Bacterial vaginosis-driven changes in cervicovaginal immunity that expand the immunological hypothesis for increased HIV susceptibility

Abstract

Bacterial vaginosis (BV) is a dysbiosis of the vaginal microbiome that is prevalent among reproductive-age females worldwide. Adverse health outcomes associated with BV include an increased risk of sexually-acquired HIV, yet the immunological mechanisms underlying this association are not well understood. To investigate BV-driven changes to cervicovaginal tract (CVT) and circulating T cell phenotypes, participants with or without BV provided vaginal tract (VT) and ectocervical (CX) tissue biopsies and PBMC samples. High-parameter flow cytometry revealed an increased frequency of cervical conventional CD4⁺ T cells (Tconv) expressing CCR5. However, we found no difference in number of CD3⁺CD4⁺CCR5⁺ cells in the CX or VT of BV+ vs BV- individuals, suggesting that BV-driven increased HIV susceptibility may not be solely attributed to increased CVT HIV target cell abundance. Flow cytometry also revealed that individuals with BV have an increased frequency of dysfunctional CX and VT CD39⁺ Tconv and CX tissue-resident CD69⁺CD103⁺ Tconv, reported to be implicated in HIV acquisition risk and replication. Many soluble immune factor differences in the CVT further support that BV elicits diverse and complex CVT immune alterations. Our comprehensive analysis expands on potential immunological mechanisms that may underlie the adverse health outcomes associated with BV including increased HIV susceptibility.

Figure 1.. BV did not alter the balance of T cell frequency in the cervicovaginal tract tissues.

Flow cytometry was used to quantify the proportions of T cells within different tissue sites as indicated. (A) The frequency of CD3⁺ among the total CD45⁺ population in ectocervix (CX), vaginal tract (VT), and peripheral mononuclear blood cells (PBMC) samples in BV− and BV+ individuals. The frequency of Tconv (CD3⁺CD4⁺CD25⁻ cells) (B) and CD3⁺CD8⁺ (C) among the total CD45⁺ population in CX, VT, and PBMC samples in BV− and BV+ individuals. The frequency of Tconv (D) and CD8⁺ (E) among the total CD3⁺ population in CX, VT, and PBMC samples in BV− and BV+ individuals. Adjusted rank regression analysis was performed to compare frequencies in each tissue between BV− and BV+ individuals. PBMC comparisons were a priori adjusted for hormonal contraceptive use, and CX and VT comparisons were a priori adjusted for hormonal contraceptive use, HSV-2 serology, HIV exposure, and semen exposure to reduce the effects of potential confounding variables on the analysis of BV-driven T cell alterations. Adjusted p-value displayed in bold when p ≤ 0.05, non-bold when 0.05 < p_adj ≤ 0.10, and “ns” for not significant when adjusted p > 0.10. Each dot represents a measurement from an individual sample. Each horizontal bar indicates the median for its respective group. For each comparison, the Ns, medians, p-values (adjusted and unadjusted), and estimated differences (adjusted and unadjusted) are provided in Supplemental Table 1.

Figure 2.. Conventional CD4⁺ T cells displayed increased markers of activation and tissue residency in the cervix of individuals with BV.

Flow cytometry was used to examine Tconv cell phenotypes within different tissue sites as indicated. (A) The frequency of CCR5⁺, (B) HLA-DR⁺, and (C) CD69⁺CD103⁺ among the total Tconv T cell population in CX, VT, and PBMC samples in BV− and BV+ individuals. Adjusted rank regression analysis was performed to compare frequencies in each tissue between BV− and BV+ individuals. PBMC comparisons were a priori adjusted for hormonal contraceptive use, and CX and VT comparisons were a priori adjusted for hormonal contraceptive use, HSV-2 serology, HIV exposure, and semen exposure to reduce the effects of potential confounding variables on the analysis of BV-driven T cell alterations. Adjusted p-value displayed in bold when p ≤ 0.05, non-bold when 0.05 < p_adj ≤ 0.10, and “ns” for not significant when adjusted p > 0.10. Each dot represents a measurement from an individual sample. Each horizontal bar indicates the median for its respective group. For each comparison, the Ns, medians, p-values (adjusted and unadjusted), and estimated differences (adjusted and unadjusted) are provided in Supplemental Table 1.

Fig 3.. The total density of T cells and HIV target cells in the cervix and vagina was not altered by BV.

(A) Representative H&E-stained vaginal tract tissue section imaged with brightfield microscopy. (B) Representative immunofluorescent stained tissue section from the same vaginal tract (VT) biopsy as Figure 3A. DAPI stain is shown in blue, the CD3 stain is shown in red, the CD4 stain is shown in cyan, and the CCR5 stain is shown in green. All fluorescent signals overlayed. Comparison of the cell density of CD3⁺ T cells, CD3⁺CD4⁺ T cells, or CD3⁺CD4⁺CD5⁺ HIV target cells between BV− and BV+ samples in the ectocervix (C) and vaginal tract (D). Wilcoxon rank sum test was performed for each comparison shown. Comparisons with p > 0.05 labeled “ns” for not significant. Each dot represents a measurement from an individual sample. Each horizontal bar indicates the median for its respective group. For each comparison, the Ns, medians, p-values (adjusted and unadjusted), and estimated differences (adjusted and unadjusted) are provided in Supplemental Table 2.

