Role of the glucocorticoid receptor and HIV-1 Vpr in inflammatory gene expression and HIV-1 LTR transcription in response to dexamethasone and progestogens

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

The Current Evidence on the Association Between the Urinary Microbiome and Urinary Incontinence in Women

Urinary incontinence (UI) is a burdensome condition with high prevalence in middle-aged to older women and an unclear etiology. Advances in our understanding of host-microbe interactions in the urogenital tract have stimulated interest in the urinary microbiome. DNA sequencing and enhanced urine culture suggest that similarly to other mucosal sites, the urinary bladder of healthy individuals harbors resident microbial communities that may play distinct roles in bladder function. This review focused on the urobiome (expanded quantitative urine culture-based or genomic sequencing-based urinary microbiome) associated with different subtypes of UI, including stress, urgency and mixed urinary incontinence, and related syndromes, such as interstitial cystitis and overactive bladder in women, contrasted to urinary tract infections. Furthermore, we examined clinical evidence for the association of the urinary microbiome with responses to pharmacotherapy for amelioration of UI symptoms. Although published studies are still relatively limited in number, study design and sample size, cumulative evidence suggests that certain Lactobacillus species may play a role in maintaining a healthy bladder milieu. Higher bacterial diversity in the absence of Lactobacillus dominance was associated with urgency UI and resistance to anticholinergic treatment for this condition. UI may also facilitate the persistence of uropathogens following antibiotic treatment, which in turn can alter the commensal/potentially beneficial microbial communities. Risk factors of UI, including age, menopausal status, sex steroid hormones, and body mass index may also impact the urinary microbiome. However, it is yet unclear whether the effects of these risks factors on UI are mediated by urinary host-microbe interactions and a mechanistic link with the female urogenital microbiome is still to be established. Strategies for future research are suggested

The injectable-only contraceptive medroxyprogesterone acetate, unlike norethisterone acetate and progesterone, regulates inflammatory genes in endocervical cells via the glucocorticoid receptor

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

Medroxyprogesterone acetate, unlike norethisterone, increases HIV-1 replication in human peripheral blood mononuclear cells and an indicator cell line, via mechanisms involving the glucocorticoid receptor, increased CD4/CD8 ratios and CCR5 levels.

High usage of progestin-only injectable contraceptives, which include the intramuscular injectables depo-medroxyprogesterone acetate (DMPA-IM, Depo-Provera) and norethisterone (NET) enanthate (NET-EN or Nur-Isterate), correlates worldwide with areas of high HIV-1 prevalence. Epidemiological data show a significant association between usage of DMPA-IM and increased HIV-1 acquisition but no such association from limited data for NET-EN. Whether MPA and NET have similar effects on HIV-1 acquisition and pathogenesis, and the relationship between these effects and the dose of MPA, are critical issues for women's health and access to suitable and safe contraceptives. We show for the first time that MPA, unlike NET, significantly increases HIV-1 replication in peripheral blood mononuclear cells (PBMCs) and a cervical cell line model. The results provide novel evidence for a biological mechanism whereby MPA, acting via the glucocorticoid receptor (GR), increases HIV-1 replication by at least in part increasing expression of the CCR5 HIV-1 coreceptor on target T-lymphocytes. MPA, unlike NET, also increases activation of T-cells and increases the CD4/CD8 ratio, suggesting that multiple mechanisms are involved in the MPA response. Our data offer strong support for different biological mechanisms for MPA versus NET, due to their differential GR activity. The dose-dependence of the MPA response suggests that significant effects are observed within the range of peak serum levels of progestins in DMPA-IM but not NET-EN users. Dose-response results further suggest that effects of contraceptives containing MPA on HIV-1 acquisition and disease progression may be critically dependent on dose, time after injection and intrinsic factors that affect serum concentrations in women

Only GR protein is detected in primary cervical epithelial cells (VEN-100).

