Studies of Chronic Aspiration

GERD Associated Asthma

Asthma

Chronic inflammatory disease

Recurrent respiratory symptoms,

wheezing, breathlessness, chest tightness, coughing,

Increase airway mucus secretion

Increase smooth muscle hyper-reactivity

Inflammatory cells involved: T lymphocyte, eosinophil, mast cells, epithelial cells

Risk Factors

Disease inception: viral infection

Enviromental exposure: allergen, pollution, tobacco smoke

Life style: living on farm, diet, antibiotic use

Comorbid condition: atopic dermis, obesity

Excerbating Factors

Allergens: Mites, trees, grasses, animal danger

→ chronic low level exposure to indoor allergens ( house dust ,cockaroach , Alternaria species)

► fur pet exposure reduce future risk

► pollen immunotherapy in school age also reduce

future risk

Infections

Chlamydis , Mycoplasma,Early childhood inceltion, infection of virus in established asthma

ExerciseCausing airway obstruction, bronchoapasm

Non steroid anti-inflammatory drug

→ the response to aspirin or NSAIDS , eve laceimation, severe

bronchospasm

→ reduced prostaglandin production for normal lung function

Psychological factors

→ Parental stress

Gastroesophageal reflux( GERD)

→ 45-60% of adult and children with asthma has GERD

→ microaspiration or irritation of esophagus with reflux bronchispasm

Normal airway function

remodeling

Epithelial injury

Matrix deposition

angiogenesis

Genetic predisposition for remodeling

repair

injury

Enviroment

Genetic desposition

Asthma

GERD( Gastro Esophageal Reflux Disease)

recurrent return of stomach content back up into the oesophagus

10% of pateirts with GERD develop Barrett’s oesphagus which increases the risk of cancer of the oesophagus

80% of patients with GERD also have a hiatal hernia

Theory proposed for pathophysiologic mechanism of GERD associated Asthma

Direct stimulation of airway inflammation by aspiration of gastric contents or airway hyperresponsiveness triggered by aspiration of minute amounts of acid into the lower airway

Gastric Fluid Acid

Food allergen digestive enzyme may promote inflammaion of airway and smooth muscle tissue

Airway Remodeling

Subepithelial fibrosis,

myofibroblast hyperplasia,

myocyte hyperplasia and hypotrophy

Epithelial damage

Globet cell metaplasia

oedema

Innate Immunity

Provide initial protection against microorganism infection

Cellular components of innate immunity:

neutrophil, macrophage and natural killer cells

Major target of innate immune response are pathogen associated

molecules pattern( PAMP)

Recognize microorganism through germline-encoded pattern

recognition( PRRs)

Mammalian Immune response

Innate immunity:

first line of host defense against pathogen, and mediated by phagocytes include macrophage and dendritic cells

Acquired immunity:

Characterized bt specificity and develops by clonal selectionfrom lymphocytesbearing antigen –specific receptors that are generated through gene rearrangement

PAMP molecule structure that are produced only by microbial pathogen

conserved molecule pattern that are essential for the survival of the microbes

shared by large groups of micro organisma

often represent molecule signature of microbes, i.e. LPS

PRRs

Recognize PAMP which is essential for the survival of

microorganisms

Express constitutively in the host cells

Germline encoded, nonclonal, express on all cells of a given type,

and independent of iminologic memory

TLR( Toll Like receptors)

A pattern recognition receptor

Highly preserved from Drosophilla to mammals

Toll : gene essential for fly dorsal ventral development

Induction immune-response to broad range of

pathogens: virus, bacteria, fungus, protozoa, helminths

ROS( reactive oxygen species), products of cell damage or

cell death

Lipoprotein, Gram positive, mycobacterial cell wall constituents

TLR1/

TLR2

TLR2/

TLR6

TLR3

TLR5

TLR7 TLR8

TLR9TLR10

TLR4

Poly(I-C) d.s. RNA

Gram negative bacterial LPS, HSP, fibrinogens

Flagellin

Imiguimoid Resiquimod

848

CpG DNA ?

