Download Trichloroethylene-mediated epigenetic changes in T cell function

Trichloroethylene-mediated
epigenetic changes in T cell function
Kathleen Gilbert
Systemic lupus erythematosus
Autoimmune disease
Multiple sclerosis
Graves disease
NIH estimates that as many as 24 million Americans
have one or more autoimmune disease.
Type 1 diabetes
Rheumatoid arthritis
This lies between the number of people with cancer
(about 10 million) and cardiovascular diseases (about
31 million).
Scleroderma
2011
RANK
SUBSTANCE NAME
1
ARSENIC
2
LEAD
3
MERCURY
4
VINYL CHLORIDE
5
POLYCHLORINATED BIPHENYLS
6
BENZENE
7
CADMIUM
8
BENZO(A)PYRENE
9
POLYCYCLIC AROMATIC HYDROCARBONS
10
BENZO(B)FLUORANTHENE
11
CHLOROFORM
12
AROCLOR 1260
13
DDT, P,P'-
14
AROCLOR 1254
15
DIBENZO(A,H)ANTHRACENE
16
TRICHLOROETHYLENE
17
CHROMIUM, HEXAVALENT
18
DIELDRIN
19
PHOSPHORUS, WHITE
20
HEXACHLOROBUTADIENE
US Agency for Toxic
Substances and Disease
Registry (ATSDR) ranks
275 chemicals at NPL
sites by combined
exposure likelihood and
negative impact on
human health
Trichloroethylene (TCE)
TCE is used as an industrial
solvent and in production of
hydrofluorocarbons
Because of improper disposal
it is a major environmental
pollutant
Million pounds
TCE is the most frequently
reported organic contaminant
in ground water (up to 34%)
and at NPL sites
EPA’s Toxic Release Inventory of TCE
On-site
Total
Off-site
Trichloroethylene (TCE) exposure
• EPA sets maximum contaminant level of drinking
water with TCE at 5 mg/L (5 ppb)
• 10% of general, non-occupationally-exposed adult
population had detectable TCE in blood (low levels);
Third NHANES (1988-1994)
• Exposure occurs through several routes including,
oral, inhalation and dermal
Trichloroethylene (TCE) exposure and
autoimmunity
• Epidemiological studies linked increased TCE exposure
(occupational and environmental) to increased incidence of
autoimmune disease (e.g. scleroderma, multiple sclerosis)
• Even in absence of overt disease can see increased
numbers of activated T cells and autoantibodies
• Occupational TCE exposure in Asia is increasingly linked to
hypersensitivity dermatitis and non-viral hepatitis
Camp Lejeune, NC
• TCE levels up to 1,400 mg/L in well water
• Contamination by off-base dry-cleaners
and on-base leakage from underground
storage tanks
• Contamination occurred between 1957
and 1987; between 0.2 – 1.0 million people
exposed at some point
• Results of health survey conducted by
ATSDR showed increased incidence of
only autoimmune disease examined
(multiple sclerosis) 2014
Trichloroethylene (TCE) exposure
“Exposure to TCE in the Millsboro, DE, drinking water between
October 2004 and October 2005 may have increased resident’s
likelihood of experiencing non-cancer health effects.” ATSDR
“An Abandoned Wastewater Treatment Plant is Oozing
Trichloroethylene”, Salem Eagle-Tribune, 2012
“Wake Forest Water Contamination: Residents Furious After
Learning State Knew of Trichloroethylene Carcinogen”,
Huffington Post, 2013
“Report finds current cleanup method at Cheyenne missile site
inadequate” Casper Star Tribune, October, 2014
Overview of in vivo experiments to test whether
adult exposure to TCE promotes autoimmune
disease in mice
Lupus-prone
female
MRL+/+ mice
TCE in drinking water:
0
0.1 mg/ml = 21 mg/kg/day
0.5 mg/ml = 80 mg/kg/day
2.5 mg/ml = 400 mg/kg/day
Pathology:
Kidney
Liver
Skin
Autoantibodies
Immune cells:
4 - 40 weeks
CD4+ T cells
CD8+ T cells
B cells
Thymocytes
Phenotype
Cytokines
Gene expression
DNA methylation
OSHA 8-hour permissible exposure limit for TCE is ~ 80 mg/kg/day
Adult TCE exposure (0.5 or 2.5 mg/ml) for
32-40 weeks induced autoimmune
hepatitis in MRL+/+ mice
Control
TCE-induced development of antiliver microsomal antibodies
4 weeks 40 weeks
100
75
50
37
25
20
TCE
15
CV
PV
PV
Gilbert et al. Tox Appl Pharm 279: 284, 2014
Griffin, Gilbert and Pumford. Toxicol Sci 57: 345, 2000
Griffin, Blossom, Jackson, Gilbert, and Pumford, Immunopharm 46:123, 2000
TCE-induced autoimmunity associated with
changes in CD4+ T cells
4
Weeks
• TCE increased production of pro-inflammatory
cytokine IFN-g by CD4+ T cells
• TCE increased expansion of activated/memory
(CD44hi, CD62Llo) CD4+ T cells32
Weeks
Trichloroethylene (mg/ml)
Griffin, Gilbert, Lamps and Pumford; Tox Sci, 57:345, 2000
How is TCE altering CD4+ T cells? Does it
involve changes in DNA methylation?
