Exogenous sex steroids regulate genital epithelial barrier function in female rhesus macaques.
Biology of reproduction
There is concern that using depot-medroxyprogesterone acetate (DMPA) for pregnancy prevention heightens HIV susceptibility. While no clinical data establishes causal link between HIV acquisition and use of this injectable progestin, prior work from our laboratory showed that DMPA comparably lowers genital levels of the cell-cell adhesion molecule desmoglein-1 (DSG1) and weakens genital epithelial barrier function in female mice and humans. We likewise saw DMPA increase mouse susceptibility to multiple genital pathogens including HIV. Herein, we sought to confirm and extend these findings by comparing genital epithelial barrier function in untreated rhesus macaques (RM) vs. RM treated with DMPA or DMPA and estrogen (E). Compared to controls, genital tissue from RM with pharmacologically relevant serum levels of medroxyprogesterone acetate displayed significantly lower DSG1 levels and greater permeability to low molecular mass molecules. Conversely, DMPA-mediated effects on genital epithelial integrity and function were obviated in RM administered DMPA and E. These data corroborate the diminished genital epithelial barrier function observed in women initiating DMPA and identify RM as a useful preclinical model for defining effects of exogenous sex steroids on genital pathogen susceptibility. As treatment with E averted DMPA-mediated loss of genital epithelial barrier function, our results also imply that contraceptives releasing progestin and E may be less likely to promote transmission of HIV and other sexually transmitted pathogens than progestin-only compounds.
View details for DOI 10.1093/biolre/ioaa105
View details for PubMedID 32542371
- ECHO: context and limitations. Lancet (London, England) 2020; 395 (10222): e21
HIV, progestins, genital epithelial barrier function, and the burden of objectivity?.
Biology of reproduction
Contributions from a diverse set of scientific disciplines will be needed to help individuals make fully informed decisions regarding contraceptive choices least likely to promote HIV susceptibility. This commentary recaps contrasting interpretations of results from the Evidence for Contraceptive Options and HIV Outcomes (ECHO) Trial, a study that compared HIV risk in women using the progestin-only injectable contraceptive depot medroxyprogesterone acetate (DMPA) vs. two other contraceptive choices. It also summarizes results from basic and translational research that establish biological plausibility for earlier clinical studies that identified enhanced HIV susceptibility in women using DMPA.
View details for DOI 10.1093/biolre/ioaa078
View details for PubMedID 32561906
Norethisterone Enanthate Increases Mouse Susceptibility to Genital Infection with Herpes Simplex Virus Type 2 and HIV Type 1.
2020; 4 (2): 72?81
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.
View details for DOI 10.4049/immunohorizons.1900077
View details for PubMedID 32047094
Depot-medroxyprogesterone acetate reduces genital cell-cell adhesion molecule expression and increases genital herpes simplex virus type 2 infection susceptibility in a dose-dependent fashion.
OBJECTIVE: Analyzing ectocervical biopsy tissue from women before and after they initiated use of depot-medroxyprogesterone acetate (DMPA), we previously reported this progestin reduces levels of the cell-cell adhesion molecule (CCAM) desmoglein-1 and increases genital mucosal permeability. We likewise saw treating mice with 1.0mg of DMPA reduced vaginal CCAM expression and increased genital pathogen susceptibility. Herein, we used dose-response studies to delimit DMPA doses and serum MPA levels in mice associated with impaired genital mucosal barrier function and enhanced susceptibility to low-dose herpes simplex virus type 2 (HSV-2) infection.STUDY DESIGN: We compared genital CCAM expression, genital mucosal permeability, and susceptibility to genital inoculation with 103 plaque-forming units of HSV-2 among mice in estrus vs. after treatment with 0.01mg, 0.1mg, 0.3mg, or 1.0mg of DMPA.RESULTS: Compared to mice in estrus, DMPA treatment in dose-dependent fashion significantly reduced desmoglein 1alpha (Dsg1a) and desmocollin-1 (Dsc1) gene expression, reduced DSG1 protein levels, and increased genital mucosal permeability to low molecular weight molecules. While no mice infected with HSV-2 in estrus died, we respectively saw 50% and 100% mortality in mice administered 0.1mg or 0.3mg of DMPA. At time of infection, mean serum MPA levels in mice administered the 0.1mg or 0.3mg doses were 3.8nM and 13.0nM respectively (values comparable to trough and peak MPA serum levels in women using DMPA).CONCLUSIONS: Mice with pharmacologically relevant serum MPA concentrations display significant changes in genital CCAM expression, genital mucosal barrier function, and HSV-2 susceptibility.
