Eosinophil-Derived Neurotoxin (EDN) As a Th2- Polarizing Alarmin

Granulocyte products act as alarmins to enhance innate and adaptive immunity

De Yang1,2 and Joost J. Oppenheim2

1Basic Research Program, SAIC-Frederick, 2Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, National Cancer Institute

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1. From granulocyte products to alarmins

2. HMGN1 as a Th1-polarizing alarmin

3. Eosinophil-derived neurotoxin (EDN) as a Th2- polarizing alarmin

4. Conclusion What are alarmins? GGranulocyteranulocyte products in immunity

Activation and/or Secondary lymphoid organs recruitment of various leukocytes including Adaptive granulocytes immune response Generation and/or Infection activation of soluble and/or mediators injury Antimicrobial peptides/proteins (AMPs: defensins cathelicidin, lactoferrin, lysozyme, BPI, elastase, PLA2, chymase, Wound EDN, ECP, HMGs, etc) healing

Innate/inflammatory response What are alarmins? Identification of the effects of β-defensin to chemoattract and activate dendritic cells

β-defensins: linking innnate and adaptive immunity through DC and T cell CCR6 (D Yang et al, Science 286:525-529; 1999)

1. Human β-defensin 2 (HBD2) is chemotactic for immature dendritic cells (iDC) and peripheral blood memory T cells.

2. β-defensin 2 utilizes CCR6 as the receptor for chemoattacting target cells.

Toll-like receptor 4-dependent activation of dendritic cells by β-defensin 2 (A Biragyn et al, Science 298:977-979; 2002)

1. Mouse β-defensin 2 induces full maturation of DCs.

2. TLR4 mediates the DC-activating effect of β-defensin 2 .

3. β-defensin 2-tumor antigen (of mouse B-cell lymphoma) fusion product enhances B-cell lymphoma-specific immune responses. What are alarmins? AMPs capable of acting as APC chemoatttractant, activator, and immune enhancer

Activity APC chemoattraction APC activation Immune enhancemen Mediator Target cells Receptor Target cells Receptor t Defensins HNP1~4 DC, M φ ? M φ ? + HBD1-4 DC, M φ CCR6, other? DC, M φ TLR2/1 + mBD2-3 DC CCR6 DC TLR4 + mBD14 DC, M φ CCR6, other? n.t. n.t. mBD29 Pre-DC CCR6 n.t. n.t. Cathelicidins LL-37 Mo, M φ FPRL1 DC, M φ P2X7 + CRAMP Mo, M φ FPR2 EARs EDN DC ? DC ? + mEAR2 DC ? Iron-binder Lactoferrin Mo, DC ? Mo, M φ ? + Cathepsin Cathepsin G Mo FPR n.t. n.t. HMGs HMGB1 DC RAGE, GPCR DC, Mo, M φ RAGE,TLR + Saposin-like Granulysin DC ? DC, M φ ? n.t.

Note: 1. Lysozyme, elafin, SLPI, PLA2, and BPI do not have the aforementioned activities. 2. N.t. = not tested. What are alarmins? The proposal of alarmin concept

Cur Opin Immunol 2005, 17:359

Innate endogenous APC activator mediators iDC mDC ↓ ‘Alarmins’

APC recruiter

Ep Eo MC Elicitation/enhancement of N Ba iDC adaptive immune responses Mφ /M0 NKT NK What are alarmins? How to determine if a given mediator is an alarmin or not?

Capacity to chemoattract DC (chemotaxis assay) . Recruitment of DC Capacity to recruit DC into sites of injection

Surface markers Capacity to induce Cytokines .Activation of DC phenotypic and functional DC CCR5 & CCR7 maturation Allogeneic MLR

Day1 Day10 Day14 Day21 .Enhancement of Spleen Ag-specific Mice cells immune response i.p. with OVA i.p. with OVA ± candidate Serum

OVA-specific OVA-specific IgG cytokine and subclasses production HMGN1 as a Th1-polarizing alarmin Why to study HMGN1?

