STRATEGIES TO SENSE PATHOGENS AND DAMAGED TISSUES

(Bottazzi, Garlanda, Doni, Mantovani Annu Rev Immunol 2010)

Decoy: Colotta et al Science 1993

MyD88 etc in TLR: J. Exp Med 1998

TL

R1

-10

LPS, CpG,

Flagellin, Poly I:C... IL-33

-

IL-1

8R

IL-1

RI

IL-1

RII

IL-1

R

AcP

IL-1β IL-1β

IL-1α

- IL-1ras S

T-2

ILRs TLRs

-

TIR domain

Ig domain Leucine - rich repeat

domain MyD88

TIRAP/MAL

TRIF

SARM

TRAM

IL-1

R

AcP

IL-1F9

IL-1F8

IL-1F6

IL-1

Rrp

2

IL-1

8B

P

IL-1

8R

AcP

IL-18

IL-1F7

IL-18

IL-1

R

AcP

IL-1

R

AcP

- Negative

regulation

? Unknown ligand

IL-1

RA

PL

-

IL-1F5

? ? ?

TIG

IRR

-1

TIR

8/

SIG

IRR

-

IL-1F5

-

G

a

r

l

a

n

d

a

(Garlanda, Anders and Mantovani Trends Immunol 2009)

decoy

IL-1

IL-18

IL-33

LPS

CpG

nucleosomes

?? IL-1F5

???

NFkB

IL-1RI

IL-18R

ST2

TIR8/

SIGIRR

TLR4

TLR9

TLR7

??

MyD88

IRAK

Intestinal inflammation

Colitis – associated cancer

TIR domain

Death domain

Conserved aa

(S447, T536)

Replaced aa

(C222, L305)

Pulmonary TB

Autoimmunity

Asthma

Kidney transplantation

Arthritis

Fungal infections

Human:

Chromosome 11, 10 exons

mRNA: 1695 bp

Protein: 410 aa

Mouse:

Chromosome 7, 9 exons

mRNA: 1450 bp

Protein: 409 aa

(Garlanda, Anders and Mantovani Trends Immunol 2009)

Regulation of inflammation by TIR8/SIGIRR

Epithelial mucosal

cells, iDC, Endothelial cells

0 10 20 30 40 50 60 700

25

50

75

100 TIR 8-/-

TIR8 +/+

days

Pe

rce

nt

su

rviv

al

Pulmonary TB- Fungal Inf-

(L.Romani)

Intestinal inflammation and

colitis – associated cancer

Weeks

Perc

en

t S

urv

ival

+/+ -/-

TI

R

D

D

TI

R

TI

R IL-1RI

MyD88

IL-1ra

IL-1

TI

R

-

TI

R

D

D

TI

R

TI

R

MyD88

TI

R

-

LPS

Pro-inflammatory cytokines

Inflammatory chemokines

Leukocyte infiltration

(Garlanda et al PNAS 2005;

Garlanda et al Cancer Res 2007;

Garlanda et al JI 2007;

Lech et al JEM 2008)

Autoimmunity

TIR

8/

SIG

IRR

IL-1

8R

IL-1

8R

AcP

IL-18

IL-1F7

IL-1

RI

IL-1β

IL-1α

IL-1

R

AcP

IL-33

T1/S

T-2

IL-1

R

AcP

TIR

8/

SIG

IRR

TIR

8/

SIG

IRR

Th17 Th2 Th1

- IL-1ra

IL-1

RII

-

-

-

-

-

IL-1

8B

P

-

(Garlanda, Anders and Mantovani Trends Immunol 2009)

