Norethisterone Enanthate Increases Mouse Susceptibility to Genital Infection with Herpes Simplex Virus Type 2 and HIV Type 1

Abstract

Norethisterone enanthate (NET-EN) and depot-medroxyprogesterone acetate (DMPA) are two forms of injectable progestin used for contraception. Whereas clinical research indicates that women using DMPA are more susceptible to HIV and other genital pathogens, causal relationships have not been determined. Providing an underlying mechanism for this connection, however, is recent work that showed DMPA weakens genital mucosal barrier function in mice and humans and respectively promotes susceptibility of wild-type and humanized mice to genital infection with HSV type 2 and HIV type 1. However, analogous effects of NET-EN treatment on antivirus immunity and host susceptibility to genital infection are much less explored. In this study, we show that compared with mice in estrus, treatment of mice with DMPA or NET-EN significantly decreased genital levels of the cell-cell adhesion molecule desmoglein-1 and increased genital mucosal permeability. These effects, however, were more pronounced in DMPA- versus NET-EN-treated mice. Likewise, we detected comparable mortality rates in DMPA- and NET-EN-treated wild-type and humanized mice after intravaginal infection with HSV type 2 or cell-associated HIV type 1, respectively, but NET-EN treatment was associated with slower onset of HSV-induced genital pathology and lower burden of systemic HIV disease. These findings reveal DMPA and NET-EN treatment of mice significantly reduces genital desmoglein-1 levels and increases genital mucosal permeability and susceptibility to genital pathogens while also implying that NET-EN generates less compromise of genital mucosal barrier function than DMPA.

FIGURE 1.. Vaginal tissue from NET-EN– and MePRDL-treated mice exhibit dissimilar expression of GR-regulated genes.

(A) Scatter plots comparing NET serum levels in untreated mice in estrus versus NET-EN–treated mice indicate the NET-EN dosing schedule devised for this study produced pharmacologically relevant drug concentrations. (B) Comparing vaginal expression of the GR-regulated gene glucocorticoid-induced leucine zipper (Tsc22d3), arrestin β 2 (Arrb2), and GR (Nr3c1) in mice in estrus and DMPA-, NET-EN–, or MePRDL-treated mice shows markedly different gene expression in mice administered an exogenous progestin versus the pure GR agonist MePRDL. Data displayed are pooled from two independent experiments with comparable results (seven animals per group); one-way ANOVA and Dunnett post hoc test provided statistical comparison; bars denote mean 6 ± SD.

FIGURE 2.. DMPA and NET-EN increase genital mucosal permeability.

(A) Representative confocal images of vaginal tissue from wild-type mice in estrus or treated with NET-EN or DMPA show these progestins increased tissue permeability to the low–molecular weight fluorescent molecule Lucifer yellow (LY) (457 Da) (green) but not the higher molecular mass dextran conjugated with Texas Red (70 kDa) (red); DAPI (blue). Scale bar, 200 μm. White lines drawn to delimit vaginal mucosal versus lamina propria tissue. (B–D) Identically treated mice were used to compare the entry of activated hPBMC into vaginal mucosal and lamina propria tissue. (B) Representative images typify the enhanced entry of CFSE-labeled hPBMC (green) into vaginal tissue of DMPA- and NET-EN–treated mice; DAPI blue. Scale bar, 100 μm. Drawn white lines demarcate mucosa from lamina propria. (C) Image analyses show that DMPA and NET-EN comparably increased hPBMC entry into vaginal epithelium. (D) Conversely, there was significantly greater hPBMC incursion into the lamina propria of DMPA- versus NET-EN–treated mice (as defined using ImageJ to calculate pixels per 10⁴ μm² of CFSE-labeled hPBMC). Data in (C) and (D) are pooled from two independent experiments in which similar results were obtained (seven animals per group). Statistical analyses were performed using one-way ANOVA and Dunnett post hoc test (bars denote mean ± SD). L, lumen.

FIGURE 3.. Treatment of mice with DMPA or NET-EN reduces vaginal DSG1 levels.

