Description
Immune modulatory effects of vaginal microbiota organic acid metabolites on lower female reproductive tract epithelial cells
David Diaz1, Anna Hearps1, David Tyssen1, J Hayward1, R Gugasyan1, D Anderson2, G Tachedjian1
1Burnet Institute, Melbourne VIC, Australia; 2Boston University, School of Medicine, Boston MA, USA
Women with diverse vaginal microbiota (e.g. bacterial vaginosis, BV), have inflammation in the lower female reproductive tract (FRT) and an increased risk of acquiring HIV in contrast to women with lactobacillus- (non-iners, LB) dominated microbiota. BV is characterised by pH>4.5, an increase in the vaginal microbiota metabolites (VMB) succinic and short chain fatty acids (SCFAs) and a dramatic depletion of the VMB lactic acid (LA) produced by LB, which acidifies the vagina to pH<4.5. Here we assessed the FRT epithelial cell immune modulatory effects of VMB at concentrations and pH present during LB dominance versus BV. Physiological levels of VMB at pH<4.5 or pH>4.5 were added apically to FRT epithelial cells in transwells ± TLR agonists or genital fluids. Cytokines were quantified by luminex-based assays. Statistical analysis was done using the Mann-Whitney U test. Treatment of epithelial cell lines with LA±SCFAs (pH<4.5, LB) significantly increased the anti-inflammatory cytokine IL-1RA. When added simultaneously to stimulation, LA±SCFAs (pH<4.5, LB) significantly inhibited TLR agonist-elicited production of inflammatory mediators IL-6, IL-8, IP-10, TNFα, RANTES, MIP3α. LA mediated these effects which was not recapitulated with media acidified to the same pH with HCl. LA treatment following 6h of TLR agonist stimulation or LA pretreatment of cells for 1h, followed by extensive cell washing and TLR agonist stimulation, inhibited inflammatory mediator production indicating a direct effect on cells. A similar anti-inflammatory effect of LA was observed when FRT epithelial cells were exposed to cervicovaginal fluid from women with BV. In contrast LA+SCFAs (pH>4.5, BV) neither elicited IL-1RA production nor altered inflammatory mediator levels induced by TLR-agonists. LA and SCFAs at physiological levels and pH had little impact on cell viability. Mixtures of VMB under conditions of LB dominance, but not BV, inhibit FRT epithelial inflammation that might explain in part the HIV protective properties of LA-producing LB. This study highlights the potential use of LA or LA-producing probiotics as adjuncts to female-initiated HIV prevention strategies.