LPS Inhalation Research Articles

Resistive spontaneous breathing exacerbated lipopolysaccharide-induced lung injury in mice

BackgroundSpontaneous respiratory mechanical force interacted with the primary lung injury and aggravated the progression of ARDS clinically. But the exact role and involved mechanism of it in the pathogenesis of ARDS animal model remained obscure. AimThis study was to investigate the effect of spontaneous respiratory mechanical force on lung injury of ARDS in mice. MethodsFemale C57BL/6 mice were subjected to resistive spontaneous breathing (RSB) by tracheal banding after 4–6 h of intranasal inhalation of LPS. Pulmonary function was examined by Buxco system, partial pressures of oxygen and carbon dioxide (PO2 and PCO2) were measured by a blood gas analyzer, and lung pathological changes were analyzed with hematoxylin and eosin staining. The levels of inflammatory markers were quantified by ELISA, total protein assay, and FACS analysis. The expression levels of mechanosensitive ion channels were detected by qRT-PCR and immunohistochemistry. ResultsThe airway resistance (Raw) was increased and the tidal volume (TV) was decreased remarkedly in RSB group. RSB treatment did not affect PO2, PCO2, pathology and inflammation levels of lung in mice. The Raw increased and ventilatory indicators decreased in RSB + ARDS compared to ARDS significantly. Besides, RSB treatment deteriorated the changes of PO2, PCO2 and level of lactic acid induced by LPS. Meanwhile, RSB significantly promoted LPS-induced pulmonary histopathological injury, and elevated the levels of IL-1β, IL-6, TNF-α and total proteins, increased neutrophils infiltration. The expression level of Piezo1 in RSB + ARDS group was remarkably reduced compared to ARDS group and consistent with the severity of pulmonary damage. ConclusionRSB exacerbated LPS-induced ARDS hypoxemia and hypercapnia, inflammation and damage. The mechanosensitive protein Piezo1 expression decreased and may play an important role in the process.

Inhaled endotoxin induces a systemic neutrophil response without affecting cardiovascular measures in a randomized cross-over exposure study

Objective The gram-negative bacterial cell wall component endotoxin (lipopolysaccharide, LPS) is a key component of particulate matter (PM). PM exposure is associated with cardiovascular morbidity and mortality. However, the contribution of individual components of PM to acute and chronic cardiovascular measures is not clear. This study examines whether systemic inflammation induced by LPS inhalation causes acute changes in cardiovascular physiology measures. Materials and Methods In this double blinded, placebo-controlled crossover study, fifteen adult volunteers underwent inhalation exposure to 20,000 EU Clinical Center Reference Endotoxin (CCRE). Peripheral blood and induced sputum neutrophils were obtained at baseline and six hours post-exposure. Blood pressure, measures of left ventricular function (ejection fraction (LVEF) and global longitudinal strain (LVGLS)), and indices of endothelial function (flow mediated dilation (FMD) and velocity time integral during hyperemia (VTIhyp)) were measured before and after treatment. Wilcoxon sign-rank tests and linear mixed models were used for statistical analysis. Results In comparison with normal saline, LPS inhalation resulted in significant increases in peripheral blood and sputum neutrophils but was not associated with significant alterations in blood pressure, LVGLS, LVEF, FMD, or VTIhyp. Discussion and Conclusions In healthy adults, systemic inflammation after LPS inhalation was not associated with acute changes in cardiovascular physiology. Larger studies are needed to investigate the effects of other PM components on inflammation induced cardiovascular dysfunction.

6-Gingerol ameliorates alveolar hypercoagulation and fibrinolytic inhibition in LPS-provoked ARDS via RUNX1/NF-κB signaling pathway

