healthy guts exclude oxygen - virology education...

Healthy guts exclude oxygen

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

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Control of microbes by our

immune systemFighting off

bad bugs

Balancing our

microbial self

Immune

system

What is gut homeostasis?

Gut microbiota of humans

What is a balanced microbial community?

Immune

system

Balancing our

microbial self

Microbial community structure in the intestine

Gut microbiota of humans Gut microbiota of mice

Obligate anaerobes(Bacteroidetes and

Firmicutes)

Fiber

Short-chain fatty

acids

Dysbiosis: shift from obligate to facultative anaerobes

Gut microbiota of humans Gut microbiota of mice


Firmicutes)

Dysbiosis

Facultative anaerobes(Proteobacteria)



Firmicutes)

Dysbiosis


Lupp et al., 2007

Stecher et al., 2007

Barman et al., 2008

Chemically-induced colitis

Salmonella infection

Frank et al., 2007 Inflammatory

bowel disease

(IBD)

High-fat

diet

Necrotizing

enterocolitis

(NEC)Toxoplasma

infection

Genetically-induced colitis

Raetz et al., 2013

Molloy et al. 2013

Haag et al. 2012

Lupp et al., 2007

Garrett et al., 2010

Martinez-Medina

et al., 2014

Irritable

bowel syndrome

(IBS)

Colorectal cancer

Krogius-Kurikka et

al. 2009

Carroll et al., 2012

Arthur et al., 2012

Antibiotics

Bohnhoff et al., 1954

Saito, 1961

Mai et al., 2011

Normann et al., 2013

Citrobacter

infectionLupp et al., 2007

HIV

enteropathyMutlu et al., 2014



Firmicutes)

Dysbiosis


Lupp et al., 2007

Stecher et al., 2007

Barman et al., 2008



Frank et al., 2007 Inflammatory

bowel disease

(IBD)

High-fat

diet

Necrotizing

enterocolitis

(NEC)Toxoplasma

infection


Raetz et al., 2013

Molloy et al. 2013

Haag et al. 2012

Lupp et al., 2007

Garrett et al., 2010

Martinez-Medina

et al., 2014

Irritable

bowel syndrome

(IBS)

Colorectal cancer

Krogius-Kurikka et

al. 2009

Carroll et al., 2012

Arthur et al., 2012

Antibiotics

Bohnhoff et al., 1954

Saito, 1961

Mai et al., 2011

Normann et al., 2013

Citrobacter

infectionLupp et al., 2007

Is there a common driver for gut dysbiosis?

HIV




Firmicutes)

Dysbiosis





Inflammatory

bowel disease

(IBD)

High-fat

diet

Necrotizing

enterocolitis

(NEC)Toxoplasma

infection


Irritable

bowel syndrome

(IBS)

Colorectal cancer

Antibiotics

Citrobacter

infection

HIV


Crypt

Clostridia

Butyrate

Complex

carbohydrates

Undifferentiated

colonocyte

Mature

colonocyte

H2O

H2O

Blood vessel

O2

O2

CO2

Streptomycin102

103

104

105

106

107

108

109

1010

“co

lon

iza

tio

n

resis

tance”

Mock

treatment

E. coli Nissle 1917

CF

U/g

co

lon

co

nte

nt

Streptomycin

(single dose)

Antibiotics drive a luminal expansion of Enterobacteriaceae

Mariana

Byndloss

Crypt

Clostridia

Butyrate

Complex

carbohydrates

Undifferentiated

colonocyte

Mature

colonocyte

H2O

H2O

Blood vessel

O2

O2

CO2

1011

1012

1013

Day 3 after

strep

**

Eub

acte

ria

(16S

rR

NA

gene

cop

ies/

g co

lon

cont

ents

)

Strep: - +

Streptomycin102

103

104

105

106

107

108

109

1010

“co

lon

iza

tio

n

resis

tance”

Mock

treatment

E. coli Nissle 1917

CF

U/g

co

lon

co

nte

nt

Streptomycin

(single dose)

Antibiotics drive a luminal expansion of Enterobacteriaceae

2017 Science 357: 570

Mariana

Byndloss

Streptomycin

(single dose)

