Gene Transcription of TLR2, TLR4, and LPS Ligands in the Endometrium of the Bitch with Cystic Endometrial Hyperplasia-Pyometra Complex.

Abstract

Cystic endometrial hyperplasia (CEH)-pyometra complex is the most frequent endometrial disorder in the bitch. Gram negative are the most frequent bacteria isolated in cases of pyometra and, their isolation are associated with more frequent and severe systemic signs. Toll-like receptors (TLRs) play an essential role in the innate immune system by initiating and directing the immune response to pathogens. TLR4 and TLR2 are recognized as receptors for components of Gram negative and Gram-positive bacteria (and lipoproteins of Gram-negative bacteria), respectively. Complex formation with bacterial co-receptor CD14 and lipoprotein binding protein (LBP), as well as MD-2 association, is necessary for maximal activation of TLR4 signaling. To the best of our knowledge, no data was reported on the expression of TLRs in the canine uterus. Therefore, the aim of this study was to evaluate the gene transcription of TLR2, TLR4 and LPS ligands in the endometrium of canine diestrous uteri without (n=7) or with pyometra (n=7). Uteri were collected during routine ovariohysterectomy (OVH) and uterine contents were cultured. Phase of the estrous cycle was determined by the observation of ovarian structures, recorded estrous date and measurement of plasma progesterone concentration by radioimmunoassay. Immediately after OVH, endometria were separated, rinsed with sterile RNAse free cold saline solution, immediately frozen in liquid nitrogen and then stored at −80°C until processed. For histological classification, according to De Bosscherre et al., (2001), tissue samples were fixed in 10% neutral phosphate buffered formalin. Gene transcription of TLR2, TLR4, CD14, MD2 and LBP were analyzed by relative real time PCR quantification and normalized with the housekeeping gene RPL27. All cases of pyometra were hyperplasic and E. coli was the only isolated bacteria. Diestrous normal uteri did not present signs of CEH and were negative for bacteriology. Gene transcription of TLR2 and TLR4 were respectively 8.3 and 2.4 times higher in pyometra cases compared to normal uteri (p< 0.001). Also, CD14, MD2 and LPB gene transcription showed a 7.4, 4.4 and 5.0 fold increase in pyometra cases compared to normal diestrous endometria, respectively (p< 0.001). This data shows that TLR2 and TLR4 are trancripted in the normal canine diestrous endometrium. It also shows that theses genes are up-regulated in E. coli pyometra cases. This up-regulation is probably ascribed to the stimulation by LPS and lipoprotein E. coli constituents. The stimulation by LPS is suggested by the observed up-regulation of CD14, LBP and MD2 transcription. (Supported by Grants POCTI/CVT/66587/2006 from Foundation for Science and Technology (FCT) and CIISA 74-Endometrial Hyperplasia. Maria Elisabete Silva was supported by a postdoctoral fellowship BPD/35031/2007 from FCT). *Mateus, L and Silva, E had equal contribution for this work. (poster)