TLR3 and TLR4 expression in healthy and diseased human endometrium

Abstract

Background: Toll-like receptors (TLRs) play an essential role in the innate immune system by initiating and directing immune response to pathogens. TLRs are expressed in the human endometrium and their regulation might be crucial for the pathogenesis of endometrial diseases.

Methods: TLR3 and TLR4 expression was investigated during the menstrual cycle and in postmenopausal endometrium considering peritoneal endometriosis, hyperplasia, and endometrial adenocarcinoma specimens (grade 1 to 3). The expression studies applied quantitative RT-PCR and immunolabelling of both proteins.

Results: TLR3 and TLR4 proteins were mostly localised to the glandular and luminal epithelium. In addition, TLR4 was present on endometrial dendritic cells, monocytes and macrophages. TLR3 and TLR4 mRNA levels did not show significant changes during the menstrual cycle. In patients with peritoneal endometriosis, TLR3 and TLR4 mRNA expression decreased significantly in proliferative diseased endometrium compared to controls. Interestingly, ectopic endometriotic lesions showed a significant increase of TLR3 und TLR4 mRNA expression compared to corresponding eutopic tissues, indicating a local gain of TLR expression. Endometrial hyperplasia and adenocarcinoma revealed significantly reduced receptor levels when compared with postmenopausal controls. The lowest TLR expression levels were determined in poor differentiated carcinoma (grade 3).

Conclusion: Our data suggest an involvement of TLR3 and TLR4 in endometrial diseases as demonstrated by altered expression levels in endometriosis and endometrial cancer.

Figure 1

TLR3 and TLR4 transcript are expressed in endometrium during the menstrual cycle. Columns indicate mean TLR3 and TLR4 mRNA quantities from endometrium in proliferative (n = 16), secretory (n = 11) and menstrual phase (n = 8) run in triplicates. The y-axis is scaled logarithmically; error bars represent the standard deviation of the mean.

Figure 2

TLR3 and TLR4 protein is localised to endometrial cells during the menstrual cycle. TLR3 protein staining in healthy late proliferative (LP) tissue was high in luminal and glandular tissue (A, brown precipitate) and lower in LP endometriotic tissue (B). Late secretory (LS) endometrium showed highly expressed TLR3 in the epithelium (C), but weakly in endometriosis (D). Intense staining of TLR4 proteins was shown in mid proliferative (MP) tissue (E). In late proliferative phase of endometriosis, TLR4 proteins were comparably lower (F). TLR4 protein was high in mid secretory (MS) normal endometrium (G), whereas it was decreased in endometriotic MS tissue (H). During the menstrual phase, both TLR3 (I) and TLR4 (J) were highly expressed. Co-immunostaining for TLR4 (green), CD14 (K, red) and CD163 (L, red) demonstrated that TLR4 proteins were expressed by CD14 positive dendritic cells and monocytes (K, yellow) and by CD163 positive macrophages (L, yellow). Localisation of TLR4 to immune cells is marked by a black arrow (J) and by white arrows (K, L).

Figure 3

TLR3 and TLR4 mRNA expression is regulated in endometriosis. The expression of TLR3 (A, C) and TLR4 mRNA (B, D) in endometrium during proliferative (n = 13, run in triplicates, A, B) and secretory phase (n = 3, C, D) was decreased in eutopic endometriotic endometrium when compared to controls. In addition, four proliferative corresponding lesions were evaluated (A, B) showing a local upregulation of both receptors on ectopic sites. Columns represent the mean ratio of TLR copy number to ACTB copy number. Error bars represent the standard deviation of the mean. * P < 0.05; ** P < 0.01.

Figure 4

TLR3 and TLR4 protein is locally induced in endometriotic lesions. No specific TLR3 protein staining was seen in eutopic endometriotic tissue (A) whereas a high glandular localisation of the protein was detected in a gland of an ectopic endometriotic lesion from the same patient (B). Similarly, TLR4 was not detectable in eutopic endometrium (C) but present in glandular epithelium of ectopic endometrium from the same women (D).

Figure 5

TLR3 and TLR4 mRNA expression is decreased in endometrial adenocarcinoma. (A-B) Columns indicate mean TLR3 (A) and TLR4 (B) mRNA levels from postmenopausal patients (PMP, n = 8), and those diagnosed with endometrial hyperplasia (HP, n = 10) and endometrial carcinoma (EnCa, n = 16). (C-D) TLR3 (C) and TLR4 (D) mRNA expression in different carcinoma grades compared to postmenopausal controls and hyperplastic endometrium: G1 (n = 5), G2 (n = 6) and G3 (n = 5). Error bars represent the standard deviation of the mean. * P < 0.05; ** P < 0.01.

Figure 6

TLR3 and TLR4 proteins are present in postmenopausal endometrium. Localisation of TLR3 in normal postmenopausal endometrium (A), endometrial hyperplasia (B), endometrial adenocarcinoma grade G1 (C), G2 (D) and G3 (E). Localisation of TLR4 protein in normal postmenopausal endometrium (F), endometrial hyperplasia (G), endometrial adenocarcinoma grade G1 (H), G2 (I) and G3(J). All stained sections indicated epithelium as the preferred localisation of TLR3 and TLR4 proteins. TLR4 protein was additionally present in immune cells (arrows).

Figure 7

TLR4 is localised to immune cells of postmenopausal endometrium. Co-Immunostaining of TLR4 with CD14 and CD163 in healthy endometrium (A, B) and in adenocarcinoma (C, D). TLR4 proteins were expressed by CD14 positive dendritic cells and monocytes (A, C) and by CD163 positive macrophages (B, D). Arrows indicate the co-localisation of TLR4 with the immune cells. Scale bar = 20 μm.