Browsing by Author "Avenant, Chanel"

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    Cross talk between the glucocorticoid receptor and the progesterone receptor in modulation of progestin responses and HIV-1 infection
    (2018) Bick, Alexis J; Hapgood, Janet; Avenant, Chanel
    Current epidemiological data showing that the use of the injectable contraceptive progestin Depotmedroxyprogesterone acetate (DMPA) is associated with increased HIV-1 acquisition is controversial. However, animal and ex vivo data reveal plausible biological mechanisms whereby MPA may increase HIV-1 acquisition. Relatively high levels of endogenous progesterone (P4) found in the luteal phase of the menstrual cycle have also been linked to increased HIV-1 acquisition in animal, clinical and ex vivo models. One of the central hypotheses of the present study was that the mechanism of MPA-induced increase in HIV-1 infection occurs via a different mechanism to that of the luteal phase. Furthermore, MPA has been shown to activate both the glucocorticoid receptor (GR) and its target, the progesterone receptor (PR) isoform B (PR-B), which are both transcription factors and regulate genes involved in immune function. Both the GR and PR are expressed in the cervix, the primary site of heterosexual HIV-1 infection. PR is regulated by endogenous estrogen (E2), of which the concentrations fluctuate throughout the menstrual cycle, and GR expression also varies in response to stress hormones, leading to conditions of varied relative levels of GR/PR. The immune-related consequences of changing the relative levels of GR and PR-B are not well understood. Therefore another hypothesis of this study was that changing the relative levels of GR/PR-B modulates HIV-1 infection and immunomodulatory gene expression in response to the GR/PR agonist, MPA. Since GR and PR-B recognize similar DNA target sequences and may regulate the same genes at the same time, the final hypothesis of the present study was that GR and PR-B reciprocally modulate each other’s activity, through possible association. To investigate the effects of exogenous hormones on HIV-1 infection and mechanisms thereof, peripheral blood mononuclear cells (PBMCs) and TZM-bl cervical cells were used as model systems for HIV-1 infection. These cells were stimulated with P4 and E2 at concentrations mimicking the menstrual cycle phases or with levels of MPA at the upper range of peak serum levels detected in DMPA users. Cells were infected with the R-tropic HIV-1 infectious molecular clone, HIV-1Bal_Renilla and luciferase assays were used to measure HIV-1 infection. Levels of HIV-1 CD4 receptor and CCR5 co-receptor protein or mRNA were measured by flow cytometry or qPCR, respectively, while activation of CD4+ T cells using the activation marker CD69 was measured by flow cytometry in PBMCs. To investigate the effects of changing GR/PR-B levels on HIV-1 infection and immune gene regulation, GR/PR levels were altered in End1/E6E7 immortalized endocervical and HeLa/TZM-bl cervical carcinoma cells by GR siRNA knockdown with or without the simultaneous over-expression of PR-B, and cells were stimulated with MPA or the GR agonist Dexamethasone. mRNA expression iii of key immunomodulatory genes in End1/E6E7 and HeLa cells was measured by qPCR. The modulation of GR activity by PR-B was assessed by promoter-reporter assay in COS1 and U2OS cells over-expressing GR and PR and stimulated with GR- and/or PR-specific ligands. Association of GR and PR-B was measured by co-immunoprecipitation in COS1 and MCF-7 cells, while co-localization of GR and PR-B was measured by confocal microscopy and super-resolution structured illumination microscopy in COS1 cells. MPA significantly increased HIV-1 infection in both PBMCs and TZM-bl cells, while luteal phase hormones did so to a lesser extent. However, MPA but not luteal phase hormones increased the ratio of CD4+/CD8+ T cells in PBMCs. MPA but not luteal phase hormones also increased CCR5 protein expression on CD4+ T cells in PBMCs and total CCR5 mRNA expression in TZM-bl cells. In addition, MPA but not luteal phase hormones increased activation of CD4+ T cells in PBMCs. Using a GR antagonist or GR siRNA, it was shown that the GR but not PR-B is required for MPA-, but not luteal phase hormone-induced increased HIV-1 infection in PBMCs and TZM-bls. The presence of PR-B altered the anti-inflammatory, GR-mediated regulation of some key immunomodulatory genes, including GILZ and IL-6, in End1/E6E7 and HeLa cells in response to MPA. In general, basal (unliganded) expression of immunomodulatory genes exhibited a pro-inflammatory profile in the presence of PR-B. Co-immunoprecipitation assays showed that GR and PR-B appeared to associate. Confocal microscopy suggested GR and PR co-localized in the nucleus in response to GR- and/or PRspecific ligands, while super-resolution microscopy showed that co-localization occurred in select regions within the nucleus. Taken together, MPA increases HIV-1 infection in a manner different from that of luteal phase hormones, most likely involving increased CD4+ T cell frequency (CD4+/CD8+ ratio), activation and increased expression of CCR5 on CD4+ T cells, and requiring the GR. Furthermore, PR-B modulates GR-mediated immune function gene regulation, via potential association and region-specific nuclear co-localization. This suggests that the relative levels of GR/PR may play an important role in determining the inflammatory and immune responses and HIV-1 infection in HIV-1 target cells, both in DMPA users and women not using hormonal contraception.
