outline - mi academy4/12/2016 cheryl rockwell, phd 8 the nrf2 activator, tbhq, causes marked...
TRANSCRIPT
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
1
Food allergy and the role of the food preservative tBHQ
Cheryl Rockwell Assistant Professor Department of Pharmacology amp Toxicology Michigan State University rockwelcmsuedu
Outline
bull Trends in food allergy
bull Basic Immunology
bull Immune response during allergy
bull What can basic research tell us Role of food preservatives
bull Summary and conclusions
Trends in Food Allergy
Branum et al 2008 NCHS Data Brief
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
2
Jackson et al 2013 NCHS Data Brief
Inverse association between poverty and food allergy
Jackson et al 2013 NCHS Data Brief
Branum et al 2008 NCHS Data Brief Branum et al 2008 NCHS Data Brief
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
3
Branum et al 2008 NCHS Data Brief
Basic Immunology
Janeway et al ldquoImmunobiology The immune system in health and diseaserdquo 2005 6th Ed Garland Science Pub
Immune system overview
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
4
Immune Response during Food Allergy
Th0
Th1
Th2
Th17
Treg
Tfh
Intracellular pathogens
Helminths nematodes
Extracellular pathogens
Prevention of autoimmunity
B cell activation
T cell differentiation
Th0
Th1
Th2
Th17
Treg
Tfh
IFNg
IL-4 IL-5 IL-13
IL-17A IL-17F IL-22 IL-21
TGFb IL-10
IL-4 IL-21
CD4+ cell
uarr TH1 differentiation
Cytotoxic T Lymphocyte
activation
darr TH2 differentiation
uarr Immunoproteasome (uarr Ag presentation)
Cytokine production by
macs (TNFa IL-1b IL-6 amp IL-12)
uarr IFNg
uarr IFNg
uarr Macrophage activation
Function and importance of IFNg
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
5
CD4+ cell
darr TH1 differentiation
uarr TH2 differentiation
B cell proliferation
uarr M2 cells ldquoalternative activationrdquo of macs
uarr IL-4
uarr IL-4
darr M1 cells macrophage
ldquohelprdquo in Th1 activity
Function and importance of IL-4
uarr IgE production in B cells (ldquoclass switchingrdquo)
Immune response to food allergen
IL-4 a mediator of immunity against parasites
What can basic research tell us Role of food preservatives
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
6
Nrf2 activators are commonly used food preservatives
bull tBHQ
bull BHA
bull BHT
Marketers such as Starbucks discover that simple sells
By Bruce Horowitz USA Today October 28 2009
ldquoConsumers these days not only want to know whats in the stuff they eat
and drink mdash they want to know whats
not In a nation bedeviled by a
whirlwind of food scares and
mounting worries about the
healthiness of a plethora of things
commonly used in processed foods
folks increasingly are demanding
cleaner food labels no artificial food
colorings (some of which have been
linked to hyperactivity in children) no
chemical additives (such as MSG) and
no chemical preservatives (such as
BHA) If they cant pronounce it
consumers dont want itrdquo
Zagorski et al 2013 Toxicological Sciences
tBHQ modulates cytokine production by human T cells
Joe Zagorski
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
7
Turley et al 2015 Cytokine
tBHQ modulates cytokine production by immune cells from human blood
Alex Turley
Turley et al 2015 Cytokine
T cells isolated from human blood are very sensitive to the effect of the food preservative tBHQ
ARE
Nrf2 Maf
Coactivators
Keap1
Nrf2 Cul3
Roc1 E2
Schematic representation of Nrf2 activation
Nrf2 Activation
Oxidative stress
Electrophilic
stimuli
15d-PGJ2
Target genes
Antioxidant
Detoxification
Metabolism
Anti-inflammatory
Proteasome ARE
Nrf2 Maf
Coactivators
Keap1
Nrf2 Cul3
Roc1 E2
Schematic representation of Nrf2 activation
Nrf2 Activation
tBHQ
Target genes
Antioxidant
Detoxification
Metabolism
Anti-inflammatory
Proteasome
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
8
The Nrf2 activator tBHQ causes marked inhibition of early IFNg production in activated splenic T cells
IFNg
BKG 0 001 01 1 10 0
20000
40000
60000
80000
ND ND
IFN
g (p
gm
l)
tBHQ (mM)
A
aCD3aCD28
VH 001 01 05 1 0
20
40
60
80
100
120
140
160
tBHQ (mM)
IFN
g m
RN
A (
V
H)
dagger dagger
Dagger
Dagger
Dagger Dagger
Wild-type Nrf2-null
B IFNg
0
10
20
30
40
50
IFN
g m
RN
A
(Fo
ld i
nd
ucti
on
o
ver
BK
G)
WT WT Null Null Nrf2
aCD3aCD28 BKG
C
TH0
TH1 TH2
IFNg IL-4 IL-5 IL-13
Differentiation of CD4 (helper) T cells
Extracellular amp intracellular
pathogens
Asthma amp Allergy
TH0
TH1 TH2
IFNg IL-4 IL-5 IL-13
Differentiation of CD4+ (helper) T cells
Extracellular amp intracellular
pathogens
Asthma amp Allergy
How do we know T cells have differentiated
bull Itrsquos tricky
bull Functional studies cytokine
bull Effect over time
ndash Short-term le24h
ndash Relatively long-term ge96h
ndash Commitment ge96h
bull Genes that regulate T cell differentiation GATA-3 T-bet
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
9
VH 001 01 05 1
IFN
(p
gm
l)
0
1000
2000
3000
4000
5000
6000 Wild-type
Nrf2-null
aCD3aCD28
tBHQ (mM)
IFNg
Dagger
Dagger
Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-4
(p
gm
l)
0
20
40
60
80
100
120
140 IL-4
Dagger Dagger Dagger Dagger
Dagger
aCD3aCD28
tBHQ (mM)
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-5
(p
gm
l)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-5
VH 001 01 05 1
IL-1
3 (
pg
ml)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-13
Dagger Dagger Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
aCD3aCD28
tBHQ (mM)
Dagger Dagger Dagger Dagger Dagger
aCD3aCD28
tBHQ (mM)
Rockwell et al 2012
VH 001 01 05 1
TN
F (
pg
ml)
0
50
100
150
200
250
aCD3aCD28
tBHQ (mM)
TNFa
Dagger
Dagger
Dagger
VH 001 01 05 1
IL-1
0 (
pg
ml)
0
500
1000
1500
2000
2500
aCD3aCD28
tBHQ (mM)
IL-10
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
10
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
T-bet (Th1 transcription factor)
Restimulated
Nrf2 activation inhibits the binding activity of T-bet the master regulator of Th1 differentiation
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
Restimulated
Nrf2 activation inhibits T-bet binding activity
IFN
(pg
ml)
0
1000
2000
3000
4000
Restimulated
VH tBHQ
IFNg
T-bet (Th1)
Dagger Dagger
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
Dagger Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
IL-4
(p
gm
l)
0
200
400
600
800
1000
1200
1400
Restimulated
VH tBHQ
IL-4
Dagger Dagger
Dagger Dagger
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
11
GA
TA
-3 m
RN
A (
Fo
ld in
du
cti
on
over
WT
VH
Co
ntr
ol)
00
05
10
15
20
25
30 Wild-type
Nrf2-null
VH 0001 001 01 1
Nrf2 activation increases GATA-3 mRNA expression
anti-CD3anti-CD28
tBHQ
Dagger
Dagger Dagger Dagger
GATA-3
Hypothesis tBHQ exacerbates immune response and clinical symptoms of anaphylaxis in response to food allergen
Why doesnrsquot everyone have immune responses to food The phenomenon of oral tolerance
1 Wk 1 Wk 1 Wk
Bleed Bleed Bleed
OVA OVA OVA OVA
Experimental design for ovalbumin transdermal exposure
1 Wk 1 Wk
OVA orally
Symptoms of anaphylaxis
Control
tBHQ (0001)
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
12
Summary
bull tBHQ inhibits production of the cytokines IL-2 and IFNg in human T cells (suggesting a possible effect on host immunity)
bullActivation of Nrf2 by the food preservative tBHQ increases production of Th2 cytokines by CD4+ T cells
bull tBHQ increases activity and expression of the Th2-regulating gene GATA-3 and this is dependent on Nrf2
bull Our preliminary data suggests that low doses of tBHQ exacerbate immune response and clinical symptoms in an animal model of food allergy
Cell-mediated Immunity
Parasite immunity
Nrf2
In the presence of Nrf2 Th1 and Th2 responses are in balance
Th1 Th2
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
13
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
bull Rockwell Lab ndash Heather Dover
ndash Jenna Bursley
ndash Alex Turley
ndash Joe Zagorski
ndash Holly Cline
bull Dr Curtis D Klaassen