108

109

1010

1011

1012

Clo

str

idia

(16S

rR

NA

copi

es/g

col

on c

onte

nts)

Strep:

**

- +

Day 3 after

strep

0

20

40

60

80

100

Rel

ativ

e ab

unda

nce

of

Clo

strid

ia fa

mili

es (

%)

Strep: - +

Day 3 after

strep

Clostridiaceae

Peptostreptococcaceae

Veillonellaceae

Ruminococcaceae

LachnospiraceaeOther:

Butyrate producers:

1011

1012

1013

Day 3 after

strep

**

Eub

acte

ria

(16S

rR

NA

gene

cop

ies/

g co

lon

cont

ents

)

Strep: - +

Crypt

Undifferentiated

colonocyte

Mature

colonocyte

H2O

H2O

Blood vessel

O2

O2

CO2

Butyrate

Complex

carbohydrates

Clostridia

Antibiotics

X

Streptomycin depletes butyrate-producing Clostridia

2017 Science 357: 570

Mariana

Byndloss

108

109

1010

1011

1012

Clo

str

idia

(16S

rR

NA

copi

es/g

col

on c

onte

nts)

Strep:

**

- +

Day 3 after

strep

0

20

40

60

80

100

Rel

ativ

e ab

unda

nce

of

Clo

strid

ia fa

mili

es (

%)

Strep: - +

Day 3 after

strep

Clostridiaceae


Veillonellaceae

Ruminococcaceae


Butyrate producers:

Crypt

Undifferentiated

colonocyte

Mature

colonocyte

H2O

H2O

Blood vessel

O2

O2

CO2

Butyrate

Complex

carbohydrates

Clostridia

Antibiotics

X

X

Streptomycin lowers butyrate levels

Cec

albu

tyra

te (μm

ol/g

)

Strep:

0.01

0.1

10

1

**

- +

Day 3 after

strep

2017 Science 357: 570

Mariana

Byndloss

108

109

1010

1011

1012

Clo

str

idia

(16S

rR

NA

copi

es/g

col

on c

onte

nts)

Strep:

**

- +

Day 3 after

strep

0

20

40

60

80

100

Rel

ativ

e ab

unda

nce

of

Clo

strid

ia fa

mili

es (

%)

Strep: - +

Day 3 after

strep

Clostridiaceae


Veillonellaceae

Ruminococcaceae


Butyrate producers:

Crypt

Undifferentiated

colonocyte

Mature

colonocyte

H2O

H2O

Blood vessel

O2

O2

CO2

Butyrate

Complex

carbohydrates

Clostridia

Antibiotics

X

X


Cec

albu

tyra

te (μm

ol/g

)

Strep:

0.01

0.1

10

1

**

- +

Day 3 after

strep

O2

O2

CO2

Butyrate

Butyrate

ATPADP

Na+

Na+

Na+

Crypt

Undifferentiated

colonocyte

Mature

colonocyte

H2O

H2O

Blood vessel

O2

O2

CO2

Butyrate

Complex

carbohydrates

Clostridia

Antibiotics

X

X


O2

O2

CO2

Butyrate

Butyrate

ATPADP

Na+

Na+

Na+

Mock-treated

Lumen Mucosa

Hypoxia

(< 1% oxygen)

10%

1%

0.1%

O2

grad

ient

Fabian Rivera-Chavez

2016 CH&M 19:443

Crypt

Blood vessel

10%

1%

0.1%

O2

grad

ient

O2

O2

Butyrate

Complex

carbohydrates

Clostridia

O2

H2O

H2O

X

X

Antibiotics

Mock-treated

Lumen Mucosa

Strep-treated

LumenMucosa

Hypoxia

(< 1% oxygen)

Streptomycin increases epithelial oxygenation

O2

O2

O2

Glucose

Glucose

Lactate

ATP

ADP

Fabian Rivera-Chavez

2016 CH&M 19:443

Crypt

Blood vessel

10%

1%

0.1%

O2

grad

ient

O2

O2

Butyrate

Complex

carbohydrates

Clostridia

O2

H2O

H2O

X

X

Antibiotics

Mock-treated

Lumen Mucosa

Lumen

Mucosa

Strep-treated +

tributyrinStrep-treated

LumenMucosa

Hypoxia

(< 1% oxygen)

