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Toll-like receptor-2 and -4 in the equine endometrium during physiologic post-breeding endometritis
Animal Reproduction Science 121S (2010) S96–S97
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Abstract
Toll-like receptor-2 and -4 in the equine endometrium during physiologic post-breeding endometritis夽 S. Eaton a,∗ , T. Raz b , C. Card c a b c
Animal Care Hospital of Williams Lake, Williams Lake BC, Canada V2G 5E8 Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, SK, Canada S7N 5B4
1. Introduction
2. Materials and methods
Physiologic post-breeding endometritis is an inflammatory response to insemination or breeding that enables the uterus to remove excess sperm, seminal plasma, debris, and bacteria. Mares resistant to endometritis are able to clear the inflammatory response rapidly enough to allow conception. Mares susceptible to endometritis have a prolonged inflammatory response, resulting in reduced conception rates (Causey, 2006). Toll-like receptors (TLR) are a family of conserved receptors that comprise part of the innate immune system and are activated by products or cellular components of bacteria, yeast, and viruses, but no information is available about their putative activation by semen. Binding of TLRs initiates a cytokine cascade which furthers the inflammatory reaction (Takeda and Akira, 2004) and TLR also form a key link between innate and humoral immune systems. In this study, we compared the expressions of TLR-4 and TLR-2 in mares inseminated with sperm or seminal plasma with their expressions in control, non-inseminated mares, as well as, the tissue levels of TLR-4 mRNA after insemination in resistant and susceptible mares.
Mares (n = 17) were examined using transrectal ultrasonography daily during estrus and for one day postovulation. Mares were categorized as resistant (R; n = 10) or susceptible (S; n = 7) to endometritis using the Streptococcus equi subsp. zooepidemicus (Strep zoo) model as described by Hughes and Loy (1969). Thereafter, all mares were given an untreated rest cycle, and on the subsequent estrous cycle were sampled (uterine double guarded swab, low volume lavage, and biopsy) when ovulation was detected (pre-treatment, non-inseminated cycle; PTCycle). Each mare was then rested for one further estrous cycle. On the subsequent ovulation mares were allocated to one of two treatment groups (randomized cross over design); Group 1: insemination with 5 ml of frozen–thawed sperm (500 × 106 ) in milk-based antibiotic-free extender (Sperm-Cycle); Group 2: insemination with 5 ml of frozen–thawed seminal plasma (SemPlasma-Cycle). Before treatment, a uterine swab was evaluated for endometritis (negative—less than 5% neutrophils per 300 cells), and, 24 h after treatment, mares were sampled (uterine swab and biopsy) as described for the PT-Cycle. Only samples from mares that were free of bacterial endometritis (negative culture, less than 15% neutrophils on low volume lavage cytology) at both pre-insemination and 24-h sample times were included in the data analysis. Mares had an untreated rest cycle before being re-enrolled in the cross over design. Uterine biopsies were flash frozen in cryovials in liquid nitrogen and then stored until analysis. Samples were processed for mRNA extraction, cDNA synthesis, and real time-PCR using previously validated methods and primers (Singh Suri et al., 2006). Friedman’s ANOVA and the Wilcoxon signed rank test were used to evaluate differ-
夽 This paper is part of the supplement entitled “Proceedings of the Tenth International Symposium on Equine Reproduction”, Guest Edited by Margaret J. Evans. ∗ Corresponding author at: 52 Campus Dr., Saskatoon SK, Canada S7N 5B4. Tel.: +1 765 426 8660; fax: +1 306 966 7159. E-mail address: [email protected] (S. Eaton). 0378-4320/$ – see front matter doi:10.1016/j.anireprosci.2010.04.067
S. Eaton et al. / Animal Reproduction Science 121S (2010) S96–S97
ences in all mares as a group and between cycles blocked by mare status for TLR-4 and TLR-2 Ct.
S97
In all mares as a group, a difference in the TLR-4 tissue expression was found between the PT-Cycle and SemPlasma-Cycle (P < 0.01) and the PT-Cycle and SpermCycle (P < 0.003). The data were further stratified by mare status. A difference was found in R mares between PT-Cycle and SemPlasma-Cycle (P < 0.03) and PT-Cycle and SpermCycle (P < 0.02). No significant differences were found in S mares for either cycle. No significant differences in TLR2 tissue expression were found in all mares as a group, or when blocked by status between PT-Cycle, Sperm-Cycle, or SemPlasma-Cycle.
