAL ANTIGENS, AND THEY EFFICIENTLY INDUCE TREGS VIA PRODUCTION OF RETINOIC ACID (RA), A VITAMIN A METABOLITE, CATALYZED BY THE ENZYME RETINALDEHYDE DEHYDROGENASE 2 (RALDH2). WE HAVE PREVIOUSLY REPORTED THAT IN THE MESENTERIC LYMPH NODE (MLN), A SECONDARY LYMPHOID TISSUE IN WHICH IMMUNE RESPONSES TO ORAL ANTIGENS ARE INDUCED, FOUR DC SUBSETS EXPRESS DIFFERENT LEVELS OF CD11B, CD103, AND PD-L1, AND WE HAVE REPORTED THAT THE CD11B(-)CD103(+)PD-L1(HIGH) SUBSET EXPRESSES THE HIGHEST LEVELS OF THE RALDH2 GENE AND INDUCES TREGS IN VITRO. WE EXAMINED TREG INDUCTION IN YOUNG AND AGED MICE USING A TREG INDUCTION MODEL BY ADMINISTERING A FOOD ANTIGEN, AND WE FOUND THAT ANTIGEN-SPECIFIC TREG INDUCTION WAS DECREASED IN AGED MICE. WE FURTHER INVESTIGATED THE MLN DCS, AND A SIGNIFICANT DECREASE IN RALDH2 GENE EXPRESSION WAS OBSERVED IN MLN DCS FROM AGED MICE. AS FACTORS, WE FOUND THAT THE PROPORTION OF THE CD11B(-)CD103(+)PD-L1(HIGH) SUBSET WAS DECREASED IN AGED MICE COMPARED WITH THAT IN YOUNG MICE AND THAT RALDH ENZYME ACTIVITY WAS DECREASED IN THE CD11B(-)CD103(+)PD-L1(HIGH) AND CD11B(+)CD103(+)PD-L1(HIGH) SUBSETS. FURTHERMORE, ANALYSIS OF THE METHYLATION OF THE RALDH2 GENE PROMOTER REGION REVEALED THAT CPG MOTIFS WERE MORE METHYLATED IN THE MLN DCS OF AGED MICE, SUGGESTING THAT RALDH2 EXPRESSION WAS SUPPRESSED BY EPIGENETIC CHANGES. FINALLY, WE FOUND THAT RA TREATMENT TENDED TO INCREASE TREG INDUCTION. THESE RESULTS SUGGEST THAT THE REGULATION OF RA PRODUCTION MAY BE INVOLVED IN THE AGE-RELATED DECREASE IN ANTIGEN-SPECIFIC TREG INDUCTION. 2020 4 407 37 ANALYSIS OF FOXP3+ REGULATORY T CELLS THAT DISPLAY APPARENT VIRAL ANTIGEN SPECIFICITY DURING CHRONIC HEPATITIS C VIRUS INFECTION. WE REPORTED PREVIOUSLY THAT A PROPORTION OF NATURAL CD25(+) CELLS ISOLATED FROM THE PBMC OF HCV PATIENTS CAN FURTHER UPREGULATE CD25 EXPRESSION IN RESPONSE TO HCV PEPTIDE STIMULATION IN VITRO, AND PROPOSED THAT VIRUS-SPECIFIC REGULATORY T CELLS (TREG) WERE PRIMED AND EXPANDED DURING THE DISEASE. HERE WE DESCRIBE EPIGENETIC ANALYSIS OF THE FOXP3 LOCUS IN HCV-RESPONSIVE NATURAL CD25(+) CELLS AND SHOW THAT THESE CELLS ARE NOT ACTIVATED CONVENTIONAL T CELLS EXPRESSING FOXP3, BUT HARD-WIRED TREG WITH A STABLE FOXP3 PHENOTYPE AND FUNCTION. OF APPROXIMATELY 46,000 GENES ANALYZED IN GENOME WIDE TRANSCRIPTION PROFILING, ABOUT 1% WERE DIFFERENTIALLY EXPRESSED BETWEEN HCV-RESPONSIVE TREG, HCV-NON-RESPONSIVE NATURAL CD25(+) CELLS AND CONVENTIONAL T CELLS. EXPRESSION PROFILES, INCLUDING CELL DEATH, ACTIVATION, PROLIFERATION AND TRANSCRIPTIONAL REGULATION, SUGGEST A SURVIVAL ADVANTAGE OF HCV-RESPONSIVE TREG OVER THE OTHER CELL POPULATIONS. SINCE NO TREG-SPECIFIC ACTIVATION MARKER IS KNOWN, WE TESTED 97 NS3-DERIVED PEPTIDES FOR THEIR ABILITY TO ELICIT CD25 RESPONSE (ASSUMING IT IS A SURROGATE MARKER), ACCOMPANIED BY HIGH RESOLUTION HLA TYPING OF THE PATIENTS. SOME REACTIVE PEPTIDES OVERLAPPED WITH PREVIOUSLY DESCRIBED EFFECTOR T CELL EPITOPES. OUR DATA OFFERS NEW INSIGHTS INTO HCV IMMUNE EVASION AND TOLERANCE, AND HIGHLIGHTS THE NON-SELF SPECIFIC NATURE OF TREG DURING INFECTION. 2009 5 6071 43 THE DNA METHYLATION INHIBITOR 5-AZACYTIDINE INCREASES REGULATORY T CELLS AND ALLEVIATES AIRWAY INFLAMMATION IN OVALBUMIN-SENSITIZED MICE. BACKGROUND: ASTHMA IS CHARACTERIZED AS A CHRONIC INFLAMMATORY DISORDER OF THE AIRWAYS ASSOCIATED WITH AN ENHANCED TH2 RESPONSE TO INHALED ALLERGENS. CD4+ T REGULATORY (TREG) CELLS ARE CONTROLLED BY THE MASTER TRANSCRIPTION FACTOR FOXP3 AND STRICTLY MAINTAIN PERIPHERAL IMMUNOTOLERANCE. EPIGENETIC REGULATION OF FOXP3 BY DNA METHYLTRANSFERASE INHIBITORS, SUCH AS 5-AZACYTIDINE (AZA), CAN GENERATE A STEADY SUPPLY OF FUNCTIONAL TREG CELLS. THEREFORE, WE PROPOSE THAT AZA CAN AUGMENT TREG CELLS IN VIVO TO PREVENT THE PATHOGENESIS OF ASTHMA. METHODS: BALB/C MICE WERE SENSITIZED WITH CHICKEN OVALBUMIN (OVA) AND TREATED WITH DIFFERENT DOSES OF AZA. AIRWAY HYPERRESPONSIVENESS TO METHACHOLINE, EOSINOPHILIA IN BRONCHOALVEOLAR LAVAGE FLUID, CIRCULATING TITERS OF OVA-SPECIFIC IGG1 AND IGE, AND STIMULATING LEVELS OF TH2 CYTOKINES FROM SPLENOCYTES WERE THEN DETERMINED. CELLULAR POPULATIONS WERE EXAMINED BY FLOW CYTOMETRY. PC61 ANTIBODY, WHICH DEPLETES CD25+ CELLS, WAS USED TO VERIFY THE ROLE OF CD25+ CELLS IN AZA-INDUCED TOLERANCE. RESULTS: ADMINISTRATION OF AZA TO OVA-SENSITIZED MICE DIMINISHED AIRWAY HYPERREACTIVITY, PULMONARY EOSINOPHILIA, LEVELS OF OVA-SPECIFIC IGG1 AND IGE IN SERUM, AND SECRETION OF TH2 CYTOKINES FROM OVA-STIMULATED SPLENOCYTES IN A DOSE-DEPENDENT MANNER. PERCENTAGES OF CD25+ AND FOXP3+ CELLS IN THE CD4+ CELL POPULATION WERE NOTABLY INCREASED IN AZA-TREATED MICE COMPARED TO SENSITIZED CONTROL MICE. FURTHERMORE, THE MAJOR SYMPTOMS OF ASTHMA WERE EXACERBATED BY DEPLETING CD25+ CELLS IN AZA-TREATED MICE. CONCLUSIONS: AZA MAY HAVE APPLICATIONS AS A NOVEL CLINICAL STRATEGY TO INCREASE THE PRODUCTION OF TREG CELLS IN ORDER TO MODULATE THE AIRWAY INFLAMMATION ASSOCIATED WITH ASTHMA. 2013 6 3998 31 LOSS OF EPIGENETIC MODIFICATION DRIVEN BY THE FOXP3 TRANSCRIPTION FACTOR LEADS TO REGULATORY T CELL INSUFFICIENCY. REGULATORY T (TREG) CELLS, DRIVEN BY THE FOXP3 TRANSCRIPTION FACTOR, ARE RESPONSIBLE FOR LIMITING AUTOIMMUNITY AND CHRONIC INFLAMMATION. WE SHOWED THAT A WELL-CHARACTERIZED FOXP3(GFP) REPORTER MOUSE, WHICH EXPRESSES AN N-TERMINAL GFP-FOXP3 FUSION PROTEIN, IS A HYPOMORPH THAT CAUSES PROFOUNDLY ACCELERATED AUTOIMMUNE DIABETES ON A NOD BACKGROUND. ALTHOUGH NATURAL TREG CELL DEVELOPMENT AND IN VITRO FUNCTION ARE NOT MARKEDLY ALTERED IN FOXP3(GFP) NOD AND C57BL/6 MICE, TREG CELL FUNCTION IN INFLAMMATORY ENVIRONMENTS WAS PERTURBED AND TGF-BETA-INDUCED TREG CELL DEVELOPMENT WAS REDUCED. FOXP3(GFP) WAS UNABLE TO INTERACT WITH THE HISTONE ACETYLTRANSFERASE TIP60, THE HISTONE DEACETYLASE HDAC7, AND THE IKAROS FAMILY ZINC FINGER 4, EOS, WHICH LED TO REDUCED FOXP3 ACETYLATION AND ENHANCED K48-LINKED POLYUBIQUITYLATION. COLLECTIVELY THIS RESULTS IN AN ALTERED TRANSCRIPTIONAL LANDSCAPE AND REDUCED FOXP3-MEDIATED GENE REPRESSION, NOTABLY AT THE HALLMARK IL-2 PROMOTER. LOSS OF CONTROLLED FOXP3-DRIVEN EPIGENETIC MODIFICATION LEADS TO TREG CELL INSUFFICIENCY THAT ENABLES AUTOIMMUNITY IN SUSCEPTIBLE ENVIRONMENTS. 2012 7 3778 35 INTERFERING WITH ALTERNATIVELY ACTIVATED MACROPHAGES BY CSF-1R INHIBITION EXERTS THERAPEUTIC CAPACITY ON ALLERGIC AIRWAY INFLAMMATION. PURPOSE: ALLERGIC ASTHMA IS A CHRONIC INFLAMMATORY DISORDER WITH AIRWAY HYPERRESPONSIVENESS AND TISSUE REMODELING AS THE MAIN PATHOLOGICAL CHARACTERISTICS. THE ETIOLOGY OF ASTHMA IS RELATIVELY COMPLICATED, INVOLVING GENETIC SUSCEPTIBILITY, EPIGENETIC REGULATION, ENVIRONMENTAL FACTORS, AND IMMUNE IMBALANCE. COLONY STIMULATING FACTOR 1 RECEPTOR (CSF-1R), HIGHLY EXPRESSED IN MYELOID MONOCYTES, PLAYS AN IMPORTANT ROLE IN REGULATING INFLAMMATION. HOWEVER, THE PATHOLOGICAL ROLE OF CSF-1R AND THE THERAPEUTIC EFFECTS OF CSF-1R INHIBITOR IN ALLERGIC AIRWAY INFLAMMATION REMAIN INDISTINCT. METHODS: THE HOUSE DUST MITE (HDM)-TRIGGERED ALLERGIC AIRWAY INFLAMMATION MODEL WAS CONDUCTED TO FULLY UNCOVER THE EFFICACIES OF CSF-1R INHIBITION, AS ILLUSTRATED BY HISTOPATHOLOGICAL EXAMINATIONS, BIOCHEMICAL ANALYSIS, ELISA, RT-PCR, WESTERN BLOTTING ASSAY, IMMUNOFLUORESCENCE, AND FLOW CYTOMETRY. FURTHERMORE, BONE MARROW-DERIVED MACROPHAGES (BMDMS) WERE DIFFERENTIATED AND POLARIZED UPON IL-4/IL-13 INDUCTION TO CLARIFY THE UNDERLYING MECHANISMS OF CSF-1R INHIBITION. RESULTS: HEREIN, WE PRESENTED THAT THE EXPRESSION OF CSF-1R WAS INCREASED IN HDM-INDUCED EXPERIMENTAL ASTHMA AND INHIBITION OF CSF-1R DISPLAYED DRAMATIC EFFECTS ON THE DISEASE SEVERITY OF ASTHMA, REFERRING TO SUPPRESSING THE SECRETION OF ALLERGIC MEDIATORS, DYSFUNCTION OF