BackgroundAlveolar hypercoagulation and fibrinolytic inhibition play a central role in refractory hypoxemia in acute respiratory distress syndrome (ARDS), but it lacks effective drugs for prevention and treatment of this pathophysiology. Our previous experiment confirmed that RUNX1 promoted alveolar hypercoagulation and fibrinolytic inhibition through NF-κB pathway. Other studies demonstrated that 6-gingerol regulated inflammation and metabolism by inhibiting the NF-κB signaling pathway. We assume that 6-gingerol would ameliorate alveolar hypercoagulation and fibrinolytic inhibition via RUNX1/ NF-κB pathway in LPS-induced ARDS. MethodsRat ARDS model was replicated through LPS inhalation. Before LPS inhalation, the rats were intraperitoneally treated with different doses of 6-gingerol or the same volume of normal saline (NS) for 12 h, and then intratracheal inhalation of LPS for 24 h. In cell experiment, alveolar epithelial cell type II (AECII) was treated with 6-gingerol for 6 h and then with LPS for another 24 h. RUNX1 gene was down-regulated both in pulmonary tissue and in cells. Tissue factor (TF), plasminogen Activator Inhibitor 1(PAI-1) and thrombin were determined by Wester-blot (WB), qPCR or by enzyme-linked immunosorbent (ELISA). Lung injury score, pulmonary edema and pulmonary collagen III in rat were assessed. NF-κB pathway were also observed in vivo and in vitro. The direct binding capability of 6-gingerol to RUNX1 was confirmed by using Drug Affinity Responsive Target Stability test (DARTS). Results6-gingerol dose-dependently attenuated LPS-induced lung injury and pulmonary edema. LPS administration caused excessive TF and PAI-1 expression both in pulmonary tissue and in AECII cell and a large amount of TF, PAI-1 and thrombin in bronchial alveolar lavage fluid (BALF), which all were effectively decreased by 6-gingerol treatment in a dose-dependent manner. The high collagen Ⅲ level in lung tissue provoked by LPS was significantly abated by 6-gingerol. 6-gingerol was seen to dramatically inhibit the LPS-stimulated activation of NF-κB pathway, indicated by decreases of p-p65/total p65, p-IKKβ/total IKKβ, and also to suppress the RUNX1 expression. RUNX1 gene knock down or RUNX1 inhibitor Ro5-3335 significantly enhanced the efficacies of 6-gingerol in vivo and in vitro, but RUNX1 over expression remarkably impaired the effects of 6-gingerol on TF, PAI-1 and on NF-κB pathway. DARTS result showed that 6-gingerol directly bond to RUNX1 molecules. ConclusionsOur experimental data demonstrated that 6-gingerol ameliorates alveolar hypercoagulation and fibrinolytic inhibition via RUNX1/NF-κB pathway in LPS-induced ARDS. 6-gingerol is expected to be an effective drug in ARDS.

Exhaled breath particles as a novel tool to study lipid composition of epithelial lining fluid from the distal lung

BackgroundSurfactant phospholipid (PL) composition plays an important role in lung diseases. We compared the PL composition of non-invasively collected exhaled breath particles (PEx) with bronchoalveolar lavage (BAL) and induced sputum (ISP) at baseline and following endotoxin (LPS) challenges.MethodsPEx and BAL were collected from ten healthy nonsmoking participants before and after segmental LPS challenge. Four weeks later, PEx and ISP were sampled in the week before and after a whole lung LPS inhalation challenge. PL composition was analysed using mass spectrometry.ResultsThe overall PL composition of BAL, ISP and PEx was similar, with PC(32:0) and PC(34:1) representing the largest fractions in all three sample types (baseline PC(32:0) geometric mean mol%: 52.1, 56.9, and 51.7, PC(34:1) mol%: 11.7, 11.9 and 11.4, respectively). Despite this similarity, PEx PL composition was more closely related to BAL than to ISP. For most lipids comparable inter-individual differences in BAL, ISP, and PEx were found. PL composition of PEx was repeatable. The most pronounced increase following segmental LPS challenge was detected for SM(d34:1) in BAL (0.24 to 0.52 mol%) and following inhalation LPS challenge in ISP (0.45 to 0.68 mol%). An increase of SM(d34:1) following segmental LPS challenge was also detectable in PEx (0.099 to 0.103 mol%). The inhalation challenge did not change PL composition of PEx.ConclusionOur data supports the peripheral origin of PEx. The lack of PL changes in PEx after inhalation challenge might to be due to the overall weaker response of inhaled LPS which primarily affects the larger airways. Compared with BAL, which always contains lining fluid from both peripheral lung and central airways, PEx analysis might add value as a selective and non-invasive method to investigate peripheral airway PL composition.Trial registrationNCT03044327, first posted 07/02/2017.

HEMIN ATTENUATED LPS-INDUCED ACUTE LUNG INJURY IN MICE VIA PROTECTING PULMONARY EPITHELIAL BARRIER AND REGULATING HO-1/NLRP3-MEDIATED PYROPTOSIS.

