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1α,25-Dihydroxyvitamin D3-induced Monocyte Antimycobacterial Activity Is Regulated by Phosphatidylinositol 3-Kinase and Mediated by the NADPH-dependent Phagocyte Oxidase*

Open AccessPublished:September 21, 2001DOI:https://doi.org/10.1074/jbc.M102876200
      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.
      D3
      1α,25-dihydroxyvitamin D3
      MDM
      monocyte-derived macrophage(s)
      PI 3-K
      phosphatidylinositol 3-kinase
      Wm
      wortmannin
      LY
      LY294002
      O⨪2
      superoxide anion
      PEG
      polyethylene glycol
      SOD
      superoxide dismutase
      cat
      catalase
      VDR
      vitamin D receptor
      PI
      l-α-phosphatidylinositol
      l-NMMA
      l-N-monomethylarginine
      LDH
      lactate dehydrogenase
      NO
      nitric oxide
      LAM
      lipoarabinomannan
      WCL
      whole cell lysates
      ECF
      concentrated extracellular filtrate
      HBSS
      Hanks' balanced saline solution
      CFU
      colony-forming units
      PMA
      phorbol 12-myristate 13-acetate
      TUNEL
      terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling
      4-hydroxy-TEMPO
      4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl
      TLR-2
      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 (
      • Kaufmann S.H.
      • van Embden J.D.
      ) 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 (
      • Day M.
      ). 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 (
      • Armstrong J.A.
      • Hart P.D.
      ). 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 (
      • Hingley-Wilson S.M.
      • Sly L.M.
      • Reiner N.E.
      • McMaster W.R.
      ). 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 (
      • Malloy P.J.
      • Feldman D.
      ). 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 (
      • Hmama Z.
      • Nandan D.
      • Sly L.
      • Knutson K.L.
      • Herrera-Velit P.
      • Reiner N.E.
      ,
      • Gniadecki R.
      ,
      • Janis A.E.
      • Kaufmann S.H.E.
      • Schwartz R.H.
      • Pardoll D.M.
      ,
      • Phillips W.
      • Hamilton J.A.
      ,
      • Duncan R.
      • McConkey E.H.
      ).
      Vitamin D3 is known to possess a variety of immunomodulatory properties including effects on both myeloid and lymphoid cells (
      • Manolagas S.C.
      • Hustmyer F.G.
      • Yu X.P.
      ). Among these are its ability to induce the differentiation of immature myeloid cells into more mature monocytes and macrophages (
      • Abe E.
      • Miyaura C.
      • Sakagami H.
      • Takeda M.
      • Konno K.
      • Yamazaki T.
      • Yoshiki S.
      • Suda T.
      ,
      • Tanaka H.
      • Abe E.
      • Miyaura C.
      • Kuribayashi T.
      • Konno K.
      • Nishii Y.
      • Suda T.
      ,
      • Kreutz M.
      • Andreesen R.
      ,
      • Schwende H.
      • Fitzke E.
      • Ambs P.
      • Dieter P.
      ,
      • Zhang D.-E.
      • Hetherington C.J.
      • Gonzalez D.A.
      • Chen H.-M.
      • Tenen D.G.
      ). 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 (
      • Davies P.D.
      ,
      • Roy S.
      • Frodsham A.
      • Saha B.
      • Hazra S.K.
      • Mascie-Taylor C.G.
      • Hill A.V.
      ,
      • Wilkinson R.J.
      • Llewelyn M.
      • Toossi Z.
      • Patel P.
      • Pasvol G.
      • Lalvani A.
      • Wright D.
      • Latif M.
      • Davidson R.N.
      ). 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 (
      • Davies P.D.
      ,
      • Crowle A.J.
      • Ross E.J.
      • May M.H.
      ). In addition, D3 has been shown to activate mononuclear phagocyte antimycobacterial activity (
      • Crowle A.J.
      • Ross E.J.
      • May M.H.
      ,
      • Rook G.A.
      • Steele J.
      • Fraher L.
      • Barker S.
      • Karmali R.
      • O'Riordan J.
      • Stanford J.
      ). 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 (
      • Fry M.J.
      ,
      • Toker A.
      • Cantley L.G.
      ,
      • Fukui Y.
      • Ihara S.
      • Nagata S.
      ). 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 (
      • Hmama Z.
      • Nandan D.
      • Sly L.
      • Knutson K.L.
      • Herrera-Velit P.
      • Reiner N.E.
      ). 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.

      DISCUSSION

      The steroid hormone vitamin D3 has been linked to host resistance to tuberculosis since the mid-19th century (
      • Davies P.D.
