Rapid gut epithelial repair at the enteroendocrine-immune intersection through microbiota-mediated rescue of tryptophan

metabolism during chronic HIV/SIV infection

4th International Workshop on Microbiome in HIV Pathogenesis, Prevention and Treatment

October 16-17, 2018Maryland, USA

Clarissa RochaDepartment of Medical Microbiology and Immunology

University of California, Davis

Front. Microbiol. 2015 Nov 18;6:1285

GUT: multi-tasking and highly functional organ

• Digestion and nutrient absorption

• Barrier to protect the internal gut microenvironment

• Protection against pathogens

• Largest immune organ in the human body

• Sense and response

• Extended metabolic capacities

• Epithelial barrier integrity

• Exclusion of pathogens

• Modulation of host metabolism

• Maturation and modulation of the immune system

GUT MICROBIOTA

GI tract: unique compartment where the nervous, endocrine and mucosal immune systems converge

Cell Mol Gastroenterol Hepatol 2018;6:133–148

Tryptophan (TRP) is a crucial mediator of the immune and endocrine system interactions

* HIV/SIV: HAND and altered TRP metabolism

HIV and the GUTMajor site for early HIV replication, dissemination and persistence

• Severe CD4+ T cell depletion

• Disruption of gut epithelial

barriers -> leaky gut

• Microbial translocation

• Gut inflammation and

impaired mucosal immunity

• Disturbed host-microbe

interactionIleum 2.5 days after SIV infection

SIV- SIV+

Can SIV infection associated gut epithelium damage be reversed/repaired at the intersection of commensal microbes?

L. plantarum (LP)B. Infantis (BI)

5 hoursIHCHost gene expressionLuminal content metabolomicsSIV uninfected

SIV infected(10 weeks)

• Ileum is tied off into 4 – 6 cm loops with intervening 1 cm spacer loops

• Loops allow for multiple bacterial species to be tested in the same animal

• Capture in vivo gut mucosal response in the natural setting

• Anaerobic microenvironment of the gut

• Bacteria can be incubated for up to 8 hours (animal still alive)

• Detect rapid changes in the gut epithelium for repair

The ligated ileal loop model

ART therapy does not repair epithelial barrier integrity in chronic SIV infection

Su

m F

luo

rescen

ce In

ten

sit

y/

Vo

lum

e (

m3)

SIV- SIV+ SIV+ART0

5,000

10,000

15,000

20,000

P=0.0010

P=0.0009

N.S.

SIV- SIV+

SIV+ ART

IleumZO-1DAPI

Chronic SIV infection

ZO-1

Exposure to LP and BI repairs gut epithelial barrier within 5 hours independent of CD4+ T cells recovery

0

5

1040

45

50

55

60

% C

D4T

cell (

LP

Ls)

SIV-

SIV+

SIV+LP+

SIV+BI+

SIV- SIV+

SIV+ LP+ SIV+ BI+

ZO-1DAPIIleum

-5 -4 -3 -2 -1 0 1 2 3 4

Tec Kinase Signaling

Role of NFAT in Regulation of the Immune Response

IL-8 Signaling

CD28 Signaling in T Helper Cells

ERK/MAPK Signaling

G Beta Gamma Signaling

NF-κB Signaling

Integrin Signaling

Phospholipase C Signaling

Fcγ Receptor-mediated Phagocytosis in Macrophages and …

PTEN Signaling

CXCR4 Signaling

Production of Nitric Oxide and Reactive Oxygen Species in…

Th1 Pathway

ILK Signaling

Acute Phase Response Signaling

PI3K Signaling in B Lymphocytes

PI3K/AKT Signaling

mTOR Signaling

Growth Hormone Signaling

Signaling by Rho Family GTPases

PPARα/RXRα Activation

Inflammasome pathway

SIV+BI+ SIV+LP+ SIV+Activation Z-score

LP and BI dampened inflammatory pathways in the gut mucosa during chronic SIV infection

How do commensals rapidly dampened inflammation and repair gut barrier during chronic SIV infection?

Metabolic profiling of gut luminal contents from chronically SIV infected animals following the exposure to LP and BI

FC p ≤ 0.05q ≤ 0.25

± 1.5

Enteric TRP metabolism is altered during chronic SIV infection

Tryptophan (TRP)

Kynurenine (KYN) Serotonin (5-HT)

TPH1IDO1

Xanthunerate (XA)Kynurenate (KA)N-formylanthranilic acid (NFAA)Picolinate(PA)Quinolinate (QA)

5-hydroxyindoleacetate (5-HIAA)

SIV- SIV+0.0

0.5

1.0

1.5

2.0

2.5

Scale

d in

ten

sit

y

TRP

SIV- SIV+0.0

0.5

1.0

1.5

Scale

d in

ten

sit

y

KYN

SIV- SIV+0

1

2

3

Scale

d in

ten

sit

y5-HIAA

***

MAOAKATSKYNUKNYU3HAO

XA KA NFAA PA QA0

5

10

1520

30

40

50

Scale

d in

ten

sit

y

SIV-

SIV+

**

*

*****

***

Accumulation of 5-HT and KYN metabolites during SIV infection

Increased IDO1 expression in the gut

SIV- SIV+0.000

0.005

0.010

0.015

IDO

1 m

RN

A

(Rela

tive t

o G

AP

DH

)

p < 0.0001

HIV infection is associated with increased IDO1 expression linked to the loss of Th17 cells and gut barrier disruption

