Vaginal lactic acid inhibits production of pro...
Transcript of Vaginal lactic acid inhibits production of pro...
Vaginal lactic acid inhibits production of
pro-inflammatory mediators from human cervicovaginalepithelial cells associated with HIV acquisition
Prof Gilda Tachedjian
3rd International Workshop on Microbiome in HIV Pathogenesis, Prevention and Treatment
Vaginal Microbiome Affects HIV Risk in Young Women in Sub-Saharan Africa
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
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
Young women in South Africa are up to8 times more likely to be infected with HIVcompared to young men
pH <4.5 pH ≥4.5
Protective Vaginal Microbiota Produces more Lactic Acid
Does lactic acid have a protective role in the FRT?
Acid Lactobacillus
(mM)
High diversity
(BV) (mM)
Lactic ~1201,2 ≤204
Acetic 2-43 ≤1201, 3
Propionic <1 2-43
Butyric <1 2-43
Succinic <14 ≤204
1Gajer, 2012 STM 4:132ra52; 2O’Hanlon, 2013 PLoS One 8:e80074;3 Mirmonsef, 2011/2012 Am J Reprod Imm. 65(3):190-5 and 67(5):391-4004Al-Mushrif, 2000, J Med Microbiol, 49 (11); 1023-30 5Ravel et al., 2011 PNAS 108:4680
L. crispatus acidifies vagina to lower pH5
Lactic Acid (LA): Protonated form has Antimicrobial,
Antiviral and Immunomodulatory Activities
Low pH
Lactic acid
Neutral pH
Lactate anion
pKa 3.89
▪ In the vagina 1.0 ± 0.2% DL-LA pH 3.5 ± 0.31
+
1O’Hanlon et al 2011 BMC Infect Dis 11; 2O’Hanlon et al 2013 PLoS One 8:e80074; 3Aldunate et al 2013 J Antimicrob
Chemother; 4Palomino et al 2017 Front Microbiol 8 907 5Graver and Wade 2011 Clin Microbiol Antimicob 10:8; 6Gong et al 2014 PLoS ONE 9:e107758; 7Isaacs et al 2013 Antimicrob Agents Chemother 57: 3806
▪ Uncharged LA mediates anti-inflammatory effects cervicovaginal epithelial cells that could protect against HIV
▪ Uncharged LA (but not H2O2) Bactericidal BV-associated bacteria but not vaginal lactobacilli2
HIV virucidal3 ex vivo tissues4
▪ Inactivates N. gonorrhoeae5, Chlamydia trachomatis6, HSV7
Uncharged form
Charged form
Cervicovaginal Epithelial Cells Provide both Physical and Immunological Barriers in the FRT
Endocervix Ectocervix and Vagina(apparent preference)
Shattock and Moore 2003 Nat Micro Reviews Carias et al 2013 J Virol 87: 11388
Physical BarriersMucousCiliary clearanceEpithelial cells
Immune Defense Epithelial cells respond to MAMPs/PAMPs: immune mediators: antiviral and pro-inflammatory (paradoxically promote HIV infection in women)
MicrobiotaCompetition, bacteriocins, organic acid metabolites (i.e. lactic acid – a major protective factor produced by vaginal lactobacilli)
Wira et al 2011 Am J Rep Immunol Stieh et al 2014 Plos Path 10:e1004440
1Libby et al 2008 Microbes Infect 10:439; 2Fichorova et al 2011 mBio; 3Doerlinger et al 2014 JID;4Anahtar et al 2015 Immunity; 5Rose et al 2012 PLoS ONE; Kyongo et al 2012 Plos ONE 7:e43951
Vaginal Bacteria Regulate Epithelial Innate Immunity in a Strain/Isolate Specific Manner
• Lactobacilli sp (e.g. L crispatus) – largely non-inflammatory while BV-associated bacteria (e.g. Atopobium vaginae, Prevotella amnii) trigger a proinflammatory cytokine response from FRT epithelial cells1,2,3,4
Does lactic acid contribute to this anti-inflammatory effect?
