Vaginal lactic acid inhibits production of pro-inflammatory mediators from human cervicovaginal epithelial cells associated with HIV acquisition Prof Gilda Tachedjian 3 rd International Workshop on Microbiome in HIV Pathogenesis, Prevention and Treatment
Vaginal Microbiome Affects HIV Risk in Young Women in Sub-Saharan Africa Young women in South Africa are up to 8 times more likely to be infected with HIV compared to young men Subclinical Genital Inflammation Vaginal Microbiome - high load and diversity of anaerobic bacteria (Prevotella) is associated with genital inflammation and an increased risk of HIV acquisition in contrast to - vaginal microbiota dominated by Lactobacillus spp. (L. crispatus) associated with lack of genital inflammation and lower risk of HIV acquisition Passmore et al 2016 Curr Opin HIV AIDS 11:156 Arnold et al Mucosal Immunology Cohen 2016 Science 353:6297 Masson et al 2015 Clin Infect Dis 61: 260 Anahtar et al 2015 Immunity 42: 965 Gosmann et al 2017 Immunity 46:1
How do lactobacilli protect against HIV?
Protective Vaginal Microbiota Produces more Lactic Acid Acid ph <4.5 ph 4.5 Lactobacillus (mm) High diversity (BV) (mm) Lactic ~120 1,2 20 4 Acetic 2-4 3 120 1, 3 Propionic <1 2-4 3 Butyric <1 2-4 3 Succinic <1 4 20 4 L. crispatus acidifies vagina to lower ph 5 Does lactic acid have a protective role in the FRT? 1 Gajer, 2012 STM 4:132ra52; 2 O Hanlon, 2013 PLoS One 8:e80074; 3 Mirmonsef, 2011/2012 Am J Reprod Imm. 65(3):190-5 and 67(5):391-400 4 Al-Mushrif, 2000, J Med Microbiol, 49 (11); 1023-30 5Ravel et al., 2011 PNAS 108:4680
Lactic Acid (LA): Protonated form has Antimicrobial, Antiviral and Immunomodulatory Activities In the vagina 1.0 ± 0.2% DL-LA ph 3.5 ± 0.3 1 Uncharged LA (but not H 2 O 2 ) Bactericidal BV-associated bacteria but not vaginal lactobacilli 2 HIV virucidal 3 ex vivo tissues 4 Inactivates N. gonorrhoeae 5, Chlamydia trachomatis 6, HSV 7 Uncharged LA mediates anti-inflammatory effects cervicovaginal epithelial cells that could protect against HIV + Uncharged form Low ph Lactic acid pka 3.89 Neutral ph Lactate anion Charged form 1 O Hanlon et al 2011 BMC Infect Dis 11; 2 O Hanlon et al 2013 PLoS One 8:e80074; 3 Aldunate et al 2013 J Antimicrob Chemother; 4 Palomino et al 2017 Front Microbiol 8 907 5 Graver and Wade 2011 Clin Microbiol Antimicob 10:8; 6 Gong et al 2014 PLoS ONE 9:e107758; 7 Isaacs et al 2013 Antimicrob Agents Chemother 57: 3806
Cervicovaginal Epithelial Cells Provide both Physical and Immunological Barriers in the FRT Physical Barriers Mucous Ciliary clearance Epithelial cells Immune Defense Epithelial cells respond to MAMPs/PAMPs: immune mediators: antiviral and proinflammatory (paradoxically promote HIV infection in women) Endocervix Ectocervix and Vagina (apparent preference) Microbiota Competition, bacteriocins, organic acid metabolites (i.e. lactic acid a major protective factor produced by vaginal lactobacilli) Shattock and Moore 2003 Nat Micro Reviews Carias et al 2013 J Virol 87: 11388 Wira et al 2011 Am J Rep Immunol Stieh et al 2014 Plos Path 10:e1004440
Vaginal Bacteria Regulate Epithelial Innate Immunity in a Strain/Isolate Specific Manner Lactobacilli sp (e.g. L crispatus) largely noninflammatory while BV-associated bacteria (e.g. Atopobium vaginae, Prevotella amnii) trigger a proinflammatory cytokine response from FRT epithelial cells 1,2,3,4 Lactobacilli but not BV-associated bacteria dampen TLR agonist response by reducing proinflammatory cytokines elicited by FRT epithelial cells 5,6 Lactobacilli: anti-inflammatory effect Does lactic acid contribute to this anti-inflammatory effect? 1 Libby et al 2008 Microbes Infect 10:439; 2 Fichorova et al 2011 mbio; 3 Doerlinger et al 2014 JID; 4 Anahtar et al 2015 Immunity; 5 Rose et al 2012 PLoS ONE; Kyongo et al 2012 Plos ONE 7:e43951
Experimental System for Evaluating Immune Modulatory Effects of Lactic acid Human Epithelial Cells: Vaginal: VK2/E6E7 Ectocervix: Ect1/E6E7 Endocervix: End1/E6E7 Primary ectocervical cells Organotypic cervicovaginal tissue model Add LA ± TLR agonist apically SFKM: TLR1/2 [Pam(3)CSK(4)] (HIV gp120, BV) TLR3 (polyic - PIC) TLR4 (LPS) (HIV gp120, BV) Soluble immune mediators relevant to HIV infection: cytokine bead array/luminex Hearps et al Mucosal Immunol 2017 10: 1480
% of untreated cells Cervicovaginal Epithelial Cells Viable in 0.3% Lactic Acid ph 3.9 TEER (Wcm 2 ) 125 100 Cell Viability VK2 Cells End Cells Ect cells Monolayer integrity 75 50 25 1000 Transepithelial electrical resistance (TEER) - Ect cells 0 0.3% L-LA 0.3% D-LA 0.3% nl-la ph 3.9 (HCl) 800 600 400 200 0 Untr. 0.3% L-LA 0.3% D-LA 0.3% ph 3.9 nl-la (HCl) Citric acid Cells pre-exposed to CVF Hearps et al Mucosal Immunol 2017 10: 1480
