Cathryn Nagler, Ph.D.

Committee on Immunology

University of Chicago

Prebiotics and food allergies

Workshop: Prebiotics – Quantifying impact on host health Probiota Americas, Chicago, IL May 31, 2016

Oral tolerance - physiological induction of

mucosal and systemic non-responsiveness to

dietary antigens

Why is the induction of tolerance to food failing

in increasing numbers of patients with food

allergies?

Antibiotic

use

Western

high fat,

low fiber

diet

Elimination of entero-

pathogens

(H. pylori,

helminths)

Vaccination/reduced

exposure to

infectious

disease

Caesarean

birth/formula

feeding

Alteration of

commensal

microbiota

“dysbiosis”

genetically

susceptible

individual

Inflammatory

Bowel

Disease

Obesity

Food Allergy

Diabetes

Autism

Asthma

Feehley et al Seminars in Immunopathology 2012, 34; 671

Dominguez-Bello, MG et al, Gastroenterology 2011, 140;1713

The gastrointestinal microbiota changes throughout life

A model of food allergy

Li et al JACI 2000, 106; 150

C3H/HeJ

Intragastric sensitization with peanut+ cholera toxin (CT)

D0 D2 D7 D14 D21 D28 D35 D36

Challenge

Sacrifice

Bashir et al J. Immunol. 2004, 172; 6978

TLR4 signaling influences susceptibility to allergic

responses to food

Does neonatal administration of oral antibiotics alter the

composition of the fecal microbiota?

Antibiotic cocktail:

kanamycin, gentamicin, colistin,

metronidazole, vancomycin

C57BL/6 mice

Daily antibiotic

gavage

Intragastric sensitization with peanut+cholera toxin (CT)