AIRWAY EPITHELIUM, AND INFILTRATION OF INFLAMMATORY CELLS. FURTHERMORE, CSF-1R INHIBITOR COULD MARKEDLY RESTRAIN THE POLARIZATION AND EXPRESSION OF TRANSCRIPTIONAL FACTORS OF ALTERNATIVELY ACTIVATED MACROPHAGES (AAMS) IN THE PRESENCE OF IL-4/IL-13 AND REDUCE THE RECRUITMENT OF CSF-1R-DOMINANT MACROPHAGES, BOTH IN ACUTE AND CHRONIC ALLERGIC AIRWAY INFLAMMATION MODEL. CONCLUSION: COLLECTIVELY, OUR FINDINGS DEMONSTRATED THE MOLECULAR PATHOLOGICAL MECHANISM OF CSF-1R IN ALLERGIC AIRWAY DISEASES AND SUGGESTED THAT TARGETING CSF-1R MIGHT BE AN ALTERNATIVE INTERVENTION STRATEGY ON THE HOMEOSTASIS OF AIRWAY IMMUNE MICROENVIRONMENT IN ASTHMA. 2022 8 5859 36 SULFORAPHANE PREVENTS AND REVERSES ALLERGIC AIRWAYS DISEASE IN MICE VIA ANTI-INFLAMMATORY, ANTIOXIDANT, AND EPIGENETIC MECHANISMS. SULFORAPHANE HAS BEEN INVESTIGATED IN HUMAN PATHOLOGIES AND PRECLINICAL MODELS OF AIRWAY DISEASES. TO PROVIDE FURTHER MECHANISTIC INSIGHTS, WE EXPLORED L-SULFORAPHANE (LSF) IN THE OVALBUMIN (OVA)-INDUCED CHRONIC ALLERGIC AIRWAYS MURINE MODEL, WITH KEY HALLMARKS OF ASTHMA. HISTOLOGICAL ANALYSIS INDICATED THAT LSF PREVENTED OR REVERSED OVA-INDUCED EPITHELIAL THICKENING, COLLAGEN DEPOSITION, GOBLET CELL METAPLASIA, AND INFLAMMATION. WELL-KNOWN ANTIOXIDANT AND ANTI-INFLAMMATORY MECHANISMS CONTRIBUTE TO THE BENEFICIAL EFFECTS OF LSF. FOURIER TRANSFORM INFRARED MICROSPECTROSCOPY REVEALED ALTERED COMPOSITION OF MACROMOLECULES, FOLLOWING OVA SENSITIZATION, WHICH WERE RESTORED BY LSF. RNA SEQUENCING IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS HIGHLIGHTED THE ANTI-INFLAMMATORY SIGNATURE OF LSF. FINDINGS INDICATED THAT LSF MAY ALTER GENE EXPRESSION VIA AN EPIGENETIC MECHANISM WHICH INVOLVES REGULATION OF PROTEIN ACETYLATION STATUS. LSF RESULTED IN HISTONE AND ALPHA-TUBULIN HYPERACETYLATION IN VIVO, AND CELLULAR AND ENZYMATIC ASSAYS INDICATED DECREASED EXPRESSION AND MODEST HISTONE DEACETYLASE (HDAC) INHIBITION ACTIVITY, IN COMPARISON WITH THE WELL-KNOWN PAN-HDAC INHIBITOR SUBEROYLANILIDE HYDROXAMIC ACID (SAHA). MOLECULAR MODELING CONFIRMED INTERACTION OF LSF AND LSF METABOLITES WITH THE CATALYTIC DOMAIN OF METAL-DEPENDENT HDAC ENZYMES. MORE GENERALLY, THIS STUDY CONFIRMED KNOWN MECHANISMS AND IDENTIFIED POTENTIAL EPIGENETIC PATHWAYS ACCOUNTING FOR THE PROTECTIVE EFFECTS AND PROVIDE SUPPORT FOR THE POTENTIAL CLINICAL UTILITY OF LSF IN ALLERGIC AIRWAYS DISEASE. 2022 9 6055 24 THE CXXC1 SUBUNIT OF THE TRITHORAX COMPLEX DIRECTS EPIGENETIC LICENSING OF CD4+ T CELL DIFFERENTIATION. DIFFERENT DYNAMICS OF GENE EXPRESSION ARE OBSERVED DURING CELL DIFFERENTIATION. IN T CELLS, GENES THAT ARE TURNED ON EARLY OR TURNED OFF AND STAY OFF HAVE BEEN THOROUGHLY STUDIED. HOWEVER, GENES THAT ARE INITIALLY TURNED OFF BUT THEN TURNED ON AGAIN AFTER STIMULATION HAS CEASED HAVE NOT BEEN DEFINED; THEY ARE OBVIOUSLY IMPORTANT, ESPECIALLY IN THE CONTEXT OF ACUTE VERSUS CHRONIC INFLAMMATION. USING THE TH1/TH2 DIFFERENTIATION PARADIGM, WE FOUND THAT THE CXXC1 SUBUNIT OF THE TRITHORAX COMPLEX DIRECTS TRANSCRIPTION OF GENES INITIALLY DOWN-REGULATED BY TCR STIMULATION BUT UP-REGULATED AGAIN IN A LATER PHASE. THE LATE UP-REGULATION OF THESE GENES WAS IMPAIRED EITHER BY PROLONGED TCR STIMULATION OR CXXC1 DEFICIENCY, WHICH LED TO DECREASED EXPRESSION OF TRIB3 AND KLF2 IN TH1 AND TH2 CELLS, RESPECTIVELY. LOSS OF CXXC1 RESULTED IN ENHANCED PATHOGENICITY IN ALLERGIC AIRWAY INFLAMMATION IN VIVO. THUS, CXXC1 PLAYS ESSENTIAL ROLES IN THE ESTABLISHMENT OF A PROPER CD4+ T CELL IMMUNE SYSTEM VIA EPIGENETIC CONTROL OF A SPECIFIC SET OF GENES. 