TLR-2 protein expression is increased after activation of the receptor by components of Gram-positive bacteria (Takeda et al., 2003). The TLR-2 was evaluated because of the large differences between R and S mares in their response to Strep zoo. However, the lack of difference in TLR-2 mRNA expression in the endometrium of R and S mares after insemination indicates that the source of their different responses is not due to TLR-2 mRNA and that TLR-2 activation may be more specific to Streptococcus zooepidemicus. We conclude that TLR-4 mRNA is increased after treatment with seminal plasma or sperm in resistant mares but not susceptible mares when compared to estrus levels. Underlying differences in innate immune responses may contribute to the responses in S and R mares after insemination
4. Discussion
Conflict of interest
3. Results
The innate immune response plays a key role in physiologic and pathologic endometritis in the mare. Sperm primarily activate the inflammatory response through complement (Troedsson et al., 1993b) but t he immediacy of the inflammatory response, the rapid involvement of phagocytic cells, and the presence of TLR in the endometrium suggested that other innate immune mechanisms besides complement are involved. TLR activation has been associated with pro-inflammatory cytokine production (Takeda and Akira, 2004) in the endometrium of humans, mice, and cattle. Initial research suggested that TLR-4 was activated primarily by lipopolysaccharide (LPS) from Gram-negative bacteria (Takeda and Akira, 2003) but other molecules such as hyaluronan and cholesteroldependent cytolysin are now known to activate it (Termeer et al., 2002; Park et al., 2004). Our study may indicate that the innate uterine immune response in the mare is a very complicated process, and that the different responses of R and S mares are likely not due to only one factor. Our findings show that R mares increase expression of TLR-4 after treatment with seminal plasma or sperm, that S mares do not, and that a component(s) in seminal plasma and haploid DNA may activate TLR-4 expression. An inability to increase expression of TLR-4 in response to challenges with seminal plasma and sperm may contribute to the initial delay in uterine clearance by S mares (Troedsson et al., 1993a) and allow bacteria to proliferate and further activate other TLR. Changes in the innate immune response through TLRs are believed to regulate pro-inflammatory cytokine production. Fumuso et al. (2003) reported an increase in IL-1, IL-6, and TNF-␣ after insemination but did not find differences in the responses of R and S mares. Increased TLR-4 mRNA would allow R mares to provide a more rapid and targeted inflammatory response to eliminate organisms and debris quickly. The process of TLR-4 activation, cytokine stimulation, and subsequent receptor upregulation is likely complex and further research is needed to identify which steps in the pathway contribute to endometritis and differ between R and S mares.
None. Acknowledgements This research was supported by the Equine Health Research Fund at the University of Saskatchewan. Daniel DeLury, Jeffrey Bergermann, and Anne-Marie Burrel assisted in sample collection. References Causey, R.C., 2006. Making sense of equine uterine infections: the many faces of physical clearance. Vet. J. 172, 405–421. Fumuso, E., Giguere, S., Wade, J., Rogan, D., Videla-Dorna, I., Bowden, R., 2003. Endometrial IL-1beta, IL-6, and TNF-alpha, mRNA expression in mares resistant or susceptible to post-breeding endometritis. Effects of estrous cycle, artificial insemination, and immunomodulation. Vet. Immunol. Immunopathol. 96, 31–41. Hughes, J., Loy, R., 1969. Investigations on the effect of intrauterine inoculations of Streptococcus zooepidemicus in the mare. Proc. Am. Assn. Equine Pract. 15, 289–292. Park, J.M., Ng, V.H., Maeda, S., Rest, R.F., Karin, M., 2004. Anthrolysin O and other gram-positive cytolysins are toll-like receptor 4 agonists. J. Exp. Med. 200, 1647–1655. Singh Suri, S., Janardhan, K.S., Parbhakar, O., Caldwell, S., Appleyard, G., Singh, B., 2006. Expression of toll-like receptor 4 and 2 in horse lungs. Vet. Res. 37, 541–551. Takeda, K., Akira, S., 2003. Toll receptors and pathogen resistance. Cell. Microbiol. 5, 143–153. Takeda, K., Akira, S., 2004. TLR signaling pathways. Semin. Immunol. 16, 3–9. Takeda, K., Kaisho, T., Akira, S., 2003. Toll-like receptors. Annu. Rev. Immunol. 21, 335–376. Termeer, C., Benedix, F., Sleeman, J., Fieber, C., Voith, U., Ahrens, T., Miyake, K., Freudenberg, M., Galanos, C., Simon, J.C., 2002. Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4. J. Exp. Med. 195, 99–111. Troedsson, M.H., Liu, I.K., Ing, M., Pascoe, J., Thurmond, M., 1993a. Multiple site electromyography recordings of uterine activity following an intrauterine bacterial challenge in mares susceptible and resistant to chronic uterine infection. J. Reprod. Fertil. 99, 307–313. Troedsson, M.H., Liu, I.K., Thurmond, M., 1993b. Immunoglobulin (IgG and IgA) and complement (C3) concentrations in uterine secretion following an intrauterine challenge of Streptococcus zooepidemicus in mares susceptible to versus resistant to chronic uterine infection. Biol. Reprod. 49, 502–506.