D0 D2 D7 D14 D21 D28 D35 D36 D-7

Weaning

Challenge

Sacrifice Dilute antibiotics in drinking water

Antibiotic treatment dramatically alters the composition of the

microbiota and increases sensitization to food

0.0

0.2

0.4

0.6

0.8

1.0

NT Abx S24-7

Prevotellaceae

Rickinellaceae

Bacteroidaceae

Others

Ruminococcaceae

Lachnospiraceae

Others

Lactobacillaceae

Others

Others

All Members

Others

Desulfovibrionales

All Members

All Members

Bacteroidetes

Firmicutes

Proteobacteria

Bacilli

Clostridia

Other Phyla/Unclassified Bacteria

Actinobacteria

Erysipelotrichi

NT Abx

Feces Ileal Contents

Ab

un

da

nce

Antibiotic treatment dramatically alters the composition of the

microbiota and increases sensitization to food

0.0

0.2

0.4

0.6

0.8

1.0

NT Abx S24-7

Prevotellaceae

Rickinellaceae

Bacteroidaceae

Others

Ruminococcaceae

Lachnospiraceae

Others

Lactobacillaceae

Others

Others

All Members

Others

Desulfovibrionales

All Members

All Members

Bacteroidetes

Firmicutes

Proteobacteria

Bacilli

Clostridia

Other Phyla/Unclassified Bacteria

Actinobacteria

Erysipelotrichi

NT Abx

Feces Ileal Contents

Ab

un

da

nce

9

10

11

12

13

NT Abx

***

NT Abx

Feces Ileal Contents

*****

*

Lo

g C

op

ies

16

S rR

NA

ge

ne

/ g

co

nte

nt

1 Clostridia

are resident bacteria

associated with the

apical mucosa

Bacteroides

are enriched among the

transient bacteria

associated with digesta

Nava & Stappenbeck 2011, Gut Microbes 2; 99

To identify allergy protective bacterial populations we

selectively colonized germ free mice with representatives of

the numerically predominant taxa in the murine colon

2

Selective colonization of gnotobiotic mice differentially

induces fecal IgA and colonic Foxp3+ Tregs

Stefka, Feehley et al PNAS 2014, 111; 13145

The composition of the colonizing commensal microbiota directs

protection against allergic responses to food

Stefka, Feehley et al PNAS 2014, 111; 13145

Clostridia colonization uniquely induces expression of a

subset of genes in the colonic epithelium

B. uniformis-induced

Clostridia-inducedCommon

GF B. uniformis Clostridia

Row Mean +3 SD-3 SD

Cyp2c55

Tmod4

Arg2

Reg3b

ProS1

Wfs1

Vegfc

Fbn1

Col16a1

Gadd45a

Pdk4

0

2

4

6

8Germ freeB. uniformisClostridia

Reg3b Reg3g

**

Re

lative

Exp

ressio

n

Stefka, Feehley et al PNAS 2014, 111; 13145

0

5

10

SmallIntestine

Colon

*****

Re

lativ

e e

xp

ressio

n Il

22

Expression of IL-22 is significantly increased upon

Clostridia colonization

GFB. uniformisClostridia

Stefka, Feehley et al PNAS 2014, 111; 13145

IL-22 is a barrier protective cytokine

Sabat, Ouyang & Wolk Nat Rev Drug Disc 2014, 13;21

Clostridia-induced IL-22 is necessary to reduce intestinal

permeability to food antigen

ELISA for Ara h 6

Arachis hypogaea

Pre 1 hr 3 hr0

20

40

60

80** ** **

Abx

AbxClostridiaAbx+IL-22Fc

NT

Ara

h 6

in

se

rum

(n

g/m

L)

C57BL/6 mice

Daily Abx gavage

D0 D6 D-7

Weaning

Challenge Clost. or IL-22Fc

Stefka, Feehley et al PNAS 2014, 111; 13145

Pre 1 hr 3 hr0

20

40

60

80

** ** **

Abx

AbxClostridiaAbx+IL-22Fc

NT

Ara

h 6

in

se

rum

(n

g/m

L)

Clostridia-induced IL-22 is necessary to reduce intestinal

permeability to food antigen

C57BL/6 mice

ELISA for Ara h 6

Arachis hypogaea

Daily Abx gavage

D0 D6 D-7

Weaning

Challenge Clostridia

Isotype or αIL-22

Pre 1 hr 3 hr0

20

40

60

80

100

*

AbxClostridia+isotype PN

AbxClostridia+aIL-22 PN

Ara

h 6

in

se

rum

(n

g/m

L)

0

5

10

SmallIntestine

Colon

*********

Germ freeB. uniformisBacteroides+AkkermansiaClostridia

Re

lativ

e e

xp

ressio

n Il2

2A consortium of Bacteroides+Akkermansia does not induce IL-

22 and enhances intestinal permeability to food antigen

Bacteroides+Akkermansia consortium (Elaine Hsiao, Cal Tech) 70% Bacteroides (uniformis, thetaiotaomicron, acidifaciens) , 25% A. muciniphila, 3% Parabacteroides

Pre 15 min 45 min0

10

20

30

1000

2000

3000 ***

Ara

h 2

in

se

rum

(n

g/m

L)

Pre 15 min 45 min0

20

40

60

80

100

2000

4000

6000

Germ free

Clostridia

B. uniformis

Bacteroides+Akkermansia

*

***

Ara

h 6

in

se

rum

(n

g/m

L)

Germ free C57BL/6

mice ELISA for Ara h 6/2

Arachis hypogaea

Colonization with B. uniformis or

consortia of Clostridia or Bacteroides

D0 D4 D6

RNA

Challenge

Peanut sensitization did not induce a Th2 skewed T cell or

antibody response in mice treated with anti-IL-22

C57BL/6 mice

Daily antibiotic

gavage Intragastric sensitization with peanut+cholera toxin (CT)

D0 D7 D14 D21 D28 D35 D36 D-7

Weaning/ Clostridia

Challenge

Sacrifice Clostridia

150µg i.p. αIL-22 (8E11, Genentech) or isotype control

Stefka, Feehley et al PNAS 2014, 111; 13145

Depletion of IL-22 primed for an IL-17 response and an

increase in peanut-specific IgG

C57BL/6 mice

Daily antibiotic

gavage Intragastric sensitization with peanut+cholera toxin (CT)

