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• W1995277882 abstract "Nuclear receptors modulate macrophage effector functions, which are imperative for clearance or survival of mycobacterial infection. The adopted orphan nuclear receptor Rev-erbα is a constitutive transcriptional repressor as it lacks AF2 domain and was earlier shown to be present in macrophages. In the present study, we highlight the differences in the relative subcellular localization of Rev-erbα in monocytes and macrophages. The nuclear localization of Rev-erbα in macrophages is subsequent to monocyte differentiation. Expression analysis of Rev-erbα elucidated it to be considerably more expressed in M1 phenotype in comparison with M2. Rev-erbα overexpression augments antimycobacterial properties of macrophage by keeping IL10 in a basal repressed state. Further, promoter analysis revealed that IL10 promoter harbors a Rev-erbα binding site exclusive to humans and higher order primates and not mouse, demonstrating a species barrier in its functionality. This direct gene repression is mediated by recruitment of co-repressors NCoR and HDAC3. In addition, our data elucidate that its overexpression reduced the survival of intracellular pathogen Mycobacterium tuberculosis by enhancing phagosome lysosome maturation, an event resulting from IL10 repression. Thus, these findings suggest that Rev-erbα bestows protection against mycobacterial infection by direct gene repression of IL10 and thus provide a novel target in modulating macrophage microbicidal properties. Nuclear receptors modulate macrophage effector functions, which are imperative for clearance or survival of mycobacterial infection. The adopted orphan nuclear receptor Rev-erbα is a constitutive transcriptional repressor as it lacks AF2 domain and was earlier shown to be present in macrophages. In the present study, we highlight the differences in the relative subcellular localization of Rev-erbα in monocytes and macrophages. The nuclear localization of Rev-erbα in macrophages is subsequent to monocyte differentiation. Expression analysis of Rev-erbα elucidated it to be considerably more expressed in M1 phenotype in comparison with M2. Rev-erbα overexpression augments antimycobacterial properties of macrophage by keeping IL10 in a basal repressed state. Further, promoter analysis revealed that IL10 promoter harbors a Rev-erbα binding site exclusive to humans and higher order primates and not mouse, demonstrating a species barrier in its functionality. This direct gene repression is mediated by recruitment of co-repressors NCoR and HDAC3. In addition, our data elucidate that its overexpression reduced the survival of intracellular pathogen Mycobacterium tuberculosis by enhancing phagosome lysosome maturation, an event resulting from IL10 repression. Thus, these findings suggest that Rev-erbα bestows protection against mycobacterial infection by direct gene repression of IL10 and thus provide a novel target in modulating macrophage microbicidal properties. Macrophages are immune system sentinels with a major role to play in both innate and adaptive immunity. They are the key effectors in antimicrobial defense, atherogenesis, autoimmunity, and many other inflammatory diseases (1Barish G.D. Downes M. Alaynick W.A. Yu R.T. Ocampo C.B. Bookout A.L. Mangelsdorf D.J. Evans R.M. A Nuclear Receptor Atlas: macrophage activation.Mol. Endocrinol. 2005; 19: 2466-2477Crossref PubMed Scopus (196) Google Scholar). Although the activation, function, classification, and plasticity of these cells have been studied extensively with regard to cellular signaling, the cytokine environment, and surface, cellular, or secretory markers, studies of the underlying molecular mechanism have mostly addressed NF-κB (2Baeuerle P.A. Henkel T. Function and activation of NF-κB in the immune system.Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4591) Google Scholar). Similarly, modulation of macrophage function and alteration of disease pathology by small molecules such as heme, lipids, or drugs such as rifampicin are well understood at the translational level of the effectors (3McLean K.J. Dunford A.J. Neeli R. Driscoll M.D. Munro A.W. Structure, function, and drug targeting in Mycobacterium tuberculosis cytochrome P450 systems.Arch. Biochem. Biophys. 2007; 464: 228-240Crossref PubMed Scopus (67) Google Scholar, 4Liu S. Suragani R.N. Wang F. Han A. Zhao W. Andrews N.C. Chen J.J. The function of heme-regulated eIF2α kinase in murine iron homeostasis and macrophage maturation.J. Clin. Invest. 2007; 117: 3296-3305Crossref PubMed Scopus (73) Google Scholar), but the transcriptional mechanism involving interactions of these ligands with transcriptional molecules and the resulting expression patterns have not been investigated. This study focuses on Rev-erbα, an adopted orphan nuclear receptor that belongs to the steroid/thyroid hormone receptor superfamily and is a known heme sensor (5Raghuram S. Stayrook K.R. Huang P. Rogers P.M. Nosie A.K. McClure D.B. Burris L.L. Khorasanizadeh S. Burris T.P. Rastinejad F. Identification of heme as the ligand for the orphan nuclear receptors REV-ERBα and REV-ERBβ.Nat. Struct. Mol. Biol. 2007; 14: 1207-1213Crossref PubMed Scopus (437) Google Scholar). It is encoded on the opposite strand of the thyroid receptor α (c-erbAα) gene (6Lazar M.A. Hodin R.A. Darling D.S. Chin W.W. A novel member of the thyroid/steroid hormone receptor family is encoded by the opposite strand of the rat c-erbAα transcriptional unit.Mol. Cell. Biol. 1989; 9: 1128-1136Crossref PubMed Scopus (263) Google Scholar). Rev-erbα modulates proinflammatory cytokines through NF-κB (7Ramakrishnan S.N. Muscat G.E. The orphan Rev-erb nuclear receptors: a link between metabolism, circadian rhythm and inflammation?.Nucl. Recept. Signal. 2006; 4: e009Crossref PubMed Google Scholar, 8Migita H. Morser J. Kawai K. Rev-erbα upregulates NF-κB-responsive genes in vascular smooth muscle cells.FEBS Lett. 2004; 561: 69-74Crossref PubMed Scopus (53) Google Scholar) and in that regard is similar to heme, its physiological ligand (5Raghuram S. Stayrook K.R. Huang P. Rogers P.M. Nosie A.K. McClure D.B. Burris L.L. Khorasanizadeh S. Burris T.P. Rastinejad F. Identification of heme as the ligand for the orphan nuclear receptors REV-ERBα and REV-ERBβ.Nat. Struct. Mol. Biol. 2007; 14: 1207-1213Crossref PubMed Scopus (437) Google Scholar). Rev-erbα is structurally unusual in that it lacks the C-terminal tail (helix 12) of the ligand binding domain, which is required for co-activator recognition, hence functions as a transcriptional repressor (9Lazar M.A. Jones K.E. Chin W.W. Isolation of a cDNA encoding human Rev-ErbA α: transcription from the noncoding DNA strand of a thyroid hormone receptor gene results in a related protein that does not bind thyroid hormone.DNA Cell Biol. 1990; 9: 77-83Crossref PubMed Scopus (28) Google Scholar). Rev-erbα may bind to its response element either as a monomer or as a homodimer. As a monomer, it binds to the consensus nuclear receptor half-site motif flanked by a 6-bp AT-rich sequence (A/T)6 PuGGTCA; as a homodimer, it binds to a direct repeat of the core motif separated by 2 bp (DR2) (10Harding H.P. Lazar M.A. The orphan receptor Rev-ErbA α activates transcription via a novel response element.Mol. Cell. Biol. 1993; 13: 3113-3121Crossref PubMed Google Scholar). Rev-erbα competes with RORα, 2The abbreviations used are: RORαRAR-related orphan receptor αRARretinoic acid receptorLXRliver X receptorPPARperoxisome proliferator-activated receptorNCoRnuclear receptor co-repressorHDAC3histone deacetylase 3PMAphorbol 12-myristate 13-acetateMDMmonocyte-derived macrophage. liver X receptor α (LXRα), and peroxisome proliferator-activated receptor α (PPARα) for these response elements and has opposing effects on transcription (11Guillaumond F. Dardente H. Giguère V. Cermakian N. Differential control of Bmal1 circadian transcription by REV-ERB and ROR nuclear receptors.J. Biol. Rhythms. 