The effects of 7β-OH Epiandrosterone on Cytokine production by human immune cells

Student thesis: Doctoral Thesis

Abstract

During inflammatory responses, tumour necrosis factor alpha (TNF-α) and interleukin-1 (IL-1b) are the major pro-inflammatory cytokines that can stimulate the production of secondary mediators such as prostaglandins (PGs) including PGE2 which are responsible for the symptoms of inflammation. 7β-hydroxy epiandrosterone (7β OHEpiA) is a naturally occurring steroid that has recently been reported to have cytoprotective actions and can prevent ischaemia-reperfusion induced cell death. This action appears to be related to the production of PGD2 and its spontaneous metabolite15-deoxy PGJ2 (15-d-PGJ2). However, previous studies have provided little information about the actions of this steroid on immune/inflammatory responses and whether any of its actions may be mediated via glucocorticoid receptors. Therefore, this study investigated the effects of 7β OH-EpiA on TNF-α and IL-1β in LPS stimulated whole human blood, isolated monocytes and the human monocytic cell line THP-1 and also its effects on the production of anti-inflammatory cytokines such as IL-4 and IL-10. Whole human blood was used directly, and responses were compared to monocytesisolated from whole human blood and to THP-1 cells which were cultured continuously. Levels of cytokines (TNF-α, IL-1β, IL-4 and IL-10) and prostaglandins (PGE2, PGD2 and 15d-PGJ2), in plasma or cell supernatants, were measured by ELISA and enzyme immunoassay (EIA) respectively. In addition to this, the effect of 7β OHEpiA on the expression of various genes (including TNF-α, TLR-4, PGDS and PTGDR2) in THP-1 cells involved in inflammatory responses was evaluated by quantitative real-time PCR.7β OH-EpiA reduced TNF-α and IL-1β production at low concentrations (<0.1 μM) in blood monocytes and THP-1 cells. In addition, the glucocorticoid analogue dexamethasone also significantly reduced the LPS-induced production of both cytokines but at higher concentrations (≥ 50 μM)7β OH-EpiA did not affect the level of the anti-inflammatory cytokines IL-4 or IL10 whereas dexamethasone significantly increased the quantity of IL-4 at concentrations ≥ 1 μM. The glucocorticoid and progesterone receptor antagonist mifepristone did not alter the inhibitory action of 7βOH-EpiA on LPS-induced TNF-α production but reversed the dexamethasone-induced suppression of TNF-α. The non-steroidal cyclooxygenase inhibitor, ketoprofen,reversed the 7β OH-EpiA-induced suppression of LPS-stimulated increases in the concentration of TNF-a. With respect to prostaglandin production, LPS increased concentrations of PGE2, PGD2 and 15d-PGJ2 in both whole human blood and isolated monocytes. 7β OH-EpiA alone selectively increased concentrations of PGD2 and 15d-PGJ2 in contrast to decreasing the level of PGE2. Dexamethasone reduced the LPS stimulated increase in production of PGE2, PGD2 and 15d-PGJ2. There were no effects of mifepristone on either the 7β OH-EpiA-induced increase in PGD2 and 15d-PGJ2 production or the decrease in PGE2 production. Ketoprofen was confirmed to directly inhibit the production of all PGs in both blood and monocytes. With respect to the actions of 7β OH-EpiA on gene expression, alone it did not affect expression of the TLR-4 gene in THP-1 cells, a small decrease in TLR4 expression was observed with both LPS and 7β OH-EpiA versus LPS alone. 7β OH-EpiA did, however, decrease the LPS-stimulated increase in TNF-a gene expression in THP-1cells. With respect to PGD2 processes, 7β OH-EpiA increased the quantity of mRNAfor the PGDS (PGD synthase) gene and increased the concentration of PTGDR2 (DP2receptor) expression.The data indicates that the actions of 7β OH-EpiA on TNF-α production are highly unlikely to be mediated via a glucocorticoid or progesterone steroid receptor. They could be mediated via the selective upregulation of prostaglandin production,specifically PGD2 or 15d-PGJ2 which were increased by 7β OH-EpiA. Coupled
Date of Award15 Jul 2019
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SupervisorDino Rotondo (Supervisor) & Alexander Gray (Supervisor)

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