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Dr Brijesh Patel Clinical Research Fellow Wellcome Trust Clinical PhD Programme

Imperial College Academic Trainees Annual Event The critical roles of TNF receptors in experimental acute lung injury. Dr Brijesh Patel Clinical Research Fellow Wellcome Trust Clinical PhD Programme Anaesthetics, Pain Medicine, and Intensive Care. ALI/ARDS. ALI/ARDS is characterised by:

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Dr Brijesh Patel Clinical Research Fellow Wellcome Trust Clinical PhD Programme

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  1. Imperial College Academic Trainees Annual EventThe critical roles of TNF receptors in experimental acute lung injury Dr Brijesh Patel Clinical Research Fellow Wellcome Trust Clinical PhD Programme Anaesthetics, Pain Medicine, and Intensive Care

  2. ALI/ARDS • ALI/ARDS is characterised by: • acute onset • hypoxaemia • bilateral chest radiographic changes • normal left atrial pressures • Has a mortality of over 35% despite lung protective ventilation strategies. • Mulitiple aetiologies can lead to ALI/ARDS. • Direct – pneumonia; aspiration pneumonitis • Indirect – non-pulmonary sepsis; trauma; transfusion related; pancreatitis • Alveolar epithelial damage is a prerequisite to pulmonary oedema formation in all aetiologies of ALI/ARDS.

  3. TNF receptor signalling p55 TNF TNF p75 TRADD TRAF2 RIP MEKK1/7 FADD NEMO/IKK MKK3 JNK Caspase 8/10 p38 Caspase 3 AP1 NFκB MAPK Apoptosis cFLIP Cell Survival

  4. Aims & Hypotheses • Do the two TNF receptors have distinct roles in experimental ALI/ARDS induced by acid aspiration? • What are the cellular mechanisms through which TNF receptor mediate their effects during experimental ALI/ARDS?

  5. Experimental model of acid aspiration Setup for murine ventilation Tracheostomy Intraperitoneal line for anaesthesia Carotid Line Airway Pressure Transducer Flow Control Inspiratory valve Expiratory valve Pneumotache Flow Transducer Experimental Protocol Acid Instillation 30 60 90 120 150 180 0 Mins GA + Setup for murine ventilation Baseline Respiratory Mechanics & Arterial Blood Gas Respiratory Mechanics Measurements ABG Vt – 8mls/kg PEEP – 3cmH2O

  6. p55KO shows improved respiratory physiology and p75KO show worse respiratory mechanics 500 *** p75KO Acid 400 DKO Acid WT Acid 300 250 PaO2:FiO2 Ratio p55KO Acid 200 200 WT Saline 100 150 Elastance Change (%) 0 + - + + + Acid +/- Mouse strain 100 DKO WT WT p75KO p55KO 50 0 60 120 150 30 90 180 Baseline Time post instillation (minutes) Significant interaction P-value (P<0.0001) by 2-way ANOVA; *** P<0.001 vs WT Acid by 1-way ANOVA with Bonferroni test; Mean±SD; N=3-6

  7. p55KO shows improved alveolar capillary barrier permeability + + - + - + + + + + Acid +/- Acid +/- Mouse strain Mouse strain DKO DKO WT p55KO WT WT WT p75KO p75KO p55KO 3 8 ** 2 BALF Protein (mg/ml) 6 * Wet:Dry Ratio 1 4 2 0 0 • P<0.05; ** P<0.01 vs WT Acid by 1-way ANOVA with Bonferroni test; Mean±SD; N=3-6

  8. Alveolar fluid clearance – an epithelial functional marker H2O Na+ Alveolus Type 2 Alveolar Epithelial Cell Type 1 Alveolar Epithelial Cell Interstitium Pulmonary capillary Aquaporins Tight Junctions ENaC Na/K ATPase

  9. Ex vivo measurement of alveolar fluid clearance ΔVinstillate = AFC = ΔCAlb = ΔF = (F30-F0)/F30 ** AFC (%/30min) ** No Drug Control Amiloride Isoproterenol ** P<0.01 vs No Drug Control by 1-way ANOVA with Bonferroni test; Mean±SD; N=3-6

  10. The p55 receptor mediates alveolar epithelial dysfunction in ALI * Alveolar Fluid Clearance (%/30minutes) * BALF RAGE (ng/ml) - - + + + + Acid +/- Acid +/- Mouse strain Mouse strain WT WT WT WT p55KO p55KO + + p75KO p75KO * P<0.05 vs WT Acid by 1-way ANOVA with Bonferroni test; Mean±SD; N=6-7

  11. Summary – Roles of TNF receptors in early ALI/ARDS • TNF receptors show opposing roles in pulmonary oedema formation in a model of direct ALI/ARDS induced by acid aspiration. • p75 TNF receptor deletion leads to worsening respiratory mechanics. • p55 TNF receptor deletion leads to reduced levels of alveolar epithelial injury as assessed by a functional marker i.e. AFC and a soluble marker i.e. RAGE. • This study provides a rationale for specific p55 TNF receptor blockade on the alveolar epithelium in ALI/ARDS states.

  12. Acknowledgements • Professor Masao Takata – Magill Chair in Anaesthesia • Dr Michael Wilson – Lecturer in Physiology • and the rest of the critical care group

  13. p75 receptor attenuates alveolar inflammation * * BALF MIP-2 (pg/ml) BALF IL-6 (pg/ml) + + + - + - + - + + + + + + + Acid +/- Acid +/- Acid +/- Mouse strain Mouse strain Mouse strain DKO DKO DKO WT WT WT WT WT WT p75KO p75KO p75KO p55KO p55KO p55KO BALF KC (pg/ml)

  14. Future work • Early phase of ALI/ARDS: • Investigate the influence of TNF p55 receptor death domain signalling in type 1 and type 2 AECs. • Extend the investigation into the longer term roles of TNF receptors in experimental ALI/ARDS: • A resolving model of experimental acid aspiration. • The role of the p75 TNF receptor in promoting resolution and repair.

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