Macrophages

NeutrophilsAntimicrobial

peptides

Natural killercells

ComplementSystem

Major Elements of Innate Immunity

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 HIVMDR-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.Malloy, P.J. and D. Feldman. 1999. American Journal of Medicine 106:355-370.

MyD88} {

TLR-4 IL-1R

IRAK

TRAF-6

NIK

IKK

NF-B

I-B

NF-B

Pi

I-B

Pi

Inflammatorygenes (chemokines,cytokines, etc)

Adaptors(Rac1, ? ceramide)

nucleus

cell membrane

p85

p110

PI 3-kinase

PI(3,4,5)-P3

Pi

Akt

NF-B

Pi

TIR domains

CD14

PI 3-PI 3-kinase mediates

D3 hormone-induced CD14 expression

ZH, Feb, 2000

CD14

p110p85

?

5’ 3’VDRE GC Box CD14

D3

VDR

?

?

Sp1 Sp1 P

RXR

RXR

Reaction catalyzed byphosphatidylinositol 3-kinase

P

P

P

P

P

P

P

lipid moieties

PtdIns(4,5)P2

1

2 3

4

56

PI 3-kinase

also PtdIns or PtdIns(4)P

Classes of PI 3-kinase enzymes

Class IClass I

Class IIClass II

Class IIIClass III

PtdInsPtdIns

PtdIns(4)PPtdIns(4)P

PtdIns(4,5)P2PtdIns(4,5)P2

PtdInsPtdIns

PtdIns(4)PPtdIns(4)P

PtdInsPtdIns

PtdIns(3)PPtdIns(3)P

PtdIns(3,4)P2PtdIns(3,4)P2

PtdIns(3,4,5)P3PtdIns(3,4,5)P3

PtdIns(3)PPtdIns(3)P

PtdIns(3,4)P2PtdIns(3,4)P2

PtdIns(3)PPtdIns(3)P

p110p110 p85p85

class IBclass IB

class IAclass IA

p110p110p101p101

CpkCpk

Vps34pVps34p

Signaling through PI 3-kinase lipid productsand their targets

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

ori

PI3P

C PMA TB TB TB/D3 D3 TB/D3 D3 TB/D3 D3 2hr 4hr 10 min 20 min 30 min

b) superoxide secretion

In vitro experimental model

O2• O2•O2•

THP-1 cells

+ PMA TB infection

plate on 7H10

a) enumerate bacteria

+ vitamin D3

c) separate membrane and cytosol

- amount of p47phox and p67 phox

Structure of 1,25-dihydroxyvitamin D3

OH

OHHO

CH2

1

25

Relative cfus recovered 4 and 7 daysafter treatment with vitamin D3

day 4

CF

Us

C D3 1-OH D3 25-OH D30.0×10-00

1.0×1007

2.0×1007

3.0×1007

4.0×1007

(100)

(36)

(89) (90)

A

N.S. N.S.

day 7

CF

Us

C D3 1-OH D3 25-OH D30.0×10-00

5.0×1007

1.0×1008

1.5×1008

2.0×1008

(100)

(20)

(100)

(94)

A

N.S.

N.S.

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

CF

Us

0.0×10 -00

5.0×1007

1.0×1008

1.5×1008

2.0×1008

vitamin D3 - + - + - +inhibitor - - LY LY Wrtm Wrtm

day 7

(100)

(26)

(71)

(61)

(71)

(63)C C

B

N.S.N.S.

CF

Us

0.0×10 -00

1.0×1007

2.0×1007

3.0×1007

4.0×1007

vitamin D3 - + - + - +inhibitor - - LY LY Wrtm Wrtm

day 4

(100)

(36)

(93)

(76)

(93)

(81)

A

B B

N.S.N.S.

