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• W1983129403 abstract "We investigated the basis for the induction of monocyte antimycobacterial activity by 1α,25-dihydroxyvitamin D3 (D3). As expected, incubation of Mycobacterium tuberculosis-infected THP-1 cells or human peripheral blood, monocyte-derived macrophages with hormone resulted in the induction of antimycobacterial activity. This effect was significantly abrogated by pretreatment of cells with either of the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, wortmannin or LY294002, or with antisense oligonucleotides to the p110 subunit of PI 3-Kα. Cells infected with M. tuberculosisalone or incubated with D3 alone produced little or undetectable amounts of superoxide anion (O⨪2). In contrast, exposure of M. tuberculosis-infected cells to D3 led to significant production of O⨪2, and this response was eliminated by either wortmannin, LY294002, or p110 antisense oligonucleotides. As was observed for PI 3-K inactivation, the reactive oxygen intermediate scavenger, 4-hydroxy-TEMPO, and degradative enzymes, polyethylene glycol coupled to either superoxide dismutase or catalase, also abrogated D3-induced antimycobacterial activity. Superoxide production by THP-1 cells in response to D3 required prior infection with liveM. tuberculosis, since exposure of cells to either killed M. tuberculosis or latex beads did not prime for an oxidative burst in response to subsequent hormone treatment. Consistent with these findings, redistribution of the cytosolic oxidase components p47phox and p67phox to the membrane fraction was observed in cells incubated with liveM. tuberculosis and D3 but not in response to combined treatment with heat-killed M. tuberculosis followed by D3. Redistribution of p47phox and p67phox to the membrane fraction in response to live M. tuberculosis and D3 was also abrogated under conditions where PI 3-K was inactivated. Taken together, these results indicate that D3-induced, human monocyte antimycobacterial activity is regulated by PI 3-K and mediated by the NADPH-dependent phagocyte oxidase. We investigated the basis for the induction of monocyte antimycobacterial activity by 1α,25-dihydroxyvitamin D3 (D3). As expected, incubation of Mycobacterium tuberculosis-infected THP-1 cells or human peripheral blood, monocyte-derived macrophages with hormone resulted in the induction of antimycobacterial activity. This effect was significantly abrogated by pretreatment of cells with either of the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, wortmannin or LY294002, or with antisense oligonucleotides to the p110 subunit of PI 3-Kα. Cells infected with M. tuberculosisalone or incubated with D3 alone produced little or undetectable amounts of superoxide anion (O⨪2). In contrast, exposure of M. tuberculosis-infected cells to D3 led to significant production of O⨪2, and this response was eliminated by either wortmannin, LY294002, or p110 antisense oligonucleotides. As was observed for PI 3-K inactivation, the reactive oxygen intermediate scavenger, 4-hydroxy-TEMPO, and degradative enzymes, polyethylene glycol coupled to either superoxide dismutase or catalase, also abrogated D3-induced antimycobacterial activity. Superoxide production by THP-1 cells in response to D3 required prior infection with liveM. tuberculosis, since exposure of cells to either killed M. tuberculosis or latex beads did not prime for an oxidative burst in response to subsequent hormone treatment. Consistent with these findings, redistribution of the cytosolic oxidase components p47phox and p67phox to the membrane fraction was observed in cells incubated with liveM. tuberculosis and D3 but not in response to combined treatment with heat-killed M. tuberculosis followed by D3. Redistribution of p47phox and p67phox to the membrane fraction in response to live M. tuberculosis and D3 was also abrogated under conditions where PI 3-K was inactivated. Taken together, these results indicate that D3-induced, human monocyte antimycobacterial activity is regulated by PI 3-K and mediated by the NADPH-dependent phagocyte oxidase. 