Metronidazole Treatment Failure and Persistent BV Lead to Increased Frequencies of Activated T- and Dendritic-Cell Subsets

Abstract

Metronidazole (MDZ) treatment failure and bacterial vaginosis (BV) recurrence rates are high among African women. This cohort study identified genital immune parameters associated with treatment response by comparing vaginal microbiota and immune cell frequencies in endocervical cytobrushes obtained from 32 South African women with symptomatic BV pre- and post-metronidazole treatment. Cervical T- and dendritic-cell subsets were phenotyped using multiparameter flow cytometry and the composition of vaginal microbial communities was characterized using 16S rRNA gene sequencing. MDZ treatment led to a modest decrease in the relative abundance of BV-associated bacteria, but colonization with Lactobacillus species (other than L. iners) was rare. At 6 and 12 weeks, MDZ-treated women had a significant increase in the frequencies of CCR5+ CD4+ T cells and plasmacytoid dendritic cells compared to the pre-treatment timepoint. In addition, MDZ non-responders had significantly higher frequencies of activated CD4 T cells and monocytes compared to MDZ responders. We conclude that MDZ treatment failure was characterized by an increased expression of activated T- and dendritic-cell subsets that may enhance HIV susceptibility. These data suggest the need to further assess the long-term impact of MDZ treatment on mucosal immune response and the vaginal microbiota.

Keywords: BV treatment; genital immune cells; vaginal microbiota.

Figure 1

Identification of cervical T- and monocyte/dendritic-cell subsets from endocervical cytobrush specimens at three time points. Representative gating plot shows the exclusion of non-lymphocytes, doublets, dead (LIVE/DEAD™), CD19, and CD56 B lymphoid cells. The resulting cells were separated into CD3+ and CD3− subsets. The CD3+ T cells were further classified into CD4+ T cells. The expression of CD38, HLA-DR, CCR5, and CCR6 on CD4+ CD3+ lymphocytes was examined. The CD3- subset was separated into dendritic cell subsets: mDCs (HLADR+ CD14− CD11c+), pDCs (HLADR+ CD123+), and monocytes (HLADR+ CD14+ CD11c+). Activated DCs were identified using their CD86 expression, while monocytes were classified as CD14+ HLADR++.

Figure 2

Frequency distribution of T- and dendritic-cell subsets in cytobrush specimens at baseline compared to 6- and 12-weeks post-treatment. Graph shows the differences over time for the frequencies of cervical (A) CD4+ T cells, (B) CD38+ CD4+ T cells, (C) HLADR+ CD4+ T cells, (D) CCR5+ CD4+ T cells, (E) CCR6+ CD4+ T cells, (F) CD14− HLADR+, (G) HLADR++ CD14+, (H) CD86+ HLADR++ CD14+, (I) HLDR+ CD14− CD11c+, (J) CD86+ CD11c+, (K) HLADR+ CD14− CD123+, and (L) CD86+ CD123+. Green color depicts CST I, blue CST III, orange CST IV-A, and red CST IV-B. The Mann–Whitney U test was used to compare the frequency distribution of T- and dendritic-cell subsets at pre- and post-treatment timepoints.

Figure 3

Composition of bacterial species and transitions within bacterial communities over the 12–week period. Box plots showing CSTs assigned to individual participants over 3 time points at baseline, 6 weeks, and 12 weeks. Green color depicts CST I, blue CST III, orange CST IV–A, and red CST IV–B.

Figure 4

Frequencies of cervical T-cell and DC subsets in women with a cervicovaginal microbiota dominated by Lactobacillus spp. (predominantly L. iners, red) and non-Lactobacillus at baseline, 6 weeks, and 12 weeks after initiating MDZ. Graphs show the differences between pre- and post-MDZ initiation for the frequencies of cervical: Graph shows the differences over time for the frequencies of cervical (A) CD4+ T cells, (B) CD38+ CD4+ T cells, (C) HLADR+ CD4+ T cells, (D) CCR5+ CD4+ T cells, (E) CCR6+ CD4+ T cells, (F) CD14− HLADR+, (G) HLADR++ CD14+, (H) CD86+ HLADR++ CD14+, (I) HLDR+ CD14− CD11c+, (J) CD86+ CD11c+, (K) HLADR+ CD14− CD123+, and (L) CD86+ CD123+. Red color depicts non-Lactobacillus dominant CST (IV-A/IV-B) and red depicts Lactobacillus dominant CST (I/III). Statistical comparisons were performed using Mann–Whitney U tests for unmatched data or the Kruskal–Wallis test with false discovery rate correction for cross-sectional data. * Indicates significant differences (p < 0.05), while ns indicates non–significant difference (p > 0.05).

Figure 5

Frequencies of cervical T-cell and DC subsets in women who cleared BV (green) and compared to women with persistent or recurrent BV after initiating MDZ. Graph shows the differences over time for the frequencies of cervical (A) CD4+ T cells, (B) CD38+ CD4+ T cells, (C) HLADR+ CD4+ T cells, (D) CCR5+ CD4+ T cells, (E) CCR6+ CD4+ T cells, (F) CD14− HLADR+, (G) HLADR++ CD14+, (H) CD86+ HLADR++ CD14+, (I) HLDR+ CD14− CD11c+, (J) CD86+ CD11c+, (K) HLADR+ CD14− CD123+, and (L) CD86+ CD123+. Clearance is depicted in green, persistent in red, and recurrence in orange. Individual associations are shown between BV status and immune subsets including CD3+, CD4+ T cells, CD38+ CD4+ T cells, HLA-DR+ CD4+ T cells, CCR5+ CD4+ T cells, CCR6+ CD4+ T cells, CD14+, CD11c+ CD14+, and CD123+ CD14+ cells.