Pulmonary epithelial barrier injury contributes to acute lung injury, accelerating exudate formation, and resulting alveolar edema. Heme oxygenase-1 (HO-1) plays an important role in ameliorating the pathological symptoms of acute lung injury (ALI). Using an ALI mouse model induced by LPS inhalation, the present study explored the potential molecular regulatory effect of hemin (a potent HO-1 inducer) against ALI epithelial damage. Lipopolysaccharide challenge triggered dysfunction of the alveolar epithelial barrier, impaired tight junctions, and disrupted alveoli fluid clearance, while these pathological changes were effectively reversed by hemin treatment. Furthermore, HO-1 elevation inhibited the activation of the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome and oxidative stress in alveolar epithelia, leading to the suppression of inflammatory responses and epithelial pyroptosis, as indicated by the decreased levels of NLRP3 and apoptosis-associated speck-like protein containing a CARD domain (ASC), repressed cleavage of caspase-1 and gasdermin D, and reduced expression levels of inflammatory cytokine IL-1β. In contrast, protoporphyrin IX zinc (II) (ZnPP, an HO-1 inhibitor) treatment had no protective effect on LPS inhalation-induced ALI in mice. In summary, HO-1 induction serves a critical role in maintaining airway epithelium homeostasis through the inhibition of NLRP3/ASC/caspase-1-mediated pyroptosis and inflammation in the occurrence of ALI.

Neutrophil Extracellular Vesicles and Airway Smooth Muscle Proliferation in the Natural Model of Severe Asthma in Horses.

Extracellular vesicles (EVs) contribute to intercellular communication through the transfer of their rich cargo to recipient cells. The EVs produced by LPS-stimulated neutrophils from healthy humans and horses increase airway smooth muscle (ASM) proliferation, but the roles of neutrophil EVs in asthma are largely unexplored. The aim of this study was to determine whether neutrophil-derived EVs isolated during the remission or exacerbation of asthma influence ASM proliferation differentially. Peripheral blood neutrophils were collected during remission and exacerbation in eight horses affected by severe asthma. The cells were cultured (±LPS), and their EVs were isolated by ultracentrifugation and characterized by laser scattering microscopy and proteomic analysis. The proliferation of ASM co-incubated with EVs was monitored in real time by electrical impedance. Two proteins were significantly upregulated during disease exacerbation in neutrophil EVs (MAST4 and Lrch4), while LPS stimulation greatly altered the proteomic profile. Those changes involved the upregulation of neutrophil degranulation products, including proteases known to induce myocyte proliferation. In agreement with the proteomic results, EVs from LPS-stimulated neutrophils increased ASM proliferation, without an effect of the disease status. The inhalation of environmental LPS could contribute to asthma pathogenesis by activating neutrophils and leading to ASM hyperplasia.

Changes of breath volatile organic compounds in healthy volunteers following segmental and inhalation endotoxin challenge

It is still unclear how airway inflammation affects the breath volatile organic compounds (VOCs) profile in exhaled air. We therefore analyzed breath following well-defined pulmonary endotoxin (lipopolysaccharide, LPS) challenges. Breath was collected from ten healthy non-smoking subjects at eight time points before and after segmental and whole lung LPS inhalation challenge. Four Tenax-TA® adsorption tubes were simultaneously loaded from an aluminum reservoir cylinder and independently analyzed by two research groups using gas chromatography—mass spectrometry. Airway inflammation was assessed in bronchoalveolar lavage (BAL) and in sputum after segmental and inhaled LPS challenge, respectively. Segmental LPS challenge significantly increased the median (interquartile range, IQR) percentage of neutrophils in BAL from 3.0 (4.2) % to 64.0 (7.3) %. The inhalation challenge increased sputum neutrophils from 33.9 (26.8) % to 78.3 (13.5) %. We observed increases in breath aldehydes at both time points after segmental and inhaled LPS challenge. These results were confirmed by an independent laboratory. The longitudinal breath analysis also revealed distinct VOC patterns related to environmental exposures, clinical procedures, and to metabolic changes after food intake. Changes in breath aldehydes suggest a relationship to LPS induced inflammation compatible with lipid peroxidation processes within the lung. Findings from our longitudinal data highlight the need for future studies to better consider the potential impact of the multiple VOCs from detergents, hygiene or lifestyle products a subject is continuously exposed to. We suspect that this very individual ‘owncloud’ exposure is contributing to an increased variability of breath aldehydes, which might limit a use as inflammatory markers in daily clinical practice.

Inflammatory cytokines can be monitored in exhaled breath particles following segmental and inhalation endotoxin challenge in healthy volunteers

