Modulation of inflammation in the female reproductive tract
Rajagopal, Shalini Priscilla
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Physiological inflammation occurs in the female reproductive tract, but pathological inflammation is implicated in reproductive pathologies such as preterm labour and endometrial cancer. Preterm labour (PTL, before 37 weeks of gestation) is the leading cause of preterm birth, neonatal mortality and perinatal morbidities. Endometrial cancer is the commonest gynaecological cancer, and its pathogenesis is characterised by chronic inflammation. The overall aims of this thesis were (i) to develop an in vitro model of myometrial-monocyte interactions to replicate the events occurring in the myometrium in preterm labour (ii) to determine the effects of potential therapeutics such as lipoxins, IL-10 and progesterone, on inflammation, and (iii) to characterise the lipoxin pathway in endometrial adenocarcinoma. Macrophages infiltrate the pregnant myometrium during labour; however the role of these cells is unclear. A myometrial-monocyte coculture model was developed either using non-pregnant primary myometrial smooth muscle cells (UtSMCs), or immortalised pregnant human myometrial cells (PHM1-41), with primary monocytes from term (38-41 weeks of gestation), non-labouring pregnant women. Cultures were stimulated with the toll-like receptor 4 agonist lipopolysaccharide (LPS), in the presence or absence of each of lipoxins, IL-10 and progesterone. A significant and synergistic increase in IL-6 and IL-8 secretion was found in the UtSMC/monocyte coculture after stimulation with LPS for 24 hours, compared to LPS-treated UtSMCs, or monocytes alone, but the increase in IL-6 and IL-8 secretion was not inhibited by lipoxin, epi-lipoxin or benzo-lipoxin. The PHM1-41/monocyte coculture both alone and in response to LPS treatment generated significantly increased IL-6 and IL-8 secretion, compared to vehicle treatment in the coculture and compared to the culture of either cell type alone. IL-1β and TNFα secretion were only detected from the PHM1/monocyte coculture, and monocytes alone. Use of a TNFα blocking antibody partially suppressed LPS-induced IL-6 and IL-8 secretion in the coculture. Coculture of PHM1/monocytes resulted in increased secretion of multiple mediators including pro-inflammatory cytokines, chemokines and growth factors compared to culture of either PHM1 cells or primary monocytes separately, both with vehicle and with LPS. IL-10 inhibited LPS-induced IL-6 and IL-8 secretion from the coculture, as did progesterone, which also inhibited GM-CSF, MCP-1 and CXCL5 secretion. Myocyte contraction, measured by PHM1-41 cells embedded in collagen was increased by primary monocyte treatment. This suggests that not only do infiltrating monocytes increase myometrial inflammation but they can induce myometrial smooth muscle contraction. In endometrial adenocarcinoma, the lipoxin synthesis enzymes, ALOX-5 and -15 and FPR2 mRNA expression were upregulated compared to proliferative phase endometrium, with FPR2, a reported lipoxin receptor, immunolocalised in endometrial adenocarcinoma tissue. Additionally, TNFα treatment of Ishikawa endometrial adenocarcinoma cells increased FPR2 mRNA expression, and upregulation of FPR2 mRNA also occurred in xenograft tumours from CD1 nude mice, compared to the Ishikawa cells from which they originated. These findings highlight FPR2 expression in endometrial adenocarcinoma, and suggest this receptor could mediate inflammatory signals, and lipoxins could be produced by ALOX-5 and ALOX-15. Collectively, these data describe the novel effects of monocytes in the regulation of myometrial smooth muscle cell inflammation, and demonstrate a mechanism by which myometrial inflammation during both term and preterm labour is triggered by infiltrating macrophages. This myocyte/monocyte inflammation is regulated in part by TNFα, and can be suppressed by both IL-10 and progesterone co-treatment. Components of the lipoxin pathway are present in endometrial adenocarcinoma, but their role in regulation of inflammation is still to be elucidated. Future research to clarify the processes, by which leukocyte recruitment is regulated at labour and the role of monocyte/macrophages in altering myocyte properties, could help to elucidate the mechanisms coupling inflammation to labour and provide more appropriate targets for the treatment of PTL.