Regulation of Type 2 Immunity by Basophils Prof. Dr. David … · Regulation of Type 2 Immunity by...

Regulation of Type 2 Immunity by BasophilsProf. Dr. David Voehringer

1The screen versions of these slides have full details of copyright and acknowledgements

Regulation of Type 2 Immunity by Basophils

Prof. Dr. David VoehringerDepartment of Infection Biology

Institute of Clinical Microbiology, Immunology and Hygiene

2

Outline of the presentation

• The concept of type 2 immunity

• Basophil development, turnover and homeostasis

• Basophil effector functions

3

Worms AllergensBacteria Viruses

Type 1 vs. Type 2 immunity

Type 1 immuneresponse

• Th1 cells (IFN-g, TNF-a)

• Opsonizing Ab (IgG2a)

• CTL, NK cells

Type 2 immuneresponse

• Th2 cells (IL-4, IL-5, IL-13)

• IgE, IgG1

• Basophils, eosinophils, mast cells



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Protective immunityagainst helminths

• >2 billion peopleinfected worldwide

• 1 million people die every year

• No vaccines yet

Positive

Allergies / Asthma

Negative

• ~15% of peoplein westernized countries suffer from allergies

• Increasing prevalence

• High costs for the health care system

Type 2 immunity – a double-edged sword

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Tissue

IgEIgG1

Th2 Eosinophilsactivation

IL-5IL-4IL-13

B cellAPC

MHC IITCR

naiveT cell

Th2

HelminthsAllergens

EliminationAllergy / Asthma

Lymph node / spleen

antigen

Basophils/Mast cells

FcRDegranulationCytokine secretion

IgEIgG1

Development of a type 2 immune response

6

EosinophilsBasophils

Paul Ehrlich (1854-1915)

Discovery of innate type 2 effector cells

Mast cells



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Basophils Mast cells Eosinophils

h in human but not mouse

Comparison between basophils, mast cells and eosinophils

•High affinity IgE receptor(FcεRI)

•Histamine

• IL-4 expression

•Carboxy-peptidase A

+++ +++ +h

•Condensed nucleus

•Lifespan of few days

++ +++ -

++ +++

+++ + +

+ +-

+ +

-

-

•Maturation inbone marrow

+ +-

•Mobilization IL-3 IL-3, SCF IL-5

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Granulocyte/Monocyte precursor(GMP)

Basophil/Mast cell commonprecursor(BMCP)

c/EBPaGATA-2

Arinobu et al., (2009) Allergology International 58: 21-28

Bone marrow/Spleen

Basophilprecursor

Basophilc/EBPa

Mast cell precursor

Mast cell

c/EBPa

Tissue

Developmental relationship

GATA-2

Eosinophil

Eosinophilprecursor

c/EBPa

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CD

49b

IgE

Detection of basophils by CD49b/IgE staining

0.82

Bone marrow

0102

103

104

105

0102 103 104 1050102 103 104 105

0.19

Spleen

0102

103

104

105

0.82

Blood

0102

103

104

105

0102 103 104 105



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Day 1-4LungL3 => L5

Day 4-10Small IntestineL6, adult

Hookworm-like life cycle

N. brasiliensis in small intestine

100µm

500 L3 larvae

Nippostrongylus brasiliensis infection

0 5 9 13

Cel

l cou

nt (x

105 )

Days after infection

Voehringer et al., (2004) Immunity20: 267

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BasophilsCD200R3/DAPI

Mast cellsc-Kit/B220

EosinophilsMBP/DAPI

Ohnmacht, C. and Voehringer, D. (2008) Blood 113: 2816

N. brasiliensis infected lung

12

200mm

FceRI/DAPI

c-KitW-sh/W-sh mice

N. brasiliensis infected small intestine




13

naive infected

BM SP

BM SP BL LU

15h

36h

84h

020406080

100

020406080

100

020406080

100

020406080

100

020406080

100

Analysis of basophil turnover in vivo


100

101

102

103

104

B

0.661.69

BrdU

IgE

with BrdU control

47.749.9

2.24 2.15e-3

0.03997.7100

101

102

103

104

100 101 102 103 104 100 101 102 103 104

CA

BM SP BL LU0

2

4

6

naiveinfected

% B

asop

hils

of t

otal

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Activating receptors on basophils