2021 10 5681 31 SHORT-CHAIN FATTY ACID-MEDIATED EPIGENETIC MODULATION OF INFLAMMATORY T CELLS IN VITRO. SHORT-CHAIN FATTY ACIDS (SCFAS) ARE MAJOR METABOLIC PRODUCTS OF INDIGESTIBLE POLYSACCHARIDES IN THE GUT AND MEDIATE THE FUNCTION OF IMMUNE CELLS TO FACILITATE HOMEOSTASIS. THE IMMUNOMODULATORY EFFECT OF SCFAS HAS BEEN ATTRIBUTED, AT LEAST IN PART, TO THE EPIGENETIC MODULATION OF IMMUNE CELLS THROUGH THE INHIBITION THE NUCLEUS-RESIDENT ENZYME HISTONE DEACETYLASE (HDAC). AMONG THE DOWNSTREAM EFFECTS, SCFAS ENHANCE REGULATORY T CELLS (T(REG)) OVER INFLAMMATORY T HELPER (TH) CELLS, INCLUDING TH17 CELLS, WHICH CAN BE PATHOGENIC. HERE, WE CHARACTERIZE THE POTENTIAL OF TWO COMMON SCFAS-BUTYRATE AND PENTANOATE-IN MODULATING DIFFERENTIATION OF T CELLS IN VITRO. WE SHOW THAT BUTYRATE BUT NOT PENTANOATE EXERTS A CONCENTRATION-DEPENDENT EFFECT ON T(REG) AND TH17 DIFFERENTIATION. INCREASING THE CONCENTRATION OF BUTYRATE SUPPRESSES THE TH17-ASSOCIATED RORGAMMATT AND IL-17 AND INCREASES THE EXPRESSION OF T(REG)-ASSOCIATED FOXP3. TO EFFECTIVELY DELIVER BUTYRATE, ENCAPSULATION OF BUTYRATE IN A LIPOSOMAL CARRIER, TERMED BLIPS, REDUCED CYTOTOXICITY WHILE MAINTAINING THE IMMUNOMODULATORY EFFECT ON T CELLS. CONSISTENT WITH THESE RESULTS, BUTYRATE AND BLIPS INHIBIT HDAC AND PROMOTE A UNIQUE CHROMATIN LANDSCAPE IN T CELLS UNDER CONDITIONS THAT OTHERWISE PROMOTE CONVERSION INTO A PRO-INFLAMMATORY PHENOTYPE. MOTIF ENRICHMENT ANALYSIS REVEALED THAT BUTYRATE AND BLIP-MEDIATED SUPPRESSION OF TH17-ASSOCIATED CHROMATIN ACCESSIBILITY CORRESPONDED WITH A MARKED DECREASE IN BZIP FAMILY TRANSCRIPTION FACTOR BINDING SITES. THESE RESULTS SUPPORT THE UTILITY AND FURTHER EVALUATION OF BLIPS AS AN IMMUNOMODULATORY AGENT FOR AUTOIMMUNE DISORDERS THAT ARE CHARACTERIZED BY CHRONIC INFLAMMATION AND PATHOGENIC INFLAMMATORY T CELLS. 2023 11 6294 24 THE PROINFLAMMATORY CYTOKINE TNFALPHA INDUCES DNA DEMETHYLATION-DEPENDENT AND -INDEPENDENT ACTIVATION OF INTERLEUKIN-32 EXPRESSION. IL-32 IS A CYTOKINE INVOLVED IN PROINFLAMMATORY IMMUNE RESPONSES TO BACTERIAL AND VIRAL INFECTIONS. HOWEVER, THE ROLE OF EPIGENETIC EVENTS IN THE REGULATION OF IL-32 GENE EXPRESSION IS UNDERSTUDIED. HERE WE SHOW THAT IL-32 IS REPRESSED BY DNA METHYLATION IN HEK293 CELLS. USING CHIP SEQUENCING, LOCUS-SPECIFIC METHYLATION ANALYSIS, CRISPR/CAS9-MEDIATED GENOME EDITING, AND RT-QPCR (QUANTITATIVE RT-PCR) AND IMMUNOBLOT ASSAYS, WE FOUND THAT SHORT-TERM TREATMENT (A FEW HOURS) WITH THE PROINFLAMMATORY CYTOKINE TUMOR NECROSIS FACTOR ALPHA (TNFALPHA) ACTIVATES IL-32 IN A DNA DEMETHYLATION-INDEPENDENT MANNER. IN CONTRAST, PROLONGED TNFALPHA TREATMENT (SEVERAL DAYS) INDUCED DNA DEMETHYLATION AT THE PROMOTER AND A CPG ISLAND IN THE IL-32 GENE IN A TET (TEN-ELEVEN TRANSLOCATION) FAMILY ENZYME- AND NF-KAPPAB-DEPENDENT MANNER. NOTABLY, THE HYPOMETHYLATION STATUS OF TRANSCRIPTIONAL REGULATORY ELEMENTS IN IL-32 WAS MAINTAINED FOR A LONG TIME (SEVERAL WEEKS), CAUSING ELEVATED IL-32 EXPRESSION EVEN IN THE ABSENCE OF TNFALPHA. CONSIDERING THAT IL-32 CAN, IN TURN, INDUCE TNFALPHA EXPRESSION, WE SPECULATE THAT SUCH FEEDFORWARD EVENTS MAY CONTRIBUTE TO THE TRANSITION FROM AN ACUTE INFLAMMATORY RESPONSE TO CHRONIC INFLAMMATION. 2019 12 1293 31 DECREASED ERK AND JNK SIGNALING CONTRIBUTE TO GENE OVEREXPRESSION IN "SENESCENT" CD4+CD28- T CELLS THROUGH EPIGENETIC MECHANISMS. AN INFLAMMATORY AND CYTOTOXIC CD4+CD28- T CELL SUBSET INFILTRATES ATHEROSCLEROTIC PLAQUES AND IS IMPLICATED IN PLAQUE RUPTURE AND MYOCARDIAL INFARCTIONS. THIS PATHOLOGIC SUBSET DEVELOPS WITH REPLICATIVE STRESS AND IS FOUND IN PATIENTS WITH CHRONIC INFLAMMATORY DISEASES SUCH AS RA AS WELL AS WITH AGING. CD4+CD28- CELLS OVEREXPRESS GENES NORMALLY SUPPRESSED BY DNA METHYLATION IN CD4+CD28+ T CELLS, SUCH AS KIR, PERFORIN, AND CD70. HOW THIS SUBSET OVER EXPRESSES METHYLATION-SENSITIVE GENES IS UNKNOWN. DNA METHYLATION PATTERNS ARE MAINTAINED IN PROLIFERATING CELLS BY DNMTS, WHICH ARE UP-REGULATED DURING MITOSIS BY THE ERK AND JNK SIGNALING PATHWAYS. WE HYPOTHESIZED THAT DEFECTS IN THESE SIGNALING PATHWAYS CONTRIBUTE TO ALTERED GENE EXPRESSION IN HUMAN CD4+CD28- CELLS THROUGH EFFECTS ON