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• W2019794639 abstract "Human immunodeficiency virus-1 (HIV-1) impairs tumor necrosis factor-α (TNF-α)-mediated macrophage apoptosis induced by Mycobacterium tuberculosis (Mtb). HIV Nef protein plays an important role in the pathogenesis of AIDS. We have tested the hypothesis that exogenous Nef is a factor that inhibits TNF-α production/apoptosis in macrophages infected with Mtb. We demonstrate that Mtb and Nef individually trigger TNF-α production in macrophages. However, TNF-α production is dampened when the two are present simultaneously, probably through cross-regulation of the individual signaling pathways leading to activation of the TNF-α promoter. Mtb-induced TNF-α production is abrogated upon mutation of the Ets, Egr, Sp1, CRE, or AP1 binding sites on the TNF-α promoter, whereas Nef-mediated promoter activation depends only on the CRE and AP1 binding sites, pointing to differences in the mechanisms of activation of the promoter. Mtb-dependent promoter activation depends on the mitogen-activated kinase (MAPK) kinase kinase ASK1 and on MEK/ERK signaling. Nef inhibits ASK1/p38 MAPK-dependent Mtb-induced TNF-α production probably by inhibiting binding of ATF2 to the TNF-α promoter. It also inhibits MEK/ERK-dependent Mtb-induced binding of FosB to the promoter. Nef-driven TNF-α production occurs in an ASK1-independent, Rac1/PAK1/p38 MAPK-dependent, and MEK/ERK-independent manner. The signaling pathways used by Mtb and Nef to trigger TNF-α production are therefore distinctly different. In addition to attenuating Mtb-dependent TNF-α promoter activation, Nef also reduces Mtb-dependent TNF-α mRNA stability probably through its ability to inhibit ASK1/p38 MAPK signaling. These results provide new insight into how HIV Nef probably exacerbates tuberculosis infection by virtue of its ability to dampen Mtb-induced TNF-α production. Human immunodeficiency virus-1 (HIV-1) impairs tumor necrosis factor-α (TNF-α)-mediated macrophage apoptosis induced by Mycobacterium tuberculosis (Mtb). HIV Nef protein plays an important role in the pathogenesis of AIDS. We have tested the hypothesis that exogenous Nef is a factor that inhibits TNF-α production/apoptosis in macrophages infected with Mtb. We demonstrate that Mtb and Nef individually trigger TNF-α production in macrophages. However, TNF-α production is dampened when the two are present simultaneously, probably through cross-regulation of the individual signaling pathways leading to activation of the TNF-α promoter. Mtb-induced TNF-α production is abrogated upon mutation of the Ets, Egr, Sp1, CRE, or AP1 binding sites on the TNF-α promoter, whereas Nef-mediated promoter activation depends only on the CRE and AP1 binding sites, pointing to differences in the mechanisms of activation of the promoter. Mtb-dependent promoter activation depends on the mitogen-activated kinase (MAPK) kinase kinase ASK1 and on MEK/ERK signaling. Nef inhibits ASK1/p38 MAPK-dependent Mtb-induced TNF-α production probably by inhibiting binding of ATF2 to the TNF-α promoter. It also inhibits MEK/ERK-dependent Mtb-induced binding of FosB to the promoter. Nef-driven TNF-α production occurs in an ASK1-independent, Rac1/PAK1/p38 MAPK-dependent, and MEK/ERK-independent manner. The signaling pathways used by Mtb and Nef to trigger TNF-α production are therefore distinctly different. In addition to attenuating Mtb-dependent TNF-α promoter activation, Nef also reduces Mtb-dependent TNF-α mRNA stability probably through its ability to inhibit ASK1/p38 MAPK signaling. These results provide new insight into how HIV Nef probably exacerbates tuberculosis infection by virtue of its ability to dampen Mtb-induced TNF-α production. The development