Classification HMGA family HMGB family HMGN family

AT hooks Box1 Box2 NBD CHUD HMGA1a/b HMGB1 HMGN1, HMGN2 HMGA1c* HMGB2 HMGN3a/b, HMGN4 HMGA2 HMGB3 Nsbp1 (NBD-45) Expression Intranuclear Abundant in embryonic tissues, differentially downregulated in adult tissues. Relative abundance: HMGBs>HMGNs>HMGAs. Extracellular ? HMGB1 can be HMGNs can be secreted by Mφ released by PBMC

Function Intranuclear Regulation of development, differentiation, and cellular processes by binding to DNA and nucleosome and subsequent modulation of the transcription of various genes. Extracellular ? HMGB1 regulates cell Can HMGNs act migration, activation, as alarmins? and acts as an alarmin HMGN1 as a Th1-polarizing alarmin HMGN1 induces recruitment of DCs into mouse peritoneal cavity (4h)

CD11c+

CD11c+/ CD11b+

CD11c+/ B220+ PBS HMGN1 CD11c+/ CD11b+/ B220+

0123456 Migration index

Cell No. in the presence of HMGN1 Migration index = Cell No. in the absence of HMGN1 (PBS) HMGN1 as a Th1-polarizing alarmin HMGN1 activation of DCs: 1. Cytokine induction

105 Sham HMGN2 104 HMGN1 LPS * 3 10 *

2 10

1 10 Concentration (pg/ml)

0 10 IL-6 IL-10 IL-12p70 TNFα HMGN1 as a Th1-polarizing alarmin HMGN1 activation of DCs: 2. Upregulation of surface markers

CD11c CD80 CD83 CD86 HLA-ABC HLA-DR

100 100 100 100 100 100

80 80 80 80 80 80

60 60 60 60 60 60 % of Max % of Max % of Max % of Max % of Max % of Max Sham 40 40 40 40 40 40

20 20 20 20 20 20

0 0 0 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 FL2-H FL1-H FL1-H FL2-H FL1-H FL2-H 100 100 100 100 100 100

80 80 80 80 80 80

60 60 60 60 60 60 % of Max % of Max % of Max % of Max % of Max % of Max HMGN1 40 40 40 40 40 40

20 20 20 20 20 20

0 0 0 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 FL2-H FL1-H FL1-H FL2-H FL1-H FL2-H

100 100 100 100 100 100

80 80 80 80 80 80

60 60 60 60 60 60 % of Max % of Max % of Max % of Max % of Max % of Max

Cell number HMGN2 40 40 40 40 40 40

20 20 20 20 20 20

0 0 0 0 0 0

0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 FL2-H10 10 10 10 10 FL1-H10 10 10 10 10 FL1-H10 10 10 10 10 FL2-H10 10 10 10 10 FL1-H10 10 10 10 10 FL2-H10 10 10

100 100 100 100 100 100

80 80 80 80 80 80

60 60 60 60 60 60 % of Max % of Max % of Max % of Max % of Max % of Max LPS 40 40 40 40 40 40

20 20 20 20 20 20

0 0 0 0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 FL2-H FL1-H FL1-H FL2-H FL1-H FL2-H

Relative fluorescence intensity (Log) HMGN1 as a Th1-polarizing alarmin HMGN1 activation of DCs: 3. Functional maturation

7 104 No DC 4 6 10 Sham HMGN1 5 104 HMGN2 4 104 LPS

3 104

2 104

1 104 H-TdR incorporation (CPM) 3 0 No DC 1:31250 1:6250 1:1250 1:250 1:50 DC:T ratio HMGN1 as a Th1-polarizing alarmin HMGN1 promotes OVA-specific Th1 immune response

OVA-specific OVA-specific splenocyte cytokine proliferation production

2000 IFNγ * 8000 PBS Alum 1000 HMGN1 0 6000 120 IL-4 * 80 40 4000 0 400 IL-10 * * 2000 200 0 Concentration (pg/ml)

H-TdR incorporation (CPM) H-TdR incorporation TNF α 3 0 40 * * 0 2 10 50 20 OVA (μg/ml) 0 PBS Alum HMGN1 HMGN1 as a Th1-polarizing alarmin Summary

• HMGN1 is capable of inducing DC activation as evidenced by induction of multiple cytokines, upregulation of surface molecules indicative of maturation, and enabling DCs to activate naïve T cells.