bull Klaassen Laboratory ndash Scott Reisman
bull Dr Bryan Copple ndash Copple Lab
bull Dr Venu Gangur ndash Tina Ortiz
ndash Radhakrishna Para
ndash Babu Gonipeta
bull Dr Jim Pestka
bull Dr Patrick Fields ndash Mingcai Zhang
ndash Manouela Ortega
Acknowledgments
This project was funded by NIH grants K99R00 ES018885 R01 ES024966 T32 ES007255 T32 GM092715
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
2
Jackson et al 2013 NCHS Data Brief
Inverse association between poverty and food allergy
Jackson et al 2013 NCHS Data Brief
Branum et al 2008 NCHS Data Brief Branum et al 2008 NCHS Data Brief
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
3
Branum et al 2008 NCHS Data Brief
Basic Immunology
Janeway et al ldquoImmunobiology The immune system in health and diseaserdquo 2005 6th Ed Garland Science Pub
Immune system overview
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
4
Immune Response during Food Allergy
Th0
Th1
Th2
Th17
Treg
Tfh
Intracellular pathogens
Helminths nematodes
Extracellular pathogens
Prevention of autoimmunity
B cell activation
T cell differentiation
Th0
Th1
Th2
Th17
Treg
Tfh
IFNg
IL-4 IL-5 IL-13
IL-17A IL-17F IL-22 IL-21
TGFb IL-10
IL-4 IL-21
CD4+ cell
uarr TH1 differentiation
Cytotoxic T Lymphocyte
activation
darr TH2 differentiation
uarr Immunoproteasome (uarr Ag presentation)
Cytokine production by
macs (TNFa IL-1b IL-6 amp IL-12)
uarr IFNg
uarr IFNg
uarr Macrophage activation
Function and importance of IFNg
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
5
CD4+ cell
darr TH1 differentiation
uarr TH2 differentiation
B cell proliferation
uarr M2 cells ldquoalternative activationrdquo of macs
uarr IL-4
uarr IL-4
darr M1 cells macrophage
ldquohelprdquo in Th1 activity
Function and importance of IL-4
uarr IgE production in B cells (ldquoclass switchingrdquo)
Immune response to food allergen
IL-4 a mediator of immunity against parasites
What can basic research tell us Role of food preservatives
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
6
Nrf2 activators are commonly used food preservatives
bull tBHQ
bull BHA
bull BHT
Marketers such as Starbucks discover that simple sells
By Bruce Horowitz USA Today October 28 2009
ldquoConsumers these days not only want to know whats in the stuff they eat
and drink mdash they want to know whats
not In a nation bedeviled by a
whirlwind of food scares and
mounting worries about the
healthiness of a plethora of things
commonly used in processed foods
folks increasingly are demanding
cleaner food labels no artificial food
colorings (some of which have been
linked to hyperactivity in children) no
chemical additives (such as MSG) and
no chemical preservatives (such as
BHA) If they cant pronounce it
consumers dont want itrdquo
Zagorski et al 2013 Toxicological Sciences
tBHQ modulates cytokine production by human T cells
Joe Zagorski
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
7
Turley et al 2015 Cytokine
tBHQ modulates cytokine production by immune cells from human blood
Alex Turley
Turley et al 2015 Cytokine
T cells isolated from human blood are very sensitive to the effect of the food preservative tBHQ
ARE
Nrf2 Maf
Coactivators
Keap1
Nrf2 Cul3
Roc1 E2
Schematic representation of Nrf2 activation
Nrf2 Activation
Oxidative stress
Electrophilic
stimuli
15d-PGJ2
Target genes
Antioxidant
Detoxification
Metabolism
Anti-inflammatory
Proteasome ARE
Nrf2 Maf
Coactivators
Keap1
Nrf2 Cul3
Roc1 E2
Schematic representation of Nrf2 activation
Nrf2 Activation
tBHQ
Target genes
Antioxidant
Detoxification
Metabolism
Anti-inflammatory
Proteasome
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
8
The Nrf2 activator tBHQ causes marked inhibition of early IFNg production in activated splenic T cells
IFNg
BKG 0 001 01 1 10 0
20000
40000
60000
80000
ND ND
IFN
g (p
gm
l)
tBHQ (mM)
A
aCD3aCD28
VH 001 01 05 1 0
20
40
60
80
100
120
140
160
tBHQ (mM)
IFN
g m
RN
A (
V
H)
dagger dagger
Dagger
Dagger
Dagger Dagger
Wild-type Nrf2-null
B IFNg
0
10
20
30
40
50
IFN
g m
RN
A
(Fo
ld i
nd
ucti
on
o
ver
BK
G)
WT WT Null Null Nrf2
aCD3aCD28 BKG
C
TH0
TH1 TH2
IFNg IL-4 IL-5 IL-13
Differentiation of CD4 (helper) T cells
Extracellular amp intracellular
pathogens
Asthma amp Allergy
TH0
TH1 TH2
IFNg IL-4 IL-5 IL-13
Differentiation of CD4+ (helper) T cells
Extracellular amp intracellular
pathogens
Asthma amp Allergy
How do we know T cells have differentiated
bull Itrsquos tricky
bull Functional studies cytokine
bull Effect over time
ndash Short-term le24h
ndash Relatively long-term ge96h
ndash Commitment ge96h
bull Genes that regulate T cell differentiation GATA-3 T-bet
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
9
VH 001 01 05 1
IFN
(p
gm
l)
0
1000
2000
3000
4000
5000
6000 Wild-type
Nrf2-null
aCD3aCD28
tBHQ (mM)
IFNg
Dagger
Dagger
Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-4
(p
gm
l)
0
20
40
60
80
100
120
140 IL-4
Dagger Dagger Dagger Dagger
Dagger
aCD3aCD28
tBHQ (mM)
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-5
(p
gm
l)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-5
VH 001 01 05 1
IL-1
3 (
pg
ml)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-13
Dagger Dagger Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
aCD3aCD28
tBHQ (mM)
Dagger Dagger Dagger Dagger Dagger
aCD3aCD28
tBHQ (mM)
Rockwell et al 2012
VH 001 01 05 1
TN
F (
pg
ml)
0
50
100
150
200
250
aCD3aCD28
tBHQ (mM)
TNFa
Dagger
Dagger
Dagger
VH 001 01 05 1
IL-1
0 (
pg
ml)
0
500
1000
1500
2000
2500
aCD3aCD28
tBHQ (mM)
IL-10
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
10
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
T-bet (Th1 transcription factor)
Restimulated
Nrf2 activation inhibits the binding activity of T-bet the master regulator of Th1 differentiation
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
Restimulated
Nrf2 activation inhibits T-bet binding activity
IFN
(pg
ml)
0
1000
2000
3000
4000
Restimulated
VH tBHQ
IFNg
T-bet (Th1)
Dagger Dagger
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
Dagger Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
IL-4
(p
gm
l)
0
200
400
600
800
1000
1200
1400
Restimulated
VH tBHQ
IL-4
Dagger Dagger
Dagger Dagger
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
11
GA
TA
-3 m
RN
A (
Fo
ld in
du
cti
on
over
WT
VH
Co
ntr
ol)
00
05
10
15
20
25
30 Wild-type
Nrf2-null
VH 0001 001 01 1
Nrf2 activation increases GATA-3 mRNA expression
anti-CD3anti-CD28
tBHQ
Dagger
Dagger Dagger Dagger
GATA-3
Hypothesis tBHQ exacerbates immune response and clinical symptoms of anaphylaxis in response to food allergen
Why doesnrsquot everyone have immune responses to food The phenomenon of oral tolerance
1 Wk 1 Wk 1 Wk
Bleed Bleed Bleed
OVA OVA OVA OVA
Experimental design for ovalbumin transdermal exposure
1 Wk 1 Wk
OVA orally
Symptoms of anaphylaxis
Control
tBHQ (0001)
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
12
Summary
bull tBHQ inhibits production of the cytokines IL-2 and IFNg in human T cells (suggesting a possible effect on host immunity)
bullActivation of Nrf2 by the food preservative tBHQ increases production of Th2 cytokines by CD4+ T cells
bull tBHQ increases activity and expression of the Th2-regulating gene GATA-3 and this is dependent on Nrf2
bull Our preliminary data suggests that low doses of tBHQ exacerbate immune response and clinical symptoms in an animal model of food allergy
Cell-mediated Immunity
Parasite immunity
Nrf2
In the presence of Nrf2 Th1 and Th2 responses are in balance
Th1 Th2
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
13
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
bull Rockwell Lab ndash Heather Dover
ndash Jenna Bursley
ndash Alex Turley
ndash Joe Zagorski
ndash Holly Cline
bull Dr Curtis D Klaassen
bull Klaassen Laboratory ndash Scott Reisman
bull Dr Bryan Copple ndash Copple Lab
bull Dr Venu Gangur ndash Tina Ortiz
ndash Radhakrishna Para
ndash Babu Gonipeta
bull Dr Jim Pestka
bull Dr Patrick Fields ndash Mingcai Zhang
