Reduced Levels and Bioactivity of Endogenous Protease Cathepsin D in Genital Tract Secretions of Postmenopausal Women

Sexually active older adults are an emerging high-risk group for HIV acquisition and transmission, ¹ yet few studies have focused on genital tract immunity in postmenopausal women as it pertains to HIV susceptibility. Endogenous proteases and antiproteases (P/A-P) are integral components of genital secretions and function during routine tissue remodeling in wound healing, menstrual cycling, and pregnancy. ² Recent data indicate that P/A-P axis might be associated with inflammation and, therefore, an important determinant of HIV susceptibility. ³ In this study, we investigated proteases cathepsins B, D, and G, and neutrophil elastase (NE) in genital secretions of postmenopausal women.

Twenty women who had regular menses for the past 3 months comprised the premenopausal group and 20 women who did not have menses for at least 1 year comprised the postmenopausal group. The age range for premenopausal women was 23–48 years (mean 35). For postmenopausal women, age range was 45–81 years, (mean 55). Each premenopausal woman provided three samples coinciding with proliferative (days 5–9), ovulatory (days 12–16), and secretory (days 19–23) stages of their menstrual cycle (with day 0 signifying start of menses). Each postmenopausal woman provided one sample. Both groups had similar percentage of bacterial vaginosis (BV) (nine visits for premenopausal versus eight visits for postmenopausal). However, three visits of Trichomonas vaginalis (TV) were all in premenopausal group with two of those with both BV and TV. Cohort characteristics and sample collection procedures are described in Ref. ¹

Cervical vaginal lavage (CVL) was assayed for cathepsins B, D, and G by ELISA (R&D Systems, Minneapolis, MN; ABCAM, Cambridge, MA; Abnova, Taipei City, Taiwan, respectively). NE concentrations were determined using a multiplex bead immunoassay system (MILLIPLEX Human Sepsis Magnetic Bead Panel 3; EMD Millipore, Billerica, MA). Protease bioactivity for cathepsins B and D was measured using fluorescence-based assays (ABCAM).

Values less than lower limit of detection were assigned the value of “1” and all values were log transformed. As premenopausal women have repeated mediator measures, all comparisons were based on generalized estimating equations models.

We observed significantly lower levels of cathepsin D in postmenopausal women than in premenopausal women (median, premenopausal 4.2 vs. postmenopausal 0 log pg/ml, p ≤ 0.0001) (Fig. 1A). Although no significant changes across menstrual cycle were observed, there was a trend toward higher levels during the proliferative phase, which has also been observed by other studies. ⁴ Cathepsin D bioactivity was also significantly reduced in postmenopausal women (median, premenopausal 3 fluorescence units (FU) vs. postmenopausal 2 FU, p = .004) (Fig. 1B) without any significant changes across menstrual cycle. In contrast, although we detected high levels and bioactivity of cathepsin B, no significant differences were observed by menopausal or menstrual status (not shown). Cathepsin G was undetectable in all samples. High levels of NE were detected and a trend toward lower values during the ovulatory stage was observed, as previously described by Tawara et al. ⁵ However, no significant changes were observed by menstrual or menopausal status (not shown). Furthermore, there were no correlations between levels or activities of proteases and BV or TV status (not shown).

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FIG. 1.

Reduced levels and bioactivity of cathepsin D in CVL from postmenopausal women. CVL from premenopausal (n = 60) and postmenopausal (n = 19) women was tested for cathepsin D by ELISA. (A) Cathepsin D levels in premenopausal women (proliferative, ovulatory, and secretory stages of menstrual cycle) compared with those in postmenopausal women. Data are presented as pg/ml of protein. Bars depict median, interquartile range, and 95% CIs. (B) Cathepsin D bioactivity was measured in CVL using a fluorescence-labeled synthetic substrate and compared between premenopausal (proliferative, ovulatory, and secretory stages of menstrual cycle) and postmenopausal women. Bars depict median, interquartile range, and 95% CIs. CI, confidence interval; CVL, cervical vaginal lavage; FU, fluorescence units.

Cathepsin D has been shown to be associated with enhanced HIV infection/replication in vaginal secretions. ⁶ However, our study found significantly reduced levels and activity of cathepsin D in postmenopausal CVL. This matches with our recently published data from the same cohort showing reduced levels of other genital immune mediators in postmenopausal women. ¹ Further studies are needed to understand mechanisms of HIV acquisition/transmission in postmenopausal women.