Crypt

Undifferentiated

colonocyte

Mature

colonocyte

H2O

H2O

Blood vessel

O2

O2

CO2

Butyrate

Complex

carbohydrates

Clostridia

Antibiotics

XTributyrin

Streptomycin increases epithelial oxygenation

10%

1%

0.1%

O2

grad

ient

2016 CH&M 19:443

Crypt

Blood vessel

10%

1%

0.1%

O2

grad

ient

O2

O2

Butyrate

Complex

carbohydrates

Clostridia

O2

H2O

H2O

X

X

Antibiotics Oxygen drives an expansion of Escherichia coli

2017 Science 357: 570

cydA cydB Cytochrome bd oxidase

(microaerobic conditions)

E. coli

Nissle 1917

cydAB

mutant

E. coli

?

Crypt

Blood vessel

10%

1%

0.1%

O2

grad

ient

O2

O2

Butyrate

Complex

carbohydrates

Clostridia

O2

H2O

H2O

X

X

Antibiotics Oxygen drives an expansion of Escherichia coli

Strep: - +

CI i

n co

lon

cont

ents

(E. c

oli w

tvs.

cyd

AB

)

0

2

4

6

8

10

12

2017 Science 357: 570



E. coli

Nissle 1917

cydAB

mutant

E. coli

cydAB

Crypt

Blood vessel

10%

1%

0.1%

O2

grad

ient

O2

O2

Butyrate

Complex

carbohydrates

Clostridia

O2

H2O

H2O

X

X

Antibiotics

Crypt

Undifferentiated

colonocyte

Mature

colonocyte

H2O

H2O

Blood vessel

O2

O2

CO2

Butyrate

Complex

carbohydrates

Clostridia

Antibiotics

XTributyrin

10%

1%

0.1%

O2

grad

ient

2017 Science 357: 570

Strep:Tributyrin:

--

+-

++

CI i

n co

lon

cont

ents

(E. c

oli w

tvs.

cyd

AB

)

0

2

4

6

8

10

12



E. coli

Nissle 1917

cydAB

mutant

Oxygen drives an expansion of Escherichia coli

Crypt

Blood vessel

10%

1%

0.1%

O2

grad

ient

O2

O2

Butyrate

Complex

carbohydrates

Clostridia

O2

H2O

H2O

X

X

Antibiotics

Crypt

Undifferentiated

colonocyte

Mature

colonocyte

H2O

H2O

Blood vessel

O2

O2

CO2

Butyrate

Complex

carbohydrates

Clostridia

Antibiotics

Tributyrin

10%

1%

0.1%

O2

grad

ient

2017 Science 357: 570

Strep:Tributyrin:

C17:

---

+--

++-

+-+

CI i

n co

lon

cont

ents

(E. c

oli w

tvs.

cyd

AB

)

0

2

4

6

8

10

12



E. coli

Nissle 1917

cydAB

mutant

Oxygen drives an expansion of Escherichia coliC17

Dysbiosis Salmonella infectionChemically-induced colitis

Inflammatory

bowel disease

(IBD)

High-fat

diet

Necrotizing

enterocolitis

(NEC)Toxoplasma

infection


Irritable

bowel syndrome

(IBS)

Colorectal cancer

Antibiotics

O2

Citrobacter

Infection

✔


Mariana Byndloss

HIV

enteropathy

Dysbiosis Salmonella infection

O2


Inflammatory

bowel disease

(IBD)

High-fat

diet

Necrotizing

enterocolitis

(NEC)Toxoplasma

infection


Irritable

bowel syndrome

(IBS)

Colorectal cancer

Antibiotics

O2

Citrobacter

Infection

✔

✔


Fabian

Rivera-Chavez

Mariana Byndloss

HIV

enteropathy


O2


Inflammatory

bowel disease

(IBD)

High-fat

diet

Necrotizing

enterocolitis

(NEC)Toxoplasma

infection


Irritable

bowel syndrome

(IBS)

Colorectal cancer

Antibiotics

O2

Citrobacter

Infection

O2✔

✔

✔


Christopher Lopez

Fabian

Rivera-Chavez

Mariana Byndloss

HIV

enteropathy

Oxygen as a driver of gut dysbiosis:

What can we learn about homeostasis?


