Abstract
Implications Pre-implantation factor (PIF) has the potential to be a candidate treatment for endometritis. The present study demonstrates anti-inflammatory effects of PIF on normal endometrium. Further work is needed to assess effects of PIF following low dose lipopolysaccharide (LPS) challenge.
Introduction
Endometritis is a postpartum uterine infection affecting 5-26% of dairy cows, caused by bacterial contamination of the uterus. LPS is an endotoxin of E. coli which is a commonly isolated pathogen of endometritis. There is a call for new endometritis treatments due to increases in antibiotic resistance. An anti-inflammatory agent may reduce the disease associated inflammation that causes infertility. Pre-implantation factor (PIF) is a pregnancy specific peptide, secreted from viable embryos. PIF has immune-modulatory roles within pregnancy and as such has been shown to modulate inflammation in mouse models of autoimmune diseases (Weiss et al., 2012). The present study aimed to assess the use of a pre-treatment of PIF on endometrial tissue explants before LPS stimulation to investigate the immune-modulatory role of PIF within a bovine model of E. coli endometritis.
Material and methods
Bovine uteri (n=12; n=6 dairy, n=6 beef animals) with stage I corpus luteum and absence of endometrial inflammation were used. All animals were tested for the presence of inflammation at collection by taking a cytobrush smear. Smears were fixed and stained using Diff-Quick to assess for the percentage of polymorphonuclear leukocytes (PMN) present compared to uterine epithelial cells. Tissue was sampled from the ipsilateral uterine horn to the corpus luteum. Weighed tissue explants were pre-treated with media alone or PIF at 50, 100 or 500 nM for 24 hrs, then with media alone, LPS (1µg/ml), PIF (50, 100 or 500 nM) or LPS (1µg/ml) with PIF (50, 100 or 500 nM) for a further 48 hrs Media samples were collected 24 and 48 hrs post LPS challenge and PGF2α, PGE2 and IL-6 measured via radioimmunoassays and ELISA. Data were expressed as secretion per mg of tissue and analysed using an analysis of variance, with treatment and cow type as main effects.
Conclusion
PIF was shown to interact with the IL-6 pathway in un-stimulated endometrial tissue at 100nM and 500nM. There was no effect of PIF on PGE2 or PGF2α secretion, or LPS stimulated IL-6 secretion. However, the latter may be due to the high dose of LPS used. A lower concentration of LPS will be used in future experiments to further test the effect of PIF.
Research is needed to establish effects of PIF on components of IL-6 pathway. Furthermore, cytokines other than IL-6 should be investigated to assess the effect of PIF on other aspects of the TLR4 stimulation pathway. Earlier sample time points should be tested due to the half -life of PIF being short, at ~45min in circulation.
Acknowledgements The authors gratefully acknowledge Aberystwyth University for DCDS studentship funding; D.C. Wathes and Z. Cheng, RVC for the donation of PGE2 and PGF2α antisera and Randall Parker Foods for sample collection
References
Weiss, L., Or, R., Jones, R.., Amunugama, R., JeBailey, L., Ramu, S., Berstein, S.., Yekhtin, Z., Almogi-Hazan, O., Shainer, R., Reibstein, I., Vortmeyer, A., Paidas, M.., Zeira, M., Slavin, S., Barnea, E.. (2012) J. Neurol Sci. 312: 146-157
Introduction
Endometritis is a postpartum uterine infection affecting 5-26% of dairy cows, caused by bacterial contamination of the uterus. LPS is an endotoxin of E. coli which is a commonly isolated pathogen of endometritis. There is a call for new endometritis treatments due to increases in antibiotic resistance. An anti-inflammatory agent may reduce the disease associated inflammation that causes infertility. Pre-implantation factor (PIF) is a pregnancy specific peptide, secreted from viable embryos. PIF has immune-modulatory roles within pregnancy and as such has been shown to modulate inflammation in mouse models of autoimmune diseases (Weiss et al., 2012). The present study aimed to assess the use of a pre-treatment of PIF on endometrial tissue explants before LPS stimulation to investigate the immune-modulatory role of PIF within a bovine model of E. coli endometritis.
Material and methods
Bovine uteri (n=12; n=6 dairy, n=6 beef animals) with stage I corpus luteum and absence of endometrial inflammation were used. All animals were tested for the presence of inflammation at collection by taking a cytobrush smear. Smears were fixed and stained using Diff-Quick to assess for the percentage of polymorphonuclear leukocytes (PMN) present compared to uterine epithelial cells. Tissue was sampled from the ipsilateral uterine horn to the corpus luteum. Weighed tissue explants were pre-treated with media alone or PIF at 50, 100 or 500 nM for 24 hrs, then with media alone, LPS (1µg/ml), PIF (50, 100 or 500 nM) or LPS (1µg/ml) with PIF (50, 100 or 500 nM) for a further 48 hrs Media samples were collected 24 and 48 hrs post LPS challenge and PGF2α, PGE2 and IL-6 measured via radioimmunoassays and ELISA. Data were expressed as secretion per mg of tissue and analysed using an analysis of variance, with treatment and cow type as main effects.
Conclusion
PIF was shown to interact with the IL-6 pathway in un-stimulated endometrial tissue at 100nM and 500nM. There was no effect of PIF on PGE2 or PGF2α secretion, or LPS stimulated IL-6 secretion. However, the latter may be due to the high dose of LPS used. A lower concentration of LPS will be used in future experiments to further test the effect of PIF.
Research is needed to establish effects of PIF on components of IL-6 pathway. Furthermore, cytokines other than IL-6 should be investigated to assess the effect of PIF on other aspects of the TLR4 stimulation pathway. Earlier sample time points should be tested due to the half -life of PIF being short, at ~45min in circulation.
Acknowledgements The authors gratefully acknowledge Aberystwyth University for DCDS studentship funding; D.C. Wathes and Z. Cheng, RVC for the donation of PGE2 and PGF2α antisera and Randall Parker Foods for sample collection
References
Weiss, L., Or, R., Jones, R.., Amunugama, R., JeBailey, L., Ramu, S., Berstein, S.., Yekhtin, Z., Almogi-Hazan, O., Shainer, R., Reibstein, I., Vortmeyer, A., Paidas, M.., Zeira, M., Slavin, S., Barnea, E.. (2012) J. Neurol Sci. 312: 146-157
| Original language | English |
|---|---|
| Publication status | Published - 2015 |
| Event | Proceedings of the British Society of Animal Science - Chester, United Kingdom of Great Britain and Northern Ireland Duration: 14 Apr 2015 → 15 Apr 2015 |
Conference
| Conference | Proceedings of the British Society of Animal Science |
|---|---|
| Country/Territory | United Kingdom of Great Britain and Northern Ireland |
| City | Chester |
| Period | 14 Apr 2015 → 15 Apr 2015 |