Cathepsin C from extracellular histone-induced M1 alveolar macrophages promotes NETosis during lung ischemia-reperfusion injury.

Primary graft dysfunction (PGD) is a severe form of acute lung injury resulting from lung ischemia/reperfusion injury (I/R) in lung transplantation (LTx), associated with elevated post-transplant morbidity and mortality rates. Neutrophils infiltrating during reperfusion are identified as pivotal contributors to lung I/R injury by releasing excessive neutrophil extracellular traps (NETs) via NETosis. While alveolar macrophages (AMs) are involved in regulating neutrophil chemotaxis and infiltration, their role in NETosis during lung I/R remains inadequately elucidated. Extracellular histones constitute the main structure of NETs and can activate AMs. In this study, we confirmed the significant involvement of extracellular histone-induced M1 phenotype of AMs (M1-AMs) in driving NETosis during lung I/R. Using secretome analysis, public protein databases, and transwell co-culture models of AMs and neutrophils, we identified Cathepsin C (CTSC) derived from AMs as a major mediator in NETosis. Further elucidating the molecular mechanisms, we found that CTSC induced NETosis through a pathway dependent on NADPH oxidase-mediated production of reactive oxygen species (ROS). CTSC could significantly activate p38 MAPK, resulting in the phosphorylation of the NADPH oxidase subunit p47phox, thereby facilitating the trafficking of cytoplasmic subunits to the cell membrane and activating NADPH oxidase. Moreover, CTSC up-regulated and activated its substrate membrane proteinase 3 (mPR3), resulting in an increased release of NETosis-related inflammatory factors. Inhibiting CTSC revealed great potential in mitigating NETosis-related injury during lung I/R. These findings suggests that CTSC from AMs may be a crucial factor in mediating NETosis during lung I/R, and targeting CTSC inhition may represent a novel intervention for PGD in LTx.

Extracellular histones promote TWIK2-dependent potassium efflux and associated NLRP3 activation in alveolar macrophages during sepsis-induced lung injury.

BACKGROUND AND AIM: Sepsis-induced acute lung injury (ALI) is a complex and life-threatening condition lacking specific and efficient clinical treatments. Extracellular histones, identified as a novel type of damage-associated molecular patterns, have been implicated in the inflammatory process of ALI. However, further elucidation is needed regarding the precise mechanism through which extracellular histones induce inflammation. The aim of this study was to investigate whether extracellular histones can activate NLRP3 inflammasome-mediated inflammation in alveolar macrophages (AMs) by affecting TWIK2-dependent potassium efflux. METHODS AND RESULTS: We conducted experiments using cecal ligation and puncture (CLP) C57BL/6 mice and extracellular histone-stimulated LPS-primed MH-S cells. The results demonstrated a significant increase in the levels of extracellular histones in the plasma and bronchoalveolar lavage fluid (BALF) of CLP mice. Furthermore, neutralizing extracellular histone mitigated lung injury and inflammation in CLP-induced ALI mice. In vitro studies confirmed that extracellular histones upregulated the expression of NLRP3 inflammasome activation-related proteins in MH-S cells, and this effect was dependent on increased potassium efflux mediated by the TWIK2 channel on the plasma membrane. Moreover, extracellular histones directly triggered a substantial influx of calcium, leading to increased Rab11 activity and facilitating the trafficking and location of TWIK2 to the plasma membrane. CONCLUSION: These findings underscore the critical role of extracellular histone-induced upregulation of TWIK2 expression on the plasma membrane of alveolar macrophages (AMs). This upregulation leads to potassium efflux and subsequent activation of the NLRP3 inflammasome, ultimately exacerbating lung inflammation and injury during sepsis.

Effect of bilateral low serratus anterior plane block on quality of recovery after trans-subxiphoid robotic thymectomy: Results of a randomized placebo-controlled trial.

