Instillation Of Lipopolysaccharide Research Articles

First-in-Class Mitogen-Activated Protein Kinase p38α: MAPK-Activated Protein Kinase-2 (MK2) Dual Signal Modulator with Anti-inflammatory and Endothelial-stabilizing Properties.

We previously identified a small molecule, UM101, predicted to bind to the substrate-binding groove of p38aMitogen-activated Protein Kinase (MAPK) near the binding site of its proinflammatory substrate, MAPK-activated protein kinase (MK2). UM101 exhibited anti-inflammatory, endothelial-stabilizing, and lung-protective effects. To overcome its limited aqueous solubility and p38a binding affinity, we designed an analog of UM101, GEn-1124, with improved aqueous solubility, stability, and p38a binding affinity. Compared with UM101, GEn-1124 has 18-fold greater p38a-binding affinity as measured by Surface Plasmon Resonance (SPR), 11-fold greater aqueous solubility, enhanced barrier-stabilizing activity in thrombin-stimulated human pulmonary artery endothelial cells (hPAEC) in vitro, and greater lung protection in vivo GEn-1124 improved survival from 10% to 40% in murine acute lung injury (ALI) induced by combined exposure to intratracheal bacterial endotoxin lipopolysaccharide (LPS) instillation and febrile-range hyperthermia (FRH) and from 0% to 50% in a mouse influenza pneumonia model. Gene expression analysis by RNASeq in TNFa-treated hPAEC showed that the gene-modifying effects of GEn-1124 were much more restricted to TNFa-inducible genes than the catalytic site p38 inhibitor, SB203580. Gene expression pathway analysis, confocal immunofluorescence analysis of p38aand MK2 subcellular trafficking, and SPR analysis of phosphorylated p38a:MK2 binding affinity supports a novel mechanism of action. GEn-1124 destabilizes the activated p38a:MK2 complex, dissociates nuclear export of MK2 and p38a, thereby promoting intranuclear retention and enhanced intranuclear signaling by phosphorylated p38a retention, and accelerated inactivation of p38-free cytosolic MK2 by unopposed phosphatases. Significance Statement We describe an analog of our first-in-class small molecule modulator of p38a/MK2 signaling targeted to a pocket near the ED substrate binding domain of p38a, which destabilizes the p38a:MK2 complex without blocking p38 catalytic activity or ablating downstream signaling. The result is a rebalancing of downstream pro- and anti-inflammatory signaling, yielding anti-inflammatory, endothelial-stabilizing, and lung-protective effects with therapeutic potential in ARDS.

Impact of time intervals on drug efficacy and phenotypic outcomes in acute respiratory distress syndrome in mice

Acute respiratory distress syndrome is a severe lung condition resulting from various causes, with life-threatening consequences that necessitate intensive care. The phenomenon can be modeled in preclinical models, notably through the use of lipopolysaccharide (LPS) instillation in mice. The phenotype induced closely recapitulates the human syndrome, including pulmonary edema, leukocyte infiltration, acute inflammation, impaired pulmonary function, and histological damage. However, the experimental designs using LPS instillations are extremely diverse in the literature. This highly complicates the interpretation of the induced phenotype chronology for future study design and hinders the proper identification of the optimal time frame to assess different readouts. Therefore, the definition of the treatment window in relation to the beginning of the disease onset also presents a significant challenge to address questions or test compound efficacy. In this context, the temporality of the different readouts usually measured in the model was evaluated in both normal and neutrophil-depleted male C57bl/6 mice using LPS-induction to assess the best window for proper readout evaluation with an optimal dynamic response range. Ventilation parameters were evaluated by whole-body plethysmography and neutrophil recruitment were evaluated in bronchoalveolar lavage fluids and in lung tissues directly. Imaging evaluation of myeloperoxidase along with activity in lung lysates and fluids were compared, along with inflammatory cytokines and lung extravasation by enzyme-linked immunoassays. Moreover, dexamethasone, the gold standard positive control in this model, was also administered at different times before and after phenotype induction to assess how kinetics affected each parameter. Overall, our data demonstrate that each readout evaluated in this study has a singular kinetic and highlights the key importance of the timing between ARDS phenotype and treatment administration and/or analysis. These findings also strongly suggest that analyzes, both in-life and post-mortem should be conducted at multiple time points to properly capture the dynamic phenotype of the LPS-ARDS model and response to treatment.