• Lactobacilli but not BV-associated bacteria dampen TLR agonist response by reducing pro-inflammatory cytokines elicited by FRT epithelial cells5,6
• Lactobacilli: anti-inflammatory effect
Experimental System for Evaluating Immune Modulatory Effects of Lactic acid
Add LA ± TLR agonist apically SFKM:
TLR1/2 [Pam(3)CSK(4)] (HIV gp120, BV)TLR3 (polyIC - PIC)TLR4 (LPS) (HIV gp120, BV)
Human Epithelial Cells:
Vaginal: VK2/E6E7Ectocervix: Ect1/E6E7Endocervix: End1/E6E7
Primary ectocervical cells Organotypic cervicovaginal tissue model
Soluble immune mediators relevant to HIV infection: cytokine bead array/luminex
Hearps et al Mucosal Immunol 2017 10: 1480
Cervicovaginal Epithelial Cells Viable in 0.3% Lactic Acid pH 3.9
0
25
50
75
100
125
Cell Viability
% o
f u
ntr
eate
d c
ells
VK2 Cells
End Cells
Ect cells
0.3%
L-LA
0.3%
D-LA
0.3%
nL-LA
pH 3.9
(HCl)
0
200
400
600
800
1000
Transepithelial electrical resistance
(TEER) - Ect cells
TE
ER
(W
cm
2)
0.3% L-LA
0.3% D-LA
0.3% nL-LA
pH 3.9 (HCl)
Untr.Citric acid
Monolayer integrity
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
Similar increasein IL-1RA with if add LA in presence of TLR agonists:polyICLPSPam3C
Anti-inflammatory Cytokine
Lactic acid elicits the production of IL-1RA from FRT epithelial cellsin 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
n≥ 4
Similar effects IL-8, TNF, RANTES
Pro-inflammatory Cytokine Pro-inflammatory Chemokine
Hearps et al Mucosal Immunol 2017 10: 1480
Low pH Alone does not Reproduce Lactic Acid’s Anti-inflammatory Effects
n≥ 5
0
40000
80000
120000
160000
Ect cells - IL-1RA
pg
/ml
Untr - +LA +HCl
+ PIC
**
HCl
Anti-inflammatory Cytokine
0
500
1000
1500
2000Ect cells - MIP3a
pg
/ml
**
+++
Untr - +LA +HCl
+ PIC
HCl
Pro-inflammatory Chemokine
Pretreated with 0.3% LA pH3.9 or HCl pH 3.9 for 1 hWashed cells, add PIC, cytokines 18 h
Hearps et al Mucosal Immunol 2017 10: 1480
L-LA and D-LA Inhibit TLR-Agonist Inflammatory Responses from Epithelial Cells: Role of Protonated LA
n≥ 4
0
10000
20000
30000
40000
50000
Ect cells - IL-1RA
pg
/ml
*****
***
**
+ PIC
Acid: L-LA D-LA nL-LA- L-LA D-LA nL-LA-
*****
Anti-inflammatory Cytokine
0
100
200
300
400
500
0
1000
2000
3000
Ect cells - MIP3a
pg
/ml pg
/ml
+ PIC
Acid: L-LA D-LA nL-LA- L-LA D-LA nL-LA-
++ ++
***
Pro-inflammatory Chemokine
IL-6, TNF, RANTES, IL-8
Hearps et al Mucosal Immunol 2017 10: 1480
L-LA elicits anti-inflammatory effect on EctocervicalEpithelial cells pre-treated with CVF and in presence of SP
0
10000
20000
30000
40000
CVF-exposed Ect cells - IL-6
pg
/mL
****
++++
Untr -
+CVF
+PIC+LA+PIC+LA
CVF – 4 h, then add LA+/- PIC
Cervicovaginal Fluid
Untreated 10% SP 10% SP + LA0.0
0.5
1.0
1.5
2.0
2.5
SP-exposed Ect Cells - IL-8
Fo
ld c
han
ge v
s u
ntr
eate
d ** +
10% SP +/- LA 12h, wash, 18-24 h
Seminal Plasma
Hearps et al Mucosal Immunol 2017 10: 1480
Lactic acid is Anti-inflammatory in a CervicovaginalTissue Model
Little change in IL-1β
ApicalBasal
No toxicity MTT, TEER
Anti-inflammatory Cytokine
3 h LA (pH 3.9) apically, Wash, incubate for 18 h
Pro-inflammatory Chemokine
pH 3.9
Hearps et al Mucosal Immunol 2017 10: 1480
n≥ 4
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
• 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)
FDR <0.05Log FC 0.5
Log2 F
old
-Change
Parallel coordinates plot
Relative to PIC
Studying lactic acid specific effects on FRT epithelial cells that could potentially provide protection against HIV and other STIs
Summary – Lactic acid dampens inflammation
• Pro-inflammatory Cytokines: IL-6, TNF-α ⬇
• Anti-inflammatory Cytokines: IL-1RA ⬆
• 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
Lactic acid elicits the production of a proinflammatory cytokine and inhibits production of inflammatory cytokines and chemokines associated with HIV transmission
Conclusion
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 virucidalactivity
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 PrEP1. LA treatment as little as 0.5 – 1 h elicits anti-inflammatory response
2. LA pretreatment able to protect subsequent inflammatory challenge3. LA protects against a range of inflammatory stimuli: TLR agonists, TNF, SP
Vaginal Environment and Impact on HIV SusceptibilityLacto
bacillu
s s
pp.
Hig
h b
acte
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HIVHIV
Aldunate et al 2015 Frontiers in Physiology 6:164
Tachedjian Lab
Johns Hopkins Univ
Burnet Institute
Acknowledgments
Boston University
Muriel Aldunate
David Tyssen
Anna Hearps
Raffi Gugasyan
David Delgado Diaz
Lois Bayigga
ThomasMoench
RichardCone
Deborah Anderson
Monash Bioinformatics Platform
David Powell and team
Daniela Srbinovski
Joshua Hayward