Ovariectomized mice and postmenopausal women exhibit analogous loss of genital epithelial integrity.
Exogenous oestrogen inhibits genital transmission of cell-associated HIV-1 in DMPA-treated humanized mice.
Journal of the International AIDS Society
2018; 21 (1)
Roughly half of all postmenopausal women are affected by the genitourinary syndrome of menopause (GSM). Symptoms of GSM, including vaginal irritation and dyspareunia, occur as reduced estrogen (E) production elicits loss of elasticity and other changes in genital tract tissue. While the use of the injectable contraceptive depot-medroxyprogesterone acetate (DMPA) likewise lowers serum E concentrations in reproductive age women and is associated with decreased genital levels of the cell-cell adhesion molecules desmoglein-1 (DSG1) and desmocollin-1 (DSC1) and impaired genital epithelial barrier function, the relevance of these findings to women in menopause is uncertain. Exploring the impact of menopause on genital epithelial integrity herein, we detected significantly lower levels of DSG1 and DSC1 in ectocervical tissue from menopausal and postmenopausal vs premenopausal women. Using ovariectomized (OVX) mice as a menopause model, we comparably saw significantly lower vaginal tissue levels of DSG1 and DSC1 in OVX mice vs. mice in estrus. Compared to estrus-stage mice and E-treated OVX mice, DMPA-treated ovary-intact mice and OVX mice also exhibited significantly reduced genital epithelial barrier function, greater susceptibility to genital herpes simplex virus type 2 infection, and delayed clearance of genital Chlamydia trachomatis infection. Current studies thus identify analogous loss of genital epithelial integrity in OVX mice and menopausal and postmenopausal women. By showing that loss of genital epithelial integrity is associated with increased mouse susceptibility to bacterial and viral pathogens, our findings also prioritize the need to resolve if reduced genital epithelial integrity in postmenopausal women is a significant risk factor for genital infection.
View details for DOI 10.1080/21688370.2020.1865760
View details for PubMedID 33427560
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
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
Medroxyprogesterone acetate and levonorgestrel increase genital mucosal permeability and enhance susceptibility to genital herpes simplex virus type 2 infection
2016; 9 (6): 1571-1583
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
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)
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
Human female genital tract infection by the obligate intracellular bacterium Chlamydia trachomatis elicits robust Type 2 immunity.
2013; 8 (3)
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
Dendritic Cell Activation and Memory Cell Development Are Impaired among Mice Administered Medroxyprogesterone Acetate Prior to Mucosal Herpes Simplex Virus Type 1 Infection
JOURNAL OF IMMUNOLOGY
2012; 189 (7): 3449-3461
While Chlamydia trachomatis infections are frequently asymptomatic, mechanisms that regulate host response to this intracellular Gram-negative bacterium remain undefined. This investigation thus used peripheral blood mononuclear cells and endometrial tissue from women with or without Chlamydia genital tract infection to better define this response. Initial genome-wide microarray analysis revealed highly elevated expression of matrix metalloproteinase 10 and other molecules characteristic of Type 2 immunity (e.g., fibrosis and wound repair) in Chlamydia-infected tissue. This result was corroborated in flow cytometry and immunohistochemistry studies that showed extant upper genital tract Chlamydia infection was associated with increased co-expression of CD200 receptor and CD206 (markers of alternative macrophage activation) by endometrial macrophages as well as increased expression of GATA-3 (the transcription factor regulating TH2 differentiation) by endometrial CD4(+) T cells. Also among women with genital tract Chlamydia infection, peripheral CD3(+) CD4(+) and CD3(+) CD4(-) cells that proliferated in response to ex vivo stimulation with inactivated chlamydial antigen secreted significantly more interleukin (IL)-4 than tumor necrosis factor, interferon-?, or IL-17; findings that repeated in T cells isolated from these same women 1 and 4 months after infection had been eradicated. Our results thus newly reveal that genital infection by an obligate intracellular bacterium induces polarization towards Type 2 immunity, including Chlamydia-specific TH2 development. Based on these findings, we now speculate that Type 2 immunity was selected by evolution as the host response to C. trachomatis in the human female genital tract to control infection and minimize immunopathological damage to vital reproductive structures.
