Transcript NF-κB is a pro inflammatory transcription factor which also controls

15dPGJ2 reduces NF-κB in peripheral blood mononuclear
cells and reduces the production of the pro inflammatory
cytokines IFN-γ and TNF-α in T helper cells
Lynne Sykes1, David MacIntyre1, Sathana Ponnampalam1, Xiao
J Yap1, TG Teoh2, and Phillip R Bennett1.
1IRDB,
Imperial College, London, United Kingdom,
2Department of Obstetrics and Gynaecology, Imperial College
Healthcare NHS Trust, London, United Kingdom
Preterm labour
• Preterm birth occurs in 8% -12% of pregnancies
• Infection/Inflammation strongly associated
with early preterm labour occurring in 80% of
SPTB < 28 weeks 1
Figure 1
Number of publications with reference to
preterm labour and inflammation 4
(Sykes et al 2012, Targeting Immune activation
for the prevention of Preterm labour)
• Inflammation leads to the activation of prolabour gene expression 2
• Association between inflammation and
neonatal brain injury 3
• Despite increased awareness of this association
(Figure 1) this has not translated into a
reduction in PTB or neonatal brain injury4
1 Goldenberg RL, Andrews WW, Hauth JC. Choriodecidual infection and preterm birth. Nutr Rev 2002;60(5 Pt 2):S19-25
2 Lindstrom TM, Bennett PR. The role of nuclear factor kappa B in human labour. Reproduction , 2005;130(5):569-81.
3 Gotsch et al. The fetal inflammatory response syndrome Clin Obstet Gynecol, 2007; 50(3):652-83.
4 Sykes et al. Targeting Immune Activation in the Prevention of Pre-term Labor. US Obstetrics & Gynecology,2011;6(2):103-9.
Inflammation and labour
NF-κB is a pro inflammatory transcription factor
which also controls the expression of labour
associated genes (MMP’s, COX-2, IL-8)1
Triggers of NF-κB activation include proinflammatory cytokines e.g. TNF- α, IL-1β 1
Figure 2
NF-κB controls the expression of labour
associated genes MMP’s COX-2, IL-8.
NF-κB is activated by pro inflammatory proteins
which can thus trigger infection induced
preterm labour.
Lindstrom TM, Bennett PR. The role of nuclear
factor kappa B in human labour. Reproduction ,
2005;130(5):569-81.
NF-κB DNA binding activity is increased in the myometrium and amnion at term and in labour
respectively 5, 6 (Chapman et al 2004 J Clin Endocrinol Metab, Allport et al 2001 Molecular Human Reprod)
Inflammation and labour
NF-κB is a pro inflammatory transcription factor
which also controls the expression of labour
associated genes (MMP’s, COX-2, IL-8)1
Triggers of NF-κB activation include proinflammatory cytokines e.g. TNF- α, IL-1β 1
Figure 2
NF-κB controls the expression of labour
associated genes MMP’s COX-2, IL-8.
NF-κB is activated by pro inflammatory proteins
which can thus trigger infection induced
preterm labour.
Lindstrom TM, Bennett PR. The role of nuclear
factor kappa B in human labour. Reproduction ,
2005;130(5):569-81.
NF-κB DNA binding activity is increased in the myometrium and amnion at term and in labour
respectively 5, 6 (Chapman et al 2004 J Clin Endocrinol Metab, Allport et al 2001 Molecular Human Reprod)
Inflammation and labour
NF-κB is a pro inflammatory transcription factor
which also controls the expression of labour
associated genes (MMP’s, COX-2, IL-8)1
Triggers of NF-κB activation include proinflammatory cytokines e.g. TNF- α, IL-1β 1
Figure 2
NF-κB controls the expression of labour
associated genes MMP’s COX-2, IL-8.
NF-κB is activated by pro inflammatory proteins
which can thus trigger infection induced
preterm labour.
Lindstrom TM, Bennett PR. The role of nuclear
factor kappa B in human labour. Reproduction ,
2005;130(5):569-81.
