Polarised bovine endometrial epithelial cells vectorially secrete prostaglandins and chemotactic factors under physiological and pathological conditions

Abstract

Epithelial cells of the endometrium secrete prostaglandins to regulate the bovine oestrous cycle and form a functional barrier to microbes. However, bacterial infection of the endometrium commonly causes infertility in dairy cattle by disrupting endometrial physiology. Epithelial cell cultures are used to study the mechanisms of physiology and pathology, but 2D cultures may not reflect the 3D complexity of the epithelium. In this study, a polarised epithelial cell transwell culture was developed, using transepithelial resistance (TER), to monitor epithelial integrity. Polarised epithelial cells were treated with oxytocin and arachidonic acid to test physiological function and with lipopolysaccharide (LPS) to mimic bacterial infection. Supernatants were analysed for prostaglandin E 2 (PGE), prostaglandin F 2a , the chemokine interleukin-8 (IL8) and the ability of supernatants to induce neutrophil migration. Confluent epithelial cells established polarity when TER was O1800 Ucm 2 and predominantly released prostaglandins basolaterally. In contrast, IL8 from epithelial cells accumulated apically and the supernatants were highly chemotactic for neutrophils. The striking exception was when the epithelial cells were treated with LPS in the apical or basolateral compartment independently, which led to the release of IL8 towards the treated compartment. Although stromal cells also accumulated PGE and IL8 in response to treatment, co-culture of stromal cells in the well below polarised epithelial cells did not influence cellular responses. In conclusion, polarised endometrial epithelial cells vectorially released prostaglandins and chemokines to reflect their respective mechanistic roles in physiology and pathology.

A three-dimensional model of primary bovine endometrium using an electrospun scaffold

“…This was sufficient to provide a multiple layer-stromal cell mass, overlaid by epithelium suitable for in vitro testing. OT+AA stimulated increased accumulation of PGF as expected, based upon previous studies using 2D monocultures, or co-cultures of epithelial and stromal cells on a transwell insert, [27,29,46]. In contrast, explants produce both PGE and PGF in response to either OT+AA or LPS treatment [26,47].…”

“…Having established a stromal cell population on the scaffold, the timing of were not in direct contact, did not alter the proliferation of either cell type [29].…”

“…Dissection and isolation of endometrial cells was performed as previously described [29,30]. Briefly, the endometrium was dissected from the uterine horn ipsilateral to the corpus luteum.…”

“…As the co-culture contained a mixture of cell types, and the standard curve for MTT optical density (OD) against cell number is specific to cell type [29], the number of cells growing in the coculture construct could not be calculated, and are reported as OD (Fig. 4(c)).…”

Innate immunity and inflammation of the bovine female reproductive tract in health and disease

“…Addition of LPS to epithelial or stromal cells in vitro stimulates phosphorylation of MAPK and the nuclear translocation of NFkB, secretion of prostaglandin F 2a , prostaglandin E 2 , IL6 and IL8, and increased expression of antimicrobial peptides MUC1, LAP and TAP (Herath et al 2006, Davies et al 2008, Cronin et al 2012. Intriguingly, the release of IL8 from polarised epithelial cells is vectorial and directed apically if the challenge is in the uterine lumen (MacKintosh et al 2013). Similar cytokine and chemokine responses are also evident for cells treated with bacterial lipopeptides, with formal proof for the importance of innate immunity established using siRNA targeting TLR1, TLR2, TLR4 and TLR6 to suppress inflammation (Cronin et al 2012, Turner et al 2014.…”