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Blood-borne myeloid cells, neutrophils and monocytes, play a central role in the development of indirect acute lung injury (ALI) during sepsis and noninfectious systemic inflammatory response syndrome. By contrast, the contribution of circulating myeloid cell-derived extracellular vesicles (EVs) to ALI is unknown, despite acute increases in their numbers during sepsis and systemic inflammatory response syndrome. Here, we investigated the direct role of circulating myeloid-EVs in ALI using a mouse isolated perfused lung system and a human cell coculture model of pulmonary vascular inflammation consisting of lung microvascular endothelial cells and peripheral blood mononuclear cells. Total and immunoaffinity-isolated myeloid (CD11b + ) and platelet (CD41 + ) EVs were prepared from the plasma of intravenous LPS-injected endotoxemic donor mice and transferred directly into recipient lungs. Two-hour perfusion of lungs with unfractionated EVs from a single donor induced pulmonary edema formation and increased perfusate concentrations of RAGE (receptor for advanced glycation end products), consistent with lung injury. These responses were abolished in the lungs of monocyte-depleted mice. The isolated myeloid- but not platelet-EVs produced a similar injury response and the acute intravascular release of proinflammatory cytokines and endothelial injury markers. In the in vitro human coculture model, human myeloid- (CD11b + ) but not platelet- (CD61 + ) EVs isolated from LPS-stimulated whole blood induced acute proinflammatory cytokine production and endothelial activation. These findings implicate circulating myeloid-EVs as acute mediators of pulmonary vascular inflammation and edema, suggesting an alternative therapeutic target for attenuation of indirect ALI.

Advancements in methods, technology, and our understanding of the pathobiology of lung injury have created the need to update the definition of experimental acute lung injury (ALI). We queried 50 participants with expertise in ALI and acute respiratory distress syndrome using a Delphi method composed of a series of electronic surveys and a virtual workshop. We propose that ALI presents as a "multidimensional entity" characterized by four "domains" that reflect the key pathophysiologic features and underlying biology of human acute respiratory distress syndrome. These domains are 1 ) histological evidence of tissue injury, 2 ) alteration of the alveolar-capillary barrier, 3 ) presence of an inflammatory response, and 4 ) physiologic dysfunction. For each domain, we present "relevant measurements," defined as those proposed by at least 30% of respondents. We propose that experimental ALI encompasses a continuum of models ranging from those focusing on gaining specific mechanistic insights to those primarily concerned with preclinical testing of novel therapeutics or interventions. We suggest that mechanistic studies may justifiably focus on a single domain of lung injury, but models must document alterations of at least three of the four domains to qualify as "experimental ALI." Finally, we propose that a time criterion defining "acute" in ALI remains relevant, but the actual time may vary based on the specific model and the aspect of injury being modeled. The continuum concept of ALI increases the flexibility and applicability of the definition to multiple models while increasing the likelihood of translating preclinical findings to critically ill patients.

Background: Respiratory complications after surgery are associated with morbidity and mortality. Acute lung injury can result from the systemic inflammatory response after acute kidney injury. The mechanisms behind this remote injury are not fully understood. In this study, a renal transplantation model was used to investigate remote lung injury and the underlying molecular mechanisms, especially the role of osteopontin (OPN)., Methods: In vitro, human lung epithelial cell line (A549) and monocyte/macrophage cell line (U937) were challenged with tumour necrosis factor-alpha (TNF-α) in combination with OPN. In vivo, the Fischer rat renal grafts were extracted and stored in 4°C University of Wisconsin preserving solution for up to 16 h, and transplanted into Lewis rat recipients. Lungs were harvested on Day 1 after grafting for further analysis., Results: Renal engraftment was associated with pathological changes and an increase in TNF-α and interleukin-1 beta in the lung of the recipient. OPN, endoplasmic reticulum (ER) stress, and necroptosis were increased in both the recipient lung and A549 cells challenged with TNF-α. Exogenous OPN exacerbated lung injury and necroptosis. Suppression of OPN through siRNA reduced remote lung injury by mitigation of ER stress, necroptosis, and the inflammatory response., Conclusions: Renal allograft transplant triggers recipient remote lung injury, which is, in part, mediated by OPN signalling. This study may provide a molecular basis for strategies to be developed to treat such perioperative complications., (Copyright © 2019 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)

