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Microbiome and atopic

disorders: lessons
learned from the
biology of natural killer
T cells
Gnotobiotic model organisms and microbiome research:
Choices, challenges and proposed solutions
December 19, 2016
Richard S. Blumberg
rblumberg@partners.org

Protective microbial factors or lack thereof:


the hygiene (or microbial) hypothesis
First coined by David P. Strachan (Br Med J 1989) to
explain the relationship between household size, birth
order and hay fever in a birth cohort during one week in
1958 and followed for 23 years
Extended by J.-F. Bach to include autoimmune diseases
(N Engl J Med 2002)
Inverse relation between asthma and growing up on a
farm correlated with exposure to a wider range of
microbes than children in the reference group (Ege, N
Engl J Med 2011)

Association between antibiotic use in first year of life and pediatric IBD (Shaw,
Am J Gastroenterol 2010)

Experimental proof of causation requires


gnotobiotic animal models to establish
that specific exposures in early life affect
susceptibility in later life
Trigger

Macpherson AJ &
McCoy KD, Muc Immunol
2015

Early Life

Later Life
Normal
phenotype
& response

Environmental
Exposure
(specific factors?)

Abnormal
phenotype
& response

Invariant natural killer T (iNKT) cells

iNKT cells recognize host and


microbial lipid antigens presented
by the MHC class I-like molecule
CD1d

NKT cells are early innate-like


responders that activate other
innate and adaptive immune cells

There are two types of NKT:


those that possess a semiinvariant
TCR
chain
(galactosylceramide),
so-called
iNKT, and those that possess a
diverse group of TCR chains
van Kaer, Nat. Rev. Immunol. 2004

CD1d dependent regulation of bacterial


colonization in the intestines and lung of
mice
Lung colonization of
SPF mice with
Pseudomonas aeruginosa

Gut monocolonization of
germ-free mice

Cd1d-/- WT
(also L. gasseri, S. aureus, P. aeruginosa)
Nieuwenhuis EES, Nature Med 2002

Nieuwenhuis EES, J Clin Invest 2009

CD1d and NKT cells are involved in the


pathogenesis of IBD
Mouse:
Oxazolone-induced colitis is CD1d-dependent (Cd1d-/-) and
NKT-mediated (Ja18-/-) (Heller, Immunity 2002)
Involves innate (IL-1), Th1 (IFN) and Th2 (IL-4, IL-13)
cytokines (Boirivant, JEM 1998; Iijima JEM 2004)
Type 2 noninvariant NKT cells can cause spontaneous
colitis in transgenic mice (Liao, Gastroenterology 2012)
Human:
Increased NKT cells in ulcerative colitis that exhibit CD1drestricted IL-13 production (Fuss, J Clin Invest 2005)
NKT cells are enriched in expression of genes associated
with IBD risk loci (Jostins, Nature 2012)

Early lifetime, but not later-life, exposure to


microbiota protects germ-free mice from high
iNKT cell infiltration in colon and lung
colon iNKT cells
iNKT cells (%)

1.5
1
0.5

GF/a: microbial exposure


in adulthood
GF/n: microbial exposure
as neonate

P < 0.05

Torsten
Olszak

Olszak T, Science 2012

Germ-free mice are highly susceptible to


colitis associated with triggers that
activate iNKT cells: normalization of
susceptibility only if microbial
programming occurs in early life
survival (%)

body weight loss


(% of initial)

80
60

GF/n
GF/a
GF

40
20
0

10
8

110

day

100
90
80
70
0

2
3
day

Mean pathology
score

100

6
4
2
0

*P, <0.01 **P, <0.001

Oxazolone-induced

colitis

iNKT cells in the current model of asthma

Iwamura, Curr Opin Immunol 2010


Umetsu, Nat Med 2003

Early life exposure to microbiota protects


GF mice from allergic asthma

1
0.5
0

GF/n

GF/a

GF/a: microbial exposure


in adulthood
GF/n: microbial exposure
as neonate

central airway resistance

eosinophils

0.30

15

0.25
0.20
0.15
0

GF/n

GF/a

(% of total cells)

1.5

Rn (cmH2O.s/mL)

(% of live
lymphocytes)

lung iNKT cells

10
5
0

GF/n GF/a

p < 0.05
p < 0.01

GF mice show increased levels of the


chemokine ligand CXCL16 due to
microbe induced epigenetic modification
of the Cxcl16 gene
colonic CXCL16

serum CXCL16

400
200
0

SPF GF GF/a GF/n

(fold increase)

(fold increase)

(pg/ml)

600

6
4
2
0

SPF GF GF/a GF/n

GF

CXCL16

lung CXCL16

SPF

2.5
2
1.5
1
0.5
0

SPF GF GF/a GF/n

GF

SPF

100
80
60
40
20
0

Weight (% of initial)

Survival (%)

B6 SPF mice germ-reduced by antibiotics


in early life exhibit increased CD1d- and
iNKT- dependent oxazolone colitis

2
3
Days

100
90
80
70

CD1d-/- (sulfatrim)
Ja18-/- (sulfatrim)
B6
B6 (sulfatrim)
P < 0.05

2
3
Days

Sphingolipid biosynthetic pathway


SPT: Serine
palmotyltransferase

3-Ketosphinganine

Sphinganine

Dihydroceramide

A B. fragilis SPT mutant was constructed


that does not produce any sphingolipid.