ndash Manouela Ortega
Acknowledgments
This project was funded by NIH grants K99R00 ES018885 R01 ES024966 T32 ES007255 T32 GM092715
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
3
Branum et al 2008 NCHS Data Brief
Basic Immunology
Janeway et al ldquoImmunobiology The immune system in health and diseaserdquo 2005 6th Ed Garland Science Pub
Immune system overview
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
4
Immune Response during Food Allergy
Th0
Th1
Th2
Th17
Treg
Tfh
Intracellular pathogens
Helminths nematodes
Extracellular pathogens
Prevention of autoimmunity
B cell activation
T cell differentiation
Th0
Th1
Th2
Th17
Treg
Tfh
IFNg
IL-4 IL-5 IL-13
IL-17A IL-17F IL-22 IL-21
TGFb IL-10
IL-4 IL-21
CD4+ cell
uarr TH1 differentiation
Cytotoxic T Lymphocyte
activation
darr TH2 differentiation
uarr Immunoproteasome (uarr Ag presentation)
Cytokine production by
macs (TNFa IL-1b IL-6 amp IL-12)
uarr IFNg
uarr IFNg
uarr Macrophage activation
Function and importance of IFNg
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
5
CD4+ cell
darr TH1 differentiation
uarr TH2 differentiation
B cell proliferation
uarr M2 cells ldquoalternative activationrdquo of macs
uarr IL-4
uarr IL-4
darr M1 cells macrophage
ldquohelprdquo in Th1 activity
Function and importance of IL-4
uarr IgE production in B cells (ldquoclass switchingrdquo)
Immune response to food allergen
IL-4 a mediator of immunity against parasites
What can basic research tell us Role of food preservatives
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
6
Nrf2 activators are commonly used food preservatives
bull tBHQ
bull BHA
bull BHT
Marketers such as Starbucks discover that simple sells
By Bruce Horowitz USA Today October 28 2009
ldquoConsumers these days not only want to know whats in the stuff they eat
and drink mdash they want to know whats
not In a nation bedeviled by a
whirlwind of food scares and
mounting worries about the
healthiness of a plethora of things
commonly used in processed foods
folks increasingly are demanding
cleaner food labels no artificial food
colorings (some of which have been
linked to hyperactivity in children) no
chemical additives (such as MSG) and
no chemical preservatives (such as
BHA) If they cant pronounce it
consumers dont want itrdquo
Zagorski et al 2013 Toxicological Sciences
tBHQ modulates cytokine production by human T cells
Joe Zagorski
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
7
Turley et al 2015 Cytokine
tBHQ modulates cytokine production by immune cells from human blood
Alex Turley
Turley et al 2015 Cytokine
T cells isolated from human blood are very sensitive to the effect of the food preservative tBHQ
ARE
Nrf2 Maf
Coactivators
Keap1
Nrf2 Cul3
Roc1 E2
Schematic representation of Nrf2 activation
Nrf2 Activation
Oxidative stress
Electrophilic
stimuli
15d-PGJ2
Target genes
Antioxidant
Detoxification
Metabolism
Anti-inflammatory
Proteasome ARE
Nrf2 Maf
Coactivators
Keap1
Nrf2 Cul3
Roc1 E2
Schematic representation of Nrf2 activation
Nrf2 Activation
tBHQ
Target genes
Antioxidant
Detoxification
Metabolism
Anti-inflammatory
Proteasome
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
8
The Nrf2 activator tBHQ causes marked inhibition of early IFNg production in activated splenic T cells
IFNg
BKG 0 001 01 1 10 0
20000
40000
60000
80000
ND ND
IFN
g (p
gm
l)
tBHQ (mM)
A
aCD3aCD28
VH 001 01 05 1 0
20
40
60
80
100
120
140
160
tBHQ (mM)
IFN
g m
RN
A (
V
H)
dagger dagger
Dagger
Dagger
Dagger Dagger
Wild-type Nrf2-null
B IFNg
0
10
20
30
40
50
IFN
g m
RN
A
(Fo
ld i
nd
ucti
on
o
ver
BK
G)
WT WT Null Null Nrf2
aCD3aCD28 BKG
C
TH0
TH1 TH2
IFNg IL-4 IL-5 IL-13
Differentiation of CD4 (helper) T cells
Extracellular amp intracellular
pathogens
Asthma amp Allergy
TH0
TH1 TH2
IFNg IL-4 IL-5 IL-13
Differentiation of CD4+ (helper) T cells
Extracellular amp intracellular
pathogens
Asthma amp Allergy
How do we know T cells have differentiated
bull Itrsquos tricky
bull Functional studies cytokine
bull Effect over time
ndash Short-term le24h
ndash Relatively long-term ge96h
ndash Commitment ge96h
bull Genes that regulate T cell differentiation GATA-3 T-bet
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
9
VH 001 01 05 1
IFN
(p
gm
l)
0
1000
2000
3000
4000
5000
6000 Wild-type
Nrf2-null
aCD3aCD28
tBHQ (mM)
IFNg
Dagger
Dagger
Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-4
(p
gm
l)
0
20
40
60
80
100
120
140 IL-4
Dagger Dagger Dagger Dagger
Dagger
aCD3aCD28
tBHQ (mM)
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-5
(p
gm
l)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-5
VH 001 01 05 1
IL-1
3 (
pg
ml)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-13
Dagger Dagger Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
aCD3aCD28
tBHQ (mM)
Dagger Dagger Dagger Dagger Dagger
aCD3aCD28
tBHQ (mM)
Rockwell et al 2012
VH 001 01 05 1
TN
F (
pg
ml)
0
50
100
150
200
250
aCD3aCD28
tBHQ (mM)
TNFa
Dagger
Dagger
Dagger
VH 001 01 05 1
IL-1
0 (
pg
ml)
0
500
1000
1500
2000
2500
aCD3aCD28
tBHQ (mM)
IL-10
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
10
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
T-bet (Th1 transcription factor)
Restimulated
Nrf2 activation inhibits the binding activity of T-bet the master regulator of Th1 differentiation
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
Restimulated
Nrf2 activation inhibits T-bet binding activity
IFN
(pg
ml)
0
1000
2000
3000
4000
Restimulated
VH tBHQ
IFNg
T-bet (Th1)
Dagger Dagger
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
Dagger Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
IL-4
(p
gm
l)
0
200
400
600
800
1000
1200
1400
Restimulated
VH tBHQ
IL-4
Dagger Dagger
Dagger Dagger
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
11
GA
TA
-3 m
RN
A (
Fo
ld in
du
cti
on
over
WT
VH
Co
ntr
ol)
00
05
10
15
20
25
30 Wild-type
Nrf2-null
VH 0001 001 01 1
Nrf2 activation increases GATA-3 mRNA expression
anti-CD3anti-CD28
tBHQ
Dagger
Dagger Dagger Dagger
GATA-3
Hypothesis tBHQ exacerbates immune response and clinical symptoms of anaphylaxis in response to food allergen
Why doesnrsquot everyone have immune responses to food The phenomenon of oral tolerance
1 Wk 1 Wk 1 Wk
Bleed Bleed Bleed
OVA OVA OVA OVA
Experimental design for ovalbumin transdermal exposure
1 Wk 1 Wk
OVA orally
Symptoms of anaphylaxis
Control
tBHQ (0001)
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
12
Summary
bull tBHQ inhibits production of the cytokines IL-2 and IFNg in human T cells (suggesting a possible effect on host immunity)
bullActivation of Nrf2 by the food preservative tBHQ increases production of Th2 cytokines by CD4+ T cells
bull tBHQ increases activity and expression of the Th2-regulating gene GATA-3 and this is dependent on Nrf2
bull Our preliminary data suggests that low doses of tBHQ exacerbate immune response and clinical symptoms in an animal model of food allergy
Cell-mediated Immunity
Parasite immunity
Nrf2
In the presence of Nrf2 Th1 and Th2 responses are in balance
Th1 Th2
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
13
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
bull Rockwell Lab ndash Heather Dover
ndash Jenna Bursley
ndash Alex Turley
ndash Joe Zagorski
ndash Holly Cline
bull Dr Curtis D Klaassen
bull Klaassen Laboratory ndash Scott Reisman
bull Dr Bryan Copple ndash Copple Lab
bull Dr Venu Gangur ndash Tina Ortiz
ndash Radhakrishna Para
ndash Babu Gonipeta
bull Dr Jim Pestka
bull Dr Patrick Fields ndash Mingcai Zhang
ndash Manouela Ortega
Acknowledgments
This project was funded by NIH grants K99R00 ES018885 R01 ES024966 T32 ES007255 T32 GM092715
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
4
Immune Response during Food Allergy
Th0
Th1
Th2
Th17
Treg
Tfh
Intracellular pathogens
Helminths nematodes
Extracellular pathogens
Prevention of autoimmunity
B cell activation
T cell differentiation
Th0
Th1
Th2
Th17
Treg
Tfh
IFNg
IL-4 IL-5 IL-13
IL-17A IL-17F IL-22 IL-21
TGFb IL-10
IL-4 IL-21