2005; 20: 391-403Crossref PubMed Scopus (506) Google Scholar, 12Fontaine C. Rigamonti E. Pourcet B. Duez H. Duhem C. Fruchart J.C. Chinetti-Gbaguidi G. Staels B. The nuclear receptor Rev-erbα is a liver X receptor (LXR) target gene driving a negative feedback loop on select LXR-induced pathways in human macrophages.Mol. Endocrinol. 2008; 22: 1797-1811Crossref PubMed Scopus (46) Google Scholar, 13Gervois P. Chopin-Delannoy S. Fadel A. Dubois G. Kosykh V. Fruchart J.C. Najïb J. Laudet V. Staels B. Fibrates increase human REV-ERBα expression in liver via a novel peroxisome proliferator-activated receptor response element.Mol. Endocrinol. 1999; 13: 400-409PubMed Google Scholar). In several instances, Rev-erbα DR2 overlaps the TATA box (14Raspé E. Duez H. Mansén A. Fontaine C. Fiévet C. Fruchart J.C. Vennström B. Staels B. Identification of Rev-erbα as a physiological repressor of apoC-III gene transcription.J. Lipid Res. 2002; 43: 2172-2179Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 15Vu-Dac N. Gervois P. Grötzinger T. De Vos P. Schoonjans K. Fruchart J.C. Auwerx J. Mariani J. Tedgui A. Staels B. Transcriptional regulation of apolipoprotein A-I gene expression by the nuclear receptor RORα.J. Biol. Chem. 1997; 272: 22401-22404Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), which suggests the possibility that Rev-erbα is a component of a basal repressive transcription complex. RAR-related orphan receptor α retinoic acid receptor liver X receptor peroxisome proliferator-activated receptor nuclear receptor co-repressor histone deacetylase 3 phorbol 12-myristate 13-acetate monocyte-derived macrophage. Mycobacterium tuberculosis latently infects one-third of the human race with incidences of active cases, which are rising alarmingly in immunocompromised individuals, especially people infected with HIV. Now with an increase in the cases of multidrug-resistant tuberculosis and with the emergence of extremely drug resistant and total drug resistant strains of M. tuberculosis, tuberculosis has become a major global public health threat. The last decade has seen a hunt for new cellular targets to control and eliminate tuberculosis infection. Nuclear receptors have emerged as decisive cellular targets with the potential of regulating host immune responses to pathogens. Nuclear receptors including PPARγ, testicular receptor 4 (TR4), and LXRs have been shown to effect M. tuberculosis survival or clearance via modulating macrophage function (16Mahajan S. Dkhar H.K. Chandra V. Dave S. Nanduri R. Janmeja A.K. Agrewala J.N. Gupta P. Mycobacterium tuberculosis modulates macrophage lipid-sensing nuclear receptors PPARγ and TR4 for survival.J. Immunol. 2012; 188: 5593-5603Crossref PubMed Scopus (141) Google Scholar, 17Almeida P.E. Silva A.R. Maya-Monteiro C.M. Töröcsik D. D'Avila H. Dezsö B. Magalhães K.G. Castro-Faria-Neto H.C. Nagy L. Bozza P.T. Mycobacterium bovis bacillus Calmette-Guerin infection induces TLR2-dependent peroxisome proliferator-activated receptor γ expression and activation: functions in inflammation, lipid metabolism, and pathogenesis.J. Immunol. 