85 results found
Koh MW, Baldi RF, Soni S, et al., 2021, Secreted extracellular cyclophilin a is a novel mediator of ventilator induced lung injury., American Journal of Respiratory and Critical Care Medicine, Vol: 204, Pages: 421-430, ISSN: 1073-449X
RATIONALE: Mechanical ventilation is a mainstay of intensive care but contributes to the mortality of patients through ventilator induced lung injury. Extracellular Cyclophilin A is an emerging inflammatory mediator and metalloproteinase inducer, and the gene responsible for its expression has recently been linked to COVID-19 infection. OBJECTIVES: Here we explore the involvement of extracellular Cyclophilin A in the pathophysiology of ventilator-induced lung injury. METHODS: Mice were ventilated with low or high tidal volume for up to 3 hours, with or without blockade of extracellular Cyclophilin A signalling, and lung injury and inflammation were evaluated. Human primary alveolar epithelial cells were exposed to in vitro stretch to explore the cellular source of extracellular Cyclophilin A, and Cyclophilin A levels were measured in bronchoalveolar lavage fluid from acute respiratory distress syndrome patients, to evaluate clinical relevance. MEASUREMENTS AND MAIN RESULTS: High tidal volume ventilation in mice provoked a rapid increase in soluble Cyclophilin A levels in the alveolar space, but not plasma. In vivo ventilation and in vitro stretch experiments indicated alveolar epithelium as the likely major source. In vivo blockade of extracellular Cyclophilin A signalling substantially attenuated physiological dysfunction, macrophage activation and matrix metalloproteinases. Finally, we found that patients with acute respiratory distress syndrome showed markedly elevated levels of extracellular Cyclophilin A within bronchoalveolar lavage. CONCLUSIONS: Cyclophilin A is upregulated within the lungs of injuriously ventilated mice (and critically ill patients), where it plays a significant role in lung injury. Extracellular Cyclophilin A represents an exciting novel target for pharmacological intervention.
Baldi RF, Soni S, Patel BV, et al., 2021, Microvesicle-Mediated Enhancement of TNF-Driven Apoptosis During VILI, International Conference of the American-Thoracic-Society (ATS), Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Soni S, Garner J, O'Dea K, et al., 2021, Intra-alveolar neutrophil-derived microvesicles are associated with disease severity in COPD, American Journal of Physiology: Lung Cellular and Molecular Physiology, Vol: 320, Pages: L73-L83, ISSN: 1040-0605
Despite advances in the pathophysiology of Chronic Obstructive Pulmonary Disease (COPD), there is a distinct lack of biochemical markers to aid clinical management. Microvesicles (MVs) have been implicated in the pathophysiology of inflammatory diseases including COPD but their association to COPD disease severity remains unknown. We analysed different MV populations in plasma and bronchoalveolar lavage fluid (BALF) taken from sixty-two patients with mild to very severe COPD (51% male; mean age: 65.9 years). These patients underwent comprehensive clinical evaluation (symptom scores, lung function, exercise testing) and the capacity of MVs to be clinical markers of disease severity was assessed. We successfully identified various MV subtype populations within BALF (leukocyte, PMN (polymorphonuclear leukocyte i.e. neutrophil), monocyte, epithelial and platelet MVs) and plasma (leukocyte, PMN, monocyte and endothelial MVs), and compared each MV population to disease severity. BALF neutrophil MVs were the only population to significantly correlate with the clinical evaluation scores including FEV1, mMRC dyspnoea score, 6-minute walk test, hyperinflation and gas transfer. BALF neutrophil MVs, but not neutrophil cell numbers, also strongly correlated with BODE index. We have undertaken, for the first time, a comprehensive evaluation of MV profiles within BALF/plasma of COPD patients. We demonstrate that BALF levels of neutrophil-derived MVs are unique in correlating with a number of key functional and clinically-relevant disease severity indices. Our results show the potential of BALF neutrophil MVs for a COPD biomarker that tightly links a key pathophysiological mechanism of COPD (intra-alveolar neutrophil activation) with clinical severity/outcome.
