STRATEGIES TO SENSE PATHOGENS AND DAMAGED TISSUES (Bottazzi, - - PowerPoint PPT Presentation

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

TLR1-10 LPS, CpG, Flagellin, Poly I:C... IL-33

• IL-18R

IL-1RI IL-1RII IL-1R AcP IL-1β IL-1β IL-1α

• IL-1ras

ST-2

ILRs TLRs

• TIR domain

Ig domain Leucine - rich repeat domain MyD88 TIRAP/MAL TRIF SARM TRAM

IL-1R AcP IL-1F9 IL-1F8 IL-1F6 IL-1Rrp2 IL-18BP IL-18R AcP IL-18 IL-1F7 IL-18 IL-1R AcP IL-1R AcP

• Negative

regulation ? Unknown ligand

IL-1RAPL

• IL-1F5

? ? ?

TIGIRR-1 TIR8/ SIGIRR

• IL-1F5
• (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

10 20 30 40 50 60 70 25 50 75 100

TIR 8-/- TIR8 +/+ days

Percent survival

Pulmonary TB- Fungal Inf- (L.Romani) Intestinal inflammation and colitis – associated cancer

Weeks

Percent Survival

+/+ -/-

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

TIR8/ SIGIRR IL-18R IL-18R AcP IL-18 IL-1F7 IL-1RI IL-1β IL-1α IL-1R AcP IL-33 T1/ST-2 IL-1R AcP TIR8/ SIGIRR TIR8/ SIGIRR

Th17 Th2 Th1

• IL-1ra

IL-1RII

• IL-18BP
• (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)

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-13R2 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

• rganized 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 collabo 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,

• ther effector molecules

C activation,

• psonization,

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.

PTX3 CD11b Nuclei PTX3 CD32 Nuclei PTX3 CD11b Nuclei PTX3 CD11b CD32

w PTX3 w/o PTX3

PTX3 CD32 Nuclei

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

• ther

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

• ther

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,

• ther 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.