D0 D7 D14 D21 D28 D35 D36 D-7

Weaning/ Clostridia

Challenge

Sacrifice Clostridia

150µg i.p. αIL-22 (8E11, Genentech) or isotype control

Stefka, Feehley et al PNAS 2014, 111; 13145

Treatment with anti-IL-22 decreases expression of anti-microbial

peptides (RegIIIb) and increases Clostridia abundance

0.0

0.5

1.0

1.5

AbxClostridia+isotypeAbxClostridia+aIL-22

Small Intestine Colon

** *

Re

lativ

e e

xp

ressio

n R

eg

3b

360 7 14 21 280.0

0.1

0.2

0.3

AbxClostridia+aIL-22AbxClostridia+isotype

Days after weaning

Clo

strid

ale

s a

bu

nd

an

ce

0 7 14 21 28 360.0

0.2

0.4

0.6

0.8

1.0AbxClostridia+aIL-22

AbxClostridia+isotype

Days after weaning

Ba

cte

roid

ale

s a

bu

nd

an

ce

0.0

0.5

1.0

1.5

AbxClostridia+isotypeAbxClostridia+aIL-22

Small Intestine Colon

** *

Re

lativ

e e

xp

ressio

n R

eg

3b

The composition of the fecal microbiota is altered

in anti-IL-22 treated mice

-0.6 -0.4 -0.2 0.0 0.2-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

PC1 (12.7% of var. expl.)

PC

2 (

5.2

% o

f va

r. e

xp

l.)

AbxClostridia+isotype

AbxClostridia+aIL-22

Day 0 7 14 21 28 36

Stefka, Feehley et al PNAS 2014, 111; 13145

What are the mechanism(s) by which

Clostridia elicit a barrier protective

response?

Commensal bacteria ferment insoluble dietary fiber

to produce short chain fatty acids

Berni Canani, Gilbert & Nagler, Curr. Opin. Allergy Clin Immunol. 2015, 15; 243

Clostridia produce high levels of the short chain fatty acids acetate and butyrate

Acetate Propionate Butyrate0

2

4

6

8

10

20

40

60

80

GFConventionalizedClostridia

****

******

***

SC

FA

co

nce

ntra

tio

n

(µm

ol/g

fe

ce

s)

Butyrate, but not acetate, induces IL-22 expression and

reduces intestinal permeability

Ileum Colon0

2

4

6

8 GFButyrateAcetate *** ***

Re

lative

exp

ressio

n Il2

2

Pre 15 min 45 min0

50

100

150 GF

Butyrate

Acetate

Ara

h 6

in s

eru

m (n

g/m

L)

Germ free

C57BL/6

mice

Ara h 6 ELISA

Arachis hypogaea

Challenge

RNA

Butyrate or acetate

gavage

D1 D3 D4

0.0

0.2

0.4

0.6

0.8

1.0

Others

Other Actinobacteria

Other proteobacteria

Tenericutes

Other/Unclassified Firmicutes

Erysipelotrichi

Other Bacilli

Lactobacillaceae

Enterococcacea

Streptococeceae

Other Clostridia

Lachnospiraceae

Ruminococcaceae

Other Bacteroidetes

Bacteroidaceae

Rickinellaceae

Prevotellaceae

S24-7

Bacteroidetes

Firmicutes

Proteobacteria

Bacilli

Clostridia

Other Phyla/Unclassified Bacteria

Actinobacteria

Erysipelotrichi

Tenericutes

AIN-76A Potato StarchWheat Bran Rice Bran Mixed Starch Fiber Blend

Ab

un

da

nc

e

Pre 15 min 1 hr 3 hr0

100

200

300

500100015002000

AIN-76A

Potato Starch

Wheat Bran

Rice Bran

Mixed Starch

Fiber Blend

**

Ara

h 6

in

se

ru

m (n

g/m

L)

High fiber diets reduce intestinal permeability to food allergens

3 week old C3H/HeJ mice

Challenge

D1

Wean on to diet

D7 D14

Ad libitum feeding

Arachis hypogaea

ELISA for Ara h 2

Bruce Hamaker, Whistler Center for Carbohydrate Research, Purdue

Tolerance to dietary allergen requires the induction

of a bacteria-induced barrier protective response

Can microbiome-modulating therapeutics be

used to treat food allergy?

