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
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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.
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