Immunomodulatory Pathwaysin Atherosclerosis

Ziad Mallat, MD, PhD

Department of Medicine, University of Cambridge,Cambridge, UK

Inserm U970

Moore KJ & Tabas I. Cell 2011

Targeting the Immuno-Inflammatory ResponseIn Atherosclerosis

Weber C et al. Nat Med 2011

Weber C et al. Nat Med 2011

Wilensky RL et al., Nat Med 2008

Serruys PW et al., Circulation 2008

Weber C et al. Nat Med 2011

Ridker PM et al. Circulation 2011

JCI 2012

Weber C et al.Nat Rev Immunol 2008

Peptide-based Therapeutic Vaccination

Therapeutic ToleranceAuto-Immunity

Larché M & Wraith DC

Nat Med 2005

Inducing tolerance to lipoproteins:

Atherosclerosis Vaccine?

ApoB peptideOVA ApoB mix

ApoB peptide-based vaccination reduces atherosclerosis by inducing a specific Treg cellresponse

OVA

P210 ApoB

ApoB

P210/P240/MDA-P210

ApoB Mix

Peptides infusion10μg/day

Follow-up

2 weeks 8 weeks

Weber C et al.Nat Rev Immunol 2008

Binder CJ et al., JCI 2004

B cell pathways in atherosclerosis

Lahoute et al. Nat Rev Cardiol, 2011;8:348-58.

Mackay FNat Rev Immunol 2009

They respond to T cell–dependent antigens and contribute to adaptive

immunity

They respond to T cell–indedependentantigens, selectivity for self antigens,

producers of natural IgM

CD20 mAb-mediated B Cell Depletion in Atherosclerosis

Decreased T cell infiltration after anti-CD20 therapy

CTR -CD20

2010

Ait-Oufella et al.

BAFF – B cell activating factor

BAFF

BAFFR

B Cell Physiology

B2 cell survivalClass switchingPlasma CellsAutoreactive B cells

Autoimmune Diseases

SLERheumatoid ArthritisSjogren’s SyndromeAnti-Phospholipid syndrome

T CellsMyeloid cells Atherosclerosis?

BAFF Levels in Human Carotid Lesions and

Relation with Plaque Phenotype

P<0.001

Lesion Size

Macrophages T lymphocytes

B cell-selective

BAFF-R deletion

White HD & Chew DP, The Lancet 2008

Acute Myocardial Infarction

Post-MI Inflammation

Blood

Ischemic

Myocardium

Pro-Inflammatory cytokines

High Proteolysis

Anti-inflammatory cytokines

Angiogenesis

ApoptosisCD11b+

Ly6G+

CCR2+Ly6Chi

7/4hi

CX3CR1lo

InflammatoryMonocytes

ResidentMonocytes

CX3CR1hi

CCR2-

Ly6Clo

7/4lo

Role of B lymphocytes in immuno-inflammatory response and tissue remodelling after myocardial infarction

Kinetics of inflammatory cell infiltration after Myocardial Infarction

Digestion

(Col I, XI, Hyaluronidase, DNase)

Gradient Density

Centrifugation

FACS

Analysis

LCAligation

7/4HI and 7/4LOW Monocytes

0

1000

2000

3000

4000

7/4LOW MI

7/4HI MI7/4HI MI sham

7/4LOW MI sham

Cel

ls/m

g tis

sue

**

***

***

***

**

Days post-MI

MIsham

Neutrophils

(CD11b+ Ly6G+ 7/4HI)