IL-8

GCP-2

NAP-2

ENA-78

GROα

GROβ

GROγ

IP-10

Mig

I-TAC

SDF-1α/β

BCA-1

BRAK

MCP-1

MCP-4

MCP-3

MCP-2

MIP-1β

MIP-1αS

MIP-1αP

RANTES

MPIF-1

HCC-1

HCC-2

HCC-4

Eotaxin

Eotaxin-2

Eotaxin-3

TARC

MDC

MIP-3α

ELC

SLC

I-309

TECK

CTACK

PARC

Lymphotactin

SCM-1β

Fractalkine

CXCL8

CXCL6

CXCL7

CXCL5

CXCL1

CXCL2

CXCL3

CXCL10

CXCL9

CXCL11

CXCL12

CXCL13

CXCL16

CXCL14

CCL2

CCL13

CCL7

CCL8

CCL4

CCL3

CCL3LI

CCL5

CCL23

CCL14

CCL15

CCL16

CCL11

CCL24

CCL26

CCL17

CCL22

CCL20

CCL19

CCL21

CCL1

CCL25

CCL27

CCL18

XCL1

XCL2

CX3CL1

CXCR1

CXCR2

CXCR3

CCR4

CCR5

Main targets

CXCR4

CXCR5

CXCR6

Unknown

CCR1

CCR3

CCR6

CCR7

CCR8

CCR9

CCR10

Unknown

XCR1

CX3CR1

CCR2

Chemokine Receptor

Neutrophil

Monocyte

Eosinophil

Basophil

NK cell

B cell

Immature

Mature

mDC

T cell

pDC

Naive

Memory

Th1

Th2

Treg

Inflammatory (RED), homeostatic (GREEN), mixed (YELLOW) (Mantovani, Bonecchi ,Locati Nature Rev Immunol 2006)

Silent vs conventional receptors

Atypical chemokine receptors

No conventional

signaling

(CTX, Ca, MAPK, …)

Signaling chemokine receptors

Signaling

(CTX, Ca, MAPK, …)

The “darc side” of the chemokine system

CXCR7

(Mantovani, Bonecchi and Locati,

Nature Rev Immunol 2006; CGFR 2009)

THE DECOY R D6: A SCAVENGER AND GATEKEEPER OF

INFLAMMATORY CC CHEMOKINE TRANSFER TO LYMPH NODES

Tissue

CCL2

Lymph

Endothelium

CCL2

D6

Lymph node

HEV Blood

(-/- data: Martinez de la Torre et EJI, 2005; PNAS 2007; Jamieson et al Nature Immunol 2005;

Liu et al J Immunol 2006. For review Mantovani, Bonecchi and Locati, Nature Rev Immunol 2006)

M. tuberculosis infection in D6-/-

M. tuberculosis (2 x 103 CFU) intranasal injection

WT (n=10)

D6-/- (n=10)

CFUs

(Di Liberto & Locati et al, J Exp Med 2008)

D6 AS A DECOY AND SCAVENGER: IN VIVO VERITAS

Colitis and colon cancer

Vetrano et al 2010

Mycobacterium tubercolosis

Di Libero Locati et al J Exp Med 2008

Martinez de la Torre Y et al, Eur J Immunol 2005

Granulomatous reaction to CFA injection

Martinez de la Torre Y et al, PNAS 2007

Fetal loss under inflammatory conditions

Jameson Nature Immunol 2005

Nibbs RJ, J Clin Invest 2007

Skin Inflammation and carcinogenesis

Whitehead Am J Res Crit C Med 2007

Otero K et al unpiblished

Allergic airway inflammation

Induction of EAE

(Liu et al JI 2006) Induction of EAE

(Liu et al JI 2006)

Negative regulators (D6; TIR8): 1. tune and balance resistance versus uncontrolled

inflammation; 2. resolution

TIR8 or D6-/-

Death

Inflammation Antimicrobial

resistance

Brakes: TIR8, D6……

Inflammation

TLR/ILR;

chemokines

Equilibrium and

long survival

Antimicrobial

resistance &

MyD88

DECOY RECEPTORS

(updated from Mantovani et al Trends Immunol 2001 )

Caspase/TRAF JAK/STAT

IL-1R TNF IL-10

JAK/STAT G protein

Argos

IL-4/IL13 Chemokines EGF Receptor

family

Core

signaling

Decoys

Decoy RII IL-1

IL-18BP IL-18

Osteoprotegerin RANK, TRAIL

DcR1, DcR2 TRAIL

DcR3 FASL, LIGHT

FDR FASL D6

Inflammatory

CC chemokines

IL-13R2

IL-13

IL-22BP

IL-22

TK

Human Genomic

clone 17 Kb Chr 3q25

Murine gene Chr 3

mRNA 1866 bp

Protein 381 aa

Protomer 43 kDa

Glycosylation 2 kDa Pentraxin domain X domain Signal

peptide

THE PROTOTYPIC LONG PENTRAXIN PTX3

Protein is

mainly

organized in

covalently

linked octamer

Inforzato et al, JBC 2008

(Garlanda et al Annu Rev Immunol 2005, 2010; Bottazzi et al Curr Op Immunol, 2006, 2008)

(Inforzato et al, in revision on J. Biol. Chem.)