NF-κB DNA binding activity is increased in the myometrium and amnion at term and in labour
respectively 5, 6 (Chapman et al 2004 J Clin Endocrinol Metab, Allport et al 2001 Molecular Human Reprod)
Inflammation and labour
NF-κB is a pro inflammatory transcription factor
which also controls the expression of labour
associated genes (MMP’s, COX-2, IL-8)1
Triggers of NF-κB activation include proinflammatory cytokines e.g. TNF- α, IL-1β 1
Figure 2
NF-κB controls the expression of labour
associated genes MMP’s COX-2, IL-8.
NF-κB is activated by pro inflammatory proteins
which can thus trigger infection induced
preterm labour.
Lindstrom TM, Bennett PR. The role of nuclear
factor kappa B in human labour. Reproduction ,
2005;130(5):569-81.
NF-κB DNA binding activity is increased in the myometrium and amnion at term and in labour
respectively 5, 6 (Chapman et al 2004 J Clin Endocrinol Metab, Allport et al 2001 Molecular Human Reprod)
Inflammation and labour
NF-κB is a pro inflammatory transcription factor
which also controls the expression of labour
associated genes (MMP’s, COX-2, IL-8)1
Triggers of NF-κB activation include proinflammatory cytokines e.g. TNF- α, IL-1β 1
Figure 2
NF-κB controls the expression of labour
associated genes MMP’s COX-2, IL-8.
NF-κB is activated by pro inflammatory proteins
which can thus trigger infection induced
preterm labour.
Lindstrom TM, Bennett PR. The role of nuclear
factor kappa B in human labour. Reproduction ,
2005;130(5):569-81.
NF-κB DNA binding activity is increased in the myometrium and amnion at term and in labour
respectively 5, 6 (Chapman et al 2004 J Clin Endocrinol Metab, Allport et al 2001 Molecular Human Reprod)
Inflammation and labour
NF-κB is a pro inflammatory transcription factor
which also controls the expression of labour
associated genes (MMP’s, COX-2, IL-8)1
Triggers of NF-κB activation include proinflammatory cytokines e.g. TNF- α, IL-1β 1
Figure 2
NF-κB controls the expression of labour
associated genes MMP’s COX-2, IL-8.
NF-κB is activated by pro inflammatory
proteins which can thus trigger infection
induced preterm labour.
Lindstrom TM, Bennett PR. The role of nuclear
factor kappa B in human labour. Reproduction ,
2005;130(5):569-81.
NF-κB DNA binding activity is increased in the myometrium and amnion at term and in labour
respectively 5, 6 (Chapman et al 2004 J Clin Endocrinol Metab, Allport et al 2001 Molecular Human Reprod)
Inflammation and neonatal morbidity
Fetal inflammatory response syndrome
Originally described in PTL/PPROM
Defined by an elevated IL-6 level in fetal plasma
Systemic activation of the fetal innate immune system 7
Figure 3. Intrauterine infection
Pictorial representation of the ascending and
transplacental routes of intrauterine infection.
Romero R, Mazor M. Infection and preterm
labor. Clin Obstet Gynaecol 1982;9:593–607
Target organs and effects:
Adrenal gland- increase in cortisol
Lungs – increase in chronic lung disease
Brain – increase in PVL, IVH
Figure 4. Target organs of the Fetal inflammatory response syndrome
7Gotsch et al. The fetal inflammatory response syndrome Clin Obstet Gynecol, 2007; 50(3):652-83.
Inflammation and neonatal morbidity
Fetal inflammatory response syndrome
Originally described in PTL/PPROM
Defined by an elevated IL-6 level in fetal plasma
Systemic activation of the fetal innate immune system 7
Figure 3. Intrauterine infection
Pictorial representation of the ascending and
transplacental routes of intrauterine infection.
Romero R, Mazor M. Infection and preterm
labor. Clin Obstet Gynaecol 1982;9:593–607
Target organs and effects:
Adrenal gland- increase in cortisol
Lungs – increase in chronic lung disease
Brain – increase in PVL, IVH
Figure 4. Target organs of the Fetal inflammatory response syndrome
7Gotsch et al. The fetal inflammatory response syndrome Clin Obstet Gynecol, 2007; 50(3):652-83.