View details for DOI 10.1371/journal.pone.0058565
View details for PubMedID 23555586
View details for PubMedCentralID PMC3603585
Exogenous sex steroids regulate genital epithelial barrier function in female rhesus macaques.
Biology of reproduction
Epidemiological studies indicate that the exogenous sex steroid medroxyprogesterone acetate (MPA) can impair cell-mediated immunity, but mechanisms responsible for this observation are not well defined. In this study, MPA administered to mice 1 wk prior to HSV type 1 (HSV-1) infection of their corneal mucosa impaired initial expansion of viral-specific effector and memory precursor T cells and reduced the number of viral-specific memory T cells found in latently infected mice. MPA treatment also dampened expression of the costimulatory molecules CD40, CD70, and CD80 by dendritic cells (DC) in lymph nodes draining acute infection, whereas coculture of such DC with T cells from uninfected mice dramatically impaired ex vivo T cell proliferation compared with the use of DC from mice that did not receive MPA prior to HSV-1 infection. In addition, T cell expansion was comparable to that seen in untreated controls if MPA-treated mice were administered recombinant soluble CD154 (CD40L) concomitant with their mucosal infection. In contrast, the immunomodulatory effects of MPA were infection site dependent, because MPA-treated mice exhibited normal expansion of virus-specific T cells when infection was systemic rather than mucosal. Taken together, our results reveal that the administration of MPA prior to viral infection of mucosal tissue impairs DC activation, virus-specific T cell expansion, and development of virus-specific immunological memory.
View details for DOI 10.4049/jimmunol.1103054
View details for Web of Science ID 000309164300021
View details for PubMedID 22942424
View details for PubMedCentralID PMC3448807
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
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
Norethisterone Enanthate Increases Mouse Susceptibility to Genital Infection with Herpes Simplex Virus Type 2 and HIV Type 1.
2020; 4 (2): 72?81
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
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.
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
Levonorgestrel and Female Genital Tract Immunity: Time for a Closer Look.
The Journal of infectious diseases
HIV and Hormonal Contraception: Bench and Bedside
JAIDS-JOURNAL OF ACQUIRED IMMUNE DEFICIENCY SYNDROMES
2017; 74 (3): E85-E86
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
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-?
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
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
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
Risk of Bacterial Vaginosis Among Women With Herpes Simplex Virus Type 2 Infection: A Systematic Review and Meta-analysis
JOURNAL OF INFECTIOUS DISEASES
2015; 212 (1): 8-17
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
Bacterial vaginosis (BV) is a perturbation of vaginal flora characterized by reduced levels of lactobacilli and concomitant overgrowth of anaerobic bacterial species. BV is highly prevalent and associated with multiple adverse outcomes, including enhanced human immunodeficiency virus transmission. Because recent reports reveal that herpes simplex virus type 2 (HSV-2) infection may increase BV risk, we initiated a systematic review and meta-analysis of the link between HSV-2 infection and BV.We searched the MEDLINE, EMBASE, and CENTRAL databases to identify articles posted before 1 December 2014. Two screeners independently reviewed the titles and abstracts of all identified articles, reviewed the full text of articles deemed potentially eligible, and extracted data from 14 cross-sectional and 3 prospective studies. Using random-effects models, we computed separate pooled estimates for cross-sectional and prospective studies.The pooled odds ratio for cross-sectional studies was 1.60 (95% confidence interval, 1.32-1.94). Stronger support for the causal effect of HSV-2 infection on BV risk was revealed by the summary relative risk for the prospective studies, which was 1.55 (95% confidence interval, 1.30-1.84), with minimal heterogeneity (I(2) = 0).These analyses imply that HSV-2 infection is an important BV risk factor. Pharmacologic HSV-2 suppression may reduce BV incidence and BV-associated adverse events.
