SOLUBLE TOLL-LIKE RECEPTOR 2 (sTLR2) IS SOLUBLE TOLL-LIKE RECEPTOR 2 (sTLR2) IS SIGNIFICANTLY ELEVATED IN HIV-1-SIGNIFICANTLY ELEVATED IN HIV-1-

INFECTED BREAST MILK AND INHIBITS HIV-1 INFECTED BREAST MILK AND INHIBITS HIV-1 INFECTION AND INFLAMMATION INFECTION AND INFLAMMATION

Ken RosenthalMcMaster University

AFRI-CAN 2013Entebbe, Uganda

Breastfeeding and HIV

Exclusively breast feed (EBF) infants are 2 to 10-fold less likely to be infected compared to infants who are non-EBF.

Exclusive BF

Non-exclusive BF

4.0%

10.1%

P=0.002

Short-lived innate factors present in breast milk inhibit vertical transmission of HIV-1 from mother-to-child.

Hypothesis

Cellular Localization of Toll-Like Receptors (TLRs)Cellular Localization of Toll-Like Receptors (TLRs)

Soluble TLR2: Breast MilkSoluble TLR2: Breast Milk

•Recognizes a wide-range of ‘danger-associated’

molecules

•Decoy receptor that limits signaling through

membrane-bound TLR2

•High concentration in breast milk

•The biological significance of sTLR2 and HIV

remains unknown

Schematic of sTLR2 cleavage and antibody binding

Increased sTLR2 in Milk of HIV-Infected Increased sTLR2 in Milk of HIV-Infected Women Correlates with p24 Levels Women Correlates with p24 Levels

HIV-N

(Ham

ilton

)

HIV-N

(Niger

ia)

HIV-P

(Niger

ia)

102

103

104

105

P=0.0062

sTLR2 pg/mL

P=0 .043

0 30000 60000 900000

2

4

6 P=0 .0186R2=0.1374

sTLR2 (pg/mL)

p24 (pg/mL)

Significantly Increased TLR Expression in Significantly Increased TLR Expression in Breast Milk Cells from HIV-Infected Nigerian Breast Milk Cells from HIV-Infected Nigerian

WomenWomen

TLR10

HIV-N

(Ham

ilton)

HIV-N

(Nig

eria)

HIV

-P (N

igeria

)0.001

0.01

0.1

1

10

100

1000

10000 P<0.0001

P<0.0012

Relative expression

(Ratio to RPL13A)

TLR1

HIV-N

(Ham

ilton)

HIV-N

(Nig

eria)

HIV-P

(Niger

ia)0.01

0.1

1

10

100P=0.0006

P<0.0001

Relative expression

(Ratio to RPL13A)

TLR3

HIV

-N (H

amilt

on)

HIV

-N (N

igeria

)

HIV

-P (N

igeria

)0.1

1

10

100

1000

10000P<0.0001

Relative expression

(Ratio to RPL13A)

TLR4

HIV-N

(Ham

ilton)

HIV-N

(Nig

eria)

HIV-P

(Nig

eria)

0.1

1

10

100

1000

relative expression

(Ratio to RPL13A)

P<0.0197

P<0.0307

TLR8

HIV

-N (H

amilt

on)

HIV

-N (N

igeria

)

HIV

-P (N

igeria

)0.01

0.1

1

10

100

1000P<0.0001

Relative expression

(Ratio to RPL13A)

TLR2

HIV-N

(Ham

ilton)

HIV-N

(Nig

eria)

HIV-P

(Niger

ia)0.1

1

10

100

1000P=0.0129

relative expression

(Ratio to RPL13A)

HIV-N

(Ham

ilton)

HIV-N

(Niger

ia)

HIV-P

(Nige

ria)

0

200

400

p<0.0249

p<0.0081

IL-15 pg/mL

HIV-N

(Ham

ilton)

HIV-N

(Niger

ia)

HIV-P

(Niger

ia)0

100

200 p<0.0128

CCL5 pg/mL

Significantly Elevated IL-15 & RANTES in Significantly Elevated IL-15 & RANTES in Breast Milk from Uninfected Compared to HIV-Breast Milk from Uninfected Compared to HIV-

infected Nigerian Women infected Nigerian Women

Significant Decrease of Inflammatory Significant Decrease of Inflammatory Mediator IL-8 by sTLR2 Mediator IL-8 by sTLR2

med

iaT2

0 110

00

500

1000

1500 TZMbl with TZMbl mediaTZMbl with TZMbl-2 media

p<0.001

TCID 50

IL-8 (pg/mL)

A B

100 TC

ID 50

500

1000

1500 TZMbl-2 media

TZMbl-2 media + TLR2 Ab

p<0.0151

IL-8 (pg/mL)

Inhibition of HIV-1 Infection by sTLR2 Inhibition of HIV-1 Infection by sTLR2 produced produced in vitroin vitro

BA

Specific Binding of sTLR2 in Human Milk Specific Binding of sTLR2 in Human Milk to HIV Proteinsto HIV Proteins

1:100 BM

Pam3CSK

CD14

gp120

gp41

p24

p17 BM

p17

nef

urea

PBS

ssRNA

CONCLUSIONSCONCLUSIONS

• Depletion of sTLR2 in human milk resulted in significantly increased IL-8 production in U937 monocytes, HEK-293-TLR2 EC’s and human intestinal EC’s.

• sTLR2-depletion of milk led to significantly (P<0.001) increased HIV infection in vitro.

• sTLR2 produced in vitro from a TLR2-tranformed cell line significantly reduced cell-free R5 HIV infection and was reversed after neutralization by anti-TLR2 Abs.

• sTLR2 significantly reduced proinflammatory cytokine production in HIV-exposed cells.

• sTLR2 was significantly increased in milk of HIV-infected Nigerian women and correlated with p24 levels.

CONCLUSIONSCONCLUSIONS

• Breast milk cells from HIV-infected Nigerian women have significantly increased expression of numerous Toll-Like Receptors (TLRs) compared to uninfected Nigerian women.

• Breast milk from uninfected Nigerian & Canadian women contains significantly increased levels of IL-15 and RANTES compared to HIV-infected breast milk.

• sTLR2 directly binds to specific HIV structural proteins.

• Overall, our data suggest that sTLR2 in human milk may be critical to infant health and prove beneficial in prevention of mother-to-child HIV transmission (PMTCT).

AcknowledgementsAcknowledgements

Rosenthal Laboratory:Bethany HenrickXiao-Dan YaoKakon NagAnna DrannikSumiti JainJen NewtonAmy Patrick

Cohort Participants in Hamilton & NigeriaHamilton Midwives & Lactation Consultants

Collaborators:Dr. Alash’le AbimikuInstitute of Human Virology

Supported by:

Questions?