Modena March 3, 2009

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A

A novel molecular marker

novel molecular marker

ove

o ecu

e

of severe asthma

Sergio Bonini Sergio Bonini

Professor of Medicine, Second University of Naples Second University of Naples INMM-CNR, ARTOV, Rome

b i i@ il

[email protected]

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Circulating nerve growth factor levels are increased in humans with allergic diseases and asthma

with allergic diseases and asthma

Bonini S, Lambiase A, Bonini S , Angelucci F, Magrini L, Manni L,Aloe L

PNAS 1996; 93: 10955-10960

•Increased NGF circulating levels in allergic diseasesIncreased NGF circulating levels in allergic diseases

•Relationship to tissue mast cells and plasma Substance P

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NGF and NGF receptors in allergic inflammation

and tissue remodelling

B

APC

Fibroblasts

B Th

Bonini S et al. PNAS 1996; 93: 10955

MC

Eos SC

Epithelial cells

P ll i i G t l J C ll Bi l 1999 145 769

Bonini S et al. PNAS 1996; 93: 10955 Lambiase A et al. JACI 1997; 100: 408

Solomon A et al. JACI 1998; 102: 454

Multipotent progenitors

Lambiase A et al. N Engl J Med 1998; 338: 1174

L bi A t l A h O hth l l 2000 188 1446

Pellegrini G et al. J Cell Biol 1999; 145: 769 Lambiase A et al IOVS 1998; 39: 1272

Lambiase A et al. Arch Ophthalmol 2000; 188: 1446

Micera A et al. PNAS 2001; 98: 6162

Manni L et al. Clin Exp Rheumatol 2003; 21: 617 Micera A et al. Clin Exp Allergy 2005; 35: 650

Mi A t l E E R 2006 83 747

Bracci-Laudiero L et al.

J N i l 2003 136 130 Micera A et al. Exp Eye Res 2006; 83: 747 Micera A et al. Mol Vis 2007; 13: 981

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Fibroblasts produce, store,

release and respond to NGF

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Study models

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Biopsies from human fibrotic lung and

ki

ti

skin scar tissues

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Coltures of lung, skin and eye fibroblasts

g

y

•

In vivo effects of NGF in humans

Lambiase et al, NEJM 1998; Bernabei et al, Lancet 1999; Lambiase et al, Arch Ophthalmol 2000; Micera et al PNAS 2001

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Micera et al

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Micera et al

PNAS 2001; 98: 6162 67 PNAS 2001; 98: 6162–67

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Micera et al

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Lambiase A, Rama P, Bonini S, Caprifoglio G, Aloe L

Topical treatment with nerve growth factor for corneal neurotrophic ulcers

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Epithelial cells produce, store,

release and respond to NGF

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NGF production by human corneal epithelial cells

NGF TrkA p75NTR 1Kb+ NG p75 500 bp 650 bp NGF = 368 bp 200 bp 300 bp 400 bp p TrkA = 246 bp p75NTR= 521 bp 100 bp

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NGF

NGF CORNEAL LEVELS CORNEAL LEVELS

FOLLOWING EPITHELIAL DEBRIDEMENT FOLLOWING EPITHELIAL DEBRIDEMENT

4000

FOLLOWING EPITHELIAL DEBRIDEMENT FOLLOWING EPITHELIAL DEBRIDEMENT

control * lesioned 3000 ) * * 2000 NGF (pg/gr ) 1000 Hours 0 2 12 24 48 96 168 Ba seline Lambiase A, IOVS 2000

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Functional effects of NGF

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Relative changes of p63 delta mRNA in human conjunctival cells primary culture after NGF stimulation

1,2

(evaluated by Real Time PCR)

1 0,8 0,6 0,4 0,2 0

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Relative changes of mucins mRNAs in human conjunctival cells primary culture after NGF stimulation

primary culture after NGF stimulation (evaluated by Real Time PCR)

control (1%FBS) 18,00 NGF (10 ng/ml) NGF (100 ng/ml) 14,00 16,00 10,00 12,00 6,00 8,00 2,00 4,00 0,00

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NGF in Tissue Remodeling

A novel function of NGF:

a regulatory role in mucus

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Mucus and Allergic Diseases

• Mucus is an aqueous solution of lipids and proteins

coating the mucosal surfaces and representing an important barrier against pathogens

• Mucin (MUC) glycoproteins represent the major (10-( ) g y p p j (

50% wt/wt) component of mucus, and the major

determinant of its visco-elastic propertiesp p

• In humans, MUC are encoded by at least 20 genes

whose protein backbone transcripts are secreted by whose protein backbone transcripts are secreted by epithelial cells and then extensively glycosilated

• In the airways MUC 5AC (preferentially secreted by

• In the airways, MUC 5AC (preferentially secreted by

goblet cells) and MUC 5B (preferentially secreted by submucosal glands) are the most represented mucins submucosal glands) are the most represented mucins, increasing in pathological states

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Regulation of mucus production

Mechanisms Differentiation of epithelial cells into

mucus producing cells (mucus cells mucus producing cells (mucus cells hyperplasia and/or metaplasia)

Hypersecretion (mucin genes

overexpression, goblet cell

degranulation, etc.)

