Bacterial infection of the female genital tract is common in cattle particularly after parturition, causing considerable disease, infertility and even mortality . The endometrium is the first line of defence against these bacteria. Key components of innate immunity are the recognition of PAMPs by TLRs, leading to increased expression of AMPs and APPs [11, 15]. In the present study endometrial samples expressed TLR s 1 to 10, whilst purified populations of epithelial cells expressed TLR s 1 to 7 and 9, and stromal cells expressed TLR s 1 to 4, 6, 7, 9 and 10. The TLRs appeared to be functional as epithelial cells secreted prostaglandin E2 in response to bacterial PAMPs. In addition, the epithelial cells expressed AMPs, such as TAP and LAP, which were increased when the cells were treated with LPS. Although, there was no evidence of localised secretion of APPs, the epithelial cells also expressed MUC-1. It appears that the epithelial cells play a critical role in the innate immune defence of the endometrium against bacteria that cause infertility.
The observation that bovine endometrial tissue expressed gene transcripts for all ten TLRs is similar to human endometrium [27, 28]. Human endometrial TLR expression is regulated in part by the stage of the cycle . In cattle, there is a progesterone concentration gradient across the uterus with the highest concentration at the tip of the horn ipsilateral to the corpus luteum and the lowest in the contralateral uterine horn . Progesterone concentrations are important because progesterone suppresses immune defences in the bovine endometrium . However, there were no obvious differences in gene transcript expression for any of the TLR s between different locations in the uterus.
Endometrial tissue samples contain several cell types including immune cells, so to test the concept that the endocrine cells have a role in immunity, we explored TLR expression in purified populations of epithelial and stromal cells, which are free of immune cell contamination in cattle . The epithelial cell expression of TLR s 1 to 7 and 9, and stromal cells expression of TLR s 1 to 4, 6, 7, 9 and 10, is similar to humans, where expression of TLR 1 to 9 has been reported in endometrial cell lines and in primary uterine epithelial cell cultures [30, 31]. The epithelial cell expression of TLR s 1, 2, 4, 6 and 9 is important because they are required to detect bacterial PAMPs . Bacterial infection is the predominant cause of uterine disease in cattle, and E. coli paves the way for other pathogens to cause damage to the endometrium, as well as disrupting endocrine function .
Bacterial PAMPs include LPS, LTA and DNA, so we tested the effects of these and the synthetic Pam3CSK4, to gain an insight into whether the cognate TLRs 1, 2, 4, 6 and 9 are functional in the epithelial cells. We chose to evaluate prostaglandin E2 concentrations because it is a clear marker of bacterial infection in the bovine endometrium, as well as having an important endocrine role for regulating ovarian cycles and implantation [14, 32]. E. coli or LPS switches epithelial cell secretion from prostaglandin F2α to predominantly prostaglandin E2, mediated by the TLR4, MD2, CD14 signalling complex expressed by epithelial cells . The predominant secretion of prostaglandin E2 rather than prostaglandin F2α is dependent on bacteria or PAMPs stimulating changes in the eicosaniod synthesis pathway, which includes phospholipases and prostaglandin synthases (Herath, unpublished data). This switch in function is important as prostaglandin F2α initiates luteolysis whereas prostaglandin E2 is luteotrophic in ruminants . Prostaglandin E2 is also an important component of the immune response to bacteria and regulates or suppresses inflammation in many tissues . In the present study, LPS, LTA and Pam3CSK4 increased prostaglandin E2 secretion and none of the ligands affected epithelial cell survival, extending earlier observations that LPS was detected by human and bovine endometrial cells [14, 35]. This stimulation of epithelial cell secretion of prostaglandin E2 by several bacterial PAMPs may explain the association between uterine disease and extended luteal phases [36, 37]. The secretion of prostaglandin E2 was greater for the LPS from O55:B5 than O111:B4 E. coli, which may reflect the greater purity of the latter preparation. On the other hand, the O55:B5 LPS provided a potent PAMP to evaluate AMP expression in subsequent experiments.
The AMPs are an important arm of the innate immune defence against bacteria and TLRs mediate their induction in many mamalian tissues [15, 38]. In the present study, bovine endometrial epithelial cells expressed several AMPs including LAP, TAP, BNBD4 and DEFB5, whilst stromal cells expressed mainly LAP and TAP. The predominant expression of TAP and LAP by the epithelial cells is similar to the bovine mammary gland . In humans, β-defensins have also been detected in the endometrium (HBD1, 2, 3 and 4), although there are some differences between studies [40, 41]. In the present study, the epithelial cell AMP expression appeared to be of functional importance as the gene expression was increased in epithelial cells treated with LPS. The epithelial cells were consistently more responsive than the stromal cells, and LPS stimulated a greater response than LTA. Similarly in humans, endometrial HBD2 mRNA expression is increased after 24 h treatment with LPS, although unlike studies with human cells we did not find that progesterone regulated AMP expression in endometrial cells [40, 41]. However, the substantial induction of epithelial LAP and TAP by LPS treatment probably reflects their defensive role against E. coli in cattle, and agrees with the concept that TLRs mediate induction of AMPs in response to PAMPs .
The concentrations of APPs, including haptoglobin and serum amyloid A, are substantially increased in the peripheral plasma of cow with bacterial infection of the endometrium . Similar to the AMPs, the APPs such as haptoglobin and serum amyloid A provide non-specific protection against microbes . Although usually produced by the liver, there is evidence of localised APP expression in the genital tract of rodents, regulated by the sex steroids [19, 20]. However, in the present study the concentrations of haptoglobin and serum amyloid A proteins were barely detectable in epithelial or stromal cells, and not affected by treatment of cells with LPS. In vivo peripheral plasma concentrations are at least 25 times those of the culture supernatants . So, it appears unlikely that localised secretion of APPs plays a major role in endometrial immunity in cattle.
MUC-1 is a glycosylated transmembrane protein commonly expressed by the epithelial cells of mucosal surfaces, including the reproductive tract, gut, testis and mammary gland [21, 43]. MUC-1 is expressed in the human and ovine endometrium and has important roles in endometrial receptivity for embryo implantation, and is a marker of endometrial health and fertility in humans [22, 23]. However, like AMPs and APPs, MUC-1 also has a role in protecting mucosal surfaces against bacteria. MUC-1 sterically inhibits microbial access to the cell surface and regulates inflammation [21, 43]. In the present study, MUC-1 expression was detected in the bovine epithelial cells and LPS increased the mRNA expression. In the human endometrium, MUC-1 is highly regulated by the ovarian sex steroids . Although luteal phase concentrations of ovarian steroids decreased MUC-1 expression, follicular phase concentrations had no effect in the present study. Further, neither steroid combination affected the increase of MUC-1 expression stimulated by LPS. This is in contrast to the reduction in LPS-stimulated prostaglandin E2 secretion associated with progesterone or oestradiol .