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Macrophages Neutrophils Antimicrobial peptides Natural killer cells Complement System Major Elements of Innate Immunity.

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Presentation on theme: "Macrophages Neutrophils Antimicrobial peptides Natural killer cells Complement System Major Elements of Innate Immunity."— Presentation transcript:

1 Macrophages Neutrophils Antimicrobial peptides Natural killer cells Complement System Major Elements of Innate Immunity

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4 Tuberculosis is a global health threat One-third of the world’s population has been exposed to tuberculosis 8-10 million new cases of pulmonary TB annually 3 million deaths annually –Snider et al., 1993 It is second to HIV as the leading cause of death among infectious diseases –WHO, 1999 Complicating factors: co-infection with HIV MDR-TB

5 Vitamin D3 and Host Defense D3 deficiency and polymorphisms in the VDR have associated with increased incidence of tuberculosis UV light induces D3 synthesis and exposure to sunlight may protect against TB D3 induces anti-tuberculous activity in macrophages ingestion of D3 associated with clinical improvement in patients with TB

6 Vitamin D3 genomic versus non-genomic signaling vitamin D3 associates with the vitamin D receptor (VDR) the complex associates with a 21 base pair vitamin D response element (VDRE) However, some genes regulated by vitamin D3 lack a VDRE, including CD14. Malloy, P.J. and D. Feldman. 1999. American Journal of Medicine 106:355-370.

7 MyD88 }{ TLR-4 IL-1R IRAK TRAF-6 NIK IKK NF-  B I-  B NF-  B Pi I-  B Pi Inflammatory genes (chemokines, cytokines, etc) Adaptors (Rac1, ? ceramide) nucleus cell membrane p85 p110 PI 3-kinase PI(3,4,5)- P 3 Pi Akt NF-  B Pi TIR domains CD14

8 PI 3- PI 3-kinase mediates D 3 hormone-induced CD14 expression ZH, Feb, 2000 CD14 p110 p85 ? 5’ 3’ VDRE GC Box CD14 D3D3 VDR ? ? Sp1 P RXR

9 Reaction catalyzed by phosphatidylinositol 3-kinase P P P P P P P lipid moieties PtdIns(4,5)P 2 1 23 4 56 PI 3-kinase also PtdIns or PtdIns(4)P

10 Classes of PI 3-kinase enzymes Class I Class I Class II Class II Class III Class III PtdInsPtdIns(4)PPtdIns(4,5)P2 PtdInsPtdIns(4)P PtdIns PtdIns(3)PPtdIns(3,4)P2PtdIns(3,4,5)P3 PtdIns(3)PPtdIns(3,4)P2 PtdIns(3)P p110 p85 class IB class IA p110  p101 Cpk Vps34p

11 Signaling through PI 3-kinase lipid products and their targets Rameh, L.E. and L.C. Cantley. 1999. J.Biol.Chem. 274:8347-8350.

12 Vitamin D3 induces PI 3-kinase activity in differentiated and TB-infected THP-1 cells ori PI3P C PMA TB TB TB/D3 D3 TB/D3 D3 TB/D3 D3 2hr 4hr 10 min 20 min 30 min

13 b) superoxide secretion In vitro experimental model O 2 THP-1 cells + PMATB infection plate on 7H10 a) enumerate bacteria + vitamin D3 c) separate membrane and cytosol - amount of p47phox and p67 phox

14 Structure of 1,25-dihydroxyvitamin D3 OH HO CH 2 1 25

15 Relative cfus recovered 4 and 7 days after treatment with vitamin D3

16 PI 3-kinase inhibitors reduce D3-mediated anti-mycobacterial activity in THP-1 cells

17 Classes of PI 3-kinase enzymes Class I Class I Class II Class II Class III Class III PtdInsPtdIns(4)PPtdIns(4,5)P2 PtdInsPtdIns(4)P PtdIns PtdIns(3)PPtdIns(3,4)P2PtdIns(3,4,5)P3 PtdIns(3)PPtdIns(3,4)P2 PtdIns(3)P p110 p85 class IB class IA p110  p101 Cpk Vps34p