Immunology of pregnancy
“Immune paradox”:
Balance between immune tolerance of the
foreign fetus along with maintaining
defence against infection
Altered bias of Th1:Th2 ratio to favour Th2
profile thought to contribute to the
maintenance of successful pregnancy
Progesterone and Prostaglandin D2
promote the Th2 profile
NF-κB is suppressed in T cells in pregnancy,
which is thought to contribute to
suppression of Th1 interleukins 8
(McCracken et al 2004 Journal of Immunology)
Figure 5. Regulation of Th1:Th2 interleukin production
A negative interaction exists between the Th1 and Th2 interleukins. IFNγ inhibits Th2,whereas IL-4 inhibits Th1 interleukins.
Adapted from O’Garra et al.
The molecular basis of T helper 1 and T helper 2 cell differentiation.
Trends in Cell Biology, 2000
The cyclopentenone prostaglandin: 15dPGJ2
Anti-inflammatory prostaglandin
•
Inhibits NF-κB and COX-2 in
amniocytes and myocytes in
vitro
•
Independent of PPAR-γ
•
Delays preterm labour in the
murine model of
inflammation induced
preterm labour
•
Improves pup mortality
Figure 6. Western blots of NF-κB and COX-2
15dPGJ2 reduces NF-κB and COX-2 in IL-1β stimulated myocytes in culture. Similar
results were seen with amniocytes.
Lindstrom et al 2005. Journal of Clin Endocrinol Metab 90: 3534-3543.
Figure 7. Murine model of infection induced preterm labour
15dPGJ2 delays LPS induced preterm delivery and increases pup survival
Pirianov et al 2009. Endocrinology 150:699-706
The cyclopentenone prostaglandin: 15dPGJ2
Anti-inflammatory prostaglandin
•
Inhibits NF-κB and COX-2 in
amniocytes and myocytes in
vitro
•
Independent of PPAR-γ
•
Delays preterm labour in the
murine model of
inflammation induced
preterm labour
•
Improves pup mortality
Figure 6. Western blots of NF-κB and COX-2
15dPGJ2 reduces NF-κB and COX-2 in IL-1β stimulated myocytes in culture. Similar
results were seen with amniocytes.
Lindstrom et al 2005. Journal of Clin Endocrinol Metab 90: 3534-3543.
Figure 7. Murine model of infection induced preterm labour
15dPGJ2 delays LPS induced preterm delivery and increases pup survival
Pirianov et al 2009. Endocrinology 150:699-706
The cyclopentenone prostaglandin: 15dPGJ2
Anti-inflammatory prostaglandin
•
Inhibits NF-κB and COX-2 in
amniocytes and myocytes in
vitro
•
Independent of PPAR-γ
•
Delays preterm labour in the
murine model of
inflammation induced
preterm labour
•
Improves pup mortality
Figure 6. Western blots of NF-κB and COX-2
15dPGJ2 reduces NF-κB and COX-2 in IL-1β stimulated myocytes in culture. Similar
results were seen with amniocytes.
Lindstrom et al 2005. Journal of Clin Endocrinol Metab 90: 3534-3543.
Figure 7. Murine model of infection induced preterm labour
15dPGJ2 delays LPS induced preterm delivery and increases pup survival
Pirianov et al 2009. Endocrinology 150:699-706
The cyclopentenone prostaglandin: 15dPGJ2
CRTH2 agonist
•
CRTH2 is the second Prostaglandin D2
receptor
•
Classic receptor for Th2 cells
•
PGD2 stimulates Th2 interleukin
production in vitro in Th2 cells 9
•
Potential for promotion of the Th2
response in pregnancy and suppression of
the pro inflammatory Th1 response .
Figure 8. Effect of Prostaglandin D2 on Th2 interleukins
A dose response increase in IL-4 and IL-10 production is seen with Prostaglandin D2 in cultured Th2
cells.
9 Xue et al, Prostaglandin D2 causes preferential induction of pro inflammatory Th2 cytokine
production through an action on CRTH2 on Th2 cells. Journal of Immunology 2005; 175; 6531-6536
The cyclopentenone prostaglandin: 15dPGJ2
CRTH2 agonist
•
CRTH2 is the second Prostaglandin D2
receptor
•
Classic receptor for Th2 cells
•
PGD2 stimulates Th2 interleukin
production in vitro in Th2 cells 9
•
Potential for promotion of the Th2
response in pregnancy and suppression of
the pro inflammatory Th1 response .
Figure 8. Effect of Prostaglandin D2 on Th2 interleukins
A dose response increase in IL-4 and IL-10 production is seen with Prostaglandin D2 in cultured Th2
cells.
