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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
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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?
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Estrogen lowers the threshold of activation in primary human
PBMCs---proliferation
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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)
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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
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Estrogen stimulated gene expression is enhanced in SLE
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• Toll-like receptor 8 (TLR8)
• Elevated expression in SLE in several studies
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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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EMSA verification of STAT1 ERE
Time (hours) 0 0.5 1 2 4
(E2)
THP-1 cells
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HealthySLE
PBMCs
STAT1
p-STAT1
actin
Estrogen (E2) stimulates STAT1 expression and is upregulated in SLE patients
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Estrogen (E2)-mediated induction of STAT1 regulates TLR8 expression
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STAT1 regulation of TLR8 expression
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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
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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
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Extracellular small RNAs are contained in exosomes
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Exosome fraction
Non-exosome fraction
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miR-21 stimulates TLR8 expression
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TLR8 expression in THP-1 cells
*
miRcontrol
miR-21
6 hours
*
miRokine
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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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