and progression of AIDS is intimately associated with loss of normal immunological functions. Nef is a 27-kDa protein expressed by HIV-1/2 3The abbreviations used are: HIV-1/2human immunodeficiency virus, types 1 and 2TNFtumor necrosis factorASK1apoptosis signal-regulating kinase 1ChIPchromatin immunoprecipitationMAPKmitogen-activated protein kinaseERKextracellular signal regulated kinaseMEK1MAPK/ERK kinase 1JNKc-Jun N-terminal kinaseMtbM. tuberculosisELISAenzyme-linked immunosorbent assayGSTglutathione S-transferaseMAP3KMAPK kinase kinaseKDkinase-deadKMkinase-dead mutantdndominant negative. early during infection from multiple spliced viral mRNAs (1Kim S.Y. Byrn R. Groopman J. Baltimore D. J. Virol. 1989; 63: 3708-3713Crossref PubMed Google Scholar). It is considered to be a factor involved in the progression to AIDS (2Greene W.C. Peterlin B.M. Nat. Med. 2002; 8: 673-680Crossref PubMed Scopus (207) Google Scholar, 3Brambilla A. Turchetto L. Gatti A. Bovolenta C. Veglia F. Santagostino E. Gringeri A. Clementi M. Poli G. Bagnarelli P. Vicenzi E. Virology. 1999; 259: 349-368Crossref PubMed Scopus (53) Google Scholar, 4Miller M.D. Warmerdam M.T. Gaston I. Greene W.C. Feinberg M.B. J. Exp. Med. 1994; 179: 101-113Crossref PubMed Scopus (479) Google Scholar, 5Spina C.A. Kwoh T.J. Chowers M.Y. Guatelli J.C. Richman D.D. J. Exp. Med. 1994; 179: 115-123Crossref PubMed Scopus (363) Google Scholar). There is a large body of literature establishing the role of Nef in lymphocyte signaling. Nef increases viral replication in lymphocytes and down-regulates the cell surface expression of CD4, major histocompatibility complex class I A and B but not C (6Lama J. Mangasarian A. Trono D. Curr. Biol. 1999; 9: 622-631Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 7Ross T.M. Oran A.E. Cullen B.R. Curr. Biol. 1999; 9: 613-621Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 8Schwartz O. Maréchal V. Le Gall S. Lemonnier F. Heard J.M. Nat. Med. 1996; 2: 338-342Crossref PubMed Scopus (877) Google Scholar, 9Schindler M. Würfl S. Benaroch P. Greenough T.C. Daniels R. Easterbrook P. Brenner M. Münch J. Kirchhoff F. J. Virol. 2003; 77: 10548-10556Crossref PubMed Scopus (143) Google Scholar, 10Cohen G.B. Gandhi R.T. Davis D.M. Mandelboim O. Chen B.K. Strominger J.L. Baltimore D. Immunity. 1999; 10: 661-671Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar, 11Hung C.H. Thomas L. Ruby C.E. Atkins K.M. Morris N.P. Knight Z.A. Scholz I. Barklis E. Weinberg A.D. Shokat K.M. Thomas G. Cell Host Microbe. 2007; 1: 121-133Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), and several other receptors, including CD28 (12Swigut T. Shohdy N. Skowronski J. EMBO J. 2001; 20: 1593-1604Crossref PubMed Scopus (174) Google Scholar), CD80, CD86 (13Chaudhry A. Das S.R. Hussain A. Mayor S. George A. Bal V. Jameel S. Rath S. J. Immunol. 2005; 175: 4566-4574Crossref PubMed Scopus (88) Google Scholar), and CCR5 (14Michel N. Allespach I. Venzke S. Fackler O.T. Keppler O.T. Curr. Biol. 2005; 15: 714-723Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Major histocompatibility complex class I down-regulation protects infected cells from killing by major histocompatibility complex class I A or B restricted cytotoxic T cells (15Collins K.L. Chen B.K. Kalams S.A. Walker B.D. Baltimore D. Nature. 1998; 391: 397-401Crossref PubMed Scopus (851) Google Scholar) and avoids killing by natural killer cells (16Geleziunas R. Xu W. Takeda K. Ichijo H. Greene W.C. Nature. 2001; 410: 834-838Crossref PubMed Scopus (292) Google Scholar). Nef also prevents apoptosis of HIV-1-infected T cells (17Wolf D. Witte V. Laffert B. Blume K. Stromer E. Trapp S. d'Aloja P. Schürmann A. Baur A.S. Nat. Med. 2001; 7: 1217-1224Crossref PubMed Scopus (250) Google Scholar). It deregulates cofilin in a PAK2-dependent manner, thereby restricting migration of T cells (18Stolp B. Reichman-Fried M. Abraham L. Pan X. Giese S.I. Hannemann S. Goulimari P. Raz E. Grosse R. Fackler O.T. Cell Host Microbe. 