<p>(<b>A</b>) Upon arrival the VEN-100 cells were rested overnight before being washed once with PBS and harvested with TRIzol®. Total RNA was isolated and 500 ng RNA was reverse-transcribed. Steroid receptor gene expression was measured by qRT-PCR with receptor-specific primers, followed by gel electrophoresis to confirm the PCR products. (<b>B</b>) VEN-100 cells were rested overnight before harvesting in 2X SDS sample buffer. COS-1 cells were transiently transfected with 1 µg/well pcDNA3 (empty vector) which served as negative control (−CTRL) or with 1 µg/well steroid receptor expression vectors (GR, PR-B, AR, MR and ERα) which served as positive controls (+CTRL). Twenty fourhrs later, the COS-1 cells were washed once and lysed with 2X SDS sample buffer. Equal volumes of cell lysate (VEN-100 and COS-1 ctrls) were analysed by Western blotting with antibodies specific for the GR and Flotillin-1 (loading control), respectively.</p

Aberrant cervical innate immunity predicts onset of dysbiosis and sexually transmitted infections in women of reproductive age.

Sexually transmitted infections (STIs) and vaginal dysbiosis (disturbed resident microbiota presenting with abnormal Nugent score or candidiasis) have been associated with mucosal inflammation and risk of HIV-1 infection, cancer and poor reproductive outcomes. To date, the temporal relationships between aberrant cervical innate immunity and the clinical onset of microbial disturbance have not been studied in a large population of reproductive age women. We examined data from a longitudinal cohort of 934 Ugandan and Zimbabwean women contributing 3,274 HIV-negative visits who had complete laboratory, clinical and demographic data. Among those, 207 women later acquired HIV, and 584 women were intermittently diagnosed with C. trachomatis (CT), N. gonorrhoeae (NG), genital herpes (HSV-2), T. vaginalis (TV), candidiasis, and abnormal intermediate (4-6) or high (7-10) Nugent score, i.e. bacterial vaginosis (BV). Immune biomarker concentrations in cervical swabs were analyzed by generalized linear and mixed effect models adjusting for site, age, hormonal contraceptive use (HC), pregnancy, breastfeeding, genital practices, unprotected sex and overlapping infections. High likelihood ratios (1.5-4.9) denoted the values of cervical immune biomarkers to predict onset of abnormal Nugent score and candidiasis at the next visits. When controlling for covariates, higher levels of β-defensin-2 were antecedent to BV, CT and HSV-2, lower anti-inflammatory ratio IL-1RA:IL-1β-to intermediate Nugent scores and candida, lower levels of the serine protease inhibitor SLPI-to candida, lower levels of the adhesion molecule ICAM-1 -to TV, and lower levels of the oxidative stress mitigator and endothelial activation marker VEGF-to NG. Changes in innate immunity following onset of dysbiosis and infections were dependent on HC use when controlling for all other covariates. In conclusion, imminent female genital tract dysbiosis or infection can be predicted by distinct patterns of innate immunity. Future research should characterize biotic and abiotic determinants of this pre-existing innate immunity state

The GR at least in part directly regulates mRNA levels of the inflammatory genes.

<p>End1/E6E7 cells were pretreated with 1 µg/ml cycloheximide (CHX) then treated for 24 hrs with 100 nM DEX, MPA, P4, NET-A or vehicle (ethanol) (CTRL), in the absence or presence of CHX. Total RNA was isolated and reverse-transcribed. Relative (<b>A</b>) GILZ (<b>B</b>) IκBα, (<b>C</b>) RANTES, (<b>D</b>) IL-6 and (<b>E</b>) IL-8 gene expressions was measured by real-time qRT-PCR and normalised to GAPDH mRNA expression. In addition, relative gene expressions were normalized to basal activity (CTRL) in order to obtain relative fold expression. Graphs represent pooled results of at least three independent experiments and are plotted as mean ± SEM. To verify that the CHX inhibited <i>de novo</i> protein synthesis, End1/E6E7 cells were pretreated with CHX then treated with 100 nM DEX or vehicle (ethanol) (CTRL) for 24 hrs. (<b>F</b>) Cells were harvested and equal volumes of lysate were analysed by Western blotting with an antibody specific for IκBα and a GAPDH specific antibody as loading control. (<b>G</b>) Western blots of four independent experiments were quantified to determine the relative GR protein expression. Statistical analysis was carried out using GraphPad Prism software (version 5) using a one-way ANOVA with a Dunnett post-test followed by a student’s t-test to compare specific conditions to each other. Statistical significance is denoted by *, ** or *** to indicate P<0.05, P<0.001 or P<0.0001, respectively.</p

MPA-induced HIV-1 replication requires the GR in TZM-bl cells.

