ros and rns: key factors in goiter and thyroiditis. · ros : definition reactive oxygen species :...
TRANSCRIPT
ROS and RNS: key factors in ROS and RNS: key factors in
goiter and goiter and thyroiditisthyroiditis..
A-C Gérard, Ph.D.
Unité de Morphologie Expérimentale (MOEX)UCL Faculté de MédecineBruxelles, Belgique
IntroductionIntroduction
Thyroid cell
ROS : definition
Reactive oxygen species : ROSReactive oxygen species : ROS
Superoxide anion O2�-
Hydrogen peroxide H2O2
Hydroxyl radical OH-
Reactive nitrogen species : RNSReactive nitrogen species : RNS
Nitric oxide NOProduced by NOSsMultiple functions
Peroxinitrite NOO-
Oxidative stress : OSOxidative stress : OS
Impaired balance between pro-and antioxidant
ROS in thyroid cells
In vitro
Basal ROS inhibition by NAC Basal ROS inhibition by NAC
Minimal oxidative load is required to maintain thyroid cell function
Minimal oxidative load is required to maintain thyroid cell function
Control NAC 0.0
0.5
1.0
1.5
2.0
2.5
*
Rel
ativ
e m
RN
Aex
pres
sion
DU
OX
/ ββ ββact
in
DUOXDUOX
Control NAC 0.0
0.5
1.0
*
Rel
ativ
e m
RN
Aex
pres
sion
TP
O/ ββ ββ
actin
TPOTPO
In vivo
DUOXDUOX
NACctrl
Basal ROS production Basal ROS production
Poncin et al, 2009, J Endocrinol 201:161-167
Protective mechanisms
Antioxidant systems present in thyroid cells:
- glutathione peroxidase
- SOD
- catalase
- thioredoxins, peroxiredoxins
CatalaseCatalase MnSODMnSOD PRDX5PRDX5
Gerard et al, 2005, Thyroid 15:205-209
Poncin et al, 2009, J Endocrinol 201:161-167
Goiter
Humans : multinodular goiterHumans : multinodular goiter
Iodine deficiency =
a global problem concerning large geographical areas
Euthyroid goiter not associated with thyroid autoimmunity or malignancy
Could result from an alternance of hyperplasia (iodine deficiency) and involution (iodine intake).
Animal models (rats and mice)Animal models (rats and mice) low iodine diet + goitrigen
Control Goiter
Imada et al, 1986, Cell Tissue Res 245:291-296
Oxidative stress in goiters
Increased oxidative stress in goiter: Likely due to H2O2 (not used for thyroid hormone synthesis)
HNEHNE
Not lethal : overproduced H2O2 may be reduced by intracellular defence mechanisms :
- Associated with increased anti-oxidant defences
0
10
20
30
40
*
*+
Cyc
linD
1 po
sitiv
e nu
clei
(%)
Ctrl
NAC 4d
ClO4
-
ClO4
-
+ NAC 4d
12d goiterctrl
ROS inhibition by NAC ROS inhibition by NAC
Oxidative load is absolutely required for thyroid cell
proliferation
Oxidative load is absolutely required for thyroid cell
proliferation
1. Goitrogenesis1. Goitrogenesis
2.Thyroiditis2.Thyroiditis
Thyroid heterogeneity : an adaptativemechanism
Important role for ROS in adaptation to iodine deficiency
Role of the immune context on thyroidcell function
Thyroid heterogeneity :
an adaptative mechanism
Thyroid heterogeneity :
an adaptative mechanism
Thyroid heterogenity
Echography
Clinical and anatomopathological aspectsClinical and anatomopathological aspects
Scintigraphy Surgical specimen
Microscopic aspectsMicroscopic aspects
Human Human Mice, autoradiography Mice, autoradiography
Active Hypofunctioning
Hot
Cold
Active
Active
Hypofunctioning
Active
HypofunctioningActive
hypofunctioning
TPOTPO DUOXDUOX PendrinPendrin
NISNIS rTSHrTSH
TgTgTg-ITg-I
Hypofunctioning
Active Active
Active
Active
Active
Hypofunctioning Hypofunctioning
Gerard et al, 2002 J Clin endocrinol Metab, 87:1291-1299
NOSIIINOSIII VEGFVEGF
Human, normal Human, normal
Mice, normal Mice, normal
hot
Relative volume of capillaries
Relative volume of capillaries
***
0
0.1
0.2
cold
A close relation exists between the thyroid cell function, the microcirculation and the expression of vasoactive factors, especially
NOSIII
A close relation exists between the thyroid cell function, the microcirculation and the expression of vasoactive factors, especially
NOSIII
Gerard et al, 2000 Eur J Endocrinol, 642:651-660
Gerard et al, 2002 J Clin endocrinol Metab, 87:1291-1299
TPOThOXspendrin
TSHr
NIS
TPO
pendrin
TSHr
NIS
TPO
pendrinThOXs
TSH+
trophic factors(paracrine/autocrine)
In case of increased needs?