Soni S, Romano R, O'Dea K, et al., 2020, Intra-alveolar neutrophil-derived microvesicles predict development of primary graft dysfunction after lung transplantation, Publisher: EUROPEAN RESPIRATORY SOC JOURNALS LTD, ISSN: 0903-1936
Koh M, Takata M, Wilson M, 2020, Cyclophilin A as a Novel Mediator in Ventilator-Induced Lung Injury, Annual Meeting on Experimental Biology, Publisher: WILEY, ISSN: 0892-6638
O'Dea K, Tan YY, Shah S, et al., 2020, Monocytes mediate homing of circulating microvesicles to the pulmonaryvasculature during low-grade systemic inflammation, Journal of Extracellular Vesicles, Vol: 9, Pages: 1-16, ISSN: 2001-3078
Microvesicles (MVs), a plasma membrane-derived subclass of extracellular vesicles, are produced and released into the circulation during systemic inflammation, yet little is known of cell/tissue-specific uptake of MVs under these conditions. We hypothesized that monocytes contribute to uptake of circulating MVs and that their increased margination to the pulmonary circulation and functional priming during systemic inflammation produces substantive changes to the systemic MV homing profile. Cellular uptake of i.v.-injected, fluorescently labelled MVs (J774.1 macrophage-derived) in vivo was quantified by flow cytometry in vascular cell populations of the lungs, liver and spleen of C57BL6 mice. Under normal conditions, both Ly6Chigh and Ly6Clow monocytes contributed to MV uptake but liver Kupffer cells were the dominant target cell population. Following induction of sub-clinical endotoxemia with low-dose i.v. LPS, MV uptake by lung-marginated Ly6Chigh monocytes increased markedly, both at the individual cell level (~2.5-fold) and through substantive expansion of their numbers (~8-fold), whereas uptake by splenic macrophages was unchanged and uptake by Kupffer cells actually decreased (~50%). Further analysis of MV uptake within the pulmonary vasculature using a combined model approach of in vivo macrophage depletion, ex vivo isolated perfused lungs and in vitro lung perfusate cell-based assays, indicated that Ly6Chigh monocytes possess a high MV uptake capacity (equivalent to Kupffer cells), that is enhanced directly by endotoxemia and ablated in the presence of phosphatidylserine (PS)-enriched liposomes and β3 integrin receptor blocking peptide. Accordingly, i.v.-injected PS-enriched liposomes underwent a redistribution of cellular uptake during endotoxemia similar to MVs, with enhanced uptake by Ly6Chigh monocytes and reduced uptake by Kupffer cells. These findings indicate that monocytes, particularly lung-marginated Ly6Chigh subset monocytes, become a dominant
Oakley C, Koh M, Baldi R, et al., 2019, Ventilation following established ARDS: a preclinical model framework to improve predictive power, Thorax, Vol: 74, Pages: 1120-1129, ISSN: 1468-3296
Background Despite advances in understanding the pathophysiology of acute respiratory distress syndrome, effective pharmacological interventions have proven elusive. We believe this is a consequence of existing preclinical models being designed primarily to explore biological pathways, rather than predict treatment effects. Here, we describe a mouse model in which both therapeutic intervention and ventilation were superimposed onto existing injury and explored the impact of β-agonist treatment, which is effective in simple models but not clinically.Methods Mice had lung injury induced by intranasal lipopolysaccharide (LPS), which peaked at 48 hours post-LPS based on clinically relevant parameters including hypoxaemia and impaired mechanics. At this peak of injury, mice were treated intratracheally with either terbutaline or tumour necrosis factor (TNF) receptor 1-targeting domain antibody, and ventilated with moderate tidal volume (20 mL/kg) to induce secondary ventilator-induced lung injury (VILI).Results Ventilation of LPS-injured mice at 20 mL/kg exacerbated injury compared with low tidal volume (8 mL/kg). While terbutaline attenuated VILI within non-LPS-treated animals, it was ineffective to reduce VILI in pre-injured mice, mimicking its lack of clinical efficacy. In contrast, anti-TNF receptor 1 antibody attenuated secondary VILI within pre-injured lungs, indicating that the model was treatable.Conclusions We propose adoption of a practical framework like that described here to reduce the number of ultimately ineffective drugs reaching clinical trials. Novel targets should be evaluated alongside interventions which have been previously tested clinically, using models that recapitulate the (lack of) clinical efficacy. Within such a framework, outperforming a failed pharmacologic should be a prerequisite for drugs entering trials.