View details for DOI 10.1016/j.contraception.2019.07.003
View details for PubMedID 31302121
Exogenous oestrogen inhibits genital transmission of cell-associated HIV-1 in DMPA-treated humanized mice.
Journal of the International AIDS Society
2018; 21 (1)
HIV affects more women than any other life-threatening infectious agent, and most infections are sexually transmitted. HIV must breach the female genital tract mucosal barrier to establish systemic infection, and clinical studies indicate virus more easily evades this barrier in women using depot-medroxyprogesterone acetate (DMPA) and other injectable progestins for contraception. Identifying a potential mechanism for this association, we learned DMPA promotes susceptibility of wild-type mice to genital herpes simplex virus type 2 (HSV-2) infection by reducing genital tissue expression of the cell-cell adhesion molecule desmoglein-1 (DSG-1) and increasing genital mucosal permeability. Conversely, DMPA-mediated increases in genital mucosal permeability and HSV-2 susceptibility were eliminated in mice concomitantly administered exogenous oestrogen (E). To confirm and extend these findings, herein we used humanized mice to define effects of systemic DMPA and intravaginal (ivag) E administration on susceptibility to genital infection with cell-associated HIV-1.Effects of DMPA or an intravaginal (ivag) E cream on engraftment of NOD-scid-IL-2Rgcnull (NSG) mice with human peripheral blood mononuclear cells (hPBMCs) were defined with flow cytometry. Confocal microscopy was used to evaluate effects of DMPA, DMPA and E cream, or DMPA and the pharmacologically active component of the cream on vaginal tissue DSG-1 expression and genital mucosal permeability to low molecular weight (LMW) molecules and hPBMCs. In other studies, hPBMC-engrafted NSG mice (hPBMC-NSG) received DMPA or DMPA and ivag E cream before genital inoculation with 106 HIV-1-infected hPBMCs. Mice were euthanized 10 days after infection, and plasma HIV-1 load quantified by qRT-PCR and splenocytes used to detect HIV-1 p24 antigen via immunohistochemistry and infectious virus via TZM-bl luciferase assay.Whereas hPBMC engraftment was unaffected by DMPA or E treatment, mice administered DMPA and E (cream or the pharmacologically active cream component) displayed greater vaginal tissue expression of DSG-1 protein and decreased vaginal mucosal permeability to LMW molecules and hPBMCs versus DMPA-treated mice. DMPA-treated hPBMC-NSG mice were also uniformly susceptible to genital transmission of cell-associated HIV-1, while no animal concomitantly administered DMPA and E cream acquired systemic HIV-1 infection.Exogenous E administration reduces susceptibility of DMPA-treated humanized mice to genital HIV-1 infection.
View details for PubMedID 29334191
Levonorgestrel and Female Genital Tract Immunity: Time for a Closer Look.
The Journal of infectious diseases
View details for PubMedID 29917111
- HIV and Hormonal Contraception: Bench and Bedside JAIDS-JOURNAL OF ACQUIRED IMMUNE DEFICIENCY SYNDROMES 2017; 74 (3): E85-E86
IL-4-secreting eosinophils promote endometrial stromal cell proliferation and prevent Chlamydia-induced upper genital tract damage.
