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Preterm labour

15dPGJ2 reduces NF- κ B in peripheral blood mononuclear cells and reduces the production of the pro inflammatory cytokines IFN- γ and TNF- α in T helper cells. Lynne Sykes 1 , David MacIntyre 1 , Sathana Ponnampalam 1 , Xiao J Yap 1 , TG Teoh 2 , and Phillip R Bennett 1 .

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Preterm labour

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  1. 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

  2. Preterm labour 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) • Preterm birth occurs in 8% -12% of pregnancies • Infection/Inflammation strongly associated with early preterm labour occurring in 80% of SPTB < 28 weeks 1 • Inflammation leads to the activation of pro-labour 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.

  3. Inflammation and labour Triggers of NF-κB activation include pro-inflammatory cytokines e.g. TNF- α, IL-1β1 NF-κB is a pro inflammatory transcription factor which also controls the expression of labour associated genes (MMP’s, COX-2, IL-8)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 respectively5, 6(Chapman et al 2004 J Clin Endocrinol Metab, Allport et al 2001 Molecular Human Reprod)

  4. Inflammation and labour Triggers of NF-κB activation include pro-inflammatory cytokines e.g. TNF- α, IL-1β1 NF-κB is a pro inflammatory transcription factor which also controls the expression of labour associated genes (MMP’s, COX-2, IL-8)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 respectively5, 6(Chapman et al 2004 J Clin Endocrinol Metab, Allport et al 2001 Molecular Human Reprod)

  5. Inflammation and labour Triggers of NF-κB activation include pro-inflammatory cytokines e.g. TNF- α, IL-1β1 NF-κB is a pro inflammatory transcription factor which also controls the expression of labour associated genes (MMP’s, COX-2, IL-8)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 respectively5, 6(Chapman et al 2004 J Clin Endocrinol Metab, Allport et al 2001 Molecular Human Reprod)

  6. Inflammation and labour Triggers of NF-κB activation include pro-inflammatory cytokines e.g. TNF- α, IL-1β1 NF-κB is a pro inflammatory transcription factor which also controls the expression of labour associated genes (MMP’s, COX-2, IL-8)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 respectively5, 6(Chapman et al 2004 J Clin Endocrinol Metab, Allport et al 2001 Molecular Human Reprod)

  7. Inflammation and labour Triggers of NF-κB activation include pro-inflammatory cytokines e.g. TNF- α, IL-1β1 NF-κB is a pro inflammatory transcription factor which also controls the expression of labour associated genes (MMP’s, COX-2, IL-8)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 respectively5, 6(Chapman et al 2004 J Clin Endocrinol Metab, Allport et al 2001 Molecular Human Reprod)

  8. Inflammation and labour Triggers of NF-κB activation include pro-inflammatory cytokines e.g. TNF- α, IL-1β1 NF-κB is a pro inflammatory transcription factor which also controls the expression of labour associated genes (MMP’s, COX-2, IL-8)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 respectively5, 6(Chapman et al 2004 J Clin Endocrinol Metab, Allport et al 2001 Molecular Human Reprod)

  9. Inflammation and neonatal morbidity 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 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 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.

  10. Inflammation and neonatal morbidity 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 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 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.

  11. 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. IFN-γ 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

  12. The cyclopentenone prostaglandin: 15dPGJ2Anti-inflammatory prostaglandin 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 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

  13. The cyclopentenone prostaglandin: 15dPGJ2Anti-inflammatory prostaglandin 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 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

  14. The cyclopentenone prostaglandin: 15dPGJ2Anti-inflammatory prostaglandin 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 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  21. 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 • Term in labour 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 pre-incubation 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 9. Peripheral blood mononuclear cells were isolated using Ficoll-Paque TM Figure 10. Detection was by flow cytometry, with gating on lymphocytes

  22. 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

  23. NF-κB activity during pregnancy and in labour in peripheral blood mononuclear cells 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 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.

  24. P-p65 β actin NS V J2 J2/x481 x481 15dPGJ2 reduces NF-κB in peripheral blood mononuclear cells A 15dPGJ2 reduces PMA/Ionomycin stimulated phospho-p65 at all gestational time points 15dPGJ2 inhibits basal phospho-p65 Independent of CRTH2 B 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 BResults 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 C

  25. P-p65 β actin NS V J2 J2/x481 x481 15dPGJ2 reduces NF-κB in peripheral blood mononuclear cells A 15dPGJ2 reduces PMA/Ionomycin stimulated phospho-p65 at all gestational time points 15dPGJ2 inhibits basal phospho-p65 Independent of CRTH2 B 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 BResults 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 C

  26. P-p65 β actin NS V J2 J2/x481 x481 15dPGJ2 reduces NF-κB in peripheral blood mononuclear cells A 15dPGJ2 reduces PMA/Ionomycin stimulated phospho-p65 at all gestational time points 15dPGJ2 inhibits basal phospho-p65 Independent of CRTH2 B 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 BResults 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 C

  27. IFN-γ and TNF-α production is reduced in pregnancy in T helper cells B A • 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.

  28. IFN-γ and TNF-α production is reduced in pregnancy in T helper cells B A • 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.

  29. 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 representative dot plot of CD4+/IFN-γ+ cells. An increase in IFN gamma is seen with PMA stimulation which reduces significantly with 15dPGJ2 treatment • 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).

  30. 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 representative dot plot of CD4+/IFN-γ+ cells. An increase in IFN gamma is seen with PMA stimulation which reduces significantly with 15dPGJ2 treatment • 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).

  31. 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 representative dot plot of CD4+/IFN-γ+ cells. An increase in IFN gamma is seen with PMA stimulation which reduces significantly with 15dPGJ2 treatment • 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).

  32. 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 representative dot plot of CD4+/IFN-γ+ cells. An increase in IFN gamma is seen with PMA stimulation which reduces significantly with 15dPGJ2 treatment • 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).

  33. 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 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). Poster T-238 Sykes et al

  34. 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 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). Poster T-238 Sykes et al

  35. 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 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). Poster T-238 Sykes et al

  36. Conclusion • The anti inflammatory prostaglandin 15dPGJ2 is capable of modulating the peripheral Th1 response, possibly via NF-κB • 15dPGJ2 does not modulate the Th2 cytokine profile of IL-4 in CRTH2 positive lymphocytes (Th2 cells) 15dPGJ2

  37. Conclusion • The anti inflammatory prostaglandin 15dPGJ2 is capable of modulating the peripheral Th1 response, possibly via NF-κB • 15dPGJ2 does not modulate the Th2 cytokine profile of IL-4 in CRTH2 positive lymphocytes (Th2 cells) 15dPGJ2

  38. Conclusion 15dPGJ2 • 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 • It therefore remains a potential anti- inflammatory agent for the prevention of both preterm labour and inflammation induced adverse neonatal outcomes

  39. Conclusion 15dPGJ2 • 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 • It therefore remains a potential anti- inflammatory agent for the prevention of both preterm labour and inflammation induced adverse neonatal outcomes

  40. 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

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