CD4+ cell
uarr TH1 differentiation
Cytotoxic T Lymphocyte
activation
darr TH2 differentiation
uarr Immunoproteasome (uarr Ag presentation)
Cytokine production by
macs (TNFa IL-1b IL-6 amp IL-12)
uarr IFNg
uarr IFNg
uarr Macrophage activation
Function and importance of IFNg
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
5
CD4+ cell
darr TH1 differentiation
uarr TH2 differentiation
B cell proliferation
uarr M2 cells ldquoalternative activationrdquo of macs
uarr IL-4
uarr IL-4
darr M1 cells macrophage
ldquohelprdquo in Th1 activity
Function and importance of IL-4
uarr IgE production in B cells (ldquoclass switchingrdquo)
Immune response to food allergen
IL-4 a mediator of immunity against parasites
What can basic research tell us Role of food preservatives
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
6
Nrf2 activators are commonly used food preservatives
bull tBHQ
bull BHA
bull BHT
Marketers such as Starbucks discover that simple sells
By Bruce Horowitz USA Today October 28 2009
ldquoConsumers these days not only want to know whats in the stuff they eat
and drink mdash they want to know whats
not In a nation bedeviled by a
whirlwind of food scares and
mounting worries about the
healthiness of a plethora of things
commonly used in processed foods
folks increasingly are demanding
cleaner food labels no artificial food
colorings (some of which have been
linked to hyperactivity in children) no
chemical additives (such as MSG) and
no chemical preservatives (such as
BHA) If they cant pronounce it
consumers dont want itrdquo
Zagorski et al 2013 Toxicological Sciences
tBHQ modulates cytokine production by human T cells
Joe Zagorski
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
7
Turley et al 2015 Cytokine
tBHQ modulates cytokine production by immune cells from human blood
Alex Turley
Turley et al 2015 Cytokine
T cells isolated from human blood are very sensitive to the effect of the food preservative tBHQ
ARE
Nrf2 Maf
Coactivators
Keap1
Nrf2 Cul3
Roc1 E2
Schematic representation of Nrf2 activation
Nrf2 Activation
Oxidative stress
Electrophilic
stimuli
15d-PGJ2
Target genes
Antioxidant
Detoxification
Metabolism
Anti-inflammatory
Proteasome ARE
Nrf2 Maf
Coactivators
Keap1
Nrf2 Cul3
Roc1 E2
Schematic representation of Nrf2 activation
Nrf2 Activation
tBHQ
Target genes
Antioxidant
Detoxification
Metabolism
Anti-inflammatory
Proteasome
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
8
The Nrf2 activator tBHQ causes marked inhibition of early IFNg production in activated splenic T cells
IFNg
BKG 0 001 01 1 10 0
20000
40000
60000
80000
ND ND
IFN
g (p
gm
l)
tBHQ (mM)
A
aCD3aCD28
VH 001 01 05 1 0
20
40
60
80
100
120
140
160
tBHQ (mM)
IFN
g m
RN
A (
V
H)
dagger dagger
Dagger
Dagger
Dagger Dagger
Wild-type Nrf2-null
B IFNg
0
10
20
30
40
50
IFN
g m
RN
A
(Fo
ld i
nd
ucti
on
o
ver
BK
G)
WT WT Null Null Nrf2
aCD3aCD28 BKG
C
TH0
TH1 TH2
IFNg IL-4 IL-5 IL-13
Differentiation of CD4 (helper) T cells
Extracellular amp intracellular
pathogens
Asthma amp Allergy
TH0
TH1 TH2
IFNg IL-4 IL-5 IL-13
Differentiation of CD4+ (helper) T cells
Extracellular amp intracellular
pathogens
Asthma amp Allergy
How do we know T cells have differentiated
bull Itrsquos tricky
bull Functional studies cytokine
bull Effect over time
ndash Short-term le24h
ndash Relatively long-term ge96h
ndash Commitment ge96h
bull Genes that regulate T cell differentiation GATA-3 T-bet
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
9
VH 001 01 05 1
IFN
(p
gm
l)
0
1000
2000
3000
4000
5000
6000 Wild-type
Nrf2-null
aCD3aCD28
tBHQ (mM)
IFNg
Dagger
Dagger
Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-4
(p
gm
l)
0
20
40
60
80
100
120
140 IL-4
Dagger Dagger Dagger Dagger
Dagger
aCD3aCD28
tBHQ (mM)
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-5
(p
gm
l)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-5
VH 001 01 05 1
IL-1
3 (
pg
ml)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-13
Dagger Dagger Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
aCD3aCD28
tBHQ (mM)
Dagger Dagger Dagger Dagger Dagger
aCD3aCD28
tBHQ (mM)
Rockwell et al 2012
VH 001 01 05 1
TN
F (
pg
ml)
0
50
100
150
200
250
aCD3aCD28
tBHQ (mM)
TNFa
Dagger
Dagger
Dagger
VH 001 01 05 1
IL-1
0 (
pg
ml)
0
500
1000
1500
2000
2500
aCD3aCD28
tBHQ (mM)
IL-10
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
10
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
T-bet (Th1 transcription factor)
Restimulated
Nrf2 activation inhibits the binding activity of T-bet the master regulator of Th1 differentiation
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
Restimulated
Nrf2 activation inhibits T-bet binding activity
IFN
(pg
ml)
0
1000
2000
3000
4000
Restimulated
VH tBHQ
IFNg
T-bet (Th1)
Dagger Dagger
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
Dagger Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
IL-4
(p
gm
l)
0
200
400
600
800
1000
1200
1400
Restimulated
VH tBHQ
IL-4
Dagger Dagger
Dagger Dagger
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
11
GA
TA
-3 m
RN
A (
Fo
ld in
du
cti
on
over
WT
VH
Co
ntr
ol)
00
05
10
15
20
25
30 Wild-type
Nrf2-null
VH 0001 001 01 1
Nrf2 activation increases GATA-3 mRNA expression
anti-CD3anti-CD28
tBHQ
Dagger
Dagger Dagger Dagger
GATA-3
Hypothesis tBHQ exacerbates immune response and clinical symptoms of anaphylaxis in response to food allergen
Why doesnrsquot everyone have immune responses to food The phenomenon of oral tolerance
1 Wk 1 Wk 1 Wk
Bleed Bleed Bleed
OVA OVA OVA OVA
Experimental design for ovalbumin transdermal exposure
1 Wk 1 Wk
OVA orally
Symptoms of anaphylaxis
Control
tBHQ (0001)
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
12
Summary
bull tBHQ inhibits production of the cytokines IL-2 and IFNg in human T cells (suggesting a possible effect on host immunity)
bullActivation of Nrf2 by the food preservative tBHQ increases production of Th2 cytokines by CD4+ T cells
bull tBHQ increases activity and expression of the Th2-regulating gene GATA-3 and this is dependent on Nrf2
bull Our preliminary data suggests that low doses of tBHQ exacerbate immune response and clinical symptoms in an animal model of food allergy
Cell-mediated Immunity
Parasite immunity
Nrf2
In the presence of Nrf2 Th1 and Th2 responses are in balance
Th1 Th2
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
13
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
bull Rockwell Lab ndash Heather Dover
ndash Jenna Bursley
ndash Alex Turley
ndash Joe Zagorski
ndash Holly Cline
bull Dr Curtis D Klaassen
bull Klaassen Laboratory ndash Scott Reisman
bull Dr Bryan Copple ndash Copple Lab
bull Dr Venu Gangur ndash Tina Ortiz
ndash Radhakrishna Para
ndash Babu Gonipeta
bull Dr Jim Pestka
bull Dr Patrick Fields ndash Mingcai Zhang
ndash Manouela Ortega
Acknowledgments
This project was funded by NIH grants K99R00 ES018885 R01 ES024966 T32 ES007255 T32 GM092715
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
5
CD4+ cell
darr TH1 differentiation
uarr TH2 differentiation
B cell proliferation
uarr M2 cells ldquoalternative activationrdquo of macs
uarr IL-4
uarr IL-4
darr M1 cells macrophage
ldquohelprdquo in Th1 activity
Function and importance of IL-4
uarr IgE production in B cells (ldquoclass switchingrdquo)
Immune response to food allergen
IL-4 a mediator of immunity against parasites
What can basic research tell us Role of food preservatives
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
6
Nrf2 activators are commonly used food preservatives
bull tBHQ
bull BHA
bull BHT
Marketers such as Starbucks discover that simple sells
By Bruce Horowitz USA Today October 28 2009
ldquoConsumers these days not only want to know whats in the stuff they eat
and drink mdash they want to know whats
not In a nation bedeviled by a
whirlwind of food scares and
mounting worries about the
healthiness of a plethora of things
commonly used in processed foods
folks increasingly are demanding
cleaner food labels no artificial food