Background: Microvesicles (MVs) are important mediators of intercellular communication, packaging a variety of molecular cargo. They have been implicated in the pathophysiology of various inflammatory diseases; yet, their role in acute lung injury (ALI) remains unknown., Objectives: We aimed to identify the biological activity and functional role of intra-alveolar MVs in ALI., Methods: Lipopolysaccharide (LPS) was instilled intratracheally into C57BL/6 mice, and MV populations in bronchoalveolar lavage fluid (BALF) were evaluated. BALF MVs were isolated 1 hour post LPS, assessed for cytokine content and incubated with murine lung epithelial (MLE-12) cells. In separate experiments, primary alveolar macrophage-derived MVs were incubated with MLE-12 cells or instilled intratracheally into mice., Results: Alveolar macrophages and epithelial cells rapidly released MVs into the alveoli following LPS. At 1 hour, the dominant population was alveolar macrophage-derived, and these MVs carried substantive amounts of tumour necrosis factor (TNF) but minimal amounts of IL-1β/IL-6. Incubation of these mixed MVs with MLE-12 cells induced epithelial intercellular adhesion molecule-1 (ICAM-1) expression and keratinocyte-derived cytokine release compared with MVs from untreated mice (p<0.001). MVs released in vitro from LPS-primed alveolar macrophages caused similar increases in MLE-12 ICAM-1 expression, which was mediated by TNF. When instilled intratracheally into mice, these MVs induced increases in BALF neutrophils, protein and epithelial cell ICAM-1 expression (p<0.05)., Conclusions: We demonstrate, for the first time, the sequential production of MVs from different intra-alveolar precursor cells during the early phase of ALI. Our findings suggest that alveolar macrophage-derived MVs, which carry biologically active TNF, may play an important role in initiating ALI., Competing Interests: Conflicts of Interest: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

The ability of the alveolar epithelium to prevent and resolve pulmonary edema is a crucial determinant of morbidity and mortality in acute lung injury (ALI). TNF has been implicated in ALI pathogenesis, but the precise mechanisms remain undetermined. We evaluated the role of TNF signaling in pulmonary edema formation in a clinically relevant mouse model of ALI induced by acid aspiration and investigated the effects of TNF p55 receptor deletion, caspase-8 inhibition, and alveolar macrophage depletion on alveolar epithelial function. We found that TNF plays a central role in the development of pulmonary edema in ALI through activation of p55-mediated death signaling, rather than through previously well-characterized p55-mediated proinflammatory signaling. Acid aspiration produced pulmonary edema with significant alveolar epithelial dysfunction, as determined by alveolar fluid clearance (AFC) and intra-alveolar levels of the receptor for advanced glycation end-products. The impairment of AFC was strongly correlated with lung caspase-8 activation, which was localized to type 1 alveolar epithelial cells by flow cytometric analysis. p55-deficient mice displayed markedly attenuated injury, with improved AFC and reduced caspase-8 activity but no differences in downstream cytokine/chemokine production and neutrophil recruitment. Caspase-8 inhibition significantly improved AFC and oxygenation, whereas depletion of alveolar macrophages attenuated epithelial dysfunction with reduced TNF production and caspase-8 activity. These results provide in vivo evidence for a novel role for TNF p55 receptor-mediated caspase-8 signaling, without substantial apoptotic cell death, in triggering alveolar epithelial dysfunction and determining the early pathophysiology of ALI. Blockade of TNF-induced death signaling may provide an effective early-phase strategy for ALI.