9 Xue et al, Prostaglandin D2 causes preferential induction of pro inflammatory Th2 cytokine
production through an action on CRTH2 on Th2 cells. Journal of Immunology 2005; 175; 6531-6536
The cyclopentenone prostaglandin: 15dPGJ2
CRTH2 agonist
•
CRTH2 is the second Prostaglandin D2
receptor
•
Classic receptor for Th2 cells
•
PGD2 stimulates Th2 interleukin
production in vitro in Th2 cells 9
•
Potential for promotion of the Th2
response in pregnancy and suppression
of the pro inflammatory Th1 response.
Figure 8. Effect of Prostaglandin D2 on Th2 interleukins
A dose response increase in IL-4 and IL-10 production is seen with Prostaglandin D2 in cultured Th2
cells.
9 Xue et al, Prostaglandin D2 causes preferential induction of pro inflammatory Th2 cytokine
production through an action on CRTH2 on Th2 cells. Journal of Immunology 2005; 175; 6531-6536
Hypothesis and aims
•
Hypothesis
15dPGJ2 represents a potential anti-inflammatory therapeutic agent for the
prevention of infection induced preterm labour and inflammation induced
neonatal brain injury.
It is not known what effects 15dPGJ2 would have on peripheral leukocytes.
•
Aims:
NF-κB
– To determine if NF-κB is activated in PBMCs at term and in labour
– To determine if 15dPGJ2 can inhibit NF-κB in PBMCs
Th1/Th2 interleukins
– To determine if the pro inflammatory cytokines IFN-γ and TNF-α are increased
at term and in labour
– To determine if 15dPGJ2 can inhibit the Th1 cytokines IFN-γ and TNF-α
– To determine if 15dPGJ2 can enhance the Th2 cytokine IL-4 via CRTH2
Hypothesis and aims
•
Hypothesis
15dPGJ2 represents a potential anti-inflammatory therapeutic agent for the
prevention of infection induced preterm labour and inflammation induced
neonatal brain injury.
It is not known what effects 15dPGJ2 would have on peripheral leukocytes.
•
Aims:
NF-κB
– To determine if NF-κB is activated in PBMCs at term and in labour
– To determine if 15dPGJ2 can inhibit NF-κB in PBMCs
Th1/Th2 interleukins
– To determine if the pro inflammatory cytokines IFN-γ and TNF-α are increased
at term and in labour
– To determine if 15dPGJ2 can inhibit the Th1 cytokines IFN-γ and TNF-α
– To determine if 15dPGJ2 can enhance the Th2 cytokine IL-4 via CRTH2
Hypothesis and aims
•
Hypothesis
15dPGJ2 represents a potential anti-inflammatory therapeutic agent for the
prevention of infection induced preterm labour and inflammation induced
neonatal brain injury.
It is not known what effects 15dPGJ2 would have on peripheral leukocytes.
•
Aims:
NF-κB
– To determine if NF-κB is activated in PBMCs at term and in labour
– To determine if 15dPGJ2 can inhibit NF-κB in PBMCs
Th1/Th2 interleukins
– To determine if the pro inflammatory cytokines IFN-γ and TNF-α are
increased at term and in labour
– To determine if 15dPGJ2 can inhibit the Th1 cytokines IFN-γ and TNF-α
– To determine if 15dPGJ2 can enhance the Th2 cytokine IL-4 via CRTH2
Experimental design
•
Blood was collected in accordance with Ethical approval from South East London Ethical
Committee Ref 10/H0805/54 (n>4 for each group)
– Non pregnant controls
– 28 weeks gestation
– Term pre labour
Figure 9.
– Term in labour
Peripheral blood mononuclear cells were
isolated using Ficoll-Paque TM
NF-κB studies:
• Whole cell lysate was used to analyse Phosphorylated p65 by Western analysis in non
stimulated or PMA (50ng/ml)/Ionomycin (0.5µg/ml) stimulated PBMCs, with or without a 2
hour incubation step of vehicle or 15dPGJ2 (32 µm)
Interleukin studies:
• PBMCs were cultured overnight and stimulated with PMA/Ionomycin for 4 hours, with a preincubation step of 2 hrs with vehicle or 15dPGJ2.