0.3% L-Lactic Acid ph 3.9 Elicits an Anti-inflammatory Effect on Cervicovaginal Epithelial Cells Anti-inflammatory Cytokine Similar increase in IL-1RA with if add LA in presence of TLR agonists: polyic LPS Pam3C Lactic acid elicits the production of IL-1RA from FRT epithelial cells in the absence or presence of TLR agonists Hearps et al Mucosal Immunol 2017 10: 1480
0.3% L-Lactic Acid ph 3.9 Protects Against TLR-agonist Mediated Inflammation Pro-inflammatory Cytokine Pro-inflammatory Chemokine n 4 Similar effects IL-8, TNF, RANTES Hearps et al Mucosal Immunol 2017 10: 1480
pg/ml Low ph Alone does not Reproduce Lactic Acid s Anti-inflammatory Effects pg/ml Anti-inflammatory Cytokine Pro-inflammatory Chemokine 160000 120000 Ect cells - IL-1RA ** 2000 1500 Ect cells - MIP3a ** 80000 1000 40000 500 +++ 0 Untr HCl - +LA +HCl 0 Untr HCl - +LA +HCl + PIC + PIC n 5 Pretreated with 0.3% LA ph3.9 or HCl ph 3.9 for 1 h Washed cells, add PIC, cytokines 18 h Hearps et al Mucosal Immunol 2017 10: 1480
pg/ml pg/ml pg/ml L-LA and D-LA Inhibit TLR-Agonist Inflammatory Responses from Epithelial Cells: Role of Protonated LA Anti-inflammatory Cytokine Pro-inflammatory Chemokine Ect cells - IL-1RA Ect cells - MIP3a 50000 40000 *** ** *** 500 400 *** 3000 30000 ** 300 2000 20000 10000 *** ** 200 100 ++ ++ 1000 Acid: 0 - L-LA D-LA nl-la - L-LA D-LA nl-la + PIC Acid: 0 0 - L-LA D-LA nl-la L-LA D-LA nl-la - + PIC n 4 IL-6, TNF, RANTES, IL-8 Hearps et al Mucosal Immunol 2017 10: 1480
L-LA elicits anti-inflammatory effect on Ectocervical Epithelial cells pre-treated with CVF and in presence of SP pg/ml Fold change vs untreated Cervicovaginal Fluid Seminal Plasma 40000 CVF-exposed Ect cells - IL-6 **** 2.5 2.0 SP-exposed Ect Cells - IL-8 ** + 30000 1.5 20000 1.0 10000 ++++ 0.5 0 Untr - +LA +PIC +CVF +PIC +LA 0.0 Untreated 10% SP 10% SP + LA CVF 4 h, then add LA+/- PIC 10% SP +/- LA 12h, wash, 18-24 h Hearps et al Mucosal Immunol 2017 10: 1480
Lactic acid is Anti-inflammatory in a Cervicovaginal Tissue Model Anti-inflammatory Cytokine Pro-inflammatory Chemokine Little change in IL-1β No toxicity MTT, TEER ph 3.9 Apical Basal 3 h LA (ph 3.9) apically, Wash, incubate for 18 h Hearps et al Mucosal Immunol 2017 10: 1480
Mechanism?
L-LA inhibits pro-inflammatory immune mediator production at a stage prior to gene transcription 1 h treatment, media removed, additional 4 h incubation, qrt-pcr - mrna n 4 Investigating NF-KB pathways Hearps et al Mucosal Immunol 2017 10: 1480
RNA-Seq Distinct Gene Expression Profile for Lactic acid vs HCl relative to PolyIC (DEGUST) Log2 Fold-Change Relative to PIC Parallel coordinates plot FDR <0.05 Log FC 0.5 Studying lactic acid specific effects on FRT epithelial cells that could potentially provide protection against HIV and other STIs
Summary Lactic acid dampens inflammation Anti-inflammatory Cytokines: IL-1RA Pro-inflammatory Cytokines: IL-6, TNF-α Chemokines: IL-8, MIP-3α, RANTES LA s immune modulatory effect mediated by protonated form and by both the L and D stereoisomers LA s effect not simply a low ph effect on cells LA pretreatment inhibits pro-inflammatory mediators elicited by TLR agonists LA s anti-inflammatory effect observed in the presence of genital secretions
Conclusion Lactic acid elicits the production of a proinflammatory cytokine and inhibits production of inflammatory cytokines and chemokines associated with HIV transmission These data might explain in part the HIV protective properties of LA producing lactobacilli, along with LA s bactericidal activity against BV-associated bacteria and potent HIV virucidal activity Lactic acid and/or lactic acid producing Lactobacillus spp. could potentially be used to reduce the risk of HIV infection as an adjunct to antiretroviral-based PrEP 1. LA treatment as little as 0.5 1 h elicits anti-inflammatory response 2. LA pretreatment able to protect subsequent inflammatory challenge 3. LA protects against a range of inflammatory stimuli: TLR agonists, TNF, SP
Lactobacillus spp. Vaginal Environment and Impact on HIV Susceptibility High bacterial diversity HIV HIV Aldunate et al 2015 Frontiers in Physiology 6:164
Acknowledgments Tachedjian Lab Burnet Institute Raffi Gugasyan Thomas Moench Richard Cone Johns Hopkins Univ Muriel Aldunate David Delgado Diaz Daniela Srbinovski David Tyssen Lois Bayigga Deborah Anderson Boston University Monash Bioinformatics Platform David Powell and team Anna Hearps Joshua Hayward