2009; 6: 174-186Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). It interacts with and regulates the activity of Src family kinases (19Trible R.P. Emert-Sedlak L. Smithgall T.E. J. Biol. Chem. 2006; 281: 27029-27038Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), class I phosphatidylinositol 3-kinase, guanine nucleotide exchange factor Vav, and calmodulin (20Greenway A.L. Holloway G. McPhee D.A. Ellis P. Cornall A. Lidman M. J. Biosci. 2003; 28: 323-335Crossref PubMed Scopus (54) Google Scholar, 21Hayashi N. Matsubara M. Jinbo Y. Titani K. Izumi Y. Matsushima N. Protein Sci. 2002; 11: 529-537Crossref PubMed Scopus (41) Google Scholar, 22Renkema G.H. Saksela K. Front. Biosci. 2000; 5: D268-D283Crossref PubMed Google Scholar). Very recently, it has been established that Nef activates bidirectional membrane trafficking in T cells, which promotes transfer of Nef from infected to bystander cells (23Muratori C. Cavallin L.E. Krätzel K. Tinari A. De Milito A. Fais S. D'Aloja P. Federico M. Vullo V. Fomina A. Mesri E.A. Superti F. Baur A.S. Cell Host Microbe. 2009; 6: 218-230Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). This offers an explanation for the detrimental effects observed in bystander cells in HIV infection. human immunodeficiency virus, types 1 and 2 tumor necrosis factor apoptosis signal-regulating kinase 1 chromatin immunoprecipitation mitogen-activated protein kinase extracellular signal regulated kinase MAPK/ERK kinase 1 c-Jun N-terminal kinase M. tuberculosis enzyme-linked immunosorbent assay glutathione S-transferase MAPK kinase kinase kinase-dead kinase-dead mutant dominant negative. Less is known about the role of Nef on signaling in macrophages. Nef reduces the expression of the mannose receptor on the macrophage cell surface by ∼50% (24Vigerust D.L. Egan B.S. Shepherd V.L. J. Leukocyte Biol. 2005; 77: 522-534Crossref PubMed Scopus (34) Google Scholar). This is likely to contribute to crippling the host immune response. Nef-expressing macrophages attract CD4+ T cells thereby promoting productive HIV infection (25Mahlknecht U. Herbein G. Trends Immunol. 2001; 22: 256-260Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Considering that extracellular Nef has been detected in supernatants from HIV-1-infected cell cultures and in the serum of AIDS patients (26Fujii Y. Otake K. Tashiro M. Adachi A. FEBS Lett. 1996; 393: 93-96Crossref PubMed Scopus (120) Google Scholar), signals delivered by exogenous Nef to immune cells are likely to be relevant to disease progression. Exogenous Nef activates the IκB kinase complex and the MAPKs JNK, ERK, and p38 in macrophages (27Mangino G. Percario Z.A. Fiorucci G. Vaccari G. Manrique S. Romeo G. Federico M. Geyer M. Affabris E. J. Virol. 2007; 81: 2777-2791Crossref PubMed Scopus (46) Google Scholar). Nef was detected inside B cells in vivo and shown to hamper B cell responses (28Qiao X. He B. Chiu A. Knowles D.M. Chadburn A. Cerutti A. Nat. Immunol. 2006; 7: 302-310Crossref PubMed Scopus (190) Google Scholar). Exogenous Nef reportedly activates NF-κB, AP-1, and c-Jun N-terminal kinase (JNK) in promonocytic cells (29Varin A. Manna S.K. Quivy V. Decrion A.Z. Van Lint C. Herbein G. Aggarwal B.B. J. Biol. Chem. 2003; 278: 2219-2227Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Exogenous Nef enters CD4+ T cells and primary macrophages by adsorptive endocytosis and activates signal transducers and activators of transcription 1 in macrophages (30Federico M. Percario Z. Olivetta E. Fiorucci G. Muratori C. Micheli A. Romeo G. Affabris E. Blood. 