Human placental mesenchymal stem cells transplantation repairs the alveolar epithelial barrier to alleviate lipopolysaccharides-induced acute lung injury

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are accompanied by high mortality rates and few effective treatments. Transplantation of human placental mesenchymal stem cells (hPMSCs) may attenuate ALI and the mechanism is still unclear. Our study aimed to elucidate the potential protective effect and therapeutic mechanism of hPMSCs against lipopolysaccharide (LPS)-induced ALI, An ALI model was induced by tracheal instillation of LPS into wild-type (WT) and angiotensin-converting enzyme 2 (ACE2) knockout (KO) male mice, followed by injection of hPMSCs by tail vein. Treatment with hPMSCs improved pulmonary histopathological injury, reduced pulmonary injury scores, decreased leukocyte count and protein levels in bronchoalveolar lavage fluid(BALF), protected the damaged alveolar epithelial barrier, and reversed LPS-induced upregulation of pro-inflammatory factors Interleukin-6 (IL-6) and Tumor necrosis factor-α(TNF-α) and downregulation of anti-inflammatory factor Interleukin-6(IL-10) in BALF. Moreover, administration of hPMSCs inhibited Angiotensin (Ang)II activation and promoted the expression levels of ACE2 and Ang (1–7) in ALI mice. Pathological damage, inflammation levels, and disruption of alveolar epithelial barrier in ALI mice were elevated after the deletion of ACE2 gene, and the Renin angiotensin system (RAS) imbalance was exacerbated. The therapeutic effect of hPMSCs was significantly reduced in ACE2 KO mice. Our findings suggest that ACE2 plays a key role in hPMSCs repairing the alveolar epithelial barrier to protect against ALI, laying a new foundation for the clinical treatment of ALI.

Modeling "Two-Hit" Severe Pneumonia in Mice: Pathological Characteristics and Mechanistic Studies.

Severe pneumonia is one of the most common critical diseases in clinical practice. Existing models for severe pneumonia have limitations, leading to limited clinical translation. In this study, a two-hit severe pneumonia mouse model was established by inducing primary pneumonia through intratracheal instillation of 800μg lipopolysaccharide (LPS), followed by intraperitoneal injection of 10mg/kg LPS. The effectiveness of various inflammatory indicators and the lung tissue damage during the time course of this model were confirmed and evaluated. At 3h post two-hit, the IL-6, TNF-α levels in peripheral blood and bronchoalveolar lavage fluid (BALF), and the white blood cells, neutrophils, and lymphocytes in BALF notably exhibited the most pronounced elevation. At 12h post two-hit, the white blood cells and neutrophils in peripheral blood significantly increased, accompanied by notable alterations in splenic immune cells and worsened pulmonary histopathological damage. Transcriptomics of lung tissue, microbiota analysis of lung and gut, as well as plasma metabolomics analyses further indicated changes in transcriptional profiles, microbial composition, and metabolites due to the two-hit modeling. These results validate that the two-hit model mimics the clinical presentation of severe pneumonia and serves as a robust experimental tool for studying the pathogenesis of severe pneumonia and developing and assessing treatment strategies.

Subchronic intranasal lipopolysaccharide exposure induces pulmonary autoimmunity and glomerulonephritis in NZBWF1 mice

Lupus, a systemic autoimmune disease shaped by gene-environment interplay, often progresses to endstage renal failure. While subchronic systemic exposure to bacterial lipopolysaccharide (LPS) triggers autoimmunity and glomerulonephritis in lupus-prone mice, it is unknown if inhaling LPS, which is common in certain occupations, can similarly trigger lupus. Here we determined how subchronic intranasal (IN) LPS instillation influences autoimmunity and glomerulonephritis development in lupusprone NZBWF1 female mice. Briefly, mice were IN-instilled with vehicle or E. coli LPS (0.8 μg/g) twice weekly for 5 wk, followed by necropsy. For systemic comparison, additional cohorts of mice were injected with LPS intraperitoneally (IP) using identical doses/timing. Lungs were assessed for inflammatory and autoimmune responses and then related to systemic autoimmunity and glomerulonephritis. IN/LPS exposure induced in the lung: i) leukocyte infiltration, ii)mRNA signatures for cytokines, chemokines, IFN-regulated, and cell death-related genes, iii) ectopic lymphoid tissue formation, and iv)diverse IgM and IgG autoantibodies (AAbs). Pulmonary effects coincided with enlarged spleens, elevated plasma IgG AAbs, and inflamed IgG-containing kidney glomeruli. In contrast, IP/LPS treatment induced systemic autoimmunity and glomerulonephritis without pulmonary manifestations. Taken together, these preclinical findings suggest the lung could serve as a critical nexus for triggering autoimmunity by respirable LPS in genetically predisposed individuals.

PDTC improves cognitive impairment in LPS-induced ARDS by regulating miR-181c/NF-κB axis-mediated neuroinflammation