Cel

ls/m

g tis

sue

0

2000

4000

6000

8000***

***

Days post-MICD3+ T lymphocytes

0

50

100

150

200

250

300MIsham

***

***

Days post-MI

Kinetics of B lymphocytes infiltration after Myocardial Infarction

Flow cytometry analysis

Sham D5 D5 after MI D14 after MI

B220 Immunostaining

B22

0Hi I

gM+

BC

ells

/mg

tissu

e

Heart

0

500

1000

1500

Days post-MI

MIsham

***

Anti-CD20 antibody treatment reduces B lymphocyte levels

200g/mouse

1 hour after MI

B22

0Hi I

gM+

B

Cel

ls/m

l Blo

od [x

104 ] PBS

Anti-CD20

0

1

23

4

5

6

Days post-MI

Blood

Anti-CD20

IgM+

CD20 mAb

Mature B cells

*

**

* *

0

50

100

150

200

250

PBS Anti-CD20

B22

0Hi I

gM+

BC

ells

/mg

tissu

e

Heart D3

0

250

500

750

1000

1250

PBS Anti-CD20B

220H

i IgM

+B

Cel

ls/m

g tis

sue

*

Heart D5

0 102 103 104 105

0102

103

104

105

0 102 103 104 105

0102

103

104

105

PBS D3 CD20 D3

B220

IgM

B22

0Hi I

gM+

B

Cel

ls/s

plee

n [x

105 ]

25

0

5

10

15

20

Spleen D14

B lymphocyte depletion limits adverse LV remodelling

0

10

20

30

40

50

PBS Anti-CD20

% In

farc

t siz

e

Infarct Size (Masson Trichrome staining)

PBS Anti-CD20D14

0

10

20

30

40

% S

F

*

Shortening Fraction

D14

0

0.1

0.2

0.3

0.4

PBS Anti-CD20

*

0

0.025

0.05

0.075

0.1

0.125

PBS Anti-CD20

*

LVID

s (m

m)

LVPW

s (m

m)

LV internal dimension in diastole LV Posterior Wall Thickness in systole

D14 D14

*

0

0.5

1

1.5

**

0

0.5

1

1.5

PBS -CD20

*

0

0.5

1

1.5

2

PBS -CD20 0

0.5

1

1.5

PBS -CD20

*

mR

NA

leve

ls(A

.U.)

Heart D14

TNF- IL1- IFN- IL18

mR

NA

leve

ls(A

.U.)

PBS -CD200

0.5

1

1.5

*

TNF-

0

0.5

1

1.5

PBS -CD20

IL1-

0

0.5

1

1.5

*

PBS -CD20

IFN-

0

0.5

1

1.5

*

PBS -CD20

IL18

Spleen D14

B lymphocyte depletion reduces systemic and local post-MI inflammation

B lymphocyte depletion alters monocyte distribution in post-MI setting

0

12

3

45

PBS -CD200

5

10

15

20

25

PBS-CD20

*

Mon

ocyt

es 7

/4H

I

Cel

ls/m

l x1

06

Mon

ocyt

es7/

4LOW

Cel

ls/m

l x1

05

Monocytes 7/4HI Monocytes 7/4Lo

Bone Marrow D3

0

2.5

5.0

7.5

10

PBS Anti-CD20 0

1

2

3

4

PBS Anti-CD20

Mon

ocyt

es 7

/4H

I

Cel

ls/m

l x1

04

Mon

ocyt

es 7

/4LO

W

Cel

ls/m

l x1

04

* *

Monocytes 7/4HI Monocytes 7/4Lo

Blood D3

plasma

0

250

500

750

1000

D0 D1 D3 D5 D7

***

MCP-1/CCL2

MCP-1/CCL2Not Detected inB cell supernatants

B lymphocyte depletion selectively reduces MCP-3 levels in post-MI

MCP-3/CCL7plasma

7/4 Hi Monocytes transmigration assay

Lower compartment :

- Medium RPMI 10% SVF

- B cells (2.106)

- -CD40 and IgM-treated B lymphocytes

Transwell insert (8 m)

7/4 Hi monocytes

Microporous col I coated membrane

B lymphocytes

4h

B lymphocytes trigger 7/4 Hi monocytes migration

Control B cells Activated B cells

MCP1 MCP3

020406080

100120140160180

Control B cells ActivatedB cells

ActivatedB cells

+MCP1

***

*** ***

ActivatedB cells

+MCP3

D-7 D0 D14

Injection splenocytes WT,B cell-depleted splenocytes ±

WT or MCP-3-deficient B cells

MI

D3

FACS Echocardiography

Exogenous administration of B lymphocytes promotes adverse LV remodelling

Rag1-/-

B cells levelsSpleen D3

SplenoWT

SplenoCD20+WT

B cells

SplenoCD20

SplenoCD20

+MCP3-/-

B cells

0

5

10

15

20

25

***

###

Exogenous administration of B lymphocytes enhances 7/4Hi monocytes mobilisationand infiltration into the ischemic heart

Exogenous administration of B lymphocytes promotes adverse LV remodelling

FAST-MI

• FAST-MI is a nationwide French registry carried out in 3059 consecutive pts

with AMI admitted in 223 CCUs

• 100 centers, which included 1036 patients, participated in the serum databank.