Toll receptors and their ligands

pIRF-3

pIRF-3

MyD88

IL-1R

TLR4

TLR-signaling pathway

TRAM

TRAF6

IKK-I

TBK1

NF-κB

IKK complex

NF-κBP PUbK48

TIRAP

NF-κB

TAB1TAB2TAK1

JNK P38

IRF-5AP-1

NEMO/IKKIKK-

IKK-

Ubk63

TRAF6IRF-5ubc13

Uev1A

IL-1R TLR4

TLR-signaling pathway

IRAK-1

IRAK-4

MyD88

JNK P38

NF-κB

P P

IRF-5

AP-1

ubc13Uev1A

Ubk63

IKK complex

NEMO/IKKIKK-

IKK-

Ubk63Pi

TAB1TAB2TAK1

MyD88

IRAK-1 IRAK-4

TRAF6IRF-5

UbK48

IB

NF-κB

IKK-i

TBK1

TRAM

TLR4

p

IRF-3

TRAF6

RIP1

Th1/2 biasingTLR activation

Activation of neutrophilic and monocytic inflammation

Cytokine production

DC/T cell

Activation

Th-1 and Th-2

activation

Protective immunity

septic shock

Asthma

COPD (chronic obstructive pulmonary disease)

ARDS (acute respiratory distress syndrom)

Clearance of infection

Bronchiolitis

The potentials of TLRs to influence respiratory disease

TLR express in

Endothelium

Fibroblast

Vascular smooth muscle

Memory T cell

Mast cell

Dendritic cell

By nature a pro-inflammatory signaling receptors

Rho/Rock pathway

Smooth muscle cell differentiation gene expresion

Rho A– small GTPase( 20kDa)

RhoA-GTP

Ca+2 sensitize forcce

RLC phosphorylation

RockRock

GPCR GPCR

Tyrosine kinase

Src, FAK…

RhoGDI

RhoA-GDP

Gene transcription

Rock

Dissociation of MLCK activity

PKC

AA

PLC PIP2

IP3

SR

Ca+2

DAG

GPCR

Gq

RockRockGqG12/13

RhoA-GTP

MyosinIIRLC20

MyosinIIRLC20 P

MLCP

Ca-CaMMLCP MYPT1 P

inhibited

+PPIC

Asthmatic epithelial cells

Reduced demosomal contact which may cause epithelial damage

More susceptable to apoptosis

Slower repair response

Increase in number of mucus- containing cells

Increase in the size of submucosal gland

Airway epithelial cell recognition of microorganisms is considered as an protective response in innate immunity

Air way epithelial cells secret large arrays of molecules that involves in inflammatory

Secretion causes chemotract and innate immunity

Damaged epithelial cells

TGF- release which promote the transformation of

fibroblast to myofibroblast

Synthesis of cytokines, remodeling of airway, chronic

inflammatipn

release of Endothelin-1: smooth muscle mitogen)

ectaxin : chemotractant for eosinophil

vascular endothelial growth factor :

promote growth of new blood vessel

Communication of asthmatic epithelium and mesenchyme by growth factors

►Susceptable to damage and repair leads to chronic inflammatory cycles

Chemokines

Eotaxin

IL-

MCA1,2,3

MIP1

RANTES

Other modulators

Cox-2

IFN-

Stem cell factor

TNF

VEGF

CytokinesIL-1IL-5IL-6IL-11GM-CSF

stimuli IL-1 IL-13

IL-5 TNF

Signal transduction IKK

JAKERK1/2

JNK

NFB

STATP38 MAPK

Stimuli and signals mediating synthesis of immune modulators in smooth muscle cells

Cytokine

Chemokine

Growth factor

EosinophilT-cell

Mast cell

Normal mouseIL-13 transgenic mouse

Airway remodling in mouse overexpressin IL-13

Increased deposition of extracellular matrix( ECM)

ECM: secret polysaccharides and proteins

i.e. proteoglycan → modify binding of cytokines to cell surface receptors

→ storage of soluble factors in matrix

In asthma patients → increase numbers cells in synthesize matrix protein

→ difference in degradation of existing proteins

→ more collegen I, III, V in submucosa membrane

increase level of tenacin, lumican, biglycan, verican…..

MMP( Metalopreteinase) : degrade ECM release cryptic information from ECM to liberate

bioactive fragments

lower MMP2, MMP-3 activity higher MMP-9 activity

higher TMP-1( tissue-specific inhibitors

of metalopreteinaase)

Comparative study of Airway smooth muscle remodeling

Gastric fluid acid ???

Ovalbumin ????

Other excerbating factors ????

Gastric fluid aspiration

8 weeks

control

8 weeks

OVA sensitize

8 weeks

1st year

Serum collection and BAL collection Trachea

Immunohistochemistry of tracheal and lung tissue

Animal sacrifice

Provocative testLung function assesment

Evaluation of pro-inflammatory cytokines and immunoglobulin

Matrix protein profile assesment

MMP profile assesment

MMP AssayCell surface protein Elisa assay

Rho/Rho kinase activity assesmentApoptotic assay

Migration assay

Primary culture of air way smooth muscle cells

In vitro study of airway smooth muscle remodeling

Treatment of gastric fluid acid, or Rho kinase inhibitor

Smooth muscle cell

Hyperplasia

Extracellular matrix protein

migration

Chemotaxis activation

Rat gastric fluid acid

apoptosis