Gut Microflora Influences Pathologyin the Kawasaki Disease (KD) Vasculitis Mouse Model

Daiko Wakita1, Yosuke Kurashima2, Yoshihiro Takasato2, Youngho Lee1, Kenichi Shimada1, Shuang Chen1,

Timothy R. Crother1, Michael C. Fishbein3, Thomas J.A. Lehman4, Hiroshi Kiyono2 and Moshe Arditi1

1Pediatrics and Infectious Diseases, Cedars-Sinai Medical Center, Los Angeles, CA. 2Division of Mucosal Immunology, The University of Tokyo, Tokyo, Japan.

3Department of Medicine Pathology and Laboratory Medicine, University of Los Angeles, Los Angeles, CA. 4Pediatric Rheumatology, Hospital for Special Surgery, New York, NY.

Daiko Wakita, PhD

Gut Microflora Influences Pathology in the Kawasaki Disease

Vasculitis Mouse Model

FINANCIAL DISCLOSURE:

No relevant financial relationship exists

Presenter Disclosure Information

Intestinal Microbiota and Disease

Health Disease

Immune system

Metabolism

IBD(inflammatory Bowel disease)

allergy

Arteriosclerosis

The Biodesign Institute at Arizona State UniversityPNAS

Changes in intestine of KD patients

A wide variety of bacteria was isolated from jejunal biopsies in the acute phase of KDas compared with those from control children

KD patients had a significantly lower incidence of Lactobacillus than disease control patients

Macrophage/dendritic cells and activated CD4+ T cells were significantly increased in the lamina propria of KD patients in the acute phase.

Coronary Arteritis 70-80% C57BL/6

Day 14

Focal, asymmetric

invasion of arterial

wall, Lymphocytic

Day 28

Circumferential

lesion with marked

proliferation of

intima/media

Day 56

Fibrous tissue,

marked

narrowing

Kawasaki Disease Vasculitis Mouse Model

Lactobacillus casei cell wall extract (LCWE) i.p.

Day 3

Mononuclear

cells in

adventitia

Control LCWE

500 mm

AoAo

Ao

AoCo

Co

Abdominal Aorta

Renal artery Iliac artery

200 mm

Control LCWE

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Max

ima

l a

ort

a d

iam

ete

r (m

m)

**

LCWE-induced KD mouse model develops abdominal aorta aneurysms

Control LCWE

Presented in The 32nd Kawasaki Disease Meeting (Kanto-area), Japan Red Cross Hospital in Tokyo, Dec 7th 2013

** P<0.01

NOD2-/- and Dectin-1-/- mice are protected from LCWE-induced KD vasculitis

FungiBacteria

NOD2

Inflammatory cytokines

Peptidoglycan

Dectin-1

β-1,3-glucan

WT NOD2-/- Dectin-1-/-

WT NOD2-/- Dectin-1-/-

0

5

10

15

Heart

ve

ss

els

Infl

am

mati

on

sc

ore

WT NOD2-/- Dectin-1-/-

0

25

50

75

100

Incid

en

ce (

%)

***

*****

**

11/12

0/12

2/7

** P<0.01, ***P<0.001

LCWE i.p.

Day7

HeartAbdominal Aorta

SPF(n=13)

GermFree

(n=13)

Germ-Free mice develop markedly decreased cardiovascular lesionsin KD mouse model

SPF GF

500 μm

SPF GF

0

2

4

6

8

10

12

He

art

ve

ss

els

Infl

am

mati

on

sco

re

SPF GF

0

10

20

30

40

50

60

70

80

Incid

en

ce (

%)

10/13

4/13

***** P<0.01 * P<0.05

(Coronary lesions)

Naive SPF GF0.0

0.3

0.6

0.9

1.2

1.5

Max

ima

l a

ort

a d

iam

ete

r (m

m)

***

*** P<0.001KD SPF

KD GF

Naïve

Germ-Free mice develop markedly decreased cardiovascular lesions in KD mouse model

200 um

(abdominal aorta lesions)

Pregnant Birth 5 wk

LCWE i.p.

6 wk

HeartAbdominal Aorta

Anti-fungal drug (Fluconazole; Fluc)

and/or

Antibiotics cocktail (Abx)(Neomycin, Ampicillin, Vancomycin, Metronidazole)

Depletion of commensal fungi and bacteria with fluconazole and antibiotic treatment

Control Abx0

1

2

3

4

5

Ba

cte

ria

l 1

6S

rD

NA

(Re

lati

ve

to

to

tal

DN

A)

***

Bacteria amount

Control Fluc0.00

0.25

0.50

0.75

1.00

Fu

ng

al

ITS

1-2

DN

A

(Re

lati

ve

to

to

tal

DN

A)

*

Fungi amount

*** P<0.001* P<0.05

Control Fluc Abx Fluc+Abx

Control Fluc Abx Fluc+Abx0

5

10

15

He

art

ve

ss

els

Infl

am

mati

on

sco

re

***

*

Fluconazole and/or antibiotics treatment decreased cardiovascular lesions in KD mouse model

* P<0.05** P<0.01

Control Fluc Abx Fluc+Abx

Control Fluc Abx Fluc+Abx

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Max

imal ao

rta d

iam

ete

r (m

m)

*

*

***

*

Fluconazole and/or antibiotics treatment decreased cardiovascular lesions in KD mouse model

* P<0.05, *** P<0.001

Bacteria

Fungi

Products

Metabolites

Gut Microflora

Inflammatory diseases

Translocation of intestinal microflora

Intestinal barrier dysfunction

Host

Intestinal permeability and disease development

LCWE injection increases intestinal permeability

Control 8 hr 20 hr0

0.2

0.4

0.6

0.8

1.0

1.2

FIT

C-D

extr

an

(u

g/m

l)

Hours after LCWE injection

LCWE (i.p.)

FITC-Dextran(p.o.)

Serum

4hr

20hr

FITC-Dextran(p.o.)

16hr

Serum

8hr

**

* P<0.05

Conclusions

LCWE-induced cardiovasculitis was decreased in germ free mice

Depletion of gut commensal fungi and bacteria diminished KD vasculitis

LCWE injection increased intestinal permeability

Gut MicrofloraLCWE

? Role of microbiome in KD pathogenesis, new diagnostic/therapuetic strategies

KD vasculitisBacteria

Fungi

Products

Metabolites

Permeability

Acknowledgement

Cedars-Sinai Medical centerPediatrics

Moshe Arditi Lab

Young Ho Lee

Shuang Chen

Timothy R. Crother

Kenichi Shimada

Wenxuan Zhang

Ganghua HuangMicheal Fishbein

University of CaliforniaLos Angeles

The University of Tokyo

Hiroshi Kiyono

Yosuke Kurashima

Yoshihiro Takasato

Grant supports

NIH AI072726 and NIH AI1070162 to Dr ArditiAHA fellowship to Dr Wakita

Thomas Lehman

Hospital for special surgery, New YorkCornell Medical College