• Surface cell staining: CRTH2 and CD4
• Intracellular staining: IFN-γ, TNF-α and IL-4
• Detection by flow cytometry
Figure 10.
Detection was by flow cytometry, with gating on lymphocytes
NF-κB activity during pregnancy and in labour in peripheral
blood mononuclear cells
•
There is no difference in NF-κB activity in
peripheral mononuclear cells of non
pregnancy women and women at 28
weeks of gestation
•
There is no increase in NF-κB activity in
women in term labour compared to term
not in labour.
Figure 11. NF-κB p65 phosphorylation in peripheral blood mononuclear
cells at different gestational time points
PBMCs were isolated with Ficoll Paque and whole cell lysate was prepared
from the lymphocyte halo. 15ug of protein was run on a western.
Densitometry analysis of Phospho p65 was performed against B actin
loading control
NF-κB activity during pregnancy and in labour in peripheral
blood mononuclear cells
•
There is no difference in NF-κB activity in
peripheral mononuclear cells of non
pregnancy women and women at 28
weeks of gestation
•
There is no increase in NF-κB activity in
women in term labour compared to term
not in labour.
Figure 11. NF-κB p65 phosphorylation in peripheral blood mononuclear
cells at different gestational time points
PBMCs were isolated with Ficoll Paque and whole cell lysate was prepared
from the lymphocyte halo. 15ug of protein was run on a western.
Densitometry analysis of Phospho p65 was performed against B actin
loading control
15dPGJ2 reduces NF-κB in peripheral blood mononuclear
cells
A
15dPGJ2 inhibits basal phospho-p65
15dPGJ2 reduces PMA/Ionomycin stimulated
phospho-p65 at all gestational time points
B
P-p65
The effect of 15dPGJ2 on Phospho-p65
β actin
Figure 12. The effect of 15dPGJ2 on NF-κB (phospho-p65 ) in
PBMCs
A Cultured PBMCS were pre-incubated with 32um of 15dPGJ2 or
vehicle control
4
2
0
on
N
C
PMA/Iono 15dPGJ2
6
B Results are summarized in the graph, n=4.
C Treated cells were stimulated with 50ng/ml of PMA and
0.5ug/ml of Ionomycin. 15ug of whole cell lysate was run on a
western
PMA/Iono
8
L+
Independent of CRTH2
Non stim
Te
rm
J2/x481 x481
L-
J2
Te
rm
V
pr
eg
na
nt
28
w
ee
ks
NS
Fold change Phospho-p65/B-Actin
**
15dPGJ2 reduces NF-κB in peripheral blood mononuclear
cells
A
15dPGJ2 inhibits basal phospho-p65
15dPGJ2 reduces PMA/Ionomycin stimulated
phospho-p65 at all gestational time points
B
P-p65
The effect of 15dPGJ2 on Phospho-p65
β actin
Figure 12. The effect of 15dPGJ2 on NF-κB (phospho-p65 ) in
PBMCs
A Cultured PBMCS were pre-incubated with 32um of 15dPGJ2 or
vehicle control
4
2
0
on
N
C
PMA/Iono 15dPGJ2
6
B Results are summarized in the graph, n=4.
C Treated cells were stimulated with 50ng/ml of PMA and
0.5ug/ml of Ionomycin. 15ug of whole cell lysate was run on a
western
PMA/Iono
8
L+
Independent of CRTH2
Non stim
Te
rm
J2/x481 x481
L-
J2
Te
rm
V
pr
eg
na
nt
28
w
ee
ks
NS
Fold change Phospho-p65/B-Actin
**
15dPGJ2 reduces NF-κB in peripheral blood mononuclear
cells
A
15dPGJ2 inhibits basal phospho-p65
15dPGJ2 reduces PMA/Ionomycin stimulated
phospho-p65 at all gestational time points
B
P-p65
The effect of 15dPGJ2 on Phospho-p65
β actin
Figure 12. The effect of 15dPGJ2 on NF-κB (phospho-p65 ) in
PBMCs
A Cultured PBMCS were pre-incubated with 32um of 15dPGJ2 or
vehicle control
4
2
0
on
N
C
PMA/Iono 15dPGJ2
6
B Results are summarized in the graph, n=4.