Purpose: This study aimed to investigate the impact of ultrasound-guided, bilateral, low level (T8-T9) deep serratus anterior plane (DSAP) blocks on postoperative recovery quality and postoperative analgesia in patients undergoing trans-subxiphoid robotic thymectomy (TRT). Methods: 39 patients undergoing TRT were randomized to receive either low DSAP block under general anesthesia (Group S) or the sham block (Group C) on each side. The primary outcome was the QoR-40 score at postoperative day (POD) 1. Secondary outcomes included numeric rating scale (NRS) scores over time, postoperative 48 hours opioid consumption, QoR-40 scores at POD 2, 30, and 90. Results: The QoR-40 scores on POD1-2 were higher in Group S than in Group C [179.1 (4.9) vs 167.7 (2.8), P < 0.01; 187.7 (4.6) vs 178.1 (3), P < 0.01, respectively]. Pain scores were significantly lower in Group S, both during resting and motion at postoperative 6h, 12h, and 24h (P < 0.05 for each). The total amount of sufentanil consumed in the first 48 h was lower in Group S than in Group C [61.4 (4.9) vs 78.9 (4.6), P < 0.001]. Conclusion: The bilateral low DSAP blocks enhanced the QoR-40 for 2 days postoperatively, relieved postsurgical pain, and reduced opioid consumption during the early postoperative period in patients undergoing TRT.

Identification of Neutrophil Extracellular Trap-Related Gene Expression Signatures in Ischemia Reperfusion Injury During Lung Transplantation: A Transcriptome Analysis and Clinical Validation.

Purpose: Ischemia reperfusion injury (IRI) unavoidably occurs during lung transplantation, further contributing to primary graft dysfunction (PGD). Neutrophils are the end effectors of IRI and activated neutrophils release neutrophil extracellular traps (NETs) to further amplify damage. Nevertheless, potential contributions of NETs in IRI remain incompletely understood. This study aimed to explore NET-related gene biomarkers in IRI during lung transplantation. Methods: Differential expression analysis was applied to identify differentially expressed genes (DEGs) for IRI during lung transplantation based on matrix data (GSE145989, 127003) downloaded from GEO database. The CIBERSORT and weighted gene co-expression network analysis (WGCNA) algorithms were utilized to identify key modules associated with neutrophil infiltration. Moreover, the least absolute shrinkage and selection operator regression and random forest were applied to identify potential NET-associated hub genes. Subsequently, the screened hub genes underwent further validation of an external dataset (GSE18995) and nomogram model. Based on clinical peripheral blood samples, immunofluorescence staining and dsDNA quantification were used to assess NET formation, and ELISA was applied to validate the expression of hub genes. Results: Thirty-eight genes resulted from the intersection between 586 DEGs and 75 brown module genes, primarily enriched in leukocyte migration and NETs formation. Subsequently, four candidate hub genes (FCAR, MMP9, PADI4, and S100A12) were screened out via machine learning algorithms. Validation using an external dataset and nomogram model achieved better predictive value. Substantial NETs formation was demonstrated in IRI, with more pronounced NETs observed in patients with PGD ≥ 2. PADI4, S100A12, and MMP9 were all confirmed to be up-regulated after reperfusion through ELISA, with higher levels of S100A12 in PGD ≥ 2 patients compared with non-PGD patients. Conclusion: We identified three potential NET-related biomarkers for IRI that provide new insights into early detection and potential therapeutic targets of IRI and PGD after lung transplantation.

Intraoperative partial pressure of arterial carbon dioxide levels and adverse outcomes in patients undergoing lung transplantation.

OBJECTIVE: Patients undergoing lung transplantation (LTx) often experience abnormal hypercapnia or hypocapnia. This study aimed to investigate the association between intraoperative PaCO2 and postoperative adverse outcomes in patients undergoing LTx. METHODS: We retrospectively reviewed the medical records of 151 patients undergoing LTx. Patients' demographics, perioperative clinical factors, and pre- and intraoperative PaCO2 data after reperfusion were collected and analyzed. Based on the PaCO2 levels, patients were classified into three groups: hypocapnia (≤35 mmHg), normocapnia (35.1-55 mmHg), and hypercapnia (>55 mmHg). Univariate and multivariable logistic regressions were used to identify independent risk factors for postoperative composite adverse events and in-hospital mortality. RESULTS: Intraoperative hypercapnia occurred in 69 (45.7%) patients, and hypocapnia in 17 (11.2%). Patients with intraoperative PaCO2 of 35.1-45 mmHg showed a lower incidence of composite adverse events (53.3%) and mortality (6.2%) (P < 0.001). There was no significant difference in composite adverse events and mortality among preoperative PaCO2 groups (P > 0.05). Compared with intraoperative PaCO2 at 35.1-45 mmHg, the risk of composite adverse events in hypercapnia group increased: the adjusted OR was 3.07 (95% confidence interval [CI]: 1.36-6.94; P = 0.007). The risk of death was significantly higher in hypocapnia group than normocapnia group, the adjusted OR was 7.69 (95% CI: 1.68-35.24; P = 0.009). Over ascending ranges of PaCO2, PaCO2 at 55.1-65 mmHg had the strongest association with composite adverse events, the adjusted OR was 6.40 (95% CI: 1.18-34.65; P = 0.031). CONCLUSION: These results demonstrate that intraoperative hypercapnia independently predicts postoperative adverse outcomes in patients undergoing LTx. Intraoperative hypocapnia shows predictive value for postoperative in-hospital mortality in LTx.