O2


O2

Inflammatory

bowel disease

(IBD)

High-fat

diet

Necrotizing

enterocolitis

(NEC)Toxoplasma

infection


O2

Irritable

bowel syndrome

(IBS)

Colorectal cancer

Antibiotics

O2

Citrobacter

Infection

O2✔

✔

✔✔

✔Sebastian E Winter

UT Southwestern

Medical Center

Christopher Lopez

Fabian

Rivera-Chavez

Mariana Byndloss

HIV

enteropathy

Peroxisome proliferator-activated receptor gamma (PPAR-γ)

PPAR-γ is mainly expressed in adipose tissue, macrophages and epithelial cells in the colon (colonocytes)

M1 macrophage

Nos2

IFN-γ

D-glucose pyruvate

O2

CO2

lactate

Anaerobic glycolysis

(Warburg effect)

iNOS

PPAR-γβ-oxidation

Nos2

STAT6

O2

CO2

Fatty

acids

M2 macrophage

O2

CO2

O2

β-oxidation

PPAR-γ

Clostridia

Butyrate

Complex

carbohydrates

WT Pparg

Moc

k

2017 Science 357: 570

Butyrate depeltion and inflammation cooperate to increase epithelial oxygenation

Pparg

(Ppargfl/flVillincre/- mice)

WT

(Ppargfl/flVillin-/- mice)

O2

O2

Inflammation


Glucose Lactate

O2

Clostridia

Butyrate

Complex

carbohydrates

PPAR-γXWT Pparg

1% D

SS

Moc

k

E. coliE. coli cydAB

1:1

E. coli cydAB

1:1

100

101

102

103WT Pparg

CI co

lon

co

nte

nts

(E. coli

wtvs.

cydA

B)

1% DSS: - +

Butyrate depeltion and inflammation cooperate to increase epithelial oxygenation

DSS 2017 Science 357: 570

Pparg

(Ppargfl/flVillincre/- mice)

WT

(Ppargfl/flVillin-/- mice)

O2

CO2

O2


β-oxidation

PPAR-γ

SCFAs

Clostridia

Butyrate

Complex

carbohydrates

How does antibiotic treatment generate an inflammatory signal?

Cec

alco

ncen

trat

ion

(μmol

/g)

Strep: - +

**

**

0.001

0.01

0.1

1

10

100

Acetate

Propionate

ADPATP

2017 Science 357: 570

Tregs

Inflammation 0

10

20

30

40

50

60

Strep: - +

CD

3+-e

nric

hed

CD

4+F

OX

P3+

cells

(% o

f tot

al C

D3+

-enr

iche

d co

loni

c ce

lls)

*

O2

CO2

O2


β-oxidation

PPAR-γ

SCFAs

Clostridia

Butyrate

Complex

carbohydrates


Tregs

Inflammation 0

10

20

30

40

50

60

Strep: - +

CD

3+-e

nric

hed

CD

4+F

OX

P3+

cells

(% o

f tot

al C

D3+

-enr

iche

d co

loni

c ce

lls)

*

0

10

20

30

40

50

Isotype

control

anti-

CD25C

D3+

-enr

iche

d C

D4+

FO

XP

3+ce

lls

(% o

f tot

al C

D3+

-enr

iche

d co

loni

c ce

lls)

*C

ecal

conc

entr

atio

n

(μmol

/g)

Strep: - +

**

**

0.001

0.01

0.1

1

10

100

Acetate

Propionate

ADPATP

2017 Science 357: 570

Streptomycin

O2

O2

Tregs

Inflammation


SCFAs Butyrate

Clostridia

Glucose Lactate

Enterobacteriaceae

X

XXO2

CI c

olon

con

tent

s

(wild

type

vs.

aer

obic

res

pira

tion

defic

ient

)

0

5

10

15

20

25

Isotype

control

anti-

CD25

WT Pparg

**

0

10

20

30

40

50

Isotype

control

anti-

CD25C

D3+

-enr

iche

d C

D4+

FO

XP

3+ce

lls

(% o

f tot

al C

D3+

-enr

iche

d co

loni

c ce

lls)