colorings (some of which have been
linked to hyperactivity in children) no
chemical additives (such as MSG) and
no chemical preservatives (such as
BHA) If they cant pronounce it
consumers dont want itrdquo
Zagorski et al 2013 Toxicological Sciences
tBHQ modulates cytokine production by human T cells
Joe Zagorski
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
7
Turley et al 2015 Cytokine
tBHQ modulates cytokine production by immune cells from human blood
Alex Turley
Turley et al 2015 Cytokine
T cells isolated from human blood are very sensitive to the effect of the food preservative tBHQ
ARE
Nrf2 Maf
Coactivators
Keap1
Nrf2 Cul3
Roc1 E2
Schematic representation of Nrf2 activation
Nrf2 Activation
Oxidative stress
Electrophilic
stimuli
15d-PGJ2
Target genes
Antioxidant
Detoxification
Metabolism
Anti-inflammatory
Proteasome ARE
Nrf2 Maf
Coactivators
Keap1
Nrf2 Cul3
Roc1 E2
Schematic representation of Nrf2 activation
Nrf2 Activation
tBHQ
Target genes
Antioxidant
Detoxification
Metabolism
Anti-inflammatory
Proteasome
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
8
The Nrf2 activator tBHQ causes marked inhibition of early IFNg production in activated splenic T cells
IFNg
BKG 0 001 01 1 10 0
20000
40000
60000
80000
ND ND
IFN
g (p
gm
l)
tBHQ (mM)
A
aCD3aCD28
VH 001 01 05 1 0
20
40
60
80
100
120
140
160
tBHQ (mM)
IFN
g m
RN
A (
V
H)
dagger dagger
Dagger
Dagger
Dagger Dagger
Wild-type Nrf2-null
B IFNg
0
10
20
30
40
50
IFN
g m
RN
A
(Fo
ld i
nd
ucti
on
o
ver
BK
G)
WT WT Null Null Nrf2
aCD3aCD28 BKG
C
TH0
TH1 TH2
IFNg IL-4 IL-5 IL-13
Differentiation of CD4 (helper) T cells
Extracellular amp intracellular
pathogens
Asthma amp Allergy
TH0
TH1 TH2
IFNg IL-4 IL-5 IL-13
Differentiation of CD4+ (helper) T cells
Extracellular amp intracellular
pathogens
Asthma amp Allergy
How do we know T cells have differentiated
bull Itrsquos tricky
bull Functional studies cytokine
bull Effect over time
ndash Short-term le24h
ndash Relatively long-term ge96h
ndash Commitment ge96h
bull Genes that regulate T cell differentiation GATA-3 T-bet
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
9
VH 001 01 05 1
IFN
(p
gm
l)
0
1000
2000
3000
4000
5000
6000 Wild-type
Nrf2-null
aCD3aCD28
tBHQ (mM)
IFNg
Dagger
Dagger
Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-4
(p
gm
l)
0
20
40
60
80
100
120
140 IL-4
Dagger Dagger Dagger Dagger
Dagger
aCD3aCD28
tBHQ (mM)
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-5
(p
gm
l)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-5
VH 001 01 05 1
IL-1
3 (
pg
ml)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-13
Dagger Dagger Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
aCD3aCD28
tBHQ (mM)
Dagger Dagger Dagger Dagger Dagger
aCD3aCD28
tBHQ (mM)
Rockwell et al 2012
VH 001 01 05 1
TN
F (
pg
ml)
0
50
100
150
200
250
aCD3aCD28
tBHQ (mM)
TNFa
Dagger
Dagger
Dagger
VH 001 01 05 1
IL-1
0 (
pg
ml)
0
500
1000
1500
2000
2500
aCD3aCD28
tBHQ (mM)
IL-10
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
10
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
T-bet (Th1 transcription factor)
Restimulated
Nrf2 activation inhibits the binding activity of T-bet the master regulator of Th1 differentiation
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
Restimulated
Nrf2 activation inhibits T-bet binding activity
IFN
(pg
ml)
0
1000
2000
3000
4000
Restimulated
VH tBHQ
IFNg
T-bet (Th1)
Dagger Dagger
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
Dagger Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
IL-4
(p
gm
l)
0
200
400
600
800
1000
1200
1400
Restimulated
VH tBHQ
IL-4
Dagger Dagger
Dagger Dagger
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
11
GA
TA
-3 m
RN
A (
Fo
ld in
du
cti
on
over
WT
VH
Co
ntr
ol)
00
05
10
15
20
25
30 Wild-type
Nrf2-null
VH 0001 001 01 1
Nrf2 activation increases GATA-3 mRNA expression
anti-CD3anti-CD28
tBHQ
Dagger
Dagger Dagger Dagger
GATA-3
Hypothesis tBHQ exacerbates immune response and clinical symptoms of anaphylaxis in response to food allergen
Why doesnrsquot everyone have immune responses to food The phenomenon of oral tolerance
1 Wk 1 Wk 1 Wk
Bleed Bleed Bleed
OVA OVA OVA OVA
Experimental design for ovalbumin transdermal exposure
1 Wk 1 Wk
OVA orally
Symptoms of anaphylaxis
Control
tBHQ (0001)
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
12
Summary
bull tBHQ inhibits production of the cytokines IL-2 and IFNg in human T cells (suggesting a possible effect on host immunity)
bullActivation of Nrf2 by the food preservative tBHQ increases production of Th2 cytokines by CD4+ T cells
bull tBHQ increases activity and expression of the Th2-regulating gene GATA-3 and this is dependent on Nrf2
bull Our preliminary data suggests that low doses of tBHQ exacerbate immune response and clinical symptoms in an animal model of food allergy
Cell-mediated Immunity
Parasite immunity
Nrf2
In the presence of Nrf2 Th1 and Th2 responses are in balance
Th1 Th2
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
13
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
bull Rockwell Lab ndash Heather Dover
ndash Jenna Bursley
ndash Alex Turley
ndash Joe Zagorski
ndash Holly Cline
bull Dr Curtis D Klaassen
bull Klaassen Laboratory ndash Scott Reisman
bull Dr Bryan Copple ndash Copple Lab
bull Dr Venu Gangur ndash Tina Ortiz
ndash Radhakrishna Para
ndash Babu Gonipeta
bull Dr Jim Pestka
bull Dr Patrick Fields ndash Mingcai Zhang
ndash Manouela Ortega
Acknowledgments
This project was funded by NIH grants K99R00 ES018885 R01 ES024966 T32 ES007255 T32 GM092715
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
6
Nrf2 activators are commonly used food preservatives
bull tBHQ
bull BHA
bull BHT
Marketers such as Starbucks discover that simple sells
By Bruce Horowitz USA Today October 28 2009
ldquoConsumers these days not only want to know whats in the stuff they eat
and drink mdash they want to know whats
not In a nation bedeviled by a
whirlwind of food scares and
mounting worries about the
healthiness of a plethora of things
commonly used in processed foods
folks increasingly are demanding
cleaner food labels no artificial food
colorings (some of which have been
linked to hyperactivity in children) no
chemical additives (such as MSG) and
no chemical preservatives (such as
BHA) If they cant pronounce it
consumers dont want itrdquo
Zagorski et al 2013 Toxicological Sciences
tBHQ modulates cytokine production by human T cells
Joe Zagorski
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
7
Turley et al 2015 Cytokine
tBHQ modulates cytokine production by immune cells from human blood
Alex Turley
Turley et al 2015 Cytokine
T cells isolated from human blood are very sensitive to the effect of the food preservative tBHQ
ARE
Nrf2 Maf
Coactivators
Keap1
Nrf2 Cul3
Roc1 E2
Schematic representation of Nrf2 activation
Nrf2 Activation
Oxidative stress
Electrophilic
stimuli
15d-PGJ2
Target genes
Antioxidant
Detoxification
Metabolism
Anti-inflammatory
Proteasome ARE
Nrf2 Maf
Coactivators
Keap1
Nrf2 Cul3
Roc1 E2
Schematic representation of Nrf2 activation
Nrf2 Activation
tBHQ
Target genes
Antioxidant
Detoxification
Metabolism
Anti-inflammatory
Proteasome
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
8
The Nrf2 activator tBHQ causes marked inhibition of early IFNg production in activated splenic T cells
IFNg
BKG 0 001 01 1 10 0
20000
40000
60000
80000
ND ND
IFN
g (p
gm
l)
tBHQ (mM)
A
aCD3aCD28
VH 001 01 05 1 0
20
40
60
80
100
120
140
160
tBHQ (mM)
IFN
g m
RN
A (
V
H)
dagger dagger
Dagger
Dagger
Dagger Dagger
Wild-type Nrf2-null
B IFNg
0
10
20
30
40
50
IFN
g m
RN
A
(Fo
ld i
nd
ucti
on
o
ver
BK
G)
WT WT Null Null Nrf2
aCD3aCD28 BKG
C
TH0
TH1 TH2
IFNg IL-4 IL-5 IL-13
Differentiation of CD4 (helper) T cells
Extracellular amp intracellular
pathogens
Asthma amp Allergy
TH0
TH1 TH2
IFNg IL-4 IL-5 IL-13
Differentiation of CD4+ (helper) T cells
Extracellular amp intracellular
pathogens
Asthma amp Allergy
How do we know T cells have differentiated