Effects of preoperative mildly elevated pulmonary artery systolic pressure on the incidence of perioperative adverse events undergoing thoracoscopic lobectomy: an observational cohort study protocol.

INTRODUCTION: Echocardiography provides a non-invasive estimation of pulmonary artery systolic pressure (PASP) and is the first diagnostic test for pulmonary hypertension. Recent studies have demonstrated that PASP of more than 30 mm Hg related to increased mortality and morbidity. However, perioperative risks and management for patients with mildly elevated PASP are not well established. This study aims to explore the association between mildly elevated PASP and perioperative adverse outcomes. METHODS AND ANALYSIS: This will be a retrospective cohort study conducted at Shanghai Pulmonary Hospital in Shanghai, China. Eligible patients are adults (≥18 years) who performed preoperative echocardiography and followed thoracoscopic lobectomy. Our primary objective is to determine the effect of preoperative mildly elevated PASP on the incidence of hypotension during surgery. Whether mildly elevated PASP is related to other perioperative adverse events (including hypoxaemia, myocardial injury, new-onset atrial fibrillation, postoperative pulmonary complications, 30-day readmission and 30-day mortality) will be also analysed. An estimated 2300 patients will be included. ETHICS AND DISSEMINATION: The study has been approved by the institutional review board of Shanghai Pulmonary Hospital (approval No: 2022LY1143). The research findings intend to be published in peer-reviewed scientific publications. TRIAL REGISTRATION NUMBER: Chinese Clinical Trial Registry (ChiCTR2200066679).

Transcriptome analysis of novel macrophage M1-related biomarkers and potential therapeutic agents in ischemia-reperfusion injury after lung transplantation based on the WGCNA and CIBERSORT algorithms.

Lung transplantation is the last effective treatment for end-stage respiratory failure, however, with ischemia-reperfusion injury (IRI) inevitably occurring in postoperative period. IRI is the major pathophysiologic mechanism of primary graft dysfunction, a severe complication that contributes to prolonged length of stay and overall mortality. The understanding of pathophysiology and etiology remain limited and the underlying molecular mechanism, as well as novel diagnostic biomarkers and therapeutic targets, urgently require exploration. Excessive uncontrolled inflammatory response is the core mechanism of IRI. In this research, a weighted gene co-expression network was established using the CIBERSORT and WGCNA algorithms in order to identify macrophage-related hub genes based on the data downloaded from the GEO database (GSE127003, GSE18995). 692 differentially expressed genes (DEGs) in reperfused lung allografts were identified, with three genes recognized as being related to M1 macrophages and validated as differentially expressed using GSE18995 dataset. Of these putative novel biomarker genes, TCRα subunit constant gene (TRAC) were downregulated, while Perforin-1 (PRF1) and Granzyme B (GZMB) were upregulated in reperfused vs. ischemic lung allografts. Furthermore, we obtained 189 potentially therapeutic small molecules for IRI after lung transplantation from the CMap database among which PD-98059 was the top molecule with the highest absolute correlated connectivity score (CS). Our study provides the novel insights into the impact of immune cells on the etiology of IRI and potential targets for therapeutic intervention. Nevertheless, further investigation of these key genes and therapeutic drugs is needed to validate their effects.

Association of preoperative spirometry tests with postoperative pulmonary complications after mediastinal mass resection: protocol for a retrospective cohort study.

