Inflammatory and antimicrobial properties differ between vaginal Lactobacillus isolates from South African women with non-optimal versus optimal microbiota

Abstract

Female genital tract (FGT) inflammation increases HIV infection susceptibility. Non-optimal cervicovaginal microbiota, characterized by depletion of Lactobacillus species and increased bacterial diversity, is associated with increased FGT cytokine production. Lactobacillus species may protect against HIV partly by reducing FGT inflammation. We isolated 80 lactobacilli from South African women with non-optimal (Nugent 4-10; n = 18) and optimal microbiota (Nugent 0-3; n = 14). Cytokine production by vaginal epithelial cells in response to lactobacilli in the presence and absence of Gardnerella vaginalis was measured using Luminex. Adhesion to vaginal epithelial cells, pH, D/L-lactate production and lactate dehydrogenase relative abundance were assessed. Lactobacilli from women with non-optimal produced less lactic acid and induced greater inflammatory cytokine production than those from women with optimal microbiota, with IL-6, IL-8, IL-1α, IL-1β and MIP-1α/β production significantly elevated. Overall, lactobacilli suppressed IL-6 (adjusted p < 0.001) and IL-8 (adjusted p = 0.0170) responses to G. vaginalis. Cytokine responses to the lactobacilli were inversely associated with lactobacilli adhesion to epithelial cells and D-lactate dehydrogenase relative abundance. Thus, while cervicovaginal lactobacilli reduced the production of the majority of inflammatory cytokines in response to G. vaginalis, isolates from women with non-optimal microbiota were more inflammatory and produced less lactic acid than isolates from women with optimal microbiota.

Microbial function and genital inflammation in young South African women at high risk of HIV infection

“…As previous studies have suggested that the cell wall, as well as the cell membrane, may influence the immunomodulatory properties of Lactobacillus species and that peptidoglycan may be directly immunosuppressive [ 14 , 24 ], we further investigated the relationships between Lactobacillus cell wall and membrane properties and inflammation using 64 Lactobacillus species that were isolated from the same women [ 25 ]. From these lactobacilli, the 22 isolates that induced the lowest levels of pro-inflammatory cytokine production by vaginal epithelial cells and the 22 isolates that induced the greatest cytokine responses were selected and their protein profiles evaluated using LC-MS/MS.…”

“…The cell wall cellular component included multiple proteins involved in adhesion (adhesion exoprotein, LPXTG-motif cell wall anchor domain protein, collagen-binding protein, mannose-specific adhesin, putative mucin binding protein, sortase-anchored surface protein, and surface anchor protein). The relative abundance of this cell wall cellular component correlated positively with the level of adhesion of the isolates to vaginal epithelial cells in vitro (rho = 0.30, p = 0.0476), which in turn correlated inversely with in vitro inflammatory cytokine production by these cells in response to the isolates [ 25 ]. Importantly, the cell wall organization biological process included multiple proteins involved in peptidoglycan biosynthesis, which has the potential to dampen immune responses in a strain-dependent manner [ 14 , 26 ].…”

“…The increase in cell wall organization and peptidoglycan biosynthesis that accompanied a reduction in local inflammation suggests an increase in Lactobacillus strains producing relatively large amounts of proteins involved in these processes. In a previous study, we showed that adhesion of lactobacilli to vaginal epithelial cells in vitro was inversely associated with cytokine responses, suggesting that direct interaction between the isolates and vaginal epithelial cells is critical for immunoregulation [ 25 ]. In addition to cell wall and membrane properties, l -lactate dehydrogenase activity was identified as a molecular function inversely associated with inflammation.…”

“…In addition to cell wall and membrane properties, l -lactate dehydrogenase activity was identified as a molecular function inversely associated with inflammation. Similarly, d -lactate dehydrogenase relative abundance and d -lactate production by the Lactobacillus isolates included in this study were also associated with inflammatory profiles in an in vitro analysis [ 25 ]. These findings support the results of previous studies showing that lactic acid has immunoregulatory properties [ 16 ].…”

“…As described previously [ 25 ], vaginal epithelial cells (VK2/E6E7 ATCC® CRL-2616™; RRID:CVCL_6471) were maintained in complete keratinocyte serum-free media (KSFM) supplemented with 0.4 mM calcium chloride, 0.05 mg/ml of bovine pituitary extract, 0.1 ng/ml human recombinant epithelial growth factor, and 50 U/ml penicillin and 50 U/ml streptomycin (Sigma-Aldrich, MO, USA). The VK2 cells were seeded into 24-well tissue culture plates, incubated at 37 °C in the presence of 5% carbon dioxide, and grown to confluency.…”