Previous animal models of acute lung injury (ALI) are limited as they only reproduce part of the complex pathobiology of clinical ALI. Here we develop a translational mouse model of ALI, which not only reflects the major clinical and pathological features but also enables investigation into ALI resolution. Anaesthetised mice underwent orotracheal instillation of hydrochloric acid. During the immediate period after instillation, mice were carefully maintained with supplemental oxygen to avoid mortality. At specified time-points, lung injury was assessed by analysis of blood gases, respiratory mechanics, bronchoalveolar lavage fluid, alveolar fluid clearance and lung histology. Animals exhibited significant weight loss, decreased oxygenation, increased respiratory elastance and pulmonary inflammation (intra-alveolar leukocyte influx/cytokine levels and histological injury scores). Moreover, mice displayed alveolar-capillary barrier dysfunction/epithelial injury as reflected by increased alveolar protein, lung wet/dry weight ratio and soluble receptor for advanced glycation end-products, as well as reduced alveolar fluid clearance. These injury parameters peaked between days 1 and 3, followed by almost complete recovery over days 5-10. Histology showed evidence of fibrosis on day 10. The results indicate that this resolving model of acid aspiration represents a powerful experimental tool to investigate the injurious, inflammatory, fibrotic, and resolving and reparative processes of ALI.

Margination and activation of monocytes within the pulmonary microcirculation contribute substantially to the development of acute lung injury in mice. The enhanced LPS-induced TNF expression exhibited by Gr-1(high) compared with Gr-1(low) monocytes within the lung microvasculature suggests differential roles for these subsets. We investigated the mechanisms responsible for such heterogeneity of lung-marginated monocyte proinflammatory response using a combined in vitro and in vivo approach. The monocyte subset inflammatory response was studied in vitro in mouse peripheral blood mononuclear cell-lung endothelial cell coculture and in vivo in a two-hit model of intravenous LPS-induced monocyte margination and lung inflammation in mice, by flow cytometry-based quantification of proinflammatory genes and intracellular phospho-kinases. With LPS stimulation in vitro, TNF expression was consistently higher in Gr-1(high) than Gr-1(low) monocytes, markedly enhanced by coculture with endothelial cells, and abrogated by p38 MAPK inhibitors. Expression of IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) was only detectable under coculture conditions, was substantially higher in Gr-1(high) monocytes, and was attenuated by p38 inhibition. Consistent with these differential responses, phosphorylation of p38 and its substrate MAPK-activated protein kinase 2 (MK2) was significantly higher in the Gr-1(high) subset. In vivo, p38 inhibitor treatment significantly attenuated LPS-induced TNF expression in "lung-marginated" Gr-1(high) monocytes. LPS-induced p38/MK2 phosphorylation was higher in lung-marginated Gr-1(high) than Gr-1(low) monocytes and neutrophils, mirroring TNF expression. These results indicate that the p38/MK2 pathway is a critical determinant of elevated Gr-1(high) subset responsiveness within the lung microvasculature, producing a coordinated proinflammatory response that places Gr-1(high) monocytes as key orchestrators of pulmonary microvascular inflammation and injury.

Elevated soluble tumor necrosis factor-α receptor (sTNFR) levels in bronchoalveolar lavage fluid (BALF) are associated with poor patient outcome in acute lung injury (ALI). The mechanisms underlying these increases are unknown, but it is possible that pulmonary inflammation and increased alveolar epithelial permeability may individually contribute. We investigated mechanisms of elevated BALF sTNFRs in two in vivo mouse models of ALI. Anesthetized mice were challenged with intratracheal lipopolysaccharide or subjected to injurious mechanical ventilation. Lipopolysaccharide instillation produced acute intra-alveolar inflammation, but minimal alveolar epithelial permeability changes, with increased BALF sTNFR p75, but not p55. Increased p75 levels were markedly attenuated by alveolar macrophage depletion. In contrast, injurious ventilation induced substantial alveolar epithelial permeability, with increased BALF p75 and p55, which strongly correlated with total protein. BALF sTNFRs were not increased in isolated buffer-perfused lungs (devoid of circulating sTNFRs) subjected to injurious ventilation. These results suggest that lipopolysaccharide-induced intra-alveolar inflammation upregulates alveolar macrophage-mediated production of sTNFR p75, whereas enhanced alveolar epithelial permeability following mechanical ventilation leads to increased BALF p75 and p55 via plasma leakage. These data provide new insights into differential regulation of intra-alveolar sTNFR levels during ALI and may suggest sTNFRs as potential markers for evaluating the pathophysiology of ALI.

