The regulatory role of adrenomedullin on macrophage activities and its association with the pathogenesis of tubal ectopic pregnancy

Abstract

Tubal ectopic pregnancy (TEP) is one of the major causes of maternal morbidity and mortality in the first trimester of pregnancy. The most important predisposing condition of TEP is salpingitis. Adrenomedullin (ADM) is an immune-modulatory peptide with potent anti-inflammatory properties. Macrophage is the major immune cell population in the oviduct of TEP. They trigger the pro-inflammatory responses by secreting various pro-inflammatory cytokines. Both ADM insufficiency and macrophage accumulation in the fallopian tubes of TEP have been reported. The hypothesis of this study is that insufficiency of ADM expression in the fallopian tubes contributes to exacerbate pro-inflammatory responses of oviductal macrophages, consequently facilitates the tubal susceptibility for embryo-tubal implantation and the occurrence of TEP. In this study, we included salpingitis as predisposing condition of TEP. Reduced ADM expression and macrophage infiltration, as well as elevated expression of implantation-related molecules such as E-cadherin, active β-catenin, leukemia inhibitory factor (LIF) and HomeoboxA (HoxA)-10 concomitantly took place in fallopian tubes of both salpingitis and TEP patients. In the oviductal macrophages from both salpingtis and TEP subjects, the expression of M1 macrophage marker inducible nitric oxide synthase (iNOS), but not M2 macrophage markers (CD163 and CD206), was observed. We also detected the expression of ADM receptors-calcitonin receptor like receptor (CRLR) and receptor activity modifying protein 2 (RAMP2) in oviductal macrophages. By using macrophages isolated from fallopian tubes of both salpingitis and TEP subjects, M1 human macrophages derived from human blood monocytes and monocytic cell line THP-1 derived macrophages, we demonstrated that ADM administration inhibited pro-inflammatory condition-induced upregulation of interleukin (IL)-6 and IL-8 in macrophages. The inhibitory effect of ADM on pro-inflammatory condition-induced IL-6 and IL-8 expression was further confirmed via CRLR knockdown by siRNA suppression. Moreover, we demonstrated that ADM treatment suppressed pro-inflammatory condition-induced activation of nuclear factor kappa B (NF-κB) signalling pathway by using THP-1 derived macrophages. A JEG-3 trophoblastic spheroids-tubal epithelial cells (OE-E6/E7) co-culture model was used to study the protective role of ADM against tubal implantation. ADM effectively blocked the oviductal macrophage-derived conditioned medium-induced JEG-3 spheroids attachment onto tubal epithelial cells. Besides, ADM administration suppressed the oviductal macrophage-derived conditioned medium-induced upregulation of implantation-related molecules in tubal epithelial cells. This effect of ADM is possibly mediated by its inhibitory effect on IL-6 and IL-8 expression in human oviductal macrophages. In conclusion, this study provided an explanation on the role of ADM in the pathogenesis of TEP by regulating macrophage activities. Our data revealed that ADM insufficiency in the fallopian tubes contributed to aggravated pro-inflammatory responses of oviductal macrophages by releasing implantation inducible pro-inflammatory cytokines, creating an adhesive and receptive tubal epithelium susceptible for embryo implantation with high levels of implantation-related molecules, further led to embryo-tubal implantation. The results of this study may indicate the possible use of ADM as a protective role against the occurrence of TEP. Further investigation is required to elucidate the detail actions of ADM in the pathogenesis of TEP.