View details for DOI 10.1093/infdis/jiv017
View details for Web of Science ID 000357818900003
View details for PubMedID 25589333
Use of Transcriptional Profiling to Delineate the Initial Response of Mice to Intravaginal Herpes Simplex Virus Type 2 Infection
2013; 26 (3): 172-179
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
Hypothesis: Chlamydia trachomatis infection of the female genital tract is controlled by Type 2 immunity
2012; 79 (6): 713-716
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
Transient Detection of Chlamydial-Specific Th1 Memory Cells in the Peripheral Circulation of Women with History of Chlamydia trachomatis Genital Tract Infection
AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY
2012; 68 (6): 499-506
Chlamydia trachomatis is an obligate intracellular bacterium sexually transmitted to more than 90 million individuals each year. As this level of infectivity implies, C. trachomatis is a successful human parasite; a success facilitated by its ability to cause asymptomatic infection. Host defense against C. trachomatis in the female genital tract is not well defined, but current dogma suggests infection is controlled largely by T(H)1 immunity. Conversely, it is well established that T(H)2 immunity controls allergens, helminths, and other extracellular pathogens that cause repetitive or persistent T cell stimulation but do not induce the exuberant inflammation that drives T(H)1 and T(H)17 immunity. As C. trachomatis persists in female genital tract epithelial cells but does not elicit over tissue inflammation, we now posit that defense is maintained by Type 2 immune responses that control bacterial growth but minimize immunopathological damage to vital reproductive tract anatomy. Evaluation of this hypothesis may uncover novel mechanisms by which Type 2 immunity can control growth of C. trachomatis and other intracellular pathogens, while confirmation that T(H)2 immunity was selected by evolution to control C. trachomatis infection in the female genital tract will transform current research, now focused on developing vaccines that elicit strong, and therefore potentially tissue destructive, Chlamydia-specific T(H)1 immunity.
View details for DOI 10.1016/j.mehy.2012.07.032
View details for Web of Science ID 000311773500003
View details for PubMedID 22986006
View details for PubMedCentralID PMC3496836
Brefeldin A, but not monensin, enables flow cytometric detection of interleukin-4 within peripheral T cells responding to ex vivo stimulation with Chlamydia trachomatis
JOURNAL OF IMMUNOLOGICAL METHODS
2012; 384 (1-2): 191-195
Development of safe and effective Chlamydia trachomatis vaccines requires better understanding of the host immune responses elicited by natural infection.Peripheral blood mononuclear cells isolated from women with or without history of genital tract chlamydial infection were stimulated with inactivated C. trachomatis elementary bodies (EB) in ELISPOT assays that enumerated frequencies of cells producing interferon (IFN)-? or interleukin (IL)-17.IFN-?-positive cells were highest among women sampled 30-60 days after diagnosis of C. trachomatis infection and treatment initiation, while the numbers of IFN-?-positive cells were equally low among uninfected women and women sampled <30 or >60 days after diagnosis of infection. Conversely, IL-17-positive cell numbers were uniformly low among all participants.Dramatically reduced numbers of Chlamydia-specific Th1 memory cells in the peripheral circulation of study participants sampled more than 2 months after diagnosis, and initiation of treatment provides new insight into the results from C. trachomatis vaccine trials, in which immunization with EB provided only short-lived protection. Our results also suggest that an effective vaccine against this weakly antigenic intracellular pathogen will need to generate immunological memory more durable than that elicited by natural infection.
