1 Young-Il Jeong, In Duk Jung, Jun Sik Lee, Chang-Min Lee Jae-Dong Lee, Yeong-Min Park Bioch. & Biophy. Res Comm 354 (2007) 1004–1009 내분비 대사 내과 R3 하상진

2 Introduction

3 Antioxidant and micronutrients  Micronutrients ; Vitamins, Trace elements, Phytochemicals Dis Mon 2006;52:

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5 Phytochemicals

6 Dis Mon 2006;52:

7  EGCG ( Epigallocatechin-3-gallate ) 1.Major polyphenolic constituent found in green tea ( other catechins ; (-)-epicatechin-3-gallate (-)-epicatechin (+)-catechin ) 2. kind of flavnols ㄱ ) Catechin and epicatechin in fruit ㄴ ) gallocatechin, epigallocatechin, and epigallocatechin gallate in grapes and tea. ㄷ ) stable in heat at low pH

8 3. Potential health benefits in green tea & EGCG 1) antioxidant effect 2) cancer chemoprevention 3) improving cardiovascular health 4) enhancing weight loss 5) protecting skin from damage caused by ionizing radiation Phytochemistry 67 (2006) 1849– Inhibition of COX-2 without affecting COX-1 expression in human prostate cancer cells

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10  Dendritic cells 1. APCs ( Ag presenting cells) 2. Immune sentinels as initiators of T cell responses against microbial pathogens & tumors 3. role during induction of T cell tolerance ( e.g cancer patients)  CD 8+ DCs induces tolerance to Ags they present : immunoregulatory enzyme indoleamine 2.3- dioxygenase

11  IDO ( indoleamine 2.3-dioxygenase ) 1. enzyme catalyzing initial & rate limiting step in catabolism of Trp along kyurenine pathway 2. unique enzyme in that it utilizes a superoxide anion radical as both a substrate and a co-factor 3. key enzyme in T cell suppression & induction of immune tolerance to tumor 4. IDO expression induction in many tumors & tolerzing APCs 5. previous study (J. Immunol, 2000, 164: 3596–3599) ; human DC expressing significant IDO activity can mediate inhibition of T cell proliferation through tryptophan depletion in microenvironment

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13  IDO ( indoleamine 2.3-dioxygenase ) 1. enzyme catalyzing initial & rate limiting step in catabolism of Trp along kyurenine pathway 2. unique enzyme in that it utilizes a superoxide anion radical as both a substrate and a co-factor 3. key enzyme in T cell suppression & induction of immune tolerance to tumor 4. IDO expression induction in many tumors & tolerzing APCs 5. previous study ( J. Immunol, 2000, 164: 3596–3599 ) ; human DC expressing significant IDO activity can mediate inhibition of T cell proliferation through tryptophan depletion in microenvironment

14  IFNγ 1.major stimulator of IDO expression in DCs 2.JAK/STAT signaling pathway mediates the induction of IDO expression by IFN- γ  PGE2 1. potential key molecule for induction of tolergenic DCs 2. inducer of IDO expression

15 Science 2002;296:1653-5

16 hypothesis IFN- γ JAK/STAT pathway IDO induction PGE2 EGCG COX-2 Prostaglandin

17 Purposes Authors want to show the modulatory effect of EGCG on the IDO expression in IFN- γ -stimulated murine BMDCs through inhibition of STAT1 activation and COX-2 expression

18 Material & Methods  Animals 1.Male 8- to 10-week-old C57BL/6 (H-2Kb and I-Ab) mice 2.OT-1 T cell receptor (TCR) transgenic mice in the C57BL/6 background  Reagents and antibodys (Abs) 1.Recombinant mouse (rm) GM-CSF, rm IL-4, and rm IFN-γ 2.anti-phospho-STAT1, anti-STAT1, and anti-a-tubulin polyclonal anti-mouse IDO Ab for western blot  Generation of BM-derived murine DCs

19  mRNA analysis by reverse transcriptase PCR (RT-PCR)  Western-blot analysis.  Enzymatic assay for IDO activity  Chromatin immunoprecipitation (ChIP) assay.  Enzyme-linked immunosorbent assay (ELISA) for PGE2  Mixed lymphocyte reaction (MLR).  statistics

20 Results

21 Fig. 1. Effect of EGCG on IDO expression in murine BMDCs stimulated by IFN- γ

22 Fig. 2. Effect of EGCG on the phosphorylation and binding of STAT1 to the IRF-1 promoter in IFN-c stimulated DCs

23 Fig. 3. Effect of EGCG on COX-2 expression and PGE2 level in BMDCs. DCs were pretreated with EGCG for 2 h as indicated concentration and then stimulated with IFN- γ

24 Fig. 4. Effect of EGCG on restoration of T cell suppression induced by IDO in vitro

25 Discussion 1. EGCG strongly inhibits the IDO expression and activity in IFN- γ stimulated BMDCs. In vitro MLR system using co-cultures of transgenic OVA specific CD8+ T cells with DCs, we demonstrated that EGCG recovers the IDO-dependent T cell suppression.  EGCG inhibits the tolerogenic function of tumor- associated DCs through the inhibition of IDO expression

26 2. Two critical sequence elements, interferon-stimulated response element (IRSE) and interferon activation sequences (GAS) are involved in the response of the IDO gene promoter to IFN- γ. Tyrosine phosphorylation of STAT1 by IFN- γ -induced reactions induced the binding of STAT1 to the GAS regions in IDO promoter as well as IRF-1 promoter Our results presented here showed that STAT1 phosphorylation was blocked by EGCG treatment.

27 hypothesis IFN- γ JAK/STAT pathway IDO induction PGE2 COX-2 Prostaglandin EGCG

28 Science 2002;296: EGCG

29 3. Overexpression of PGE2 by tumor is also caused immune suppression particularly by interfering with an immune attack. PGE2 was described to up-regulate functional IDO during DC maturation  In previous studies, reduction of IDO expression in DCs may be derive from the suppressive activity of EGCG on COX-2/PGE2.

30 Conclusion  EGCG could inhibit IDO expression by down-regulation of STAT1 activation and COX-2 expression in IFN- γ -stimulated murine DCs.  This study suggests that EGCG is worthy of a new pharmacological agent for IDO regulation and a therapeutic adjuvant for DCs-related diseases.