ABSTRACT Background Cognitive impairment is a severe complication of acute respiratory distress syndrome (ARDS). Emerging studies have revealed the effects of pyrrolidine dithiocarbamate (PDTC) on improving surgery-induced cognitive impairment. The major aim of the study was to investigate whether PDTC protected against ARDS-induced cognitive dysfunction and to identify the underlying mechanisms involved. Methods The rat model of ARDS was established by intratracheal instillation of lipopolysaccharide (LPS), followed by treatment with PDTC. The cognitive function of rats was analyzed by the Morris Water Maze, and pro-inflammatory cytokines were assessed by quantitative real-time PCR, enzyme-linked immunosorbent assay, and western blot assays. A dual-luciferase reporter gene assay was performed to identify the relationship between miR-181c and its target gene, TAK1 binding protein 2 (TAB2). Results The results showed that PDTC improved cognitive impairment and alleviated neuroinflammation in the hippocampus in LPS-induced ARDS model. Furthermore, we demonstrated that miR-181c expression was downregulated in the hippocampus of the ARDS rats, which was restored by PDTC treatment. In vitro studies showed that miR-181c alleviated LPS-induced pro-inflammatory response by inhibiting TAB2, a critical molecule in the nuclear factor (NF)-κB signaling pathway. Conclusion PDTC improves cognitive impairment in LPS-induced ARDS by regulating miR-181c/NF-κB axis-mediated neuroinflammation, providing a potential opportunity for the treatment of this disease.

Astragali Radix-Salviae Miltiorrhizae Radix et Rhizoma alleviates acute lung injury in rats by regulating autophagy via PI3K/Akt/mTOR pathway

This study aims to reveal the effects of the herb pair Astragali Radix-Salviae Miltiorrhizae Radix et Rhizoma(AR-SMRR) on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR) pathway and autophagy in the lung tissue of the rat model of acute lung injury(ALI). Fifty adult male SD rats were randomized into sham, model, autophagy inhibition(intraperitoneal injection of chloroquine at 10 mg·kg~(-1)), autophagy induction(intraperitoneal injection of rapamycin at 15 mg·kg~(-1)), and AR-SMRR(5 g·kg~(-1), gavage) groups. The rats in the sham group received intratracheal instillation of normal saline, and those in other groups received intratracheal instillation of lipopolysaccharide(LPS, 5 mg·kg~(-1)) for the modeling of ALI. Seven days before the operation, the rats in the sham and model groups were administrated with normal saline, and those in other groups with corresponding drugs. Specimens were collected 24 h after modeling. The pathological changes of the lung tissue were observed under a light microscope. The lung wet/dry weight ratio and the lactate dehydrogenase(LDH) activity and total protein concentration in the bronchoalveolar lavage fluid(BALF) were measured. Western blot was employed to measure the protein levels of microtubule-associated protein 1-light chain 3(LC3), beclin-1, p62, PI3K, Akt, and mTOR. Compared with the sham group, the model group showed increased histopathological score of the lung tissue, lung wet/dry weight ratio, and LDH activity and protein concentration in BALF. Autophagy inhibition further increased these indicators compared with the model group, while autophagy induction and AR-SMRR lowered the levels. In addition, AR-SMRR up-regulated the protein levels of LC3-Ⅱ and beclin-1, down-regulated the expression of p62, and inhibited the expression of p-PI3K, p-Akt, and p-mTOR in the lung tissue of ALI rats. The findings suggest that AR-SMRR can alleviate the lung injury and edema in the rat model of ALI induced by LPS by enhancing autophagy via down-regulating PI3K/Akt/mTOR signaling pathway.

Transcriptomic Insights into Different Stimulation Intensity of Electroacupuncture in Treating COPD in Rat Models.

Electroacupuncture (EA), with varying stimulation intensities, has demonstrated therapeutic potentials in both animal and clinical studies for the treatment of chronic obstructive pulmonary disease (COPD). However, a comprehensive investigation of the intensity-related effects, particularly 1mA and 3mA of EA, and the underlying mechanisms remains lacking. A COPD rat model was established by prolonged exposure to cigarette smoke and intermittent intratracheal instillation of lipopolysaccharide. EA treatment was administered at acupoints BL13 (Feishu) and ST36 (Zusanli), 20 minutes daily for 2 weeks, with intensities of 1mA and 3mA. EA effectiveness was evaluated by pulmonary function, histopathological change, serum level of inflammatory cytokines, and level of oxidative stress markers in serum and lung tissues. Transcriptome profiling and weighted gene co-expression network analysis (WGCNA) were performed to reveal gene expression patterns and identify hub genes. Real-time quantitative PCR (RT-qPCR) and Western blot (WB) were performed to detect the mRNA and protein expression levels, respectively. EA at both 1mA and 3mA exerted differing therapeutic effects by improving lung function and reducing inflammation and oxidative stress in COPD rats. Transcriptome analysis revealed distinct expression patterns between the two groups, functionally corresponding to shared and intensity-specific (1mA and 3mA) enriched pathways. Eight candidate genes were identified, including Aqp9, Trem1, Mrc1, and Gpnmb that were downregulated by EA and upregulated in COPD. Notably, Msr1 and Slc26a4 exclusively downregulated in EA-1mA, while Pde3a and Bmp6 upregulated solely in EA-3mA. WGCNA constructed 5 key modules and elucidated the module-trait relationship, with the aforementioned 8 genes being highlighted. Additionally, their mRNA and protein levels were validated by RT-qPCR and WB. Our results demonstrated that 1mA and 3mA intensities induce distinct gene expression patterns at the transcriptional level, associated with shared and 1mA vs 3mA-specific enriched pathways. Genes Mrc1, Gpnmb, Trem1, and Aqp9 emerge as promising targets, and further studies are needed to elucidate their functional consequences in COPD.

