Functional and phenotypic characterisation of alveolar and circulating neutrophil subsets in acute respiratory distress syndrome (ARDS)
CCCF ePoster library. Juss J. Oct 27, 2015; 117319; P73 Disclosure(s): This work was funded by a non-commercial grant from GSK, The Wellcome Trust, Papworth Hospital, The British Lung Foundation and the NIHR Cambridge Biomedical Research Centre.
Dr. Jatinder Juss
Dr. Jatinder Juss
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P73


Topic: Basic/Translational Science


Functional and phenotypic characterisation of alveolar and circulating neutrophil subsets in acute respiratory distress syndrome (ARDS)



Jatinder Juss, D. House, J. Herre, M. Begg, J. Herre, D. Storisteanu, K. Hoenderdos, G. Bradley, M. Lennon, C. Summer, E. Hessel, A. Condliffe, E. Chilvers

Anaesthesia, St. Michae;'s Hospital, Toronto, Canada | 2Refractory Respiratory Inflammation Discovery Performance Unit, GSK, Stevenage, United Kingdom (Great Britain) | Respiratory Medicine, Addenbrooke's Hospital, Cambridge, United Kingdom (Great Britain) | Refractory Respiratory Inflammation Discovery Performance Unit, GSK, Stevenage, United Kingdom (Great Britain) | Respiratory Medicine, Addenbrooke's Hosptial, Cambridge, United Kingdom (Great Britain) | Medicine, University of Cambridge, Cambridge, United Kingdom (Great Britain) | Medicine, University of Cambridge, Cambridge, United Kingdom (Great Britain) | Refractory Respiratory Inflammation Discovery Performance Unit, GSK, Stevenage, United Kingdom (Great Britain) | Refractory Respiratory Inflammation Discovery Performance Unit, GSK, Stevenage, United Kingdom (Great Britain) | Medicine, University of Cambridge, Cambridge, United Kingdom (Great Britain) | Refractory Respiratory Inflammation Discovery Performance Unit, GSK, Stevenage, United Kingdom (Great Britain) | Medicine, University of Cambridge, Cambri

Introduction: The acute respiratory distress syndrome (ARDS) is a leading cause of mortality in Intensive Care Unit (ICU) patients. It is characterised by diffuse alveolar inflammation, resulting in intractable hypoxemia [1]. To date there are no effective pharmacological strategies for this condition; management remains largely supportive with optimisation of ventilator parameters [2], judicious fluid balance, and treatment of underlying causes.


Neutrophil (PMN) alveolitis is a key histopathological hallmark of ARDS[3];clear association exists between the intensity of alveolar PMN infiltration and disease severity.
Inappropriate accumulation/activation of PMNs within the alveoli is proposed to cause unrestrained release of histotoxic oxygen radicals and pro-inflammatory cytokines which is thought to underpin the complex pathophysiology of ARDS. PMN longevity increases dramatically in ARDS, whilst the precise molecular mechanisms governing this are incompletely understood, previous studies identified GM-CSF as a major pro-survival mediator in ARDS. The cytoprotective effect of GM-CSF is Class I phosphoinositide 3-kinase (PI3K)-dependent[4].

Given

the technical challenges inherent in obtaining purified alveolar PMNs (alvPMNs) in ARDS, the phenotype and functional activity of alveolar-sequestered PMNs in ARDS are largely unknown. Historically, mouse models have been used as surrogates for alvPMNs, however, rodent PMNs differ markedly from their human counterparts.

W

e used

genome-wide transcriptional analysis as a powerful tool to identify novel targets relevant to altered PMN functions.



Objectives: ARDS alvPMNs exist in a unique microenvironment and may display fundamentally distinct properties relative to autologous bloodPMNs during inflammation. The objective of this study is to provide a comprehensive functional/phenotypic characterization of highly purified ARDS alvPMNs and bloodPMNs and perform genome-wide transcriptomic analysis of purified ARDS bloodPMNs.



Methods:

Mechanically ventilated patients (n=23) fulfilling the Berlin criteria for ARDS underwent bronchoalveolar lavage within 48 hours of ARDS onset. ARDS alvPMNs were isolated to 92±3.3% (%±SD) purity using a negative-selection immunomagnetic bead technique and studied in a simultaneous and parallel manner with age- and gender-matched healthy volunteer (HV)bloodPMNs. This paired sampling approach provided a robust experimental design in controlling for confounders.

PMN apoptosis, phagocytosis and basal activation were quantified by flow cytometry, and oxidase responses by chemiluminescence. Cytokine and transcriptional profiling utilized multiplex and GeneChip arrays.




Results:

The mean age of patients was 56.0 ±16.4 years (±SD); eight (34.7%), eleven (47.8%) and four (17.39%) patients had mild, moderate and severe ARDS respectively. ARDS alvPMNs andbloodPMNs were distinct from healthy circulating PMNs (Fig 1), with increased CD11b and reduced CD62L expression, delayed apoptosis and constitutively primed oxidase responses. Incubating control PMNs with disease bronchoalveolar lavage recapitulated the aberrant functional phenotype and this could be reversed by PI3K inhibitors. In contrast, the pro-survival phenotype of patient PMNs was recalcitrant to PI3K inhibition. RNA transcriptomic analysis revealed modified immune, cytoskeletal and cell death pathways in patient PMNs, aligning closely to sepsis and burns data sets but not with PI3K signatures (Fig 1).



Conclusion: We present the first well-controlled comprehensive functional and phenotypic characterization of highly pure alvPMNs and bloodPMNs from ARDS patients. ARDS alvPMNs and bloodPMNs display a distinct primed, pro-survival profile and transcriptional signature. The enhanced respiratory burst was PI3K-dependent, but delayed apoptosis and the altered transcriptional profile were not. These findings cast doubt over the utility of P3K inhibition in ARDS and highlight the importance of evaluating novel therapeutic strategies in patient-derived cells.

References:

1. Ashbaugh DG, Bigelow DB, Petty TL, Levine BE. Acute respiratory distress in adults. Lancet. 1967;290:319-23

2. Amato MBP, Barbas CSV, Medeiros DM, Magaldi RB, Schettino GP, Lorenzo-Filho G, Kairalla RA, Deheinzelin D, Munoz C, Oliveira R, Takagake TY,
Carvalho RR. Effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome. New England Journal of Medicine.
1998;338:347-354

3. Thille AW, Esteban A, Fernandez-Segoviano P, Rodriguez JM, Aramburu JA, Vargas-Errazuriz P, Martin-Pellicer A, Lorente JA, Frutos-Vivar F. Chronology of histological lesions in acute respiratory distress syndrome with diffuse alveolar damage: a prospective cohort study of clinical autopsies. Lancet Respir Med 2013;1:395-401.

4. Juss JK, Hayhoe RP, Owen CE, Bruce I, Walmsley SR, Cowburn AS, Kulkarni S, Boyle KB, Stephens L, Hawkins PT, Chilvers ER, Condliffe AM. Functional redundancy of class I phosphoinositide 3-kinase (PI3K) isoforms in signaling growth factor-mediated human neutrophil survival. PLoSOne. 2012;7:




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