Functional reserve?Functional reserve?
When stimulated by TSH, hypofunctioning follicles are re-activated:- Epithelium becomes thicker- Tg globules disappear- NOSIII is expressed- microcirculation is increased
When stimulated by TSH, hypofunctioning follicles are re-activated:- Epithelium becomes thicker- Tg globules disappear- NOSIII is expressed- microcirculation is increased
12d goiterctrl
NOSIIINOSIII
Gerard et al, 2000 Eur J Endocrinol, 642:651-660
Tg globulesTg globules
Gérard et al, 2004, Eur J Endocrinol150:73-80
Mechanisms underlying goiterdevelopment as an adaptation to environmental changes are
better understood
Mechanisms underlying goiterdevelopment as an adaptation to environmental changes are
better understood
Conclusion
Increase in thyroid vasculature and
thyroid blood flow
Increase in thyroid vasculature and
thyroid blood flowIodine deficiency : TSH-controlled growth
ECMECM
CrinopexyCrinopexy releaserelease
ECMECM
CrinopexyCrinopexy releaserelease
CrinopexyCrinopexy releaserelease
TGFTGFββ
FGFFGF
dilationdilation
ETETBB
ETETAA
constrictionconstriction
ETET
NOSIII
NOSIII
NONOdilationdilation
VEGFVEGF++
TPOThOXspendrin
TSHr
NIS
TPO
pendrin
VEGFR1VEGFR1
VEGFVEGF
++
VEGFR2VEGFR2
TSHr
NIS NONO
dilationdilation
NOSIII
Independentangiofollicular unitsIndependent
angiofollicular units
Close relationship between endothelial and follicular
cells
Close relationship between endothelial and follicular
cells
Control of OS Control of OS NO
ROS
NO
ROS
H2O2
Gerard et al, 2002 J Clin endocrinol Metab, 87:1291-1299 ; 2000 Eur J Endocrinol, 642:651-660; 2004, Eur J Endocrinol 150:73-80; 2003, J. Endocrinol. 177:269-277;2005, Thyroid 15:205-209
Important role for ROS in
adaptation to iodine
deficiency
Important role for ROS in
adaptation to iodine
deficiency
What happens during the early stages of goiter development?
What role is played by iodine deficiency?
2) Endothelial cell proliferation occurs very early, before epithelial cells
1) Increase in the thyroid blood flow and enlargement of the thyroid gland may occur without any changes in TSH levels
**
TP
O/ ββ ββ-actin1
2
3
0
Function
Goitrigen treatmentDay 0 Day 6 Day 12 Day 24
Tg-I
Until day 6 : no change
From day 12 :stimulation by TSH
Until day 6 : no change
From day 12 :stimulation by TSH
*
*
0.5
1.0
1.5
2.0
TS
H (
ng)
0
Goitrigen treatment (days)0 1 2 3 4 6 0 6 12 24
Gerard et al, 2008, Am J Pathol178:748-760
Day 0
31P
127I
Day 24Day 1
127I Detection by Nano-SIMS127I Detection by Nano-SIMS
Gerard et al, 2008, Am J Pathol 178:748-760
Thy
roid
blo
od fl
ow (
% o
f day
0)
0 4 8 12 16 20 24
75
100
125
150
175
200
225
Goitrigen treatment (days)
*
**
+
+
+
Vascularisation
2
2 TSH dependentTSH dependent
11 TSH independentTSH independent
Gerard et al, 2008, Am J Pathol 178:748-760
6 days
Day 12 Day 24
ctrl 6 12 24
*
*
*
+
Day 6Day 3 Day 4
Day 0 Day 1 Day 2
2
2
1
1
VEGF
0 1 2 3 4 6
1
2
3
4
5
6
7
8
9
10
**
*
Rel
ativ
e m
RN
Aex
pres
sion
VE
GF
/GA
PD
H
Goitrigen treatment (days)
Gerard et al, 2008, Am J Pathol178:748-760
2 TSH dependentTSH dependent
1 TSH independentTSH independent
Two phases of angiogenesis:
1
2
TSH independentTSH independent
TSH dependentTSH dependent
During goiter development:During goiter development:
mR
NA
exp
ress
ion
VE
GF
/act
in
ctrl 2 h 4 h 6 h0.0
0.5
1.0
1.5
2.0 *
ctrl 2 h 4 h 6 h0.00
0.05
0.10
0.15
0.20 *
*
Pro
tein
exp
ress
ion
VE
GF
/act
in
VEGF
actin
43 kDa
42 kDa
Role of iodide
Iodide deprivation is associated with increased VEGF-A synthesis and secretion
Iodide deprivation is associated with increased VEGF-A synthesis and secretion
Gerard et al, Am J Physiol EndocrinolMetab, 2009
VE
GF
(pg
/ml)
2h 4h 6h0
30
60
90
120
150
180
*+
*+
+
Control Without NaI5.0 µµµµm
ControlWithout NaI
5.0 µµµµm
5.0 µµµµm
5.0 µµµµm
131P 127I131P 127I
In vitro
What is the pathway linking iodide deficiency and VEGF-A synthesis?