View details for DOI 10.1111/aji.12008
View details for Web of Science ID 000310959300007
View details for PubMedID 22934581
View details for PubMedCentralID PMC3493686
Recalcitrance of bacterial vaginosis among herpes-simplex-virus-type-2-seropositive women
JOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH
2012; 38 (1): 77-83
Intracellular cytokine staining (ICS) assay optimization should include selection of suitable cytokine secretion inhibitors. Here, peripheral blood mononuclear cells (PBMC) from women with proven history of C. trachomatis genital tract infection were used to compare the ability of brefeldin A (BFA) and monensin (MN) to concurrently trap interferon-? (IFN-?), tumor necrosis factor (TNF), interleukin (IL)-4, and IL-17 within T cells responding to ex vivo stimulation with chlamydial antigen. While flow cytometric analyses showed similar intracellular levels of TNF, IFN-?, and IL-17 among T cells treated with BFA or both BFA and MN, markedly more IL-4 was found inside T cells treated with BFA compared to those that received MN or BFA and MN. The latter findings oppose current ICS recommendations informing that ICS results are unaffected by concomitant use of BFA and MN, and also suggests that MN may be an unsuitable cytokine secretion inhibitor for ICS assays designed to measure intracellular IL-4 accumulation.
View details for DOI 10.1016/j.jim.2012.07.018
View details for Web of Science ID 000309572300025
View details for PubMedID 22850275
View details for PubMedCentralID PMC3444442
Limitations of the criteria used to diagnose histologic endometritis in epidemiologic pelvic inflammatory disease research.
Pathology, research and practice
2011; 207 (11): 680-685
The multifactorial etiology of bacterial vaginosis (BV) impedes development of effective treatment and prevention strategies. Herein, we evaluated the effects of herpes simplex virus type 2 (HSV-2), a suspected BV risk factor, on vaginal flora composition.? Correlations between HSV-2 infection and BV were prospectively explored among 12 HSV-2-seropositive women with asymptomatic BV who were asked to collect daily vaginal swab specimens for Gram stain analysis of vaginal flora and determination of HSV-2 shedding frequencies during the 1month before and after metronidazole therapy.Unlike prior longitudinal studies that reported rapid fluctuations in vaginal flora composition and frequent episodes of spontaneously resolving BV, we found that 99.4% (310/312) of vaginal smears collected before initiation of metronidazole were consistent with a diagnosis of BV. Effectiveness of metronidazole therapy was also much lower than previously reported in studies not restricting enrollment to HSV-2-seropositive women; we observed a BV recurrence rate of 89% in the first month after completion of therapy while the median time to this recurrence occurred only 14days after treatment.Our study demonstrates BV recalcitrance among HSV-2-infected women and provides additional evidence for a linkage between this chronic viral infection and abnormal vaginal flora. Additional work will be needed to define mechanisms responsible for this correlation and to determine if vaginal flora health of HSV-2-infected women is improved by medications that suppress HSV-2 shedding.
View details for DOI 10.1111/j.1447-0756.2011.01697.x
View details for Web of Science ID 000298882800012
View details for PubMedID 22136755
View details for PubMedCentralID PMC3253171
Endometrial leukocyte subpopulations associated with Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis genital tract infection.
American journal of obstetrics and gynecology
2011; 205 (4): 324 e1-7
While endometrial neutrophils and plasma cells are criteria used to diagnose histologic endometritis in epidemiologic pelvic inflammatory disease (PID) research, plasma cell misidentification and nonspecificity may limit the accuracy of these criteria. Herein, we examined: (1) the identification of endometrial plasma cells with conventional methyl green pyronin-based methodology versus plasma cell-specific (CD138) immunostaining, (2) the prevalence of endometrial plasma cells among women at low risk for PID, and (3) endometrial leukocyte subpopulations among women diagnosed with acute or chronic histologic endometritis by conventional criteria. We observed an absence of CD138+ cells in 25% of endometrial biopsies in which plasma cells had been identified by conventional methodology, while additional immunohistochemical analyses revealed indistinguishable inflammatory infiltrates among women diagnosed with acute or chronic endometritis by conventional criteria. Among women considered at lower risk for PID development, flow cytometric analyses detected plasma cells in 30% of endometrial biopsy specimens, suggesting that these cells, even when accurately identified, only nonspecifically identify upper genital tract inflammatory processes. Combined, our findings underscore the limitations of the criteria used to diagnose histologic endometritis in PID-related research and suggest that satisfactory understanding of PID pathogenesis, treatment, and prevention is hindered by continued use of these criteria.