INTRODUCTION: Patients with a mediastinal mass are at risk of pulmonary complications in the perioperative period. Preoperative spirometry tests are recommended in patients scheduled for thoracic surgery. Our objective is to investigate the association between preoperative spirometry results and the incidence of postoperative pulmonary complications in patients following mediastinal mass resection, which may determine the usefulness of spirometry tests in the prediction of the perioperative respiratory risk. METHODS AND ANALYSIS: This protocol describes a retrospective cohort study of patients with mediastinal masses in Shanghai Pulmonary Hospital between 1 September 2021 and 1 September 2022, with a planned sample size of 660 patients. The primary aim of this study is to explore the association between preoperative spirometry results and the occurrence of postoperative pulmonary complications after mediastinal mass resection. Logistic regression analysis will be used to calculate the adjusted incidence rate difference and incidence rate ratios (with 95% CIs). ETHICS AND DISSEMINATION: The study was approved by the ethics committee of Shanghai Pulmonary Hospital (K21-372Y). The results of the study will be submitted to a peer-reviewed biomedical journal for publication and presented at relevant conferences.

Ultrasound-Guided Bilateral Sequential Thoracic Paravertebral Block for Simultaneous Bilateral Uniportal Video-Assisted Thoracoscopic Surgery: Study Protocol for a Randomized Controlled Trial.

Purpose: Simultaneous bilateral pulmonary resection via uniportal video-assisted thoracoscopic surgery (UVATS) was safe and feasible for the treatment of bilateral multiple pulmonary nodules. But, it should be noted that considerable postoperative pain at the bilateral surgical site was a crucial issue. The safety and efficacy of bilateral thoracic paravertebral block (TPVB) have been reported for postoperative analgesia. But, whether bilateral sequential TPVB can be safely and effectively used in simultaneous bilateral UVATS remains unknown. Therefore, this study aimed to determine the analgesic efficacy and safety of bilateral sequential TPVB after simultaneous bilateral UVATS. Study Design and Methods: In this study, 80 participants scheduled for UVATS will be randomly allocated to the bilateral sequential TPVB group (G2) and the control group (G1). The patient of G2 will be performed bilateral TPVB at 2 time-points: before the start of the first side of pulmonary resection and before the start of the contralateral pulmonary resection. G1 will only receive standard analgesia protocol. The primary outcome is the numeric rating scale score during coughing at 24 h postoperatively. The secondary outcomes include the Prince Henry Pain Score scores, sufentanil consumption, postoperative nausea and vomiting, levels of inflammatory factors, and the Quality of Recovery-40 scores at different time points, as well as chronic pain at postoperative day (POD) 90. Discussion: This is the first prospective trial to determine the safety and effectiveness of ultrasound-guided bilateral sequential TPVB for postoperative analgesia following simultaneous bilateral UVATS. This study also intended to evaluate the effect of this intervention on postoperative quality of recovery and inflammation levels. The final results will provide clinical evidence related to bilateral sequential TPVB, and promote the application of that acting as a more appropriate analgesic method for simultaneous bilateral UVATS.

Shock lung is not "wet" but characterized as necroptotic inflammation in a mouse model of hypotension.

OBJECTIVES: Hypotension episodes before or after donor brain death are assumed to trigger hypoxia-reoxygenation, causing diffuse alveolar-capillary damage via necrosis. However, alveolar-capillary membranes have direct access to oxygen in alveoli. We hypothesized hypotension-induced lung injury is not diffuse alveolar-capillary damage but interstitial inflammation resulting from nonhypoxic lung ischemia and systemic responses to hypoxic extrapulmonary ischemia. METHODS: The 4-hour hypotension model was established by subjecting C57BL/6J mice to 4-hour hypotension at 15 ± 5 mm Hg of mean artery pressure and resuscitated with whole shed blood and norepinephrine. Nonhypoxic lung ischemia model was established by 4-hour left pulmonary artery ligation. At 24 hours postprocedure, an arterial blood gas analysis and a gastroduodenal occult blood test were conducted. Lung samples were assessed for histology, cytokine transcripts, regulated cell death, and alveolar-capillary permeability. RESULTS: The 4-hour hypotension model had an intraoperative mortality rate of 17.7% (41/231) and a stress-ulcer bleeding rate of 15.3% (29/190). No signs of alveolar flooding were observed in both models. Four-hour hypotension without stress ulcer showed normal oxygenation and permeability but increased interstitial infiltration, transcription of Tnf and Il1b, phosphorylation of MLKL and RIPK3, and cleaved caspase 3 compared with 4-hour pulmonary artery ligation and naïve control. Animals that developed stress ulcer presented with worse pulmonary infiltration, intracellular edema, and oxygenation but just slightly increased permeability. Immunoblotting showed significant upregulations of protein expression and phosphorylation of MLKL and RIPK3, cleaved Caspase-3, but not its prototype in 4-hour hypotension with stress ulcer. CONCLUSIONS: Hypotensive lung injury is essentially a nonhypoxic ischemia-reperfusion injury enhanced by systemic responses. It is predominated by necroptosis-induced inflammation rather than necrosis-induced diffuse alveolar-capillary damage.

