1. 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

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

4. Aims & Hypotheses 1.Do the two TNF receptors have distinct roles in experimental ALI/ARDS induced by acid aspiration? 2.What are the cellular mechanisms through which TNF receptor mediate their effects during experimental ALI/ARDS?

5. Experimental model of acid aspiration Carotid Line Tracheostomy Intraperitoneal line for anaesthesia Airway Pressure Transducer Flow Transducer Pneumotache Flow Control Inspiratory valve Expiratory valve Setup for murine ventilation Experimental Protocol Baseline Respiratory Mechanics & Arterial Blood Gas Acid Instillation 0306090120150180 ABG Mins Respiratory Mechanics Measurements GA + Setup for murine ventilation Vt – 8mls/kg PEEP – 3cmH 2 O

6. p55KO shows improved respiratory physiology and p75KO show worse respiratory mechanics 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 0 50 100 150 200 250 30 60 90 120150 180 Elastance Change (%) Time post instillation (minutes) Baseline WT Saline WT Acid p55KO Acid p75KO Acid DKO Acid 0 100 200 300 400 500 PaO 2 :FiO 2 Ratio *** Acid +/- + + + + - Mouse strain WT p55KO p75KO DKO WT

7. 0 2 4 6 8 Wet:Dry Ratio ** Acid +/- + + + + - Mouse strain WT p55KO p75KO DKO WT p55KO shows improved alveolar capillary barrier permeability BALF Protein (mg/ml) 0 1 2 3 * Acid +/- + + + + - Mouse strain WT p55KO p75KO DKO WT 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 Tight JunctionsENaCNa/K ATPase Aquaporins Alveolus Type 2 Alveolar Epithelial Cell Type 1 Alveolar Epithelial Cell Pulmonary capillary Interstitium Na + H2OH2O

9. Ex vivo measurement of alveolar fluid clearance No Drug Control Amiloride Isoproterenol AFC (%/30min) ** ΔV instillate = AFC = ΔC Alb = ΔF = (F 30 -F 0 )/F 30 ** P<0.01 vs No Drug Control by 1-way ANOVA with Bonferroni test; Mean±SD; N=3-6

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

11. Summary – Roles of TNF receptors in early ALI/ARDS 1.TNF receptors show opposing roles in pulmonary oedema formation in a model of direct ALI/ARDS induced by acid aspiration. 2.p75 TNF receptor deletion leads to worsening respiratory mechanics. 3.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. 4.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

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

15. Future work 1.Early phase of ALI/ARDS: Investigate the influence of TNF p55 receptor death domain signalling in type 1 and type 2 AECs. 2.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.