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    Differential effects of progestogens on HIV-1 replication and host gene expression in primary PBMCs and cervical tissue explants
    (2015) Ray, Roslyn Michelle; Hapgood, Janet P; Avenant, Chanel
    The synthetic progestogens, medroxyprogesterone acetate (MPA) and norethisterone enanthate (NET-EN),are widely used in developing countries as injectable contraceptives, where disease burden is high.Levonorgestrel (LNG) is a common progestogen used in oral contraceptives and intrauterine devices. Some studies suggest that MPA, unlike NET, increases HIV-1 acquisition in women, while few studies have reported on the effects of LNG on HIV-1 acquisition. Whether MPA, NET and LNG differentially affect HIV-1infection and the expression of key genes relevant to HIV-1 acquisition via differential molecular mechanisms is key to understanding choice of progestogen contraceptive in young women at high risk forHIV-1 acquisition. The central hypothesis of this study is that the differential effects on host gene expression and HIV-1replication by the different progestogens is due to their differential selectivity to towards different steroid receptors, in particular the glucocorticoid receptor (GR). In order to investigate this hypothesis, the regulation of selected genes was investigated in cervical tissue explants from premenopausal, HIV-1negative, and contraception negative women and peripheral blood mononuclear cells (PBMCs) from women, by real time quantitative PCR, western blotting and Luminex assays, in response to physiologically relevant doses of progestogens. Infection assays were performed in the absence and presence of HIV-1using HIV-1BAL-RENILLA or HIVpNL4.3 infectious molecular clones (IMCs). The GR antagonist RU486 or GR siRNA knockdown was used to determine the role of the GR in modulating ligand-specific effects. PBMCs and primary cervical explants were chosen as useful models to assess the direct effects of these progestogens in both the systemic and in the local mucosal immune environments. In PBMCs, MPA like dexamethasone (DEX, a GR specific agonist), showed anti-inflammatory effects, decreasing pro-inflammatory interleukin (IL) 6, IL8 and regulated upon activation, normal T cell expressed and secreted(RANTES) levels and increasing anti-inflammatory glucocorticoid interacting leucine zipper (GILZ) gene expression levels, while NET, progesterone (P4) and LNG did not, after 48 hours. In primary ectocervical tissue explants, DEX, cortisol, MPA and P4 significantly repressed IL6 while only DEX, cortisol and MPA significantly repressed IL8 and increased GILZ gene expression levels after 48 hours. Steroid receptor expression was characterised in both PBMCs and ectocervical explants. GR was the only detectable steroid receptor protein expressed in PBMCs, while ectocervical explants expressed all the steroid receptors. The progesterone and estrogen receptor levels were higher in ectocervical explants from donors that were in the follicular phase compared to ectocervical explants from donors in the luteal phase of the menstrual cycle. In PBMCs, results suggested that differential gene expression by MPA versus NET and P4is mediated via the GR after 48 hours. Furthermore, it was observed that MPA and DEX, unlike NET, LNG and P4 increases HIV-1 replication in viable PBMCs, in the majority of donors. The increase in HIV-1replication in the MPA treated PBMC samples correlated significantly with an increased in IL6 mRNA levels.