Diagnostic and Therapeutic Implications of Ex Vivo Lung Perfusion in Lung Transplantation: Potential Benefits and Inherent Limitations.

Ex vivo lung perfusion (EVLP), a technique in which isolated lungs are continually ventilated and perfused at normothermic temperature, is emerging as a promising platform to optimize donor lung quality and increase the lung graft pool. Over the past few decades, the EVLP technique has become recognized as a significant achievement and gained much attention in the field of lung transplantation. EVLP has been demonstrated to be an effective platform for various targeted therapies to optimize donor lung function before transplantation. Additionally, some physical parameters during EVLP and biological markers in the EVLP perfusate can be used to evaluate graft function before transplantation and predict posttransplant outcomes. However, despite its advantages, the clinical practice of EVLP continuously encounters multiple challenges associated with both intrinsic and extrinsic limitations. It is of utmost importance to address the advantages and disadvantages of EVLP for its broader clinical usage. Here, the pros and cons of EVLP are comprehensively discussed, with a focus on its benefits and potential approaches for overcoming the remaining limitations. Directions for future research to fully explore the clinical potential of EVLP in lung transplantation are also discussed.

Exploring predisposing factors and pathogenesis contributing to injuries of donor lungs.

INTRODUCTION: Lung transplantation (LTx) remains the only therapeutic strategy for patients with incurable lung diseases. However, its use has been severely limited by the narrow donor pool and potential concerns of inferior quality of donor lungs, which are more susceptible to external influence than other transplant organs. Multiple insults, including various causes of death and a series of perimortem events, may act together on donor lungs and eventually culminate in primary graft dysfunction (PGD) after transplantation as well as other poor short-term outcomes. AREAS COVERED: This review focuses on the predisposing factors contributing to injuries to the donor lungs, specifically focusing on the pathogenesis of these injuries and their impact on post-transplant outcomes. Additionally, various maneuvers to mitigate donor lung injuries have been proposed. EXPERT OPINION: The selection criteria for eligible donors vary and may be poor discriminators of lung injury. Not all transplanted lungs are in ideal condition. With the rapidly increasing waiting list for LTx, the trend of using marginal donors has become more apparent, underscoring the need to gain a deeper understanding of donor lung injuries and discover more donor resources.

[Research progress on the mechanism of role of podoplanin in sepsis].

Podoplanin (PDPN) is a small transmembrane mucin-like glycoprotein which is expressed on the surface of lymphatic endothelial cells, glomerular podocytes, type-I alveolar epithelial cells and some tumor cells. PDPN plays crucial function in variety of physiological and pathological processes such as embryonic development, immunoreaction, inflammation and cancer. C-type lectin-like receptor 2 (CLEC2) is mainly expressed on the platelet which specific ligand is PDPN. The interaction between PDPN and CLEC2 has received extensive attention. In this review, we summarized recent researches on the role of in sepsis and elaborated the possible mechanisms and some potential therapies for sepsis by targeting PDPN, which may provide theoretical basis for the mechanism and treatment of sepsis.

The Role of Podoplanin in the Immune System and Inflammation.

Podoplanin is a small cell-surface mucin-like glycoprotein that participates in multiple physiological and pathological processes. Podoplanin exerts an important function in the immune response and is upregulated in fibroblasts, macrophages, T helper cells, and epithelial cells during inflammation. Herein, we summarize the latest knowledge on the functional expression of podoplanin in the immune system and review the contribution of podoplanin to several inflammatory diseases. Furthermore, we discuss podoplanin as a novel therapeutic target for various inflammatory diseases.

Inhibition of O-glycosylation aggravates GalN/LPS-induced liver injury through activation of ER stress.