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Abstract
Toll-like receptor-2 and -4 in the equine endometrium during physiologic post-breeding endometritis夽 S. Eaton a,∗ , T. Raz b , C. Card c a b c
Animal Care Hospital of Williams Lake, Williams Lake BC, Canada V2G 5E8 Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, SK, Canada S7N 5B4
1. Introduction
2. Materials and methods
Physiologic post-breeding endometritis is an inflammatory response to insemination or breeding that enables the uterus to remove excess sperm, seminal plasma, debris, and bacteria. Mares resistant to endometritis are able to clear the inflammatory response rapidly enough to allow conception. Mares susceptible to endometritis have a prolonged inflammatory response, resulting in reduced conception rates (Causey, 2006). Toll-like receptors (TLR) are a family of conserved receptors that comprise part of the innate immune system and are activated by products or cellular components of bacteria, yeast, and viruses, but no information is available about their putative activation by semen. Binding of TLRs initiates a cytokine cascade which furthers the inflammatory reaction (Takeda and Akira, 2004) and TLR also form a key link between innate and humoral immune systems. In this study, we compared the expressions of TLR-4 and TLR-2 in mares inseminated with sperm or seminal plasma with their expressions in control, non-inseminated mares, as well as, the tissue levels of TLR-4 mRNA after insemination in resistant and susceptible mares.
Mares (n = 17) were examined using transrectal ultrasonography daily during estrus and for one day postovulation. Mares were categorized as resistant (R; n = 10) or susceptible (S; n = 7) to endometritis using the Streptococcus equi subsp. zooepidemicus (Strep zoo) model as described by Hughes and Loy (1969). Thereafter, all mares were given an untreated rest cycle, and on the subsequent estrous cycle were sampled (uterine double guarded swab, low volume lavage, and biopsy) when ovulation was detected (pre-treatment, non-inseminated cycle; PTCycle). Each mare was then rested for one further estrous cycle. On the subsequent ovulation mares were allocated to one of two treatment groups (randomized cross over design); Group 1: insemination with 5 ml of frozen–thawed sperm (500 × 106 ) in milk-based antibiotic-free extender (Sperm-Cycle); Group 2: insemination with 5 ml of frozen–thawed seminal plasma (SemPlasma-Cycle). Before treatment, a uterine swab was evaluated for endometritis (negative—less than 5% neutrophils per 300 cells), and, 24 h after treatment, mares were sampled (uterine swab and biopsy) as described for the PT-Cycle. Only samples from mares that were free of bacterial endometritis (negative culture, less than 15% neutrophils on low volume lavage cytology) at both pre-insemination and 24-h sample times were included in the data analysis. Mares had an untreated rest cycle before being re-enrolled in the cross over design. Uterine biopsies were flash frozen in cryovials in liquid nitrogen and then stored until analysis. Samples were processed for mRNA extraction, cDNA synthesis, and real time-PCR using previously validated methods and primers (Singh Suri et al., 2006). Friedman’s ANOVA and the Wilcoxon signed rank test were used to evaluate differ-
夽 This paper is part of the supplement entitled “Proceedings of the Tenth International Symposium on Equine Reproduction”, Guest Edited by Margaret J. Evans. ∗ Corresponding author at: 52 Campus Dr., Saskatoon SK, Canada S7N 5B4. Tel.: +1 765 426 8660; fax: +1 306 966 7159. E-mail address: [email protected] (S. Eaton). 0378-4320/$ – see front matter doi:10.1016/j.anireprosci.2010.04.067
S. Eaton et al. / Animal Reproduction Science 121S (2010) S96–S97
ences in all mares as a group and between cycles blocked by mare status for TLR-4 and TLR-2 Ct.
S97
In all mares as a group, a difference in the TLR-4 tissue expression was found between the PT-Cycle and SemPlasma-Cycle (P < 0.01) and the PT-Cycle and SpermCycle (P < 0.003). The data were further stratified by mare status. A difference was found in R mares between PT-Cycle and SemPlasma-Cycle (P < 0.03) and PT-Cycle and SpermCycle (P < 0.02). No significant differences were found in S mares for either cycle. No significant differences in TLR2 tissue expression were found in all mares as a group, or when blocked by status between PT-Cycle, Sperm-Cycle, or SemPlasma-Cycle.
