Megan Raetz, Sun -hee Hwang, Cara Wilhelm, Donna Kirkland ... · Supplementary Figure 2. Different...

Supplementary Information

Parasite-induced Th1 cells and intestinal dysbiosis cooperate in IFN--dependent

elimination of Paneth cells

Megan Raetz, Sun-hee Hwang, Cara Wilhelm, Donna Kirkland, Alicia Benson, Carolyn Sturge,

Julie Mirpuri, Shipra Vaishnava, Baidong Hou, Anthony L. DeFranco, Christopher J Gilpin,

Lora V. Hooper, Felix Yarovinsky

Nature Immunology doi:10.1038/ni.2508

0 3 5 7 1 0 1 4

0 .0

0 .5

1 .0

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2 .0

Fo

ld c

ha

ng

e

0 3 5 7 1 0 1 4

1 0 -2

1 0 -1

1 0 0

1 0 1

1 0 2

1 0 3

1 0 4

1 0 5

Fo

ld c

ha

ng

e

Firmicutes

Supplementary Figure 1

c d Proteobacteria/Bacteroidetes

(ratio)

Proteobacteria

(total)

Bacteroidetes

(total)

16

S r

RN

A c

op

ies

/lu

me

n

0 3 5 7 1 0 1 4

1 0 1

1 0 2

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1 0 1 0

16

S r

RN

A c

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ies

/lu

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n

0 3 5 7 1 0 1 4

1 0 1

1 0 2

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1 0 7

1 0 8

1 0 9

1 0 1 0

NS

** **

NS NS

*

*

NS

NS

NS

a b

Time after infection (d) Time after infection (d)


Supplementary Figure 1. Mucosal responses to T. gondii result in severe intestinal dysbiosis

caused by the expansion of Proteobacteria and loss of Bacteroidetes. qRT-PCR analysis of the

absolute (a-b) and the relative amounts (c-d) of Proteobacteria, Bacteroidetes, and Firmicutes in

the lumens of the small intestines of WT mice infected orally with 20 cysts per mouse of the

ME49 strain. * P< 0.05; ** P< 0.001. The results are representative of at least ten independent

experiments.


naive T. gondii infected

(day 7 post infection)


naive T. gondii infected

(day 7 post infection)

T. gondii infected

naive

b

a c Nature Immunology doi:10.1038/ni.2508

Supplementary Figure 2. Different structures of intestinal lumen microbiota in naïve and

T. gondii infected mice. (a) The number of operational taxonomic units (OTUs) in naïve and T.

gondii infected mice (Day 7) is based on 3% divergence, and the number of sequences

considered per sample is standardized. The observations from the first comparison are shown.

(b) Taxonomic representation of statistically and biologically consistent differences between

naïve and T. gondii infected luminal bacteria. Differences are represented by the color of the

most abundant class (Red indicating luminal bacteria in naïve mice, and green intestinal luminal

bacteria in T. gondii infected mice). The diameter of each circle is proportional to the taxon’s

abundance. Taxa are color coded according the group that they are associated with, and group

associations with higher taxonomic levels are illustrated using a shaded region. Taxonomic

levels lower than classes are labeled with a letter, and then listed to the right. (c) Biplot of the

principle coordinates analysis (PCoA) based on weighted UniFrac metrics. Ellipses represent the

95% confidence interval around group centroids, and arrows indicate the direction and relative

magnitude of the correlation of Escherichia and Shigella to PCoA axes.