Voehringer (2009) Trends in Parasitology 25: 551

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Activation of basophils by IgE

FceRIag2

b

ITAM

IgE

Antigen• Affinity for IgE: 10-10 M

• Isoforms:─ abg2: Mast cells, basophils

─ ag2: Monocytes, eosinophils, neutrophils

Degranulation

Cytokine release



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JAK2

bc

GM-CSF

GM-CSFRa

Cytokine instruction of basophils

IL-3

bcIL-3Ra

MyD88

NFkB

ERKp38JNK

ST2 IL1-RAcP

IL-33IL-18

IL-18Ra IL-18Rb

• Lipid metabolism• Cell adhesion• Cell communication

STAT5

JAK2

SHIP

Hck/LynPI3KSyk

FcRg2

JAK2

IL-4IL-6

• Proliferation• Survival• Activation

• Degranulation• Lipid mediators• Cytokines

TSLP

TSLPR

IL-7Ra

?

Pim-1 • SOCS1/• SOCS3/• CIS

Negativefeedback

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PAF

IgG1-mediated anaphylaxis

IgE-mediatedchronic allergicinflammation

Worm expulsion

Basophil effector functions

Voehringer (2009) Trends in Parasitology 25: 551

MHC-IIlow

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Generation and analysis of basophil-deficient mice



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Mcpt-8

Mcpt-4

Mcpt-1

Mcpt-7

Mcpt-6

Mcpt-2

Mcpt-5

Basophils Mast cells

HPRT

Mouse mast cell protease 8 is expressed in basophils

Ohnmacht et al., (2010) Immunity 33: 364

Mcp

t-8 e

xpre

ssio

n

0.00

0.05

0.10

0.15

0.20

0.25

20

0.82 1.52e-3

0.19 0.015

0.050.82

blood

spleen

bone marrow

IgE

control Mcpt8Cre

CD

49b

Constitutive deletion of basophils in Mcpt8Cre mice


0102

103

104

105

0102

103

104

105

0102 103 104 105 0102 103 104 105

0102

103

104

105

0102 103 104 105

0102

103

104

105

0102 103 104 105

0102

103

104

105

0102 103 104 105

0102

103

104

105

0102 103 104 105

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Small intestine

Mast cells are not deleted in Mcpt8Cre mice


3.19

Mcpt8Cre

0102

103

104

105

0102 103 104 105

2.86

control

c-K

it

IgE

Peritoneum0

102

103

104

105

0102 103 104 105

c-KitW-shMcpt8Cre Control

Skin



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Effector function of basophils

100µm

Worm expulsion?

Th2 polarization

? ?

Systemic anaphylaxis

?Chronic allergic dermatitis

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Are basophils required for Th2 polarization?

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Are DCs or basophils required for Th2 polarization?

?Basophils

Dendriticcells

naive

Th2

Th2

Allergen



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CD11c-Cre R-DTA

x

∆DC mice

Caton et al., (2007) J Exp Med 204: 1653

Cre BGHpA

CD11c-BAC (RP24 361C4)CD11c exon 1

Generation of DC-deficient mice

Ohnmacht et al., (2009) J Exp Med 206: 549

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OT-II (Thy1.1)

CFSE+Transfer

ProliferationIL-4 expression

Popliteal LN

Papain+OVA

Footpad

Are DCs or basophils required for Th2 polarization?


control

Mcpt8Cre

ΔDC

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coun

ts

CFSE

control Mcpt8Cre ΔDC

DCs are required for Th2 polarization


∆DCcontrol Mcpt8Cre

Thy 1

.1

IL-4

% u

ndiv

ided

OT-

II

P=0.01

05

1015202530

% IL

-4+

of O

T-II P<0.01

0

10

20

30

40



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Role of innate IL-4 for Th2 polarization

Th2 IL-4IL-5IL-13IL-25

NaïveCD4+T cell

IL-4

DC

???