bull Itrsquos tricky
bull Functional studies cytokine
bull Effect over time
ndash Short-term le24h
ndash Relatively long-term ge96h
ndash Commitment ge96h
bull Genes that regulate T cell differentiation GATA-3 T-bet
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
9
VH 001 01 05 1
IFN
(p
gm
l)
0
1000
2000
3000
4000
5000
6000 Wild-type
Nrf2-null
aCD3aCD28
tBHQ (mM)
IFNg
Dagger
Dagger
Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-4
(p
gm
l)
0
20
40
60
80
100
120
140 IL-4
Dagger Dagger Dagger Dagger
Dagger
aCD3aCD28
tBHQ (mM)
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-5
(p
gm
l)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-5
VH 001 01 05 1
IL-1
3 (
pg
ml)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-13
Dagger Dagger Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
aCD3aCD28
tBHQ (mM)
Dagger Dagger Dagger Dagger Dagger
aCD3aCD28
tBHQ (mM)
Rockwell et al 2012
VH 001 01 05 1
TN
F (
pg
ml)
0
50
100
150
200
250
aCD3aCD28
tBHQ (mM)
TNFa
Dagger
Dagger
Dagger
VH 001 01 05 1
IL-1
0 (
pg
ml)
0
500
1000
1500
2000
2500
aCD3aCD28
tBHQ (mM)
IL-10
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
10
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
T-bet (Th1 transcription factor)
Restimulated
Nrf2 activation inhibits the binding activity of T-bet the master regulator of Th1 differentiation
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
Restimulated
Nrf2 activation inhibits T-bet binding activity
IFN
(pg
ml)
0
1000
2000
3000
4000
Restimulated
VH tBHQ
IFNg
T-bet (Th1)
Dagger Dagger
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
Dagger Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
IL-4
(p
gm
l)
0
200
400
600
800
1000
1200
1400
Restimulated
VH tBHQ
IL-4
Dagger Dagger
Dagger Dagger
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
11
GA
TA
-3 m
RN
A (
Fo
ld in
du
cti
on
over
WT
VH
Co
ntr
ol)
00
05
10
15
20
25
30 Wild-type
Nrf2-null
VH 0001 001 01 1
Nrf2 activation increases GATA-3 mRNA expression
anti-CD3anti-CD28
tBHQ
Dagger
Dagger Dagger Dagger
GATA-3
Hypothesis tBHQ exacerbates immune response and clinical symptoms of anaphylaxis in response to food allergen
Why doesnrsquot everyone have immune responses to food The phenomenon of oral tolerance
1 Wk 1 Wk 1 Wk
Bleed Bleed Bleed
OVA OVA OVA OVA
Experimental design for ovalbumin transdermal exposure
1 Wk 1 Wk
OVA orally
Symptoms of anaphylaxis
Control
tBHQ (0001)
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
12
Summary
bull tBHQ inhibits production of the cytokines IL-2 and IFNg in human T cells (suggesting a possible effect on host immunity)
bullActivation of Nrf2 by the food preservative tBHQ increases production of Th2 cytokines by CD4+ T cells
bull tBHQ increases activity and expression of the Th2-regulating gene GATA-3 and this is dependent on Nrf2
bull Our preliminary data suggests that low doses of tBHQ exacerbate immune response and clinical symptoms in an animal model of food allergy
Cell-mediated Immunity
Parasite immunity
Nrf2
In the presence of Nrf2 Th1 and Th2 responses are in balance
Th1 Th2
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
13
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
bull Rockwell Lab ndash Heather Dover
ndash Jenna Bursley
ndash Alex Turley
ndash Joe Zagorski
ndash Holly Cline
bull Dr Curtis D Klaassen
bull Klaassen Laboratory ndash Scott Reisman
bull Dr Bryan Copple ndash Copple Lab
bull Dr Venu Gangur ndash Tina Ortiz
ndash Radhakrishna Para
ndash Babu Gonipeta
bull Dr Jim Pestka
bull Dr Patrick Fields ndash Mingcai Zhang
ndash Manouela Ortega
Acknowledgments
This project was funded by NIH grants K99R00 ES018885 R01 ES024966 T32 ES007255 T32 GM092715
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
7
Turley et al 2015 Cytokine
tBHQ modulates cytokine production by immune cells from human blood
Alex Turley
Turley et al 2015 Cytokine
T cells isolated from human blood are very sensitive to the effect of the food preservative tBHQ
ARE
Nrf2 Maf
Coactivators
Keap1
Nrf2 Cul3
Roc1 E2
Schematic representation of Nrf2 activation
Nrf2 Activation
Oxidative stress
Electrophilic
stimuli
15d-PGJ2
Target genes
Antioxidant
Detoxification
Metabolism
Anti-inflammatory
Proteasome ARE
Nrf2 Maf
Coactivators
Keap1
Nrf2 Cul3
Roc1 E2
Schematic representation of Nrf2 activation
Nrf2 Activation
tBHQ
Target genes
Antioxidant
Detoxification
Metabolism
Anti-inflammatory
Proteasome
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
8
The Nrf2 activator tBHQ causes marked inhibition of early IFNg production in activated splenic T cells
IFNg
BKG 0 001 01 1 10 0
20000
40000
60000
80000
ND ND
IFN
g (p
gm
l)
tBHQ (mM)
A
aCD3aCD28
VH 001 01 05 1 0
20
40
60
80
100
120
140
160
tBHQ (mM)
IFN
g m
RN
A (
V
H)
dagger dagger
Dagger
Dagger
Dagger Dagger
Wild-type Nrf2-null
B IFNg
0
10
20
30
40
50
IFN
g m
RN
A
(Fo
ld i
nd
ucti
on
o
ver
BK
G)
WT WT Null Null Nrf2
aCD3aCD28 BKG
C
TH0
TH1 TH2
IFNg IL-4 IL-5 IL-13
Differentiation of CD4 (helper) T cells
Extracellular amp intracellular
pathogens
Asthma amp Allergy
TH0
TH1 TH2
IFNg IL-4 IL-5 IL-13
Differentiation of CD4+ (helper) T cells
Extracellular amp intracellular
pathogens
Asthma amp Allergy
How do we know T cells have differentiated
bull Itrsquos tricky
bull Functional studies cytokine
bull Effect over time
ndash Short-term le24h
ndash Relatively long-term ge96h
ndash Commitment ge96h
bull Genes that regulate T cell differentiation GATA-3 T-bet
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
9
VH 001 01 05 1
IFN
(p
gm
l)
0
1000
2000
3000
4000
5000
6000 Wild-type
Nrf2-null
aCD3aCD28
tBHQ (mM)
IFNg
Dagger
Dagger
Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-4
(p
gm
l)
0
20
40
60
80
100
120
140 IL-4
Dagger Dagger Dagger Dagger
Dagger
aCD3aCD28
tBHQ (mM)
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-5
(p
gm
l)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-5
VH 001 01 05 1
IL-1
3 (
pg
ml)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-13
Dagger Dagger Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
aCD3aCD28
tBHQ (mM)
Dagger Dagger Dagger Dagger Dagger
aCD3aCD28
tBHQ (mM)
Rockwell et al 2012
VH 001 01 05 1
TN
F (
pg
ml)
0
50
100
150
200
250
aCD3aCD28
tBHQ (mM)
TNFa
Dagger
Dagger
Dagger
VH 001 01 05 1
IL-1
0 (
pg
ml)
0
500
1000
1500
2000
2500
aCD3aCD28
tBHQ (mM)
IL-10
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
10
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
T-bet (Th1 transcription factor)
Restimulated
Nrf2 activation inhibits the binding activity of T-bet the master regulator of Th1 differentiation
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
Restimulated
Nrf2 activation inhibits T-bet binding activity
IFN
(pg
ml)
0
1000
2000
3000
4000
Restimulated
VH tBHQ
IFNg
T-bet (Th1)
Dagger Dagger
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
Dagger Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
IL-4
(p
gm
l)
0
200
400
600
800
1000
1200
1400
Restimulated
VH tBHQ
IL-4
Dagger Dagger
Dagger Dagger
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
11
GA
TA
-3 m
RN
A (
Fo
ld in
du
cti
on
over
WT
VH
Co
ntr
ol)
00
05
10
15
20
25
30 Wild-type
Nrf2-null
VH 0001 001 01 1
Nrf2 activation increases GATA-3 mRNA expression
anti-CD3anti-CD28
tBHQ
Dagger
Dagger Dagger Dagger
GATA-3
Hypothesis tBHQ exacerbates immune response and clinical symptoms of anaphylaxis in response to food allergen
Why doesnrsquot everyone have immune responses to food The phenomenon of oral tolerance
1 Wk 1 Wk 1 Wk
Bleed Bleed Bleed
OVA OVA OVA OVA
Experimental design for ovalbumin transdermal exposure
1 Wk 1 Wk
OVA orally
Symptoms of anaphylaxis
Control
tBHQ (0001)
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
12
Summary
bull tBHQ inhibits production of the cytokines IL-2 and IFNg in human T cells (suggesting a possible effect on host immunity)
bullActivation of Nrf2 by the food preservative tBHQ increases production of Th2 cytokines by CD4+ T cells