0 7 0 7

0

2 0

4 0

6 0

8 0

1 0 0

c u ltu re -b a s e d

q P C R -b a s e d

% E

nte

rob

ac

teri

ac

ea

e

naive naive Infected, day 7 Infected, day 7 Infected, day 7 a

c

Supplementary Figure 3 B

acte

ria

per

g fe

ces

b *

109

1010

1011

1012

1013 **

**


naiv

e

Infe

cte

d, d

ay 7

Enterobacteriaceae Eubacteria SYTO62 d


Supplementary Figure 3. Mucosal responses to T. gondii result in dysbiosis caused by the

expansion of Enterobacteriaceae. (a) The growth of intestinal bacteria on CHROMagar (top) or

blood agar (bottom) plates isolated from the lumens of the small intestines of naïve and T.

gondii-infected (d7) mice (aerobic conditions). The results shown are for two naïve (left) and

three infected (right) mice. (b) Quantifications of growth of intestinal bacteria on blood agar

isolated from the lumens of the small intestines of naïve and T. gondii-infected (d7) mice under

anaerobic conditions. (c) Identification of bacteria grown on the blood agar plates

(Supplementary Fig. 3a) accomplished using culture-based (re-growth on the selective

CHROMagar plates) or qRT-PCR techniques. * P< 0.05; ** P< 0.01. (d) The distribution of

Proteobacteria (Enterobacteriaceae) in the lumens of the small intestines was analyzed by in

situ hybridization with Enterobacteriaceae -specific (green) and Eubacteria-specific (red) probes

in naive (left panel) or infected orally (right panel) mice on day 7 post infection. SYTO-62 (blue)

was used for counterstaining. The results are representative of at least ten independent

experiments.


naiv

e (

day 0

)

naiv

e (

day 3

)

naiv

e (

day 5

)

naiv

e (

day 7

)

naiv

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day 1

0)

naiv

e (

day 1

4)

0

2

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6

8

1 0

Fo

ld c

ha

ng

e

naiv

e (

day 0

)

naiv

e (

day 3

)

naiv

e (

day 5

)

naiv

e (

day 7

)

naiv

e (

day 1

0)

naiv

e (

day 1

4)

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

Fo

ld c

ha

ng

e


Bacteroidetes Proteobacteria

Co-housed with T. gondii

infected mice

Co-housed with T. gondii

infected mice

Supplementary Figure 4. T. gondii infection triggers dysbiosis which is not transmissible to

naïve mice. Naïve WT mice were co-housed with the T. gondii-infected mice for the indicated

periods of time, and the relative amounts of Proteobacteria and Bacteroidetes loads in the

lumens of the small intestines were measured by qRT-PCR. The results are representative of

three independent experiments. Please note that T. gondii cysts are found predominantly in the

central nervous system and muscle tissue of mice. T. gondii is not present in feces of affected

mice, and the parasite can be transmitted only through ingestion of infected tissues. The presence

of the parasite in feces is restricted to feline hosts, in which T. gondii undergoes the sexual part

of its life cycle.


0 3 5 7 1 0 1 4

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1 .5

NS ** ** NS NS NS ** ** NS NS

NS ** ** NS NS NS ** ** * NS

NS ** ** * NS NS NS NS NS NS

Supplementary Figure 5 L

yz1

(fo

ld)

De

fcr1

(fo

ld)

De

fa-r

s1

(fo

ld)

De

fa21

(fo

ld)

CR

2 (

fold

)

Re

gII

I-

(fo

ld)




WT, Day 0 WT, Day 3

WT, Day 7

WT, Day 5

WT, Day 10 WT, Day 14

a

b


Supplementary Figure 5. Loss of Paneth cells during mucosal responses to T. gondii. (a)

The relative expression levels of Lyz1, Defcr1 (alpha-defensin-1), Defa-rs1, Defa21, and CR2

were measured in the small intestines of mice on days 3, 5, 7, 10, and 14 post infection together

with the pan-epithelial antimicrobial RegIII-. The results are representative of three independent

experiments. * P< 0.05; ** P< 0.001. (b) Histological visualization of Paneth cells by Alcian

blue staining in the small intestines of naïve or T. gondii-infected mice on days 3, 5, 7, 10, and

14 post-infection. The blue arrows indicate Paneth cells and the black arrows point toward bases

of the crypts lacking these cells. The results are representative of three independent experiments.