Particles in exhaled air (PEx) are generated when collapsed small airways reopen during breathing. PEx can be noninvasively collected by particle impaction, allowing the analysis of undiluted epithelial lining fluid (ELF). We used the endotoxin (LPS) challenge model to proof the concept that PEx can be used to monitor inflammatory changes in the lung. In this pilot study PEx were collected from ten healthy nonsmoking subjects using the PExA® instrument twice before and twice after a segmental LPS challenge (5, 21 h). Following a 4-week washout period, PEx were collected during the week before and 5 h after a whole lung LPS inhalation challenge. PEx biomarkers were compared to blood, bronchoalveolar lavage (BAL) following segmental challenge and induced sputum (ISP) following inhalation challenge. A clear LPS-induced inflammatory response was detectable in BAL fluid, ISP and blood. Albumin and surfactant–protein D were detectable in all PEx samples. While most baseline cytokines were close to or below the detection limit, the median (IQR) IL-6 and IL-8 concentrations in PEx increased significantly after segmental (0.04 (0.03; 0.06) fg/ng PEx; 0.10 (0.08; 0.17) fg/ng PEx) and inhalation LPS challenge (0.19 (0.15; 0.23) fg/ng PEx; 0.32 (0.23; 0.42) fg/ng PEx). Using a highly sensitive analysis platform, we were able to detect a cytokine response in PEx during the early phase of LPS-induced inflammation. This will broaden the spectrum of applications for this noninvasive method to monitor inflammatory processes in the lung, including its use in clinical trials for respiratory drug development.Trial registration: The study has been registered on 07.02.2017 at Clinicaltrials.gov (NCT03044327).

Innate Lymphoid Cells Are Required to Induce Airway Hyperreactivity in a Murine Neutrophilic Asthma Model.

RationaleNon-allergic asthma is driven by multiple endotypes of which neutrophilic and pauci-granulocytic asthma have been best established. However, it is still puzzling what drives inflammation and airway hyperreactivity (AHR) in these patients and how it can be treated effectively. Recently, a potential role of the innate immune system and especially the innate lymphoid cells (ILC) has been proposed.ObjectiveIn this study, we investigated the effects of LPS inhalation on airway inflammation and AHR as a potential model for elucidating the pathogenesis of non-allergic asthma.MethodsWild-type (BALB/c), SCID, IL-17A-/-, and Rag2-/- γC-/- mice were endonasally exposed to lipopolysaccharide (LPS, 2 µg) on four consecutive days. Twenty-four hours after the last exposure, AHR to methacholine was assessed. Cytokine levels and ILC subpopulations were determined in lung tissue. Cellular differential analysis was performed in BAL fluid.Main ResultsIn this study, we developed a murine model for non-allergic neutrophilic asthma. We found that repeated endonasal applications of low-dose LPS in BALB/c mice led to AHR, BAL neutrophilia, and a significant increase in lung ILC3 as well as a significant increase in lung chemokines KC and MIP-2 and cytokines IL-1β, IL-17A, IL-22, and TNF. The adoptive transfer of ILC in Rag2-/- γC-/- mice showed that ILC played a causal role in the induction of AHR in this model. Antagonising IL-1β, but not IL-17A or neutrophils, resulted in a partial reduction in LPS-induced AHR.ConclusionIn conclusion, we report here a murine model for neutrophilic asthma where ILC are required to induce airway hyperreactivity.

Regulation of MHC Class I Expression in Lung Epithelial Cells during Inflammation.

Lung infections are a perennial leading cause of death worldwide. The lung epithelium comprises three main cell types: alveolar type I (AT1), alveolar type II (AT2), and bronchiolar cells. Constitutively, these three cell types express extremely low amounts of surface MHC class I (MHC I) molecules, that is, <1% of levels found on medullary thymic epithelial cells (ECs). We report that inhalation of the TLR4 ligand LPS upregulates cell surface MHC I by ∼25-fold on the three subtypes of mouse lung ECs. This upregulation is dependent on Nlrc5, Stat1, and Stat2 and caused by a concerted production of the three IFN families. It is nevertheless hampered, particularly in AT1 cells, by the limited expression of genes instrumental in the peptide loading of MHC I molecules. Genes involved in production and response to cytokines and chemokines were selectively induced in AT1 cells. However, discrete gene subsets were selectively downregulated in AT2 or bronchiolar cells following LPS inhalation. Genes downregulated in AT2 cells were linked to cell differentiation and cell proliferation, and those repressed in bronchiolar cells were primarily involved in cilium function. Our study shows a delicate balance between the expression of transcripts maintaining lung epithelium integrity and transcripts involved in Ag presentation in primary lung ECs.

