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.

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.

SIRP-alpha-IL-6 axis induces immunosuppressive macrophages in non-small-cell lung cancer.

Signal regulatory protein-alpha (SIRPα) and IL-6 participate in the induction of tumor immune suppressive environment and facilitate tumor growth. In this study, we found that SIRPα was significantly elevated in macrophages of non-small cell lung cancer (NSCLC) tissues, which was positively correlated to the expression of CD163, PD-1, IL-6, and lung cancer progression. SIRPα in peripheral blood mononuclear cells (PBMCs) of NSCLC patients was also associated with CD163, PD-1, and plasma IL-6. Blockade of SIRPα signaling in SIRPα ± and SIRPα-/- mice attenuated lung cancer growth and reduced IL-6 expression in LLC cells-transplanted murine lung cancer model. Co-targeting SIRPα and IL-6 additively suppressed the expression of IL-6 and activation of STAT3, accompanied with a reduced population of pro-tumorigenic CD206+ M2 subtype of macrophages, PD-1+ tumor-associated macrophages (TAMs), and PD-1+CD8+ T cells in tumor tissues of anti-IL-6 antibody (aIL-6)-treated mice deficient in SIRPα. Further in vitro studies showed that blockade of SIRPα signaling by anti-SIRPα effectively improved phagocytosis of human PBMCs. IL-6 treatment improved polarization of M2 subtypes and the expression of PD-1 in bone marrow-derived macrophages (BMDMs); whereas both aIL-6 and STAT3 inhibitor C188-9 suppressed the expression of PD-1 and SIRPα in BMDMs. M2 cell-biased polarization was also reduced in aIL-6 or C188-9 treated BMDMs. Thereby, SIRPα and IL-6 form a positive feedback loop and regulate each other through STAT3 signaling in macrophages. The increased SIRPα/IL-6 axis may promote immune suppressive environment and lung cancer growth, which may be a potential target for clinical treatment.

Lomerizine attenuates LPS-induced acute lung injury by inhibiting the macrophage activation through reducing Ca2+ influx.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening lung diseases with high mortality rates, predominantly attributable to acute and severe pulmonary inflammation. Lomerizine (LMZ) is a calcium channel blocker previously used in preventing and treating migraine. Here, we found that LMZ inhibited inflammatory responses and lung pathological injury by reducing pulmonary edema, neutrophil infiltration and pro-inflammatory cytokine production in lipopolysaccharide (LPS)-induced ALI mice. In vitro experiments, upon treating with LMZ, the expression of interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α was attenuated in macrophages. The phosphorylation of p38 MAPK, ERK1/2, JNK, and NF-κB p65 was inhibited after LMZ treatment. Furthermore, LPS-induced Ca2+ influx was reduced by treating with LMZ, which correlated with inhibition of pro-inflammatory cytokine production. And L-type Ca2+ channel agonist Bay K8644 (BK) could restore cytokine generation. In conclusion, our study demonstrated that LMZ alleviates LPS-induced ALI and is a potential agent for treating ALI/ARDS.

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).

Future carbon storages of ecosystem based on land use change and carbon sequestration practices in a large economic belt.

Assessments of ecosystem carbon storage are needed to form the scientific basis for carbon policies. Due to lack of data, there are few accurate, large-scale, and long-term predictions of ecosystem carbon storage. This study used the Distributed Land-Use Change Prediction (DLUCP) model with ten socioeconomic and two climate change scenarios for a total of 20 combinations that take into account population increase, technology innovation, climate change, and Grain for Green Project to make high-resolution predictions of land use change in the Yangtze River Economic Belt. Low and high carbon sequestration practices were considered to predict future carbon densities. Land use change data, carbon densities data, and the InVEST model were used to predict changes in ecosystem carbon storage from now to 2070. The results show a slight increase (1.88-4.17%) in carbon storage in the study area only based on land use change. Grain for Green Project has the largest impact on carbon storage among population increase, technology innovation, climate scenarios, and Grain for Green Project, which increases carbon storage by 4.17%. After the implementation of carbon sequestration practices, there is an increase in carbon storages from 28.51 to 56.77% in the study area from now to 2070, and increasing carbon storages of forest in each stream and carbon storage of cropland in downstream are efficient ways to achieve carbon neutralization.

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.

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.

Climate factors affect N2O emissions by influencing the migration and transformation of nonpoint source nitrogen in an agricultural watershed.

Precipitation can affect the residence time of nitrogen compounds, and temperature can influence nitrogen transformation in soil. Therefore, we hypothesized that climate factors can affect the emissions of N2O, an important greenhouse gas produced via nitrogen transformation, by influencing the migration and transformation of nonpoint source nitrogen in soil. To test this hypothesis and quantify the effect of climate factors on N2O emissions, the SWAT model and the modified SWAT-N2O coupler were used to study the effect of climate factors on the migration and transformation of nonpoint source nitrogen and N2O emissions in an agricultural watershed from 2009 to 2018. Temperature affected N2O emissions more significant than precipitation, and N2O emissions increased with temperature and reached a plateau when the average monthly temperature was 23.0 °C. The N2O emissions first increased rapidly with precipitation due to the increase in moisture. However, when the average monthly precipitation reached 78.8 mm, the N2O emissions began to decrease because the residence time of nitrogen compounds in soil were reduced due to fast removal via runoff, which inhibits N2O emissions. Under the context of climate change with three scenarios (RCP2.6, RCP4.5, RCP8.5), temperature would increase gradually while precipitation would not change significantly from 2021 to 2080, as a result, the changes would increase N2O emissions by 6.7%, 32.3%, and 70.7%, respectively. This study quantifies the feedback of N2O emissions to climate change in croplands, providing a scientific basis for climate change mitigation and agricultural management.