View details for DOI 10.1016/j.prp.2011.08.007
View details for PubMedID 21996319
View details for PubMedCentralID PMC3215901
Detection of AA-type amyloid protein in labial salivary glands
MEDICINA ORAL PATOLOGIA ORAL Y CIRUGIA BUCAL
2011; 16 (2): E149-E152
The objective of the study was to characterize endometrial inflammation associated with common genital tract pathogens.The design of the study was the immunohistochemical characterization of the endometrial leukocyte subpopulations from 37 controls and 45 women infected with Chlamydia trachomatis, Neisseria gonorrhoeae, or Trichomonas vaginalis.Compared with uninfected women, endocervical infection with C trachomatis, N gonorrhoeae, or T vaginalis was associated with significant increases in endometrial T cells, B cells, plasma cells, and polymorphonuclear leukocytes. Even more substantial increases in T cell, B cell, and plasma cell numbers were detected among women infected endocervically and endometrially with C trachomatis.Because lower genital tract C trachomatis, N gonorrhoeae, or T vaginalis infections were associated with comparable increases in the same endometrial leukocyte subpopulations, our results suggest the underappreciated involvement of T vaginalis in upper genital tract inflammatory processes. The more robust inflammatory infiltrate associated with C trachomatis endometrial ascension may offer insight into host inflammatory responses associated with pelvic inflammatory disease development.
View details for DOI 10.1016/j.ajog.2011.05.031
View details for PubMedID 21777898
View details for PubMedCentralID PMC3204313
A caveat for T cell transfer studies: generation of cytotoxic anti-Thy1.2 antibodies in Thy1.1 congenic mice given Thy1.2+ tumors or T cells.
Journal of leukocyte biology
2011; 89 (2): 291-300
Among the diverse forms of amyloidosis, secondary type is the most frequent one. Diagnosis of amyloid deposition is based on the identification of the fibrillary protein amyloid by means of Congo Red (CR) or crystal violet (CV) stains, but these techniques do not differentiate between the different types of amyloid fibrils. The aim of this study was to identify by immunofluorescence (IF) AA amyloid a pathological fibrillar low-molecular-weight protein formed by cleavage of serum amyloid A (SAA) protein in labial salivary gland (LSG) biopsies from patients with secondary amyloidosis.98 LSG were studied, 65 were from patients with secondary amyloidosis and 33 from subjects with chronic inflammatory diseases without evidence of this anomaly. All sections were stained with hematoxylin and eosin (H &E), CV, CR and IF using anti-AA antibodies. Positive and negative controls were used for all techniques.CV and CR demonstrated that the amyloid substance was found mainly distributed periductally (93.8%), followed by periacinar and perivascular locations (p <0.001); however, the IF demonstrated that amyloid AA substance predominates in the periacinar area (73.8%), followed by periductal and perivascular locations (p <0.001). IF has a sensitivity of 83%, 100% of specificity, 100% of predictive positive value and 75% of predictive negative value.The results of this study confirm the efficacy of the LSG biopsy as a highly reliable method for diagnosis of secondary amyloidosis.
View details for DOI 10.4317/medoral.16.e149
View details for Web of Science ID 000288682100004
View details for PubMedID 21196885
Chlamydia trachomatis infection control programs: lessons learned and implications for vaccine development.
Infectious diseases in obstetrics and gynecology
2011; 2011: 754060-?