      ), and the active metabolite, 1α,25-dihydroxyvitamin D3 has been shown to induce antimycobacterial activity in macrophages (
      • Crowle A.J.
      • Ross E.J.
      • May M.H.
      ,
      • Rook G.A.
      • Steele J.
      • Fraher L.
      • Barker S.
      • Karmali R.
      • O'Riordan J.
      • Stanford J.
      ). In addition, it has been found that the antimycobacterial efficacy of cholecalciferol metabolites correlated with their binding affinities to the VDR (
      • Rook G.A.
      • Steele J.
      • Fraher L.
      • Barker S.
      • Karmali R.
      • O'Riordan J.
      • Stanford J.
      ). Other evidence for the role of D3 in host resistance to tuberculosis has emerged recently. For example, D3 deficiency has been linked to increased susceptibility to tuberculosis, and evidence suggests that vitamin D receptor polymorphisms confer differential resistance to mycobacterial diseases including leprosy and tuberculosis (
      • Roy S.
      • Frodsham A.
      • Saha B.
      • Hazra S.K.
      • Mascie-Taylor C.G.
      • Hill A.V.
      ,
      • Wilkinson R.J.
      • Llewelyn M.
      • Toossi Z.
      • Patel P.
      • Pasvol G.
      • Lalvani A.
      • Wright D.
      • Latif M.
      • Davidson R.N.
      ,
      • Bellamy R.
      • Ruwende C.
      • Corrah T.
      • McAdam K.P.
      • Thursz M.
      • Whittle H.C.
      • Hill A.V.
      ). Whereas it is clear from these observations that D3 enhances host resistance to tuberculosis and other mycobacterial diseases, the underlying mechanism accounting for D3-mediated antimycobacterial activity in human cells and its regulation have been obscure.
      The findings of the present study indicate that monocyte antimycobacterial activity induced by 1α,25-dihydroxyvitamin D3 is mediated by the NADPH-dependent phagocyte oxidase and that this process is regulated by phosphatidylinositol 3-kinase. The conclusion that D3-induced antimycobacterial activity is due to activation of the phagocyte oxidative burst is based on three lines of evidence: (i) D3 treatment of M. tuberculosis-infected cells leads to a burst of O⨪2 production (Fig. 5, A and C); (ii) the reactive oxygen intermediate scavenger, 4-hydroxy-TEMPO, and enzymes that degrade reactive oxygen intermediates (PEG-cat and PEG-SOD) dramatically reduce the D3-mediated antimycobacterial effect in both THP-1 cells and MDM (Fig. 7,A and B); and (iii) the phagosome oxidase components, p47phox and p67phox, translocate to the membrane fraction in M. tuberculosis-infected cells upon treatment with D3, as would be expected during assembly of a functional oxidase (Fig. 8 A).
      Increased production of superoxide by infected cells in response to D3 was rapid and transient, whereas we were only able to detect a decrease in M. tuberculosis CFUs at either 2, 4, or 7 days following D3 treatment. This apparently delayed effect could reflect the production of low, undetectable levels of superoxide for longer periods of time. However, we believe a more likely possibility is that D3-induced antimycobacterial activity mediated by the NADPH oxidase acts synergistically by rendering M. tuberculosis more susceptible to other host factors that contribute to decreased CFUs observed over a longer time course. This model is consistent with the recent finding that NADPH oxidase-dependent killing of Salmonella in vivo was confined to the first few hours after phagocytosis. Reactive oxygen intermediates were essential to control infection but contributed at the very early stages of a multiphasic host response (
      • Vazquez-Torres A.
      • Jones-Carson J.
      • Mastroeni P.
      • Ischiropolous H.
      • Fang F.C.
      ).
      The conclusion that the oxidative burst is the principal mechanism by which D3 mediates antimycobacterial activity in human mononuclear phagocytes is consistent with genetic evidence linking the phagocyte oxidase to host resistance to mycobacteria. Thus, patients with chronic granulomatous disease, a heterogeneous group of genetic disorders involving functional inactivation of the phagocyte oxidase (
      • Babior B.M.
      ), show increased susceptibility to recurrent and life-threatening bacterial and fungal infections including infections due to mycobacteria (
      • Ohga S.
      • Ikeuchi K.
      • Kadoya R.
      • Okada K.
      • Miyazaki C.
      • Suita S.
      • Ueda K.
      ,
      • Allen D.M.
      • Chng H.H.
      ,
      • Chusid M.J.
      • Parrillo J.E.
      • Fauci A.S.
      ,
      • Gonzalez B.
