Estrogen-mediated TLR8 expression via STAT1 facilitates endogenous miRokine ligand activation by exosomes containing miR-21

Nicholas Young, PhDDepartment of Internal Medicine

Division of Rheumatology and Immunology

June 25th 2015Federation of Clinical Immunology Societies

No relevant financial or competing interests specific to this project to declare

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Background

Females are generally more resistant to infection than males

Longer life spans in many female species ranging from nematodes to mammals

Estrogenic effects could lead to a heightened immuno-reactive state

Survival advantage in the defense against infection

Can estrogen (E2) enhance an immune response?

2

4

Estrogen lowers the threshold of activation in primary human

PBMCs---proliferation

Estrogen (E2) influence of genetic expression

Several receptors for E2 in cells

Primary route if influence is through ERaand b

E2 enters cells and binds receptors

Receptor dimers translocate to the nucleus and bind EREs

Enhance transcription of genes

MANY more E2-induced genes yet to be identified

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Weatherman et al. Nature Chemical Biology 2, 175 - 176 (2006)doi:10.1038/nchembio0406-175 (modified)

Hypothesis

Estrogen may lower the threshold of immune system stimulation by influencing gene expression

Heightened female immune response

If dysregulated, could drive autoimmune development in diseases such as lupus

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Background

Systemic Lupus Erythematosus (SLE)

Hormonal influence

SLE incidence is highest in females with the highest physiological levels of estrogen

Childbearing age women is 9:1 to males

Drops closer to 2:1 during childhood or post menopause

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Lupus and the Lung G Diaz-Fuentes, MD Pulmonary &

Critical Division Bronx Lebanon Hospital Center

Estrogen stimulated gene expression is enhanced in SLE

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• Toll-like receptor 8 (TLR8)

• Elevated expression in SLE in several studies

Toll-like receptors (TLRs)

TLRs detect highly conserved components of pathogens

TLR 3 and 7-9 are expressed on intracellular compartments such as endosomes

These pathways all result in the downstream induction of characteristic pro-inflammatory cytokine responses

TLR8 binds single-stranded RNA (viruses)

Dysregulation of TLR8 could contribute to autoimmune inflammatory state ML MacKichan. Toll bridge to immunity. Immune molecules hold

promise for vaccine adjuvant discovery. IAVI Rep. 2005 Sep-

Oct;9(4):1-5.

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Rela

tive

TR

L8

exp

ressio

n

Healthy SLE

Media E2

-p-ERa

-hsp90

-TLR8

E2: Media E2 E2 Media

K562 Daudi

-b-actin

-TLR8

0

0.5

1

1.5

2

0.951

1.051.1

1.151.2

1.251.3

1.351.4

1.45

Media E2 TT

* p < 0.05

*** p < 0.005

***

*

1 1.2 1.8 1

1 12.4 2.4 1

1 2

Re

lative

TL

R8

exp

ressio

n

TLR8 expression is elevated in SLE patients and stimulated by estrogen

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CHIP-seq Identification of a positive estrogen response binding peak proximal to the Stat1 genetic locus

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12

EMSA verification of STAT1 ERE

Time (hours) 0 0.5 1 2 4

(E2)

THP-1 cells

HealthySLE

PBMCs

STAT1

p-STAT1

actin

Estrogen (E2) stimulates STAT1 expression and is upregulated in SLE patients

Estrogen (E2)-mediated induction of STAT1 regulates TLR8 expression

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STAT1 regulation of TLR8 expression

Time (hours) Time (hours)

* p < 0.03, **p < 0.008

***

*

Rela

tive

TR

L8

exp

ressio

n

Rela

tive

TR

L8

exp

ressio

n

PBMCs stimulated with estrogen and TLR8 agonist induce TLR8

expression and display gender-biased responses---RTPCR

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miRNAs are secreted by cancer cells in exosomes and can reach and bind TLR7

(in mice) or TLR8 (in humans) in the endosomes of surrounding immune cells

Fabbri M et al. PNAS 2012;109:12278-12279

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J Immunol. 2010 Jun 15;184(12):6773-81. doi: 10.4049/jimmunol.0904060. Epub 2010 May 17.

Pan W1, Zhu S, Yuan M, Cui H, Wang L, Luo X, Li J, Zhou H, Tang Y, Shen N.

miR-21 is upregulated in mice and in SLE patients

Paxgene expression Exosome expression

0.00

0.50

1.00

1.50

miR-21

HealthySLE

n.s. n.s.

No difference in miR-21 levels in cells or exosomes in SLE

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cytokine

chemokine

myokine

adipokine

miRokine

hormiR

miRmone

Extracellular small RNAs are contained in exosomes

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Exosome fraction

Non-exosome fraction

miR-21 stimulates TLR8 expression

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TLR8 expression in THP-1 cells

*

miRcontrol

miR-21

6 hours

*

miRokine

0.0E+00

2.0E+05

4.0E+05

6.0E+05

8.0E+05

1.0E+06

1.2E+06

1.4E+06

1.6E+06

1.8E+06

01

22

43

64

86

07

28

49

61

08

12

01

32

14

4

0.0E+00

2.0E+05

4.0E+05

6.0E+05

8.0E+05

1.0E+06

1.2E+06

1.4E+06

1.6E+06

1.8E+06

01

22

43

64

86

07

28

49

61

08

12

01

32

14

4

0.0E+00

2.0E+05

4.0E+05

6.0E+05

8.0E+05

1.0E+06

1.2E+06

1.4E+06

1.6E+06

1.8E+06

01

22

43

64

86

07

28

49

61

08

12

01

32

14

4

Con

ce

ntr

atio

n (

pa

rtic

les / m

L)

Con

ce

ntr

atio

n (

pa

rtic

les / m

L)

Con

ce

ntr

atio

n (

pa

rtic

les / m

L)

Size (nm) Size (nm)

miR control miR-21 R-848

R-848 and miR-21 stimulate exosome formation

miR-21 R-848miR control

Size (nm)1.5E+07

3.5E+07

5.5E+07

7.5E+07

9.5E+07

1.2E+08

miR-scramble miR-21 R-848

*

Are

a U

nder

Curv

e *

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SLE Pathogenesis

Exosome production

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nicholas.young@osumc.edu

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