2001; 98: 2752-2761Crossref PubMed Scopus (87) Google Scholar). Although the stimulatory effects of exogenous Nef on NF-κB, AP-1, and JNK have been observed in U937 cells, Ma et al. (31Ma W. Mishra S. Gajanayaka N. Angel J.B. Kumar A. J. Biol. Chem. 2009; 284: 7578-7587Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar) have reported that intracellular Nef, expressed through transduction of primary monocytes and promonocytic THP1 cells with retroviral nef gene, inhibits lipopolysaccharide-induced interleukin-12 p40 transcription by inhibiting JNK. The effects of Nef clearly appear to be context-specific. HIV-infected macrophages transfer Nef to B cells through long cellular protrusions, resulting in inhibition of immunoglobulin class switching (32Xu W. Santini P.A. Sullivan J.S. He B. Shan M. Ball S.C. Dyer W.B. Ketas T.J. Chadburn A. Cohen-Gould L. Knowles D.M. Chiu A. Sanders R.W. Chen K. Cerutti A. Nat. Immunol. 2009; 10: 1008-1017Crossref PubMed Scopus (226) Google Scholar). Mycobacterium tuberculosis (Mtb) co-infection occurs in a large number of HIV-positive subjects, and mortality rates are very high (33Nunn P. Williams B. Floyd K. Dye G. Elzinga G. Raviglione M. Nat. Rev. Immunol. 2005; 5: 819-826Crossref PubMed Scopus (198) Google Scholar). Alveolar macrophages serve as reservoirs of Mtb. Apoptosis of macrophages is believed to be essential for efficient control of Mtb infection, and increased apoptosis is a hallmark of bronchoalveolar lavage and lung tissue specimens of Mtb-infected patients (34Klingler K. Tchou-Wong K.M. Brändli O. Aston C. Kim R. Chi C. Rom W.N. Infect. Immun. 1997; 65: 5272-5278Crossref PubMed Google Scholar, 35Keane J. Balcewicz-Sablinska M.K. Remold H.G. Chupp G.L. Meek B.B. Fenton M.J. Kornfeld H. Infect. Immun. 1997; 65: 298-304Crossref PubMed Google Scholar). In vitro HIV infection of alveolar macrophages has been reported to reduce both macrophage apoptosis as well as release of the apoptosis-inducing cytokine TNF-α in response to challenge with Mtb (36Patel N.R. Zhu J. Tachado S.D. Zhang J. Wan Z. Saukkonen J. Koziel H. J. Immunol. 2007; 179: 6973-6980Crossref PubMed Scopus (87) Google Scholar). Taken together with the observation that Nef attenuates HIV-induced macrophage apoptosis (37Olivetta E. Federico M. Exp. Cell Res. 2006; 312: 890-900Crossref PubMed Scopus (48) Google Scholar), this provided the motivation to test whether Nef could modulate apoptosis in Mtb-infected macrophages. The viral burden is high in tissue compartments such as lymph nodes, even during the clinically latent stage of the disease (38Pantaleo G. Graziosi C. Demarest J.F. Butini L. Montroni M. Fox C.H. Orenstein J.M. Kotler D.P. Fauci A.S. Nature. 1993; 362: 355-358Crossref PubMed Scopus (1573) Google Scholar). There is close interaction between infected lymphocytes and macrophages, in these compartments, raising the possibility that exogenous Nef could influence signaling in bystander macrophages. In this report we have tested the effects of exogenous Nef on signaling in Mtb-infected macrophages. We provide evidence that Nef attenuates apoptosis of Mtb-infected macrophages. This is most likely due to an attenuation of TNF-α production by Nef. Nef and Mtb both elicit TNF-α production. However, there is a net inhibitory effect when macrophages are challenged simultaneously with Nef and Mtb, probably due to mutual cross-regulation of signaling pathways. Escherichia coli BL21 (DE3) and E. coli DH5α strains were grown in Luria-Bertani (LB) Miller media. M. tuberculosis H37Rv was grown in Middlebrook 7H9 broth or Middlebrook 7H10 solid medium supplemented with albumin-dextrose-catalase and 0.05% Tween 80. Standard procedures