Targeting transitioning lung monocytes/macrophages as treatment strategies in lung disease related to environmental exposures

BackgroundEnvironmental/occupational exposures cause significant lung diseases. Agricultural organic dust extracts (ODE) and bacterial component lipopolysaccharide (LPS) induce recruited, transitioning murine lung monocytes/macrophages, yet their cellular role remains unclear.MethodsCCR2 RFP+ mice were intratracheally instilled with high concentration ODE (25%), LPS (10 μg), or gram-positive peptidoglycan (PGN, 100 μg) for monocyte/macrophage cell-trafficking studies. CCR2 knockout (KO) mice and administration of intravenous clodronate liposomes strategies were employed to reduce circulating monocytes available for lung recruitment following LPS exposure. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected. Pro-inflammatory and/or pro-fibrotic cytokines, chemokines, and lung extracellular matrix mediators were quantitated by ELISA. Infiltrating lung cells including monocyte/macrophage subpopulations, neutrophils, and lymphocytes were characterized by flow cytometry. Lung histopathology, collagen content, vimentin, and post-translational protein citrullination and malondialdehyde acetaldehyde (MAA) modification were quantitated. Parametric statistical tests (one-way ANOVA, Tukey’smultiple comparison) and nonparametric statistical (Kruskal–Wallis, Dunn’s multiple comparison) tests were used following Shapiro–Wilk testing for normality.ResultsIntratracheal instillation of ODE, LPS, or PGN robustly induced the recruitment of inflammatory CCR2+ CD11cintCD11bhi monocytes/macrophages and both CCR2+ and CCR2− CD11c−CD11bhi monocytes at 48 h. There were also increases in CCR2+ CD4+ and CD8+ T cells and NK cells. Despite reductions in LPS-induced lung infiltrating CD11cintCD11bhi cells (54% reduction), CCR2 knockout (KO) mice were not protected against LPS-induced inflammatory and pro-fibrotic consequences. Instead, compensatory increases in lung neutrophils and CCL2 and CCL7 release occurred. In contrast, the depletion of circulating monocytes through the administration of intravenous clodronate (vs. vehicle) liposomes 24 h prior to LPS exposure reduced LPS-induced infiltrating CD11cintCD11bhi monocyte-macrophage subpopulation by 59% without compensatory changes in other cell populations. Clodronate liposome pre-treatment significantly reduced LPS-induced IL-6 (66% reduction), matrix metalloproteinases (MMP)-3 (36%), MMP-8 (57%), tissue inhibitor of metalloproteinases (61%), fibronectin (38%), collagen content (22%), and vimentin (40%). LPS-induced lung protein citrullination and MAA modification, post-translational modifications implicated in lung disease, were reduced (39% and 48%) with clodronate vs. vehicle liposome.ConclusionHighly concentrated environmental/occupational exposures induced the recruitment of CCR2+ and CCR2− transitioning monocyte-macrophage and monocyte subpopulations and targeting peripheral monocytes may reduce the adverse lung consequences resulting from exposures to LPS-enriched inhalants.

Dynasore Alleviates LPS-Induced Acute Lung Injury by Inhibiting NLRP3 Inflammasome-Mediated Pyroptosis.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are clinically severe respiratory disorders without available pharmacological therapies. Dynasore is a cell-permeable molecule that inhibits GTPase activity and exerts protective effects in several disease models. However, whether dynasore can alleviate lipopolysaccharide (LPS)-induced ALI is unknown. This study investigated the effect of dynasore on macrophage activation and explored its potential mechanisms in LPS-induced ALI in vitro and in vivo. Bone marrow-derived macrophages (BMDMs) were activated classically with LPS or subjected to NLRP3 inflammasome activation with LPS+ATP. A mouse ALI model was established by the intratracheal instillation (i.t.) of LPS. The expression of PYD domains-containing protein 3 (NLRP3), caspase-1, and gasdermin D (GSDMD) protein was detected by Western blots. Inflammatory mediators were analyzed in the cell supernatant, in serum and bronchoalveolar lavage fluid (BALF) by enzyme-linked immunosorbent assays. Morphological changes in lung tissues were evaluated by hematoxylin and eosin staining. F4/80, Caspase-1 and GSDMD distribution in lung tissue was detected by immunofluorescence. Dynasore downregulated nuclear factor (NF)-κB signaling and reduced proinflammatory cytokine production in vitro and inhibited the production and release of interleukin (IL)-1β, NLRP3 inflammasome activation, and macrophage pyroptosis through the Drp1/ROS/NLRP3 axis. Dynasore significantly reduced lung injury scores and proinflammatory cytokine levels in both BALF and serum in vivo, including IL-1β and IL-6. Dynasore also downregulated the co-expression of F4/80, caspase-1 and GSDMD in lung tissue. Collectively, these findings demonstrated that dynasore could alleviate LPS-induced ALI by regulating macrophage pyroptosis, which might provide a new therapeutic strategy for ALI/ARDS.

