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

How do lactobacilli protect against HIV?

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


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


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


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


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


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-

*****


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-

++ ++

***


IL-6, TNF, RANTES, IL-8


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


Lactic acid is Anti-inflammatory in a CervicovaginalTissue Model

Little change in IL-1β

ApicalBasal

No toxicity MTT, TEER


3 h LA (pH 3.9) apically, Wash, incubate for 18 h


pH 3.9


Mechanism?

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


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

rial d

ivers

ity

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

vaginal lactic acid inhibits production of pro...

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