2009; 183: 1337-1345Crossref PubMed Scopus (132) Google Scholar, 18Tontonoz P. Nagy L. Alvarez J.G. Thomazy V.A. Evans R.M. PPARγ promotes monocyte/macrophage differentiation and uptake of oxidized LDL.Cell. 1998; 93: 241-252Abstract Full Text Full Text PDF PubMed Scopus (1610) Google Scholar); the role of Rev-erbα in M. tuberculosis infection has not been addressed, although it has been reported to modulate macrophage function, and its ligand heme has been shown to exhibit antimicrobial properties. Among cytokines, IL10 is a master regulator of macrophage plasticity and function; it antagonizes the expression of co-stimulatory molecules, blocks the release of proinflammatory cytokines, and inhibits phagolysosome maturation and key events in apoptosis (19O'Leary S. O'Sullivan M.P. Keane J. IL-10 blocks phagosome maturation in Mycobacterium tuberculosis-infected human macrophages.Am. J. Respir. Cell Mol. Biol. 2011; 45: 172-180Crossref PubMed Scopus (173) Google Scholar, 20Oswald I.P. Wynn T.A. Sher A. James S.L. Interleukin 10 inhibits macrophage microbicidal activity by blocking the endogenous production of tumor necrosis factor α required as a costimulatory factor for interferon γ-induced activation.Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 8676-8680Crossref PubMed Scopus (323) Google Scholar, 21Rojas M. Olivier M. Gros P. Barrera L.F. García L.F. TNF-α and IL-10 modulate the induction of apoptosis by virulent Mycobacterium tuberculosis in murine macrophages.J. Immunol. 1999; 162: 6122-6131PubMed Google Scholar). IL10 ameliorates immunopathology and prevents host injury, but also has been reported to impede clearance of several pathogens such as Mycobacterium spp., Plasmodium spp., Listeria monocytogenes, and Leishmania major (22Couper K.N. Blount D.G. Riley E.M. IL-10: the master regulator of immunity to infection.J. Immunol. 2008; 180: 5771-5777Crossref PubMed Scopus (1525) Google Scholar). Despite the pleiotropic effects of IL10, its regulation at the level of signal transduction, epigenetics, and transcription factor binding has been addressed in a limited fashion, mostly in regard to IL10 gene activation (23Saraiva M. O'Garra A. The regulation of IL-10 production by immune cells.Nat. Rev. Immunol. 2010; 10: 170-181Crossref PubMed Scopus (2018) Google Scholar). Understanding the molecular events and associated transcription factors that constitute basal repression of IL10 is a requirement for design of newer strategies for infectious disease intervention. In this study, we demonstrate that Rev-erbα binds in vitro and in vivo to the human IL10 putative Rev-erbα DR2 response element, which is preceded by an A/T-rich sequence. Rev-erbα forms a repressive complex by associating with NCoR-HDAC3 upon heme binding and keeps IL10 in a basal repressed state. This repression of human IL10 provides microbicidal phenotype characterized by increased phagolysosome maturation and creation of a macrophage niche that reduces survival of the intracellular parasite M. tuberculosis. Further, the proficiency of Rev-erbα to regulate human IL10 selectively and precisely makes it a valuable target for pharmacological exploitation in infection and tumor regression. Thus, this study will allow us to understand a hitherto unknown mechanism for direct gene regulation of human IL10 by Rev-erbα and utilize the ligand binding domain of Rev-erbα to design small molecules with microbicidal properties. THP-1 (National Centre for Cell Science (NCCS), India) cells were maintained in RPMI 1640 medium (Gibco) with 10% FBS (Gibco), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). MG132 (carbobenzoxy-Leu-Leu-leucinal, Sigma). Peripheral blood mononuclear cells were isolated from the blood of healthy volunteers by Ficoll-Hypaque density centrifugation. Recombinant human GM-CSF and M-CSF and cytokines (eBioscience) were used for differentiation of monocytes into macrophages. pCMV-XL5-Rev-erbα construct was supplied by OriGene. Full-length Rev-erbα was PCR-amplified