Sea Cucumber and Blueberry Extracts Suppress Inflammation and Reduce Acute Lung Injury through the Regulation of NF-κB/MAPK/JNK Signaling Pathway in Lipopolysaccharide-Treated C57BL/6 Mice.

Acute lung injury (ALI) represents a life-threatening condition with high morbidity and mortality despite modern mechanical ventilators and multiple pharmacological strategies. Therefore, there is a need to develop efficacious interventions with minimal side effects. The anti-inflammatory activities of sea cucumber (Cucumaria frondosa) and wild blueberry (Vaccinium angustifolium) extracts have been reported recently. However, their anti-inflammatory activities and the mechanism of action against ALI are not fully elucidated. Thus, the present study aims to understand the mechanism of the anti-inflammatory activity of sea cucumber and wild blueberry extracts in the context of ALI. Experimental ALI was induced via intranasal lipopolysaccharide (LPS) instillation in C57BL/6 mice and the anti-inflammatory properties were determined by cytokine analysis, histological examination, western blot, and qRT-PCR. The results showed that oral supplementation of sea cucumber extracts repressed nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, thereby downregulating the expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF) in the lung tissue and in the plasma. Wild blueberry extracts also suppressed the expression of IL-4. Furthermore, the combination of sea cucumber and wild blueberry extracts restrained MAPK signaling pathways by prominent attenuation of phosphorylation of NF-κB, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) while the levels of pro-inflammatory cytokines were significantly suppressed. Moreover, there was a significant and synergistic reduction in varying degrees of ALI lesions such as distorted parenchyma, increased alveoli thickness, lymphocyte and neutrophil infiltrations, fibrin deposition, pulmonary emphysema, pneumonia, intra-alveolar hemorrhage, and edema. The anti-inflammatory effect of the combination of sea cucumber and wild blueberry extracts is associated with suppressing MAPK and NF-κB signaling pathways, thereby significantly reducing cytokine storm in LPS-induced experimental ALI.

Obesity-induced downregulation of miR-192 exacerbates lipopolysaccharide-induced acute lung injury by promoting macrophage activation

BackgroundMacrophage activation may play a crucial role in the increased susceptibility of obese individuals to acute lung injury (ALI). Dysregulation of miRNA, which is involved in various inflammatory diseases, is often observed in obesity. This study aimed to investigate the role of miR-192 in lipopolysaccharide (LPS)-induced ALI in obese mice and its mechanism of dysregulation in obesity.MethodsHuman lung tissues were obtained from obese patients (BMI ≥ 30.0 kg/m2) and control patients (BMI 18.5–24.9 kg/m2). An obese mouse model was established by feeding a high-fat diet (HFD), followed by intratracheal instillation of LPS to induce ALI. Pulmonary macrophages of obese mice were depleted through intratracheal instillation of clodronate liposomes. The expression of miR-192 was examined in lung tissues, primary alveolar macrophages (AMs), and the mouse alveolar macrophage cell line (MH-S) using RT-qPCR. m6A quantification and RIP assays helped determine the cause of miR-192 dysregulation. miR-192 agomir and antagomir were used to investigate its function in mice and MH-S cells. Bioinformatics and dual-luciferase reporter gene assays were used to explore the downstream targets of miR-192.ResultsIn obese mice, depletion of macrophages significantly alleviated lung tissue inflammation and injury, regardless of LPS challenge. miR-192 expression in lung tissues and alveolar macrophages was diminished during obesity and further decreased with LPS stimulation. Obesity-induced overexpression of FTO decreased the m6A modification of pri-miR-192, inhibiting the generation of miR-192. In vitro, inhibition of miR-192 enhanced LPS-induced polarization of M1 macrophages and activation of the AKT/ NF-κB inflammatory pathway, while overexpression of miR-192 suppressed these reactions. BIG1 was confirmed as a target gene of miR-192, and its overexpression offset the protective effects of miR-192. In vivo, when miR-192 was overexpressed in obese mice, the activation of pulmonary macrophages and the extent of lung injury were significantly improved upon LPS challenge.ConclusionsOur study indicates that obesity-induced downregulation of miR-192 expression exacerbates LPS-induced ALI by promoting macrophage activation. Targeting macrophages and miR-192 may provide new therapeutic avenues for obesity-associated ALI.Graphical