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    The injectable-only contraceptive medroxyprogesterone acetate, unlike norethisterone acetate and progesterone, regulates inflammatory genes in endocervical cells via the glucocorticoid receptor
    (Public Library of Science, 2014) Govender, Yashini; Avenant, Chanel; Verhoog, Nicolette J D; Ray, Roslyn M; Grantham, Nicholas J; Africander, Donita; Hapgood, Janet P
    Clinical studies suggest that the injectable contraceptive medroxyprogesterone acetate (MPA) increases susceptibility to infections such as HIV-1, unlike the injectable contraceptive norethisterone enanthate (NET-EN). We investigated the differential effects, molecular mechanism of action and steroid receptor involvement in gene expression by MPA as compared to NET and progesterone (P4) in the End1/E6E7 cell line model for the endocervical epithelium, a key point of entry for pathogens in the female genital mucosa. MPA, unlike NET-acetate (NET-A) and P4, increases mRNA expression of the anti-inflammatory GILZ and IκBα genes. Similarly, MPA unlike NET-A, decreases mRNA expression of the pro-inflammatory IL-6, IL-8 and RANTES genes, and IL-6 and IL-8 protein levels. The predominant steroid receptor expressed in the End1/E6E7 and primary endocervical epithelial cells is the glucocorticoid receptor (GR), and GR knockdown experiments show that the anti-inflammatory effects of MPA are mediated by the GR. Chromatin-immunoprecipitation results suggest that MPA, unlike NET-A and P4, represses pro-inflammatory cytokine gene expression in cervical epithelial cells via a mechanism involving recruitment of the GR to cytokine gene promoters, like the GR agonist dexamethasone. This is at least in part consistent with direct effects on transcription, without a requirement for new protein synthesis. Dose response analysis shows that MPA has a potency of ∼24 nM for transactivation of the anti-inflammatory GILZ gene and ∼4-20 nM for repression of the pro-inflammatory genes, suggesting that these effects are likely to be relevant at injectable contraceptive doses of MPA. These findings suggest that in the context of the genital mucosa, these GR-mediated glucocorticoid-like effects of MPA in cervical epithelial cells are likely to play a critical role in discriminating between the effects on inflammation caused by different progestins and P4 and hence susceptibility to genital infections, given the predominant expression of the GR in primary endocervical epithelial cells.
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    Mechanisms of glucocorticoid pro-inflammatory effects on CCL20: crosstalk and synergy
    (2018) Nhundu, Tawanda; Hapgood, Janet P; Avenant, Chanel
    Glucocorticoids (GCs) are steroid hormones widely prescribed to treat inflammatory disorders and are regarded as anti-inflammatory molecules. GCs classically induce the expression of antiinflammatory genes, while repressing pro-inflammatory genes via its endogenous cell receptor; the glucocorticoid receptor (GR). Emerging evidence, however, suggests that the mechanisms of GR action are more complex than previously assumed, with many reports of pro-inflammatory actions of GCs via the GR. While chronic exposure to GCs has been noted as anti-inflammatory, reports suggest that acute exposure can increase peripheral immune responses. Specifically, the GCs have been shown to positively regulate the innate immune response, which may be important in preventing the local, affected area from being immunocompromised. Furthermore, the GR can crosstalk with cell signalling pathways involved in pro-inflammatory responses, such as the TNFα pathway, to reciprocally modulate the expression of pro-inflammatory genes. The mechanisms behind the GR’s pro-inflammatory actions and crosstalk with inflammatory inducers are not well understood. GC’s pro-inflammatory actions are attributed to GC insensitivity in asthma patients. The insensitivity is attributed to long-term GC usage, and the increase in Th17 neutrophilic airway infiltration. A proposed hypothesis for the increase in neutrophils in the airways was that it was due to an increase in expression of chemokines by epithelial cells due to GC exposure. The pro-inflammatory, chemoattractant cytokine C-C motif chemokine ligand 20 (CCL20) has been previously shown to be induced by GCs and pro-inflammatory inducers in human bronchial cells, with positive modulation of their responses occurring with co-stimulation. The present study investigated whether the GC dexamethasone (dex) and the pro-inflammatory inducer TNFα could induce CCL20 expression in a variety of human epithelial cell lines, and a simian fibroblast cell line. Using Real-Time Quantitative Reverse Transcription PCR (qRT-PCR), it was confirmed that dex can induce CCL20 mRNA expression, and modulate the TNFα-induced expression in some, but not all cell lines. Moreover, in the HeLa cell line, there was an apparent synergistic response between dex and TNFα, and modulation of the CCL20 response was observed between dex and the pro-inflammatory inducers phorbol 12-myristate 13-acetate (PMA), interferon γ (IFNγ) and lipopolysaccharide (LPS). The GR was shown to be required for the GC induction and modulation of CCL20 mRNA expression. Using promoter-reporter assays, the results showed that the NFκB binding site was necessary for the activation by the pro-inflammatory inducers, but not dex, while the STAT binding region was necessary for the IFNγ activation. Interestingly, lack of the STAT binding site on the promoter-reporter construct caused IFNγ to have repressive effects on CCL20 activation. Stimulation of cells by the pro-inflammatory inducers in the presence or absence of dex had no effect on the total levels of the p65 subunit of NFκB, while dex did appear to cause GR turnover as expected. The results show that dex, via the GR, is able to crosstalk with different pro-inflammatory inducers to induce and potentiate CCL20 mRNA expression and promoter activation. The mechanisms of CCL20 induction and crosstalk with the GR may be different for each proinflammatory inducer, however. Regulation of CCL20 expression is complex, with many transcription factors converging on the promoter region to modulate its expression. This thesis shows that the NFκB binding site is important for the overall induction level of the promoter, however it is not necessary for the dex induced activation. The potentiation of the dex response by pro-inflammatory inducers may be due to the GR interacting with the AP-1 and C/EBP transcription factors, which have been shown to positively interact to increase gene expression. The potentiation of the dex response does not require the activation of NFκB, as IFNγ does not activate the transcription factor, yet can potentiate the dex response.