<p>TZM-bl cells transfected with 10 nM non-silencing control (NSC) or siRNA targeting the human GR (siGR) for 48 hours. (a) Cells were re-seeded into 96-well plates for 24 hours, followed by stimulation for 24 hours with 100 nM MPA or vehicle (0.1% v/v EtOH), then infected with 10 IU/mL HIV-1_{BaL_Renilla} (“HIV-BaL”) or equivalent volume of virus control (“Uninfected”). Cells were harvested 72 hours later for luciferase (infection) and for MTT (cell viability). The results were pooled from 3 independent experiments where each point was in quadruplicate and are represented as mean ± SEM. Relative infection was calculated as luciferase (RLU) divided by average absorbance at 595nm (MTT) for the quadruplicates. Infection was plotted relative to HIV-1_{BaL_Renilla} vehicle control set to 1. Statistical comparisons were carried out using a two-way ANOVA with Tukey’s multiple comparisons post-test, with **** denoting p<0.001. (b) Cells seeded and transfected in parallel were harvested 48 hours after transfection in SDS sample buffer. Lysates were analyzed for GR levels by western blotting using GAPDH as a loading control. A representative western blot is shown.</p

MPA, unlike NET, increases CD4 and CCR5 mRNA levels in TZM-bls, via a GR-dependent mechanism.

<p>TZM-bl cells were stimulated for 24 hours with indicated ligands (a-b) 100 nM MPA, 100 nM NET or vehicle control (EtOH) (0.1% v/v ethanol) or (c-d) 10 nM MPA or (e-f) 100 nM MPA, 100 nM RU486 (RU), or combinations thereof (RU/MPA). RNA was isolated, cDNA was synthesised and relative CCR5, CD4 mRNA levels were determined by real time qPCR, normalized to GAPDH and relative fold change in expression was determined by setting vehicle control to 1. Results are shown from 6 independent experiments for each panel and is represented as mean ± SEM. Statistical significance was determined by using (a-b, e-f) one-way ANOVA with a post hoc Tukey tests between conditions or (c-d) using a parametric unpaired t-test comparing the vehicle to 10 nM MPA with * or ** denoting p<0.05 or p<0.01 respectively.</p

MPA, unlike NET, dose-dependently increases R5 HIV-1 replication in the TZM-bl cervical cell line, via a GR-dependent mechanism.

<p>TZM-bl cells were stimulated in parallel for 24 hours with the various ligands, or combinations thereof or vehicle control (0.1% v/v EtOH), at the concentrations indicated, before infection with 20 IU/mL R5 HIV-1_BaL-Renilla. Samples were harvested 48 hours later for <i>Renilla</i> luciferase and MTT viability assays. Each condition was performed at least in triplicate. Relative infection was calculated by normalising RLU against corresponding MTT values. This value for vehicle was set to 1 for (a) and (b), while for (c), data were analysed relative to the maximal response generated by MPA which was set to 100%. Statistical analysis was performed using (a) a parametric one-way ANOVA with Dunnett’s multiple comparisons post test when comparing to vehicle or (b) a non-parametric Kruskal-Wallis one-way ANOVA with a subsequent unpaired t test comparing vehicle and MPA or comparing MPA and MPA/RU486. (c) A non-linear regression line was generated to calculate EC₅₀ and statistical analysis was performed using a parametric one-way ANOVA with Tukey multiple comparisons post-test comparing all conditions. Significant differences are shown by *, ** or *** denoting p<0.05, p<0.01 or p<0.001, respectively. The data (a-c) are represented as mean ± SEM and show the results of nine, five and six independent experiments, respectively, with each point performed in triplicate at least. <b>(d-e) The GR is the predominant steroid receptor protein expressed in the TZM-bl cell line</b>. Cell lysates were prepared and the steroid receptor mRNA and protein levels were detected by qRT-PCR and western blotting, respectively. (d) indicates that TZM-bl cells express detectable GR, AR and MR mRNA while (e) shows that TZM-bl cells express detectable GR and AR protein.</p