using a forward primer with a ClaI restriction site and a reverse primer with a KpnI restriction site and then cloned into pCMV2-FLAG vector. pAdeno-X cloning of Rev-erbα was performed according to the manufacturer's instructions (Clontech). Adenoviral particles were produced, titrated, and stored at −70 °C. pSC301 GFP vector capable of expressing in Mycobacterium sp. and in Escherichia coli was kindly provided by Dr. Yossef Av-Gay. GFP-H37Rv and H37Ra were made by electroporation and selection as described previously (24Cowley S.C. Av-Gay Y. Monitoring promoter activity and protein localization in Mycobacterium spp. using green fluorescent protein.Gene. 2001; 264: 225-231Crossref PubMed Scopus (47) Google Scholar). THP-1 cells obtained from the NCCS and maintained in RPMI 1640 with 10% FBS and penicillin/streptomycin were plated at a density of 1 × 106/well in 6-well plates and stimulated with phorbol 12-myristate 13-acetate (PMA) (30 ng/ml) for 6 h. After 6 h, the medium was replaced by fresh complete RPMI 1640 with PMA plus either IFNγ (20 ng/ml) and LPS (100 ng/ml) or IL4 (20 ng/ml) for another 18 h (supplemental Fig. 1). Cells treated with only PMA were taken as controls. Peripheral blood was drawn from healthy volunteers. Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation. The isolated mononuclear cells were then resuspended in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated FCS and plated at 5 × 106 cells/well for 2 h (37 °C/5% CO2) to allow monocyte adherence. After 2 h, nonadherent lymphocytes were removed by PBS washes, and fresh complete medium was then added to the wells. After 24 h, adherent cells were washed with PBS, detached from the wells by scrapping with rubber scrapper, and counted after trypan blue dye staining. The cells (>85% monocytes as determined by flow cytometric analysis after staining with anti-CD14 mAbs) were plated either in 96-well plates at the density of 2 × 105 cells/well or in 12-well plates at the concentration of 1 × 106 cells/well and were cultured for in complete RPMI 1640 along with either GM-CSF (50 ng/ml) for M1-type cells or M-CSF (50 ng/ml) for M2-type cells for 5–6 days at 37 °C in 5% CO2 to promote their full differentiation into monocyte-derived macrophages (MDMs). After 5–6 days, these MDMs (>95% CD14) were stimulated by IFNγ (50 ng/ml) and LPS (10 ng/ml) or IL4 (50 ng/ml) for another 24 h (supplemental Fig. 2) (25Bouhlel M.A. Derudas B. Rigamonti E. Dièvart R. Brozek J. Haulon S. Zawadzki C. Jude B. Torpier G. Marx N. Staels B. Chinetti-Gbaguidi G. PPARγ activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties.Cell Metab. 2007; 6: 137-143Abstract Full Text Full Text PDF PubMed Scopus (983) Google Scholar, 26Cassol E. Cassetta L. Rizzi C. Alfano M. Poli G. M1 and M2a polarization of human monocyte-derived macrophages inhibits HIV-1 replication by distinct mechanisms.J. Immunol. 2009; 182: 6237-6246Crossref PubMed Scopus (145) Google Scholar). siRNA and/or plasmids were transfected into the cells by using Lipofectamine Plus reagent according to the manufacturer's instructions (Invitrogen). Overexpression of Rev-erbα was performed by infecting primary human monocyte-derived macrophages with adenoviral particles (500–1000 plaque-forming units/cell) in Opti-MEM for 2–4 h followed by the addition of fresh medium and additional incubation of 24 h. Oligonucleotides were annealed and then end-labeled using T4-polynucleotide kinase and [γ-32P]ATP. pCMV-XL5-Rev-erbα was transcribed in vitro by T7-polymerase and subsequently translated using the TnT-coupled transcription and translation system (Promega) according to the manufacturer's protocol. A DNA protein binding assay was performed (27Vu-Dac N. Schoonjans K. Kosykh V. Dallongeville J. Fruchart J.C. Staels B. Auwerx J. Fibrates increase human apolipoprotein A-II expression through activation of the peroxisome proliferator-activated receptor.J. Clin. Invest. 