New insights into the innate immune defences of the equine endometrium: in situ and in vitro expression pattern of beta-defensin

Abstract

Summary: Equine endometrial diseases are a frequent cause of subfertility causing financial loss. Degenerative and inflammatory diseases are often associated with alterations of the innate immunity and beta-defensin has been identified within the equine endometrium. The aim of this study was to further characterize the expression of beta-defensin protein within in the equine endometrium by immunohistochemistry. For in situ investigations, endometrial samples were collected from 26 mares once at different stages of the oestrus cycle and from 3 mares repeatedly during the same oestrus cycle (days 0, 5, 10, 13, 16, 19 and 21; day 0 is defined as ovulation day). In vitro examinations were performed on cultured epithelial cells obtained from endometrial specimens of 15 mares. The following results were obtained: Endometrial tissue sections of the 26 mares were either without pathological alterations (n = 3) or showed varying degrees of endometritis, endometrosis and/or angiosclerosis. Beta-defensin protein was mainly expressed within the cytoplasm of epithelial cells lining the luminal surface (100 %, 26 mares) and glandular ducts (69 %, 18 mares) and rarely within the nucleus of these cell populations (8 % and 19 %, respectively). The vast majority of cells showed either a cytoplasmic or a nuclear immunostaining. A simultaneous cytoplasmic and nuclear immunoreaction within the same cell was seldom observed (3 mares, up to 1% of the analyzed cells). Mid and basal glands without endometrosis were rarely immunopositive (nuclear immunostaining: 5 mares; cytoplasmic labelling: 1 mare). In contrast, endometrotic glands often contained small to moderate numbers of epithelial cells with a cytoplasmic immunosignal (15 of 19 cases; 79 %). In regard to the repeatedly collected biopsies of three mares, the percentage of immunopositive cells and the calculated immunoreactive scores were highly variable over the course of the oestrus cycle and between individual mares. In regard to cultured endometrial epithelial cells (n =15 mares), a positive beta-defensin immunosignal was observed in almost all cells (97 % of 521 analyzed cells) and was mainly located solely within the cytoplasm (76% of the analyzed cells), less frequently within the cytoplasm and nucleus (17 %) and rarely only within the nucleus (4 %). These results lead to the following conclusions: the antimicrobial peptide beta-defensin can be detected within the equine endometrium, the almost complete lack of beta-defensin immunostaining of unaltered glands could predispose to bacterial colonization of glandular lumina, the frequent beta-defensin immunoreaction of endometrotic glands indicates their functional alteration also in regard to the synthesis of beta-defensin, the variations in regard to the beta-defensin immunostaining between individual mares suggest a complex regulation of the beta-defensin expression, possibly under the influence of genetic factors, the beta-defensin immunostaining of cultured epithelial cells is similar to the in situ situation and therefor...

The Healthy and Diseased Equine Endometrium: A Review of Morphological Features and Molecular Analyses

“…The luminal epithelium contains always positive cells [17,21] and 71-77% of examined biopsies display staining of GD [17,21]. In comparison, labelling of healthy mid and basal glands is observed in 23% and 4% of investigated biopsies, respectively [21]. The immunoreaction of LE cells and GD is predominantly located in the cytoplasm [17,21].…”

“…Components of the innate immunity include the antimicrobial peptide β-defensin [17,21], pattern recognition receptors such as Toll-like receptors [23,24] and Nucleotide-binding and oligomerization domain (NOD) receptors [58], the immunomodulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO 1) [25], and proinflammatory cytokines [19,59,60]. The immunostaining patterns for β-defensin and IDO 1 in non-endometrotic areas and Toll-like receptor 4 (TLR 4) in different cell populations [23,24] are depicted in Figure 3.…”

“…The immunostaining patterns for β-defensin and IDO 1 in non-endometrotic areas and Toll-like receptor 4 (TLR 4) in different cell populations [23,24] are depicted in Figure 3. Endometrial biopsies immunostained for β-defensin; IDO 1; and TLRs 2, 4, and 6 (21-29 per study) are obtained from mares of varying ages, and they were collected during different stages of the endometrial cycle [17,21,[23][24][25]. Only some biopsies are devoid of any alterations (2-3 per study), whereas the remaining have one to several endometrial diseases [17,21,[23][24][25].…”

“…Immunostaining for β-defensin is mainly observed in LE cells and glandular ducts (GD), whereas epithelial cells of endometrial glands are rarely positive [17,21]. The luminal epithelium contains always positive cells [17,21] and 71-77% of examined biopsies display staining of GD [17,21]. In comparison, labelling of healthy mid and basal glands is observed in 23% and 4% of investigated biopsies, respectively [21].…”

“…In recent years, a major research focus in equine endometrial pathology was to reveal disease-associated cellular and molecular mechanisms [15][16][17][18][19][20][21][22][23][24][25][26][27][28]. This will help to gain further insights into the pathogenesis of endometrial diseases [15][16][17][18][19][20][21][22][23][24][25][26][27][28], and it may contribute to the development of effective prophylactic regimes and novel treatment options. Some of the identified biological parameters have the potential to be used as biomarkers for endometrial health and disease.…”