Inflammatory Cytokine Profiles of Semen Influence Cytokine Responses of Cervicovaginal Epithelial Cells

Abstract

Genital inflammatory cytokine responses increase HIV risk. Since male partner semen is a complex mixture of immune-modulatory prostaglandins and cytokines, we hypothesized that exposure to semen may influence genital inflammation in women. Here, we investigated cytokine response kinetics of cervical cells following stimulation with seminal plasma from HIV-negative and HIV-positive men characterized as having low or high concentrations of inflammatory cytokines. Irrespective of the HIV status or semen cytokine profile, in vitro stimulation of cervical cells with seminal plasma resulted in significantly elevated concentrations of secreted IL-6, IL-8, TNF-β, MCP-1, GM-CSF, and VEGF within 8 h of stimulation, which tended to decline by 24 h, although this was only significant for TNF-β. Consistent with this, cervical cells responded to seminal plasma with increases in IL-8 and IL-1β mRNA expression of 10-fold. These findings suggest that the impact of semen on local female genital cytokines is likely transient. Although these findings suggest that the impact of semen on local female genital cytokines may not be sustained long-term, this heightened genital inflammation may have implications for HIV risk in women.

Keywords: HIV; HeLa; cytokines; genital inflammation; interleukins; semen.

Figure 1

Unsupervised hierarchical clustering confirmed selection of semen from the HIV-negative (A) and HIV-positive (B) men by cytokine concentration; and to cluster men according to the similarities of their semen cytokine expression profiles (using Qlucore Omics Explorer). (A) HIV-negative men with the lowest concentrations of 23 cyokines (n = 5; turquoise blocks) clustered separately from HIV-negative men with the highest concentrations of these cytokines (n = 5; blue blocks). (B) HIV-positive men with the lowest concentrations of 23 cyokines (n = 5; turquoise blocks) clustered separately from HIV-positive men with the highest concentrations of these cytokines (n = 5; blue blocks). Cytokine concentrations are indicated using a color scale, ranging from green (low) to red (high). The dendrogram above the heat map illustrates degrees of relatedness between cytokine profiles evident within semen from the various men. In this tree, shorter and longer horizontal branch path lengths between pairs of cytokines, respectively reflect greater and lower degrees of similarity between the expression profiles of the assessed cytokines.Cytokines shown in bold indicate those that were measured in the in vitro experiments in this study.

Figure 2

Effects of seminal plasma on cytokine mRNA expression by HeLa cells. Quantitative real-time PCR was used to determine the mRNA expression of (C) IL-6, (A) IL-8, (B) IL-1β, (E) GM-CSF, (D) MIP3α, and (F) VEGF by HeLa cells treated with 1:50 dilution of seminal plasma from HIV-negative (white bars) or HIV-positive individuals (black bars) for 8 h (n = 4) and 24 h (n = 3). The mRNA levels were normalized by housekeeping gene Human cyclophilin A. Data are represented as the mean ± SEM of duplicate wells in each experiment. Wilcoxon matched pairs (non-parametric) and standard matched pairs t-tests (parametric) were used to compare the control and treatment groups. *p < 0.05.

Figure 3

Effects of seminal plasma on cytokine secretion by HeLa cells. Hela cells were exposed to seminal plasma from HIV-negative (white bars) or HIV-infected individuals (black bars). A 1:50 dilution of seminal plasma from men with extreme inflammatory profiles (highest vs. lowest percentile) were used to stimulated HeLa cells for 8 and 24 h. Cytokine levels was measured in the 8 and 24 h post-stimulation in Hela cells supernatants by Luminex. Seminal plasma (1:50) in serum free culture media was used, while control was serum free culture media. Data are represented as the mean ± SEM of duplicate wells in each experiment. Wilcoxon matched pairs (non-parametric) and a standard matched pairs t-tests (parametric) were used to compare the control and treatment groups. *p < 0.05; **p < 0.01. (A) IL-6, (B) IL-8, (C) TNF-α, (D) MCP-1, (E) GM-CSF, and (F) VEGF.