C Treated cells were stimulated with 50ng/ml of PMA and
0.5ug/ml of Ionomycin. 15ug of whole cell lysate was run on a
western
PMA/Iono
8
L+
Independent of CRTH2
Non stim
Te
rm
J2/x481 x481
L-
J2
Te
rm
V
pr
eg
na
nt
28
w
ee
ks
NS
Fold change Phospho-p65/B-Actin
**
IFN-γ and TNF-α production is reduced in pregnancy in
T helper cells
A
•
•
B
The percentage of Th1 cells producing IFN-γ and TNF-α (non stimulated and PMA
stimulated) in pregnancy was reduced
There was no increase seen in labouring women
Figure 13. The percentage of T helper cells producing the Th1 interleukins IFN-γ and TNF-α during pregnancy and in labour
Cultured PBMCS were stimulated with 50ng/ml of PMA and 0.5ug/ml of Ionomycin. Cells were stained with CD4 and either IFN -γ or TNF-α
antibodies analysed by flow cytometry n=4.
IFN-γ and TNF-α production is reduced in pregnancy in
T helper cells
A
•
•
B
The percentage of Th1 cells producing IFN-γ and TNF-α (non stimulated and PMA
stimulated) in pregnancy was reduced
There was no increase seen in labouring women
Figure 13. The percentage of T helper cells producing the Th1 interleukins IFN-γ and TNF-α during pregnancy and in labour
Cultured PBMCS were stimulated with 50ng/ml of PMA and 0.5ug/ml of Ionomycin. Cells were stained with CD4 and either IFN -γ or TNF-α
antibodies analysed by flow cytometry n=4.
15dPGJ2 suppresses the Th1 cytokines IFN-γ and TNF-α
NS
PMA
PMA/15dPGJ2
CD4-APC
IFN-γ- FITC
Figure 14. The effect of 15dPGJ2 on PMA/Ionomycin stimulated IFN-γ
A) A representative dot plot of CD4+/IFN-γ+ cells. An increase in IFN gamma is seen
with PMA stimulation which reduces significantly with 15dPGJ2 treatment
B) Histogram showing the effect of 15dPGJ2 on IFN gamma in CD4 positive cells (T
helper cells)
Figure 15. The effect of 15dPGJ2 on IFN-γ and TNF-α production in T helper cells
A reduction in mean fluorescence intensity of IFN-γ was seen at all gestations (p<0.05 in non pregnant, 28 weeks and at term).
A reduction was also seen in TNF-α production, but this did not reach statistical significance (n=4).
15dPGJ2 suppresses the Th1 cytokines IFN-γ and TNF-α
NS
PMA
PMA/15dPGJ2
CD4-APC
IFN-γ- FITC
Figure 14. The effect of 15dPGJ2 on PMA/Ionomycin stimulated IFN-γ
A) A representative dot plot of CD4+/IFN-γ+ cells. An increase in IFN gamma is seen
with PMA stimulation which reduces significantly with 15dPGJ2 treatment
B) Histogram showing the effect of 15dPGJ2 on IFN gamma in CD4 positive cells (T
helper cells)
Figure 15. The effect of 15dPGJ2 on IFN-γ and TNF-α production in T helper cells
A reduction in mean fluorescence intensity of IFN-γ was seen at all gestations (p<0.05 in non pregnant, 28 weeks and at term).
A reduction was also seen in TNF-α production, but this did not reach statistical significance (n=4).
15dPGJ2 suppresses the Th1 cytokines IFN-γ and TNF-α
NS
PMA
PMA/15dPGJ2
CD4-APC
IFN-γ- FITC
Figure 14. The effect of 15dPGJ2 on PMA/Ionomycin stimulated IFN-γ
A) A representative dot plot of CD4+/IFN-γ+ cells. An increase in IFN gamma is seen
with PMA stimulation which reduces significantly with 15dPGJ2 treatment
B) Histogram showing the effect of 15dPGJ2 on IFN gamma in CD4 positive cells (T
helper cells)
Figure 15. The effect of 15dPGJ2 on IFN-γ and TNF-α production in T helper cells
A reduction in mean fluorescence intensity of IFN-γ was seen at all gestations (p<0.05 in non pregnant, 28 weeks and at term).
A reduction was also seen in TNF-α production, but this did not reach statistical significance (n=4).