Dingding
An

B. fragilis sphingolipids regulate host iNKT


cell homeostasis in colon

SPF

GF

BFWT BFSPT

An,Cell2014

Only early-life colonization of GF mice with B.


fragilis rescues iNKT cell number and colitis
phenotype

B. fragilis produce a glycosphingolipid


molecule, GSL-Ff717, that inhibits iNKT cell
proliferation in early life
OH OH

OH OH

O
OH

HN
HO
O

OH
C16(OH)H32
C15(OH)H30

GSL-Bf717

Inhibition

HO
O

C25H51
OH

HN

C14H29
OH

KRN7000

Activation

GSL-Bf717 represents a new class of microbial


immunomodulatory molecules
Sungwhan Oh

Microbiota regulate iNKT during early life and


later life susceptibility to colitis and asthma

Gensollen , Science 2016

Pathways demonstrating temporal restriction


of immune development during early life
IgE

Cahenzli J, Cell Host Microbe 2013

Treg (skin and lungs)

Scharschmidt T, Immunity 2015


Gollwitzer ES, Nature Med 2014

Also, intestinal epithelial cell tolerance to TLR (Chasin C, Cell Host Microbe 2010) and
CD71+ erythrocyte regulation of myeloid responses to microbes (Elahi S, Nature 2013)

Summary
Vision
Atopic as well as numerous complex diseases will be shown to
originate from inappropriate microbial exposures during early life
through pathways that are developmentally regulated and thus
only amenable to manipulation in early life

Future
Identification of the developmentally regulated pathways and
their determinant microbial factors as well as the later life
environmental triggers that activate the pathophysiologic
responses leading to disease

Opportunities
Early life manipulation of the pathways involved or later life
elimination of the stimulating factors might allow for the
prevention and/or treatment of atopic and complex diseases

Challenges
Confirming the existence of these temporally restricted pathways
in humans and defining methods to prevent and/or treat disease

Eosinophilic Esophagitis
EoE during
years 1-5 of life
associated with
antibiotics in
year 1 (Radano
MC, JACI 2014)

Identification of
a diseasespecific EoE
tissue
transcriptome
characterized
by upregulation
of eotaxin-3,
periostin, IL-13
(Blanchard R,
JACI 2007)
Rothenberg ME, Gastroenterology 2009

Failure to induce experimental EoE with corn or peanut


derived allergens in Cd1d-/- mice
Identifies a role for NKT cells in experimental EoE

iNKT cell pathway is increased in


EoE in an age dependent manner

Relative expression (

Ct)

LTC4S
64

**

32

***

16
8
4
2
1
0.5

1-6 y

EoE

6-18 y

1-18 y

Non-EoE

Lexmond W, Am J Gastro 2014, Clin Exp Immunol 2013

Successful treatment of early-onset EoE with


allergen elimination associated with
normalization of iNKT cell pathway

EoE
Response

Eotaxin-3
105
104

** **

NS
NS

103
102
101
100
10-1

EoE
Response

ct)

Periostin
105
104

** **

NS
NS

103
102
101
100

10-1

EoE
Response

Non
EoE

50

EoE
Response
ct)

100

Relative expression (

**
**
* NS **

Non
EoE

32
16

NS
NS
NS

8
4
2
1
0.5

EoE
Response

Non
EoE

64

LTC4S
Relative expression (

ct)
Relative expression (

Esophageal eosinophilia

* *

NS
NS

EoE
Response

150

30
10

Non
EoE

ct)

EoE
Response

10-1

Relative expression (

100

Non
EoE

Tissue eosinophil count /hpf

0.5

Non
EoE

101

*NS *

40

Non
EoE

*NS *

NS

102

Relative expression (

ct)

NS

16

ct)
Relative expression (

V24

CD1d

CXCL16

Acknowledgments
Torsten Olszak
Miguel Pinilla Vera
Rebecca Baron
Shankar Iyer
Thomas Gensollen
(Brigham and Women's Hospital,
Harvard Medical School)
Sebastian Zeissig
(Technical University of Dresden)
Julia Richter
Andre Franke
(Christian-Albrechts University Kiel )

Dennis Kasper
Dingding An
Sungwhan Oh
(Channing Laboratory,
Harvard Medical School)

Edda Fiebiger
Willem Lexmond
(Childrens Hospital,
Harvard Medical School)