Soni S, Tirlapur N, O'Dea KP, et al., 2019, Microvesicles as new therapeutic targets for the treatment of the acute respiratory distress syndrome (ARDS), EXPERT OPINION ON THERAPEUTIC TARGETS, Vol: 23, Pages: 931-941, ISSN: 1472-8222
Soni S, O'Dea K, Tan YY, et al., 2019, ATP redirects cytokine trafficking and promotes novel membrane TNF signalling via microvesicles, FASEB Journal, ISSN: 0892-6638
Cellular stress or injury induces release of endogenous danger signals such as ATP, which plays a central role in activating immune cells. ATP is essential for the release of nonclassically secreted cytokines such as IL-1β but, paradoxically, has been reported to inhibit the release of classically secreted cytokines such as TNF. Here, we reveal that ATP does switch off soluble TNF (17 kDa) release from LPS-treated macrophages, but rather than inhibiting the entire TNF secretion, ATP packages membrane TNF (26 kDa) within microvesicles (MVs). Secretion of membrane TNF within MVs bypasses the conventional endoplasmic reticulum– and Golgi transport–dependent pathway and is mediated by acid sphingomyelinase. These membrane TNF–carrying MVs are biologically more potent than soluble TNF in vivo, producing significant lung inflammation in mice. Thus, ATP critically alters TNF trafficking and secretion from macrophages, inducing novel unconventional membrane TNF signaling via MVs without direct cell-to-cell contact. These data have crucial implications for this key cytokine, particularly when therapeutically targeting TNF in acute inflammatory diseases.—Soni, S., O’Dea, K. P., Tan, Y. Y., Cho, K., Abe, E., Romano, R., Cui, J., Ma, D., Sarathchandra, P., Wilson, M. R., Takata, M. ATP redirects cytokine trafficking and promotes novel membrane TNF signaling via microvesicles.
Wilson M, Takata M, 2019, Mechanical Ventilation in Mice: Does Longer Equal Better?, AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY, Vol: 60, Pages: 137-138, ISSN: 1044-1549
Koh MW, Takata M, Wilson MR, 2019, Cyclophilin A as a Novel Mediator in Acute Lung Injury, International Conference of the American-Thoracic-Society, Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Garner J, Soni S, O'Dea K, et al., 2018, Late Breaking Abstract - Intra-alveolar neutrophil-derived microvesicles: a biomarker of COPD severity, 28th International Congress of the European-Respiratory-Society (ERS), Publisher: EUROPEAN RESPIRATORY SOC JOURNALS LTD, ISSN: 0903-1936
Du W, Takata M, Wilson MR, 2018, Activation of lung-marginated monocytes in initiating ventilator-induced extra-pulmonary inflammation, British-Journal-of-Anaesthesia (BJA) Research Forum, Publisher: ELSEVIER SCI LTD, Pages: E25-E26, ISSN: 0007-0912
Nakamura H, Wilson MR, Yao K, et al., 2018, Modulation Of Mechanical Ventilation-Induced Alveolar Epithelial Cell Death Signaling By Underlying Lung Inflammation, American Thoracic Society 2018, Publisher: American Thoracic Society
Soni S, Wilson MR, Dea KPO, et al., 2018, Microvesicles Mediate Long-Range Membrane TNF Signalling, Inducing Inflammatory Changes Consistent with Acute Lung Injury, International Conference of the American-Thoracic-Society, Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Wilson MR, Petrie JE, Shaw MW, et al., 2017, High fat feeding protects mice from ventilator-induced lung injury, via neutrophil-independent mechanisms, Critical Care Medicine, Vol: 45, Pages: e831-e839, ISSN: 1530-0293
Objective: Obesity has a complex impact on acute respiratory distress syndrome patients, being associated with increased likelihood of developing the syndrome but reduced likelihood of dying. We propose that such observations are potentially explained by a model in which obesity influences the iatrogenic injury that occurs subsequent to intensive care admission. This study therefore investigated whether fat feeding protected mice from ventilator-induced lung injury.Design: In vivo study.Setting: University research laboratory.Subjects: Wild-type C57Bl/6 mice or tumor necrosis factor receptor 2 knockout mice, either fed a high-fat diet for 12–14 weeks, or age-matched lean controls.Interventions: Anesthetized mice were ventilated with injurious high tidal volume ventilation for periods up to 180 minutes.Measurements and Main Results: Fat-fed mice showed clear attenuation of ventilator-induced lung injury in terms of respiratory mechanics, blood gases, and pulmonary edema. Leukocyte recruitment and activation within the lungs were not significantly attenuated nor were a host of circulating or intra-alveolar inflammatory cytokines. However, intra-alveolar matrix metalloproteinase activity and levels of the matrix metalloproteinase cleavage product soluble receptor for advanced glycation end products were significantly attenuated in fat-fed mice. This was associated with reduced stretch-induced CD147 expression on lung epithelial cells.Conclusions: Consumption of a high-fat diet protects mice from ventilator-induced lung injury in a manner independent of neutrophil recruitment, which we postulate instead arises through blunted up-regulation of CD147 expression and subsequent activation of intra-alveolar matrix metalloproteinases. These findings may open avenues for therapeutic manipulation in acute respiratory distress syndrome and could have implications for understanding the pathogenesis of lung disease in obese patients.