1995; 96: 741-750Crossref PubMed Scopus (364) Google Scholar). For all competition experiments, cold oligonucleotides were added after labeled probe. ChIP was performed according to standard protocol (28Wang J. Yin L. Lazar M.A. The orphan nuclear receptor Rev-erbα regulates circadian expression of plasminogen activator inhibitor type 1.J. Biol. Chem. 2006; 281: 33842-33848Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). ChIP and re-ChIP-purified DNA fragments along with input DNA were PCR-amplified using primers specific to putative Rev-erbα response elements on the IL10 promoter (+1 to −350). The following primers were used for the detection of ChIP products: ChFwd IL10, 5′-AAT CAA CTT TTT TTA ATT GAG AAG CT-3′ and ChRev IL10, 5′-GCC TTC TTT TGC AAG TCT GTC T-3′; ChRevDR2(+), forward, 5′-GGT GGA CTA CAA ATC CCG ACA GTC TT-3′ and ChRevDR2(+) reverse, 5′-TGG GAC AGA GGG CTC TGC GC-3′; β-actin forward, 5′-ACT GTT ACC CTC AAA AGC AG-3′ and β-actin reverse, 5′-GTG GGT CAC TAG GGA GAG ACC-3′. M. tuberculosis H37Ra and H37Rv cultures were grown in Middlebrook 7H9 medium (Difco Laboratories) supplemented with 0.2% glycerol, 0.05% Tween 80 (Sigma) and 10% Middlebrook oleic acid albumin dextrose catalase (BD Biosciences) to log phase. Cultures of log phase were used for infection. In brief, cultures were centrifuged for 5 min and resuspended in RPMI 1640 medium containing 10%FBS. For single bacterial cell preparation, culture was syringe-passed carefully for 20–30 times for 3–5 min and then used for infection. To ascertain the multiplicity of infection, 1 × 106 PMA-treated THP-1 macrophages were infected with different dilutions of bacteria for 4 h and then washed three times with medium to remove any unphagocytosed bacteria. Cells were again incubated for 24 h in complete RPMI medium, and then acid-fast staining or confocal imaging was performed to count the cell-associated mycobacteria (29Riendeau C.J. Kornfeld H. THP-1 cell apoptosis in response to Mycobacterial infection.Infect. Immun. 2003; 71: 254-259Crossref PubMed Scopus (181) Google Scholar). On the basis of this screening, multiplicity of infection of 1:5 was used for H37Ra and H37Rv infection. At this multiplicity of infection, most macrophages confirmed the presence of 5–10 bacilli per cell. Infected M1-MDMs with knockdown of Rev-erbα or ectopically overexpressed Rev-erbα were lysed with 0.06% SDS after 24 h of incubation, and bacterial suspensions were used with the LIVE/DEAD BacLight bacterial viability and counting kit as per the manufacturer's instructions (Invitrogen). The percentage of live and dead bacteria was determined by flow cytometry (BD FACSCalibur, BD Biosciences) after staining with SYTO9 and propidium iodide. For CFU determination, after macrophage solubilization, the bacterial suspensions were serially diluted, 50 μl of each sample was plated, and CFU were counted. Final calculations included the dilution factor and the volume of diluted sample for plating. Anti-IL10 neutralizing antibody (purified Rat anti-human IL10, BD Biosciences) or isotype control was used 30 min prior to infection with H37Ra or H37Rv. Analysis of cell surface marker expression was performed by surface staining of cells at a density of 105 for 30 min at 4 °C by using the appropriate isotype controls with phycoerythrin-conjugated anti-CD86, anti-CD80, anti-CD40, and anti-CD-206 and FITC-conjugated anti-CD-68 (eBioscience/BD Biosciences). Samples were analyzed on a FACSCalibur using CellQuest software (BD Biosciences). Cell culture supernatants were harvested after 36 h of siRNA transfection or 24 h of viral transduction