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    Modulation of GR transcriptional signalling by HIV-1 Vpr insights into regulation by progestins
    (2012) Grantham, N J; Hapgood, Janet P; Avenant, Chanel
    It has been 30 years since HIV was first discovered, yet the molecular mechanisms whereby the virus mediates its pathogenic effects have not yet been completely elucidated. The glucocorticoid receptor (GR) is a ligand-activated host transcription factor, which mediates anti-inflammatory effects in response to stimulation with glucocorticoids (GC). One of the HIV-1 accessory proteins, Vpr, is highly immunosuppressive and contributes to suppression of the immune system thereby creating an environment favourable for viral proliferation. Vpr has been previously reported to act as a GR co-activator on glucocorticoid response element (GRE) containing promoters. Thus, the GR appears likely to play a role in HIV-1 pathogenesis. Contraceptive usage is also likely to affect HIV-1 pathogenesis as some hormonal contraceptives can bind to and activate the GR. Progesterone (P4) regulates the female reproductive system and the synthetic progestins medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-A) are extensively used as injectable contraceptives. MPA has been shown to act as a partial or full GR agonist and recent evidence indicates that injectable MPA increases HIV-1 acquisition and transmission. The molecular mechanisms of this remain unclear, but may involve decreasing the thickness of the vaginal epithelium as well as actions via the GR that affect gene expression in the cervo-vaginal environment and/or elsewhere. This study aims to investigate the actions of GC's, P4, MPA and NET-A via the GR in the absence and presence of Vpr protein towards gaining some insight into the potential interplay between the host GR, contraceptive use, HIV-1 pathogenesis, and the mechanisms thereof.
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    Modulation of the progesterone receptor by progestogens, antiretroviral drugs and the glucocorticoid receptor
    (2021) Enfield, Kim; Hapgood, Janet P; Avenant, Chanel
    Hormonal contraceptives (HCs) and hormonal replacement therapies (HRTs) are widely used globally by women. However, the relative activity of some of the progestogens used in HCs and HRT has not been determined via their target steroid receptor (SR), the progesterone receptors and its isoforms (PR-A and PR-B). This thesis involves an in vitro investigation of some of the factors that affect PR activity and that may thus impact progestogen responses in women on HC or HRT. These include progestogen-specific effects via PR-A and PR-B, progestogen metabolism, the role of antiretroviral drugs (ARVs) and crosstalk with the glucocorticoid receptor (GR). The PR isoforms play important roles in multiple physiological processes, including cognition, regulation of inflammation, mitochondrial function, neurogenesis, female reproduction and disease. Due to the gender disparity of higher prevalence of HIV infection among women compared to same-aged men, there is increased interest in developing treatments designed to protect women from HIV infection. These include HIV pre-exposure prophylactics (PrEPs) and multipurpose prevention technologies (MPTs). Furthermore, many HIV -infected women are using combined antiretroviral therapy (cART) to treat AIDS as well as an HC to prevent unwanted pregnancy. Importantly, the effects of the combination of a progestogen with ARVs on the activity of the PR is unexplored. In addition, there are many studies which provide evidence that SRs interact and influence each other's activity. Therefore, the presence of the ubiquitously GR may also influence PR activity. To characterise the progestogen-induced activity of the PRs, dose-response analysis was performed using promoter-reporter genes and varying doses of progesterone (P4) and five widely-used progestogens namely; norethisterone (NET), etonogestrel (ETG), levonorgestrel (LNG), medroxyprogesterone acetate (MPA) and nestorone (NES), in parallel relative to the reference progestogen promegestone (R5020). The effects of experimental conditions and progestogen metabolism on PR activity were investigated in U2OS cells using two different transient transfection conditions to express PR-B. The effect of model system on PR activity was investigated by comparing the progestogen-induced PR-B responses obtained in U2OS cells to those obtained in MDA-MB-231 cells stably transfected with expression vectors for PR-A or PR-B. Progestogen-induced PR activity was also investigated on endogenous genes by quantitative