The specialized role of mouse Gr-1(high) monocytes in local inflammatory reactions has been well documented, but the trafficking and responsiveness of this subset during systemic inflammation and their contribution to sepsis-related organ injury has not been investigated. Using flow cytometry, we studied monocyte subset margination to the pulmonary microcirculation during subclinical endotoxemia in mice and investigated whether marginated monocytes contribute to lung injury in response to further septic stimuli. Subclinical low-dose i.v. LPS induced a rapid (within 2 h), large-scale mobilization of bone marrow Gr-1high monocytes and their prolonged margination to the lungs. With secondary LPS challenge, membrane TNF expression on these premarginated monocytes substantially increased, indicating their functional priming in vivo. Zymosan challenge produced small increases in pulmonary vascular permeability, which were markedly enhanced by the preadministration of low-dose LPS. The LPS-zymosan-induced permeability increases were effectively abrogated by pretreatment (30 min before zymosan challenge) with the platelet-activating factor antagonist WEB 2086 in combination with the phosphatidylcholine-phospholipase C inhibitor D609, suggesting the involvement of platelet-activating factor/ceramide-mediated pathways in this model. Depletion of monocytes (at 18 h after clodronate-liposome treatment) significantly attenuated the LPS-zymosan-induced permeability increase. However, restoration of normal LPS-induced Gr-1high monocyte margination to the lungs (at 48 h after clodronate-liposome treatment) resulted in the loss of this protective effect. These results demonstrate that mobilization and margination of Gr-1high monocytes during subclinical endotoxemia primes the lungs toward further septic stimuli and suggest a central role for this monocyte subset in the development of sepsis-related acute lung injury.

Ventilator-induced lung injury plays a crucial role in the outcome of patients with acute lung injury. Previous studies have shown a role for the cytokine tumor necrosis factor-α (TNF) in stretch-induced alveolar neutrophil recruitment, but the involvement of TNF in stretch-induced pulmonary edema is unclear. We investigated the effects of TNF through its individual p55 and p75 receptors on early pulmonary edema formation during high stretch ventilation, before neutrophil infiltration. Anesthetized wild-type or TNF receptor single/double knockout mice were ventilated with high tidal volume (~38 ml/kg) for 2 h or until they developed arterial hypotension. Pulmonary edema was assessed by physiological parameters including respiratory mechanics and blood gases, and by lavage fluid protein, lung wet:dry weight ratio, and lung permeability measurements using fluorescence-labeled albumin. High stretch ventilation in wild-type and TNF receptor double knockout animals induced similar pulmonary edema, and only 25-30% of mice completed the protocol. In contrast, the p55 receptor knockout mice were strongly protected from edema formation, with all animals completing the protocol. Myeloperoxidase assay indicated that this protective effect was not associated with decreased pulmonary neutrophil sequestration. The p75 receptor knockout mice, however, displayed increased susceptibility to edema formation, and no animals survived the full 2 h. These results demonstrate a novel role for TNF signaling (independent from its effects on neutrophil recruitment) specifically through the p55 receptor, in promoting high stretch-induced pulmonary edema, whereas p75 signaling may play an opposing role. [ABSTRACT FROM AUTHOR]