were used for cloning and analysis of DNA, PCR, and transformation. Enzymes used to manipulate DNA were from Roche Applied Science. All constructs made by PCR were sequenced to verify their integrity. Antibodies against ASK1, Sp1, FosB, and ATF2 were from Santa Cruz Biotechnology (Santa Cruz, CA); Rac1, PAK1, ERK1/2, p38 MAPK, phospho-ERK1/2, phospho p38 MAPK, and phospho PAK1 antibodies were from Cell Signaling Technology (Beverly, MA). N-terminal His-tagged SF2 Nef in pET 15b was a gift from Dr. Yong Hui Zheng (Michigan State University). SF2 Nef in pET15b was transformed in E. coli BL21 (DE3). Expression was carried out in the presence of 0.1 mm isopropyl-β-d-thiogalactopyranoside at 37 °C for 4 h. Cells were disrupted by sonication in 10 mm Tris-HCl (pH 8.0) containing 1 mm MgCl2, 1 mm Pefabloc, 1 μg/ml leupeptin, and 1 μg/ml pepstatin. Protein was purified from the post-sonicate supernatant by chromatography on nickel-nitrilotriacetic acid-agarose. HA-tagged wild-type ASK1 and a catalytically inactive mutant (K709M) of ASK1(ASK1, KM) were obtained from Prof. Hidenori Ichijo, University of Tokyo. The dominant negative mutant of p38 MAPK (p38(agf)) was obtained from Dr. Roger Davis, University of Massachusetts Medical School, Worcester, MA. Dominant-negative MEK1 was a gift from Dr. D. J. Templeton, Case Western Reserve University, Cleveland, OH. Kinase-dead pCMV-PAK1 (K299R) (PAK1-KD) was a gift from Dr. Jeffrey Frost, University of Texas Southwestern Medical Center, Dallas, TX. Using genomic DNA from RAW264.7 cells as template, the TNF-α promoter region (−185 to +69) was amplified by PCR using the sense and antisense primers 5′-ATAGGTACCCCCCAACTTTCCAAACCC-3′ and 5′-AAAGATCTAGCTATTTCCAAGATGTTC-3′, respectively. The resulting TNF-α promoter region (wild type) was cloned into the vector pGL3-basic (Promega) harboring the promoter-less luciferase gene, using asymmetric KpnI and BglII sites (underlined). CRE, Sp1, AP1, and Ets binding sites were mutated by overlap extension PCR. THP-1 cells were obtained from the National Center for Cell Science, Pune, India, cultured and differentiated with phorbol 12-myristate 13-acetate as described by Maiti et al. (39Maiti D. Bhattacharyya A. Basu J. J. Biol. Chem. 2001; 276: 329-333Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Adherent cells were 95% viable as determined by trypan blue dye exclusion. Differentiated THP-1 cells were either left untreated or treated with recombinant Nef (0.1–1 μg/ml) or with M. tuberculosis H37Rv (multiplicity of infection of 10) or with Nef and M. tuberculosis for 2 h or for the indicated periods of time. Unless otherwise stated, Nef was used at 1 μg/ml (the concentration at which the inhibitory effect of Nef was maximal). Cells were washed once, fresh medium was added, and incubations were carried out for the indicated time periods. For the detection of histone by ELISA, cells (6 × 104) were plated in 96-well plates. After treatments, cell death was measured by the detection of histones in the cell supernatant using the Cell Death ELISA Plus kit (Roche Applied Science) according to the manufacturer's protocol. Cells were lysed after treatment in buffer containing 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% (v/v) Triton X-100, 1% sodium deoxycholate, 0.5 mm sodium pyrophosphate, 1 mm sodium β-glycerophosphate, 1 mm Na3VO4, and 1 μg/ml leupeptin. The supernatant (equivalent to 200 μg of protein) was incubated overnight at 4 °C with rabbit polyclonal ASK1 antibody. Protein A/G Plus-agarose was added and incubated at 4 °C for an additional 3 h. The beads were washed twice with lysis buffer and twice with kinase buffer (25 mm Tris-HCl, pH 7.5, containing 5 mm sodium β-glycerophosphate, 2 mm dithiothreitol, 