15dPGJ2 suppresses the Th1 cytokines IFN-γ and TNF-α
NS
PMA
PMA/15dPGJ2
CD4-APC
IFN-γ- FITC
Figure 14. The effect of 15dPGJ2 on PMA/Ionomycin stimulated IFN-γ
A) A representative dot plot of CD4+/IFN-γ+ cells. An increase in IFN gamma is seen
with PMA stimulation which reduces significantly with 15dPGJ2 treatment
B) Histogram showing the effect of 15dPGJ2 on IFN gamma in CD4 positive cells (T
helper cells)
Figure 15. The effect of 15dPGJ2 on IFN-γ and TNF-α production in T helper cells
A reduction in mean fluorescence intensity of IFN-γ was seen at all gestations (p<0.05 in non pregnant, 28 weeks and at term).
A reduction was also seen in TNF-α production, but this did not reach statistical significance (n=4).
15dPGJ2 does not increase the Th2 cytokine IL-4
Figure 16. Representative dot plot of PMA stimulated IL-4 in CRTH2+ cells
•
PMA stimulates IL-4 in
CRTH2 positive cells
•
No increase in IL-4
production with 15dPGJ2
Poster T-238 Sykes et al
Figure 17. The effect of 15dPGJ2 on IL-4 production in CRTH2 + lymphocytes
No increase in IL-4 production was seen with the CRTH2 agonist 15dPGJ2 (n=4).
15dPGJ2 does not increase the Th2 cytokine IL-4
Figure 16. Representative dot plot of PMA stimulated IL-4 in CRTH2+ cells
•
PMA stimulates IL-4 in
CRTH2 positive cells
•
No increase in IL-4
production with 15dPGJ2
Poster T-238 Sykes et al
Figure 17. The effect of 15dPGJ2 on IL-4 production in CRTH2 + lymphocytes
No increase in IL-4 production was seen with the CRTH2 agonist 15dPGJ2 (n=4).
15dPGJ2 does not increase the Th2 cytokine IL-4
Figure 16. Representative dot plot of PMA stimulated IL-4 in CRTH2+ cells
•
PMA stimulates IL-4 in CRTH2
positive cells
•
No increase in IL-4
production with 15dPGJ2
Poster T-238 Sykes et al
Figure 17. The effect of 15dPGJ2 on IL-4 production in CRTH2 + lymphocytes
No increase in IL-4 production was seen with the CRTH2 agonist 15dPGJ2 (n=4).
Conclusion
•
The anti inflammatory
prostaglandin 15dPGJ2 is
capable of modulating the
peripheral Th1 response,
possibly via NF-κB
15dPGJ2
•
15dPGJ2 does not modulate
the Th2 cytokine profile of IL-4
in CRTH2 positive lymphocytes
(Th2 cells)
Conclusion
•
The anti inflammatory
prostaglandin 15dPGJ2 is
capable of modulating the
peripheral Th1 response,
possibly via NF-κB
15dPGJ2
•
15dPGJ2 does not modulate
the Th2 cytokine profile of IL-4
in CRTH2 positive lymphocytes
(Th2 cells)
Conclusion
•
15dPGJ2, consistent with the
local effects seen on myocytes
and amniocytes, also has
peripheral anti-inflammatory
effects by reducing NF-κB and
modulating the Th1:Th2 ratio
15dPGJ2
•
It therefore remains a potential
anti- inflammatory agent for
the prevention of both preterm
labour and inflammation
induced adverse neonatal
outcomes
Conclusion
•
15dPGJ2, consistent with the
local effects seen on myocytes
and amniocytes, also has
peripheral anti-inflammatory
effects by reducing NF-κB and
modulating the Th1:Th2 ratio
15dPGJ2
•
It therefore remains a potential
anti- inflammatory agent for
the prevention of both preterm
labour and inflammation
induced adverse neonatal
outcomes
Thanks to.........
The co-authors;
David MacIntyre, Research Associate
Sathana Ponnampalam, Clinical Researcher
Xiao J Yap, MSc project
The support from our research group;
Dr Yooni Lee, Sung Hye Kim,
Dr Manju Chandiramani, Dr Jenny Loudon, Dr Vasso Terzidou
My supervisors;
Mr TG Teoh and Professor Phillip Bennett
To Wellbeing of Women for funding this research