1α,25-dihydroxyvitamin D3 monocyte-derived macrophage(s) phosphatidylinositol 3-kinase wortmannin LY294002 superoxide anion polyethylene glycol superoxide dismutase catalase vitamin D receptor l-α-phosphatidylinositol l-N-monomethylarginine lactate dehydrogenase nitric oxide lipoarabinomannan whole cell lysates concentrated extracellular filtrate Hanks' balanced saline solution colony-forming units phorbol 12-myristate 13-acetate terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl toll-like receptor 2 As the leading cause of death from any single bacterial infection in the world, tuberculosis represents a global health problem of paramount importance. Current estimates are that one-third of the world's population is infected with the etiological agentMycobacterium tuberculosis (1Kaufmann S.H. van Embden J.D. Trends Microbiol. 1993; 1: 2-5Abstract Full Text PDF PubMed Scopus (48) Google Scholar) and that the incidence of new cases with active disease is anticipated to rise from the current 7 million per year to 10 million per year by 2015 (2Day M. New Sci. 1998; 2127: 21Google Scholar). These statistics highlight the importance of developing new, more effective anti-tuberculous drugs, an effort dependent on acquiring more insights into host-pathogen interactions that determine the outcome of infection. M. tuberculosis primarily infects mononuclear phagocytes, where it resides and multiplies within a host-derived phagosome (3Armstrong J.A. Hart P.D. J. Exp. Med. 1975; 142: 1-16Crossref PubMed Scopus (345) Google Scholar). Its success as a pathogen is largely attributable to its ability to evade or resist the multiplicity of antimicrobial mechanisms available to this host cell. The macrophage is not only the primary site for M. tuberculosis growth but also ordinarily provides the primary line of host defense against invading pathogens in its role as an effector of innate immunity. Macrophages use varied strategies to kill and destroy invading organisms, including production of reactive nitrogen and oxygen intermediates, phagosome maturation and acidification, fusion with lysosomes, exposure to defensins and host cell apoptosis (4Hingley-Wilson S.M. Sly L.M. Reiner N.E. McMaster W.R. Mod. Aspects Immunobiol. 2000; 1: 96-101Google Scholar). Augmentation of any of these processes during macrophage activation may contribute to control of disease. Vitamin D3 is a steroid hormone that regulates several cellular and physiological responses. The classical mechanism of action of D31 involves genomic signaling where hormone binds to the vitamin D receptor (VDR), a ligand-dependent transcription factor. The D3·VDR complex then translocates to the nucleus, where it directly regulates transcription by binding to the vitamin D response element consensus sequence located upstream of D3-activated genes (5Malloy P.J. Feldman D. Am. J. Med. 1999; 106: 355-370Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Recently, nongenomic signaling in response to D3 (i.e. cellular responses brought about independent of de novo transcription from a classical vitamin D response element) has been recognized to regulate important cellular processes (6Hmama Z. Nandan D. Sly L. Knutson K.L. Herrera-Velit P. Reiner N.E. J. Exp. Med. 1999; 190: 1583-1594Crossref PubMed Scopus (164) Google Scholar, 7Gniadecki R. Biochem. Pharmacol. 1998; 56: 1273-1277Crossref PubMed Scopus (40) Google Scholar, 8Janis A.E. Kaufmann S.H.E. Schwartz R.H. Pardoll D.M. Science. 