DNA METHYLATION. WE REPORT THAT SIGNALING THROUGH THE ERK AND JNK PATHWAYS IS DECREASED IN CD4+CD28- RELATIVE TO CD4+CD28+ CELLS FROM THE SAME INDIVIDUALS AND THAT ERK AND JNK PATHWAY INHIBITION DECREASES DNMT1 AND -3A LEVELS, WHICH IN TURN, CAUSES DEMETHYLATION AND OVEREXPRESSION OF THE TNFSF7 (CD70) GENE. WE ALSO REPORT THAT CD4+CD28- T CELLS OVEREXPRESS PP5, A STRESS-INDUCED INHIBITOR OF THE ERK AND JNK SIGNALING PATHWAYS THAT MAY CONTRIBUTE TO THE SIGNALING DEFECTS. WE CONCLUDE THAT DECREASED ERK AND JNK SIGNALING IN THE CD4+CD28- SUBSET, ARISING WITH REPLICATIVE STRESS, CAN LEAD TO THE OVEREXPRESSION OF NORMALLY SUPPRESSED GENES THROUGH EFFECTS ON DNMTS AND CONSEQUENTLY, CHROMATIN STRUCTURE. 2010 13 984 39 CHRONIC PSYCHOLOGICAL STRESS ALTERS GENE EXPRESSION IN RAT COLON EPITHELIAL CELLS PROMOTING CHROMATIN REMODELING, BARRIER DYSFUNCTION AND INFLAMMATION. CHRONIC STRESS IS COMMONLY ASSOCIATED WITH ENHANCED ABDOMINAL PAIN (VISCERAL HYPERSENSITIVITY), BUT THE CELLULAR MECHANISMS UNDERLYING HOW CHRONIC STRESS INDUCES VISCERAL HYPERSENSITIVITY ARE POORLY UNDERSTOOD. IN THIS STUDY, WE EXAMINED CHANGES IN GENE EXPRESSION IN COLON EPITHELIAL CELLS FROM A RAT MODEL USING RNA-SEQUENCING TO EXAMINE STRESS-INDUCED CHANGES TO THE TRANSCRIPTOME. FOLLOWING CHRONIC STRESS, THE MOST SIGNIFICANTLY UP-REGULATED GENES INCLUDED ATG16L1, COQ10B, DCAF13, NAT2, PTBP2, RRAS2, SPINK4 AND DOWN-REGULATED GENES INCLUDING ABAT, CITED2, CNNM2, DAB2IP, PLEKHM1, SCD2, AND TAB2. THE PRIMARY ALTERED BIOLOGICAL PROCESSES REVEALED BY NETWORK ENRICHMENT ANALYSIS WERE INFLAMMATION/IMMUNE RESPONSE, TISSUE MORPHOGENESIS AND DEVELOPMENT, AND NUCLEOSOME/CHROMATIN ASSEMBLY. THE MOST SIGNIFICANTLY DOWN-REGULATED PROCESS WAS THE DIGESTIVE SYSTEM DEVELOPMENT/FUNCTION, WHEREAS THE MOST SIGNIFICANTLY UP-REGULATED PROCESSES WERE INFLAMMATORY RESPONSE, ORGANISMAL INJURY, AND CHROMATIN REMODELING MEDIATED BY H3K9 METHYLATION. FURTHERMORE, A SUBPOPULATION OF STRESSED RATS DEMONSTRATED VERY SIGNIFICANTLY ALTERED GENE EXPRESSION AND TRANSCRIPT ISOFORMS, ENRICHED FOR THE DIFFERENTIAL EXPRESSION OF GENES INVOLVED IN THE INFLAMMATORY RESPONSE, INCLUDING UPREGULATION OF CYTOKINE AND CHEMOKINE RECEPTOR GENE EXPRESSION COUPLED WITH DOWNREGULATION OF EPITHELIAL ADHERENS AND TIGHT JUNCTION MRNAS. IN SUMMARY, THESE FINDINGS SUPPORT THAT CHRONIC STRESS IS ASSOCIATED WITH INCREASED LEVELS OF CYTOKINES AND CHEMOKINES, THEIR DOWNSTREAM SIGNALING PATHWAYS COUPLED TO DYSREGULATION OF INTESTINAL CELL DEVELOPMENT AND FUNCTION. EPIGENETIC REGULATION OF CHROMATIN REMODELING LIKELY PLAYS A PROMINENT ROLE IN THIS PROCESS. RESULTS ALSO SUGGEST THAT SUPER ENHANCERS PLAY A PRIMARY ROLE IN CHRONIC STRESS-ASSOCIATED INTESTINAL BARRIER DYSFUNCTION. 2022 14 5153 27 PPP2R2B HYPERMETHYLATION CAUSES ACQUIRED APOPTOSIS DEFICIENCY IN SYSTEMIC AUTOIMMUNE DISEASES. CHRONIC INFLAMMATION CAUSES TARGET ORGAN DAMAGE IN PATIENTS WITH SYSTEMIC AUTOIMMUNE DISEASES. THE FACTORS THAT ALLOW THIS PROTRACTED RESPONSE ARE POORLY UNDERSTOOD. WE ANALYZED THE TRANSCRIPTIONAL REGULATION OF PPP2R2B (B55SS), A MOLECULE NECESSARY FOR THE TERMINATION OF THE IMMUNE RESPONSE, IN PATIENTS WITH AUTOIMMUNE DISEASES. ALTERED EXPRESSION OF B55SS CONDITIONED RESISTANCE TO CYTOKINE WITHDRAWAL-INDUCED DEATH (CWID) IN PATIENTS WITH AUTOIMMUNE DISEASES. THE IMPAIRED UPREGULATION OF B55SS WAS CAUSED BY INFLAMMATION-DRIVEN HYPERMETHYLATION OF SPECIFIC CYTOSINES LOCATED WITHIN A REGULATORY ELEMENT OF PPP2R2B PREVENTING CTCF BINDING. THIS PHENOTYPE COULD BE INDUCED IN HEALTHY T CELLS BY EXPOSURE TO TNF-ALPHA. OUR RESULTS REVEAL A GENE WHOSE EXPRESSION IS AFFECTED BY AN ACQUIRED DEFECT, THROUGH AN EPIGENETIC MECHANISM, IN THE SETTING OF SYSTEMIC AUTOIMMUNITY. BECAUSE FAILURE TO REMOVE ACTIVATED T CELLS THROUGH CWID COULD CONTRIBUTE TO AUTOIMMUNE PATHOLOGY, THIS MECHANISM ILLUSTRATES A VICIOUS CYCLE THROUGH WHICH AUTOIMMUNE INFLAMMATION CONTRIBUTES TO ITS OWN PERPETUATION. 