bull tBHQ increases activity and expression of the Th2-regulating gene GATA-3 and this is dependent on Nrf2
bull Our preliminary data suggests that low doses of tBHQ exacerbate immune response and clinical symptoms in an animal model of food allergy
Cell-mediated Immunity
Parasite immunity
Nrf2
In the presence of Nrf2 Th1 and Th2 responses are in balance
Th1 Th2
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
13
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
bull Rockwell Lab ndash Heather Dover
ndash Jenna Bursley
ndash Alex Turley
ndash Joe Zagorski
ndash Holly Cline
bull Dr Curtis D Klaassen
bull Klaassen Laboratory ndash Scott Reisman
bull Dr Bryan Copple ndash Copple Lab
bull Dr Venu Gangur ndash Tina Ortiz
ndash Radhakrishna Para
ndash Babu Gonipeta
bull Dr Jim Pestka
bull Dr Patrick Fields ndash Mingcai Zhang
ndash Manouela Ortega
Acknowledgments
This project was funded by NIH grants K99R00 ES018885 R01 ES024966 T32 ES007255 T32 GM092715
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
8
The Nrf2 activator tBHQ causes marked inhibition of early IFNg production in activated splenic T cells
IFNg
BKG 0 001 01 1 10 0
20000
40000
60000
80000
ND ND
IFN
g (p
gm
l)
tBHQ (mM)
A
aCD3aCD28
VH 001 01 05 1 0
20
40
60
80
100
120
140
160
tBHQ (mM)
IFN
g m
RN
A (
V
H)
dagger dagger
Dagger
Dagger
Dagger Dagger
Wild-type Nrf2-null
B IFNg
0
10
20
30
40
50
IFN
g m
RN
A
(Fo
ld i
nd
ucti
on
o
ver
BK
G)
WT WT Null Null Nrf2
aCD3aCD28 BKG
C
TH0
TH1 TH2
IFNg IL-4 IL-5 IL-13
Differentiation of CD4 (helper) T cells
Extracellular amp intracellular
pathogens
Asthma amp Allergy
TH0
TH1 TH2
IFNg IL-4 IL-5 IL-13
Differentiation of CD4+ (helper) T cells
Extracellular amp intracellular
pathogens
Asthma amp Allergy
How do we know T cells have differentiated
bull Itrsquos tricky
bull Functional studies cytokine
bull Effect over time
ndash Short-term le24h
ndash Relatively long-term ge96h
ndash Commitment ge96h
bull Genes that regulate T cell differentiation GATA-3 T-bet
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
9
VH 001 01 05 1
IFN
(p
gm
l)
0
1000
2000
3000
4000
5000
6000 Wild-type
Nrf2-null
aCD3aCD28
tBHQ (mM)
IFNg
Dagger
Dagger
Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-4
(p
gm
l)
0
20
40
60
80
100
120
140 IL-4
Dagger Dagger Dagger Dagger
Dagger
aCD3aCD28
tBHQ (mM)
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-5
(p
gm
l)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-5
VH 001 01 05 1
IL-1
3 (
pg
ml)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-13
Dagger Dagger Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
aCD3aCD28
tBHQ (mM)
Dagger Dagger Dagger Dagger Dagger
aCD3aCD28
tBHQ (mM)
Rockwell et al 2012
VH 001 01 05 1
TN
F (
pg
ml)
0
50
100
150
200
250
aCD3aCD28
tBHQ (mM)
TNFa
Dagger
Dagger
Dagger
VH 001 01 05 1
IL-1
0 (
pg
ml)
0
500
1000
1500
2000
2500
aCD3aCD28
tBHQ (mM)
IL-10
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
10
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
T-bet (Th1 transcription factor)
Restimulated
Nrf2 activation inhibits the binding activity of T-bet the master regulator of Th1 differentiation
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
Restimulated
Nrf2 activation inhibits T-bet binding activity
IFN
(pg
ml)
0
1000
2000
3000
4000
Restimulated
VH tBHQ
IFNg
T-bet (Th1)
Dagger Dagger
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
Dagger Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
IL-4
(p
gm
l)
0
200
400
600
800
1000
1200
1400
Restimulated
VH tBHQ
IL-4
Dagger Dagger
Dagger Dagger
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
11
GA
TA
-3 m
RN
A (
Fo
ld in
du
cti
on
over
WT
VH
Co
ntr
ol)
00
05
10
15
20
25
30 Wild-type
Nrf2-null
VH 0001 001 01 1
Nrf2 activation increases GATA-3 mRNA expression
anti-CD3anti-CD28
tBHQ
Dagger
Dagger Dagger Dagger
GATA-3
Hypothesis tBHQ exacerbates immune response and clinical symptoms of anaphylaxis in response to food allergen
Why doesnrsquot everyone have immune responses to food The phenomenon of oral tolerance
1 Wk 1 Wk 1 Wk
Bleed Bleed Bleed
OVA OVA OVA OVA
Experimental design for ovalbumin transdermal exposure
1 Wk 1 Wk
OVA orally
Symptoms of anaphylaxis
Control
tBHQ (0001)
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
12
Summary
bull tBHQ inhibits production of the cytokines IL-2 and IFNg in human T cells (suggesting a possible effect on host immunity)
bullActivation of Nrf2 by the food preservative tBHQ increases production of Th2 cytokines by CD4+ T cells
bull tBHQ increases activity and expression of the Th2-regulating gene GATA-3 and this is dependent on Nrf2
bull Our preliminary data suggests that low doses of tBHQ exacerbate immune response and clinical symptoms in an animal model of food allergy
Cell-mediated Immunity
Parasite immunity
Nrf2
In the presence of Nrf2 Th1 and Th2 responses are in balance
Th1 Th2
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
13
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
bull Rockwell Lab ndash Heather Dover
ndash Jenna Bursley
ndash Alex Turley
ndash Joe Zagorski
ndash Holly Cline
bull Dr Curtis D Klaassen
bull Klaassen Laboratory ndash Scott Reisman
bull Dr Bryan Copple ndash Copple Lab
bull Dr Venu Gangur ndash Tina Ortiz
ndash Radhakrishna Para
ndash Babu Gonipeta
bull Dr Jim Pestka
bull Dr Patrick Fields ndash Mingcai Zhang
ndash Manouela Ortega
Acknowledgments
This project was funded by NIH grants K99R00 ES018885 R01 ES024966 T32 ES007255 T32 GM092715
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
9
VH 001 01 05 1
IFN
(p
gm
l)
0
1000
2000
3000
4000
5000
6000 Wild-type
Nrf2-null
aCD3aCD28
tBHQ (mM)
IFNg
Dagger
Dagger
Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-4
(p
gm
l)
0
20
40
60
80
100
120
140 IL-4
Dagger Dagger Dagger Dagger
Dagger
aCD3aCD28
tBHQ (mM)
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
VH 001 01 05 1
IL-5
(p
gm
l)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-5
VH 001 01 05 1
IL-1
3 (
pg
ml)
0
200
400
600
800
1000
1200
1400
1600
1800
2000 IL-13
Dagger Dagger Dagger Dagger
Dagger
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
aCD3aCD28
tBHQ (mM)
Dagger Dagger Dagger Dagger Dagger
aCD3aCD28
tBHQ (mM)
Rockwell et al 2012
VH 001 01 05 1
TN
F (
pg
ml)
0
50
100
150
200
250
aCD3aCD28
tBHQ (mM)
TNFa
Dagger
Dagger
Dagger
VH 001 01 05 1
IL-1
0 (
pg
ml)
0
500
1000
1500
2000
2500
aCD3aCD28
tBHQ (mM)
IL-10
The effect of Nrf2 activation on magnetically-isolated CD4+ T cells at 96 hr
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
10
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
T-bet (Th1 transcription factor)
Restimulated
Nrf2 activation inhibits the binding activity of T-bet the master regulator of Th1 differentiation
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
Restimulated
Nrf2 activation inhibits T-bet binding activity
IFN
(pg
ml)
0
1000
2000
3000
4000
Restimulated
VH tBHQ
IFNg
T-bet (Th1)
Dagger Dagger
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
Dagger Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
IL-4
(p
gm
l)
0
200
400
600
800
1000
1200
1400
Restimulated
VH tBHQ
IL-4
Dagger Dagger
Dagger Dagger
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
11
GA
TA
-3 m
RN
A (
Fo
ld in
du
cti
on
over
WT
VH
Co
ntr
ol)
00
05
10
15
20
25
30 Wild-type
Nrf2-null
VH 0001 001 01 1
Nrf2 activation increases GATA-3 mRNA expression
anti-CD3anti-CD28
tBHQ
Dagger
Dagger Dagger Dagger
GATA-3
Hypothesis tBHQ exacerbates immune response and clinical symptoms of anaphylaxis in response to food allergen
Why doesnrsquot everyone have immune responses to food The phenomenon of oral tolerance
1 Wk 1 Wk 1 Wk
Bleed Bleed Bleed
OVA OVA OVA OVA
Experimental design for ovalbumin transdermal exposure
1 Wk 1 Wk
OVA orally
Symptoms of anaphylaxis
Control
tBHQ (0001)
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
12
Summary
bull tBHQ inhibits production of the cytokines IL-2 and IFNg in human T cells (suggesting a possible effect on host immunity)