18 Subunits of PI 3-kinase, class IA p85 N- p110 N- SH3 bcr homology SH2 p110 binding SH2 and activation and activation -C -C  and  isoforms  and  isoforms Hu, P. et al. 1993. Mol. Cell. Biol. 13:7677-7687. Wymann, M.P. and L. Pirola. 1998. Biochim. Biophys. Acta 1436:127-150. p85 ras binding binding p85 ras binding bindingbasic/ leu zip 4 conserved regions of homology (1-4) 1234

19 Antisense mRNA to PI 3-kinase reduces D3-mediated anti-mycobacterial activity

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21 A nitric oxide inhibitor does not reduce vitamin D3-mediated anti-mycobacterial activity

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23 ROI scavenger or degradative enzymes 4-hydroxy-tempo N O CH 3 H 2 O 2 H2O2H2O2 2 H 2 O 2 2 H 2 O PEG-catalase PEG-SOD - scavenge and reduce oxidative burst products + O 2 + 2 H + O

24 The oxidative burst mediates anti-mycobacterial activity in THP-1 cells

25 PI 3-kinase inhibitors reduce O 2 secretion by M. tuberculosis-infected, D3-treated THP-1 cells

26 Antisense mRNA to PI 3-kinase reduces O 2 secretion by TB-infected, D3-treated THP-1 cells

27 Diagrammatic representation of the NADPH oxidase gp91 p22 Rap1A p67 p47 rac2 P P P p40 ACTIVATED p67 p47 p40 OH rac2 RESTING Babior, B.M. 1999. Blood 93:1464-1476.

28 p47phox and p67phox in membrane and cytosol membrane cytosol  p47  p67  p47  p67 C PMA TB2 TB4 +D3 HK2 HK4 +D3 D3 TB HK only

29 Effect of PI 3-K inhibitors on p47phox and p67phox membrane translocation + LY294002 + wortmannin  p47  p67  p47  p67 PMA TB4 D3 +D3 + LY +TB +D3 +D3 +D3 TB PMA LY LY +TB +LY LY TB PMA TB4 D3 +D3 + W +TB +D3 +D3 +D3 TB PMA W W +TB +W W TB

30 Summary Vitamin D3-mediated anti-mycobacterial activity is: 1)mediated by the oxidative burst 2)regulated by PI 3-kinase PI 3-kinase regulates the Mtb-primed, D3-triggered oxidative burst.

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32 Acknowledgements Dr. Neil Reiner Dr. William Nauseef Dr. Martin Lopez Dr. Reiner’s research group

33 Vitamin D3-induced Anti-mycobacterial Activity Requires A Phosphatidylinositol 3- kinase-Mediated Oxidative Burst L.M. Sly, M. Lopez, W.M. Nauseef & Neil E. Reiner Division of Infectious Diseases Department of Medicine University of British Columbia

34 M. tuberculosis pathogenesis Susceptible individuals can acquire infection by inhaling fewer than 10 bacilli –Riley, 1962 Initial encounter of tubercle bacillus is usually with an alveolar macrophage –Dannenberg and Rook, 1994 Risk of development of clinically evident disease is ~10% –Comstock, 1982

35 Vitamin D: a steroid hormone with pleiotropic actions ZH, Feb, 2000 D 3 hormone regulates calcium homeostasis in intestine, bone and kidney D 3 acts on the immune system: Inhibition of T-cell proliferation and IL-2 expression D 3 regulates the myeloid cell differentiation: Expression of Cdk inhibitor p21: growth arrest Expression of CD14 and CD11b

36 Nitrite is produced by RAW cells but not by THP-1 cells

37 Pathways for phosphoinositide synthesis Rameh, L.E. and L.C. Cantley. 1999. J.Biol.Chem. 274:8347-8350.

38 Vitamin D3 restricts M. tuberculosis growth in THP-1 cells

39 PI 3-kinase inhibitors reduce D3-mediated anti-mycobacterial activity in PBMCs

40 D3-triggered O 2 secretion by THP-1 cells is primed by infection with live M. tuberculosis

41 The oxidative burst mediates anti-mycobacterial activity in PBMCs

42 PI 3-kinase inhibitors reduce O 2 secretion by M. tuberculosis-infected, D3-treated PBMCs

43 Sly et al. Figure 3 A C B

44 Sly et al. Figure 2

45 D3-triggered O 2 secretion by THP-1 cells is primed by treatment with LAM


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