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Figure legends

477

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Fig. 1. Anti-allergic activity of LP22A3 in OVA-induced allergic mice.

479

LP22A3 (1 ×108 cfu/mouse/day) were orally administered to BALB/c mice for 17 days.

26

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mice were sensitized with 10 μg OVA + 1 mg Al(OH)3 adjuvant dissolved in 300 μL

481

phosphate-buffered saline (PBS) by intraperitoneal injection. The sensitizations were

482

continued once every 5 days for 2 more times. Five days after the last intraperitoneal

483

sensitization, mice were challenged with OVA by intravenous injection with 3 μg OVA

484

dissolved in 100 μL PBS to trigger an allergy response. (A) Experimental design. Total

485

IgE (B), OVA specific IgE (C), and TGF-β (D) contents were measured by ELISA. Values

486

represent means ± SD (n = 6). Items with different letter were significantly different (p

487

< 0.05).

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Fig. 2. Effect of exogenous TGF-β on PCA reaction.

490

Values represent means ± SD (n = 3). Items with different letter were significantly

491

different (p < 0.05).

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Fig. 3. Effect of LP22A3 administration on DNCB-induced atopic mice

494

Mice were orally administered with LP22A3 (1 x 108 cfu/mouse/day) or prednisolone (3

495

mg/mouse/day) for 7 weeks. DNCB was applied to the dorsal skin to induce AD lesions.

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DNCB (1%, 200 µl) in the mixture of acetone and ethanol (2:3, v/v) was applied. After

497

the first application, 0.5% DNCB (150 µl) in the mixture (acetone:Olive oil = 3:1) was

27

498

applied once every two days for 3 weeks. Mice were orally administrated with

499

LP22A3(1×108 cfu/day) dissolved in 100 μl 0.5% CMC solution. (A) Experimental

500

design. (B) Total IgE contents were measured by ELISA. (C) The TEWL values were

501

measured using a Tewameter TM300. (D) Changes in atopic dermatitis symptoms at the

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dorsal skin lesion on week 0, 3 and 7. (E) Change in skin epidermal thickness elicited by

503

DNCB. Values represent means ± SD (n = 4). Items with different letter were

504

significantly different (p < 0.05).

505

506

Fig. 4. Effect of different timing of LP22A3 treatment on degranulation of RBL-2H3 cells

507

in co-culture system of Caco-2/RBL-2H3 cells.

508

LP22A3 (1 x 108 cfu/mL) was applied into apical side of Caco-2/RBL-2H3 cells after (A)

509

and before (B) the sensitization of anti DNP-IgE antibody. Values represent means ±

510

SD (n = 3). **P<0.01, *P<0.05 versus anti DNP-IgE/DNP-BSA group.

511

512

Fig. 5. Effect of exogenous TGF-β on β-hexosaminidase release from RBL-2H3 cells.

513

**P<0.01, *P<0.05 versus anti DNP-IgE/DNP-BSA group.

514

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Fig. 6. Effect of exogenous TGF-β on IgE contents binding RBL-2H3 cells.

28

516

RBL-2H3 cells were sensitized with anti DNP-IgE (1 μg/mL) for 2 h, and then TGF-β

517

(50, 100 pg/ml) was added and incubated for another 6 h. The cells were collected and

518

the amount of IgE bound to RBL-2H3 cells was measured by Western blotting. Values

519

represent means ± SD (n = 3).

29

LP22A (1 x 108 cfu/mouse/day)

OVA/Al(OH)3 i.p.

-7

-1

Balb/c (♀)

5 weeks old

OVA i.v.

10

15 17

(Days)

14

Cardiac

puncture

Tail vain sampling

16

: Control

Total IgE (g/ml)

14

: LP22A

12

: OVA

10

10

15

20

OVA specific IgE (g/ml)

TGF- (pg/ml)

200

180

160

140

120

100

80

60

40

20

OVA

LP22A3 (1x108 cfu/ml)

Up

Mizuno et al., Fig. 1

Anti TNP-IgE

intravenously

PiCl

epicutaneously

TGF-

intravenously

Cardiac puncture

2h

Ear edema

30 min

Ear edema

Balb/c (♀)

5 weeks old

Ear edema (m)

100

80

60

40

20

TGF- (pg/ml)

150

100

50

Anti TNP-IgE (2 g/mouse)

PiCl (1.6%)

TGF-(40 ng/mouse)

Up

Mizuno et al., Fig. 2

TEWL measurement

Hair removal

on the back

of mice

Tail vain sampling

(Weeks)

-1

Applying 1% DNCB

in dorsal skin

Nc/Nga (♂)

4 weeks old

Applying 0.5% DNCB in dorsal

skin once every two days

Cardiac puncture

Tissue sampling

LP22A (1 x 108 cfu/mouse/day)

3000

2500

: DNCB

2000

: LP22A3 + DNCB

: Prednisolone + DNCB

1500

1000

500

**

TEWL (g•m-2 •h-1)

Total IgE (ng•ml-1)

: Control

70

60

50

40

30

**

20

10

**

Week

Control

0 week

3 week

7 week

DNCB

Prednisolone

DNCB

LP22A3

DNCB

**

Control

DNCB

500 m

500 m

Prednisolone

DNCB

LP22A3

DNCB

500 m

Thickness (m)

150

500 m

a,b

100

b,c

50

Up

Mizuno et al., Fig. 3

Anti DNP-IgE

DNP-BSA

LP22A3

Over night

6h

Caco-2

Caco-2

RBL-2H3 YY Y Y Y

RBL-2H3 YY Y Y Y

RBL-2H3

-Hexosaminidase

release (%)

120

100

80

60

40

20

**

Anti DNP-IgE

DNP-BSA

LP22A3 (1x108 cfu/ml)

Anti DNP-IgE

LP22A3

6h

DNP-BSA

4h

Caco-2

RBL-2H3 YY Y Y Y

RBL-2H3

RBL-2H3

-Hexosaminidase

release (%)

120

100

80

60

40

20

**

Anti DNP-IgE

DNP-BSA

LP22A3 (1x108 cfu/ml)

Up

Mizuno et al., Fig. 4

Anti DNP-IgE

2ｈ

O/N

DNP-BSA

TGF-

-hexosaminidase

assay

6ｈ

-Hexosaminidase release (%)

RBL-2H3

140

120

100

80

60

**

40

20

**

Anti DNP-IgE

DNP-BSA

TGF- (pg/ml)

50

100

200

Up

Mizuno et al., Fig. 5

Anti DNP-IgE

O/N

2ｈ

TGF-

6ｈ

Western blot

RBL-2H3

IgE

-actin

IgE/-actin

IgE

TGF- (pg/ml)

50

100

Up

Mizuno et al., Fig. 6

...