0.1 mm Na3VO4, and 10 mm MgCl2). The pellet was washed once with kinase buffer without protease inhibitors. The beads were then incubated in 20 μl of kinase buffer in the presence of 2.5 μCi of [γ-32P]ATP (specific activity, 6000 Ci/mmol) with 1 μg of myelin basic protein as substrate at 30 °C for 15 min. The reaction was stopped by adding protein gel denaturing buffer. After SDS-PAGE, gels were dried and subjected to autoradiography. To assess the activation of Rac1 and formation of Rac1-GTP in response to stimulation with Nef, affinity precipitation was performed with a GST fusion protein corresponding to the p21-binding domain (PBD) of PAK1 (GST-PBD) that specifically binds to and precipitates Rac-GTP from cell lysates (40Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). The presence of Rac1 in the precipitate was assessed using Rac1 antibody. For immunoprecipitations, cells were lysed, clarified by centrifugation, and then immunoprecipitated using appropriate antibody. Proteins were separated by SDS-PAGE and then transferred electrophoretically to polyvinylidene difluoride membranes. Blots after blocking were incubated overnight at 4 °C with primary antibody in Tris-buffered saline-Tween 20 (1%, v/v) with 5% (w/v) bovine serum albumin. After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) in blocking buffer for 1 h at room temperature. Blots were developed by chemiluminescence. TransFactor assays were carried out as described previously (41Pathak S.K. Basu S. Bhattacharyya A. Pathak S. Kundu M. Basu J. J. Biol. Chem. 2005; 280: 42794-42800Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Briefly, nuclear extracts from cells were prepared using the TransFactor extraction kit (Clontech). After centrifugation for 5 min at 20,000 × g at 4 °C, supernatants were assayed for the presence of the respective transcription factors by addition of equal amounts of lysates to wells precoated with DNA-binding consensus sequences. The presence of any particular transcription factor in the nucleus was then assessed by using the Mercury TransFactor kit (Clontech) according to the manufacturer's instructions. Plates were read at 655 nm. Cells were transfected with luciferase reporter plasmids. After treatments, cells were washed once with phosphate-buffered saline and scraped into luciferase lysis buffer (50 mm Tris-HCl, pH 8, 70 mm K2HPO4, 0.1% Nonidet P-40, 2 mm MgCl2, 1 mm dithiothreitol, 20 μg/ml aprotinin, 10 μg/ml pepstatin, 10 μg/ml leupeptin). The lysates were rapidly mixed, and insoluble material was pelleted by centrifugation at 4 °C. The supernatant was removed and stored at −80 °C. For promoter activation analysis, luciferase activity assays were performed in a luminometer, and the results were normalized for transfection efficiencies by assay of β-galactosidase activity. Chromatin immunoprecipitation (ChIP) assays were carried out using the Upstate Biotechnology ChIP assay kit. Briefly, cells after treatment were fixed by addition of formaldehyde (1%) to the culture medium for 10 min at 37 °C, washed in phosphate-buffered saline, scraped, lysed in lysis buffer (10 mm Tris-HCl, pH 8.0, 1% SDS, 0.5 mm Pefabloc, 2 μg/ml pepstatin A, and 2 μg/ml aprotinin) for 10 min at 4 °C, and sonicated to generate DNA fragments with an average size of 1 kb. The debris was removed by centrifugation. One-third of the lysate was used as DNA input control. The remaining two-thirds of the lysate were diluted 10-fold with a dilution buffer (10 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.01% SDS, 1% Triton X-100, 1 mm EDTA), precleared with salmon sperm DNA/protein A/G-agarose slurry, followed by incubation of the supernatant with appropriate antibody (1.5–2 μg) overnight at 4 °C. Immunoprecipitated