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Macrophages

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

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Macrophages

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

  2. 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

  3. 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

  4. 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.

  5. IL-1R TLR-4 CD14 cell membrane p85 TIR domains } { MyD88 p110 Adaptors (Rac1, ? ceramide) PI 3-kinase IRAK Pi PI(3,4,5)- P3 TRAF-6 NIK Akt Pi Pi IKK Pi I-B I-B NF-B NF-B Inflammatory genes (chemokines, cytokines, etc) NF-B nucleus

  6. PI 3- RXR RXR ? VDR PI 3-kinase mediates D3 hormone-induced CD14 expression CD14 D3 ? p110 p85 Sp1 Sp1 P ? 3’ 5’ VDRE GC Box CD14 ZH, Feb, 2000

  7. Reaction catalyzed byphosphatidylinositol 3-kinase lipid moieties P P 6 5 P P PI 3-kinase 1 4 P P 2 3 P PtdIns(4,5)P2 also PtdIns or PtdIns(4)P

  8. Classes of PI 3-kinase enzymes class IA PtdIns(3)P PtdIns(3,4)P2 PtdIns(3,4,5)P3 Class I PtdIns PtdIns(4)P PtdIns(4,5)P2 p85 p110 class IB p101 p110g PtdIns(3)P PtdIns(3,4)P2 Class II PtdIns PtdIns(4)P Cpk Vps34p PtdIns(3)P Class III PtdIns

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

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

  11. In vitro experimental model b) superoxide secretion + PMA TB infection + vitamin D3 THP-1 cells c) separate membrane and cytosol a) enumerate bacteria plate on 7H10 - amount of p47phox and p67 phox O2• O2• O2•

  12. Structure of 1,25-dihydroxyvitamin D3 OH 1 CH2 OH HO 25

  13. Relative cfus recovered 4 and 7 daysafter treatment with vitamin D3

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

  15. Classes of PI 3-kinase enzymes class IA PtdIns(3)P PtdIns(3,4)P2 PtdIns(3,4,5)P3 Class I PtdIns PtdIns(4)P PtdIns(4,5)P2 p85 p110 class IB p101 p110g PtdIns(3)P PtdIns(3,4)P2 Class II PtdIns PtdIns(4)P Cpk Vps34p PtdIns(3)P Class III PtdIns

  16. Subunits of PI 3-kinase, class IA p85 N- -C SH3 bcr homology SH2 p110 binding SH2 and activation a and b isoforms p110 N- -C 1 2 3 4 p85 ras binding binding basic/ 4 conserved regions of homology (1-4) leu zip a,b and d 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.

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

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

  19. ROI scavenger or degradative enzymes O • 4-hydroxy-tempo CH3 N CH3 CH3 CH3 O 2 O2• PEG-SOD H2O2 H +2 H+ + O2 PEG-catalase - scavenge and reduce oxidative burst products 2 H2O2 2 H2O + O2

  20. The oxidative burst mediatesanti-mycobacterial activity in THP-1 cells

  21. PI 3-kinase inhibitors reduce O2• secretion byM. tuberculosis-infected, D3-treated THP-1 cells

  22. Antisense mRNA to PI 3-kinase reduces O2• secretion by TB-infected, D3-treated THP-1 cells

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

  24. p47phox and p67phoxin membrane and cytosol membrane TB HK only C PMATB2TB4+D3HK2 HK4 +D3 D3 a p47 a p67 cytosol TB HK only C PMATB2TB4+D3HK2 HK4 +D3 D3 a p47 a p67

  25. Effect of PI 3-K inhibitors on p47phox and p67phox membrane translocation + LY294002 LY TB TB PMA LY LY+TB+LY PMATB4D3+D3 +LY +TB +D3 +D3 +D3 a p47 a p67 + wortmannin W TB TB PMA W W+TB+W PMATB4D3+D3 +W +TB +D3 +D3 +D3 a p47 a p67

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

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

  28. 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

  29. 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

  30. Vitamin D: a steroid hormone with pleiotropic actions D3 hormone regulates calcium homeostasis in intestine, bone and kidney D3 acts on the immune system: Inhibition of T-cell proliferation and IL-2 expression D3 regulates the myeloid cell differentiation: Expression of Cdk inhibitor p21: growth arrest Expression of CD14 and CD11b ZH, Feb, 2000

  31. Nitrite is produced by RAW cellsbut not by THP-1 cells

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

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

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

  35. D3-triggered O2• secretion by THP-1 cells is primed by infection with live M. tuberculosis

  36. The oxidative burst mediatesanti-mycobacterial activity in PBMCs

  37. PI 3-kinase inhibitors reduce O2• secretion byM. tuberculosis-infected, D3-treated PBMCs

  38. A B C Sly et al. Figure 3

  39. Sly et al. Figure 2

  40. D3-triggered O2• secretion by THP-1 cells is primed by treatment with LAM

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