Tirlapur N, O'Dea K, Soni S, et al., 2017, Pathological Stretch Of Endothelial Cells Activates Marginated Monocytes To Release Microvesicles In An In Vitro Model Of Ventilator-Induced Lung Injury, International Conference of the American-Thoracic-Society (ATS), Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Oakley CM, Wilson M, O'Dea K, et al., 2017, Tnfr1 Inhibition Mitigates Susceptibility To Ventilator-Induced Lung Injury In Mice With Established Ards, International Conference of the American-Thoracic-Society (ATS), Publisher: American Thoracic Society, ISSN: 1073-449X
Takata M, Wilson MR, 2017, If We Ask a Mouse about Biotrauma, Will It Give Us a Sensible Answer?, ANESTHESIOLOGY, Vol: 126, Pages: 766-767, ISSN: 0003-3022
Wilson MR, Wakabayashi K, Bertok S, et al., 2017, Inhibition of TNF receptor p55 by a domain antibody attenuates the initial phase of acid-induced lung injury in mice, Frontiers in Immunology, Vol: 8, ISSN: 1664-3224
Background: Tumor necrosis factor-α (TNF) is strongly implicated in the development ofacute respiratory distress syndrome (ARDS), but its potential as a therapeutic target has beenhampered by its complex biology. TNF signals through two receptors, p55 and p75, whichplay differential roles in pulmonary edema formation during ARDS. We have recentlyshown that inhibition of p55 by a novel domain antibody (dAb™) attenuated ventilator36induced lung injury. In the current study we explored the efficacy of this antibody in mousemodels of acid-induced lung injury, to investigate the longer consequences of treatment.Methods: We employed two acid-induced injury models, an acute ventilated model and aresolving spontaneously breathing model. C57BL/6 mice were pretreated intratracheally orintranasally with p55-targeting dAb or non-targeting ‘dummy’ dAb, 1 or 4 hours before acidinstillation.Results: Acid instillation in the dummy dAb group caused hypoxemia, increased respiratorysystem elastance, pulmonary inflammation and edema in both the ventilated and resolvingmodels. Pretreatment with p55-targeting dAb significantly attenuated physiological markersof ARDS in both models. p55-targeting dAb also attenuated pulmonary inflammation in theventilated model, with signs that altered cytokine production and leukocyte recruitmentpersisted beyond the very acute phase.Conclusions: These results demonstrate that the p55-targeting dAb attenuates lung injury andedema formation in models of ARDS induced by acid aspiration, with protection from asingle dose lasting up to 24 hours. Together with our previous data, the current study lends support towards the clinical targeting of p55 for patients with, or at risk of ARDS.
Takata M, Wilson M, 2017, Editorial View: If we ask a mouse about biotrauma, will it give us a sensibleanswer?, Anesthesiology, ISSN: 1528-1175
Acuterespiratory distress syndrome (ARDS) is a major cause of mortalityin critical care, but to date no specific treatment exists.There is growing concern about our failure to translate from bench to bedside within the acute lung injury research community, and the crucial importance of better modelling in preclinical studiesto identify targetswithmore predictivepoweris increasingly appreciated. Mechanical ventilation, while being a vital tool forsupport of ARDSpatients, produces or worsens lung injury. This ‘ventilator-induced lung injury’(VILI)hassubstantive negative impact on theoutcomeof ARDS.Increasing tidal volumes are associated with enhanced releaseof local and systemic inflammatory mediatorsinpatients, andanimal models demonstrated that excessive tidal volumes inducelung inflammation,edema and physiological dysfunction. Such findings have lent support to the ‘biotrauma’ hypothesis, i.e. VILIpromotes the release of inflammatorymediators,which play a criticalrole in the progression of injury of the lungs as well asother systemic organs .In this issueof Anesthesiology, Lex and Uhlig  investigatewhether this biotraumacan be studied in so-called ‘one-hit’ models of VILI in mice. Their resultsprovide useful information for physiologists to better design mouse VILI experiments, but more importantly, provoke a series of important questionsthat areessentialfor cliniciansdesiring to interpret animal VILI models for future clinical translation.
