Characterization of host immunity during persistent vaginal colonization by Group B Streptococcus

Abstract

Streptococcus agalactiae (Group B Streptococcus, GBS) is a Gram-positive bacterium, which colonizes the vaginal tract in 10-30% of women. Colonization is transient in nature, and little is known about the host and bacterial factors controlling GBS persistence. Gaining insight into these factors is essential for developing therapeutics to limit maternal GBS carriage and prevent transmission to the susceptible newborn. In this work, we have used human cervical and vaginal epithelial cells, and our established mouse model of GBS vaginal colonization, to characterize key host factors that respond during GBS colonization. We identify a GBS strain that persists beyond a month in the murine vagina, whereas other strains are more readily cleared. Correspondingly, we have detected differential cytokine production in human cell lines after challenge with the persistent strain vs. other GBS strains. We also demonstrate that the persistent strain more readily invades cervical cells compared with vaginal cells, suggesting that GBS may potentially use the cervix as a reservoir to establish long-term colonization. Furthermore, we have identified interleukin-17 production in response to long-term colonization, which is associated with eventual clearance of GBS. We conclude that both GBS strain differences and concurrent host immune responses are crucial in modulating vaginal colonization.

Figure 1. GBS strain persistence in the murine vaginal tract

CD1 mice (n=10/group) were colonized with 1×10⁷ CFU of GBS clinical isolates CJB111, COH1, or A909 in the vaginal lumen. GBS persistence was determined by swabbing the vagina and plating recovered bacteria. (A) Percentage of mice with detectable GBS within the vaginal lumen via swabbing. Limit of detection is 50 CFU. (B) Mean recovered GBS CFU of same mice as in (A). (C–E) Reproductive tract tissues were isolated 4 weeks post-inoculation with GBS strains. Bacterial load was quantified by plating serially diluted tissue homogenates. In vivo experiments were conducted independently at least twice and data from one representative experiment is shown. Data was analyzed using Kruskal-Wallis with Dunn’s multiple comparisons post-test. *, P<0.05; **, P<0.01.

Figure 2. Interaction of GBS strains with human female reproductive epithelial cells

(A–C) Adherence, (D–F) Invasion, or (G–I) Intracellular survival of GBS CJB111, COH1, or A909 with indicated epithelial cells, MOI = 10 for A–F and MOI = 1 for G–I. For H–I, significance given is CJB111 in comparison to both COH1 and A909. Experiments were repeated at least twice with four replicates and data from a representative experiment is shown. Data was analyzed by one-way ANOVA with Bonferroni’s multiple comparisons post-test for (A–F) and two-way repeated measures ANOVA with Bonferroni’s multiple comparisons post-test for (G–I). *, P<0.05; **, P<0.01; ***,P<0.001; ****,P<0.0001.

Figure 3. GBS cytokine induction in human female reproductive epithelial cells

(A–C) Transcript abundance of IL-1β, IL-6, IL-23, and IL-36γ in human epithelial cells was determined using quantitative RT-PCR following infection with CJB111, COH1, or A909 (MOI = 10). Fold change was calculated using GAPDH and then normalized to media controls as described Methods. Statistical values are in reference to CJB111. (D–F) Protein expression of IL-1β and (G–I) IL-36γ in human epithelial cell lysates was determined as described in Materials and Methods 4 hours post-infection with CJB111, COH1, or A909 at an MOI = 10. Statistical values are in reference to media controls. Data is one representative experiment of at least 2 independent experiments performed in 4 replicates at minimum. Data was analyzed by one-way ANOVA with Bonferroni’s multiple comparisons post-test for (A–C) and two-way repeated measures ANOVA with Bonferroni’s multiple comparisons post-test for (D–I).*, P<0.05; **, P<0.01; ***,P<0.001; ****,P<0.0001.

Figure 4. GBS colonization and cytokine production in the murine reproductive tract

KC/GROα (A), MIP-2 (B), IL-1β (C), IL-6 (D), and IL-23 (E) levels in vaginal homogenates from mice 2 days post-inoculation, or IL-17A (F) levels at 4 weeks post-inoculation, were quantified by ELISA. Data is the combined results of two independent experiments (n = 10–20 per group). (G–I) GBS load in reproductive tract tissue homogenates collected from mice 2 days post-inoculation (n = 10 per group). Lines represent median values of each group. Data was analyzed by Kruskal-Wallis with Dunn’s multiple comparisons post-test.*, P<0.05; **, P<0.01.

Figure 5. Presence of IL-17 within the reproductive tract is associated with reduced clearance of GBS CJB111

(A,E) Total IL-17A+ cells present in reproductive tract tissues collected from mice (n = 4–6 per group) at indicated time points post-inoculation with CJB111 were quantified by flow cytometry as described in Methods. Cleared mice (black bars) were separated from colonized mice (white bars) if GBS counts were below the limit of detection at the time of sacrifice (50 CFU/tissue). (B–D,F–H) Surface markers Ly6G, CD11b, and CD4 present on populations identified within total IL-17A+ cells from (A,E). Representative data from one of two independent experiments is shown. Immunohistochemistry of the vagina (I) and uterus (J) of GBS-colonized mice with IL-17+ cells visualized with DAB (brown) and counter-stained with hematoxylin (blue). Magnification = 200×. (K) Mice were colonized with CJB111, and one day later, received daily treatment of rIL-17 within the vaginal lumen. Data shown is from 3 days post-inoculation with CJB111, and is the result of 3 independent experiments combined (total n = 38 per group).Lines represent median values of each group. Data was analyzed by Mann-Whitney.*, P<0.05; **, P<0.01.