DNA methylation can regulate the activity of self-reactive CD4+ T
cells that mediate autoimmune disease. Studies by others:
•
Activated CD4+ T cells treated with 5-aza-2’ deoxycytidine (DNA
methyltransferase inhibitor) become autoreactive
•
Distinct DNA methylation patterns can be detected in CD4+ T
cells from patients with a certain autoimmune disease, e.g. SLE
• Twin discordance in certain autoimmune diseases correlate with
patterns of DNA methylation in PBMC
• Bisphenol A: CD4 immunotoxicant changes methylation-sensitive
coat color in agouti mice
Expression of retrotransposons can be used
as possible sign of DNA demethylation
Transcriptional
Repression
CH3
LTR
Host gene #1 (may enhance or suppress expression)
CH3
LTR
Retrotransposon
Transcription ( gene expression of IAP and MuErv)
Demethylation event
Escape from suppression
LTR
LTR
IAP (Intracisternal A particle); MuErv (Murine endogenous retrovirus)
12-week adult exposure to TCE decreased
expression of retrotransposons in CD4+ T cells
Iap
Muerv
Gilbert et al. Tox Sci 127:169, 2012
Overview of in vivo experiments to test
developmental effects of TCE
TCE (0.01 mg/ml) GD0 – PND0
Gestational Exposure
PND49
TCE (0.01 mg/ml) PND0 – PND49
Early Life Exposure
PND49
0.01 mg/ml = 2 mg/kg/day
Immune cells
Phenotype
Cytokines
Gene expression
DNA methylation
Developmental exposure to TCE decreased
expression of retrotransposons in CD4+ T
cells
fold change
2.00
Iap
MuERV
1.50
0.00
Dnmt3a
1.50
Control
1.00
0.50
2.00
*
*
1.00
TCE
Dnmt1
2.00
Control
TCE
Iap
MuERV
1.50
0.50
0.00
0.00
Dnmt3a
1.50
1.00
0.50
2.00
1.00
*
*
0.50
0.00
Control
Control
TCE
TCE
Kinetics experiment to test effects of TCE
Lupus-prone
female
MRL+/+ mice
Pathology:
TCE in drinking water:
0
0.5 mg/ml = 80 mg/kg/day
Kidney
Liver
Skin
Autoantibodies
Immune cells:
4, 10, 16, 22, 28, 34
and 40 weeks
CD4+ T cells
CD8+ T cells
B cells
Thymocytes
Gene expression:
Iap, Ifng
DNA methylation
OSHA 8-hour permissible exposure limit for TCE is ~ 80 mg/kg/day
Compared naïve and effector/memory CD4+ T
cells from control and TCE-treated mice
Effector/memory
CD44hiCD62Llo
Sample_047.fcs
10
FL1
CD44
10
10
10
4
3
2
Naive
CD44loCD62Lhi
1
0
10
0
10
10
1
2
10
FL2
CD62L
10
3
10
4
Does TCE alter expression of Infg in CD4+ T
cells by altering DNA methylation
associated with the promoter ?
Isolated effector/memory and naïve populations of CD4+ T
4
cells from MRL+/+mice treated with TCE
(0.5 mg/ml):
Weeks
Stimulated with mitogen in vitro to generate IFN-g
Examined Ifng expression
32
Conducted selected methylation mapping
by sequencing
Weeks
bisulfite-converted DNA of promoters of functionally
important genes: Ifng, Il4, Cd70, Cdknl1, Foxp3
Trichloroethylene (mg/ml)
118440745
0
118441459
118441432
118441384
118441344
118441333
118441326
118441304
118441222
118441007
118440996
118440988
118440868
118440848
118440834
% m ethy lation
Did TCE alter DNA methylation in Infg
promoter?
Example of Ifng
methylation
pattern (4 weeks)
4 w eeks memory
120
100
80
60
Cont r ol
TCE
Control
TCE
40
20
Conclusion
• TCE alters expression of methylation-dependent
Iap in CD4 + T cells after adult or developmental
exposure
• TCE-induced alteration in the expression of Iap and
other genes (e.g. Ifng) is time-dependent
• Epigenetic mechanisms may contribute to the long
term effects of TCE on CD4+ T cells
Acknowledgements
UAMS :Sarah Blossom, PhD
Funding: EPA (R82941701-0)
Ashley Nelson
NIH (1R01ES017286)
Meagan Kreps
Organic Compounds Property
Contamination
Joe Griffin, PhD
Jason Doss, DVM
Laura Lamps, MD
Leah Hennings, DVM
UAF: Neil Pumord PhD
VAMC: Craig Cooney, PhD
Class Action Settlement (CV 1992002603)
Arkansas Biosciences Institute