Thy1.1 congenic B6.PL mice were used to simultaneously monitor Thy1.2+ E.G7-OVA tumors transplanted in the a.c. of the eye and i.v.-transferred tumor-specific Thy1.2+ CTLs to determine mechanisms that inhibit the tumoricidal activity of CTL responses in mice with established ocular tumors. Transferred CTLs were systemically deleted in mice with established ocular tumors. However, this deletion was not a unique mechanism of immune evasion by ocular tumors. Rather, development of Thy1.2+ tumors in the eye or skin of B6.PL mice generated cytotoxic anti-Thy1.2 antibodies that eliminated a subsequent Thy1.2+ T cell transfer. Anti-Thy1.2 immune responses in B6.PL mice were influenced by the route of antigen administration, as the serum concentration of cytotoxic anti-Thy1.2 antibodies was 92-fold greater in mice with eye tumors in comparison with mice with skin tumors. In addition, anti-Thy1.2 immune responses were detected in B6.PL mice given naïve Thy1.2+ T cells i.p. but not i.v. Anti-Thy1.2 responses were augmented in B6.PL mice with ocular Thy1.2+ EL-4 tumors that did not express OVA, suggesting immunodominance of OVA antigen over Thy1.2. Thy1.1+ T cells given i.p. was not immunogenic in Thy1.2 congenic mice. These data reaffirm that the introduction of antigens in the a.c. induces robust antibody responses. Experimentation using allotypic differences in Thy1 between donor cells and recipient mice must consider cytotoxic anti-Thy1 antibody generation in the interpretation of results.
View details for DOI 10.1189/jlb.0610333
View details for PubMedID 20959413
View details for PubMedCentralID PMC3024899
CTL Induction of Tumoricidal Nitric Oxide Production by Intratumoral Macrophages Is Critical for Tumor Elimination
JOURNAL OF IMMUNOLOGY
2010; 185 (11): 6706-6718
Chlamydia trachomatis control efforts that enhance detection and treatment of infected women may paradoxically increase susceptibility of the population to infection. Conversely, these surveillance programs lower incidences of adverse sequelae elicited by genital tract infection (e.g., pelvic inflammatory disease and ectopic pregnancy), suggesting enhanced identification and eradication of C. trachomatis simultaneously reduces pathogen-induced upper genital tract damage and abrogates formation of protective immune responses. In this paper, we detail findings from C. trachomatis infection control programs that increase our understanding of chlamydial immunoepidemiology and discuss their implications for prophylactic vaccine design.
View details for DOI 10.1155/2011/754060
View details for PubMedID 22144851
View details for PubMedCentralID PMC3227443
17-beta estradiol promotion of herpes simplex virus type 1 reactivation is estrogen receptor dependent.
Journal of virology
2010; 84 (1): 565-572
To characterize mechanisms of CTL inhibition within an ocular tumor microenvironment, tumor-specific CTLs were transferred into mice with tumors developing within the anterior chamber of the eye or skin. Ocular tumors were resistant to CTL transfer therapy whereas skin tumors were sensitive. CTLs infiltrated ocular tumors at higher CTL/tumor ratios than in skin tumors and demonstrated comparable ex vivo effector function to CTLs within skin tumors indicating that ocular tumor progression was not due to decreased CTL accumulation or inhibited CTL function within the eye. CD11b(+)Gr-1(+)F4/80(-) cells predominated within ocular tumors, whereas skin tumors were primarily infiltrated by CD11b(+)Gr-1(-)F4/80(+) macrophages (Ms), suggesting that myeloid derived suppressor cells may contribute to ocular tumor growth. However, CD11b(+) myeloid cells isolated from either tumor site suppressed CTL activity in vitro via NO production. Paradoxically, the regression of skin tumors by CTL transfer therapy required NO production by intratumoral Ms indicating that NO-producing intratumoral myeloid cells did not suppress the effector phase of CTL. Upon CTL transfer, tumoricidal concentrations of NO were only produced by skin tumor-associated Ms though ocular tumor-associated Ms demonstrated comparable expression of inducible NO synthase protein suggesting that NO synthase enzymatic activity was compromised within the eye. Correspondingly, in vitro-activated Ms limited tumor growth when co-injected with tumor cells in the skin but not in the eye. In conclusion, the decreased capacity of Ms to produce NO within the ocular microenvironment limits CTL tumoricidal activity allowing ocular tumors to progress.