OBJECTIVE: O-glycosylation is the most common post-translational modification of proteins, which is involved in many pathophysiological processes including inflammation. Acute liver injury is characterized by an excessive, uncontrolled inflammatory response, but the effects of aberrant O-glycosylation on acute liver injury are yet to explore. Here we aimed to investigate the role of defective O-glycosylation in D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced acute liver damage in mice. MATERIAL AND METHODS: Experimental mice were administrated with an O-glycosylation inhibitor (benzyl-a-GalNac, 5 mg/kg) at 24 h before administration of GalN/LPS. At 12 h after GalN/LPS administration, mice were sacrificed to collect blood and liver samples for further analysis. RESULTS: We found that benzyl-a-GalNac treatment-induced abundant expression of Tn antigen, which is an immature O-glycan representing abnormal O-glycosylation. Benzyl-a-GalNac pretreatment exacerbated considerably GalN/LPS-induced liver damage in mice, evidenced by significantly reduced survival rates, more severe histological alterations, and notable elevation of multiple inflammatory cytokines and chemokines. Mechanistically, benzyl-a-GalNac could trigger endoplasmic reticulum (ER) stress in the liver of mice, demonstrated by the elevated expression of glucose-regulated protein 78 (GRP78) and C/EBP-homologous protein (CHOP), both of which are hallmarks for ER stress. Inhibition of ER stress by 4-phenylbutyric acid (4-PBA) markedly abrogated benzyl-a-GalNac-mediated enhanced hepatotoxicity and systemic inflammation in GalN/LPS-treated mice. CONCLUSIONS: This study demonstrated that inhibition of O-glycosylation caused by benzyl-a-GalNac aggravated GalN/LPS-induced liver damage and systemic inflammation, which may be due to activation of ER stress.

The Controversy About the Effects of Different Doses of Corticosteroid Treatment on Clinical Outcomes for Acute Respiratory Distress Syndrome Patients: An Observational Study.

Background: Corticosteroid usage in acute respiratory distress syndrome (ARDS) remains controversial. We aim to explore the correlation between the different doses of corticosteroid administration and the prognosis of ARDS. Methods: All patients were diagnosed with ARDS on initial hospital admission and received systemic corticosteroid treatment for ARDS. The main outcomes were the effects of corticosteroid treatment on clinical parameters and the mortality of ARDS patients. Secondary outcomes were factors associated with the mortality of ARDS patients. Results: 105 ARDS patients were included in this study. Corticosteroid treatment markedly decreased serum interleukin-18 (IL-18) level (424.0 ± 32.19 vs. 290.2 ± 17.14; p = 0.0003) and improved arterial partial pressure of oxygen/fraction of inspired oxygen (PaO2/FiO2) (174.10 ± 65.28 vs. 255.42 ± 92.49; p < 0.0001). The acute physiology and chronic health evaluation (APACHE II) score (16.15 ± 4.41 vs. 14.88 ± 4.57, p = 0.042) decreased significantly on the seventh day after systemic corticosteroid treatment. Interestingly, the serum IL-18 decreased significantly (304.52 ± 286.00 vs. 85.85 ± 97.22, p < 0.0001), whereas the improvement of PaO2/FiO2 (24.78 ± 35.03 vs. 97.17 ± 44.82, p < 0.001) was inconspicuous after systemic corticosteroid treatment for non-survival patients, compared with survival patients. Furthermore, the receiver operating characteristic (ROC) model revealed, when equivalent methylprednisolone usage was 146.5 mg/d, it had the best sensitivity and specificity to predict the death of ARDS. Survival analysis by Kaplan-Meier curves presented the higher 45-day mortality in high-dose corticosteroid treatment group (logrank test p < 0.0001). Multivariate Cox regression analyses demonstrated that serum IL-18 level, APACHE II score, D-dimer, and high-dose corticosteroid treatment were associated with the death of ARDS. Conclusion: Appropriate dose of corticosteroids may be beneficial for ARDS patients through improving the oxygenation and moderately inhibiting inflammatory response. The benefits and risks should be carefully weighed when using high-dose corticosteroid for ARDS. Trial registration: This work was registered in ClinicalTrials.gov. Name of the registry: Corticosteroid Treatment for Acute Respiratory Distress Syndrome. Trial registration number: NCT02819453. URL of trial registry record: https://register.clinicaltrials.gov.

Rapid postmortem ventilation improves donor lung viability by extending the tolerable warm ischemic time after cardiac death in mice.