Proceedings of the National Academy of Sciences of the United States of America
Genital Chlamydia trachomatis infections in women typically are asymptomatic and do not cause permanent upper genital tract (UGT) damage. Consistent with this presentation, type 2 innate and TH2 adaptive immune responses associated with dampened inflammation and tissue repair are elicited in the UGT of Chlamydia-infected women. Primary C. trachomatis infection of mice also causes no genital pathology, but unlike women, does not generate Chlamydia-specific TH2 immunity. Herein, we explored the significance of type 2 innate immunity for restricting UGT tissue damage in Chlamydia-infected mice, and in initial studies intravaginally infected wild-type, IL-10(-/-), IL-4(-/-), and IL-4R?(-/-) mice with low-dose C. trachomatis inoculums. Whereas Chlamydia was comparably cleared in all groups, IL-4(-/-) and IL-4R?(-/-) mice displayed endometrial damage not seen in wild-type or IL-10(-/-) mice. Congruent with the aberrant tissue repair in mice with deficient IL-4 signaling, we found that IL-4R? and STAT6 signaling mediated IL-4-induced endometrial stromal cell (ESC) proliferation ex vivo, and that genital administration of an IL-4-expressing adenoviral vector greatly increased in vivo ESC proliferation. Studies with IL-4-IRES-eGFP (4get) reporter mice showed eosinophils were the main IL-4-producing endometrial leukocyte (constitutively and during Chlamydia infection), whereas studies with eosinophil-deficient mice identified this innate immune cell as essential for endometrial repair during Chlamydia infection. Together, our studies reveal IL-4-producing eosinophils stimulate ESC proliferation and prevent Chlamydia-induced endometrial damage. Based on these results, it seems possible that the robust type 2 immunity elicited by Chlamydia infection of human genital tissue may analogously promote repair processes that reduce phenotypic disease expression.
View details for PubMedID 28765368
Comment on 'Effects of injectable progestogen contraception versus the copper intrauterine device on HIV acquisition: sub-study of a pragmatic randomised controlled trial'.
The journal of family planning and reproductive health care
View details for PubMedID 28756404
- Setting Sights on Chlamydia Immunity's Central Paradigm: Can We Hit a Moving Target? Infection and immunity 2017; 85 (7)
Dendritic cell function and pathogen-specific T cell immunity are inhibited in mice administered levonorgestrel prior to intranasal Chlamydia trachomatis infection.
2016; 6: 37723-?
The growing popularity of levonorgestrel (LNG)-releasing intra-uterine systems for long-acting reversible contraception provides strong impetus to define immunomodulatory properties of this exogenous progestin. In initial in vitro studies herein, we found LNG significantly impaired activation of human dendritic cell (DCs) and their capacity to promote allogeneic T cell proliferation. In follow-up studies in a murine model of intranasal Chlamydia trachomatis infection, we analogously found that LNG treatment prior to infection dramatically reduced CD40 expression in DCs isolated from draining lymph nodes at 2 days post infection (dpi). At 12 dpi, we also detected significantly fewer CD4(+) and CD8(+) T cells in the lungs of LNG-treated mice. This inhibition of DC activation and T cell expansion in LNG-treated mice also delayed chlamydial clearance and the resolution of pulmonary inflammation. Conversely, administering agonist anti-CD40 monoclonal antibody to LNG-treated mice at 1 dpi restored lung T cell numbers and chlamydial burden at 12 dpi to levels seen in infected controls. Together, these studies reveal that LNG suppresses DC activation and function, and inhibits formation of pathogen-specific T cell immunity. They also highlight the need for studies that define in vivo effects of LNG use on human host response to microbial pathogens.
View details for DOI 10.1038/srep37723
View details for PubMedID 27892938
View details for PubMedCentralID PMC5125275
Medroxyprogesterone acetate and levonorgestrel increase genital mucosal permeability and enhance susceptibility to genital herpes simplex virus type 2 infection
2016; 9 (6): 1571-1583
Depot-medroxyprogesterone acetate (DMPA) is a hormonal contraceptive especially popular in areas with high prevalence of HIV and other sexually transmitted infections (STI). Although observational studies identify DMPA as an important STI risk factor, mechanisms underlying this connection are undefined. Levonorgestrel (LNG) is another progestin used for hormonal contraception, but its effect on STI susceptibility is much less explored. Using a mouse model of genital herpes simplex virus type 2 (HSV-2) infection, we herein found that DMPA and LNG similarly reduced genital expression of the desmosomal cadherin desmoglein-1? (DSG1?), enhanced access of inflammatory cells to genital tissue by increasing mucosal epithelial permeability, and increased susceptibility to viral infection. Additional studies with uninfected mice revealed that DMPA-mediated increases in mucosal permeability promoted tissue inflammation by facilitating endogenous vaginal microbiota invasion. Conversely, concomitant treatment of mice with DMPA and intravaginal estrogen restored mucosal barrier function and prevented HSV-2 infection. Evaluating ectocervical biopsy tissue from women before and 1 month after initiating DMPA remarkably revealed that inflammation and barrier protection were altered by treatment identically to changes seen in progestin-treated mice. Together, our work reveals DMPA and LNG diminish the genital mucosal barrier; a first-line defense against all STI, but may offer foundation for new contraceptive strategies less compromising of barrier protection.