complexes were collected by pulldown assay with salmon sperm DNA/protein A/G-agarose beads. The precipitates were extensively washed and incubated in elution buffer (25 mm Tris-HCl, pH 8, 10 mm EDTA, 0.5% SDS) at 60 °C for 15 min. Cross-linking of protein-DNA complexes was reversed at 65 °C for 4 h, followed by ethanol precipitation overnight and centrifugation at 14,000 rpm for 20 min at 4 °C. The pellet was air-dried and reconstituted in Tris-EDTA buffer followed by treatment with 100 μg/ml proteinase K in PK buffer (50 mm Tris-HCl, pH 8, 25 mm EDTA, 1.25% SDS, and glycogen) for 1 h at 45 °C. DNA was extracted twice with phenol/chloroform and precipitated with ethanol. Pellets were resuspended in Tris-EDTA buffer and subjected to PCR amplification using TNF promoter-specific primers 5′-ATAGGTACCCCCCAACTTTCCAAACCC-3′ and 5′-AAAGATCTAGCTATTTCCAAGATGTTC-3′. TNF-α mRNA stability was assessed 16 h after treatment of cells with either M. tuberculosis, or M. tuberculosis and Nef by the addition of actinomycin D (5 μg/ml) for different periods of time. Total RNA was extracted using an RNeasy kit (Qiagen), and cDNA was prepared using first strand synthesis kit (Roche Applied Science) and quantification of TNF-α mRNA was carried out by quantitative real-time PCR on an ABI 7500 Fast detection system using SYBR green PCR master mix (Applied Biosystems). The sense and antisense primers used for TNF-α were 5′-GAGTGACAAGCCTGTAGCCCATGTTGTAGC-3′ and 5′-CTGGGAGTAGATGAGGTACAGGCCCTCTGA-3′, respectively. The sense and antisense primers used for glyceraldehyde-3-phosphate dehydrogenase were 5′-GATGGGATTTCCATTGATGACA-3′ and 5′-CCACCCATGGCAAATTCC-3′, respectively. Conditioned medium was removed 24 h after treatments with M. tuberculosis or Nef or a combination of the two, and assayed for TNF-α by ELISA using the TNF-α assay kit (BD Biosciences). Data are represented as means ± S.D. of three separate experiments. Student's t test was performed to test statistical significance. Exogenous Nef was able to inhibit apoptosis induced in differentiated THP-1 cells by M. tuberculosis as measured by histone ELISA (Fig. 1A). M. tuberculosis-induced macrophage apoptosis is known to depend on TNF-α, with HIV being able to dampen this process (36Patel N.R. Zhu J. Tachado S.D. Zhang J. Wan Z. Saukkonen J. Koziel H. J. Immunol. 2007; 179: 6973-6980Crossref PubMed Scopus (87) Google Scholar). In view of this, we tested the effect of Nef on M. tuberculosis-induced TNF-α release. TNF-α was released from differentiated THP-1 cells challenged with either M. tuberculosis or with recombinant Nef (Fig. 1B) in conformity with earlier reports on the effect of Nef or M. tuberculosis on macrophages (42Olivetta E. Percario Z. Fiorucci G. Mattia G. Schiavoni I. Dennis C. Jäger J. Harris M. Romeo G. Affabris E. Federico M. J. Immunol. 2003; 170: 1716-1727Crossref PubMed Scopus (128) Google Scholar, 43Barthel R. Tsytsykova A.V. Barczak A.K. Tsai E.Y. Dascher C.C. Brenner M.B. Goldfeld A.E. Mol. Cell. Biol. 2003; 23: 526-533Crossref PubMed Scopus (71) Google Scholar). However, when macrophages were challenged simultaneously with M. tuberculosis and Nef, there was significant decrease in TNF-α production (Fig. 1B). This suggested that M. tuberculosis and Nef were perhaps cross-inhibiting the respective signaling pathways associated with TNF-α production. Production of TNF-α in cells of the monocytic lineage in response to stimulus, is regulated at the transcriptional and post-transcriptional level depending both on cell type and stimulus (44Swantek J.L. Cobb M.H. Geppert T.D. Mol. Cell. Biol. 1997; 17: 6274-6282Crossref PubMed Google Scholar, 45Raabe T. Bukrinsky M. Currie R.A. J. Biol. Chem. 1998; 273: 974-980Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 