Regulatory factorsg y Bacteria; Pollutants and chemicals;; ;

Biological products; Cytokine (IL-1β

IL-4,IL-6, IL-9, IL-13, TNFα, IFNγ);, , , , , γ);

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9000

NGF LEVELS IN PLASMA AND TEARS OF VKC PATIENTS

NGF LEVELS IN PLASMA AND TEARS OF VKC PATIENTS

7000 9000 control 5000 VKC 3000 300 NGF p g /ml 1000 200 100 0 Plasma Tear

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Study models

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Transgenic mice overexpressing NGF at

the level of Clara cells

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Effects of NGF on human epithelial cell

coltures

E ith li l diff ti ti

– Epithelial differentiation

– Mucin gene expression

– Mucin protein content and release by

goblet cells g

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Hyperinnervation of the Airways in

Transgenic Mice Overexpressing

Nerve Growth Factor

Gary W. Hoyle, Regina M. Graham, Jeffrey B. Finkelstein, Kim Phoung Thi Nguyen David Gozal and Mitchell Freidman Kim-Phoung Thi Nguyen, David Gozal and Mitchell Freidman

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Goblet cells in gut of NGF transgenic mice

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Modulation of mucus production in a

transgenic mouse model of asthma

Groups

Mice

Treatment

1 (PBS/NGF-tg) NGF-tg PBS/OVA 2 (OVA/NGF-tg) NGF-tg OVA/OVA 3 (wt) C57/BL6 (wt) PBS/PBS 4 (OVA) C57/BL6 (wt) OVA/OVA 5 (PBS/NGF-tg)( g) NGF-tgg PBS 6 (OVA/NGF-tg) NGF-tg OVA

Bonini S. et al, J Allergy Clini Immunol 2006 ;117:S250 Bonini Se, Lambiase A, Renz H et al. in preparation

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Mucins expression in NGF transgenic mice

10 20 30 40 n e e xp res si o n A 4 5 6 7 e x p re ssi o n D -10 0 10

MUC1 MUC4 MUC5ac

ta rg e t g e n

Rest analysis: NGF-tg mice vs Wt

0 1 2 3

MUC1 MUC4 MUC5ac

ta rg e t ge ne e

Rest analysis: OVA/NGF-tg mice vs. OVA

E MUC5ac M 3 4 5 6 7 n e e xp ress io n B 20 40 60 80 m uc 5A C , O D 0 1 2 3

MUC1 MUC4 MUC5ac

ta

rg

e

t ge

n

Rest analysis; OVA mice vs Wt

4 5 6 7 e x p ressi o n C NGF-TG WT Sensitization/Challenge 0 PBS OVA PBS OVA m F 0 1 2 3

MUC1 MUC4 MUC5ac

ta

rge

t ge

ne

e

Rest analysis: OVA/NGF-tg mice vs PBS/NGF-tg mice

U C5ac , X600 F M U Isotype NGF-tg OVA- NGF-tg

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NGF promotes epithelial cell differentiation to goblet cells

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Mucus metaplasia

d i

Epithelium

and mucin gene expression

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Collaborations

Stefano Bonini, Alessandra Micera, Alessandro Lambiase

Eye Clinic, University Campus Biomedicus Rome, Italy Eye Clinic, University Campus Biomedicus Rome, Italy

Megon Bresciani, Marie France Lalibertè, François Lalibertè,

CHU Arnaud de Villeneuve, CHU Arnaud de Villeneuve, France CHU Arnaud de Villeneuve, CHU Arnaud de Villeneuve, France

Claudia Gramiccioni, University of Foggia, Institute of Respiratory

Diseases, Italy Diseases, Italy

Harald Renz, Dept. of Clinical Chemistry and Molecular

Diagnostics, Philipps-University Marburg, Germany Diagnostics, Philipps University Marburg, Germany

Francesca Levi-Schaffer, Dept. of Pharmacology, School of

Pharmacy Hadassah Med School Hebrew Univ Jerusalem Pharmacy, Hadassah Med. School, Hebrew Univ., Jerusalem, Israel

Abraham Solomon Joseph Frucht–Pery Jacob Pe’er

Abraham Solomon, Joseph Frucht–Pery, Jacob Pe er

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