0

20

40

60

80

100

EHCF EHCF+LGG RHF SF AAF

% o

f ch

ildre

n a

cqu

irin

g t

ole

ran

ce

afte

r 1

2 m

on

ths

Berni Canani R, et al. The Journal of Pediatrics 2013, 163; 771

Supplementation of extensively hydrolyzed casein formula (EHCF)

with Lactobacillus GG accelerates acquisition of tolerance

in children with cow’s milk allergy

casein rice soy amino acids

P<0.001 P<0.001

P<0.001

P<0.001

Visit 1

Visit 2

6 months

• Full clinical evaluation

Visit 3

12 months

• Full clinical evaluation

• SPT and APT

• Oral food challenge (OFC)

• Full clinical evaluation

• SPT and APT

• Oral food challenge (OFC)

The cow’s milk allergic (CMA) infant microbiome exhibits

significantly increased diversity

Berni Canani, Sangwan, Stefka et al ISMEJ 2016, 10; 742

The composition of the fecal microbiota is altered in

cow’s milk allergic (CMA) infants

Berni Canani, Sangwan, Stefka et al ISMEJ 2016, 10; 742

Demographic variables do not explain differences in bacterial abundance

Lachnospiraceae−60 −20 0 20 40 60 80 120 160

Age of Solid Food

Introduction

Bodyweight

Exclusive Breastfeeding

Period

Age at Sampling

Allergy

Mode of Birth

Sex

*

Berni Canani, Sangwan, Stefka et al ISMEJ 2016, 10; 742

Treatment with EHCF plus LGG is associated with increased

levels of fecal butyric acid and enhanced tolerance to CMA

Tolerance to CMA

at 12 months

EHCF 0/7

EHCF + LGG 5/12*

Berni Canani, Sangwan, Stefka et al ISMEJ 2016, 10; 742

Genus level differential feature selection revealed a relationship between the abundance of bacterial strains, increased fetal butyrate concentation

and acquisition of tolerance

Berni Canani, Sangwan, Stefka et al ISMEJ 2016, 10; 742

food

IL-22

M. Velasquez-Manoff,

Nature 2015, 518: S4

Is sensitization to a food allergen increased in

mice colonized with an allergic infant microbiota?

Healthy infant

microbiota

Cow’s milk

allergic (CMA)

infant microbiota

Sensitize with b lactoglobulin (BLG)/CT

Non-allergic

Allergic

A “humanized” pre-clinical food allergy model to screen microbiome-modulating therapeutics

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70

-10

-8

-6

-4

-2

0

Healthy Colonized

Allergic Colonized

50mg BLGgavage

50mg BLGgavage

* * * * * *

†=3

Time (min.)

DT

°C

Healthy CMA0

10

20

30

40*

BL

G-s

pe

cifi

c Ig

E (n

g/m

L)

n=6 n=7

A “humanized” pre-clinical food allergy model to

screen microbiome-modulating therapeutics

Healthy infant

microbiota

Cow’s milk

allergic (CMA)

infant microbiota

Sensitize with b-lactoglobulin/CT

Non-allergic

Allergic

Healthy infant

microbiota

Cow’s milk

allergic (CMA)

infant microbiota

Sensitize with b-lactoglobulin/CT

Non-allergic

Allergic

• Candidate therapeutic bacterial species (or mixtures)

• “Pre-biotic” dietary fiber (to expand butyrate producing Clostridia)

• Encapsulated nanoformulations of butyrate

Acknowledgments

University of Chicago

Yang-Xin Fu

Aly Khan

Digestive Diseases Research Center

Eugene Chang

Betty Theriault

Dion Antonopoulos

Institute for Molecular Engineering

Jeff Hubbell

Argonne National Laboratories

Jack Gilbert

Naseer Sangwan

Taylor Feehley

Vince Guzzetta

Caitlin Yee

Emily Beltran

Songhua Quan

Pedro Belda-Ferre

Cat Plunkett

Andrew Stefka

Rosita Aitoro

Diana Bouhassira

Prabhanshu Tripathi

Goo-young Seo

Severine Cao

Stefano Guandalini

Roberto Berni Canani & team

University of Naples

Liang Zhou (Northwestern)

Wenjun Ouyang (Genentech)

Bruce Hamaker

Bin Zhang

(Purdue/Whistler Center for

Carbohydrate Research)

Sarkis Mazmanian (Cal Tech)

Elaine Hsiao (Cal Tech)

Kathy McCoy (University of

Bern)