Regulation of Antimicrobial Peptides in Human Endocervical Epithelial Cells by IL-1β, Endotoxin and Vitamin D.

INTRODUCTION: Antimicrobial peptides (AMPs), including trappin-2/elafin and cathelicidin, are a first line defense peptides released from epithelial cells in response to pathogens. Vit D also plays an integral role in the acquired and innate immune responses and a correlation between Vit D deficiency and infection is well established. However, the cellular impact of Vit D on AMPs in the reproductive tract has not been fully defined. We hypothesize that pregnant women at risk of SPTB express altered AMPs profiles and that this may be linked to Vit D deficiency and existing inflammation/infection. The aim of this study was to describe AMP expression in a human endocervical cell line (END1/E6E7) and determine the impact of Vit D in the presence and absence of the pro-inflammatory cytokine IL-1β and the bacterial product LPS on AMP profiles.
METHODS: END1/E6E7 cells were cultured to ∼80% confluence and assessed for AMP (elafin, hBD2, SLPI, cathelicidin) mRNA expression by qPCR. Cells were treated ± 1,25-dihydroxyvitamin D3 (Vit D), IL-1β or LPS or with IL-1β/ LPS pre-treatment followed by Vit D. Cell culture supernatants were assessed for AMP protein expression by ELISA (normalised to whole cell protein). AMP mRNA copy number was normalised to GAPDH. Data was analysed using Kruskal-Wallis analysis of variance and Mann-Whitney U test for individual comparisons (significance taken as P<0.05; all treatment groups n=6).
RESULTS: All AMPs were expressed in unstimulated END1/E6E7. Basal expression of SLPI and elafin mRNA was relatively high (mean copy number ± SEM; SLPI: 2183666 ± 417498; elafin: 2612389 ± 328253); hBD2 and cathelicidin had low mRNA expression (hBD2: 1318 ± 239; cathelicidin: 1505 ± 158). IL-β increased elafin and hBD2 mRNA (196± 14%; 577± 149%, P<0.01) and protein (239 ± 23%; 1374 ± 167%, P<0.01) expression from basal levels as did LPS (P<0.01). Neither IL-1β nor LPS had any effect on SLPI or cathelicidin. Vit D did not alter elafin, SLPI or hBD2 expression but enhanced cathelicidin mRNA and protein expression (P<0.01 and P<0.05 respectively). Vit D also increased cathelicidin mRNA and protein expression in cells pre-treated with IL-1β or LPS (P<0.01).
CONCLUSIONS: AMPs were differentially stimulated by inflammation and Vit D in END1 cells. Further investigation into the specific functional roles of the AMPS in this system and the potential for Vit D treatment to enhance cathelicidin defenses against pathogens is warranted.