1989; 244: 713-716Crossref PubMed Scopus (406) Google Scholar, 9Phillips W. Hamilton J.A. J. Immunol. 1989; 142: 2445-2449PubMed Google Scholar, 10Duncan R. McConkey E.H. Eur. J. Biochem. 1982; 123: 535-538Crossref PubMed Scopus (72) Google Scholar). Vitamin D3 is known to possess a variety of immunomodulatory properties including effects on both myeloid and lymphoid cells (11Manolagas S.C. Hustmyer F.G. Yu X.P. Kidney Int. Suppl. 1990; 29: 9-16PubMed Google Scholar). Among these are its ability to induce the differentiation of immature myeloid cells into more mature monocytes and macrophages (12Abe E. Miyaura C. Sakagami H. Takeda M. Konno K. Yamazaki T. Yoshiki S. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4990-4994Crossref PubMed Scopus (1026) Google Scholar, 13Tanaka H. Abe E. Miyaura C. Kuribayashi T. Konno K. Nishii Y. Suda T. Biochem. J. 1982; 204: 713-719Crossref PubMed Scopus (396) Google Scholar, 14Kreutz M. Andreesen R. Blood. 1990; 76: 2457-2461Crossref PubMed Google Scholar, 15Schwende H. Fitzke E. Ambs P. Dieter P. J. Leukocyte Biol. 1996; 59: 555-561Crossref PubMed Scopus (450) Google Scholar, 16Zhang D.-E. Hetherington C.J. Gonzalez D.A. Chen H.-M. Tenen D.G. J. Immunol. 1994; 153: 3276-3285Crossref PubMed Google Scholar). Several lines of evidence indicate that D3 regulates host resistance to M. tuberculosis. D3 deficiency and vitamin D receptor polymorphisms have been linked to increased susceptibility toM. tuberculosis and Mycobacterium leprae (17Davies P.D. Tubercle. 1985; 66: 301-306Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 18Roy S. Frodsham A. Saha B. Hazra S.K. Mascie-Taylor C.G. Hill A.V. J. Infect. Dis. 1999; 179: 187-191Crossref PubMed Scopus (194) Google Scholar, 19Wilkinson R.J. Llewelyn M. Toossi Z. Patel P. Pasvol G. Lalvani A. Wright D. Latif M. Davidson R.N. Lancet. 2000; 355: 618-621Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). In this regard, D3 productionin vivo is promoted by exposure to ultraviolet light, and this may provide a link between exposure to sunlight and antimycobacterial resistance mechanisms (17Davies P.D. Tubercle. 1985; 66: 301-306Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 20Crowle A.J. Ross E.J. May M.H. Infect. Immun. 1987; 55: 2945-2950Crossref PubMed Google Scholar). In addition, D3 has been shown to activate mononuclear phagocyte antimycobacterial activity (20Crowle A.J. Ross E.J. May M.H. Infect. Immun. 1987; 55: 2945-2950Crossref PubMed Google Scholar, 21Rook G.A. Steele J. Fraher L. Barker S. Karmali R. O'Riordan J. Stanford J. Immunology. 1986; 57: 159-163PubMed Google Scholar). Until now, the molecular basis through which D3 regulates host resistance to M. tuberculosis has not been identified. Class I phosphatidylinositol 3-kinase (PI 3-K) is a lipid kinase that phosphorylates the 3′-position of the inositol ring of phosphatidylinositol (PI) and its derivatives. It is composed of an 85-kDa Src homology 2 domain containing regulatory subunit (p85) and a 110-kDa catalytic subunit (p110). It is a multifunctional signaling molecule that has been implicated in a wide range of cellular processes including nuclear signaling, vesicle transport, organization of the cytoskeleton, cell growth, transformation, and survival (22Fry M.J. Biochim. Biophys. Acta Mol. Basis Dis. 1994; 1226: 237-268Crossref PubMed Scopus (174) Google Scholar, 23Toker A. Cantley L.G. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1229) Google Scholar, 24Fukui Y. Ihara S. Nagata S. J Biochem. (Tokyo). 