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

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 American Journal of Medicine 106:

7. } { 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

8. 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

9. 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

10. 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

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

12. 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 min min min
PI3P
ori

13. 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•

14. Structure of 1,25-dihydroxyvitamin D3OH 1
CH2 OH HO 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 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

18. 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
p ras binding binding
basic/
4 conserved regions of homology (1-4)
leu zip
a,b and d isoforms
Hu, P. et al Mol. Cell. Biol. 13:
Wymann, M.P. and L. Pirola Biochim. Biophys. Acta 1436:

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

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

23. 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

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

25. 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

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

27. 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 Blood 93:

28. 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

29. 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

30. 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.

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
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

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 J.Biol.Chem. 274:

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

43. A B C
Sly et al.
Figure 3

44. Sly et al.
Figure 2

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