Transcriptomic characterization of the human segmental endotoxin challenge model

Segmental instillation of lipopolysaccharide (LPS) by bronchoscopy safely induces transient airway inflammation in human lungs. This model enables investigation of pulmonary inflammatory mechanisms as well as pharmacodynamic analysis of investigational drugs. The aim of this work was to describe the transcriptomic profile of human segmental LPS challenge with contextualization to major respiratory diseases. Pre-challenge bronchoalveolar lavage (BAL) fluid and biopsies were sampled from 28 smoking, healthy participants, followed by segmental instillation of LPS and saline as control. Twenty-four hours post instillation, BAL and biopsies were collected from challenged lung segments. Total RNA of cells from BAL and biopsy samples were sequenced and analysed for differentially expressed genes (DEGs). After challenge with LPS compared with saline, 6316 DEGs were upregulated and 241 were downregulated in BAL, but only one DEG was downregulated in biopsy samples. Upregulated DEGs in BAL were related to molecular functions such as “Inflammatory response” or “chemokine receptor activity”, and upregulated pro-inflammatory pathways such as “Wnt-"/“Ras-"/“JAK-STAT” “-signaling pathway”. Furthermore, the segmental LPS challenge model resembled aspects of the five most prevalent respiratory diseases chronic obstructive pulmonary disease (COPD), asthma, pneumonia, tuberculosis and lung cancer and featured similarities with acute exacerbations in COPD (AECOPD) and community-acquired pneumonia. Overall, our study provides extensive information about the transcriptomic profile from BAL cells and mucosal biopsies following LPS challenge in healthy smokers. It expands the knowledge about the LPS challenge model providing potential overlap with respiratory diseases in general and infection-triggered respiratory insults such as AECOPD in particular.

Ferroptosis mediates airway epithelial E-cadherin dysfunction in LPS-induced acute lung injury

BackgroundLoss of E-cadherin in the airway epithelial cells is a critical contributor to the development of ALI/ARDS. Yet the underlying mechanisms are largely unknown. Increasing evidences have revealed the significance of ferroptosis in the pathophysiological process of ALI/ARDS. The aim of this study was to investigate the role of ferroptosis in dysregulation of airway epithelial E-cadherin in ALI/ARDS. MethodsBALB/c mice were subjected to intratracheal instillation of lipopolysaccharide (LPS) to establish an ALI model. Two inhibitors of ferroptosis, liproxstatin-1 (Lip-1, at the dose of 10 mg/kg and 30 mg/kg) and ferrostatin-1 (Fer-1, at the dose of 1 mg/kg and 5 mg/kg), were respectively given to the mice through intraperitoneal injection after LPS challenge. The expression of ferroptotic markers, full-length E-cadherin and soluble E-cadherin (sE-cadherin) were both detected. ResultsLPS exposure dramatically down-regulated pulmonary expression of E-cadherin in mice, with profound loss of membrane E-cadherin in the airway epithelial cells and increased secretion of sE-cadherin in the airway lumen. At the same time, we found that the mitochondrial of airway epithelial cells in LPS-exposed mice exhibited significant morphological alterations that are hallmark features of ferroptosis, with smaller volume and increased membrane density. Other makers of ferroptosis were also detected, including increased cytoplasmic levels of iron and lipid peroxidates (MDA), as well as decreased GPX4 expression. 30 mg/kg of Lip-1 not only showed potent protective effects against the LPS-induced injury, inflammation, edema of the lung in those mice, but also rescued airway epithelial E-cadherin expression and decreased the release of sE-cadherin through inhibiting ferroptosis. While no noticeable changes induced by LPS were observed in mice treated with Lip-1 at 10 mg/kg nor Fer-1 at 1 mg/kg or 5 mg/kg. ConclusionsTaken together, these data demonstrated that ferroptosis mediates airway epithelial E-cadherin dysfunction in LPS-induced ALI.

Acute Amelioration of Inflammatory Activity Caused by Endothelin-2 Deficiency during Acute Lung Injury.

In acute lung injury (ALI), a severe insult induces a hyperinflammatory state in the lungs. The mortality rate of severe ALI remains high, and novel mechanistic insights are required to improve therapeutic strategies. Endothelin-2 (Edn2), the least studied isoform of endothelin, is involved in lung physiology and development and can be affected by various factors. One of them is inflammation, and another isoform of endothelin, endothelin-1 (Edn1), affects lung inflammatory responses. Considering the importance of Edn2 in the lungs and how Edn2 works through the same receptors as Edn1, we postulated that Edn2 may affect inflammatory responses that are central to ALI pathophysiology. In this study, we performed 24 hours intratracheal lipopolysaccharide (LPS) instillation or PBS control as an in vivo ALI model in eight-week-old conditional Edn2 knockout mice (Edn2-iKO), with Edn2-floxed mice as controls. Bronchoalveolar lavage (BAL) fluid and tissue were collected after exsanguination and analyzed for its cellular, molecular, functional, and histological inflammatory phenotypes. We found that Edn2-iKO mice displayed a reduced pro-neutrophilic inflammatory phenotype even after acute LPS treatment, shown by the reduction in the overall protein concentration and neutrophil count in bronchoalveolar lavage fluids. Further investigation revealed a reduction in mRNA interferon gamma (IFNγ) level of Edn2-iKO lungs and suppression of its downstream signaling, including phosphorylated level of STAT1 and IL-1β secretion, leading to reduced NFĸB activation. To conclude, Edn2 deletion suppressed acute lung inflammation by reducing neutrophil-mediated IFNγ/STAT1/IL-1β/NFĸB signaling cascade. Targeting Edn2 signaling may be beneficial for the development of novel treatment options for ALI.