View details for DOI 10.1038/mi.2016.22
View details for Web of Science ID 000386773800021
View details for PubMedID 27007679
View details for PubMedCentralID PMC5035233
Intravaginal Chlamydia trachomatis Challenge Infection Elicits T(H)1 and T(H)17 Immune Responses in Mice That Promote Pathogen Clearance and Genital Tract Damage
2016; 11 (9)
While ascension of Chlamydia trachomatis into the upper genital tract of women can cause pelvic inflammatory disease and Fallopian tube damage, most infections elicit no symptoms or overt upper genital tract pathology. Consistent with this asymptomatic clinical presentation, genital C. trachomatis infection of women generates robust TH2 immunity. As an animal model that modeled this response would be invaluable for delineating bacterial pathogenesis and human host defenses, herein we explored if pathogen-specific TH2 immunity is similarly elicited by intravaginal (ivag) infection of mice with oculogenital C. trachomatis serovars. Analogous to clinical infection, ascension of primary C. trachomatis infection into the mouse upper genital tract produced no obvious tissue damage. Clearance of ivag challenge infection was mediated by interferon (IFN)-?-producing CD4+ T cells, while IFN-? signaling blockade concomitant with a single ivag challenge promoted tissue damage by enhancing Chlamydia-specific TH17 immunity. Likewise, IFN-? and IL-17 signaling blockade or CD4+ T cell depletion eliminated the genital pathology produced in untreated controls by multiple ivag challenge infections. Conversely, we were unable to detect formation of pathogen-specific TH2 immunity in C. trachomatis-infected mice. Together, our work revealed C. trachomatis infection of mice generates TH1 and TH17 immune responses that promote pathogen clearance and immunopathological tissue damage. Absence of Chlamydia-specific TH2 immunity in these mice newly highlights the need to identify experimental models of C. trachomatis genital infection that more closely recapitulate the human host response.
View details for DOI 10.1371/journal.pone.0162445
View details for Web of Science ID 000383255600046
View details for PubMedID 27606424
View details for PubMedCentralID PMC5015975
Fluorescent labeling reliably identifies Chlamydia trachomatis in living human endometrial cells and rapidly and accurately quantifies chlamydial inclusion forming units
JOURNAL OF MICROBIOLOGICAL METHODS
2015; 119: 79-82
Chlamydia replication requires host lipid acquisition, allowing flow cytometry to identify Chlamydia-infected cells that accumulated fluorescent Golgi-specific lipid. Herein, we describe modifications to currently available methods that allow precise differentiation between uninfected and Chlamydia trachomatis-infected human endometrial cells and rapidly and accurately quantify chlamydial inclusion forming units.