AdMSC-derived exosomes alleviate acute lung injury via transferring mitochondrial component to improve homeostasis of alveolar macrophages

Rationale: Aberrant activation of macrophages with mitochondria dismiss was proved to be associated with pathogenesis of ALI (acute lung injury). Exosomes from adipose-derived mesenchymal stem cells (AdMSC-Exos) have been distinguished by their low immunogenicity, lack of tumorigenicity, and high clinical safety, but their role in treating ALI and the mechanism involved need to be defined. In this study, we sought to investigate whether the mitochondrial donation from AdMSC-Exos provides profound protection against LPS-induced ALI in mice, accompanied by improvement of macrophage mitochondrial function.Methods: C57BL/6 mice were orotracheally instilled with LPS (1 mg/kg). AdMSC-Exos were administered via the tail vein 4 h after LPS inhalation. Flow cytometry, H&E, Quantitative Real-Time PCR, immunofluorescence (IF), confocal microscopy imaging was conducted to investigate lung tissue inflammation and macrophage mitochondrial function. And further observe the transfer of exosomes and the effect on mitochondrial function of MH-S cells through in vitro experiments.Results: AdMSC-Exos can transfer the stem cell-derived mitochondria components to alveolar macrophages in a dose-dependent manner. Likely through complementing the damaged mitochondria, AdMSC-Exos exhibited the ability to elevate the level of mtDNA, mitochondrial membrane potential (MMP), OXPHOS activity and ATP generation, while reliving mROS stress in LPS-challenged macrophages. Restoring mitochondrial integrity via AdMSC-Exos treatment enabled macrophages shifting to anti-inflammatory phenotype, as featured with the down-regulation of IL-1β, TNF-α and iNOS secretion and increase in production of anti-inflammatory cytokines IL-10 and Arg-1. As we depleted alveolar macrophages using clodronate liposomes, the protective role for AdMSC-Exos was largely abrogated.Conclusions: AdMSC-Exos can effectively donate mitochondria component improved macrophages mitochondrial integrity and oxidative phosphorylation level, leading to the resumption of metabolic and immune homeostasis of airway macrophages and mitigating lung inflammatory pathology.

The impact of airborne endotoxin exposure on rheumatoid arthritis-related joint damage, autoantigen expression, autoimmunity, and lung disease

Airborne biohazards are risk factors in the development and severity of rheumatoid arthritis (RA) and RA-associated lung disease, yet the mechanisms explaining this relationship remain unclear. Lipopolysaccharide (LPS, endotoxin) is a ubiquitous inflammatory agent in numerous environmental and occupational air pollutant settings recognized to induce airway inflammation. Combining repetitive LPS inhalation exposures with the collagen induced arthritis (CIA) model, DBA1/J mice were assigned to either: sham (saline injection/saline inhalation), CIA (CIA/saline), LPS (saline/LPS 100 ng inhalation), or CIA + LPS for 5 weeks. Serum anti-citrullinated (CIT) protein antibody (ACPA) and anti-malondialdehyde-acetaldehyde (MAA) antibodies were strikingly potentiated with co-exposure (CIA + LPS). CIT- and MAA-modified lung proteins were increased with co-exposure and co-localized across treatment groups. Inhaled LPS exacerbated arthritis with CIA + LPS > LPS > CIA versus sham. Periarticular bone loss was demonstrated in CIA and CIA + LPS but not in LPS alone. LPS induced airway inflammation and neutrophil infiltrates were reduced with co-exposure (CIA + LPS). Potentially signaling transition to pro-fibrotic processes, there were increased infiltrates of activated CD11c+CD11b+ macrophages and transitioning CD11c+CD11bint monocyte-macrophage populations with CIA + LPS. Moreover, several lung remodeling proteins including fibronectin and matrix metalloproteinases as well as complement C5a were potentiated with CIA + LPS compared to other treatment groups. IL-33 concentrations in lung homogenates were enhanced with CIA + LPS with IL-33 lung staining driven by LPS. IL-33 expression was also significantly increased in lung tissues from patients with RA-associated lung disease (N = 8) versus controls (N = 7). These findings suggest that patients with RA may be more susceptible to developing interstitial lung disease following airborne biohazard exposures enriched in LPS.

Triptolide dose-dependently improves LPS-induced alveolar hypercoagulation and fibrinolysis inhibition through NF-κB inactivation in ARDS mice