What is the pathway linking iodide deficiency and VEGF-A synthesis?
HIF-1αααα protein detectionHIF-1αααα protein detection
120 kDa
42 kDa
HIF-1αααα
Actin
ctrl 2 h 4 h 6 h CoCl20
2
4
6
8
**
Pro
tein
exp
ress
ion
HIF
-1αα αα/
actin
ctrl 2 h 4 h 6 h CoCl20
2
4
6
8
**
ctrl 2 h 4 h 6 h CoCl20
2
4
6
8
**
Gerard et al, Am J Physiol EndocrionlMetab, 2009
Control 2h
10 µm
Without NaI 2h
25 µm
25 µm 25 µm
25 µm
ctrl 1h 2h 4h 6h DFO0
25
50
75
100
125
HIF
-1αα αα
cc (
pg/m
l)
*
*
*
HIF-1αααα mRNA detection HIF-1αααα mRNA detection
mR
NA
exp
ress
ion
HIF
-1αα αα/
actin
ctrl 2 h 4 h 6 h0.00.20.40.60.81.01.21.4
HIF-1αααα protein is stabilized by iodide
deficiency.
HIF-1αααα protein is stabilized by iodide
deficiency.
ROS production (DCFH-DA) ROS production (DCFH-DA)
Ctrl 2h
20 µm
20 µm
Ctrl 1h Without NaI1 h
20 µm
Without NaI2 h
20 µm
NAC - NAC +0.0
0.2
0.4
0.6
Pro
tein
exp
ress
ion
VE
GF
/act
in
*
NAC - NAC +0
2
4
6
8
Pro
tein
exp
ress
ion
HIF
-1αα αα/
actin
*
ROS inhibition by NAC ROS inhibition by NAC
Control Without NaI
NAC – NAC +0.00.51.01.52.02.5
mR
NA
exp
ress
ion
VE
GF
/act
in
*
ROS production is involved in the activation of the HIF-1/VEGF-A
pathway
ROS production is involved in the activation of the HIF-1/VEGF-A
pathway
Gerard et al, Am J PhysiolEndocrinolMetab, 2009
2 Na+
I-
HIF-1ββββ
HRE
DUOX
ROS
I-
VEGFmRNA
NIS
Endothelial cells
Thyrocyte
TPO
HIF-1αααα
Na+
II--
VEGF
angiogenesis
HIF-1ββββ
HIF-1αααα
1. Goitrogenesis1. Goitrogenesis
2.Thyroiditis2.Thyroiditis
Thyroid heterogeneity : an adaptativemechanism
Important role for ROS in adaptation to iodine deficiency
Role of the immune context on thyroidcell function
Role of immune context
on thyroid cell function
Role of immune context
on thyroid cell function
Hashimoto’s thyroiditis
Autoimmune disease characterized by:
- Inflammatory infiltration
- Progressive thyroid cell destruction
HypothyroidismHypothyroidism
Cytokine profile is highly variable:
- Prevalence of Th1 cytokines (IFNγγγγ, TNF-αααα,ββββ, IL-1αααα,ββββ, IL-2, PDGF)
- But also Th2 and Th3 cytokines
Highly variable outcomes:
- patients can recover from hypothyroidism towards euthyroidism
- alternating hypo- and hyperthyroidism
1) Are Th1 cytokines lethal for thyroid cells ?
2) Why is thyroiditis so fluctuating ?