real-time PCR (real-time qPCR) in the MDA-MB-231 PR-B-stably expressing (MDA-PR-B+) cells. Once the progestogen responses via the PR were characterised, PR-B activity in the presence of three ARVs namely; tenofovir disoproxil fumarate (TDF), dapivirine (DPV) and maraviroc (MVC), was investigated next using promoter-reporter assays and real-time qPCR in MDA-PR-B+ cells. The mechanism of ARV action on PR-B activity was investigated by competitive binding assays, as well as by determination of PR phosphorylation levels. The effect of the GR on progestogen-induced PR-B activity in terms of potency, efficacy and biocharacter was investigated using promoter-reporter assays, real-time qPCR on select endogenous genes and efficacy on multiple genes in a PCR array. Using GR-specific siRNA knockdown in MDA-PR-B+ cells, the progestogen-induced PR responses were compared for higher and lower GR levels. The mechanism of the effect of the GR on PR activity was further investigated by co-immunoprecipitation studies and the degree of PR phosphorylation in the presence of higher and lower GR levels was also compared. Results show that in the presence of the progestogens investigated, in vitro biological responses via PR-B can vary significantly in biocharacter and absolute values for efficacies and potencies. Progestogen-specific responses were found on the same synthetic promoter, as well as on the same endogenous gene in the same cell. These progestogen-specific responses negatively correlated with progestogen-specific metabolism in U2OS cells. Furthermore, the progestogen-specific responses also varied in a gene- and model system-specific manner. The majority of the progestogeninduced responses via PR-B were significantly more efficacious, but less potent that the progestogen-induced responses via PR-A, highlighting the importance of the relative expression of PR-B vs PR-A protein levels in determining the resultant progestogen-specific response. It was shown for the first time that the ARVs, MVC and TDF activated PR-B transcription in the absence of progestogens to result in increased transcription in promoter-reporter assays and increased mRNA for endogenous genes. MVC and TDF exhibited no direct binding to PR-B; however, novel data from this thesis showed increased PR-B phosphorylation at Ser294 with TDF but not MVC. DPV activated two PR-regulated genes in the absence of progestogens and competed for binding of P4 to PR-B, while resulting in no effect on PR-B phosphorylation. Using GR-specific siRNA, it was determined by promoter-reporter assays that the presence of the GR had an inhibitory effect on PR-B efficacy. This surprising observation was supported by the global and promoter-specific gene expression changes observed for some genes using a PCR array with and without GR knockdown. The mechanism by which the GR affects the PR-B activity was determined to be most likely by association with PR-B in the same protein complex, probably resulting in a significant decrease in PR-B phosphorylation. Taken together, the in vitro differences between progestogen actions via the PR suggest that the absolute values of the progestogen-induced efficacies and potencies are likely to vary in vivo in a progestogen-, promoter-, model system- and isoformspecific manner. While obtaining such data in vivo is not possible, the in vitro data from the present study show proof-of-concept of potential significant cell-, gene- and progestogen-specific PR-B effects, as well as PR isoform-specific effects. Additionally, this study shows that potential novel off-target immunomodulatory effects of MVC, TDF and DPV occur in vitro and these are most likely mediated by different mechanisms of PR-B activation. Lastly, this study shows that in cells where both the GR and PR-B are co-expressed, the resultant PR response is likely to be inhibited on select genes as a result of reduced PR-B phosphorylation. The extent to which the presence of the GR affected the determination of progestogen-, model system-, promoter- and isoform-specific responses of progestogens via the PR in this study was not investigated for all models and genes, but is likely to be an unavoidable confounding factor. However, since all cells that express the PR also express the GR in vivo to the best of the author's knowledge, characterising PR activity in the absence of detectable GR expression is unlikely to be physiologically relevant. Collectively these data suggest that multiple factors influence PR activity and these need to be taken into account, especially given the treatments available and under investigation, which are designed to specifically target the PR.