View details for DOI 10.1016/j.mimet.2015.10.003
View details for Web of Science ID 000366442400013
View details for PubMedID 26453947
View details for PubMedCentralID PMC4993108
Medroxyprogesterone acetate impairs human dendritic cell activation and function
2015; 30 (5): 1169-1177
Does medroxyprogesterone acetate (MPA) impair human dendritic cell (DC) activation and function?In vitro MPA treatment suppressed expression of CD40 and CD80 by human primary DCs responding to Toll-like receptor 3 (TLR3) agonist stimulation (i.e. DC activation). Moreover, this MPA-mediated decrease in CD40 expression impaired DC capacity to stimulate T cell proliferation (i.e. DC function).MPA is the active molecule in Depo-Provera(®) (DMPA), a commonly used injectable hormonal contraceptive (HC). Although DMPA treatment of mice prior to viral mucosal tissue infection impaired the capacity of DCs to up-regulate CD40 and CD80 and prime virus-specific T cell proliferation, neither DC activation marker expression nor the ability of DCs to promote T cell proliferation were affected by in vitro progesterone treatment of human DCs generated from peripheral blood monocytes.This cross-sectional study examined MPA-mediated effects on the activation and function of human primary untouched peripheral blood DCs.Human DCs isolated from peripheral blood mononuclear cells by negative immunomagnetic selection were incubated for 24 h with various concentrations of MPA. After an additional 24 h incubation with the TLR3 agonist polyinosinic:polycytidylic acid (poly I:C), flow cytometry was used to evaluate DC phenotype (i.e. expression of CD40, CD80, CD86, and HLA-DR). In separate experiments, primary untouched human DCs were sequentially MPA-treated, poly I:C-activated, and incubated for 7 days with fluorescently labeled naïve allogeneic T cells. Flow cytometry was then used to quantify allogeneic T cell proliferation.Several pharmacologically relevant concentrations of MPA dramatically reduced CD40 and CD80 expression in human primary DCs responding to the immunostimulant poly I:C. In addition, MPA-treated DCs displayed a reduced capacity to promote allogeneic CD4(+) and CD8(+) T cell proliferation. In other DC: T cell co-cultures, the addition of antibody blocking the CD40-CD154 (CD40L) interaction mirrored the decreased T cell proliferation produced by MPA treatment, while addition of recombinant soluble CD154 restored the capacity of MPA-treated DCs to induce T cell proliferation to levels produced by non-MPA-treated controls.While our results newly reveal that pharmacologically relevant MPA concentrations suppress human DC function in vitro, additional research is needed to learn if DMPA similarly inhibits DC maturation and function in the human female genital tract.Identification of a mechanism by which MPA impairs human DC activation and function increases the biological plausibility for the relationships currently suspected between DMPA use and enhanced susceptibility to genital tract infection.Funding provided by the NIH (grant R01HD072663) and The Ohio State University College of Medicine. The authors have no con?icts of interest to declare.
View details for DOI 10.1093/humrep/dev035
View details for Web of Science ID 000354792100017
View details for PubMedID 25740884
View details for PubMedCentralID PMC4481667
Use of Transcriptional Profiling to Delineate the Initial Response of Mice to Intravaginal Herpes Simplex Virus Type 2 Infection
2013; 26 (3): 172-179
Intravaginal (ivag) infection of mice with herpes simplex virus type 2 (HSV-2) causes genital tissue damage, quickly followed by development of fatal encephalopathy. To delineate initial host responses generated by HSV-2 infection, here oligonucleotide microarrays compared gene expression in vaginal tissue from uninfected mice and mice 1, 2, 3, 4, 5, 6, or 7 days after ivag infection with 10(4) pfu HSV-2. While comparison of mRNA expression in uninfected and HSV-infected vaginal tissue detected few changes during the first 2 days post infection (dpi), there were 156 genes whose expression was first significantly altered 3 dpi that remained significantly modified at all later time points examined. These 156 genes were significantly enriched in canonical pathways associated with interferon (IFN) signaling, activation of IFN elements by intracellular pattern recognition receptors, and antiviral immunity induced by cytosolic RIG-like receptors. Evaluation of this gene set with the National Center for Biotechnology Information Gene and INTERFEROME databases corroborated pathway analysis, as function of most (53%) were linked to IFN-mediated host immunity. In the final set of experiments, ivag administration of the Toll-like receptor 3 agonist polyinosinic: polycytidylic acid (poly I:C) 24?h before ivag HSV-2 infection reduced the incidence of genital pathology and encephalopathy, while these poly I:C-treated mice were subsequently protected from ocular HSV-2 challenge lethal to uninfected controls. The latter results imply that the exuberant antiviral immunity produced in our experimental model is simply formed too late to prevent viral replication and dissemination, and that poly I:C-induced formation of an antiviral state protecting against primary ivag infection also permits development of HSV-specific protective immunity.
View details for DOI 10.1089/vim.2012.0093
View details for Web of Science ID 000320506100002
View details for PubMedID 23638732
View details for PubMedCentralID PMC3676663