1998; 124: 1-7Crossref PubMed Scopus (42) Google Scholar). It was recently found that PI 3-K is required for D3-induced cell differentiation in the human macrophage cell lines THP-1 and U937 and in peripheral blood monocytes (6Hmama Z. Nandan D. Sly L. Knutson K.L. Herrera-Velit P. Reiner N.E. J. Exp. Med. 1999; 190: 1583-1594Crossref PubMed Scopus (164) Google Scholar). In the latter study, vitamin D3 was observed to activate PI 3-K, and D3-induced expression of the monocyte differentiation markers, CD14 and CD11b, required PI 3-K. Furthermore, D3treatment induced the formation of a vitamin D receptor·PI 3-K complex, representing a novel nongenomic signaling pathway activated by D3. In light of these results, this study examined whether PI 3-K is involved in regulating D3-induced antimycobacterial activity and investigated the mechanistic basis for this effector function. The results obtained show that D3-induced monocyte resistance to M. tuberculosis is regulated by PI 3-K and that this effect is due to activation of the phagocyte NADPH oxidase. RPMI 1640 and HBSS were from Stem Cell Technologies (Vancouver, Canada). Wortmannin, LY294002,l-NMMA, 1α,25-dihydroxyvitamin D3, 1α-hydroxyvitamin D3, and 25-hydroxyvitamin D3 were from Calbiochem.l-α-Phosphatidylinositol, phenylmethylsulfonyl fluoride, leupeptin, pepstatin A, aprotonin, microcystin, vanadate, ferricytochrome c, superoxide dismutase, latex beads, 4-hydroxy-TEMPO, PEG-catalase, and PEG-superoxide dismutase were from Sigma. Protein A-agarose, Griess reagent, and electrophoresis reagents were from Bio-Rad. [γ-32P]ATP was from Amersham Pharmacia Biotech. Phosphorothioate-modified oligonucleotides were synthesized by Life Technologies, Inc. Mannose-capped lipoarabinomannan was provided by D. John T. Belisle. Mouse monoclonal anti-p85 UB93–3 was from Upstate Biotechnology Inc. (Lake Placid, NY). Mouse monoclonal anti-p110α was from Transduction Laboratories. Anti-p47phoxand anti-p67phox antibodies were prepared as described previously (25Allen L.A. DeLeo F.R. Gallois A. Toyoshima S. Suzuki K. Nauseef W.M. Blood. 1999; 93: 3521-3530Crossref PubMed Google Scholar). The human promonocytic cell line THP-1 and murine macrophage-like cell line RAW264.7 were from the American Type Culture Collection. Cell lines were cultured in RPMI 1640 supplemented with 10% fetal calf serum (Hyclone), 2 mml-glutamine and maintained between 2 and 10 × 105 cells/ml. The human myeloid cell line THP-1 was used as a model for M. tuberculosis infection studies because of its similarity to human MDM (26Stokes R.W. Doxsee D. Cell. Immunol. 1999; 197: 1-9Crossref PubMed Scopus (97) Google Scholar) in M. tuberculosis infection models and its availability for use. Peripheral blood mononuclear cells were isolated as described previously (27Liu M.K. Herrera-Velit P. Brownsey R.W. Reiner N.E. J. Immunol. 1994; 153: 2642-2652PubMed Google Scholar). Mononuclear cells were allowed to adhere for 45 min at 37 °C in a humidified atmosphere with 5% CO2. Nonadherent cells were removed with three washes with HBSS. Adherent cells were maintained for 3 days at 37 °C in a humidified atmosphere with 5% CO2 prior to use for infections or treatments. A virulent strain of M. tuberculosis (H37Rv) was grown to late log phase in Middlebrook 7H9 with OADC (Difco). Aliquots were frozen at −70 °C, and representative samples were thawed and evaluated for colony-forming units (CFUs) on Middlebrook 7H10 agar with OADC (Difco Laboratories). Heat-killed bacteria were prepared by heating at 80 °C for 2 h. Formaldehyde-fixed bacteria were prepared by fixation in 30% formaldehyde in methanol for 30 min. UV-irradiated bacteria were prepared by UV irradiation for 16 h. Each treatment reduced the viability of M. tuberculosis by greater than 5 logs. Cells were infected as described previously (28Hmama Z. Gabathuler R. Jefferies W.A. de Jong G. Reiner N.E. J. Immunol. 