(A) Comparing vaginal Dsg1a and Dsc1 gene expression in mice in estrus versus DMPA- and NET-EN-treated mice shows significantly lower levels of these cell–cell adhesion molecules in progestin-treated mice; one-way ANOVA and Dunnett’s post hoc tests provided statistical analyses; bars denote mean ± SD. (B) These groups were also used in DSG1 protein studies, and representative images typify lower levels of DSG1 in DMPA- and NET-EN-treated mice; DSG1a (green); DAPI (blue). Scale bar, 100 μm. (C) Image analyses show both progestins significantly reduced DSG1, but DMPA caused greater reduction; one-way ANOVA and Dunnett post hoc test were used for statistical analyses; bars indicate mean ± SD. (D) Significantly greater kallikrein-related 7, 13, and 14 levels were detected in vaginal tissue from DMPA- versus NET-EN–treated mice; bars denote mean ± SD; unpaired Student t tests used for statistical analyses. Data in (A), (C), and (D) were pooled from two independent studies with similar results (five to eight mice per group). Dsc1, desmocollin; Dsg1a, desmoglein-1α; L, lumen.

FIGURE 4.. DMPA and NET-EN treatment increases mouse susceptibility to genital HSV-2 infection.

(A) Wild-type mice in estrus and mice treated with DMPA or NET-EN were euthanized 24 h after ivag infection with 3 × 10⁶ PFUs of HSV-1q–GFP, and representative images illustrate widespread dissemination of virus only in vaginal tissue from progestin-treated mice. Scale bar, 50 μm. DAPI (blue); HSV-1q–GFP (green). (B–D) In these panels, identically treated groups of mice were used to assess morbidity and mortality generated by ivag infection with 10⁴ PFUs of HSV-2 333. (B) All progestin-treated mice developed genital pathology, but (C) HSV-2 infection induced greater pathology in DMPA- versus NET-EN–treated mice; AUC were calculated for individual mice treated with DMPA or NET-EN and compared using unpaired t test. (D) Survival curves depict the 100% mortality induced by genital HSV-2 infection of DMPA- and NET-EN–treated mice and that no mice infected in estrus succumbed; data representative of two independent studies with comparable results (five mice per group in each study); log-rank tests compared cumulative survival incidence after HSV-2 infection (estrus versus DMPA or NET-EN, p < 0.0001) (DMPA versus NET-EN, p = 0.018). AUC, area under the curve; HSV-1q–GFP, HSV-1–expressing green-fluorescent protein; L, lumen.

FIGURE 5.. Treatment of humanized mouse with DMPA or NET-EN increases susceptibility to genital infection with cell-associated HIV-1.

(A) Estrus-stage, DMPA-treated, and NET-EN–treated hPBMC-NSG (i.e., humanized) mice were monitored daily after ivag infection with 2 × 10⁶ HIV-1–_Ba-L–infected hPBMC. Although all DMPA-treated mice succumbed to infection by 9 days postinfection (dpi),~ 65% of the NET-EN-treated mice were alive at 14 dpi. (B and C) Other identically treated humanized mice were ivag inoculated with 10⁶ HIV-1–infected hPBMC and euthanized at 10 dpi to assess systemic HIV-1 infection and viral load. (B) Quantitative BTZM-bl luciferase assays show higher HIV levels detected in splenocyte cultures from DMPA- versus NET-EN–treated mice (splenocytes from uninfected mice and media-diluted HIV-1–_Ba-L were used as positive and negative controls, respectively). (C) RT-qPCR assays found significantly higher HIV-1 RNA copy number in plasma of DMPA- versus NET-EN–treated mice; data depicted is representative of two independent experiments that produced similar results (three mice per group) (bars denote mean ± SD). Statistical analyses were performed with one-way ANOVA and Kruskal–Wallis post hoc test (B) or unpaired Mann–Whitney U test (C); log rank tests were used to compare cumulative survival incidence after HIV-1 infection (estrus versus DMPA, p = 0.001; estrus versus NET-EN, p = 0.138).