*


0

10

20

30

40

50

60

Strep: - +

CD

3+-e

nric

hed

CD

4+F

OX

P3+

cells

(% o

f tot

al C

D3+

-enr

iche

d co

loni

c ce

lls)

*

Cec

alco

ncen

trat

ion

(μmol

/g)

Strep: - +

**

**

0.001

0.01

0.1

1

10

100

Acetate

Propionate

Streptomycin

O2

O2

Tregs

Inflammation


SCFAs Butyrate

Clostridia

Glucose Lactate

Enterobacteriaceae

X

XXO2

CI c

olon

con

tent

s

(wild

type

vs. a

erob

ic r

espi

ratio

n de

ficie

nt)

0

5

10

15

20

25

Isotype

control

anti-

CD25

WT Pparg

**

1000-fold

10-fold

Colonization

resistance

NO3-

NO

NO3-

Nitrate

iNOS

ROS

Nos2

NO2-

Nitrite

narKnarGHJInarUnarZYWVnapFDAGHBC

Respiration drives post-antibiotic expansion of Enterobacteriaceae

?

Nos2

iNOS

Col

onoc

yte

Nos

2 m

RN

A

(fol

d-ch

ange

)

0

2

4

6

8*

Strep: - +

Day 3 after

strep

Streptomycin

O2

O2

Tregs

Inflammation


SCFAs Butyrate

Clostridia

Glucose Lactate

Enterobacteriaceae

X

XXO2

CI c

olon

con

tent

s

(wild

type

vs. a

erob

ic r

espi

ratio

n de

ficie

nt)

0

5

10

15

20

25

Isotype

control

anti-

CD25

WT Pparg

**

Respiration drives post-antibiotic expansion of Enterobacteriaceae

1000-fold

10-fold

?

Nos2

iNOS

NO3-

102

103

104

100

101

***

WT Pparg +

anti-CD25

CI in

fe

ce

s

(E. co

li w

tvs

.cyd

AB

napA

narG

narZ

) 105

E. coli cydAB

napA

narG

narZ

What is gut homeostasis?

Immune

system

Balancing our

microbial self

Nos2O2

CO2

O2


β-oxidation

PPAR-γ

SCFAsButyrate

Tregs

Inflammation

ADPATP

PPAR-γ signalling and Tregscooperate

to maintain gut homeostasis

anaerobiosis

Virtuous

PPAR-γ

cycle

Balancing our

microbial self

Immune

system

Can we remediate dysbiosis by reinstating epithelial control to limit the

flow of oxygen into the gut lumen?

Inflammatory

bowel disease

(IBD)Ulcerative colitis

DSS-induced colitis5-aminosalicylic acid

(5-ASA)

DSS induces colitis by triggering ER stressDSS

ER stress

DSS-induced colitis5-aminosalicylic acid

(5-ASA)O2

O2

CO2

Butyrate

Butyrate

ATPADP

Na+

Na+

Na+

O2

O2

Glucose

Glucose

Lactate

ATP

ADP

O2

Undifferentiated

colonocyte

10%

1%

0.1%O

2g

radie

nt

Exc

essi

ve in

test

inal

epith

elia

l rep

air

resp

onse

Co

lon

ic c

ryp

t h

yp

erp

lasia

DSS

ER stressDSS induces colonic crypt hyperplasia

5-aminosalicylic acid

(5-ASA)

O2

O2

Blood vessel

Undifferentiated

colonocyte

10%

1%

0.1%O

2g

radie

nt

Co

lon

ic c

ryp

t h

yp

erp

lasia

DSS

ER stress


(5-ASA)

Mock 2.5% DSSE

xces

sive

inte

stin

al

epith

elia

l rep

air

resp

onse

DSS increases epithelial oxygenation

E. coli

102

103

104

105

106

107

Mock 2.5% DSSE

. col

i in

colo

n co

nten

ts

CF

U/g

)