Uncontrolled donation after cardiac death (uDCD) contributes little to ameliorating donor lung shortage due to rapidly progressive warm ischemia after circulatory arrest. Here, we demonstrated that nonhypoxia improves donor lung viability in a novel uDCD lung transplant model undergoing rapid ventilation after cardiac death and compared the evolution of ischemia-reperfusion injury to mice that underwent pulmonary artery ligation (PAL). The tolerable warm ischemia time at 37°C was initially determined in mice using a modified PAL model. The donor lung following PAL was also transplanted into syngeneic mice and compared with those that underwent rapid ventilation or no ventilation at 37°C before transplantation. Twenty-four hours following reperfusion, lung histology, [Formula: see text]/[Formula: see text] ratio, and inflammatory mediators were measured. Four hours of PAL had little impact on [Formula: see text]/[Formula: see text] ratio and acute lung injury score in contrast to significant injury induced by 5 h of PAL. Four-hour PAL lungs showed an early myeloid-dominant inflammatory signature when compared with naïve lungs and substantially injured 5 h PAL lungs. In the context of transplantation, unventilated donor lungs showed severe injury after reperfusion, whereas ventilated donor lungs showed minimal changes in [Formula: see text]/[Formula: see text] ratio, histologic score, and expression of inflammatory markers. Taken together, the tolerable warm ischemia time of murine lungs at 37°C can be extended by maintaining alveolar ventilation for up to 4 h. Nonhypoxic lung undergoing warm ischemia-reperfusion injury shows an early transcriptional signature of myeloid cell recruitment and extracellular matrix proteolysis before blood-gas barrier dysfunction and significant tissue damage.

Extracellular histones aggravate inflammation in ARDS by promoting alveolar macrophage pyroptosis.

Extracellular histones have been discovered to play a pathogenic role in ARDS, but the underlying mechanisms are yet to be fully defined. Alveolar macrophage (AM) is essential for the initiation and progression of lung inflammation; of note, AM pyroptosis has been suggested contributing to ARDS-associated inflammation. Here we aimed to investigate whether extracellular histones promote ARDS by triggering AM pyroptosis. The BALF samples of ARDS patients were collected and AMs were further isolated. Extracellular histones, AM pyroptosis, and pyroptosis-associated mediators were measured. Furthermore, the effects of extracellular histones on AM pyroptosis and the underlying mechanisms were investigated. It showed that extracellular histones were markedly elevated in the BALF of ARDS patients and correlated with the increased AM pyroptosis. ARDS patient's BALF induced pronounced pyroptosis in cultured human monocytes, which could be prevented by neutralizing extracellular histones with heparin. In addition, exogenous histones induced pyroptosis of MH-S cells in a dose- and time-dependent manner, which acted through the NLRP3 inflammasome signaling pathway. Inhibition of NLRP3 inflammasome signaling substantially reduced cell pyroptosis. In a murine model of LPS-induced ARDS, extracellular histones were increased in the BALF and its increase was associated with enhanced AM pyroptosis and exaggerated lung inflammation. Blockade of extracellular histones or NLPR3 inflammasome equally inhibited macrophage pyroptosis, whereas targeting histones appeared more effective in alleviating lung inflammation. This study suggested that extracellular histones promote AM pyroptosis through NLRP3 inflammasome pathway, which in turn aggravates lung inflammation in ARDS. Pharmacological manipulation of extracellular histones or AM pyroptosis may become promising strategies for the treatment of ARDS.

[Extracellular histones are involved in lipopolysaccharide-induced alveolar macrophage injury by activating the TWIK2-NLRP3 pathway].