BackgroundAlveolar hypercoagulation and fibrinolysis inhibition were associated with the refractory hypoxemia and the high mortality in patient with acute respiratory distress syndrome (ARDS), and NF-κB pathway was confirmed to contribute to the process. Triptolide (TP) significantly inhibited NF-κB pathway and thus depressed accessive inflammatory response in ARDS. We speculate that TP could improve alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS via NF-κB inactivation. PurposeThe aim of this experiment was to explore the efficacy and potential mechanism of TP on alveolar hypercoagulation and fibrinolysis inhibition in LPS-induced ARDS in mice. Methods50 μl of LPS (5 mg/ml) was inhalationally given to C57BL/6 mice to set up ARDS model. Male mice were randomly accepted with LPS, LPS + TP (1 μg/kg, 10 μg/kg, 50 μg/kg respectively), or with NEMO Binding domain peptide (NBD), an inhibitor of NF-κB. TP (1 μg/kg, 10 μg/kg, 50 μg/kg) were intraperitoneally injected or 10 μg/50 μl of NBD solution were inhaled 30 min before LPS inhalation. A same volume of normal saline (NS) substituted for TP in mice in control. The endpoint of experiment was at 8 hours after LPS stimulation. Pulmonary tissues were taken for hematoxylin-eosin (HE) staining, wet / dry ratio and for lung injury scores (LIS). Tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 in lung tissue were detected by Western-blotting and by quantitative Real-time PCR(qPCR) respectively. Concentrations of TF, PAI-1, thrombin-antithrombin complex (TAT), procollagen peptide type Ⅲ (PⅢP) and activated protein C (APC) in bronchoalveolar lavage fluid (BALF) were measured by ELISA. NF-κB activation and p65-DNA binding activity in pulmonary tissue were simultaneously determined. ResultsLPS stimulation resulted in pulmonary edema, neutrophils infiltration, obvious alveolar collapse, interstitial congestion, with high LIS, which were all dose-dependently ameliorated by Triptolide. LPS also dramatically promoted the expressions of TF and PAI-1 either in mRNA or in protein in lung tissue, and significantly stimulated the secretions of TF, PAI-1, TAT, PⅢP but inhibited APC production in BALF, which were all reversed by triptolide treatment in dose-dependent manner. TP dose-dependently inhibited the activation of NF-κB pathway induced by LPS, indicated by the changes of phosphorylations of p65 (p-p65), p-IKKα/β and p-IκBα, and weakened p65-DNA binding activity. TP and NBD had same efficacies either on alveolar hypercoagulation and fibrinolysis inhibition or on NF-κB signalling pathway in ARDS mice. ConclusionsTP dose-dependently improves alveolar hypercoagulation and fibrinolysis inhibition in ARDS mice through inhibiting NF-κB signaling pathway. Our data demonstrate that TP is expected to be an effective selection in ARDS.

Emodin improves alveolar hypercoagulation and inhibits pulmonary inflammation in LPS-provoked ARDS in mice via NF-κB inactivation

BackgroundAlveolar hypercoagulation and pulmonary inflammation are important characteristics and they regulate each other in acute respiratory distress syndrome (ARDS). NF-κB pathway has been confirmed to be involved in regulation of this crosstalk. Emodin, a traditional Chinese herb, shows potent inhibitory effect on NF-κB pathway, but whether it is effective in alveolar hypercoagulation and pulmonary inflammation in ARDS remains to be elucidated. PurposeThe aim of this experiment was to evaluate the efficacy of emodin on LPS-provoked alveolar hypercoagulation and excessive pulmonary inflammation in ARDS, and its potential mechanism. MethodsMice ARDS was set up through LPS (40 μl, 4 mg/ml) inhalation. Male mice were randomly received with BPS, LPS only, LPS+ emodin (5 mg/kg, 10 mg/kg, 20 mg/kg, respectively) and BAY65-1942, an inhibitor of IKKβ. After 48 h of LPS stimulation, pulmonary pathological injury, expressions of Tissue factor (TF), plasminogen activator inhibitor (PAI)-1, activated protein C (APC), collagen Ⅰ, collagen III, interleukin (IL) 8, IL-1β and tumor necrosis factor (TNF)-α in lung tissues, as well as concentrations of antithrombin III (AT III), procollagen peptide type III (PIIIP), soluble thrombomodulin (sTM), thrombin antithrombin complex (TAT), myeloperoxidase (MPO) and the percentage of inflammatory cells in bronchoalveolar lavage fluid (BALF) were all determined. NF-κB pathway activation as well as NF-κB DNA binding activity in pulmonary tissue were simultaneously checked. ResultsLPS stimulation resulted in obvious lung injury, excessive inflammatory cells infiltration, which all were dose-dependently ameliorated by emodin. Expressions of TF, PAI-1, collagen Ⅰ and collagen III as well as IL-8, IL-1β and TNF-α in pulmonary tissue were all elevated while APC decreased under LPS provocation, which were all reversed by emodin treatment in dose-dependent manner. LPS promoted the secretions of PIIIP, sTM, TAT and inhibited AT III production in BALF, and resulted in high levels of MPO and the percentage of inflammatory cells in BALF, all of which were significantly and dose-dependently attenuated while AT III production was increased by emodin. Meanwhile, emodin effectively inhibited NF-κB pathway activation and attenuated p65 DNA binding activity induced by LPS inhalation. Emodin and BAY-65-1942 had similar impacts in this experiment. ConclusionsEmodin improves alveolar hypercoagulation and fibrinolytic inhibition and depresses excessive pulmonary inflammation in ARDS mice in dose-dependent manner via NF-κB inactivation. Our data demonstrate that emodin is expected to be an effective drug in alveolar hypercoagulation and pulmonary inflammation in ARDS.