1998; 161: 4882-4893PubMed Google Scholar). Briefly, prior to infection, THP-1 cells were seeded at a density of 105/cm2 in either six-well flat bottom or 10-cm diameter tissue culture plates (Becton Dickinson, Franklin Lakes, NJ) and allowed to adhere and differentiate in the presence of 20 ng/ml PMA at 37 °C in a 5% CO2 humidified atmosphere for 24 h. Cells were washed, and medium without PMA was replenished 4 h prior to the addition of bacteria. Prior to infection, bacteria were opsonized with fresh serum as described previously (28Hmama Z. Gabathuler R. Jefferies W.A. de Jong G. Reiner N.E. J. Immunol. 1998; 161: 4882-4893PubMed Google Scholar). Monolayers were infected with opsonized M. tuberculosis at a 50:1 ratio. Latex beads were opsonized as for M. tuberculosis and incubated with cells at a ratio of 20:1. After 4 h, noningested bacilli or beads were removed by washing three times with HBSS, and medium was replenished. This resulted in infection of 80–90% of cells with 1–5 bacteria/cell. Infection was evaluated by Kinyoun staining (BBL Microbiology Systems, Cockeysville, MD). For latex beads, ∼90% of cells contained beads with 1–7 beads per cell. Immediately after infection, macrophages were treated with inhibitors at final concentrations as follows: LY, 14 μm; Wm, 50 nm, and l-NMMA, 8 μm. Cells were incubated with these inhibitors for 15 min at 37 °C, 5% CO2 prior to the addition of D3. 4-Hydroxy-TEMPO (0.1 mm), PEG-SOD (100 units/ml), or PEG-cat (100 units/ml) were added during the 4-h infection, and reagents remaining in the culture medium were washed away from treated cells along with noningested bacteria. Heat-inactivated PEG-SOD and PEG-cat were used as negative controls for the active enzymes. Vitamin D3 (1 μm) was added to monolayers after treatment with inhibitors and was left in the medium for the remainder of the experiment. In vitro PI 3-K assays were performed as described previously (6Hmama Z. Nandan D. Sly L. Knutson K.L. Herrera-Velit P. Reiner N.E. J. Exp. Med. 1999; 190: 1583-1594Crossref PubMed Scopus (164) Google Scholar). Colony-forming units were determined as described previously (20Crowle A.J. Ross E.J. May M.H. Infect. Immun. 1987; 55: 2945-2950Crossref PubMed Google Scholar). Bacilli were plated immediately after infection (time 0) and at 2 and 4 or 4 and 7 days after infection. Organisms were released in cold phosphate-buffered saline, 0.1% Triton X-100, serially diluted in Middlebrook 7H9, and 20 μl of three dilutions were plated in triplicate on Middlebrook 7H10 agar. CFUs were counted after 14 days of incubation and maintained for 21 days to ensure that no additional CFUs became visible. Phosphorothioate-modified oligonucleotides were prepared and incorporated into cells as described previously (6Hmama Z. Nandan D. Sly L. Knutson K.L. Herrera-Velit P. Reiner N.E. J. Exp. Med. 1999; 190: 1583-1594Crossref PubMed Scopus (164) Google Scholar, 29Volinia S. Hiles I. Ormondroyd E. Nizetic D. Antonacci R. Rocchi M. Waterfield M.D. Genomics. 1994; 24: 472-477Crossref PubMed Scopus (98) Google Scholar). Briefly, phosphorothioate-modified oligonucleotides to the α-isoform of the p110 subunit were synthesized and high pressure liquid chromatography-purified by Life Technologies, Inc. Oligonucleotides were phosphorothioate-modified to prevent intracellular degradation and were purified to remove incomplete synthesis products. 21-mers were produced to the human α isoform of the p110 subunit of PI 3-K, including the presumed translation initiation site in both sense and antisense directions with the following sequences: sense (5′-ATG CCT CCA AGA CCA TCA TCA-3′) and antisense (5′-TGA TGA TGG TCT TGG AGG CAT-3′). THP-1 cells (5 × 106) were resuspended in 500 μl of RPMI containing 2.5% LipofectAMINE (Life Technologies) and 5 μm phosphorothioate-modified oligonucleotides and incubated on a rotary shaker for 4 h at 37 °C prior to adherence and differentiation. NO3− Assays—Nitrite secreted by cells was measured using the Griess reagent according to the manufacturer's protocol (Sigma). Superoxide assays were performed in triplicate as described previously (30DeLeo F.R. Jutila M.A. Quinn M.T. J. Immunol. Methods. 