bullActivation of Nrf2 by the food preservative tBHQ increases production of Th2 cytokines by CD4+ T cells
bull tBHQ increases activity and expression of the Th2-regulating gene GATA-3 and this is dependent on Nrf2
bull Our preliminary data suggests that low doses of tBHQ exacerbate immune response and clinical symptoms in an animal model of food allergy
Cell-mediated Immunity
Parasite immunity
Nrf2
In the presence of Nrf2 Th1 and Th2 responses are in balance
Th1 Th2
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
13
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
bull Rockwell Lab ndash Heather Dover
ndash Jenna Bursley
ndash Alex Turley
ndash Joe Zagorski
ndash Holly Cline
bull Dr Curtis D Klaassen
bull Klaassen Laboratory ndash Scott Reisman
bull Dr Bryan Copple ndash Copple Lab
bull Dr Venu Gangur ndash Tina Ortiz
ndash Radhakrishna Para
ndash Babu Gonipeta
bull Dr Jim Pestka
bull Dr Patrick Fields ndash Mingcai Zhang
ndash Manouela Ortega
Acknowledgments
This project was funded by NIH grants K99R00 ES018885 R01 ES024966 T32 ES007255 T32 GM092715
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
10
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
T-bet (Th1 transcription factor)
Restimulated
Nrf2 activation inhibits the binding activity of T-bet the master regulator of Th1 differentiation
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
T-b
et
Bin
din
g A
cti
vit
y(A
BS
4
50n
m)
00
02
03
04
05
WT
KO
Restimulated
Nrf2 activation inhibits T-bet binding activity
IFN
(pg
ml)
0
1000
2000
3000
4000
Restimulated
VH tBHQ
IFNg
T-bet (Th1)
Dagger Dagger
Dagger
Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
Dagger Dagger
Rockwell et al 2012
BKG VH tBHQ
GA
TA
-3 B
ind
ing
Acti
vit
y(A
BS
4
50n
m)
000
002
004
006
008 GATA-3
Restimulated
Nrf2 activation promotes GATA-3 binding activity
IL-4
(p
gm
l)
0
200
400
600
800
1000
1200
1400
Restimulated
VH tBHQ
IL-4
Dagger Dagger
Dagger Dagger
Rockwell et al 2012
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
11
GA
TA
-3 m
RN
A (
Fo
ld in
du
cti
on
over
WT
VH
Co
ntr
ol)
00
05
10
15
20
25
30 Wild-type
Nrf2-null
VH 0001 001 01 1
Nrf2 activation increases GATA-3 mRNA expression
anti-CD3anti-CD28
tBHQ
Dagger
Dagger Dagger Dagger
GATA-3
Hypothesis tBHQ exacerbates immune response and clinical symptoms of anaphylaxis in response to food allergen
Why doesnrsquot everyone have immune responses to food The phenomenon of oral tolerance
1 Wk 1 Wk 1 Wk
Bleed Bleed Bleed
OVA OVA OVA OVA
Experimental design for ovalbumin transdermal exposure
1 Wk 1 Wk
OVA orally
Symptoms of anaphylaxis
Control
tBHQ (0001)
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
12
Summary
bull tBHQ inhibits production of the cytokines IL-2 and IFNg in human T cells (suggesting a possible effect on host immunity)
bullActivation of Nrf2 by the food preservative tBHQ increases production of Th2 cytokines by CD4+ T cells
bull tBHQ increases activity and expression of the Th2-regulating gene GATA-3 and this is dependent on Nrf2
bull Our preliminary data suggests that low doses of tBHQ exacerbate immune response and clinical symptoms in an animal model of food allergy
Cell-mediated Immunity
Parasite immunity
Nrf2
In the presence of Nrf2 Th1 and Th2 responses are in balance
Th1 Th2
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
13
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
bull Rockwell Lab ndash Heather Dover
ndash Jenna Bursley
ndash Alex Turley
ndash Joe Zagorski
ndash Holly Cline
bull Dr Curtis D Klaassen
bull Klaassen Laboratory ndash Scott Reisman
bull Dr Bryan Copple ndash Copple Lab
bull Dr Venu Gangur ndash Tina Ortiz
ndash Radhakrishna Para
ndash Babu Gonipeta
bull Dr Jim Pestka
bull Dr Patrick Fields ndash Mingcai Zhang
ndash Manouela Ortega
Acknowledgments
This project was funded by NIH grants K99R00 ES018885 R01 ES024966 T32 ES007255 T32 GM092715
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
11
GA
TA
-3 m
RN
A (
Fo
ld in
du
cti
on
over
WT
VH
Co
ntr
ol)
00
05
10
15
20
25
30 Wild-type
Nrf2-null
VH 0001 001 01 1
Nrf2 activation increases GATA-3 mRNA expression
anti-CD3anti-CD28
tBHQ
Dagger
Dagger Dagger Dagger
GATA-3
Hypothesis tBHQ exacerbates immune response and clinical symptoms of anaphylaxis in response to food allergen
Why doesnrsquot everyone have immune responses to food The phenomenon of oral tolerance
1 Wk 1 Wk 1 Wk
Bleed Bleed Bleed
OVA OVA OVA OVA
Experimental design for ovalbumin transdermal exposure
1 Wk 1 Wk
OVA orally
Symptoms of anaphylaxis
Control
tBHQ (0001)
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
12
Summary
bull tBHQ inhibits production of the cytokines IL-2 and IFNg in human T cells (suggesting a possible effect on host immunity)
bullActivation of Nrf2 by the food preservative tBHQ increases production of Th2 cytokines by CD4+ T cells
bull tBHQ increases activity and expression of the Th2-regulating gene GATA-3 and this is dependent on Nrf2
bull Our preliminary data suggests that low doses of tBHQ exacerbate immune response and clinical symptoms in an animal model of food allergy
Cell-mediated Immunity
Parasite immunity
Nrf2
In the presence of Nrf2 Th1 and Th2 responses are in balance
Th1 Th2
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
13
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
bull Rockwell Lab ndash Heather Dover
ndash Jenna Bursley
ndash Alex Turley
ndash Joe Zagorski
ndash Holly Cline
bull Dr Curtis D Klaassen
bull Klaassen Laboratory ndash Scott Reisman
bull Dr Bryan Copple ndash Copple Lab
bull Dr Venu Gangur ndash Tina Ortiz
ndash Radhakrishna Para
ndash Babu Gonipeta
bull Dr Jim Pestka
bull Dr Patrick Fields ndash Mingcai Zhang
ndash Manouela Ortega
Acknowledgments
This project was funded by NIH grants K99R00 ES018885 R01 ES024966 T32 ES007255 T32 GM092715
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
12
Summary
bull tBHQ inhibits production of the cytokines IL-2 and IFNg in human T cells (suggesting a possible effect on host immunity)
bullActivation of Nrf2 by the food preservative tBHQ increases production of Th2 cytokines by CD4+ T cells
bull tBHQ increases activity and expression of the Th2-regulating gene GATA-3 and this is dependent on Nrf2
bull Our preliminary data suggests that low doses of tBHQ exacerbate immune response and clinical symptoms in an animal model of food allergy
Cell-mediated Immunity
Parasite immunity
Nrf2
In the presence of Nrf2 Th1 and Th2 responses are in balance
Th1 Th2
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
During Nrf2 activation CD4+ T cell differentiation is skewed toward Th2
Nrf2
tBHQ
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
13
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
bull Rockwell Lab ndash Heather Dover
ndash Jenna Bursley
ndash Alex Turley
ndash Joe Zagorski
ndash Holly Cline
bull Dr Curtis D Klaassen
bull Klaassen Laboratory ndash Scott Reisman
bull Dr Bryan Copple ndash Copple Lab
bull Dr Venu Gangur ndash Tina Ortiz
ndash Radhakrishna Para
ndash Babu Gonipeta
bull Dr Jim Pestka
bull Dr Patrick Fields ndash Mingcai Zhang
ndash Manouela Ortega
Acknowledgments
This project was funded by NIH grants K99R00 ES018885 R01 ES024966 T32 ES007255 T32 GM092715
Food allergy and the role of the food
preservative tBHQ
4122016
Cheryl Rockwell PhD
13
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
In the absence of Nrf2 CD4+ T cell differentiation is skewed toward Th1
bull Rockwell Lab ndash Heather Dover
ndash Jenna Bursley
ndash Alex Turley
ndash Joe Zagorski
ndash Holly Cline
bull Dr Curtis D Klaassen
bull Klaassen Laboratory ndash Scott Reisman
bull Dr Bryan Copple ndash Copple Lab
bull Dr Venu Gangur ndash Tina Ortiz
ndash Radhakrishna Para
ndash Babu Gonipeta
bull Dr Jim Pestka
bull Dr Patrick Fields ndash Mingcai Zhang
ndash Manouela Ortega
Acknowledgments
This project was funded by NIH grants K99R00 ES018885 R01 ES024966 T32 ES007255 T32 GM092715