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
2
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
8
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
3
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
6
Lupus and the Lung G Diaz-Fuentes, MD Pulmonary &
Critical Division Bronx Lebanon Hospital Center
Estrogen stimulated gene expression is enhanced in SLE
10
• 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.
14
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
15
CHIP-seq Identification of a positive estrogen response binding peak proximal to the Stat1 genetic locus
11
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
14
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
20
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
18
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
20
cytokine
chemokine
myokine
adipokine
miRokine
hormiR
miRmone
Extracellular small RNAs are contained in exosomes
21
Exosome fraction
Non-exosome fraction
miR-21 stimulates TLR8 expression
22
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 *
24
SLE Pathogenesis
Exosome production
25
27