1996; 198: 35-49Crossref PubMed Scopus (24) Google Scholar) by measuring the superoxide dismutase inhibitable reduction of ferricytochrome c. To separate membrane and cytosol fractions, 107 cells were resuspended in 1 ml of cold 5 mm HEPES, pH 7.4, and placed on ice for 20 min. Cell suspensions were homogenized on ice by 20 passages through a 22-gauge needle. Nuclei and intact cells were removed by centrifugation at 800 rpm for 2 min in a Microfuge. Sodium chloride was added to the supernatant to a final concentration of 0.15 m. Lysates were centrifuged at 15,000 rpm for 30 min. The resulting supernatant contained cytosolic proteins. The pellet was solubilized in 5 mm HEPES, pH 7.4, 1% Triton X-100, 0.15 mNaCl, 10% glycerol and incubated with rotation at 4 °C for 1 h. The resulting suspension was filter-sterilized through a 0.22-μm filter and contained membrane and membrane-associated proteins. 5′-nucleotidase and lactate dehydrogenase (LDH) assays were done to monitor for membrane contamination of cytosol and cytosolic contamination of membrane fractions, respectively. 5′-Nucleotidase activity was measured by the cleavage of phosphate from 5′-AMP (1.25 mm) in reaction buffer containing 10 mm HEPES, pH 7.4, 0.15m NaCl, 2 mm KCl, 2 mmMgCl2 for 15 min at room temperature. Liberated phosphate was measured by adding malachite green in 0.01% Tween 20 and allowing color to develop for 15 min at room temperature. The amount of phosphate released was determined from a standard curve. LDH assays were performed using an LDH assay kit from Sigma. SDS-polyacrylamide gel electrophoresis was performed by the method of Laemmli et al. (31Laemmli U.K. FEMS Microbiol. Rev. 1970; 227: 680-685Google Scholar). Membranes were developed by ECL as described previously (32Nandan D. Reiner N.E. Infect. Immun. 1995; 63: 4495-4500Crossref PubMed Google Scholar). Apoptosis was evaluated using the TUNEL assay (Calbiochem) including controls provided with the kit and incubation with actinomycin D as a positive control. Data in graphs are expressed as means ± S.E. Statistical analyses for superoxide and nitrite assays were done by an unpaired Student's ttest. Comparisons for CFUs were done by analysis of variance for each time point. Differences were considered significant at a level ofp < 0.05. Incubation of M. tuberculosis-infected THP-1 cells with the bioactive metabolite of vitamin D3 resulted in the induction of antimycobacterial activity. In contrast, no effect was observed using either the D3 precursor, 1-hydroxyvitamin D3 (1-OH D3) or an inactive analog, 25-hydroxyvitamin D3 (25-OH D3) (Fig.1 A). Whereas D3had no effect on the growth of M. tuberculosis in broth culture (data not shown), the number of CFUs recovered from THP-1 cells 4 and 7 days after treatment with D3 was 36 ± 3 and 20 ± 4%, respectively, compared with untreated, control cells (Fig. 1 A). Vitamin D3 has previously been shown to initiate a signaling pathway in human mononuclear phagocytes involving activation of PI 3-K (6Hmama Z. Nandan D. Sly L. Knutson K.L. Herrera-Velit P. Reiner N.E. J. Exp. Med. 1999; 190: 1583-1594Crossref PubMed Scopus (164) Google Scholar). Similarly, D3 treatment of M. tuberculosis-infected THP-1 cells activated PI 3-K (Fig.1 B, lanes 5, 7, and9 versus lane 4). To examine the role of PI 3-K in D3-induced antimycobacterial activity, the growth of M. tuberculosis was evaluated in D3-treated cells in the presence and absence of the PI 3-K inhibitors, LY and Wm, as well as in cells treated with antisense phosphorothioate-modified oligonucleotides to the p110 subunit of PI 3-Kα. Treatment with either LY