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    Molecular mechanism of action of the glucocorticoid receptor:Role of ligand-dependent receptor phosphorylation and half-life in determination of ligand-specific transcriptional activity. : Contents Pages
    (2009) Avenant, Chanel; Hapgood, Janet P
    Glucocorticoids mediate their effects by binding to the glucocorticoid receptor (GR), resulting in modulation of transcription of target genes via direct binding to DNA or tethering via proteinprotein interactions. A central question is what determines the rank order of ligand-selective transcription with different GR ligands for the same gene in the same cell. Using a panel of twelve GR ligands, including agonists, partial agonists and antagonists, the relationship between the extent of GR phosphorylation at S226, GR turnover and transcriptional response, was investigated using a variety of biochemical approaches. Using a phospho-S226-specific GR antibody, ligand-selective S226 phosphorylation was shown to occur in both COS-1 and U2OS cells, while GR phosphorylation at S226 was shown to inhibit maximal transactivation and transrepression efficacy. Attempts to identify the kinases responsible for this interaction were inconclusive but suggested a combination of kinases is responsible for the in vivo phosphorylation of the hGR in these cells. Similarly the rate of GR degradation was different for the different ligands. Interestingly, both ligand-selective GR phosphorylation and half-life were found to correlate with efficacy for transactivation and transrepression of model synthetic reporter genes, where agonists resulted in the greatest extent of phosphorylation and the fastest vii rate of GR turnover, suggesting a link between these functions. Furthermore experiments where transcription was blocked suggest that GR turnover does not require transcription. However, using a S226A GR mutant, as well as in experiments where GR turnover was blocked, it was established that neither phosphorylation of the GR at S226 nor GR degradation rate determines the rank order of ligand-selective GR transactivation. The mechanisms whereby GR phosphorylation influence GR-mediated transcription was further investigated using a triple phosphorylation deficient mutant. It was shown that phosphorylation at one or more of residues S203/S211/S226 is required for transactivation of a MMTV promoter but does not affect unliganded or agonist-induced GR degradation and acetylation. Additionally, it was shown that phosphorylation at S203/S211/S226 is not the sole determinant of co-activator p300 recruitment to the GR. Interestingly, GR-mediated transrepression via AP-1 is less sensitive to GR phosphorylation than GR-mediated transactivation, indicating different mechanisms in the role of GR phosphorylation on transactivation vs. transrepression. Pull-down and chromatin immunoprecipitation assays showed that phosphorylation of the GR at one or more of these residues are required for interaction of the GR with the co-activator GRIP-1 in vitro and for maximal recruitment of GR and GRIP-1 to the MMTV promoter in intact cells. Cellular fractionation showed that phosphorylation at these residues is not however required for GR nuclear localisation. Taken together these results support the conclusion that phosphorylation at one or more of S203/S211/S226 of the hGR is required for maximal transactivation response to enable GRIP-1 recruitment to the hGR.
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    The progestin-only contraceptive medroxyprogesterone acetate, but not norethisterone acetate, enhances HIV-1 Vpr-mediated apoptosis in human CD4+ T cells through the glucocorticoid receptor
    (Public Library of Science, 2013) Tomasicchio, Michele; Avenant, Chanel; Toit, Andrea Du; Ray, Roslyn M; Hapgood, Janet P
    The glucocorticoid receptor (GR) regulates several physiological functions, including immune function and apoptosis. The HIV-1 virus accessory protein, viral protein R (Vpr), can modulate the transcriptional response of the GR. Glucocorticoids (GCs) and Vpr have been reported to induce apoptosis in various cells, including T-cells. We have previously shown that the injectable contraceptive, medroxyprogesterone acetate (MPA) is a partial to full agonist for the GR, unlike norethisterone acetate (NET-A). We investigated the functional cross talk between the GR and Vpr in inducing apoptosis in CD4 + T-cells, in the absence and presence of GCs and these progestins, as well as progesterone. By using flow cytometry, we show that, in contrast to NET-A and progesterone, the synthetic GR ligand dexamethasone (Dex), cortisol and MPA induce apoptosis in primary CD4 + T-cells. Furthermore, the C-terminal part of the Vpr peptide, or HIV-1 pseudovirus, together with Dex or MPA further increased the apoptotic phenotype, unlike NET-A and progesterone. By a combination of Western blotting, PCR and the use of receptor- selective agonists, we provide evidence that the GR and the estrogen receptor are the only steroid receptors expressed in peripheral blood mononuclear cells. These results, together with the findings that RU486, a GR antagonist, prevents Dex-, MPA- and Vpr-mediated apoptosis, provide evidence for the first time that GR agonists or partial agonists increase apoptosis in primary CD4 + T-cells via the GR. We show that apoptotic induction involves differential expression of key apoptotic genes by both Vpr and GCs/MPA. This work suggests that contraceptive doses of MPA but not NET-A or physiological doses of progesterone could potentially accelerate depletion of CD4 + T-cells in a GR-dependent fashion in HIV-1 positive women, thereby contributing to immunodeficiency. The results imply that choice of progestin used in contraception may be critical to susceptibility and progression of diseases such as HIV-1.
