Expression of Toll-Like Receptors in Uteri and Conceptuses of Ewes.

Abstract

Toll-like Receptors (TLRs) are type I transmembrane proteins involved in the innate immune response to pathogen-associated molecular pattern (PAMPs) molecules. They are pattern recognition receptors which, after binding to their ligands, initiate signaling cascades by means of inflammation, phagocytosis, activation of complement, release of inflammatory cytokines, apoptosis and activation of transcription factors. TLRs are expressed primarily in antigen processing and presenting cells, although they have been detected in endothelial cells, fibroblasts and epithelium of multiple tissues including the ovary and uterus. Influences of the conceptus on the innate immune system have been described for humans and mice; therefore, we investigated differences in expression of TLRs in cyclic and pregnant ewes. Expression of TLR was analyzed in uterine endometria from ewes on Days 10, 12, 14 and 16 of the estrous cycle and endometria and conceptuses from ewes on Days 10, 12, 14, 16, 18 and 20 of pregnancy (n=4 or 5 ewes/day; Day 0 is the day of onset of estrus). Expression of mRNAs for TLR1-TLR9 was quantified by real-time quantitative PCR. Relative quantification of gene expression was evaluated using a comparative CT method by subtracting the expression of alpha tubulin (TUBA1) CT value of each sample from the target gene CT value and data were analyzed to detect differences due to Day, Pregnancy Status and Day X Pregnancy Status interaction using PROC GLM of SAS. Expression of all TLR were affected by Day (P<0.01) except for TLR4 and TLR8 (P>0.05). Expression of TLR2, TLR3, TLR6, TLR7 and TLR8 was different between cyclic and pregnant (P<0.01) ewes and there were Day X Pregnancy Status effects for TLR2, TLR6, TLR7 (P<0.01) and TLR8 and TLR9 (P<0.05). Immunoreactive TLR7- and TLR8-like proteins were detected in uterine luminal epithelium, superficial glandular epithelium and stroma between Days 10 and 16 of the estrous cycle. Conversely, in pregnancy, endometrial expression of TLR7- and TLR8-like proteins decreased between Days 10 to Day 16, but then increased between Days 18 and 20 with abundant expression in the conceptus trophectoderm. Additionally, intraepithelial lymphocytes present mainly in the deep uterine glands expressed TLR7 on all days studied. Results of this study indicate that TLRs are differentially expressed in most uterine cell types as well as resident immune cells during the estrous cycle, and that the pattern of expression is modified during the peri-implantation period of pregnancy in ewes. (poster)