notch and tle-dependent histone deacetylation explain il-12 … · notch and tle-dependent histone...

Notch and TLE-dependent histone deacetylation explain IL-12 p70 inhibition by zymosan Yolanda Alvarez, Cristina Municio, Etzel Hugo, Jimmy Zhu, Sara Alonso, Xiaoyu Hu, Nieves Fernández, and Mariano Sánchez Crespo

Four Supplemental Figures

Figure Legends for Supplemental Data

Supplemental Fig. 1. Effect of different ligands on IFN-β production and induction of IRF-1, IRF-3, and IRF-8. DC were treated for 1 hour with 1 mg/ml zymosan or left untreated and at the end of this period were incubated in the presence of 1000 U/ml of either IFN-α or IFN-γ, 10 μg/ml Poli I:C, 10 μg/ml LPS or combination thereof. IFN-β protein was assayed in the media at 24 hours and the mRNA of IFN-β was assayed by RT-PCR in total RNA extracted after 4 hours of incubation (A). DC were stimulated with different additions for the times indicated. After this time, nuclear extracts were obtained and used for the binding assay of IRF-3 activity to consensus sequences using TransAM® assay. The specificity of binding was confirmed by carrying out the reaction in the presence of an excess of wild type (WT) and mutated probe. Positive and negative controls have been included as indicated (B). The expression of the mRNA encoding IRF-1 and IRF-8 in samples obtained 4 hours after the addition of the stimuli is shown in (C). Results are representative of at least three independent experiments and are expressed as mean ± S.D. * P < 0.05 Supplemental Fig. 2. Dectin-1 blockade does not reverse the effect of zymosan on IL-12 p70 production elicited by LPS and IFN-γ. The effect of preincubation with 10 μg/ml anti-dectin-1 mAb on IL-12 p70 production elicited by LPS + IFN-γ is shown in (A). The effect of zymosan addition prior to the stimulation with LPS and IFN-γ in bone-marrow derived DC from dectin-1+/+ and dectin-1-/- mice is shown in (B). Panel A shows a representative experiment of three with identical trend. Panel B shows mean ± S.D. of three independent experiments. Supplemental Fig. 3. Effect of different additions on the expression of the mRNA encoding hes1 and hey1. DC were incubated with zymosan or left untreated for 1 hour. At the end of this time, different ligands were added to the medium. Two hours thereafter, total mRNA was extracted and used for the assay of hes1 and hey1 mRNA by real-time RT-PCR. Data represent mean values of two independent experiments with duplicate samples. Supplemental Fig. 4. Diagram of the mechanism of inhibition of IL-12 p35 by zymosan. Zymosan is bound by a number of receptors on DC (TLR2 and dectin-1 have been selected for the sake of clarity, but receptors as DC-SIGN, CR3, the mannose receptor, and dectin-2 might also be involved). This binding generates signals mediated

Notch and histones in the IL-12/IL-23 balance

1

by Syk, Ca2+, PKC, MAPKs, and IKKs that produce binding of RBP-Jκ and NICD to the promoters of Notch family proteins. This allows the induction of Hes1, Hes5, Hey1, and TLE proteins. These proteins bind to the il12a promoter and counteract κB- and IFN-γ-activated sequence (GAS)-mediated transcription by recruiting deacetylases and allowing the removal of acetyl groups from histones and/or transcription factors. PKC can contribute to counter signals from IFN-γ receptor (IFNGR1) that are inhibitory by acting on Notch intracellular domain (NICD) protein expression. Ac, acetyl; DAC, diacylglycerol; HDA, histone deacetylase; H3, histone H3; IP3, inositol trisphosphate; P, phosphate; PLCγ, phospholipase Cγ; RBP-J (recombining binding protein suppressor of hairless). The core sequence of the Runt site is in red. The E-box palindrome is in italics.


2

A

Supplemental Figure 1

IFN

-β

(p

g/10

6 cel

ls)

Con

trol

IFN

-γ

IFN

-α

LPS

Pol

i I:C

Pol

i I:C

+ IF

N-α

Pol

i I:C

+ IF

N-γ

LPS

+ IF

N-α

LPS

+ IF

N-γ

LPS

+ P

oli I

:C

IFN

-β m

RN

A (A

U)

020406080

100120140160180200

ZymosanMedium

0.000.050.100.150.200.250.300.350.400.45

IRF-

3 bi

ndin

g ac

tivity

(opt

ical

den

sity

450

nm

)

Con

trol

LPS

+ IF

N-α

LP

S +

IFN

-γ

Con

trol

LPS

+ IF

N-α

LP

S +

IFN

-γ

- Con

trol

+ C

ontro

lC

ontro

l + W

T pr

obe

+ C

ontro

l+

Mut

ated

pro

be

1 Hour 2 Hours

0.0

0.5

1.0

1.5

2.0

2.5

3.0

ZymosanMedium

Con

trol

IFN

-γ

IFN

-α

LPS

Pol

i I:C

Pol

i I:C

+ IF

N-α

Pol

i I:C

+ IF

N-γ

LPS

+ IF

N-α

LPS

+ IF

N-γ

LPS

+ P

oli I

:C

IRF-

1 m

RN

A (A

U)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

IRF-

8 m

RN

A (A

U)

B

C

*****

**

*

**

*

*** *

**

* * *


3

IL-1

2 p7

0 (n

g/10

6 cel

ls)

0.00.20.40.60.81.01.21.41.61.82.0

Zymosan (mg/ml)0 0.5 1

WTDectin-1 KO

Zymosan (mg/ml) 0 0.05 0.1 0.5 1 2

IL-1

2 p7

0 (n

g/10

6 cel

ls)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4 ControlAnti-Dectin-1 mAb

A

B



4

Med

ium

IFN

-γ

LPS

Pol

i I:C

LPS

+ IF

N-γ

Pol

i I:C

+ IF

N-γ

LPS

+ P

oli I

:C

Preincubation with zymosanA


02468

101214161820

hey1

mR

NA

(AU

)

0

2

4

6

8

10

hes1

mR

NA

(AU

)

Med

ium

IFN

-γ

LPS

Pol

i I:C

LPS

+ IF

N-γ

Pol

i I:C

+ IF

N-γ

LPS

+ P

oli I

:C

0.0

0.1

0.2

0.3

hey1

mR

NA

(AU

)

0.0

0.1

0.2

0.3

0.4

hes1

mR

NA

(AU

)


5

Hes

NIC

D

Syk

Dectin-1

PLC γ-Y-P

DAGDAG

PKC

ITAM

MAPK

TLR9TLR2

MyD

88

DD

MAL

Kinase

IRAK

4K

inaseIR

AK4

IRAK

4K

inaseIR

AK4

Kinase

IRAK

4IR

AK4

PP

P IP3

PP

P

PP

P IP3

CARD9BCL10MALT

TRAF6

hes1 promoter il12a promoter

IKKβ

IKKα

IKK γ

INFγINFγ

INFγR1INFγR1 INFγR1INFγR1

RBP-Jκ

JAK-1JAK-1JAK-2JAK-2

STAT2

STAT

1

Y701

Y701

Hey

1H

es1

TLE

accgcacgtg

HDA

H3H3

AcPP

HDAP

Ac

κB siteGASE-boxRunt site



6

notch and tle-dependent histone deacetylation explain il-12 … · notch and tle-dependent histone...

Documents