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    Role of the glucocorticoid receptor and HIV-1 Vpr in inflammatory gene expression and HIV-1 LTR transcription in response to dexamethasone and progestogens
    (2015) Govender, Yashini; Hapgood, Janet P; Avenant, Chanel
    The relationship between progestin-only injectable contraception and risk of HIV-1 acquisition is controversial. Most clinical data suggests that the injectable contraceptive medroxyprogesterone acetate (MPA), unlike norethisterone enanthate (NET-EN), increases susceptibility to infections such as HIV-1. The first part of this thesis investigated the differential effects, molecular mechanisms of action and steroid receptor involvement in gene expression by MPA as compared to NET and progesterone (P4) in the End1/E6E7 and HeLa cell line models for the endocervical epithelium, a key point of entry for pathogens in the lower female genital tract (FGT). Quantitative real-time PCR analysis showed that MPA, unlike NET-acetate (NET-A) and P4, increases mRNA expression of the anti-inflammatory GILZ and IκBα genes. Similarly, MPA unlike NET-A, decreases mRNA expression of the pro-inflammatory IL-6, IL-8 and RANTES genes, and IL-6 and IL-8 protein levels. The predominant steroid receptor expressed in the cervical cell lines and primary endocervical epithelial cells is the glucocorticoid receptor (GR), and GR siRNA experiments show that the anti-inflammatory effects of MPA are mediated by the GR. Chromatin-immunoprecipitation results suggest that MPA, unlike NET-A and P4, represses pro-inflammatory cytokine gene expression in cervical epithelial cells via a mechanism involving recruitment of the GR to cytokine gene promoters, like the GR agonist dexamethasone (DEX). This is at least in part consistent with direct effects on transcription, without a requirement for new protein vi synthesis. This is the first study to show direct proof for a GR-mediated mechanism of action in anti-inflammatory effects of MPA. Dose response analysis shows that MPA has a potency of ~24 nM for transactivation of the anti-inflammatory GILZ gene and ~4 - 20 nM for repression of the pro-inflammatory genes, suggesting that these effects are likely to be relevant at injectable contraceptive doses of MPA. These findings suggest that MPA effects on genital mucosal immune function and susceptibility to infections are likely to be very different to those of NET and P4, when mediated by the GR The second part of this thesis investigated the effects of the virion associated HIV-1 protein, Vpr, on GR-regulated inflammatory genes in the presence of the ligands.