Classes of PI 3-kinase enzymes

Class IClass I

Class IIClass II

Class IIIClass III

PtdInsPtdIns

PtdIns(4)PPtdIns(4)P

PtdIns(4,5)P2PtdIns(4,5)P2

PtdInsPtdIns

PtdIns(4)PPtdIns(4)P

PtdInsPtdIns

PtdIns(3)PPtdIns(3)P

PtdIns(3,4)P2PtdIns(3,4)P2

PtdIns(3,4,5)P3PtdIns(3,4,5)P3

PtdIns(3)PPtdIns(3)P

PtdIns(3,4)P2PtdIns(3,4)P2

PtdIns(3)PPtdIns(3)P

p110p110 p85p85

class IBclass IB

class IAclass IA

p110p110p101p101

CpkCpk

Vps34pVps34p

Subunits of PI 3-kinase, class IA

p85 N- p85 N-

p110 N-p110 N-

SH3 SH3 bcr homologybcr homology SH2 p110 binding SH2 SH2 p110 binding SH2 and activationand activation

-C-C

-C-C

and and isoforms isoforms

and and isoforms isoforms

Hu, P. Hu, P. et al.et al. 1993. Mol. Cell. Biol. 13:7677-7687. 1993. Mol. Cell. Biol. 13:7677-7687.

Wymann, M.P. and L. Pirola. 1998. Biochim. Biophys. Acta 1436:127-150.Wymann, M.P. and L. Pirola. 1998. Biochim. Biophys. Acta 1436:127-150.

p85 ras p85 ras binding binding bindingbinding

basic/basic/leu zipleu zip

4 conserved regions 4 conserved regions of homology (1-4)of homology (1-4)

11 22 33 44

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

CF

Us

0.0×10 -00

5.0×1007

1.0×1008

1.5×1008

2.0×1008

vitamin D3 - + - + - +oligo - - S S A A

day 7

(100)

(21)

(89)

(20)

(94)(83)

A

BN.S. N.S.

N.S.

CF

Us

0.0×10 -00

1.0×1007

2.0×1007

3.0×1007

4.0×1007

vitamin D3 - + - + - +oligo - - S S A A

day 4

(100)

(37)

(94)

(38)

(96)(78)

A

BN.S.

N.S.

N.S.

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

CF

Us

0.0×10 -00

5.0×1007

1.0×1008

1.5×1008

2.0×1008

cell type THP-1 cells RAW cells vitamin D3 - + - + - + - + inhibitor - - N N - - N N

day 7

(100)

(20)

(97)

(18)

(100)

(11)

(100)

(71)

A

N.S.N.S.

N.S.A

B

CF

Us

0.0×10 -00

1.0×1007

2.0×1007

3.0×1007

4.0×1007

day 4

cell type THP-1 cells RAW cells vitamin D3 - + - + - + - + inhibitor - - N N - - N N

(100)

(42)

(91)

(41)

(100)

(34)

(100)

(81)

A

N.S.

A

N.S.

B

N.S.

ROI scavenger or degradative enzymes

4-hydroxy-tempoN

O•

CH3

CH3 CH3

CH3

H2 O2• H2O2

2 H2O2 2 H2OPEG-catalase

PEG-SOD

- scavenge and reduce oxidative burst products

+ O2

+ O2+2 H+

O

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

CF

Us

0.0×10 -00

5.0×1007

1.0×1008

1.5×1008

2.0×1008

(100)

(14)

(109)

(88)

(102)

(77)

(104)

(72)

vitamin D3 - + - + - + - + inhibitor - - T T C C S S

day 7

A

B B

B

N.S. N.S. N.S.

CF

Us

0.0×10 -00

1.0×1007

2.0×1007

3.0×1007

4.0×1007

(100)

(35)

(103)

(83)

(99)

(79)

(100)

(74)

vitamin D3 - + - + - + - + inhibitor - - T T C C S S

day 4

A

N.S.N.S. N.S.

B BB

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

nm

ole

s s

up

ero

xid

e p

rod

uc

ed

/1

06 m

ac

rop

ha

ge

s / h

ou

r

0

25

50

75

100

125

TB infection + - + + + + +vitamin D3 - + + - + - +inhibitor none LY294002 wortmannin

A

B

B

N.S. N.S.

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

nm

ole

s s

up

ero

xid

e p

rod

uc

ed

/1

06 m

ac

rop

ha

ge

s / h

ou

r

0

10

20

30

40

50

60

70

80

90

100

TB infection + + - + + - + + -vitamin D3 - + + - + + - + +mRNA none sense antisense

C

DN.S.

N.S.