Cholesterol 25-hydroxylase promotes efferocytosis and resolution of lung inflammation.

Alveolar macrophages (AM) play a central role in initiation and resolution of lung inflammation, but the integration of these opposing core functions is poorly understood. AM expression of cholesterol 25-hydroxylase (CH25H), the primary biosynthetic enzyme for 25-hydroxycholesterol (25HC), far exceeds the expression of macrophages in other tissues, but no role for CH25H has been defined in lung biology. As 25HC is an agonist for the antiinflammatory nuclear receptor, liver X receptor (LXR), we speculated that CH25H might regulate inflammatory homeostasis in the lung. Here, we show that, of natural oxysterols or sterols, 25HC is induced in the inflamed lung of mice and humans. Ch25h-/- mice fail to induce 25HC and LXR target genes in the lung after LPS inhalation and exhibit delayed resolution of airway neutrophilia, which can be rescued by systemic treatment with either 25HC or synthetic LXR agonists. LXR-null mice also display delayed resolution, suggesting that native oxysterols promote resolution. During resolution, Ch25h is induced in macrophages upon their encounter with apoptotic cells and is required for LXR-dependent prevention of AM lipid overload, induction of Mertk, efferocytic resolution of airway neutrophilia, and induction of TGF-β. CH25H/25HC/LXR is, thus, an inducible metabolic axis that programs AMs for efferocytic resolution of inflammation.

Inhibition of Pendrin by a small molecule reduces Lipopolysaccharide-induced acute Lung Injury.

Rationale: Pendrin is encoded by SLC26A4 and its mutation leads to congenital hearing loss. Additionally, pendrin is up-regulated in inflammatory airway diseases such as chronic obstructive pulmonary disease, allergic rhinitis, and asthma. In this study, the effects of a novel pendrin inhibitor, YS-01, were investigated in an LPS-induced acute lung injury (ALI) mice model, and the mechanism underlying the effect of YS-01 was examined.Methods: Lipopolysaccharide (LPS, 10 mg/kg) was intranasally instilled in wild type (WT) and pendrin-null mice. YS-01 (10 mg/kg) was administered intra-peritoneally before or after LPS inhalation. Lung injury parameters were assessed in the lung tissue and bronchoalveolar lavage fluid (BALF). Pendrin levels in the BALF of 41 patients with acute respiratory distress syndrome (ARDS) due to pneumonia and 25 control (solitary pulmonary nodule) patients were also measured.Results: LPS instillation induced lung injury in WT mice but not in pendrin-null mice. Pendrin expression was increased by LPS stimulation both in vitro and in vivo. YS-01 treatment dramatically attenuated lung injury and reduced BALF cell counts and protein concentration after LPS instillation in WT mice. Proinflammatory cytokines and NF-κB activation were suppressed by YS-01 treatment in LPS-induced ALI mice. In BALF of patients whose ARDS was caused by pneumonia, pendrin expression was up-regulated compared to that in controls (mean, 24.86 vs. 6.83 ng/mL, P < 0.001).Conclusions: A novel pendrin inhibitor, YS-01, suppressed lung injury in LPS-induced ALI mice and our data provide a new strategy for the treatment of inflammatory airway diseases including sepsis-induced ALI.

Radiolabelling and immunohistochemistry reveal platelet recruitment into lungs and platelet migration into airspaces following LPS inhalation in mice

Platelets are under investigation for their role in host defence and inflammatory lung diseases and have been demonstrated to be recruited to the lung. However, the mechanisms and consequences of platelet recruitment into lungs are poorly understood. We have utilised a murine model to investigate the mechanisms of platelet involvement in lung inflammation induced by intranasal administration of LPS. Our aim was to characterise lung platelet recruitment following LPS inhalation in mice using immunohistochemistry, and non-invasive and invasive radiolabelled platelet tracking techniques. Intranasal administration of LPS caused an increase in lung platelet staining in lung tissue and elicited the recruitment of radiolabelled platelets into the lung. Prior to these responses in the lung, we observed an earlier decrease in blood platelet counts, temporally associated with platelet recruitment to the liver and spleen. Non-invasive measurements of thoracic radioactivity reflected changes in blood counts rather than extravascular lung platelet recruitment. However, both in situ counting of radiolabelled platelets and immunostaining for platelet surface markers showed LPS-induced increases in extravascular platelets into lung airspaces suggesting that some of the platelets recruited to the lung enter air spaces. Intranasal administration of LPS activates the innate immune response which includes a fall in peripheral blood platelet counts with subsequent platelet recruitment to the lung, spleen and liver, measured by immunohistochemistry and radiolabelling techniques.

