Suppl. Fig. 1

(1)

Suppl. Fig. 1:

Time course of T cell infiltration in the several regions after MOG^35-55 immunization. (A, B) Immunofluores- cence of CD3 (red) in the several regions during the preclinical phase (A) and clinical phase (B) of EAE. DRG and TG are costained with Nissl. Others are costained with DAPI. Scale bar: 100 µm. SDH: spinal dorsal horn;

SVH: spinal ventral horn; DRG: dorsal root ganglion; TG: trigeminal ganglion; AP: area postrema; cc: central canal; VII: facial motor nucleus; X: dorsal motor nucleus; XII: hypoglossal nucleus; RPa: Raphe pallidus; IO:

inferior olive; pyr: pyramidal tract.

Nucleus caudalis Nucleus caudalis

VII

AP

cc X XII VII

AP

cc X

100 µm XII

SDH SDH

SVH

DRG

TG DRG

TG

Day 18Day 5

A

B

RPa

pyr IO

RPa

pyr IO

(2)

Suppl. Fig. 2:

Images show Iba1 (microglia) in the L5 spinal dorsal horn 5 days after MOG^35-55 immunization. Microglia in the spinal dorsal horn are not activated even after MOG^35-55 immunization. Broken lines indicate the border between white and gray matter in the spinal dorsal horn.

Iba1

Saline (day 5)

100 µm 10 µm 100 µm 10 µm

EAE (day 5)

(3)

Suppl. Fig. 3:

Time course of neutrophil infiltration in the several regions after MOG^35-55 immunization.. (A, B) Immunofluo- rescence of ELA2 (red) in the several region during the preclinical phase (A) and clinical phase (B). DRG and TG are costained with Nissl. Others are costained with DAPI. Scale bar: 100 µm. SDH: spinal dorsal horn;

SVH: spinal ventral horn; DRG: dorsal root ganglion; TG: trigeminal ganglion; AP: area postrema; cc: central canal; VII: facial motor nucleus; X: dorsal motor nucleus; XII: hypoglossal nucleus; RPa: Raphe pallidus; IO:

inferior olive; pyr: pyramidal tract.

SDH SDH

SVH

DRG

TG TG

VII VII

AP

cc X XII

RPa

pyr IO

AP

cc X XII

100 µm

Day 5Day 18

Nucleus caudalis

A

B

RPa

pyr IO

(4)

Suppl. Fig. 4:

Quantitative PCR analyses of CatD mRNA in neutrophils. Neutrophils were stimulated MOG^35-55 (25 µg/mL) for 6 hours. CatD mRNA in neutrophils were not changed after MOG^35-55 stimulation. n=3 mice per group, unpaired t-test.

CatD mRNA (relative expression) WT

MOG

^35-55

- +

P =0.6775

0.0

0.5

1.0

1.5

(5)

Suppl. Fig. 5:

Forced expression of mouse TLR4 and MD-2 in HEK293T. HEK293T are cotransfected with mouse TLR4-flag and MD-2 plasmids. (A) The immunoblot for flag and MD-2 in mTLR4-flag⁺/mMD-2⁺ HEK293T cells. The protein for TLR4 was detected by anti-DDDDK (flag) antibody. (B) Thr gel shows semi-quantitative PCR for mouse TLR4 and MD-2 in mTLR4-flag⁺/mMD-2⁺. Amplicon of Tlr4, Md-2 and Gapdh was 709 bp, 241 bp and 123 bp, respectively.

B A

TLR4-flag

+MD-2

+

Marker TLR4-flag

+MD-2

+

TLR4-flag

+MD-2

+

Cont Cont Cont

709 bp 241 bp 123 bp 1500

Tlr4 Md-2 Gapdh

(bp) 1000 500

100 TLR4-flag

+MD2

+

Control

TLR4-flag

+MD2

+

Control marker Marker

(kDa)

(kDa) 75

100 150 250

TLR4-flag

MD-2

β-actin

1520 25 37 50