TLR-2 protein expression is increased after activation of the receptor by components of Gram-positive bacteria (Takeda et al., 2003). The TLR-2 was evaluated because of the large differences between R and S mares in their response to Strep zoo. However, the lack of difference in TLR-2 mRNA expression in the endometrium of R and S mares after insemination indicates that the source of their different responses is not due to TLR-2 mRNA and that TLR-2 activation may be more specific to Streptococcus zooepidemicus. We conclude that TLR-4 mRNA is increased after treatment with seminal plasma or sperm in resistant mares but not susceptible mares when compared to estrus levels. Underlying differences in innate immune responses may contribute to the responses in S and R mares after insemination
4. Discussion
Conflict of interest
3. Results
The innate immune response plays a key role in physiologic and pathologic endometritis in the mare. Sperm primarily activate the inflammatory response through complement (Troedsson et al., 1993b) but t he immediacy of the inflammatory response, the rapid involvement of phagocytic cells, and the presence of TLR in the endometrium suggested that other innate immune mechanisms besides complement are involved. TLR activation has been associated with pro-inflammatory cytokine production (Takeda and Akira, 2004) in the endometrium of humans, mice, and cattle. Initial research suggested that TLR-4 was activated primarily by lipopolysaccharide (LPS) from Gram-negative bacteria (Takeda and Akira, 2003) but other molecules such as hyaluronan and cholesteroldependent cytolysin are now known to activate it (Termeer et al., 2002; Park et al., 2004). Our study may indicate that the innate uterine immune response in the mare is a very complicated process, and that the different responses of R and S mares are likely not due to only one factor. Our findings show that R mares increase expression of TLR-4 after treatment with seminal plasma or sperm, that S mares do not, and that a component(s) in seminal plasma and haploid DNA may activate TLR-4 expression. An inability to increase expression of TLR-4 in response to challenges with seminal plasma and sperm may contribute to the initial delay in uterine clearance by S mares (Troedsson et al., 1993a) and allow bacteria to proliferate and further activate other TLR. Changes in the innate immune response through TLRs are believed to regulate pro-inflammatory cytokine production. Fumuso et al. (2003) reported an increase in IL-1, IL-6, and TNF-␣ after insemination but did not find differences in the responses of R and S mares. Increased TLR-4 mRNA would allow R mares to provide a more rapid and targeted inflammatory response to eliminate organisms and debris quickly. The process of TLR-4 activation, cytokine stimulation, and subsequent receptor upregulation is likely complex and further research is needed to identify which steps in the pathway contribute to endometritis and differ between R and S mares.
None. Acknowledgements This research was supported by the Equine Health Research Fund at the University of Saskatchewan. Daniel DeLury, Jeffrey Bergermann, and Anne-Marie Burrel assisted in sample collection. References Causey, R.C., 2006. Making sense of equine uterine infections: the many faces of physical clearance. Vet. J. 172, 405–421. Fumuso, E., Giguere, S., Wade, J., Rogan, D., Videla-Dorna, I., Bowden, R., 2003. Endometrial IL-1beta, IL-6, and TNF-alpha, mRNA expression in mares resistant or susceptible to post-breeding endometritis. Effects of estrous cycle, artificial insemination, and immunomodulation. Vet. Immunol. Immunopathol. 96, 31–41. Hughes, J., Loy, R., 1969. Investigations on the effect of intrauterine inoculations of Streptococcus zooepidemicus in the mare. Proc. Am. Assn. Equine Pract. 15, 289–292. Park, J.M., Ng, V.H., Maeda, S., Rest, R.F., Karin, M., 2004. Anthrolysin O and other gram-positive cytolysins are toll-like receptor 4 agonists. J. Exp. Med. 200, 1647–1655. Singh Suri, S., Janardhan, K.S., Parbhakar, O., Caldwell, S., Appleyard, G., Singh, B., 2006. Expression of toll-like receptor 4 and 2 in horse lungs. Vet. Res. 37, 541–551. Takeda, K., Akira, S., 2003. Toll receptors and pathogen resistance. Cell. Microbiol. 5, 143–153. Takeda, K., Akira, S., 2004. TLR signaling pathways. Semin. Immunol. 16, 3–9. Takeda, K., Kaisho, T., Akira, S., 2003. Toll-like receptors. Annu. Rev. Immunol. 21, 335–376. Termeer, C., Benedix, F., Sleeman, J., Fieber, C., Voith, U., Ahrens, T., Miyake, K., Freudenberg, M., Galanos, C., Simon, J.C., 2002. Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4. J. Exp. Med. 195, 99–111. Troedsson, M.H., Liu, I.K., Ing, M., Pascoe, J., Thurmond, M., 1993a. Multiple site electromyography recordings of uterine activity following an intrauterine bacterial challenge in mares susceptible and resistant to chronic uterine infection. J. Reprod. Fertil. 99, 307–313. Troedsson, M.H., Liu, I.K., Thurmond, M., 1993b. Immunoglobulin (IgG and IgA) and complement (C3) concentrations in uterine secretion following an intrauterine challenge of Streptococcus zooepidemicus in mares susceptible to versus resistant to chronic uterine infection. Biol. Reprod. 49, 502–506.