The role of microvesicles in systemic inflammatory signalling in acute respiratory distress syndrome

Patients with acute respiratory distress syndrome (ARDS) have an unacceptably high mortality surpassing 40%. Most patients with ARDS die from multi-organ failure rather than respiratory failure, presumably due to systemic propagation of inflammation between the lungs and peripheral organs. Numerous systemic inflammatory mediators have been identified as potential therapeutic targets for ARDS. However, few specific therapies have been successfully developed and treatment remains primarily limited to supportive care. Microvesicles (MVs) are tiny particles released from cells during cell activation and stress. MVs represent a unique form of inter-cellular communication through their ability to act as ‘biological ferries’ carrying inflammatory mediators as their cargoes in a packaged, lipid-encapsulated environment resistant to neutralisation in the systemic circulation. We hypothesised that leukocyte-derived MVs play a critical role in systemic inflammatory signalling in ARDS. Through initially focusing on periphery-to-lung signalling, we developed an in vitro bioassay of pulmonary microvascular inflammation and investigated the bioactivity of neutrophil-derived MVs. We found that inflammatory neutrophil-derived MVs induced significant acute pulmonary microvascular inflammation, in a peripheral blood mononuclear cell-dependent manner. We then focused on lung-to-periphery signalling through developing a novel human in vitro model of ventilator-induced pulmonary microvascular inflammation. We found, for the first time, that pathological cyclic stretch induced acute leukocyte activation and release of leukocyte-derived MVs. We developed methodology to separate MV subtypes from the mixed total MV population generated, and demonstrated that these stretch-induced leukocyte-derived MVs are capable of inducing remote organ endothelial injury. Finally, we investigated circulating MV profiles in plasma samples of patients with ARDS. We found significantly increased baseline neutrophil- and endothelial-derived MV levels in patients with hyperinflammatory ARDS. Our findings suggest MVs play a crucial role in the early phase of ARDS pathophysiology and could represent a paradigm shift in our understanding of systemic inflammatory signalling in ARDS.Open Acces

Evaluation of flow cytometry gating strategy for MVs in plasma.

<p>Suspensions of neutrophil-derived MVs and MV-depleted healthy volunteer plasma were stained with antibodies, individually or after addition of MVs to plasma (volume ratio: 1:4). Larger events were excluded using 1.3 μm beads (not shown) and a gate (<b>R1</b>) for neutrophil-derived MVs was drawn based on CD66b+, CD11b+ double-staining and a cut-off boundary defined by detergent-mediated selective lysis of MVs. Isotype-matched control staining provided confirmation of the detergent-lysis method for identification of non-MV background events.</p

Burns patient demographics, assessment scores and outcome, with survivor and non-survivor comparisons.

<p>Burns patient demographics, assessment scores and outcome, with survivor and non-survivor comparisons.</p

Flow cytometric identification of MV sub-types.

<p>Dots plots of burns patient plasma stained for MVs using fluorophore-conjugated monoclonal antibodies to cell-surface markers (<b>A</b>). Plots show events that have been gated on forward light scatter using 1.3 μm calibration beads to define the upper size limit. For CD45/CD14 MV analysis, total CD45+ events are shown in the larger region 1 (<b>R1</b>) and CD45+/CD14+ events in the smaller sub-region 2 (<b>R2</b>). MV identification is confirmed by sensitivity to detergent lysis (Triton X-100, 0.1%)(<b>B</b>). Matched antibody isotype and fluorophore control plots are shown for comparison (<b>C</b>).</p

Spearman rank correlation coefficients for MV subpopulations in burns patients.

<p>Spearman rank correlation coefficients for MV subpopulations in burns patients.</p

Comparison of circulating MV levels in burns and sepsis patient survivors and non-survivors.

<p>Levels of MV subtypes were compared between burns patients that recovered (survivors, n = 10) or died in burns ICU (non-survivors, n = 5) (<b>A</b>) and sepsis patients that recovered (n = 11) or died (n = 4) in the general ICU (<b>B</b>). Data are log-transformed and analyzed by t tests (for burns: leukocyte-, granulocyte- and monocyte-derived MVs; sepsis: leukocyte- and granulocyte-derived MVs; mean ± SD) or Mann-Whitney U tests (for the remainder; median ± interquartile range). Levels of total leukocyte- and granulocyte-derived MVs were higher in burns non-survivors than survivors, **<i>p</i> < 0.01.</p

Comparison of circulating MV levels in burns patients on day 0 and day 2.

<p>Plasma from burns patients on admission day 0 and post-admission day 2 was analyzed by flow cytometry to quantify MVs of different cellular origin: CD45+ leukocyte-derived (<b>A</b>), CD105+ endothelia-derived (<b>B</b>), CD66b+/CD11b+ granulocyte-derived (<b>C</b>), and CD45+/CD14+ monocyte-derived (<b>D</b>). Data from individual patients are log-transformed and analyzed by paired t test (<b>A</b>, <b>C</b> and <b>D</b>) or Wilcoxon signed rank test (<b>B</b>). *p < 0.05.</p

Circulating MV levels were elevated in burns patients.

<p>Plasma from healthy volunteers (HV), burns patients on day of admission, and sepsis patients was analyzed by flow cytometry to quantify MVs of different cellular origin: CD45+ leukocyte-derived (<b>A</b>), CD105+ endothelial-derived (<b>B</b>), CD66b+/CD11b+ granulocyte-derived (<b>C</b>), and CD45+/CD14+ monocyte-derived (<b>D</b>). Data are log-transformed and analyzed by one-way ANOVA with Tukey’s tests (<b>A</b>, <b>C</b> and <b>D;</b> mean ± SD) or Kruskal-Wallis with Dunn’s tests (<b>B;</b> median ± interquartile range). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p

Receiver operator characteristic curves for circulating leukocyte MVs in burns patients.

<p>Receiver operating characteristic (ROC) curves and corresponding area under the curve values for: leukocyte-derived MVs, neutrophil-derived MVs and CRP (<b>A</b>), and for BOBI, ABSI and TBSA (<b>B</b>). *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p