and analyzed for IL10 using a commercial kit (BD Biosciences) in accordance with the manufacturer's instructions. Total RNA was isolated by the TRIzol method from macrophages and monocytes, and 1 μg was reverse-transcribed (Fermentas) according to the manufacturer's protocol and subsequently amplified by PCR using specific primers. GAPDH mRNA was used as a loading control. The relative abundance of gene was calculated using the formula 2−ΔCT, where ΔCT is calculated as the difference between target gene and GAPDH CT values. For immunoblotting, 40 μg of total protein extract was resolved by SDS-PAGE on a 10% acrylamide gel (Bio-Rad) and transferred to a nitrocellulose membrane. β-Actin, tubulin, and lamin-B (Santa Cruz Biotechnology, Santa Cruz, CA) were used as loading controls for the whole cell extract, cytoplasmic fraction, and nuclear fraction. THP-1 cells were treated with PMA (30 ng/ml) and grown on poly-l-lysine-coated coverslips. THP-1, PMA-treated THP-1 cells, and transfected PMA-treated THP-1 cells were then fixed with 4% paraformaldehyde and processed for immunostaining. Rev-erbα (Santa Cruz Biotechnology) was stained using mouse-anti-Rev-erbα or mouse anti-FLAG tag followed by Texas Red conjugated with goat-anti-mouse antibody. Tubulin was stained with Tubulin Tracker, and DAPI (Sigma) was used as a nuclear stain. For immunostaining of Rev-erbα in M1-MDM and M2-MDM, goat-anti-mouse FITC was used as secondary antibody. For co-localization of mycobacteria with lysosomes, GFP-H37Ra and GFP-H37Rv were used to infect macrophages. Cells were then incubated for another 24 h followed by the addition of 100 nm LysoTracker for 30 min at 37 °C and fixation with 4% paraformaldehyde. The coverslips were washed thoroughly with PBS and mounted on slides with antifade reagent (Invitrogen, Molecular Probes). The stained cells were observed with an LSM 510 Meta Carl Zeiss confocal microscope and Nikon A1R confocal microscope. The co-localization of LysoTracker and GFP-H37Ra and H37Rv was quantified by selecting a region of interest and determining the overlap coefficient (30Manders E.M.M. Verbeek F.J. Aten J.A. Measurement of co-localization of objects in dual-colour confocal images.J. Microscopy. 1993; 169: 375-382Crossref PubMed Scopus (1517) Google Scholar). For total reactive oxygen species measurement, the cells were incubated with 10 μm DHR123 probe for 30 min at 37 °C. The cells were harvested and subjected to flow cytometry to determine the levels of reactive oxygen species. The project was approved by the Ethics Committee of the Government Medical College and Hospital (GMCH), Sector 32, Chandigarh, India (GMCH/TA-1 [19]/2011/Agenda Number 2) and Ethics and Biosafety Committee of the Institute of Microbial Technology (IMTECH), Sector 39A, Chandigarh, India (01/2011/IMT/IEC-Blood; 12/2010IMT/IBSC). The study was conducted strictly in accordance with the Ethical Guidelines for Biomedical Research on Human Subjects by the Central Ethics Committee on Human Research, Indian Council of Medical Research-2000 and those as contained in the Declaration of Helsinki. Each subject was provided with written information about the study, and written consent on the consent form was obtained from each healthy volunteer prior to his/her induction in the study. Information to healthy volunteers and consent forms were in languages (English, Hindi, and Punjabi) familiar to the volunteers. Rev-erbα has been shown to express in various cell types. It can modulate adipocyte and myocyte differentiation, which led us to question of whether Rev-erbα affects monocyte differentiation to macrophages. THP-1 (THP-1 cells treated with PMA) macrophages are a very well recognized and widely used model for a differentiated tissue macrophage that closely