View details for DOI 10.4049/jimmunol.0903411
View details for Web of Science ID 000284311500036
View details for PubMedID 21041723
View details for PubMedCentralID PMC3152256
Delayed processing of blood increases the frequency of activated CD11b+CD15+granulocytes which inhibit T cell function
JOURNAL OF IMMUNOLOGICAL METHODS
2009; 341 (1-2): 68-75
Correlations between estrogen and herpes simplex virus (HSV) reactivation from latency have been suggested by numerous clinical reports, but causal associations are not well delineated. In a murine HSV-1 corneal infection model, we establish 17-beta estradiol (17-betaE) treatment of latently infected ovariectomized mice induces viral reactivation, as demonstrated by increased viral load and increased immediate-early viral gene expression in the latently infected trigeminal ganglia (TG). Interestingly, the increased HSV reactivation occurred in the absence of inhibition of viral specific CD8(+) T-cell effector function. 17-betaE administration increased HSV reactivation in CD45(+) cell-depleted TG explant cultures, providing further support that leukocyte-independent effects on latently infected neurons were responsible for the increased reactivation. The drug-induced increases in HSV copy number were not recapitulated upon in vivo treatment of latently infected estrogen receptor alpha-deficient mice, evidence that HSV reactivation promoted by 17-betaE was estrogen receptor dependent. These findings provide additional framework for the emerging conceptualization of HSV latency as a dynamic process maintained by complex interactions among multiple cooperative and competing host, viral, and environmental forces. Additional research is needed to confirm whether pregnancy or hormonal contraceptives containing 17-betaE also promote HSV reactivation from latency in an estrogen receptor-dependent manner.
View details for DOI 10.1128/JVI.01374-09
View details for PubMedID 19846508
View details for PubMedCentralID PMC2798450
We tested whether granulocytes, which contaminate PBMC isolates after prolonged blood storage at room temperature, are responsible for inhibited T cell function in aged blood. We extend previous observations by characterizing these contaminating granulocytes as CD11b+ CD15+ cells comparable to activated CD11b+ CD15+ granulocytes induced by incubation of blood with FMLP. Granulocyte contamination of PBMC was observed within 6-8 h after venipuncture and room temperature storage (2.3 fold increase), and increased 11.3-fold by 24-26 h in comparison to PBMC from fresh blood. Refrigerated 22-26 hour storage of blood exacerbated granulocyte contamination (84-fold increase). In contrast, granulocyte contamination was markedly reduced if blood was diluted in RPMI-1640 medium (3.9-fold increase) or PBS (1.8-fold increase) prior to 22-26 hour room temperature storage. Granulocyte contamination significantly correlated with reduced CD3zeta chain expression, a marker of T cell dysfunction. Correspondingly, T cell proliferation following PHA stimulation was significantly decreased in PBMC with contaminating granulocytes from aged blood (77% of control) or FMLP treated blood (44% of control). Minimizing granulocyte contamination in PBMC of aged blood by cell sorting, or by reducing granulocyte activation by diluting blood in PBS prior to storage, increased CD3zeta chain expression and increased T cell proliferation following stimulation. These data indicate that granulocytes inhibit T cell function in aged blood. Therefore, preventing granulocyte activation in blood specimens is critical to maintain optimal T cell function. This may be accomplished by limiting the time from venipuncture to PBMC isolation to <8 h and may be extended to 26 h by simply diluting blood in PBS prior to room temperature storage.
View details for DOI 10.1016/j.jim.2008.10.019
View details for Web of Science ID 000264037000007
View details for PubMedID 19041316