Grape Seed Proanthocyanidin Ameliorates LPS-induced Acute Lung Injury By Modulating M2a Macrophage Polarization Via the TREM2/PI3K/Akt Pathway

Acute lung injury (ALI) is an acute and progressive pulmonary inflammatory disease that is difficult to cure and has a poor prognosis. Macrophages, which have various phenotypes and diverse functions, play an essential role in the pathogenesis of ALI. Grape seed proanthocyanidin (GSP) has received much attention over several decades, and many biological activities such as anti-apoptotic, antioxidant, and anti-inflammatory have been identified. This study aimed to determine the effect of GSP on lipopolysaccharide (LPS)-induced ALI. In this study, we established an ALI mouse model by tracheal instillation of LPS, and by pre-injection of GSP into mice to examine the effect of GSP on the ALI mouse model. Using H&E staining, flow cytometry, and ELISA, we found that GSP attenuated LPS-induced lung pathological changes and decreased inflammatory cytokine expression in ALI mice. In addition, GSP reduced the recruitment of monocyte-derived macrophages to the lung and significantly promoted the polarization of primary mouse lung macrophages from M1 to M2a induced by LPS. In vitro, GSP also decreased the expression levels of inflammatory cytokines such as TNF-α, IL-6, IL-1β, and M1 macrophage marker iNOS induced by LPS in MH-S cells, while increasing the expression levels of M2a macrophage marker CD206. Bioinformatics analysis identified TREM2 and the PI3K/Akt pathway as candidate targets and signaling pathways that regulate M1/M2a macrophage polarization in ALI, respectively. Furthermore, GSP activated PI3K/Akt and increased TREM2 expression in vivo and in vitro. Meanwhile, GSP's impact on M2a polarization and inflammation suppression was attenuated by the PI3K inhibitor LY294002 or siRNA knockdown TREM2. In addition, GSP-enhanced PI3K/Akt activity was prevented by TREM2 siRNA. In conclusion, this study demonstrated that GSP could ameliorate LPS-induced ALI by modulating macrophage polarization from M1 to M2a via the TREM2/PI3K/Akt pathway.

Mitochondria of lung venular capillaries mediate lung-liver cross talk in pneumonia.

Failure of the lung's endothelial barrier underlies lung injury, which causes the high mortality acute respiratory distress syndrome (ARDS). Multiple organ failure predisposes to the mortality, but mechanisms are poorly understood. Here, we show that mitochondrial uncoupling protein 2 (UCP2), a component of the mitochondrial inner membrane, plays a role in the barrier failure. Subsequent lung-liver cross talk mediated by neutrophil activation causes liver congestion. We intranasally instilled lipopolysaccharide (LPS). Then, we viewed the lung endothelium by real-time confocal imaging of the isolated, blood-perfused mouse lung. LPS caused alveolar-capillary transfer of reactive oxygen species and mitochondrial depolarization in lung venular capillaries. The mitochondrial depolarization was inhibited by transfection of alveolar Catalase and vascular knockdown of UCP2. LPS instillation caused lung injury as indicated by increases in bronchoalveolar lavage (BAL) protein content and extravascular lung water. LPS or Pseudomonas aeruginosa instillation also caused liver congestion, quantified by liver hemoglobin and plasma aspartate aminotransferase (AST) increases. Genetic inhibition of vascular UCP2 prevented both lung injury and liver congestion. Antibody-mediated neutrophil depletion blocked the liver responses, but not lung injury. Knockdown of lung vascular UCP2 mitigated P. aeruginosa-induced mortality. Together, these data suggest a mechanism in which bacterial pneumonia induces oxidative signaling to lung venular capillaries, known sites of inflammatory signaling in the lung microvasculature, depolarizing venular mitochondria. Successive activation of neutrophils induces liver congestion. We conclude that oxidant-induced UCP2 expression in lung venular capillaries causes a mechanistic sequence leading to liver congestion and mortality. Lung vascular UCP2 may present a therapeutic target in ARDS.NEW & NOTEWORTHY We report that mitochondrial injury in lung venular capillaries underlies barrier failure in pneumonia, and venular capillary uncoupling protein 2 (UCP2) causes neutrophil-mediated liver congestion. Using in situ imaging, we found that epithelial-endothelial transfer of H2O2 activates UCP2, depolarizing mitochondria in venular capillaries. The conceptual advance from our findings is that mitochondrial depolarization in lung capillaries mediates liver cross talk through circulating neutrophils. Pharmacologic blockade of UCP2 could be a therapeutic strategy for lung injury.