NKTMast cell

Eos

Innate helper cells

AnalyzeTh2 polarization

Nb

Nb

TCR-Ca-/-

IL-4/IL-13/TCR-Ca-/-

IL-4eGFP

Transfer naïveCD4 T cells

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Innate IL-4/IL-13 regulates Th2 accumulation in the lung

LUNG

LYMPHNODE

IL4eGFP

CD

4

4get CD4 in TCR-Ca-/-

4get CD4 inIL-4/IL-13/Ca-/-

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Basophils in anaphylactic responses



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IgG1-mediated systemic anaphylaxis

Galli/Franco comment in Immunity2008

BasophilsTsujimura et al., (2008)Immunity 28: 581

NeutrophilsJönsson et al., (2011) J Clin Invest. 121: 1484

MacrophagesArias et al., (2011) JACI 127: 1552

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IgE-anti-TNP

Basophils are not required for systemic anaphylaxis

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Basophils in chronic allergic inflammationof the skin



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IgE-anti-TNPi.v.

d-1 d0

TNP-OVA (left ear)OVA (right ear)

Analysis of ear swelling

Basophils are requiredfor chronic allergic inflammation

1 2 3 4 5 6

Basophil transfer


Days after TNP-OVA

1 2 3 4 5 6

Ear

sw

ellin

g

Wild-type mice

1 2 3 4 5 6

Mcpt8Cre miceBaso-depleted miceFcRg-deficient mice

Obata et al., (2007) Blood 110: 913Mukai et al., (2005) Immunity 23: 191

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Basophil response against helminths and ticks

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Eosinophils Basophils Th2 cells

Cel

l cou

nts

(x10

6 )C

ell c

ount

s (x

104 )

1 2 3 4 7 9 11 14 1 2 3 4 7 9 11 14 1 2 3 4 7 9 11 14

days after infection

Lung

mLN

Primary response to N. brasiliensiscontrolMcpt8Cre


05

10152025

0

2

4

6

8

0.0

0.2

0.4

0.6

05

10152025

0

50

100

150

200

0.0

0.2

0.4

0.6



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1 2 3 4 5 6 7 8 9 10

50

100

Num

ber o

f wor

ms

in th

e in

test

ine

Days after infection

Primary infection

Secondary infection

Expulsion kinetics of N. brasiliensis

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nd ndWor

ms

/ mou

se

Primary Infection

Protective immunity against N. brasiliensis


020406080

100120140

Wor

ms

/ mou

se

ND

Secondary Infection

0

10

20

30

40

39

Ih2

1° infection

Th2 BBasophils

FcR

Long-livedPlasma cell

IL-4

Worm expulsion

Activationof tissue cells

• Relm-b

• Mucus

• Smooth muscle activity

• Epithelial cell shedding

IgEIgG1

IL-13IL-4/IL-13

2° infection



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Protective immunity against ticks

1° infestation 2° infestation

Basophils absent

Wada et al., (2010) J. Clin. Invest. 120: 2867

2° infestation

Basophils present

Mast cell

basophil

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1. Basophils have a lifespan of ~60h in steady-state conditions

2. In our hands Th2 polarization (helminths, papain, OVA/alum) depends on DCs and not on basophils

3. IgE- or IgG1-mediated anaphylaxis occurs without basophils

4. Basophils are required for IgE-mediated chronic allergic inflammation of the skin

5. Basophils contribute to protection against helminths and ticks

Summary

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Acknowledgements

• R. M. Locksley (UCSF)

• B. Reizis (Columbia University)

• J. Lee (Mayo Clinic Scottsdale)

• H. Karasuyama (University of Tokyo)

• Caspar Ohnmacht

• Christian Schwartz

• Marc Panzer

• Isabell Schiedewitz

UniversitätsklinikumErlangen

[email protected]



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