Searching for Lower Female Genital Tract Soluble and Cellular Biomarkers: Defining Levels and Predictors in a Cohort of Healthy Caucasian Women

Abstract

BackgroundHigh concentrations of pro-inflammatory cytokines have been previously observed in the genital fluids of women enrolled in microbicide trials and may explain observed increased HIV transmission in some of these trials. Although the longitudinal nature of these studies allows within-subject comparisons of post-product levels to baseline levels, the fact that the physiologic variations of these cytokines and other markers of immune activation are not fully defined in different populations, makes it difficult to assess changes that can be directly attributed to microbicide use as opposed to other biological and behavioural factors.MethodsCervicovaginal lavage samples were collected from 30 healthy Caucasian and assayed for concentrations of ten cytokines/chemokines, total protein content and two antimicrobial proteins using a multiplex immunoassay and ELISA. Cellular markers were characterized by flow cytometry on mononuclear cells collected from the endocervix using flocked swabs. Bacterial quantification was performed using quantitative PCR.ResultsEctopy, menstrual cycle phase, prostate-specific antigen and presence of leucocytes in endocervical cells' supernatant were associated with the concentrations of cyto-/chemokines in cervicovaginal secretions. Approximately 3% of endocervical cells collected were monocytes of which a median of 52% (SD = 17) expressed both CD4 and CCR5 markers. Approximately 1% of the total cells were T-cells with a median of 61% (SD = 10) CD4 and CCR5 expression. Around 5% of the monocytes and 16% of the T-cells expressed the immune activation marker HLA-DR. Higher percentages of T-cells were associated with greater quantities of IL-1RA, GM-CSF and elafin.ConclusionWe demonstrate the presence of selected soluble and cellular immune activation markers and identify their predictors in the female genital tract of healthy women. Future clinical trials should consider ectopy, sexual activity, menstrual cycle phase and presence of bacterial species as possible confounders when evaluating the possible inflammatory effects of microbicide compounds.

Comparison of Follicular and Luteal Phase Mucosal Markers of HIV Susceptibility in Healthy Women

“…45 No significant differences in the antimicrobial activity of the CVL The secretion of cervical mucus is significantly influenced by endogenous reproductive hormones, with cervical mucus volume peaking with surges in preovulatory serum E2. [46][47][48] The biologic rationale for differences in antimicrobial activity of the CVL is based on data from small cohorts showing that secreted cytokines, chemokines, and other cationic antimicrobial polypeptides in the vagina have significant alterations in concentrations based on the phase of the menstrual cycle with immune factors such as IL-6, IL-1b, IL-1RA, and MIP-1b being significantly higher in the FOL phase versus the LUT phase, 19,27 while IL-1a and b-defensin were significantly elevated in the LUT phase. 27 Keller et al found that immune mediators, SLPI, a and b defensins, lysozyme, and lactoferrin, were significantly lower at mid-cycle ovulation compared with both the FOL and LUT phases.…”

“…[46][47][48] The biologic rationale for differences in antimicrobial activity of the CVL is based on data from small cohorts showing that secreted cytokines, chemokines, and other cationic antimicrobial polypeptides in the vagina have significant alterations in concentrations based on the phase of the menstrual cycle with immune factors such as IL-6, IL-1b, IL-1RA, and MIP-1b being significantly higher in the FOL phase versus the LUT phase, 19,27 while IL-1a and b-defensin were significantly elevated in the LUT phase. 27 Keller et al found that immune mediators, SLPI, a and b defensins, lysozyme, and lactoferrin, were significantly lower at mid-cycle ovulation compared with both the FOL and LUT phases. 20 Secretion of mannosebinding lectin, a molecule in the complement system that plays a critical role in host protection, from vaginal epithelial cells is increased in the LUT phase of the menstrual cycle.…”

“…26,50 Genital tract cytokines have also been shown to vary based on clinical factors, such as cervical ectopy, inflammation, and sexual activity. 27,51 An important functional assay, which may incorporate host contributions of cytokines, chemokines, and vaginal microflora, is the antimicrobial activity of the CVL as this helps to model how these factors may potentially work in concert to either protect or enhance infection. Our data are consistent with a cross-sectional study of premenopausal women, sampled either in the FOL (n = 26) or LUT (n = 27) phase of the menstrual cycle, showing no difference between HIV inhibition of the CVL and the menstrual cycle phases.…”

“…18 However, there is a paucity of data regarding the effect of the menstrual cycle on immune cell recruitment and activation in the lower reproductive tract, including the ectocervix and vagina, 19,20 with some studies showing no changes in CD4 and CCR5-positive cells, based on the menstrual cycle, in ectocervical explants. 21 Previous studies regarding the effect of endogenous hormones on mucosal HIV targets in the lower genital tract have been done primarily with explants obtained from women undergoing elective surgeries such as hysterectomies [21][22][23][24][25] or among small cohorts of women in a longitudinal 19,[26][27][28] or cross-sectional design. 29 While surgical explants offer convenience and supply tissue without invasive biopsy procedures, women undergoing indicated surgery invariably have a host of medical comorbidities and concomitant medication use, which may affect study endpoints.…”

Cross-Sectional Analysis of Selected Genital Tract Immunological Markers and Molecular Vaginal Microbiota in Sub-Saharan African Women, with Relevance to HIV Risk and Prevention

“…CVL samples and vaginal swabs were shipped in batches using a temperature-monitored dry shipper to the central laboratory at the Institute of Tropical Medicine (ITM) in Antwerp, Belgium, where they were stored at Ϫ80°C before analysis for soluble markers of inflammation or use in the CVL antiviral assay. The concentrations of the cytokines interleukin-1␣ (IL-1␣), IL-1␤, IL-6, and IL-12(p70), the CC chemokine MIP-1␤, the CXC chemokines gamma interferon (IFN-␥)-induced protein (IP-10) and IL-8, and the growth factors granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in CVL samples were analyzed at the ITM in Antwerp, Belgium, using the Bio-Plex human cytokine assay kit (Bio-Rad Laboratories NV-SA, Nazareth, Belgium) as previously described (11). Fluorescence data for nine soluble immune mediators were collected using a Bio-Plex array reader, and Bio-Plex Manager 5.0 software was used to calculate cytokine concentrations with a weighted five-parameter logistic curve-fitting method on the 4-fold dilution series of the standard provided with the kit at the ITM.…”