Visualization of the rhPTX3 protein by TEM

Shape of the PTX3 protein

Transmission Electron Microscopy (TEM)…

Shape of the PTX3 protein

…and Small Angle X-ray Scattering (SAXS).

(Inforzato et al, in revision on J. Biol. Chem.)

SAXS analysis of rhPTX3 and an ab initio model of the PTX3 protein

THE PENTRAXIN SUPERFAMILY

(Garlanda, Bottazzi and Mantovani

Annu Rev Immunol 2005)

"The serum obtained from human

beings and monkeys during the acute

phase of diverse infections contains a

protein which is precipitable by the C

polysaccharide of pneumococcus"

Abernathy and Avery, J. Exp. Med., 1941

Tillet and Francis, J. Exp. Med, 1930

THE PENTRAXIN SUPERFAMILY

(Garlanda, Bottazzi and Mantovani

Annu Rev Immunol 2005)

SELECTIVE SUSCEPTIBILITY OF

PTX3 -/- MICE TO A.FUMIGATUS

A.FUMIGATUS CLP L. MONOCYTOGENES

(Garlanda et al Nature 2002)

REGULATION OF PTX3 PRODUCTION IN MYELOMONOCYTIC CELLS

HA MATRIX

DEPOSITION

Mø myeloid

DC

TLR AGONISTS

(e.g. LPS)

PRIMARY INFLAMMATORY

CYTOKINES (IL-1, TNF)

HUMORAL INNATE IMMUNITY

(C; OPSONIZATION VIA FcgR;

NEUTRALIZATION)

IL-10 IFNg - +

TUNING

INFLAMMATION

(Bottazzi et al Annu Rev Immunol 2010)

Modified from Biswas and Mantovani Nature Immunol 2010

The yin-yang of macrophage polarization

M1 M2-like

- intracellular pathogens

- tissue damage

- tumor resistance - parasite encapsulation

- tissue remodeling

- tumor promotion

- immunoregulation

Ligands recognized by PTX3

- Microbial ligands: OMPA, galactomannan and more

- Apoptotic cells

- Complement: C1q, Factor H

- FcgR

- ECM

Outer Membrane Protein A

(Bottazzi et al JBC; Jeannin et al Immunity 2005; Rovere et al Blood 2004; Garlanda et al Nature 2002; Lu et al Nature 2009)

Synergy between Ficolin-2 and PTX3 boosts innate immune recognition

and complement deposition

Ma Y J et al. J. Biol. Chem. 2009, L-ficolin; see also Gout et al J Immunol 2011, M-Ficolin

C4 deposition depends on Ficolin-2 and Ficolin-2/PTX3 collaboration

Ficolin-2/PTX3 cross-talk on A. fumigatus Ficolin-2 isolation from

human plasma by

immobilized PTX3

Classical pathway Antibodies, Pentraxins

Lectin pathway MBL, Ficolins

Alternative pathway C3b-microorganisms

C1q,

C1r,

C1s MASPs

C3b

C4, C2

Factor B,

Factor D,

Properdin C3 and C5

convertases C3a, C5a

C5b-C9

C3b

Inflammation Membrane

attack complex,

lysis

Opsonization

Amplification loop

PENTRAXINS (CRP, SAP, PTX3)

Factor H

C1q

Ficolin

MBL

CRP

SAP

PTX3

Figure 2

(Bottazzi, Garlanda, Doni,

Mantovani, Annu Rev Immunol 2010)

(Jaillon et al, JEM 2007)

Localization of PTX3 in human Neutrophil granules

Localization of PTX3 in Neutrophil Extracellular Traps (NETs)

(Jaillon et al, JEM 2007)

Box 2 | Neutrophil extracellular traps

Neutrophils in the activation and regulation of innate and adaptive immunity

Mantovani A, Cassatella MA, Costantini C, Jaillon S.

Nat Rev Immunol. 2011 Jul 25;11(8):519-31. Review.

Figure 1 | Neutrophils crosstalk with immune and non-immune cells in

inflamed tissues and lymph nodes.

Neutrophils in the activation and regulation of innate and adaptive immunity

Mantovani A, Cassatella MA, Costantini C, Jaillon S.

Nat Rev Immunol. 2011 Jul 25;11(8):519-31. Review.

