Objectives: Human Endogenous Retroviruses (HERVs) were originally derived from germ cell infection by exogenous retroviruses and comprise around eight per cent of the human genome. HERVs are highly expressed in the placenta, where HERV-W (syncytin 1) has been demonstrated to perform a fusogenic function. Due to their retroviral origin, placental syncytin 1 has been suggested to also be involved in modulating the maternal immune system. The placenta constantly sheds microvesicles (MV) into the maternal circulation, demonstrated to cause innate immune cell activation associated with normal pregnancy. In pre-eclampsia, there is both increased placental MV shedding and a heightened pro-inflammatory immune response. It was therefore hypothesised that HERVs shed via placental MV play a role in feto-maternal immune interactions and thus may be an important factor in the pathogenesis of preeclampsia (PE). More specifically, it was hypothesised that syncytin 1-positive MV activate monocytes through toll-like receptor 4 (TLR-4). The aim of this study was to determine if syncytin 1 is released from the placenta via MV and exerts an immunological effect. Methods: HERV mRNA and protein expression was measured in placenta and the BeWo choriocarcinoma cell line by qPCR, western blotting (WB) and immunostaining. Glycosylation of syncytin 1 protein was determined by PNGase F treatment followed by WB. MV shed by first trimester, term normal and PE placental explants as well as BeWo cells were isolated by ultracentrifugation. Morphology of these microvesicles was examined by electron microscopy. Syncytin 1 protein and RNA was detected in microvesicles by WB and PCR. Activation and priming of PBMCs to respond to lipopolysaccharide (LPS) by syncytin 1-positive MV and recombinant syncytin 1 was examined through cytokine production by ELISA and multiplex. Antagonism of TLR-4 by LPS-RS was used to determine involvement of the receptor. The role of syncytin 1 in MV activation was examined by siRNA knockdown. Results: HERVs are highly expressed in placental tissue. Syncytin 1 is a glycosylated protein and its expression is altered in PE. MV shed from the BeWo choriocarcinoma cell line and from first trimester and term placental explants, express HERV protein and RNA. Syncytin 1 positive MV and recombinant syncytin protein cause activation of PBMCs. Greatest activation is stimulated by PE MV. Normal MV exhibit a neutral or suppressive effect on subsequent LPS challenge to PBMCs. PE MV exacerbate the response to LPS. Antagonism of TLR-4 on PBMCs and knockdown of syncytin 1 content in MV reduces activation by placental MV.Conclusions: The findings of this thesis suggest that syncytin 1 protein expressed by the placenta is shed into the maternal circulation via MV, and can activate immune cells through TLR-4. Syncytin 1-positive microvesicles may play a role in endotoxin tolerance of innate immune cells in pregnancy. The increased activation by PE MV implies that in addition to the increased microvesicle load in this pathology, a factor intrinsic to PE MV is responsible for increased inflammation. These studies implicate microvesicle-bound syncytin 1 in the regulation of immunotolerance during pregnancy.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:553523 |
| Date | January 2011 |
| Creators | Holder, Elizabeth |
| Contributors | Tower, Clare; Aplin, John |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/the-role-of-placental-human-endogenous-retroviruses-and-shed-microvesicles-on-the-maternal-immune-system(a4dfe0ac-c938-4768-99d6-7f132c5aecf9).html |
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