LPS-induced Lung Platelet Recruitment Occurs Independently from Neutrophils, PSGL-1, and P-Selectin.

Platelets are recruited to inflammatory foci and contribute to host defense and inflammatory responses. Compared with platelet recruitment in hemostasis and thrombosis, the mechanisms of platelet recruitment in inflammation and host defense are poorly understood. Neutrophil recruitment to lung airspaces after inhalation of bacterial LPS requires platelets and PSGL-1 in mice. Given this association between platelets and neutrophils, we investigated whether recruitment of platelets to lungs of mice after LPS inhalation was dependent on PSGL-1, P-selectin, or interaction with neutrophils. BALB/c mice were administered intranasal LPS (O55:B5, 5 mg/kg) and, 48 hours later, lungs were collected and platelets and neutrophils quantified in tissue sections by immunohistochemistry. The effects of functional blocking antibody treatments targeting the platelet-neutrophil adhesion molecules, P-selectin or PSGL-1, or treatment with a neutrophil-depleting antibody targeting Ly6G, were tested on the extent of LPS-induced lung platelet recruitment. Separately in Pf4-Cre × mTmG mice, two-photon intravital microscopy was used to image platelet adhesion in live lungs. Inhalation of LPS caused both platelet and neutrophil recruitment to the lung vasculature. However, decreasing lung neutrophil recruitment by blocking PSGL-1, P-selectin, or depleting blood neutrophils had no effect on lung platelet recruitment. Lung intravital imaging revealed increased adhesion of platelets in the lung microvasculature which was not associated with thrombus formation. In conclusion, platelet recruitment to lungs in response to LPS occurs through mechanisms distinct from those mediating neutrophil recruitment, or the occurrence of pulmonary emboli.

Selective Inactivation of Intracellular BiP/GRP78 Attenuates Endothelial Inflammation and Permeability in Acute Lung Injury

The role of Endoplasmic Reticulum Chaperone and Signaling Regulator BiP/GRP78 in acute inflammatory injury, particularly in the context of lung endothelium, is poorly defined. In his study, we monitored the effect of SubAB, a holoenzyme that cleaves and specifically inactivates BiP/GRP78 and its inactive mutant SubAA272B on lung inflammatory injury in an aerosolized LPS inhalation mouse model of acute lung injury (ALI). Analysis of lung homogenates and bronchoalveolar lavage (BAL) fluid showed that LPS-induced lung inflammation and injury were significantly inhibited in SubAB- but not in SubAA272B-treated mice. SubAB-treated mice were also protected from LPS-induced decrease in lung compliance. Gene transfer of dominant negative mutant of BiP in the lung endothelium protected against LPS-induced lung inflammatory responses. Consistent with this, stimulation of endothelial cells (EC) with thrombin caused an increase in BiP/GRP78 levels and inhibition of ER stress with 4-phenylbutyric acid (4-PBA) prevented this response as well as increase in VCAM-1, ICAM-1, IL-6, and IL-8 levels. Importantly, thrombin-induced Ca2+ signaling and EC permeability were also prevented upon BiP/GRP78 inactivation. The above EC responses are mediated by intracellular BiP/GRP78 and not by cell surface BiP/GRP78. Together, these data identify intracellular BiP/GRP78 as a novel regulator of endothelial dysfunction associated with ALI.

AIBP augments cholesterol efflux from alveolar macrophages to surfactant and reduces acute lung inflammation.

Acute respiratory distress syndrome (ARDS) is characterized by an excessive pulmonary inflammatory response. Removal of excess cholesterol from the plasma membrane of inflammatory cells helps reduce their activation. The secreted apolipoprotein A-I binding protein (AIBP) has been shown to augment cholesterol efflux from endothelial cells to the plasma lipoprotein HDL. Here, we find that AIBP was expressed in inflammatory cells in the human lung and was secreted into the bronchoalveolar space in mice subjected to inhalation of LPS. AIBP bound surfactant protein B and increased cholesterol efflux from alveolar macrophages to calfactant, a therapeutic surfactant formulation. In vitro, AIBP in the presence of surfactant reduced LPS-induced p65, ERK1/2 and p38 phosphorylation, and IL-6 secretion by alveolar macrophages. In vivo, inhalation of AIBP significantly reduced LPS-induced airspace neutrophilia, alveolar capillary leak, and secretion of IL-6. These results suggest that, similar to HDL in plasma, surfactant serves as a cholesterol acceptor in the lung. Furthermore, lung injury increases pulmonary AIBP expression, which likely serves to promote cholesterol efflux to surfactant and reduce inflammation.