Distinct Immune Responses Elicited From Cervicovaginal Epithelial Cells by Lactic Acid and Short Chain Fatty Acids Associated With Optimal and Non-optimal Vaginal Microbiota

Abstract

Non-optimal vaginal microbiota, as observed in bacterial vaginosis (BV), is typically characterized by a depletion of beneficial lactobacilli and an abundance of numerous anaerobes. These non-optimal conditions are associated with subclinical cervicovaginal inflammation and an increased risk of HIV infection compared to women colonized with optimal vaginal microbiota dominated by lactobacilli. Lactic acid (LA) is a major organic acid metabolite produced by vaginal lactobacilli that elicits anti-inflammatory effects from cervicovaginal epithelial cells and is dramatically depleted during BV. However, it is unclear if LA retains its anti-inflammatory activity in the presence of vaginal microbiota metabolites comprising short chain fatty acids (SCFAs) and succinic acid, which are also produced by an optimal vaginal microbiota. Furthermore, the immunomodulatory effect of SCFAs and succinic acid on cervicovaginal epithelial cells at higher concentrations present during BV is unknown. Here we report that in the presence of physiologically relevant concentrations of SCFAs and succinic acid at pH 3.9 (as found in women with lactobacillus-dominated microbiota) LA induced an anti-inflammatory state in cervicovaginal epithelial cells and inhibited inflammation elicited by the toll-like receptor (TLR) agonists polyinosinic:polycytidylic acid and Pam3CSK4. When cervicovaginal epithelial cells were treated with a vaginal microbiota metabolite mixture representative of BV, containing a lower concentration of LA but higher concentrations of SCFA/succinic acid at pH 7, no anti-inflammatory was observed. Rather, the vaginal microbiota metabolite mixture representative of BV dysregulated the immune response of cervicovaginal epithelial cells during prolonged and sustained treatments. This was evidenced by increased basal and TLR-induced production of pro-inflammatory cytokines including tumor necrosis factor-α, but decreased basal production of chemokines including RANTES and IP-10. Further characterization of individual components of the BV vaginal microbiota mixture suggested that acetic acid is an important vaginal microbiota metabolite capable of eliciting diverse immunomodulatory effects on a range of cervicovaginal epithelial cell targets. These findings indicate that elevated levels of SCFAs are a potential source of cervicovaginal inflammation in women experiencing BV, and support the unique anti-inflammatory properties of LA on cervicovaginal epithelial cells as well as a role for LA or LA-producing lactobacilli to reverse genital inflammation associated with increased HIV risk.

Keywords: bacterial vaginosis; cervicovaginal epithelium; inflammation; lactic acid; short chain fatty acids; succinic acid; vaginal microbiota metabolites.

Figure 1

Eubiotic vaginal microbiota metabolite mixture containing L-LA elicits a similar anti-inflammatory effect on Ect cells compared to L-LA alone. Ect cells seeded in transwells were apically treated with a mixture of vaginal microbiota metabolites at pH 3.9 recapitulating vaginal eubiosis (Eub) and L-LA alone in the absence or presence of the TLR agonists PIC (TLR3) and Pam (TLR1/2) as indicated. Cells were stimulated for 1 h and supernatant collected from the apical compartment after an additional 18 h of culture. Production of IL-1RA (A), IL-6 (B), IL-8 (C), IP-10 (D), RANTES (E), and MIP-3α (F) was quantified using a luminex multiplex assay. Graphs show mean and standard error of the mean from n = 4 independent assays. Mann-Whitney tests were used to estimate statistical significance between treatment groups. + denotes a significant difference (p < 0.05) as compared to untreated (UT) cells. P ≤ 0.05 and ≤ 0.01 are denoted as * and **, respectively. L-LA, L-isomer of lactic acid; TLR, toll-like receptor; PIC, polyinosinic:polycytidylic acid; Pam, Pam3CSK4.

Figure 2

A BV vaginal microbiota metabolite combination does not alter the production of immune mediators from Ect cells treated in a transwell system. Ect cells seeded in transwells were apically treated with a mixture of vaginal microbiota metabolites at pH 7 recapitulating bacterial vaginosis (BV; 6 mM L-LA, 100 mM acetic acid, 20 mM succinic acid, 4 mM propionic acid, and 4 mM butyric acid) in the absence or presence of TLR agonists PIC (TLR3) and Pam (TLR1/2) as indicated. Cells were stimulated for 1 h and supernatant collected from the apical compartment after an additional 18 h of culture. Production of IL-1RA (A), IL-6 (B), IL-8 (C), IP-10 (D), RANTES (E), and MIP-3α (F) was quantified using a luminex multiplex assay. Graphs show mean and standard error of the mean from n = 4 independent assays. Mann-Whitney tests were used to estimate statistical significance between treatment groups. + denotes a significant difference (p < 0.05) as compared to untreated (UT) cells. TLR, toll-like receptor; PIC, polyinosinic:polycytidylic acid; Pam, Pam3CSK4.

Figure 3

Prolonged treatment of Ect cells with BV vaginal microbiota metabolite mixture at pH 7 elicits a dysfunctional inflammatory response. Ect cells seeded in a plate format were stimulated with a mixture of vaginal microbiota metabolites recapitulating BV (6 mM L-LA, 100 mM acetic acid, 20 mM succinic acid, 4 mM propionic acid and 4 mM butyric acid) for 24 h at pH 7 in the absence or presence of stimulation with the TLR agonists PIC and Pam and production of IL-6 (A), IL-8 (B), TNFα (C), IP-10 (D), RANTES (E), and MIP-3α (F) was quantified from supernatants. Graphs show mean and standard error of the mean from n = 4 independent assays. P ≤ 0.05 is denoted as * as determined by Mann-Whitney test. + denotes a significant difference (p < 0.05) as compared to untreated (UT) cells. BV, bacterial vaginosis; TLR, toll-like receptor; PIC, polyinosinic:polycytidylic acid; Pam, Pam3CSK4.

Figure 4

Acetic acid alone increases basal and TLR-elicited TNF production from lower female reproductive tract epithelial cells. Ect (A), VK2 (B), and primary cervicovaginal epithelial cells (C) seeded into plates were treated with 100 mM acetic acid (Acetic) at pH 7 for 24 h in the absence or presence of stimulation with the TLR agonists PIC and Pam as indicated and production of TNFα was quantified from supernatants. Graphs show mean and standard error of the mean from n = 4 independent assays. P ≤ 0.05 are denoted as * as determined by Mann-Whitney test. + denotes a significant difference (p < 0.05) as compared to untreated (UT) cells. TLR, toll-like receptor; PIC, polyinosinic:polycytidylic acid; Pam, Pam3CSK4.

Figure 5

Acetic acid alone inhibits TLR-elicited IL-6, IP-10, and RANTES production from lower female reproductive tract epithelial cells. Ect (left panels), VK2 (center panels), and primary cervicovaginal epithelial cells (right panels) seeded into plates were treated with 100 mM acetic acid (Acetic) at pH 7 for 24 h in the absence or presence of stimulation with the TLR agonist PIC. Production of IL-6 (A–C), IP-10 (D–F), and RANTES (G–I) was quantified from supernatants. Graphs show mean and standard error of the mean from n = 4 independent assays. P ≤ 0.05 are denoted as * (as determined by Mann-Whitney test). + denotes a significant difference (p < 0.05) as compared to untreated (UT) cells. PIC, polyinosinic:polycytidylic acid.