      • Moreno S.
      • Burdach R.
      • Valenzuela M.T.
      • Henriquez A.
      • Ramos M.I.
      • Sorensen R.U.
      ).
      Vitamin D3 has been reported to stimulate directly human peripheral blood monocytes to generate an oxidative burst based upon the finding that it induced peripheral blood monocytes to secrete hydrogen peroxide (
      • Cohen M.S.
      • Mesler D.E.
      • Snipes R.G.
      • Gray T.K.
      ). In the present study, either D3 orM. tuberculosis acting alone triggered only modest oxidative burst activity. In respect to M. tuberculosis, this finding is consistent with previous reports showing only low levels of phagocyte oxidase activation in response to infection with mycobacteria (
      • Brett S.J.
      • Butler R.
      ,
      • Gordon A.H.
      • Hart P.D.
      ). In contrast to the results obtained with either agent acting alone, in the presence ofM. tuberculosis infection the oxidative burst in response to D3 was markedly augmented. These findings indicate that M. tuberculosis infection acted to prime cells to respond to D3, and this priming was specific for viable M. tuberculosis, since it was not observed in cells that had ingested either dead bacilli or latex beads.
      The precise mechanism for this priming effect by viable bacilli is unclear but probably involves the recruitment of one or more signaling pathways that act in conjunction with PI 3-K to prompt assembly of a functional oxidase complex. The finding that M. tuberculosis LAM was able to condition cells for an enhanced oxidative response to D3 (Fig. 9) provides a potential mechanism to account for the priming observed in response to infection with live M. tuberculosis. LAM is known to have pleiotropic effects including effects on multiple signaling pathways in macrophages (
      • Reiner N.E.
      ,
      • Knutson K.L.
      • Hmama Z.
      • Herrera-Velit P.
      • Rochford R.
      • Reiner N.E.
      ,
      • Maiti D.
      • Bhattacharyya A.
      • Basu J.
      ). The requirement for infection with live bacilli may reflect a need for de novo synthesis of LAM within cells, for the intracellular release and trafficking of LAM, or both. At the same time, although it was possible to show that LAM had the property of oxidase priming, these results do not establish that it is the actual in vivo priming factor or that it is the only priming factor elaborated by viable M. tuberculosis within cells.
      In activated murine macrophages, antimycobacterial activity has been shown to be due, at least in part, to the inducible synthesis of nitric oxide, and studies in inducible nitric oxide synthase knock out mice have suggested that this is an effector arm of the host response to M. tuberculosis (
      • Chan J.
      • Xing Y.
      • Magliozzo R.S.
      • Bloom B.R.
      ,
      • MacMicking J.D.
      • North R.J.
      • LaCourse R.
      • Mudgett J.S.
      • Shah S.K.
      • Nathan C.F.
      ). In the experiments reported above, detectable levels of nitrite were not produced by D3-treated, M. tuberculosis-infected THP-1 cells or MDM, and the NO inhibitor l-NMMA did not affect the induction of antimycobacterial activity by D3 in THP-1 cells (Fig. 4 A). This was in direct contrast to parallel observations made using the murine macrophage-like cell line RAW 274.1, which when infected with M. tuberculosis produced significant amounts of nitrite in response to D3 (Fig. 4 B) and in which the antimycobacterial effect of D3 was markedly attenuated by prior treatment with l-NMMA (Fig. 4 A). These results suggest that at least two independent mechanisms operate to induce antimycobacterial activity in D3-treated macrophages. In murine cells, inducible nitric oxide synthase operates independently of PI 3-K, whereas in human macrophages the NADPH-dependent phagocyte oxidative burst appears to be predominant and regulated by PI 3-K. Moreover, these findings are consistent with a recent report that examined the mechanisms involved in the antimycobacterial activity of toll-like receptor 2-activated macrophages (
      • Thoma-Uszynski S.
      • Stenger S.
      • Takeuchi O.
      • Ochoa M.T.
      • Engele M.
      • Sieling P.A.
      • Barnes P.F.
      • Rollinghoff M.
      • Bolcskei P.L.
      • Wagner M.
      • Akira S.
      • Norgard M.V.
      • Belisle J.T.
      • Godowski P.J.
      • Bloom B.R.
      • Modlin R.L.
      ). In these studies, TLR-2-activated murine macrophages demonstrated NO-dependent antimycobacterial activity, whereas TLR-2-activated antimycobacterial activity in human macrophages was NO-independent. No effector mechanism for TLR-2-activated antimycobacterial activity was defined in human cells (
      • Thoma-Uszynski S.