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.

Ultrasound-assisted C3F8-filled PLGA nanobubbles for enhanced FGF21 delivery and improved prophylactic treatment of diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is a serious cardiac complication of diabetes that currently lacks specific treatment. Fibroblast growth factor 21 (FGF21) has been proved to have cardioprotective effect in DCM. However, the insufficient cardiac delivery effect of FGF21 limits its application in DCM. Therefore, to improve the therapeutic efficacy of FGF21 in DCM, an effective drug delivery system is urgently required. In this study, perfluoropropane (C3F8) and polyethylenimine (PEI)-doped poly (lactic-co-glycolic acid) (PLGA) nanobubbles (CPPNBs) were synthesized via double-emulsion evaporation and FGF21 was efficiently absorbed (CPPNBs@FGF21) via the electrostatic incorporation effect. CPPNBs@FGF21 could effectively deliver FGF21 to the myocardial tissue through the cavitation effect under low-frequency ultrasound (LFUS). The as-prepared CPPNBs@FGF21 could efficiently load FGF21 after doping with the cationic polymer PEI, and displayed uniform dispersion and favorable biosafety. After filling with C3F8, CPPNBs@FGF21 could be used for distribution monitoring through ultrasound imaging. Moreover, CPPNBs@FGF21 significantly downregulated the expression of ANP, CTGF, and caspase-3 mRNA via the action of LFUS owing to increased FGF21 release, therefore exhibiting enhanced inhibition of myocardial hypertrophy, apoptosis, and interstitial fibrosis in DCM mice. In conclusion, we established an effective protein delivery nanocarrier for the diagnosis and prophylactic treatment of DCM. STATEMENT OF SIGNIFICANCE: Diabetic cardiomyopathy (DCM) is a serious cardiac complication of diabetes that currently lacks effective clinical treatments. Fibroblast growth factor 21 (FGF21) can protect cardiomyocytes from diabetic damage, but insufficient cardiac drug delivery limits the application of FGF21 in DCM. In this study, perfluoropropane (C3F8) and polyethylenimine (PEI)-doped poly (lactic-co-glycolic acid) (PLGA) nanobubbles loaded with FGF21 (CPPNBs@FGF21) were developed for the prophylactic treatment of DCM. CPPNBs@FGF21 could effectively deliver the FGF21 to the myocardial tissue through the cavitation effect of low-frequency ultrasound (LFUS). Our results indicated that CPPNBs@FGF21 combined with LFUS could significantly down-regulate the expressions of ANP, CTGF, and caspase-3 mRNA, and as a result, it prevented the myocardial hypertrophy, apoptosis, and interstitial fibrosis of DCM mice. Overall, we established an effective protein delivery nanocarrier for the diagnosis and prophylactic treatment of DCM.

Carbon nanocage-based nanozyme as an endogenous H2O2-activated oxygenerator for real-time bimodal imaging and enhanced phototherapy of esophageal cancer.

Intelligent phototherapy by theranostic nanosystems that can be activated by a tumor microenvironment has high sensitivity and specificity. However, hypoxia and low drug accumulation in tumors greatly limit its clinical application. Herein, we have designed a cage-like carbon-manganese nanozyme, which effectively relieves tumor hypoxia and delivers numerous photosensitizers (PSs) to the tumor site, for real-time imaging and enhanced phototherapy of esophageal cancer. Specifically, bovine serum albumin (BSA) was used as a template and reducing agent for preparing a BSA-MnO2 nanozyme; then a BSA-MnO2/IR820@OCNC (BMIOC) nanosystem was successfully synthesized by crosslinking BSA-MnO2 on the surface of IR820-loaded carboxylated carbon nanocages (OCNCs). Abundant PSs were successfully delivered to tumor sites via hollow OCNCs, and the final loading rate of IR820 reached 42.8%. The intratumor BMIOC nanosystem can be initiated by a tumor microenvironment to switch on its magnetic resonance (MR) imaging signal, and photothermal therapy (PTT) and photodynamic therapy (PDT) functions. Notably, the BSA-MnO2 nanozyme, with intrinsic catalase (CAT)-like activity, catalyzed endogenous H2O2 for oxygen generation to overcome tumor hypoxia and enhance PDT, thereby leading to more efficient therapeutic effects in combination with OCNC-elevated PTT. In addition, the H2O2-activated and acid-enhanced properties enable our nanosystem to be specific to tumors, protecting normal tissues from damage. By integrating a high drug loading capacity, a hypoxia regulation function, an enlarged phototherapy effect, and bimodal imaging into a nanozyme-mediated nanoreactor, this work realizes a "one for all" system and represents promising clinical translation for efficient esophageal cancer theranostics.