Wilson MR, Takata M, John AE, et al., 2016, Loss of epithelial Gq and G11 signaling inhibits TGFβ production but promotes IL-33–mediated macrophage polarization and emphysema, Science Signaling, Vol: 9, Pages: 1-17, ISSN: 1945-0877
Heterotrimeric guanine nucleotide–binding protein (G protein) signaling links hundreds of Gprotein–coupled receptors (GPCRs) with four G protein signaling pathways. Two of these,one mediated by Gq and G11 (Gq/11) and the other by G12 and G13 (G12/13), are implicated inthe force-dependent activation of transforming growth factor–β (TGFβ) in lung epithelialcells. Reduced TGFβ activation in alveolar cells leads to emphysema, whereas enhancedTGFβ activation promotes acute lung injury and idiopathic pulmonary fibrosis. Therefore,precise control of alveolar TGFβ activation is essential for alveolar homeostasis. Here, weinvestigated the involvement of the Gq/11and G12/13 pathways in epithelial cells in generatingactive TGFβ and regulating alveolar inflammation. Mice deficient in both Gαq and Gα11developed inflammation that was primarily caused by alternatively activated (M2-polarized)macrophages, enhanced matrix metalloprotease 12 (MMP12) production, and age-relatedalveolar airspace enlargement consistent with emphysema. Mice with impaired Gq/11signaling had reduced stretch-mediated generation of TGFβ by epithelial cells and enhancedmacrophage MMP12 synthesis, but were protected from the effects of ventilator-induced lunginjury. Furthermore, synthesis of the cytokine interleukin-33 (IL-33) was increased in thesealveolar epithelial cells, resulting in the M2-type polarization of alveolar macrophages independently of the effect on TGFβ. Our results suggest that alveolar Gq/11 signalingmaintains alveolar homeostasis, and likely independently increases TGFβ activation inresponse to mechanical stress of the epithelium and decreases epithelial IL-33 synthesis.Together, these findings suggest that disruption of Gq/11 signaling promotes inflammatoryemphysema but protects against mechanically induced lung injury.
Soni S, Wilson MR, O'Dea KP, et al., 2016, Alveolar macrophage-derived microvesicles mediate acute lung injury, Thorax, Vol: 71, Pages: 1020-1029, ISSN: 1468-3296
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.
Halford P, Woods S, Wilson M, et al., 2016, Murine pulmonary cell response to varying degrees of mechanical ventilation and its modification by lipopolysaccharide, Meeting of the Difficult-Airway-Society, Publisher: Oxford University Press (OUP), Pages: E933-E934, ISSN: 1471-6771
Wilson M, Petrie J, Shaw M, et al., 2016, High Fat Feeding Protects Mice From Ventilator-Induced Lung Injury Via A Neutrophil-Independent Mechanism, International Conference of the American-Thoracic-Society (ATS), Publisher: AMER THORACIC SOC, ISSN: 1073-449X
Soni S, Yoshida M, Woods S, et al., 2015, Microvesicles derived from alveolar macrophages mediate epithelial activation via a TNF dependant mechanism, Publisher: EUROPEAN RESPIRATORY SOC JOURNALS LTD, ISSN: 0903-1936
Patel BV, Tatham KC, Wilson MR, et al., 2015, In vivo compartmental analysis of leukocytes in mouse lungs, American Journal of Physiology-Lung Cellular and Molecular Physiology, Vol: 309, Pages: L639-L652, ISSN: 1522-1504
The lung has a unique structure consisting of three functionally different compartments (alveolar, interstitial, and vascular) situated in an extreme proximity. Current methods to localize lung leukocytes using bronchoalveolar lavage and/or lung perfusion have significant limitations for determination of location and phenotype of leukocytes. Here we present a novel method using in vivo antibody labelling to enable accurate compartmental localization/quantification and phenotyping of mouse lung leukocytes. Anesthetized C57BL/6 mice received combined in vivo intravenous and intratracheal labelling with fluorophore-conjugated anti-CD45 antibodies, and lung single cell suspensions were analyzed by flow cytometry. The combined in vivo intravenous and intratracheal CD45 labelling enabled robust separation of the alveolar, interstitial, and vascular compartments of the lung. In naive mice, the alveolar compartment consisted predominantly of resident alveolar macrophages. The interstitial compartment, gated by events negative for both intratracheal and intravenous CD45 staining, showed two conventional dendritic cell populations, as well as a Ly6C(lo) monocyte population. Expression levels of MHCII on these interstitial monocytes were much higher than the vascular Ly6C(lo) monocyte populations. In mice exposed to acid-aspiration induced lung injury, this protocol also clearly distinguished the three lung compartments showing the dynamic trafficking of neutrophils and exudative monocytes across the lung compartments during inflammation and resolution. This simple in vivo dual labelling technique substantially increases the accuracy and depth of lung flow cytometric analysis, facilitates a more comprehensive examination of lung leukocyte pools, and enables the investigation of previously poorly defined 'interstitial' leukocyte populations during models of inflammatory lung diseases.
