- Title
- Epigenetic regulation of labour promoting pro and anti-inflammatory genes in the human decidual stromal cells (hDSCs): involvement of bromodomains and extra-terminal (BET) proteins
- Creator
- Ajgaonkar, Sandeep
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2024
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- The decidua is the endometrium of pregnancy, contacting the outermost layer of the fetal membranes. The decidua composes of 70% of stromal cells and 30% of bone marrow-derived cells. The decidual stromal cells (DSCs) exhibit immunomodulatory properties, allowing the semi-allogenic foetus to develop in the mother’s womb throughout the pregnancy. The activity of the immune cells is modulated, among others, by indoleamine-2,3 dioxygenase 1 (IDO1), an enzyme produced by decidual stromal cells (DSCs) promoting immune tolerance. Furthermore, the DSCs, exhibit an anti-inflammatory phenotype during pregnancy. At the end of gestation, a proinflammatory transformation occurs, stimulating labour. In the case of intrauterine infection, the inflammatory phenotype of the DSCs is assumed early, which may result in preterm labour. The mechanism underlying these processes are poorly understood. My PhD research addresses the decidual expression and regulation of pro- and anti-inflammatory genes in response to sub-clinical infection. I isolated, purified and characterised DSCs from term pregnant women. I treated the cells with lipopolysaccharide (LPS) to model a infectious insult and determined the effects on the expression of a panel of pro-inflammatory genes (IL6, CXCL8/IL8, PTGS1, PTGS2, PTGES, TNF-a) and anti-inflammatory genes (IL10, IDO1). I examined the involvement of histone acetylation and the acetyl-histone binding BET (Bromodomain and Extra-terminal) family transcription factors in regulating the selected genes in the LPS-treated primacy DSC cultures. I treated the cell with epigenetic probes (+)-JQ1, (-)-JQ1 and I-BET-762 under conditions recommended by SGC (Structural Genomics Consortium) for studying BET involvement. I also assessed BET involvement by determining the expression of the BET family members- BRD2, BRD3, BRD4S and BRD4L in the cultured DSCs. I found that both the proinflammatory and the anti-inflammatory genes in the panel were upregulated by LPS, indicating a balanced response. The constitutively expressed genes (PTGS1 and PGES) were largely unaffected. The BET-inhibiting epigenetic probes blocked the upregulation, suggesting that the BET transcription factors(s) are critically important players in the DSC response to LPS. In agreement with this, I found robust expression of two BET family members, BRD2 and BRD4L, in DSCs, potentially mediating the actions of LPS and the epigenetic probes. The TNFa gene, however, was unresponsive to BET inhibitors, demonstrating that a BET independent pathway of inflammatory gene regulation also exists. Chromatin precipitation (ChIP) analysis showed varying levels of histone-3 and histone-4 acetylation at the promoter regions of the LPS-induced genes, but there was no consistent change in response to treatments. Furthermore, no BRD2 or BRD4 binding was detected at the promoters under any condition studied. My results demonstrate that the BET transcription factors are major regulators of the DSC responses in infection. Their actions, however, likely involve chromatin loci distant from the promoters of the regulated genes. My research is identified BET inhibitors as a new class of drugs that may control the labour-associated proinflammatory phenotype conversion of DSCs before labour, which has potential therapeutic benefit. In preliminary study, I addressed a further important aspect of DSC regulation in pregnancy: the possibility of long term, persistent effects of a transient infectious insult changing DSC responses to subsequent stimulation. A proinflammatory ‘reprogramming’ of DSCs by a subclinical infection may sensitise the decidua to a second exposure, resulting in adverse pregnancy outcomes such as preterm birth. I performed priming-restimulation experiments, which suggested the possibility of DSCs forming an innate immune memory enhancing the response to a second stimulation. I also conducted a flow-cytometry analysis of the prime DSCs, which indicated that a subset of the cells changed phenotype and became activated by LPS, supporting a reprogramming effect. Further studies are planned to substantiate these possibilities.
- Subject
- epigenetic; histone; infection; preterm birth; bromodomains
- Identifier
- http://hdl.handle.net/1959.13/1516924
- Identifier
- uon:57049
- Rights
- Copyright 2024 Sandeep Ajgaonkar
- Language
- eng
- Full Text
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