O2

Stephanie Cevallos

O2

O2

Blood vessel

Undifferentiated

colonocyte

10%

1%

0.1%O

2g

radie

nt

Co

lon

ic c

ryp

t h

yp

erp

lasia

DSS

ER stress


(5-ASA)

Mock 2.5% DSSE

xces

sive

inte

stin

al

epith

elia

l rep

air

resp

onse

5-ASA restores epithelial hypoxia

E. coli

2.5% DSS +

5-ASA

102

103

104

105

106

107

Mock 2.5% DSSE

. col

i in

colo

n co

nten

ts

CF

U/g

)

Control 5-ASA

O2

O2O2

O2

Blood vessel

Undifferentiated

colonocyte

10%

1%

0.1%O

2g

radie

nt

Co

lon

ic c

ryp

t h

yp

erp

lasia

E. coliNR

cydAB

iNOS

NO3-

DSS

ER stress


(5-ASA)

Exc

essi

ve in

test

inal

epith

elia

l rep

air

resp

onse

102

103

104

105

106

107

Mock 2.5% DSSE

. col

i in

colo

n co

nten

ts

CF

U/g

)

Control 5-ASA


DSS-induced colitis

E. coli cydAB

napA

narG

narZ

O2

O2

Blood vessel

Undifferentiated

colonocyte

10%

1%

0.1%O

2g

radie

nt

Co

lon

ic c

ryp

t h

yp

erp

lasia

E. coli

DSS

ER stress


(5-ASA)

Exc

essi

ve in

test

inal

epith

elia

l rep

air

resp

onse

102

103

104

105

106

107

Mock 2.5% DSSE

. col

i in

colo

n co

nten

ts

CF

U/g

)

Control 5-ASA


DSS-induced colitis

E. coli cydAB

napA

narG

narZ

1

10

100

1000

10000

102

103

101

1Mock DSS

CI i

n fe

ces

(wtv

s.cy

dAB

napA

narG

narZ

)

WT

DSS

Pparg

104

iNOS

NO3- O2

ControlNR

cydAB5-ASA

Nos2O2

CO2

O2


β-oxidation

PPAR-γ

SCFAsButyrate

Tregs

Inflammation

ADPATP

Gut homeostasis

anaerobiosis

Can we remediate dysbiosis by

reinstating epithelial control to

limit the flow of oxygen into the

gut lumen?

Nos2

O2

O2

Tregs

Inflammation


SCFAs Butyrate

Glucose Lactate

iNOS

XX

Nos2O2

CO2

O2


β-oxidation

PPAR-γ

SCFAsButyrate

Tregs

Inflammation

ADPATP

Gut homeostasis

Gut dysbiosis

anaerobiosis




gut lumen?


(5-ASA)

Gut dysbiosis: a microbial signature of

epithelial dysfunction

Nos2

O2

O2

Tregs

Inflammation


SCFAs Butyrate

Glucose Lactate

iNOS

XX

Nos2O2

CO2

O2


β-oxidation

PPAR-γ

SCFAsButyrate

Tregs

Inflammation

ADPATP

Gut homeostasis

Gut dysbiosis

anaerobiosis




gut lumen?


(5-ASA)

Torsten

Sterzenbach

Univ. Osnabrück,

Germany

Thank you

Collaborators

Renée Tsolis, Garry Adams,

Volkmar Heinrich, Charles

Bevins, Carlito Lebrilla, Oliver

Fiehn, Stephen McSorley,

Valley Stewart, John Roth

Helene

Andrews-

Polymenis

Texas A&M University

Sebastian E Winter

UT Southwestern

Medical Center

Robert A Kingsley

Quadram Institute,

UK

Çagla Tükel

Temple University

Manuela Raffatellu

UC San Diego

Marijke

Keestra-Gounder

University of Colorado

Denver

Tracy Nicholson

USDA, Ames

Robert Crawford

CSU Sacramanto

Parameth

Thiennimitr

Chiang

Mai Univ.

Thailand

Takeshi Haneda

Kitasato Univ.,

Japan

Renato Santos

UFMG, Brazil

Franziska Faber

Univ. Würzburg,

Germany

Christopher

Lopez,

Vanderbilt

Fabian

Rivera-Chavez,

Harvard

healthy guts exclude oxygen - virology education...

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