Is it in relation with a variation in the production of pro- and anti inflammatory cytokines ?
1) Are Th1 cytokines lethal for thyroid cells ?
2) Why is thyroiditis so fluctuating ?
Is it in relation with a variation in the production of pro- and anti inflammatory cytokines ?
0
10
20
30
40
50
% r
ed
TSH - + +IL-1α/IFNγ - - +
Cell viability
The Th1 cytokines IL-1αααα and IFNγγγγ are not lethal for thyroid cells.
The Th1 cytokines IL-1αααα and IFNγγγγ are not lethal for thyroid cells.
TPOTPO DUOXDUOX
+ §+
Normal tissues
Gérard et al, 2006, Am J Physiol Endocrinol Metab291:E242-E253
TPOTPO
110 kDa
NS
0
1000
2000
3000
*����
°
Arb
itrar
y un
its/µ
g pr
ot
Graves’ disease
110 kDa
0
1500
3000
4500
6000
7500
9000
Arb
itary
units
/µgp
rot
Hot nodules
TSH - + + +IL-1α/IFNγ - - + +L-NAME - - - +
Gérard et al, 2006, Am J Physiol Endocrinol Metab291:E242-E253
TSH - + + +IL-1α/IFNγ - - + +L-NAME - - - +
Th1 Th2
TPOTPO
110 kDa
02468
1012
TP
O p
rote
inex
pres
sion
(arb
itrar
yun
its/µ
g pr
otei
n)
*+
IL-1αααα/IFNγγγγ - + +IL-4 - - +
ThyroglobulinThyroglobulin
01234
*
+
Tg
(ng
/ml)
5
IL-1αααα/IFNγ γ γ γ - + +IL-4 - - +
IL-10Th1 Th3
TPOTPO
IL-1αααα/IFNγγγγ - + +IL-10 - - +
0
5
10
15
20
**
TP
O p
rote
inex
pres
sion
(arb
itrar
yun
its/µ
g pr
otei
ns)
110 kDa
ThyroglobulinThyroglobulin
0102030405060
* *+T
g (n
g/m
l)
IL-1αααα/IFNγ γ γ γ - + +IL-10 - - +
IL-1αααα/IFNγγγγ - -IL-10 - +
Poncin et al, 2008, Endocrinology 149:1534-1542
IL-4
DUOXDUOX
Conclusions
- Do not induce cell death
- Alter TPO and DUOX expression
IL-1αααα/IFNγγγγ (Th1) cytokinesIL-1αααα/IFNγγγγ (Th1) cytokines
- Mediates partially the Th1 cytokine effects
NONO
- Blocks Th1 cytokine-induced inhibitory effects on TPO, DUOX and Tg
IL-4 (Th2) cytokineIL-4 (Th2) cytokine
- Inhibit thyroid cell function
- Do not prevent inhibitory effects of Th1 cytokines
IL-10, TGF-ββββ (Th3) cytokinesIL-10, TGF-ββββ (Th3) cytokines
Immune context may explain the clinical
variability of Hashimoto’s thyroiditis
Immune context may explain the clinical
variability of Hashimoto’s thyroiditis
Direct action of Th1 cytokines could impair
thyroid hormone synthesis
Direct action of Th1 cytokines could impair
thyroid hormone synthesis
General conclusions
- Oxidative stress/load is strictly required for thyroid cell function and
proliferation.
- Mechanisms underlying goiter development are better understood.
- The link between iodine deficiency and hypervascularisation occurs
through a ROS - HIF-1 – VEGF pathway.
ROS are physiologically required for thyroid cell
function and adaptation to environmental changes
ROS are physiologically required for thyroid cell
function and adaptation to environmental changes
- Functional variability in thyroiditis outcomes may occur through changes in the Th1 – Th2 – Th3 cytokine balance
Acknowledgments
M.F. van den Hove (Cell Biology Unit, UCL)
Ch. Daumerie (Endocrinology Unit, UCL)
J. Rahier, M.C. Nolleveaux (Anatomopathology unit, UCL)
C. Mestdagh (clinique Saint Jean, Brussels)
F. Miot, S. Costagliola(IRIBHN, ULB)
J.J.M. deVijlder(Academic Medical Center, University of Amsterdam)
J.N Audinot (Centre Gabriel Lippmann, Luxembourg)
P. Sonveaux (Pharmacotherapy unit, UCL)
F. Soncin (Institut de Biologie de Lille, IBL)
Acknowledgments
MOEX, unité de Morphologie Expérimentale
Prof. B. Lengelé