INNATE IMMUNE RESPONSE

Cellular

innate

immunity

Cytokines,

chemokines,

other effector molecules

C activation,

opsonization,

agglutination

hrs

min TLR

SCAVENGER R

DECTIN-1

CD14

MØ/DC

Microbe, eg

OMPA,

galactomannan

Microbe

COMPLEXITY AND COMPLEMENTARITY OF

CELLULAR AND HUMORAL INNATE IMMUNITY

PMN

Humoral

amplification of

innate immunity

PTX3

(Garlanda et al Nature 2002;

Jeannin, Bottazzi et al

Immunity, 2005; Jaillon et al

J Exp Med 2007)

PAMP

Eg OMPA

JH Lu et al. Nature 000, 1-4 (2008) doi:10.1038/nature07468

Crystal structure of SAP–FccRIIa complex.

PT

X3

CD

11b N

ucle

i

PT

X3

CD

32 N

ucle

i

PT

X3 C

D11b N

ucle

i

PT

X3

CD

11b C

D32

w

PT

X3

w/o

PT

X3

PT

X3 C

D32 N

ucle

i

w PTX3

w/o PTX3

RECRUITMENT OF ACTIVE CD11b AND CD32 IN THE PHAGOCITIC CUP BY PTX3

ROLE OF PTX3 IN FITC-CONIDIA PHAGOCYTOSIS BY ALVEOLAR NEUTROPHILS

A.fumigatus

conidia

FITC-conidia

+

FITC

8X107

i.t.

4h

BAL FITC intensity in leucocytes

(CD45+) and neutrophils

(CD11b+ Ly6G+)

(Moalli et al)

MECHANISM OF ACTION FOR PTX3 OPSONIZING ACTIVITY

(Moalli et al Blood 2010)

PTX3: LESSONS FROM GENE TARGETING

NON-REDUNDANT FOR:

-FEMALE FERTILITY (ASSEMBLY OF HA-RICH MATRIX; TSG6)

-RESISTANCE AGAINST SELECTED PATHOGENS: AN ANTE-ANTIBODY

(A. fumigatus, P Brasiliensis, P. aeruginosa, K pneumoniae, CMV, Influenza)

-TUNING INFLAMMATION AND IMMUNITY

A UNIQUE, SOLUBLE, MULTIFUNCTIONAL

PATTERN RECOGNITION RECEPTOR

(eg Garlanda et al Nature 2002; Salustri et al Development 2004; Garlanda et al Annu Rev Immunol 2005;

Bottazzi et al Curr Opinion Immunology 2006; Jeannin et al Immunity, 2005; Soares et al Microbes Inf 2006

Romani et al Blood 2006; Reading et al J Immuno 2007; Jaillon et al JEM 2007)

INNATE IMMUNITY - INFLAMMATION

ASN220

Chr 3q25

mRNA 1866 bp

Prot 381 aa

PTX signature

(HxCxS/TWxS)

Ex 1 Ex 2 Ex 3

SP NTD PTX

OmpA Mannans

other

C1q Factor H

FcγR CD11b

? Ficolin-2

Microbe recognition

C activation Opsonization Regulation of inflammation

Repertoire synergism

(Bottazzi et al Annu Rev Immunol 2010)

PTX3 binds P-selectin

(Deban et al., Nat. Immunol. 2010)

PTX3 binds P-selectin via C-terminal (pentraxin)

domain, in a glycosylation-dependent

manner

Kd 360 ± 30 nM (mean of four experiments)

Model of acid-induced acute lung injury (ARDS)

PTX3 attenuates in vivo leukocyte recruitment

(Deban et al., Nat. Immunol. 2010)

In four models of P-selectin-dependent recruitment (pleurisy, ALI/ARDS, thrombin mesenteric inflammation for intravital microscopy, atherosclerosis) and in cancer, genetic deficiency of PTX3 is associated with increased recruitment of PMN or macrophages (TAM)

(Deban et al Nature Immunol 2010 in press; Garlanda et al unpublished data)

Impairment of tethering and rolling on activated endothelium

PTX3

PSGL-1

Psel

Activated endothelium

TLRs TNF-α IL-1

+ IL-10

PTX3

Macrophages

Dendritic cells

Monocytes

PTX3 production in tissues

SYSTEMIC INFLAMMATION

LOCAL INFLAMMATION

PTX3- mediated regulation of leukocyte recruitment

(Deban et al., Nat. Immunol. 2010)

INNATE IMMUNITY - INFLAMMATION

ASN220

Chr 3q25

mRNA 1866 bp

Prot 381 aa

PTX signature

(HxCxS/TWxS)