      • Stenger S.
      • Takeuchi O.
      • Ochoa M.T.
      • Engele M.
      • Sieling P.A.
      • Barnes P.F.
      • Rollinghoff M.
      • Bolcskei P.L.
      • Wagner M.
      • Akira S.
      • Norgard M.V.
      • Belisle J.T.
      • Godowski P.J.
      • Bloom B.R.
      • Modlin R.L.
      ). Our results presented here suggest that one possible effector mechanism is oxygen-dependent killing via activation of the phagocyte oxidase. This model is supported further by the findings that, when ligand-activated, the D3 receptor (
      • Hmama Z.
      • Nandan D.
      • Sly L.
      • Knutson K.L.
      • Herrera-Velit P.
      • Reiner N.E.
      ) and TLR-2 (
      • Arbibe L.
      • Mira J.-P.
      • Teusch N.
      • Kline L.
      • Mausumee G.
      • Mackman N.
      • Godowski P.J.
      • Ulevitch R.J.
      • Knaus U.G.
      ) have in common the property of binding to PI 3-K. As we have shown, in M. tuberculosis-infected, D3-treated cells, this results in activation of the phagocyte oxidase. It seems highly likely that this would occur as well in TLR-2-activated cells infected with mycobacteria.
      Recently, there has been considerable interest in whether macrophage apoptosis is involved in the host response to mycobacteria and, if so, whether apoptosis could affect mycobacterial viability. Although results from various studies have been inconsistent (
      • Santucci M.B.
      • Amicosante M.
      • Cicconi R.
      • Montesano C.
      • Casarini M.
      • Giosuè S.
      • Bisetti A.
      • Colizzi V.
      • Fraziano M.
      ,
      • Kremer L.
      • Estaquier J.
      • Brandt E.
      • Ameisen J.C.
      • Locht C.
      ,
      • Placido R.
      • Mancino G.
      • Amendola A.
      • Mariani F.
      • Vendetti S.
      • Piacentini M.
      • Sanduzzi A.
      • Bocchino M.L.
      • Zembala M.
      • Colizzi V.
      ,
      • Durrbaum-Landmann I.
      • Gercken J.
      • Flad H.D.
      • Ernst M.
      ,
      • Keane J.
      • Remold H.G.
      • Kornfeld H.
      ,
      • Klingler K.
      • Tchou-Wong K.M.
      • Brandli O.
      • Aston C.
      • Kim R.
      • Chi C.
      • Rom W.N.
      ), we nevertheless considered the possibility that D3-induced antimycobacterial activity could be due to the induction of apoptosis. Rates of apoptosis in THP-1 cells incubated with D3 alone for either 1 or 4 days were at background levels (1–2%). In cells infected with M. tuberculosis alone, apoptosis was detected at frequencies of 13 and 15%, respectively, at 1 and 4 days postinfection, and these rates were not affected by the addition of D3. Similar results were found using human MDM. Treatment with D3 alone for 1 or 4 days resulted in levels of apoptosis similar to untreated cells (4 or 6%, respectively). Cells infected with M. tuberculosis alone showed apoptosis in 17 and 20% of cells at 1 and 4 days postinfection, and these rates were not affected by the addition of vitamin D3. These findings indicate that apoptosis is unlikely to be involved in the induction of antimycobacterial activity by D3.
      The second principal conclusion drawn from this study is that PI 3-K regulates the antimycobacterial action of D3. Several lines of evidence support this argument including the following: (i) PI 3-K is activated by D3 in THP-1 cells (Fig. 1 B; see Ref.
      • Hmama Z.
      • Nandan D.
      • Sly L.
      • Knutson K.L.
      • Herrera-Velit P.
      • Reiner N.E.
      ); (ii) D3-induced antimycobacterial activity is abrogated by PI 3-K inhibitors LY and Wm in both THP-1 cells and MDM independent of any cytotoxic effect on the cells (Fig. 2, Band C); (iii) D3-induced antimycobacterial activity is abrogated by antisense mRNA to class I PI 3-K (Fig.3 B); (iv) O⨪2 production by M. tuberculosis-infected THP-1 cells and MDM in response to D3 is inhibited by PI 3-K inhibitors LY and Wm (Fig. 5,A and C); (v) D3-induced O⨪2production by M. tuberculosis-infected THP-1 cells is inhibited by antisense mRNA to class I PI 3-K (Fig.5 B); and (vi) pretreatment of THP-1 cells with the PI 3-K inhibitors LY or Wm prevents p47phox and p67phoxtranslocation to the membrane fraction upon D3 treatment (Fig. 8, C and D).