Network pharmacology and transcriptomics to determine Danggui Yifei Decoction mechanism of action for the treatment of chronic lung injury

Ethnopharmacological relevanceSeveral children with pneumonia (especially severe cases) have symptoms of cough and expectoration during the recovery stage after standard symptomatic treatment, which eventually results in chronic lung injury. Danggui yifei Decoction (DGYFD), a traditional Chinese formula, has shown clinical promise for the treatment of chronic lung injury during the recovery stage of pneumonia, however, its mechanism of action is yet to be deciphered. Aim of this studyTo investigate the therapeutic mechanism of DGYFD for the treatment of chronic lung injury by integrating network pharmacology and transcriptomics. Materials and methodsBALB/c mice were used to establish the chronic lung injury mouse model by intratracheal instillation of lipopolysaccharide (LPS). Pathological analysis of lung tissue, lung injury histological score, lung index, protein levels in bronchoalveolar lavage fluid (BALF), immunohistochemical staining, blood rheology, inflammatory cytokines, and oxidative stress levels were used to evaluate the pharmacological effects of DGYFD. Chemical components of DGYFD were identified using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Integrated network pharmacology together with transcriptomics was used to predict potential biological targets. Western blot analysis was used to verify the results. ResultsIn this study, we demonstrated that DGYFD could improve lung injury pathological changes, decreases lung index, down-regulate NO and IL-6 levels, and regulate blood rheology. In addition, DGYFD was able to reduce the protein levels in BALF, up-regulate the expression levels of occludin and ZO-1, improve the ultrastructure of lung tissues, and reverse the imbalance of AT I and AT II cells to repair the alveolar-capillary permeability barrier. Twenty-nine active ingredients of DGYFD and 389 potential targets were identified by UPLC-MS/MS and network pharmacology, and 64 differentially expressed genes (DEGs) were identified using transcriptomics. GO and KEGG analysis revealed that the MAPK pathway may be the molecular target. Further, we found that DGYFD inhibits phosphorylation levels of p38 MAPK and JNK in chronic lung injury mouse models. ConclusionsDGYFD could regulate the imbalance between the excessive release of inflammatory cytokines and oxidative stress, repair the alveolar-capillary permeability barrier and improve the pathological changes during chronic lung injury by regulating the MAPK signaling pathway.

Yinma Jiedu Granule attenuates LPS-induced acute lung injury in rats via suppressing inflammation level.

Yinma Jiedu Granule (YMJD) is a traditional Chinese patent medicine (CPM), which has been proved to have anti-inflammatory effects and therapeutical effects on obstructive pulmonary disease. The purpose of the current investigation is to find out if YMJD can alleviate acute lung injury (ALI) induced by lipopolysaccharide (LPS) in rats and its underlying mechanisms. Rats were treated with either vehicle or YMJD for 14 consecutive days, and 2h after the last administration, the rat model of ALI was induced by the intratracheal instillation of LPS. High performance liquid chromatography (HPLC) was applied for the fingerprint analysis of YMJD. The efficacy and molecular mechanisms were investigated. The results showed that treatment with YMJD improved the general state of rats, reduced weight loss and serum lactate (LA) levels, attenuated pulmonary edema and pathological damage of the lung tissue. Moreover, we found that YMJD effectively decreased the infiltration of white blood cells (WBC), lymphocytes (LYM), mononuclear cells (MON) and neutrophils (NEUT) in bronchoalveolar lavage fluid (BALF), reduced the concentration of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in the lung tissue. Additionally, we found that YMJD could significantly increase the activity of superoxide dismutase (SOD) and reduce the malondialdehyde (MDA) level in the lung tissue. By employing RNA-sequencing, we have identified that JAK2/STAT1 is an important pathway that is involved in the lung protection of YMJD, and further Western blot assay verified that YMJD could effectively inhibit the activation of the JAK2/STAT1 pathway. YMJD could attenuate LPS-induced ALI through suppressing inflammation and oxidative stress in the lung tissue of rats, associating with the inhibition of JAK2/STAT1 activation. These findings provide evidence for the clinical use of YMJD for treatment of inflammatory pulmonary diseases like ALI.

OMIP-92: Characterization of rat macrophage subsets, lymphocytes, and granulocytes in bronchoalveolar lavage fluid.

Airway inflammation is a defense mechanism against inhaled agents characterized by infiltration of circulating immune cells. Given the inconsistent cellular identification across pre-clinical rat model, we have developed a flow cytometry panel of six colors to characterize macrophages subsets, lymphocytes and granulocytes in bronchoalveolar lavage fluid (BAL). Rats were challenged with intratracheal instillation of lipopolysaccharide (LPS). BAL were harvested 24 h after one LPS exposure in rats. This flow cytometry panel involve the description of macrophage subsets, T and B lymphocytes and neutrophils, which are central to airway immune responses, as based on scientific literature. By using a relatively small number of parameters to identify multiple cell types, additional parameters can be used for project/disease-specific activation markers.