2019 15 1966 35 EPIGENETIC ALTERATION OF PRKCDBP IN COLORECTAL CANCERS AND ITS IMPLICATION IN TUMOR CELL RESISTANCE TO TNFALPHA-INDUCED APOPTOSIS. PURPOSE: PRKCDBP IS A PUTATIVE TUMOR SUPPRESSOR IN WHICH ALTERATION HAS BEEN OBSERVED IN SEVERAL HUMAN CANCERS. WE INVESTIGATED EXPRESSION AND FUNCTION OF PRKCDBP IN COLORECTAL CELLS AND TISSUES TO EXPLORE ITS CANDIDACY AS A SUPPRESSOR IN COLORECTAL TUMORIGENESIS. EXPERIMENTAL DESIGN: EXPRESSION AND METHYLATION STATUS OF PRKCDBP AND ITS EFFECT ON TUMOR GROWTH WERE EVALUATED. TRANSCRIPTIONAL REGULATION BY NF-KAPPAB SIGNALING WAS DEFINED BY LUCIFERASE REPORTER AND CHROMATIN IMMUNOPRECIPITATION ASSAYS. RESULTS: PRKCDBP EXPRESSION WAS HARDLY DETECTABLE IN 29 OF 80 (36%) PRIMARY TUMORS AND 11 OF 19 (58%) CELL LINES, AND ITS ALTERATION CORRELATED WITH TUMOR STAGE AND GRADE. PROMOTER HYPERMETHYLATION WAS COMMONLY FOUND IN CANCERS. PRKCDBP EXPRESSION INDUCED THE G(1) CELL-CYCLE ARREST AND INCREASED CELLULAR SENSITIVITY TO VARIOUS APOPTOTIC STRESSES. PRKCDBP WAS INDUCED BY TNFALPHA, AND ITS LEVEL CORRELATED WITH TUMOR CELL SENSITIVITY TO TNFALPHA-INDUCED APOPTOSIS. PRKCDBP INDUCTION BY TNFALPHA WAS DISRUPTED BY BLOCKING NF-KAPPAB SIGNALING WHILE IT WAS ENHANCED BY RELA TRANSFECTION. THE PRKCDBP PROMOTER ACTIVITY WAS INCREASED IN RESPONSE TO TNFALPHA, AND THIS RESPONSE WAS ABOLISHED BY DISRUPTION OF A KAPPAB SITE IN THE PROMOTER. PRKCDBP DELAYED THE FORMATION AND GROWTH OF XENOGRAFT TUMORS AND IMPROVED TUMOR RESPONSE TO TNFALPHA-INDUCED APOPTOSIS. CONCLUSIONS: PRKCDBP IS A PROAPOPTOTIC TUMOR SUPPRESSOR WHICH IS COMMONLY ALTERED IN COLORECTAL CANCER BY PROMOTER HYPERMETHYLATION, AND ITS GENE TRANSCRIPTION IS DIRECTLY ACTIVATED BY NF-KAPPAB IN RESPONSE TO TNFALPHA. THIS SUGGESTS THAT PRKCDBP INACTIVATION MAY CONTRIBUTE TO TUMOR PROGRESSION BY REDUCING CELLULAR SENSITIVITY TO TNFALPHA AND OTHER STRESSES, PARTICULARLY UNDER CHRONIC INFLAMMATORY MICROENVIRONMENT. 2011 16 3948 31 LNCRNA-CD160 DECREASES THE IMMUNITY OF CD8(+) T CELLS THROUGH EPIGENETIC MECHANISMS IN HEPATITIS B VIRUS INFECTION. THE TRANSFER AND DEVELOPMENT OF CHRONIC HEPATITIS B VIRUS (HBV) INFECTION IS ASSOCIATED WITH THE T CELL IMMUNE RESPONSE, THEREFORE INVESTIGATING THE KEY REGULATORS OF CELL IMMUNE RESPONSE IS NEEDED TO IMPROVE CHRONIC HBV TREATMENT. BLOOD SAMPLES FROM PATIENTS WITH CHRONIC HBV INFECTION WERE USED TO CONFIRM THE CORRELATION BETWEEN HBV INFECTION STAGE AND CD160 RECEPTOR EXPRESSION LEVELS IN CD8(+) T CELLS, THE CD8(+) T CELLS ARE USED TO RESEARCH THE MECHANISM OF T CELL IMMUNE RESPONSE MODULATION, MOREOVER, C3H/HEN MICE WITH REDUCED CD160 EXPRESSION LEVELS WERE USED TO INVESTIGATE THE ASSOCIATION BETWEEN LONG NON-CODING (LNC)RNA-CD160 AND HBV INFECTION. LONG NON-CODING (LNC)RNA-CD160 AND HISTONE-MODIFICATION ENZYME GENE HISTONE DEACETYLASE 11 (HDAC11) EXPRESSION LEVELS WERE NEGATIVELY ASSOCIATED WITH CD160 EXPRESSION. LNCRNA-CD160 CAN INHIBIT THE SECRETION OF IFN-GAMMA AND TNF-ALPHA THROUGH HDAC11 RECRUITMENT AND BIND TO HDAC11 TO FORM A COMPLEX ON THE PROMOTERS OF IFN-GAMMA AND TNF-ALPHA. THE HDAC11, IFN-GAMMA AND TNF-ALPHA FORM A COMPLEX AND ENHANCE THE METHYLATION OF H3K9ME1, CHROMATIN CHANGES INTO THE HETEROCHROMATIN AND THE TRANSCRIPTION OF IFN-GAMMA AND TNF-ALPHA IS BLOCKED; MOREOVER, THE HDAC11/IFN-GAMMA/TNF-ALPHA COMPLEX CAN ALSO INHIBIT THE SECRETION OF IFN-GAMMA AND TNF-ALPHA IN CD160(-) CD8(+) T CELLS AND SUPPRESSES THE FUNCTION OF CD8(+) T CELLS. FURTHERMORE, SMALL INTERFERING RNA TARGETING LNCRNA-CD160 CAN BLOCK HBV INFECTION PROGRESSION. LNCRNA-CD160 ACTS AS AN IMMUNE SUPPRESSIVE FACTOR AND IS EXPRESSED AT A HIGH LEVEL IN PERIPHERAL BLOOD CD8(+) T CELLS OF HBV INFECTED PATIENTS. FURTHERMORE, HIGH EXPRESSION LEVELS OF LNCRNA-CD160 CAN CONTRIBUTE TO THE INHIBITION OF IFN-GAMMA AND TNF-ALPHA SECRETION IN CD8(+) T CELLS AND DECREASE THE IMMUNE RESPONSE OF CD8(+) T CELLS. THEREFORE, LNCRNA-CD160 MAY BECOME A NEW TARGET FOR IMMUNOTHERAPY OF CHRONIC HBV INFECTION IN THE FUTURE AND MAY PROVIDE A NEW THERAPEUTIC STRATEGY FOR THE TREATMENT OF HBV INFECTION. 