resembles native monocyte-derived macrophage differentiation (supplemental Fig. 1). This model system was utilized to first investigate the localization of Rev-erbα in both monocytes and macrophages. Interestingly, subcellular localization patterns were found to be distinct. In monocytes, Rev-erbα was exclusively cytoplasmic, whereas in macrophages, it was predominantly nuclear; this discrete subcellular localization of Rev-erbα in monocytes and macrophages was confirmed by confocal microscopy and immunoblotting (Fig. 1A and supplemental Fig. 3A). Ectopic expression of Rev-erbα in monocytes and differentiated macrophages led to a similar localization pattern (Fig. 1B). Noise-to-signal ratio was ascertained for anti-Rev-erbα antibody by staining the cells with Rev-erbα knockdown background. Using FACS analysis, we determined surface expression of macrophage differentiation marker CD68, co-stimulatory molecules CD80 and CD86, and CD40 in THP-1 monocytes and THP-1 derived macrophages in Rev-erbα knockdown backgrounds and compared them with a THP-1 macrophage control (Fig. 1C). Upon silencing Rev-erbα in THP-1-derived macrophages, no change in expression of differentiation marker or co-stimulatory molecule was observed, suggesting that Rev-erbα had no effect on PMA-induced THP-1 monocyte-to-macrophage differentiation or activation (Fig. 1C). There was a striking difference in the abundance of Rev-erbα in M1- and M2-polarized THP-1 macrophages, with less Rev-erbα (mRNA and protein) present in the alternative M2 macrophages (Fig. 1D, upper and middle panel). Further, to determine whether proteasomal activity is involved in this difference at the level of both transcription and protein stability, PMA-induced THP-1 cells and M1- and M2-polarized THP-1 macrophages were treated with MG132, a specific inhibitor of 26 S proteasome. Noticeably, MG132 promoted Rev-erbα accumulation in M2-polarized THP-1 macrophages as evident by ubiquitination of Rev-erbα in M2- but not M1-polarized THP-1 macrophages (Fig. 1D, lower panel and supplemental Fig. 3B). To corroborate the findings in the primary cells, human MDMs were utilized, and an endogenous Rev-erbα staining was performed as mentioned above (Fig. 1E and supplemental Fig. 3C). A similar observation of nuclear localization of Rev-erbα was also found in MDMs. Also, real-time PCR analysis of Rev-erbα mRNA expression was performed on human MDMs programmed into M1 and M2 macrophages (supplemental Fig. 3D).Thus, cytoplasmic localization of Rev-erbα, a nuclear receptor, explains its inability to modulate monocyte-macrophage differentiation, but its nuclear localization after differentiation suggests that it may regulate some of the genes and modulate macrophage function. Our previous study has illustrated the role of nuclear receptors in modulating macrophage effector functions in mycobacterial clearance and survival (16Mahajan S. Dkhar H.K. Chandra V. Dave S. Nanduri R. Janmeja A.K. Agrewala J.N. Gupta P. Mycobacterium tuberculosis modulates macrophage lipid-sensing nuclear receptors PPARγ and TR4 for survival.J. Immunol. 2012; 188: 5593-5603Crossref PubMed Scopus (141) Google Scholar). Given that Rev-erbα finds enhanced expression in bactericidal M1 macrophages, we investigated its antimycobacterial role. We employed an RNAi approach to knock down Rev-erbα in M1 macrophages followed by infection with avirulent M. tuberculosi" @default.
• W1995277882 created "2016-06-24" @default.
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• W1995277882 date "2013-04-01" @default.
• W1995277882 modified "2023-10-18" @default.
• W1995277882 title "Human IL10 Gene Repression by Rev-erbα Ameliorates Mycobacterium tuberculosis Clearance" @default.
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