46Udalova I.A. Knight J.C. Vidal V. Nedospasov S.A. Kwiatkowski D. J. Biol. Chem. 1998; 273: 21178-21186Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Among the several possible steps at which Nef and M. tuberculosis could exert cross-inhibitory effects, we tested the possibility that this could be at the level of activation of the TNF-α promoter. It was observed that exogenous Nef and M. tuberculosis could individually activate TNF-α promoter-driven luciferase expression (Fig. 1C). However, in macrophages challenged simultaneously with Nef and M. tuberculosis, there was a significant decrease in TNF-α promoter-driven luciferase expression, compared with either entity used alone (Fig. 1C). Heat treatment abrogated the ability of Nef to induce TNF-α (Fig. 1, B and C), suggesting that the effect was attributable specifically to the protein, rather than any contaminant. The ability of Nef to down-regulate Mtb-dependent TNF-α production was not attributable to endotoxin contamination, because pretreatment of Nef with polymyxin B did not alter this activity (Fig. 1, B and C). We next reasoned that, in the event of a cross-inhibitory mechanism being at work, M. tuberculosis and Nef were likely to operate through different mechanisms to activate the TNF-α promoter. Sequences in the proximal 200 bp and the distal (−627 to −487 bp) of the TNF-α promoter are remarkably conserved in mouse and human and encompass binding sites for multiple transcription factors (46Udalova I.A. Knight J.C. Vidal V. Nedospasov S.A. Kwiatkowski D. J. Biol. Chem. 1998; 273: 21178-21186Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 47Yao J. Mackman N. Edgington T.S. Fan S.T. J. Biol. Chem. 1997; 272: 17795-17801Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 48Newell C.L. Deisseroth A.B. Lopez-Berestein G. J. Leukocyte Biol. 1994; 56: 27-35Crossref PubMed Scopus (117) Google Scholar, 49Pope R.M. Leutz A. Ness S.A. J. Clin. Invest. 1994; 94: 1449-1455Crossref PubMed Scopus (165) Google Scholar, 50Geist L.J. Hopkins H.A. Dai L.Y. He B. Monick M.M. Hunninghake G.W. Am. J. Respir. Cell Mol. Biol. 1997; 16: 31-37Crossref PubMed Scopus (34) Google Scholar). Previous studies have shown that the TNF-α gene is regulated in a cell type-specific manner (51Tsai E.Y. Yie J. Thanos D. Goldfeld A.E. Mol. Cell. Biol. 1996; 16: 5232-5244Crossref PubMed Scopus (178) Google Scholar). In addition, within the same cell type, the TNF-α gene is regulated in a stimulus-specific manner (52Falvo J.V. Uglialoro A.M. Brinkman B.N. Merika M. Parekh B.S. Tsai E.Y. King H.C. Morielli A.D. Peralta E.G. Maniatis T. Thanos D. Goldfeld A.E. Mol. Cell. Biol. 2000; 20: 2239-2247Crossref PubMed Scopus (146) Google Scholar) through the action of distinct sets of transcription factors. We therefore tested the hypothesis that M. tuberculosis and Nef each induce TNF-α transcription through different sets of transcription factors. Both the human and the murine proximal TNF-α promoter contain, among other sites, multiple NFAT/ETS, Egr, Sp1, CRE, and AP1 binding sites. We mutated the −180 Ets, −117 Ets, −84 Ets, −76 Ets, Egr, Sp1, CRE, and AP1 sites (Fig. 1D) on the TNF-α promoter to evaluate the contributions of these binding sites in M. tuberculosis or Nef-induced TNF-α activation. M. tuberculosis-driven TNF-α promoter activation was" @default.
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• W2019794639 title "Exogenous Nef Is an Inhibitor of Mycobacterium tuberculosis-induced Tumor Necrosis Factor-α Production and Macrophage Apoptosis" @default.
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• W2019794639 doi "https://doi.org/10.1074/jbc.m109.073320" @default.
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