• HMGN1 can induce the recruitment of DCs.

• HMGN1 is able to promote OVA-specific immune response, favoring the polarization of T cells into Th1 (IFNγ-producing) direction.

• How HMGN1 attracts and/or activates is currently under investigation. Eosinophil-derived neurotoxin (EDN) as a Th2-polarizing alarmin EDN is capable of inducing DC migration and recruitment

140 EDN: a 16-kDa cationic glycoprotein EDN 120 hpRNase belonging to EAR superfamily. Angiogenin 100 mEAR2 bpRNase 80 Source: eosinophils, PMN, Mφ, and certain Boyden epithelial cells. 60 chamber 40 Structure: 136 a.a. folded into two lobes, 20 each consisting of three anti-parallel β- DC migration (No./HPF) 0 sheets. 10 100 1000 5000 Concentration (ng/ml) Activity: RNase; antimicrobial (helminth,

viruses). 20 Total infiltrating cells DCs (CD11c+/I-A/I-E+ ) 15 Air-pouch 10 model × 5 /pouch) 4 Cells ( 10 0 PBS Ang mEAR2

Dose (1 μg/air pouch) Eosinophil-derived neurotoxin (EDN) as a Th2-polarizing alarmin EDN induces maturation of human DCs

Surface Cytokine Allogeneic marker production MLR

IL-6 10 3 )

3 Sham 10 2 CD80 10 1 15 EDN LPS IL-10 10 3 CD83 10 2 10 10 1

2 10 IL-12p70

Cell number CD86 5 10 1 Concentration (pg/ml)

3 HLA-ABC 10 TNF α

H-TdR incororation (CPM x 10 0 2 3 1:6250 1:1250 10 1:250 0 10 1 HLA-DR - 200 2000 1000 Log F.I. EDN LPS DC: T cell ratio Eosinophil-derived neurotoxin (EDN) as a Th2-polarizing alarmin EDN promotes OVA-specific Th2 response

OVA-specific cytokines OVA-specific Abs

100 IL-2 Day 10 0 104 10 IL-4 103 0 102

100 IL-5 1 0 10 IgG1 100 400 IL-6 IgG2a 0 Day 21 IgG2b 104 200 IL-10 IgG3 0 Anti-OVA IgG titer 3 Cytokine (pg/ml) 10

40 IL-13 102 0 101

100 IFNγ 0 0 10 EDN - + EDN -+ Eosinophil-derived neurotoxin (EDN) as a Th2-polarizing alarmin How does EDN act? EDN activates NF-κB and multiple MAPKs in DCs

EDN

01 5102030 (min)

NF-κB I-κBα

p-JNK

MAPKs p-p38

p-Erks Eosinophil-derived neurotoxin (EDN) as a Th2-polarizing alarmin How does EDN act? EDN induction of DC cytokines depends on MyD88

Human DCs Mouse DCs

MyD88 +/+

) s Chariot II 300 MyD88 -/- C 200

D Chariot II + hMyD88 7 gripNA

1 200

( 100

- 100

0 0 100 1000 0 EDN Pam3 EDN LPS Concentration (ng/ml) Concentration (1 μg/ml) Eosinophil-derived neurotoxin (EDN) as a Th2-polarizing alarmin How does EDN act? EDN triggers the activation of TLR2

NF-κB activation in HEK293 cells IL-6 production by mouse DC

) 40 TLR2 400 U TLR3 TLR2+/+ L -/-

R TLR4+MD2 ) TLR2

30 s

n( 300

20 0 v i 200

(

B 10 100

I N 0 0 None PGN PolyI:C LPS EDN Pam3 EDN LPS Eosinophil-derived neurotoxin (EDN) as a Th2-polarizing alarmin How does EDN act? EDN activation of TLR2 is independent of TLR1 or TLR6

