Studies of Chronic Aspiration GERD Associated Asthma

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 Exercise Causing 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 Enviroment Genetic desposition Asthma remodeling injury Epithelial injury angiogenesis repair Matrix deposition Genetic predisposition for remodeling

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

Toll receptors and their ligands Lipoprotein, Gram positive, mycobacterial cell wall constituents Poly(I-C) d. s. RNA Gram negative bacterial LPS, HSP, fibrinogens Imiguimoid Resiquimod Flagellin Cp. G DNA 848 TLR 9 TLR 7 TLR 5 TLR 1/ TLR 2 TLR 6 TLR 3 TLR 4 ? TLR 8 TLR 10

TLR-signaling pathway IL-1 R My. D 88 TLR 4 TIRAP TRAM IRF-5 TRAF 6 ubc 13 Uev 1 A TAB 1 TAK 1 TAB 2 JNK P 38 TRAF 6 Ubk 63 NEMO/IKK IKK- complex IKK- P P IRF-5 AP-1 NF-κB Ub. K 48 NF-κB IKK-I TBK 1 p IRF-3

TLR-signaling pathway IL-1 R TLR 4 My. D 88 IRAK-1 IRAK-4

Ubk 63 My. D 88 IRAK-1 IRAK-4 IRF-5 TRAF 6 TAB 1 TAK 1 TAB 2 Pi ubc 13 Uev 1 A Ubk 63 NEMO/IKK IKK- IKK complex JNK P 38 Ub. K 48 P P I B NF-κB AP-1 IRF-5

TLR 4 TRAM TRAF 6 RIP 1 IKK-i TBK 1 p IRF-3 NF-κB

The potentials of TLRs to influence respiratory disease Activation of neutrophilic and monocytic inflammation Protective immunity septic shock Asthma Activation Cytokine production Th 1/2 biasing TLR activation COPD (chronic obstructive pulmonary disease) ARDS (acute respiratory distress DC/T cell syndrom) Clearance of infection Th-1 and Th-2 activation Bronchiolitis

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( 20 k. Da) Ca+2 sensitize forcce Rho. A-GTP RLC phosphorylation

GPCR G 12/13 PLC GPCR G q IP 3 Rock AA Tyrosine kinase DAG SR Rock Src, FAK… Ca+2 Dissociation of MLCK activity PKC Rho. A-GDP Rho. A-GTP Rho. GDI Myosin. IIRLC 20 Gene transcription PIP 2 MLCP Ca-Ca. M MYPT 1 inhibited Myosin. IIRLC 20 P P +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

Stimuli and signals mediating synthesis of immune modulators in smooth muscle cells stimuli IL-1 Signal transduction JAK IKK NF B Chemokines Eotaxin IL- MCA 1, 2, 3 MIP 1 RANTES IL 13 IL-5 ERK 1/2 TNF JNK STAT P 38 MAPK Other modulators Cox-2 IFN- Stem cell factor TNF VEGF Cytokines IL-1 IL-5 IL-6 IL-11 GM-CSF

Cytokine Chemokine Growth factor T-cell Mast cell Eosinophil

Airway remodling in mouse overexpressin IL-13 Normal mouse IL-13 transgenic mouse

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 MMP 2, 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 ? ?

1 st year 8 weeks control 8 weeks Gastric fluid aspiration 8 weeks OVA sensitize Lung function assesment Provocative test Animal sacrifice Immunohistochemistry of tracheal and lung tissue Matrix protein profile assesment Trachea MMP profile assesment Serum collection and BAL collection Evaluation of pro-inflammatory cytokines and immunoglobulin

In vitro study of airway smooth muscle remodeling Primary culture of air way smooth muscle cells Treatment of gastric fluid acid, or Rho kinase inhibitor Cell surface protein Elisa assay MMP Assay Migration assay Apoptotic assay Rho/Rho kinase activity assesment

Extracellular matrix protein Chemotaxis activation Rat gastric fluid acid Smooth muscle cell Hyperplasia migration apoptosis