Enteric TRP metabolism is altered during chronic SIV infection

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SIV- SIV+0.0

0.2

0.4

0.6

0.8

MA

OA

mR

NA

(Rela

tive t

o G

AP

DH

)p = 0.3

SIV- SIV+ 0

1

2

3

TP

H1

(FC

rela

tive t

o G

AD

PH

)

p = 0.03

5-HT

SIV- SIV+

No significant differences in the expression of TPH1 and MAOA

SIV- SIV+0

50

100

150

5-H

T n

g/ g

tis

su

e

p = 0.0008

No significant differences in EC cells numbers

Intestinal mucosal 5-HT is increased during chronic SIV infection due to decreased SERT-mediated 5-HT uptake

Serotonin

EC cells: major producers of 5-HT in the gut

SIV- SIV+ no Tx SIV+ Tx0.000

0.005

0.010

0.015

0.020

SE

RT

mR

NA

(Rela

tive t

o G

AP

DH

)

p = 0.0003

p = 0.001

Pre 2wk PI 10wk PI0

10

20

30

LP

L C

D4+

SE

RT

+ (

%) SIV+

SIV-

*

Pre 2wk PI 10wk PI0

5

10

15

20

LP

L C

D8+

SE

RT

+ (

%) SIV+

SIV-

*

5000 10000 15000

-15

-10

-5

0

5

Pearson r = -0.5299P = 0.0422

Chronic Viral Load(SIV RNA copies/ug

Total RNA)

SE

RT

Exp

ressio

n

Rela

tive t

o U

nin

fecte

d

2 4 6 8 10

-8

-6

-4

-2

0

2

Pearson r = -0.6232P = 0.0405

IFN (pg/ml serum)S

ER

T E

xp

ressio

n

Rela

tive t

o U

nin

fecte

d500 1000 1500

-8

-6

-4

-2

0

2

Pearson r = -0.6276P = 0.0217

MCP1 (pg/ml serum)

SE

RT

Exp

ressio

n

Rela

tive t

o U

nin

fecte

d

Intestinal mucosal 5-HT is increased during chronic SIV infection due to decreased SERT-mediated 5-HT reuptake

Decreased SERT expression correlates with biomarkers of SIV disease progression

• Serotonin reuptake transporter (SERT)• Removes intraluminal serotonin

LP and BI shift the metabolism of TRP towards Indolelactate (ILA) biosynthesis during SIV infection

SIV+ vs. SIV - SIV+ LP vs. SIV+ SIV+BI vs. SIV+

LP and BI shift the metabolism of TRP towards Indolelactate (ILA) biosynthesis during SIV infection

SIV

+ L

P v

s.

SIV

+

SIV

+ B

I vs.

SIV

+

-3

-2

-1

0

IDO

1 F

old

Ch

an

ge

p = 0.02

p = 0.3

SIV- SIV+ SIV+ LP SIV+ BI0.0

0.5

1.0

1.5

2.0

5

10

15

ILA

(S

cale

d in

ten

sit

y)

p = 0.008

p < 0.0001

SIV

LP

BI

5−hydroxyindoleacetate

kynurenate

N−formylanthranilic acid

xanthurenate

quinolinate

picolinate

3−indoxyl sulfate −2

−1

0

1

2

3

4

SIV

+ vs

. SIV

-

SIV

+ LP

vs.

SIV

+

SIV

+ B

I vs.

SIV

+

Increased levels of ILA (indolic acid derivative of TRP)Lower levels of KYN metabolites Decreased IDO1 expression

DMSO ILA 50uM0

5

10

15

CD

4+

IL17+

(%

)

DMSO ILA 50uM0

2

4

6

CD

8+

IL17+

(%

)

DMSO ILA 50uM0

5

10

15

CD

4+

CD

8+

IL17+

(%

)

DMSO ILA 50uM0

10

20

30

40

CD

4+

IL22+

(%

)

p = 0.06

DMSO ILA 50uM0

10

20

30

40

50

CD

4+

CD

8+

IL22+

(%

) p = 0.03

DMSO ILA 50uM0

2

4

6

8

10

CD

8+

IL22+

(%

)

Immune modulation effects of ILA

ILA induce the production of IL-22 by CD4+CD8+ LPLs isolated from the gut

Increased expression of IL-22 related genes in vivo after LP and BI exposure

PROPOSED MODEL

SUMMARY

• LP and BI rapidly restores (5 hours) SIV-induced gut barrier damage and dampens inflammation during SIV infection

• LP and BI rescue the TRP metabolism from SIV-mediated effects and drive towards ILA biosynthesis

• Rapid gut barrier repair through enteric immune-epithelial-commensal axis

• Damaged gut epithelium in SIV infection can be rapidly restored by leveraging commensal metabolites to modulate immune-epithelial interactions

• Novel targets for repair and protection of the gut immunity in HIV disease

Acknowledgements

Dandekar LAB

Satya Dandekar

Guochun Jiang

Katti Horng

Mariana Weber

Shuang Hu

Juan Arredondo

Sam Sankaran-Walters

Irina Grishina

Lauren Hirao

Chris Gaulke

Marco LabMaria MarcoBen Golomb

CNPRCJeff RobertsWilhelm Von MorgenlandLaurie BrignoloKari ChristieStaff

NIH/NIAIDRISECHRP

Cecilia GiuliviIan McHardy