Diagrammatic representation of the NADPH oxidase

gp91 gp91p22 p22Rap1A Rap1A

p67

p47

rac2 PPP

p40

ACTIVATED

p67p47

p40

OH

OH

OH

rac2

RESTING

Babior, B.M. 1999. Blood 93:1464-1476.

p47phox and p67phoxin membrane and cytosol

membrane

cytosol

p47

p67

p47

p67

C PMA TB2 TB4 +D3 HK2 HK4 +D3 D3

C PMA TB2 TB4 +D3 HK2 HK4 +D3 D3

TB HK only

TB HK only

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

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.

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

ZH, Feb, 2000

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

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

nit

rite

co

nc

en

tra

tio

n(m

icro

mo

lar)

0

10

20

30

40

vitamin D3 - + - + - + - +L-NMMA - - + + - - + + day 4 day 7

A

N.S.B

A

C

B

Pathways for phosphoinositide synthesis

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

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

cfu

s (l

og

sca

le)

0 1 2 3 4 5 6 7 81.0×1006

1.0×1007

1.0×1008

1.0×1009

controlvitamin D31-OH vitamin D325-OH vitamin D3

time (days)

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

CF

Us

0.0×10 -00

2.5×1006

5.0×1006

7.5×1006

1.0×1007

vitamin D3 - + - + - +inhibitor - - LY LY Wrtm Wrtm

day 2

(100)

(46)

(98)(90) (93)

(88)

A

N.S.C N.S.

C

CF

Us

0.0×10 -00

3.0×1006

6.0×1006

9.0×1006

1.2×1007

vitamin D3 - + - + - +inhibitor - - LY LY Wrtm Wrtm

day 4

(100)

(42)

(95) (91) (91)(85)

A

CN.S. N.S.C

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

nm

ole

s s

up

ero

xid

e p

rod

uc

ed

/1

06 m

ac

rop

ha

ge

s /

ho

ur

0

25

50

75

100

125

150

infection none TB LB HKvitamin D3 - + - + - + - +

A

N.S.N.S.

The oxidative burst mediatesanti-mycobacterial activity in PBMCs

CF

Us

0.0×10 -00

3.0×1006

6.0×1006

9.0×1006

1.2×1007

1.5×1007

(100)

(32)

(99) (92) (97)

(80)(87)

(78)

vitamin D3 - + - + - + - +

inhibitor - - T T C C S S

day 4

C

D

D D

N.S. N.S.

N.S.

CF

Us

0.0×10 -00

2.5×1006

5.0×1006

7.5×1006

1.0×1007

(100)

(47)

(104)(92) (100)

(77)

(98)(97)

vitamin D3 - + - + - + - + inhibitor - - T T C C S S

day 2

C

N.S. D N.S. N.S.

D

D

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

nm

ole

s s

up

ero

xid

e p

rod

uc

ed

/1

06 m

ac

rop

ha

ge

s /

ho

ur

0

20

40

60

80

100

TB infection + - + + + + +vitamin D3 - + + - + - +inhibitor none LY294002 wortmannin

E

FF

N.S. N.S.

Sly et al.

Figure 3

0 2 4 6 80.0×10 -00

3.0×1006

6.0×1006

9.0×1006

1.2×1007

1.5×1007 sodCIsodCI + D3WmWm + D3LYLY + D3

time (hours)

CF

Us

/ 10

6

mac

roph

ages

0 2 4 6 80.0×10 -00

3.0×1006

6.0×1006

9.0×1006

1.2×1007

1.5×1007 sodCIIsodCII + D3WmWm + D3LYLY + D3

time (hrs)

CF

Us

/ 10

6

mac

roph

ages

0 2 4 6 80.0×10 -00

2.0×1006

4.0×1006

6.0×1006

8.0×1006

1.0×1007 sodCI/sodCIIsodCI/sodCII + D3WmWm + D3LYLY + D3

time (hrs)

CF

Us

/ 10

6

mac

roph

ages

A

C

B

Sly et al.Figure 2

0 2 4 6 81.0×1000

1.0×1002

1.0×1004

1.0×1006

1.0×1008

wtwt + D3sodCIsodCI + D3sodCIIsodCII + D3sodCI/sodCIIsodCI/sodCII + D3

time (hours)

CF

Us

/ 10

6 m

acro

phag

es

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

nm

ole

s s

up

ero

xid

e p

rod

uc

ed

/1

06 m

ac

rop

ha

ge

s/h

ou

r

0

25

50

75

100

125

150

cell fractionvitamin D3

- LAM WCL ECF - + - + - + - +

A

A

N.S.