Nakamura, H

Patel, BV

Romano, R

Takata, M

Yao, K

American Journal of Respiratory and Critical Care Medicine

English

Spiral - Imperial College Digital Repository

Abstract 8317Distinct Differences in Alveolar Epithelial Cell Death Signalling Between Models of ARDSType: Scientific AbstractTopic: 01. Acute Lung Injury, ARDS / Adult / Cellular/Molecular Investigation / Respiratory Cell and Molecular Biology (RCMB)Authors: B.V. Patel1, K. Yao2, H. Nakamura2, R. Romano2, M. Takata2; 1Imperial College London - London/GB, 2Imperial College London -2nh/GBAbstract BodyBackground: Acute respiratory distress syndrome (ARDS) is characterized by the formation of alveolar edema as a result of a disrupted alveolar-capillary barrier. A functional alveolar epithelium is critical to the prevention and resolution of alveolar edema through alveolar fluid clearance.Apoptosis has been implicated in alveolar epithelial cell (AEC) dysfunction1. Apoptosis is mediated by caspases, and activated via extrinsic pathwaysinvolving caspase-8 mediated death-inducing signaling complex or intrinsic pathways through caspase-9 mediated apoptosome formation. These‘initiator’ caspases converge to activate ‘executioner’ caspase-3/7, leading to programmed cell death. We hypothesize that different ARDS mousemodels show specific and distinct patterns of AEC caspase activation/apoptotic activity.Methods: Anaesthetized C57BL/6 mice were orotracheally instilled with 50µl of 0.1 M hydrochloric acid or 50µl of 1mg/ml (50µg) of lipopolysaccharide(LPS). Lungs and blood were harvested from uninjured mice and mice at days 1, 2, and 4 after the instillation of acid or LPS. After formation of a singlecell suspension, we applied a novel flow cytometric protocol using a Fluorochrome Labeled Inhibitor of Caspase Activity (FLICA) probe to measurecaspase-3/7, -8 and -9 activities specifically in AECs, endothelial cells and leukocytes.Results: The LPS model showed greater pulmonary leukocyte infiltration whilst no upregulation in caspase activities were observed in AECs orendothelium. In sharp contrast, acid-aspiration model showed little change in leukocytes infiltration, but much higher caspases activities were seenspecifically in AECs.Acid-induced ARDS (N=4-6) LPS-induced ARDS (N=3)Acidversus LPSP-valueNumbers of lung leukocytes (Neutrophil; CD45+CD11b+Ly6C-Ly6G+, Ly6Chi monocyte; CD45+CD11b+Ly6C+Ly6G-)Time postinjury Day 0 Day 1 Day 2 Day 4 Day 1 Day 2 Day 4Neutrophil(x105)1.7±0.5 3.0±0.6 2.1±0.9 1.3±0.5 35.0±1.8♯♯ 22.0±19.0 29.6±11.1♯ ***Ly6Chimonocyte(x105)1.3±0.9 2.0±1.0 2.6±1.7 1.0±0.7 3.8±2.7 5.9±2.5 3.7±4.3 *Caspase activity in AEC identified as CD45- CD31- EpCAM- T1alpha+ (Median Fluorescent Intensity)Caspase-8 52.0±26.1 203.7±56.3♯♯ 281.4±85.0♯♯♯ 63.1±6.4 90.4±32.2 38.2±19.8 18.4±5.1 ***Caspase-9 69.2±45.0 233.7±68.0♯♯ 161.6±64.2 56.5±11.4 94.1±38.6 59.1±36.0 24.3±8.3 ***Caspase-3 49.1±20.2 120.2±55.7 235.6±81.6♯♯♯ 78.2±29.6 42.8±10.2 39.4±16.4 22.7±7.9 ***Mean±SD; ♯♯♯P<0.001, ♯♯P<0.01, ♯P<0.05 vs Day 0; ***P<0.001, *P<0.05 by Two Way ANOVAConclusion: We found, for the first time, that activation profiles of apoptosis signalling in AECs display entirely different patterns between twofrequently-used ARDS mouse models: significant caspase activations occurs in AECs with acid-, but not with LPS-induced acute lung injury. Ourresults suggest that different aetiologies and different pathophysiological processes determine the degree of cell-death signalling activation in AECs,which may significantly influence the resolution and outcomes of ARDS.References: 1. Patel et al. J Immunol, 2013;190 Print27/03/2017, 07)53Page 1 of 1

Distinct Differences In Alveolar Epithelial Cell Death Signalling Between Models Of Ards

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Distinct Differences In Alveolar Epithelial Cell Death Signalling Between Models Of Ards

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