or Wm inhibited PI 3-K activity in M. tuberculosis-infected, D3-treated THP-1 cells (Fig.2 A, lanes 4 and 6 versus lane 2). Treatment with either LY or Wm also significantly reduced the antimycobacterial effect of D3 at both day 4 and day 7 in THP-1 cells and day 2 and day 4 in human peripheral blood MDM (Fig. 2, B and C). The effect of LY or Wm was independent of effects on viability of the THP-1 cells. LDH activity was measured in culture supernatants of M. tuberculosis-infected, D3-treated cells 24 h after D3 treatment. Cells were treated with LY, Wm, or diluent. LDH activity at 25 °C was 124.4 units in diluent-treated or LY-treated culture supernatants and 125.9 units in Wm-treated culture supernatants. Analysis of apoptosis in similarly treated cells by TUNEL assay revealed 19.1% TUNEL-positive cells in control samples, 18.5% in LY-treated cells, and 17.2% in Wm-treated cells. Treatment with antisense, but not sense, oligonucleotides to the p110α subunit of PI 3-K eliminated the expression of this protein subunit in M. tuberculosis-infected, D3-treated THP-1 cells (Fig.3 A, lane A versus lane S). Further, treatment of M. tuberculosis-infected THP-1 cells with p110 antisense oligonucleotides also significantly attenuated the antimycobacterial action of D3. At day 4, M. tuberculosis growth was restored from 37 ± 3 to 78 ± 18% of control cells, and at day 7 growth was restored from 21 ± 2 to 77 ± 9% of control cells (Fig. 3 B). Treatment with antisense oligonucleotides to the p110 subunit of PI 3-Kα did not alter either the infection rate or M. tuberculosis growth in non-D3-treated THP-1 cells (Fig. 3 B). Furthermore, the reconstitution of CFUs by antisense to the p110α isoform was as effective as was the use of either nonspecific inhibitor, LY or Wm. Taken together, these findings suggest that the antimycobacterial action of D3 is regulated by class I PI 3-K, p85/p110α. The potential role of NO in mediating the effects of D3 inM. tuberculosis-infected cells was examined using the inducible nitric oxide synthase inhibitor l-NMMA and by direct measurement of nitrite production. Treatment ofM. tuberculosis-infected THP-1 cells withl-NMMA did not reduce the antimycobacterial action of D3 (Fig. 4 A), and neither THP-1 cells nor human MDM produced nitrite in response to D3 (data not shown). In contrast, D3-induced antimycobacterial activity in M. tuberculosis-infected, murine RAW 274.1 cells was significantly reduced by pretreatment with l-NMMA. Consistent with the implication that NO contributed to D3-induced antimycobacterial activity in murine cells, D3 treatment of infected RAW cells resulted in significant secretion of nitrite (Fig. 4 B), and this was markedly reduced in the presence of l-NMMA (Fig. 4 B). Also, in contrast to human macrophages, PI 3-K inhibitors did not affect the induction of antimycobacterial activity in infected RAW cells (data not shown). These findings demonstrate that the antimycobacterial action of D3 is NO-independent in human macrophages, whereas the converse is true in murine cells. Moreover, in the latter, this process is independent of any effects of PI 3-K. To examine further mechanisms accounting for the antimycobacterial action of D3, superoxide anion production by THP-1 cells and MDM was measured. M. tuberculosis-infected macrophages secreted large amounts of O⨪2 when treated with D3, and this effect was inhibited by LY and Wm (Fig.5, A and C) as well as by antisense phosphorothioate-modified oligonucleotides to the p110α subunit of PI 3-K (Fig. 5 B). Superoxide production occurred within the first 1 h following treatment with D3. There was no detectable superoxide produced by macrophages during the remainder of the experiment. M. tuberculosis infection alone induced the production of 20 ± 3 nmol of O⨪2/106 THP-1 