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 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

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

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.

} { IL-1R TLR-4 MyD88 IRAK NIK IKK nucleus CD14 cell membrane p85 TIR domains } { p110 MyD88 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

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

Reaction catalyzed by phosphatidylinositol 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

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

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

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

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

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

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

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

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

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.

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

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

ROI scavenger or degradative enzymes • 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

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

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

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

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

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

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

Vitamin D3-mediated anti-mycobacterial activity is: 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.

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

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

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

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

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

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

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

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

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

The oxidative burst mediates anti-mycobacterial activity in PBMCs

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

A B C Sly et al. Figure 3

Sly et al. Figure 2

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