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

David J Delgado-Diaz,Joshua A Hayward,Raffi Gugasyan,David Tyssen,Gilda Tachedjian,Anna C Hearps

doi:10.3389/fcimb.2019.00446

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.

Highlights

Our previous studies examined the immunomodulatory effect of Lactic acid (LA) alone on cervicovaginal epithelial cells (Hearps et al, 2017); LA exists in combination with other organic acid metabolites from female reproductive tract-resident bacteria in the vaginal lumen (Aldunate et al, 2015)
To determine if L-LA maintains its anti-inflammatory effect when combined with other organic acid metabolites, cervicovaginal epithelial cells were treated in transwell inserts with a combination of vaginal microbiota metabolites relevant to eubiosis with and without toll-like receptors (TLRs) stimulation for 1 h, shown previously to be sufficient for L-LA to elicit its anti-inflammatory effects (Hearps et al, 2017), and cytokine and chemokine production were analyzed 18 h post-treatment
We describe for the first time the effect of L-LA on dampening TLR-elicited induced protein 10 (IP-10) production which is of special interest given the association of this cytokine with increased human immunodeficiency virus (HIV) transmission in women (Masson et al, 2015)

Summary

Introduction

The lower female reproductive tract epithelium plays important roles as a physical and immunological barrier against infection (Anderson et al, 2014); its function is influenced by many factors including the effects of commensal vaginal microbiota and their metabolic products (O’Hanlon et al, 2011, 2013; Ravel et al, 2011; Aldunate et al, 2013; Anderson et al, 2014; Hearps et al, 2017; Tachedjian et al, 2017). Lactic acid (LA) is an organic acid metabolite predominately produced by lactobacilli (Boskey et al, 2001) and is present in the lower female reproductive tract at a concentration of ∼110 mM (∼1.0% ± 0.2% w/v) and a pH < 4.5 in women with lactobacillus-dominated microbiota (Boskey et al, 2001; O’Hanlon et al, 2013). LA has antimicrobial activities against human immunodeficiency virus (HIV) (Aldunate et al, 2013), herpes simplex virus (Conti et al, 2009; Isaacs and Xu, 2013), Neisseria gonorrhoeae (Graver and Wade, 2011), Chlamydia trachomatis (Gong et al, 2014), and bacteria associated with bacterial vaginosis (BV) (O’Hanlon et al, 2011), and inhibits the infection of epithelial cells with Chlamydia trachomatis in vitro (Edwards et al, 2019)

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