cells/h, and similar values were observed for treatment with D3 alone. However, infection with M. tuberculosis followed by treatment with D3 was observed to result in the production of significantly more O⨪2 (88 nmol of O⨪2/106 macrophages/h). The amount of O⨪2 secreted by infected THP-1 cells in response to D3 was reduced to 41 ± 14 and 33 ± 13 nmol of O⨪2/106 macrophages/h by pretreatment with LY or Wm, respectively (Fig. 5 A). Antisense oligonucleotides to PI 3-K also reduced O⨪2 secretion by M. tuberculosis-infected, D3-treated THP-1 cells from 94 ± 3 to 17 ± 2 nmol/106 macrophages/h, while sense oligonucleotides had no effect (Fig. 5 B). Similar results were obtained using MDM (Fig. 5 C).M. tuberculosis infection alone induced the production of 28 ± 1 nmol of O⨪2/106macrophages/h, and D3 alone induced production of 16 ± 2 nmol of O⨪2/106 macrophages/h. However, infection followed by D3 treatment resulted in the production of significantly higher amounts of O⨪2(78 nmol of O⨪2/106 macrophages/h) by MDM. This amount was reduced to 36 ± 2 and 36 ± 3 nmol of O⨪2/106 macrophages/h by pretreatment with either LY or Wm, respectively. Taken together, these results suggest an interaction between M. tuberculosis infection and D3 in triggering the phagocyte oxidative burst in human MDM as well as in THP-1 cells and that this process is regulated by PI 3-K. Superoxide production was measured in cells treated with either live M. tuberculosis, latex beads, or killed M. tuberculosis alone or in combination with D3. As individual stimuli, live M. tuberculosis, latex beads, heat-killed M. tuberculosis, formaldehyde-fixed M. tuberculosis, UV-irradiatedM. tuberculosis, and D3 elicited comparable and only minimal amounts of O⨪2 production (less than 30 nmol of O⨪2/106 macrophages/h) by THP-1 cells (Fig. 6). However, infection with live M. tuberculosis primed cells for a marked increase in O⨪2 production in response to D3, and this was not observed with either killedM. tuberculosis or latex beads. These findings indicate that phagocytosis per se is not sufficient to prime cells and that viable M. tuberculosis actively contribute to the observed potentiation of the oxidative burst in response to D3. To assess the role of the phagocyte oxidative burst in the antimycobacterial action of D3, 4-hydroxy-TEMPO, PEG-SOD, and PEG-cat were used to examine whether killing of M. tuberculosiswas related to the production of reactive oxygen intermediates. Whereas none of these compounds alone affected the growth of M. tuberculosis in untreated cells, each of them significantly attenuated the ability of D3 to induce resistance toM. tuberculosis in THP-1 cells and MDM (Fig. 7, A and B). Heat-inactivated PEG-SOD or PEG-cat, however, had no effect on D3-induced anti-mycobacterial activity (data not shown). These results indicate that removal of reactive oxygen intermediates, either by scavenging (4-hydroxy-TEMPO) or enzymatically (PEG-cat or PEG-SOD) significantly nullifies the antimycobacterial action of D3. Phagosome oxidase activity requires recruitment of the cytosolic components, p47phox and p67phox, to the membrane for assembly with flavocytochrome b 558(33Clark R.A. Volpp B.D. Leidal K.G. Nauseef W.M. J. Clin. Invest" @default.
• W1983129403 created "2016-06-24" @default.
• W1983129403 creator A5039788418 @default.
• W1983129403 creator A5074839791 @default.
• W1983129403 creator A5089063045 @default.
• W1983129403 creator A5090033549 @default.
• W1983129403 date "2001-09-01" @default.
• W1983129403 modified "2023-10-18" @default.
• W1983129403 title "1α,25-Dihydroxyvitamin D3-induced Monocyte Antimycobacterial Activity Is Regulated by Phosphatidylinositol 3-Kinase and Mediated by the NADPH-dependent Phagocyte Oxidase" @default.
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