Ex 1 Ex 2 Ex 3

SP NTD PTX

OmpA Mannans

other

C1q Factor H

FcγR P-selectin Ficolin-2

Microbe recognition

C activation Opsonization Regulation of leukocyte

recruitment and inflammation

Repertoire synergism

(Bottazzi et al Annu Rev Immunol 2010)

PTX3 TRANSLATION

• Diagnostic/prognostic (ELISA,genetics)

• Therapy

PTX3 IN HUMAN PATHOLOGY

• Inflammatory/vascular ( acute myocardial infarction; heart

failure etc; ALI/ARDS; small vessel vasculitis; eclampsia; renal

pathology)

• Infectious (sepsis; meningococcal infection; dengue;

leptospirosis; TB; malaria; monitoring fungal infections in

leukemia)

• Genetic polymorphisms (TB; P. aeruginosa; pregnancy)

Plasmatic PTX3 levels correlate with evolution of disease and

response to antifungal treatment

Effect of PTX3 in mice with

invasive aspergillosis

Lethally irradiated C3HHeJ mice were

infused with 2x106 T-cell depleted

allogeneic bone marrow cells from BALBc

mice a week before infection with 2x107

Aspergillus conidia. Mice were treated for

5 days before (Pre) or after (Post) the

infection or in concomitance with the

infection.

Dosage:

PTX3 0.04 or 0.2 mg/kg

L-AMB 1 mg/kg

D-AMB 2 mg/kg

PTX3 increases

the therapeutic

effect of L-AMB

and D-AMB

Survival rates and lung fungal

burdens of rats infected with

A. fumigatus and treated with

PTX3 (n = 16).

(A) daily treatment 3 days before

the day of infection and then

for an additional 3 days.

(B) (B) tratment started the day of

infection and then daily for an

additional 3 days.

____ 1.5 mg/kg PTX3;

------- 0.15 mg/kg PTX3; …….

saline.

(C, D) Fungal burden

(galactomannan GMI).

Synergism -

regulation

Activated Mø, PMN, DC

Cellular innate immunity

Cytokines, chemokines,

other effector molecules

days min

epithelia

hr

Microbes-damaged tissues

Cellular sensors: TLR and coreceptors; NLR; RLR; capturing R

DC Mø PMN Endothelium

Humoral, fluid phase, innate immunity

C activation; opsonization; agglutination and

neutralization; regulation of inflammation

PTX3

Properdin

Ficolins

PTX3, C,

M-Ficolin,

SAA3

Pentraxins (CRP, SAP, PTX3)

Collectins (C1q, SP-A, SP-D, MBL)

Ficolins, SAAs

Constitutive and inducible humoral sensors

Figure 6

(Bottazzi, Garlanda, Doni, Mantovani, Annu Rev Immunol 2010)

Antibodies PTX3

Glycosilation-dependent

regulation of inflammation

PTX3 as functional ancestor of antibodies

(ante-antibody)

Agglutination and

neutralization

Opsonization

(FcgR)

Complement

activation

(Bottazzi et al Annu Rev Immunol 2010)

Istituto Clinico Humanitas/ University of Milan Livija Deban Marina Sironi Federica Moalli Sonia Valentino Ivan Cuccovillo Cecilia Garlanda Andrea Doni Barbara Bottazzi Universidade Federal de Minas Gerais, Belo Horizonte Remo Castro Russo Istituto Mario Negri Antonio Bastone Marco Gobbi Vanessa Zambelli Margherita Scanziani Consorzio Mario Negri Sud Gino Evangelista

University of Verona Carlo Laudanna Gabriela Constantin Tecnogen S.p.A Marica Sassano Sigma-Tau Research and Development Giovanni Salvatori Peter Garred Nicole Thielens

Sigma Tau S.p.A. Dr. Giovanni Salvatori

Dr. Ragnar Lindstedt

Istituto clinico “Humanitas”

Università di Milano Prof. Alberto Mantovani

Dr. Barbara Bottazzi

Acknowledgments

Tecnogen S.p.A. Dr. Antonio Verdoliva

Dr. Vincenzo Rivieccio

University of Manchester Prof. Tony Day

All the guys in Day’s group

Dr. Tom Jowitt

Prof. Karl Kadler

Dr. David Holmes

Dr. Clair Baldock

Funding bodies: Federazione Italiana per la Ricerca sul Cancro (FIRC)

Istituto clinico “Humanitas”

Sigma Tau S.p.A.

Tecnogen S.p.A.