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    Synergism between immune activators and progestogens: differential effects on gene expression and HIV-1 replication and the role of the glucocorticoid receptor
    (2018) Moliki, Johnson Mosoko; Hapgood, Janet P.; Avenant, Chanel
    Globally, women account for more than half of the 36.9 million people living with HIV-1. In some regions particularly Sub-Saharan Africa, women of child-bearing age are at great risk of being infected with HIV-1 than men of the same age group. Transmission is mainly through penetrative vaginal intercourse with an infected male partner. Current evidence suggests that women using certain types of progestin-only hormonal contraceptive methods have a higher risk of HIV-1 infection. In addition, there is evidence suggesting that genital tract infections (GTIs), which have high prevalence in SubSaharan Africa, also modify the risk of HIV-1 infection in women. Incidentally, regions were progestogen-only hormonal contraceptives use and genital tract infections are prevalent also have with high rates of HIV-1 prevalence in women. This raises the question whether medroxyprogesterone acetate (MPA), norethisterone enanthate (NET) or levonorgestrel (LNG), the active compounds in progestogen-only hormonal contraceptives, cooperate with inflammation associated with GTIs to further elevate the risk of HIV-1 acquisition in women. Current evidence suggests MPA, LNG and NET differentially the expression of immune function genes relevant for HIV-1 infection in the female genital tract (FGT). In addition, there is evidence suggest that MPA and LNG impairs the integrity of mucosal barrier of the FGT. This study investigated the central hypothesis that MPA, NET and LNG acting alone or in synergy with immune activators [tumour necrosis factor-alpha (TNF) and lipopolysaccharide (LPS)] via the glucocorticoid receptor (GR) regulate the expression of genes of relevant for maintaining the integrity of genital tract mucosal surfaces, mucosal permeability, select immune function genes and HIV-1 replication. This hypothesis was investigated in the endocervical epithelial End1/E6E7 cell, ectocervical tissues explants from pre-menopausal women, primary genital tract epithelial cells, peripheral blood mononuclear cells (PBMCs) and the TZM-bl indicator cell line. Gene expression analysis in responses to progestogen alone or in combination with immune activators was performed by real-time PCR, ELISA, Luminex assays and western blotting. The role of the GR was investigated using RU486 or GR siRNA knockdown. Mucosal barrier integrity and permeability was assessed by confocal microscopy and transepithelial electrical resistance measurements. For infection assays, TZM-bl cells were exposed to HIV-1Bal-Rellina infectious molecular clones (IMCs) before treatment progestogen alone or in combination with immune activators. In addition, TZM-bl cells were preconditioned with supernatants from PBCs treated alone with progestogen or in combination with immune activators before exposed to HIV-1Bal-Rellina IMCs. In the absence of TNF, MPA at physiologically relevant doses acted via the GR to downregulate claudin-4 mRNA expression in End1/E6E7 cells, with MPA behaving like a partial GR agonist when compared to dexamethasone (DEX). Similarly, MPA acted via the GR to selectively upregulate CCL20 mRNA expression in End1/E6E7 cells, with MPA behaving like a full GR agonist when compared to hydrocortisone (CORT). It was also observed that MPA upregulated toll-like receptor (TLR)2 mRNA expression, but reduced interleukin (IL)6 and IL1β mRNA expression in End1/E6E7 cells. Neither NET not LNG regulated claudin-4 and immune function expression in End1/E6E7 cells. The addition of TNF, on the other hand, did not alter the effect of MPA on claudin-4 expression, suggesting that there is no cooperativity between MPA and TNF in regulating this gene. Neither NET nor LNG was found to cooperate with TNF to regulate claudin-4 expression in End1/E6E7 cells. However, MPA unlike NET acted synergistically via the GR with TNF or LPS to upregulate C-C motif chemokine ligand (CCL)20 expression in End1/E6E7 cells. This was not an isolated event as MPA was also found to enhance TNF-induced expression of TNF receptor 2 (TNFRSF1B). The occurred against the backdrop of MPA but not NET repressing TNF-induced expression of IL6, IL1β and CCL5. In ectocervical tissues, MPA like NET did not regulate basal claudin-4 expression, but unlike NET downregulated desmoglein-1 expression. This suggest that MPA may increase the permeability of the endocervix and ectocervix via different mechanisms. This study was unable to established whether MPA and TNF synergistically upregulate CCL20 in ectocervical tissues. In PBMCs, however, MPA selectively enhanced LPS-induced expression of CCL20, but suppressed LPS-induced expression of IL6, IL8, IL1β and CCL5. Finally, MPA unlike NET was found to act alone or additively with TNF or LPS to increase HIV-1 replication in TZM-bl cells. While this suggest MPA directly affects HIV-1 replication, it was observed that the effects may also be indirect as secretions from PBMCs cotreated with MPA and LPS but not NET and LPS enhanced HIV-1 replication in TZM-bl cells. Taken together, the results shown in this study provides new insight into plausible mechanisms by MPA but not NET acting via the GR may enhance the susceptibility of the endocervix to HIV-1. Firstly, they suggest that MPA unlike NET may increase the permeability of the endocervix via a mechanism that is different from the ectocervix. Secondly, they suggest that the upregulation of select immune mediators and innate immune receptors by MPA but not NET in the endocervix in the absence of immune activation may render the endocervix vulnerable to HIV-1 infection. Thirdly, that MPA unlike NET is more likely to synergise with immune activators to further upregulate the expression of select immune function, but not tight junction genes in the endocervix. Collectively, this suggest MPA unlike NET can cooperate with GTIs to further increase the risk of HIV-1 acquisition in women residing in high risk regions. In this setting, NET-EN but not DMPA-IM would be a safer choice of injectable progestin-only contraceptive.