Figure 4.. Conventional CD4⁺ T cells in the cervix showed signs of dysfunction in individuals with BV.

Flow cytometry was used to examine Tconv cell phenotypes within different tissue sites as indicated. (A) The frequency of CD39⁺, (B) CD101 ⁺, and (C) T Cell Factor-1⁺ (TCF-1) among the total Tconv T cell population in CX, VT, and PBMC samples in BV− and BV+ individuals. Adjusted rank regression analysis was performed to compare frequencies in each tissue between BV− and BV+ individuals. PBMC comparisons were a priori adjusted for hormonal contraceptive use, and CX and VT comparisons were a priori adjusted for hormonal contraceptive use, HSV-2 serology, HIV exposure, and semen exposure to reduce the effects of potential confounding variables on the analysis of BV-driven T cell alterations. Adjusted p-value displayed in bold when p ≤ 0.05, non-bold when 0.05 < p_adj ≤ 0.10, and “ns” for not significant when adjusted p > 0.10. Each dot represents a measurement from an individual sample. Each horizontal bar indicates the median for its respective group. For each comparison, the Ns, medians, p-values (adjusted and unadjusted), and estimated differences (adjusted and unadjusted) are provided in Supplemental Table 1.

Figure 5.. Th17 cells exhibited increased markers of activation and tissue residency in cervical samples from individuals with BV.

Flow cytometry was used to examine Th17 phenotypes within different tissue sites as indicated. (A) The frequency of Th17 T cells, defined as CD161⁺CCR6⁺ Tconv, among the total Tconv T cell population in CX, VT, and PBMC samples in BV− and BV+ individuals. (B) HLA-DR ⁺, (C) CD69⁺CD103⁺, (D) CD101 ⁺, and (E) CCR5⁺ frequencies among the total Th17 population in CX, VT, and PBMC samples in BV− and BV+ individuals. Adjusted rank regression analysis was performed to compare frequencies in each tissue between BV− and BV+ individuals. PBMC comparisons were a priori adjusted for hormonal contraceptive use, and CX and VT comparisons were a priori adjusted for hormonal contraceptive use, HSV-2 serology, HIV exposure, and semen exposure to reduce the effects of potential confounding variables on the analysis of BV-driven T cell alterations. Adjusted p-value displayed in bold when p ≤ 0.05, non-bold when 0.05 < p_adj ≤ 0.10, and “ns” for not significant when adjusted p > 0.10. Each dot represents a measurement from an individual sample. Each horizontal bar indicates the median for its respective group. For each comparison, the Ns, medians, p-values (adjusted and unadjusted), and estimated differences (adjusted and unadjusted) are provided in Supplemental Table 1.

Figure 6.. Cervical CD8⁺ T cells displayed a dysfunctional phenotype in individuals with BV.

Flow cytometry was used to examine CD8⁺ T cell phenotypes within different tissue sites as indicated. (A) The frequency of CD39⁺, (B) Granzyme B⁺, and (C) T-bet⁺ among the total CD8⁺ T cell population in CX, VT, and PBMC samples in BV− and BV+ individuals. Adjusted rank regression analysis was performed to compare frequencies in each tissue between BV− and BV+ individuals. PBMC comparisons were a priori adjusted for hormonal contraceptive use, and CX and VT comparisons were a priori adjusted for hormonal contraceptive use, HSV-2 serology, HIV exposure, and semen exposure to reduce the effects of potential confounding variables on the analysis of BV-driven T cell alterations. Adjusted p-value displayed in bold when p ≤ 0.05, non-bold when 0.05 < p_adj ≤ 0.10, and “ns” for not significant when adjusted p > 0.10. Each dot represents a measurement from an individual sample. Each horizontal bar indicates the median for its respective group. For each comparison, the Ns, medians, p-values (adjusted and unadjusted), and estimated differences (adjusted and unadjusted) are provided in Supplemental Table 1.

Figure 7.. BV was associated with reduced chemokine concentrations and increased inflammatory cytokine concentrations in CVT fluid.

Luminex was used to quantify cytokines and chemokines from CVT fluid or serum samples. Adjusted estimated difference (BV positive – BV negative) for CVT fluid cytokines (A) and serum (B) that met the criteria for quantification of cytokine concentrations (greater than or equal to 80% of samples were detectable). The adjusted 95% confidence interval is shown for all comparisons. Significant results when adjusted p ≤ 0.05 comparing BV− vs BV+ are colored in orange and non-significant differences (p > 0.05) comparing BV− vs BV+ are purple. Vertical dashed line at x=0 for reference. Serum comparisons were a priori adjusted for hormonal contraceptive use, and CVT fluid comparisons were a priori adjusted for hormonal contraceptive use, HSV-2 serology, HIV exposure, and semen exposure to reduce the effects of potential confounding variables on the analysis of BV-driven T cell alterations. Statistical analyses can be found in Supplemental Table 3.