      The finding that the antimycobacterial action of D3 is regulated by PI 3-K represents another important example of nongenomic signaling by this and other steroid hormones. The long standing paradigm for D3 action has been recognized to involve genomic signaling in which hormone binds to the VDR. The D3·VDR complex then translocates to the nucleus, where the ligand-activated transcription factor VDR directly regulates transcription by binding to vitamin D response elements in D3-activated genes (
      • Malloy P.J.
      • Feldman D.
      ). However, numerous reports have shown that D3 also acts via nongenomic signaling, where cellular responses are brought about independent of de novotranscription from a classical vitamin D response element (
      • Bhatia M.
      • Kirkland J.B.
      • Meckling-Gill K.A.
      ,
      • Berry D.M.
      • Antochi R.
      • Bhatia M.
      • Meckling-Gill K.A.
      ,
      • Darwish H.M.
      • DeLuca H.F.
      ). Moreover, it was recently shown that D3 treatment of human monocytes results in the rapid activation of PI 3-K leading to monocyte differentiation (
      • Hmama Z.
      • Nandan D.
      • Sly L.
      • Knutson K.L.
      • Herrera-Velit P.
      • Reiner N.E.
      ). In the latter report, a novel nongenomic mechanism of action of D3 was shown to be regulated by a VDR·PI 3-K signaling complex. Similar findings were also recently reported for signaling by estrogen, where hormone treatment induced the formation of a signaling complex including the estrogen receptor-α and the p85α regulatory subunit of PI 3-K (
      • Simoncini T.
      • Hafezi-Moghadam A.
      • Brazil D.P.
      • Ley K.
      • Chin W.W.
      • Liao J.K.
      ).
      PI 3-K has been shown to regulate a wide range of physiological processes including movement of organelle membranes, cytoskeletal rearrangement, cell proliferation, apoptosis, etc. (
      • Toker A.
      • Cantley L.G.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      ). Importantly, PI 3-K has also been shown to regulate the oxidative burst. For example, PI 3-K has been shown to be required for triggering the NADPH oxidase in response to particulate stimuli through activating Fcα and Fcγ receptors in neutrophils (
      • Lang M.L.
      • Kerr M.A.
      ). Similarly, based upon effects of the PI 3-K inhibitor wortmannin, a role for this enzyme in regulating the production of superoxide by human neutrophils in response to soluble stimuli such as fMLP (
      • Santoro P.
      • Cacciapuoti C.
      • Palumbo A.
      • Graziano D.
      • Annunziata S.
      • Capasso L.
      • Formisano S.
      • Ciccimarra F.
      ) and PMA (
      • Yang M.
      • Wu W.
      • Mirocha C.J.
      ) has also been suggested. In the present study, PI 3-K was found to be required to trigger the oxidative burst in response to D3 in M. tuberculosis-infected cells. However, it appears that activation of PI 3-kinase alone is insufficient to bring about full oxidase activation, since treatment of THP-1 cells with D3 alone activates PI 3-K (Fig.1 B, lanes 6, 8, and10 versus lanes 5,7, and 9; see Ref.
      • Hmama Z.
      • Nandan D.
      • Sly L.
      • Knutson K.L.
      • Herrera-Velit P.
      • Reiner N.E.
      ), but at best this elicits only modest superoxide production (Fig. 5, AC). In contrast, prior infection of cells with live M. tuberculosis primed cells for an enhanced D3-induced oxidative burst, and this was not recapitulated by either latex beads or killed M. tuberculosis(Fig. 6). Consistent with these findings, only in cells infected with live bacilli did D3 induce translocation of the oxidase components p47phox and p67phox to the membrane fraction for oxidase assembly (Fig. 8). These findings suggest that liveM. tuberculosis or some factor unique to the phagosome containing viable M. tuberculosispotentiates phagocytes to undergo a vigorous oxidative burst in response to D3.
      In summary, this report identifies a key mechanism of D3-induced host defense against tuberculosis and a key regulatory pathway required to bring about this effector mechanism. The findings demonstrate that the antimycobacterial activity of D3 in human macrophages is due to activation of the NADPH oxidase and the production of reactive oxygen intermediates. Furthermore, they show that PI 3-K is necessary but not sufficient for assembly of a functional oxidase in response to D3 inM. tuberculosis-infected cells. This represents another novel example of nongenomic signaling by vitamin D3. Ongoing studies concerned with the basis forM. tuberculosis priming and the additional regulatory pathways involved should provide additional insight into host resistance to mycobacterial disease and the associated inflammatory consequences.

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