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
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)