• Outcome events were defined as all-cause death, recurrent AMI and

incident stroke

• The 24-month follow-up of mortality was complete for 95% of patients

170 events occurred during follow-up

Impact of circulating levels of BAFF/CCL7 on 24 months-survival, recurrent myocardial infarction and incident stroke in patients with acute MI

B Lymphocytes Trigger CCL7-Dependent Monocyte Mobilisation and Promote

Adverse Ventricular Remodelling afterAcute Myocardial Infarction

B cell depleting and CCL7-targeting therapies may be cardioprotective

Zouggari Y et al., Nature Medicine, In Press

B2 cell depleting agents

Anti-IL1beta

PLA2 inhibitors

Peptide-based vaccination

University of CambridgeBritish Heart Foundation

Andy SAGEXuan LIDeirdre MURPHYLauren BAKERJames HARRISONLeanne MASTERS

Inserm U970Alain TEDGUIHafid AIT-OUFELLAOlivier HERBINPatrick BRUNEVAL

Jean-Sébastien SILVESTREYasmine ZOUGGARI

CeMM, Medical Universityof Vienna, AustriaChristoph J. BINDER

AP-HP, Pierre et Marie Curie UniversityTabassome SIMON

Duke University, USAThomas F. TEDDER

University of Utrecht,The NetherlandsGerard PASTERKAMPUCSF, USA

Israel F. CHARO

Rader DJ & Daugherty A. Nature 2008

O

O

C R1

O

O

C R2

CH2

CH

CH2O

O

P O

O-

R Fatty Acids

Phosphate Group

Glycerol Head

PLA2

PLA2 enzymes hydrolyze phospholipids at the

sn-2 position to generate lysophospholipids and

fatty acids

sn-1

sn-2

Kolodgie et al., Arterioscler Thromb Vasc Biol 2006

sPLA2 inhibitor acts synergistically with statinto decrease atherosclerosis

apoE-/- mice treated with varespladib (A-002) +/- pravastatin

Shaposhnik et al. J Lipid Res, 2009, 50: 623–629.

Effects of 1-H-indole-3-glyoxamide (A-002, (Varespladib, Anthera Pharmaceuticals) on concentration of sPLA2 (PLASMA study): a phase II double-blind, randomised,

placebo-controlled trial

Rosenson et al. Lancet, Vol 373 February 21, 2009

393 patients randomly assigned received placebo (n=79) or the sPLA2 inhibitor, A-002: 50 mg (n=79), 100 mg (n=80), 250 mg (n=78), or 500 mg (n=77) twice daily, for 8 weeks.

Primary endpoint:change in sPLA2 IIA concentration or activity from baseline to week 8

Results: Dose dependent reduction in sPLA2-IIA concentration in the A-002 groups (from 69±2% in the 50 mg group to 95 ± 8% in the 500 mg group), significantly different from placebo (p<0.0001)

B lymphocyte depletion limits adverse LV remodelling

Apoptotic cells number (Tunel)

0

5

10

15

20

PBS Anti-CD20

Tune

l cel

ls/fi

eld

*

PBS Anti-CD20D14

Capillary Density (BS1 lectin, WGA)

0

0.5

1

1.5

2

capi

llarie

s/m

yocy

te *PBS Anti-CD20 D14

Arteriolar Density (-actin)

0

5

10

15

20

PBS Anti-CD20

Arte

riole

s/m

m2

PBS Anti-CD20 D14

0

123456

WT MCP3KO

**

Monocytes 7/4HI

Blood D0

0

2

4

6

8

10

*

WT MCP3KO

Blood D14 BM D14

Cel

l/fem

urx1

05

WT MCP3KO0

2468

10121416 *

MCP-3 deficiency preserves LV function after acute MI

Shortening Fraction

0

10

20

30

40

% S

F

*D14