5 4

g/ml) WT 3 (n

α 2

TNF 1 TLR1-/- 0 4 TLR2-/- 3

ng/ml) 2 TLR6-/- IL-6 ( 1

0 Sham Pam3 FSL-1 EDN LPS Eosinophil-derived neurotoxin (EDN) as a Th2-polarizing alarmin How does EDN act? EDN induced signal transduction pathways

EDN

? TLR2

α MyD88 β γ

I- Bα↓ MAPKs (Erk, p38)↑ PI3K↑

NF- B, AP-1, etc. Akt ↑

Chemoattraction & recruitment Cytokines DC Maturation

Enhancement of Th2 response Eosinophil-derived neurotoxin (EDN) as a Th2-polarizing alarmin EDN enhancement of anti-OVA Th2 response is mediated by TLR2

Anti-OVA IgG Splenocyte cytokine

104 Primary 200 IL-5 103 100 IgG1 0 102 IgG2a 800 IL-6 101 400 0 100 80 IL-13 105 Secondary 40 104 0 Cytokine (pg/ml) Anti-OVA IgG (ng/ml) 3 IgG1 200 IFNγ 10 IgG2a 100 102 0 20 IL-17 101 10 0 10 0 --++ EDN EDN --++ +/+ -/- TLR2 TLR2 TLR2+/+ TLR2-/- Eosinophil-derived neurotoxin (EDN) as a Th2-polarizing alarmin OVA-loaded DCs treated with EDN in vitro promote Th2 response in vivo

DC conditioning OVA-specific T cell in vitro response in vivo

500 IL-1 β IFN γ 300 250 200 100 0 0 IL-6 IL-5 20000 * 200 10000 100 pg/ml) 0 0 3000 IL-10 IL-6 800 1500 400 0 0 DC cytokine ( 800 IL-12 p70 IL-10 300 400 200 100 0 0 30000 TNF α IL-13 OVA-specific splenocyte cytokine (pg/ml) OVA-specific splenocyte 90 15000 60 30 0 0 Sham EDN LPS Sham EDN LPS Eosinophil-derived neurotoxin (EDN) as a Th2-polarizing alarmin Summary

1. EDN acts as an alarmin by inducing DC recruitment, activation, and promoting antigen-specific immune responses.

2. EDN activation of DCs is mediated by TLR2.

3. EDN selectively promotes Th2 immune response, in which both DCs and TLR2 are critical.

4. EDN’s effect on immune response may contribute to Th2 polarization associated with allergic and/or certain parasite infection. Conclusions and perspectives Granulocyte products as alarmins to enhance and regulate innate and adaptive immunity

Mφ /M0 Ep NKT NK

Alarmins .Defensin .HMGB1 Th1 IFNγ Endogenous . HMGN1 iDC Tm mDC IL-4 .Lactoferrin activator 8 Th2 IL-5 .Cathelicidin 4 B IL-13 .EDN Te

PC Th17 IL-17 APC recruiter IgM IgG, IgA, IgE Ep N Eo MC Ba iDC CTL

1. The mechanism(s) of alarmins’ action (receptor and signaling pathway) 2. Can blockade of alarmins ameliorate inflammation? 3. The potential utilization of alarmins as adjuvants for vaccination or antitumor immunotherapeutic intervention? Acknowledgments

LMI, CIP, NCI Univ. of Kiel, Germany Qian Chen Jens M. Schröder Kahori Kurosaka Oleg Chertov LAD, NIAID Zhenhua Liu Helene Rosenberg O.M. Zack Howard Poonan Tewary LI, NEI Osaka Univ., Japan Gonzalo de la Rosa Shao Bo Su Shizuo Akira Joost J. Oppenheim Rachel Caspi

LM, NCI DM, UAB Yuri V. Postnikov Ping Zhang Michael Bustin Suzanne Michalek