2020 17 3468 40 HYPOXIA-INDUCED DNA HYPERMETHYLATION IN HUMAN PULMONARY FIBROBLASTS IS ASSOCIATED WITH THY-1 PROMOTER METHYLATION AND THE DEVELOPMENT OF A PRO-FIBROTIC PHENOTYPE. BACKGROUND: PULMONARY FIBROSIS IS A DEBILITATING AND LETHAL DISEASE WITH NO EFFECTIVE TREATMENT OPTIONS. UNDERSTANDING THE PATHOLOGICAL PROCESSES AT PLAY WILL DIRECT THE APPLICATION OF NOVEL THERAPEUTIC AVENUES. HYPOXIA HAS BEEN IMPLICATED IN THE PATHOGENESIS OF PULMONARY FIBROSIS YET THE PRECISE MECHANISM BY WHICH IT CONTRIBUTES TO DISEASE PROGRESSION REMAINS TO BE FULLY ELUCIDATED. IT HAS BEEN SHOWN THAT CHRONIC HYPOXIA CAN ALTER DNA METHYLATION PATTERNS IN TUMOUR-DERIVED CELL LINES. THIS EPIGENETIC ALTERATION CAN INDUCE CHANGES IN CELLULAR PHENOTYPE WITH PROMOTER METHYLATION BEING ASSOCIATED WITH GENE SILENCING. OF PARTICULAR RELEVANCE TO IDIOPATHIC PULMONARY FIBROSIS (IPF) IS THE OBSERVATION THAT THY-1 PROMOTER METHYLATION IS ASSOCIATED WITH A MYOFIBROBLAST PHENOTYPE WHERE LOSS OF THY-1 OCCURS ALONGSIDE INCREASED ALPHA SMOOTH MUSCLE ACTIN (ALPHA-SMA) EXPRESSION. THE INITIAL AIM OF THIS STUDY WAS TO DETERMINE WHETHER HYPOXIA REGULATES DNA METHYLATION IN NORMAL HUMAN LUNG FIBROBLASTS (CCD19LU). AS IT HAS BEEN REPORTED THAT HYPOXIA SUPPRESSES THY-1 EXPRESSION DURING LUNG DEVELOPMENT WE ALSO STUDIED THE EFFECT OF HYPOXIA ON THY-1 PROMOTER METHYLATION AND GENE EXPRESSION. METHODS: CCD19LU WERE GROWN FOR UP TO 8 DAYS IN HYPOXIA AND ASSESSED FOR GLOBAL CHANGES IN DNA METHYLATION USING FLOW CYTOMETRY. REAL-TIME PCR WAS USED TO QUANTIFY EXPRESSION OF THY-1, ALPHA-SMA, COLLAGEN I AND III. GENOMIC DNA WAS BISULPHITE TREATED AND METHYLATION SPECIFIC PCR (MSPCR) WAS USED TO EXAMINE THE METHYLATION STATUS OF THE THY-1 PROMOTER. RESULTS: SIGNIFICANT GLOBAL HYPERMETHYLATION WAS DETECTED IN HYPOXIC FIBROBLASTS RELATIVE TO NORMOXIC CONTROLS AND WAS ACCOMPANIED BY INCREASED EXPRESSION OF MYOFIBROBLAST MARKERS. THY-1 MRNA EXPRESSION WAS SUPPRESSED IN HYPOXIC CELLS, WHICH WAS RESTORED WITH THE DEMETHYLATING AGENT 5-AZA-2'-DEOXYCYTIDINE. MSPCR REVEALED THAT THY-1 BECAME METHYLATED FOLLOWING FIBROBLAST EXPOSURE TO 1% O2. CONCLUSION: THESE DATA SUGGEST THAT GLOBAL AND GENE-SPECIFIC CHANGES IN DNA METHYLATION MAY PLAY AN IMPORTANT ROLE IN FIBROBLAST FUNCTION IN HYPOXIA. 2012 18 4866 32 ORTHOGONAL CRISPR SCREENS TO IDENTIFY TRANSCRIPTIONAL AND EPIGENETIC REGULATORS OF HUMAN CD8 T CELL FUNCTION. THE CLINICAL RESPONSE TO ADOPTIVE T CELL THERAPIES IS STRONGLY ASSOCIATED WITH TRANSCRIPTIONAL AND EPIGENETIC STATE. THUS, TECHNOLOGIES TO DISCOVER REGULATORS OF T CELL GENE NETWORKS AND THEIR CORRESPONDING PHENOTYPES HAVE GREAT POTENTIAL TO IMPROVE THE EFFICACY OF T CELL THERAPIES. WE DEVELOPED POOLED CRISPR SCREENING APPROACHES WITH COMPACT EPIGENOME EDITORS TO SYSTEMATICALLY PROFILE THE EFFECTS OF ACTIVATION AND REPRESSION OF 120 TRANSCRIPTION FACTORS AND EPIGENETIC MODIFIERS ON HUMAN CD8+ T CELL STATE. THESE SCREENS NOMINATED KNOWN AND NOVEL REGULATORS OF T CELL PHENOTYPES WITH BATF3 EMERGING AS A HIGH CONFIDENCE GENE IN BOTH SCREENS. WE FOUND THAT BATF3 OVEREXPRESSION PROMOTED SPECIFIC FEATURES OF MEMORY T CELLS SUCH AS INCREASED IL7R EXPRESSION AND GLYCOLYTIC CAPACITY, WHILE ATTENUATING GENE PROGRAMS ASSOCIATED WITH CYTOTOXICITY, REGULATORY T CELL FUNCTION, AND T CELL EXHAUSTION. IN THE CONTEXT OF CHRONIC ANTIGEN STIMULATION, BATF3 OVEREXPRESSION COUNTERED PHENOTYPIC AND EPIGENETIC SIGNATURES OF T CELL EXHAUSTION. CAR T CELLS OVEREXPRESSING BATF3 SIGNIFICANTLY OUTPERFORMED CONTROL CAR T CELLS IN BOTH IN VITRO AND IN VIVO TUMOR MODELS. MOREOVER, WE FOUND THAT BATF3 PROGRAMMED A TRANSCRIPTIONAL PROFILE THAT CORRELATED WITH POSITIVE CLINICAL RESPONSE TO ADOPTIVE T CELL THERAPY. FINALLY, WE PERFORMED CRISPR KNOCKOUT SCREENS WITH AND WITHOUT BATF3 OVEREXPRESSION TO DEFINE CO-FACTORS AND DOWNSTREAM FACTORS OF BATF3, AS WELL AS OTHER THERAPEUTIC TARGETS. THESE SCREENS POINTED TO A MODEL WHERE BATF3 INTERACTS WITH JUNB AND IRF4 TO REGULATE GENE EXPRESSION AND ILLUMINATED SEVERAL OTHER NOVEL TARGETS FOR FURTHER INVESTIGATION. 