OBJECTIVE: To explore the role and mechanism of extracellular histones involved in lipopolysaccharide (LPS)-induced alveolar macrophage injury. METHODS: The mouse alveolar macrophage cell line (MH-S) was cultured in vitro and passaged, and the cells were cultured to 80% of cells for cell proliferation. The cells were stimulated with 1 mg/L LPS for 3 hours and 50 mg/L exogenous histones for 3, 6, 12, and 24 hours, respectively (LPS+histones 3, 6, 12, 24 h groups), and other groups included phosphate buffered saline (PBS) control group (PBS group), LPS alone stimulation group (LPS group), the exogenous histones alone stimulation group (histones group) and heparin pretreatment histones group (heparin+LPS+histones group). The cells in each group were challenged with different reagent, the expression of lactate dehydrogenase (LDH) and inflammatory factors in the supernatant were detected by enzyme linked immunosorbent assay (ELISA), and the change of intracellular K+ concentration was detected by FluxORTM II green potassium channel. The proteins such as potassium channel protein (TWIK2), inflammasome (NLRP3), and apoptosis associated speck like protein containing a CARD (ASC) were determined by Western Blot. RESULTS: Compared with the PBS group, the levels of LDH and inflammatory factors such as interleukin (IL-1ß, IL-18) and tumor necrosis factor-α (TNF-α) were significantly increased after LPS stimulation group. Compared with the LPS group, the levels of LDH and inflammatory factors were significantly increased after the treatment with exogenous histones, and reached a peak after 3 hours of the histones stimulation [LDH (U/L): 123.10±1.83 vs. 85.32±1.66, IL-1ß (mg/L): 40.75±2.60 vs. 18.78±1.37, IL-18 (mg/L): 49.94±2.45 vs. 30.19±1.82, TNF-α (mg/L): 36.51±1.56 vs. 20.84±1.61, all P < 0.01]. Western Blot results showed that compared with the LPS group, NLRP3, ASC and TWIK2 protein expression were significantly up-regulated in the LPS+histones group (NLRP3/GAPDH: 0.80±0.02 vs. 0.57±0.02, ASC/GAPDH: 0.57±0.02 vs. 0.38±0.01, TWIK2/GAPDH: 0.65±0.01 vs. 0.41±0.01, all P < 0.01), and the expression of the above proteins were significantly down-regulated after heparin pretreatment (NLRP3/GAPDH: 0.28±0.02 vs. 0.80±0.02, ASC/GAPDH: 0.25±0.02 vs. 0.57±0.02, TWIK2/GAPDH: 0.35±0.01 vs. 0.65±0.01, all P < 0.01), indicating that histones could activate NLRP3 through TWIK2 to participate in inflammatory reaction. In addition, intracellular K+ concentration in LPS+histones group decreased significantly compared with the LPS group (fluorescence intensity: 35.48±2.53 vs. 83.92±3.11, P < 0.01). Compared with LPS+histones group, K+ concentration increased significantly after pretreatment with heparin (fluorescence intensity: 72.10±1.78 vs. 35.48±2.53, P < 0.01), indicating that extracellular histones could cause K+ massive efflux through TWIK2, and thus mediate NLRP3 activation and participate in inflammatory injury of alveolar macrophages. CONCLUSIONS: Extracellular histones can cause inflammatory damage in alveolar macrophages, and its mechanism may be related to the activation of NLRP3 by extracellular histones activation of TWIK2 channel to promote K+ efflux.

Extracellular histones play a pathogenic role in primary graft dysfunction after human lung transplantation.

Primary graft dysfunction (PGD) causes early mortality and late graft failure after lung transplantation. The mechanisms of PGD are not fully understood but ischemia/reperfusion (I/R) injury may be involved. Extracellular histones have recently been identified as major contributors to I/R injury. Hence, we investigated whether extracellular histones are associated with PGD after lung transplantation. In total, 65 lung transplant patients were enrolled into this study. Blood samples were collected from patients before and serially after transplantation (24 h, 48 h, and 72 h) and measured for extracellular histones, myeloperoxidase (MPO), lactate dehydrogenase (LDH), and multiple cytokines. Besides, the patients' sera were cultured with human pulmonary artery endothelial cells (HPAEC) and human monocyte cell line (THP1) cells, respectively, and cellular viability and cytokine production were determined. Heparin or anti-histone antibody were used to study the effects of histone-neutralized interventions. The results showed that extracellular histones increased markedly after lung transplantation, peaked by 24 h and tended to decrease thereafter, but still retained high levels up to 72 h. Extracellular histones were more abundant in patients with PGD (n = 8) than patients without PGD (n = 57) and linearly correlated with MPO, LDH, and most detected cytokines. Ex vivo studies showed that the patients' sera collected within 24 h after transplantation were very damaging to HPAEC cells and promoted cytokine production in cultured THP1 cells, which could be largely prevented by heparin or anti-histone antibodies. These data suggested a pathogenic role for extracellular histones in PGD after lung transplantation. Targeting extracellular histones may serve as a preventive and therapeutic strategy for PGD following lung transplantation.