Patel BV, Tatham KC, Wilson MR, et al., 2015, In Vivo Compartmental Labeling of the Mouse Lung, American Thoracic Society, Publisher: ATS Journals, Pages: A3926-A3926, ISSN: 1073-449X
ShareModerators: G.N. Maksym, PhD, R.S. Harris, MD, J.C. Sieren, PhDSession Info: Mini Symposium, [C18] STATE OF PLAY IN RESPIRATORY STRUCTURE AND FUNCTION: SEEING IS BELIEVINGDay/Date: Tuesday, May 19, 2015Session Time: 9:30 AM - 11:30 AMRoom: Mile High Ballroom 2C/3C (Lower Level)Location: Colorado Convention CenterIn Vivo Compartmental Labeling of the Mouse Lung, [Publication Page: A3926]B.V. Patel, MBBS MRCP FRCA PhD, K.C. Tatham, MBBS, M.R. Wilson, PhD, K.P. O'Dea, PhD, M. Takata, MDLondon/UKRationaleCurrent methods for compartmental analyses of lungs using bronchoalveolar lavage (BAL) and/or lung perfusion have significant limitations for accurate determination of location and phenotype of lung leukocytes. BAL retrieves only <25% of intra-alveolar cells (1) and lung perfusion fails to remove vascular marginated cell populations (2). We present a novel method of in vivo antibody labelling to facilitate the accurate compartmental investigation of mouse lung leukocytes by flow cytometry.MethodsAnesthetized C57BL6 mice underwent tracheostomy and venous cannulation. An anti-CD45 antibody (PE-conjugated) was injected intravenously and allowed to circulate for 5 minutes. Mice were then exsanguinated and lungs immediately flooded intratracheally with another anti-CD45 antibody (PE-Cy5-conjugated). Lungs were harvested, and single cell suspensions prepared for flow cytometric analysis using a panel of myeloid markers. This in vivo labelling protocol was also applied to mice exposed to acid-aspiration induced lung injury (3). To confirm the separation between the vascular and interstitial cell populations, a group of mice underwent intravenous labelling alone followed by lung perfusion for 15 minutes using an isolated-perfused lung apparatus (2).ResultsThe combined in vivo intravenous and intratracheal CD45 labelling enabled robust separation of the alveolar, interstitial, and vascular compartments of the lung by flow cytometry, with <0.3% of leukocytes staining
Woods SJ, Waite AAC, O'Dea KP, et al., 2015, Kinetic profiling of in vivo lung cellular inflammatory responses to mechanical ventilation, AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY, Vol: 308, Pages: L912-L921, ISSN: 1040-0605
Lax S, Wilson MR, Takata M, et al., 2014, Using a non-invasive assessment of lung injury in a murine model of acute lung injury, BMJ Open Respiratory Research, Vol: 1, ISSN: 2052-4439
Arterial oxygen saturation has not been assessed sequentially in conscious mice as a direct consequence of an in vivo murine model of acute lung injury. Here, we report daily changes in arterial oxygen saturation and other cardiopulmonary parameters by using infrared pulse oximetry following intratracheal lipopolysaccharide (IT-LPS) for up to 9 days, and following IT-phosphate buffered saline up to 72 h as a control. We show that arterial oxygen saturation decreases, with maximal decline at 96 h post IT-LPS. Blood oxygen levels negatively correlate with 7 of 10 quantitative markers of murine lung injury, including neutrophilia and interleukin-6 expression. This identifies infrared pulse oximetry as a method to non-invasively monitor arterial oxygen saturation following direct LPS instillations.
This data is extracted from the Web of Science and reproduced under a licence from Thomson Reuters. You may not copy or re-distribute this data in whole or in part without the written consent of the Science business of Thomson Reuters.