2023 19 5918 26 TARGETING BMI-1 IN B CELLS RESTORES EFFECTIVE HUMORAL IMMUNE RESPONSES AND CONTROLS CHRONIC VIRAL INFECTION. INEFFECTIVE ANTIBODY-MEDIATED RESPONSES ARE A KEY CHARACTERISTIC OF CHRONIC VIRAL INFECTION. HOWEVER, OUR UNDERSTANDING OF THE INTRINSIC MECHANISMS THAT DRIVE THIS DYSREGULATION ARE UNCLEAR. HERE, WE IDENTIFY THAT TARGETING THE EPIGENETIC MODIFIER BMI-1 IN MICE IMPROVES HUMORAL RESPONSES TO CHRONIC LYMPHOCYTIC CHORIOMENINGITIS VIRUS. BMI-1 WAS UPREGULATED BY GERMINAL CENTER B CELLS IN CHRONIC VIRAL INFECTION, CORRELATING WITH CHANGES TO THE ACCESSIBLE CHROMATIN LANDSCAPE, COMPARED TO ACUTE INFECTION. B CELL-INTRINSIC DELETION OF BMI1 ACCELERATED VIRAL CLEARANCE, REDUCED SPLENOMEGALY AND RESTORED SPLENIC ARCHITECTURE. DELETION OF BMI1 RESTORED C-MYC EXPRESSION IN B CELLS, CONCOMITANT WITH IMPROVED QUALITY OF ANTIBODY AND COUPLED WITH REDUCED ANTIBODY-SECRETING CELL NUMBERS. SPECIFICALLY, BMI-1-DEFICIENCY INDUCED ANTIBODY WITH INCREASED NEUTRALIZING CAPACITY AND ENHANCED ANTIBODY-DEPENDENT EFFECTOR FUNCTION. USING A SMALL MOLECULE INHIBITOR TO MURINE BMI-1, WE COULD DEPLETE ANTIBODY-SECRETING CELLS AND PROHIBIT DETRIMENTAL IMMUNE COMPLEX FORMATION IN VIVO. THIS STUDY DEFINES BMI-1 AS A CRUCIAL IMMUNE MODIFIER THAT CONTROLS ANTIBODY-MEDIATED RESPONSES IN CHRONIC INFECTION. 2022 20 2340 29 EPIGENETIC REGULATION OF LEUKOCYTE INFLAMMATORY MEDIATOR PRODUCTION DICTATES STAPHYLOCOCCUS AUREUS CRANIOTOMY INFECTION OUTCOME. STAPHYLOCOCCUS AUREUS IS A COMMON CAUSE OF SURGICAL-SITE INFECTIONS, INCLUDING THOSE ARISING AFTER CRANIOTOMY, WHICH IS PERFORMED TO ACCESS THE BRAIN FOR THE TREATMENT OF TUMORS, EPILEPSY, OR HEMORRHAGE. CRANIOTOMY INFECTION IS CHARACTERIZED BY COMPLEX SPATIAL AND TEMPORAL DYNAMICS OF LEUKOCYTE RECRUITMENT AND MICROGLIAL ACTIVATION. WE RECENTLY IDENTIFIED UNIQUE TRANSCRIPTIONAL PROFILES OF THESE IMMUNE POPULATIONS DURING S. AUREUS CRANIOTOMY INFECTION. EPIGENETIC PROCESSES ALLOW RAPID AND REVERSIBLE CONTROL OVER GENE TRANSCRIPTION; HOWEVER, LITTLE IS KNOWN ABOUT HOW EPIGENETIC PATHWAYS INFLUENCE IMMUNITY TO LIVE S. AUREUS. AN EPIGENETIC COMPOUND LIBRARY SCREEN IDENTIFIED BROMODOMAIN AND EXTRATERMINAL DOMAIN-CONTAINING (BET) PROTEINS AND HISTONE DEACETYLASES (HDACS) AS CRITICAL FOR REGULATING TNF, IL-6, IL-10, AND CCL2 PRODUCTION BY PRIMARY MOUSE MICROGLIA, MACROPHAGES, NEUTROPHILS, AND GRANULOCYTIC MYELOID-DERIVED SUPPRESSOR CELLS IN RESPONSE TO LIVE S. AUREUS. CLASS I HDACS (C1HDACS) WERE INCREASED IN THESE CELL TYPES IN VITRO AND IN VIVO DURING ACUTE DISEASE IN A MOUSE MODEL OF S. AUREUS CRANIOTOMY INFECTION. HOWEVER, SUBSTANTIAL REDUCTIONS IN C1HDACS WERE OBSERVED DURING CHRONIC INFECTION, HIGHLIGHTING TEMPORAL REGULATION AND THE IMPORTANCE OF THE TISSUE MICROENVIRONMENT FOR DICTATING C1HDAC EXPRESSION. MICROPARTICLE DELIVERY OF HDAC AND BET INHIBITORS IN VIVO CAUSED WIDESPREAD DECREASES IN INFLAMMATORY MEDIATOR PRODUCTION, WHICH SIGNIFICANTLY INCREASED BACTERIAL BURDEN IN THE BRAIN, GALEA, AND BONE FLAP. THESE FINDINGS IDENTIFY HISTONE ACETYLATION AS AN IMPORTANT MECHANISM FOR REGULATING CYTOKINE AND CHEMOKINE PRODUCTION ACROSS DIVERSE IMMUNE CELL LINEAGES THAT IS CRITICAL FOR BACTERIAL CONTAINMENT. ACCORDINGLY, ABERRANT EPIGENETIC REGULATION MAY BE IMPORTANT FOR PROMOTING S. AUREUS PERSISTENCE DURING CRANIOTOMY INFECTION. 2023