Day 7

Day 5

Day 3

Supplementary Figure 6 Nature Immunology doi:10.1038/ni.2508

Supplementary Figure 6. Ultra structural analysis of Paneth cells in naïve and T. gondii-

infected mice reveals mitochondrial structural changes during infection. WT mice were

infected orally with 20 cysts per mouse of the ME49 strain, and the small intestines were

prepared for electron microscopic analysis on days 3, 5, and 7 post-infection. Two representative

images are shown for each day post-infection. The blue arrows indicate Paneth cells and the

black arrows point toward missing Paneth cells. The yellow arrows indicate Paneth cell

mitochondria with structural changes. For comparison, a structural analysis of Paneth cells in

naïve mice is shown in Figure 2d.


a

b


s e g m e n ts o f s m a ll in te s tin e

pa

ras

ite

s/

1 u

g g

DN

A

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6

0

5 .0 1 0 4

1 .0 1 0 5

1 .5 1 0 5

2 .0 1 0 5

** **

**

**

NS NS

NS NS

NS NS NS

NS NS

*

* *

Supplementary Figure 7. T. gondii infected IFN- deficient mice do not loose Paneth cells.

WT and IFN--/- mice were infected orally with 20 cysts per mouse of the ME49 strain of T.

gondii. (a) The small intestine were divided into sixteen equal segments and the T. gondii loads

in each segment of the small intestines were analyzed on day 7 post infection by quantitative RT-

PCR. * P< 0.05; ** P< 0.01. (b) Histological analysis of the small intestine from IFN--/- mice

was performed on day 7 post-infection together with the samples shown in Figure 5.


0 .0

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WT

CD4

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a

IFN--/-

CD4

WT

CD8

IFN--/-

CD8

-

WT

CD4

IFN--/-

CD4

WT

CD8

IFN--/-

CD8

-

Adoptive

transfer:

Adoptive

transfer: WT

CD4

IFN--/-

CD4

WT

CD8

IFN--/-

CD8

- WT

CD4

IFN--/-

CD4

WT

CD8

IFN--/-

CD8

-

b


**

* NS NS

NS NS NS

***

NS NS NS

***

* NS NS

***

Lyz1

(fo

ld)

De

fcr1

(fo

ld)

CR

2 (

fold

)

IFN

- (

fold

)

0 .0

0 .5

1 .0

1 .5

2 .0

0 .0

0 .5

1 .0

1 .5

2 .0

WT

CD4

IFN--/-

CD4 -

0 .0

0 .5

1 .0

1 .5

2 .0

IFN--/- WT

Adoptive

transfer: - WT

CD4

IFN--/-

CD4 -

IFN--/- WT

- WT

CD4

IFN--/-

CD4 -

IFN--/- WT

-

c NS

*** ***

NS

*** ***

NS

*** ***

Lyz1

(fo

ld)

De

fcr1

(fo

ld)

CR

2 (

fold

)


Supplementary Figure 8. CD4 T cell IFN- mediates loss of Paneth cells. (a) CD4 or CD8 T

cells were isolated from WT or IFN--/- mice and were adoptively transferred (5106 cells) to

RAG1-/- mice 4 weeks prior to T. gondii infection. The control and reconstituted RAG1-/- mice

were infected orally with 20 cysts per mouse of the ME49 strain. The relative expression levels

of (a) IFN-, (b) Lyz1, Defcr1 (alpha-defensin-1), and CR2 were measured in the small

intestines of mice on day 7 post-infection * P< 0.05; ** P< 0.01; *** P< 0.001. The results are

representative of two independent experiments. (c) WT or IFN--/- CD4 T cells were adoptively

transferred (5106

cells) to IFN--/- mice. On the same day control and reconstituted mice were

infected orally with 20 cysts per mouse of the ME49 strain of T. gondii. The relative expression

levels of Lyz1, Defcr1 (alpha-defensin-1), and CR2 were measured in the small intestines of

mice on day 7 post-infection by qRT-PCR. The results are representative of two independent

experiments.


Megan Raetz, Sun -hee Hwang, Cara Wilhelm, Donna Kirkland ... · Supplementary Figure 2. Different...

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