A Functional Assessment of Fetal Liver and Monocyte-Derived Macrophages in the Lung Alveolar Environment.

It is becoming clear that every organ is seeded by a population of fetal liver-derived macrophages that are replaced at different rates by monocyte-derived macrophages. Using the Ms4a3tdTomato reporter mouse that reports on monocyte-derived alveolar macrophages (Mo-AMs) and our ability to examine AM function using our multichannel intravital microscopy, we examined the fetal-liver derived alveolar macrophage (FL-AM) and Mo-AM populations within the same mouse under various environmental conditions. The experiments unveiled that AMs migrated from alveolus to alveolus and phagocytosed bacteria identically regardless of ontogenic origin. Using 50 PFU of influenza A virus (IAV) determined using the Madin-Darby canine kidney (MDCK) cell line, we noted that both populations were susceptible to IAV-induced immunoparalysis, which also led to impaired phagocytosis of secondary bacterial infections. Both FL-AMs and Mo-AMs were trained by ß-glucan to resist IAV-induced paralysis. Over time (40 wk), Mo-AMs began to outperform FL-AMs, although both populations were still sensitive to IAV. Our data also show that clodronate depletion of AMs leads to replenishment, but by FL-AMs, and these macrophages do show some functional impairment for a limited time. Overall, the system is designed such that new macrophages rapidly assume the function of tissue-resident macrophages when both populations are examined in an identical environment. These data do differ from artificial depletion methods that compare Mo-AMs and FL-AMs.

An In Vitro Alveolar Model Allows for the Rapid Assessment of Particles for Respiratory Sensitization Potential.

Dust, both industrial and household, contains particulates that can reach the most distal aspects of the lung. Silica and nickel compounds are two such particulates and have known profiles of poor health outcomes. While silica is well-characterized, nickel compounds still need to be fully understood for their potential to cause long-term immune responses in the lungs. To assess these hazards and decrease animal numbers used in testing, investigations that lead to verifiable in vitro methods are needed. To understand the implications of these two compounds reaching the distal aspect of the lungs, the alveoli, an architecturally relevant alveolar model consisting of epithelial cells, macrophages, and dendritic cells in a maintained submerged system, was utilized for high throughput testing. Exposures include crystalline silica (SiO2) and nickel oxide (NiO). The endpoints measured included mitochondrial reactive oxygen species and cytostructural changes assessed via confocal laser scanning microscopy; cell morphology evaluated via scanning electron microscopy; biochemical reactions assessed via protein arrays; transcriptome assessed via gene arrays, and cell surface activation markers evaluated via flow cytometry. The results showed that, compared to untreated cultures, NiO increased markers for dendritic cell activation, trafficking, and antigen presentation; oxidative stress and cytoskeletal changes, and gene and cytokine expression of neutrophil and other leukocyte chemoattractants. The chemokines and cytokines CCL3, CCL7, CXCL5, IL-6, and IL-8 were identified as potential biomarkers of respiratory sensitization.

High content analysis of in vitro alveolar macrophage responses can provide mechanistic insight for inhaled product safety assessment.

Assessing the safety of inhaled substances in the alveolar region of the lung requires an understanding of how the respired material interacts with both physical and immunological barriers. Human alveolar-like macrophages in vitro provide a platform to assess the immunological response in the airways and may better inform the understanding of a response to an inhaled challenge being adaptive or adverse. The aim of this study was to determine if a morphometric phenotyping approach could discriminate between different inhaled nicotine products and indicate the potential mechanism of toxicity of a substance. Cigarette smoke (CS) and e-liquids extracted into cell culture medium were applied to human alveolar-like macrophages in mono-culture (ImmuONE™) and co-culture (ImmuLUNG™) to test the hypothesis. Phenotype profiling of cell responses was highly reproducible and clearly distinguished the different responses to CS and e-liquids. Whilst the phenotypes of untreated macrophages were similar regardless of culture condition, macrophages cultured in the presence of epithelial cells were more sensitive to CS-induced changes related to cell size and vacuolation processes. This technique demonstrated phenotypical observations typical for CS exposure and indicative of the established mechanisms of toxicity. The technique provides a rapid screening approach to determine detailed immunological responses in the airways which can be linked to potentially adverse pathways and support inhalation safety assessment.

Assessment of Statin Treatment for Pulmonary Alveolar Proteinosis without Hypercholesterolemia: A 12-Month Prospective, Longitudinal, and Observational Study.

Background: Pulmonary alveolar proteinosis (PAP) is a rare disorder which is characterized by the accumulation of excessive surfactant lipids and proteins in alveolar macrophages and alveoli. Oral statin therapy has been reported to be a novel therapy for PAP with hypercholesterolemia. We aimed to evaluate the safety and efficacy of oral statin therapy for PAP without hypercholesterolemia. Methods: In a prospective real-world observational study, 47 PAP patients without hypercholesterolemia were screened. Oral statin was initiated as therapy for these PAP patients with 12 months of follow-up. Results: Forty PAP patients completed the study. 26 (65%) of 40 PAP patients responded to statin therapy according to the study criteria. Partial pressure of arterial oxygen (PaO2) and percentage of diffusion capacity predicted (DLCO%) significantly increased while disease severity score (DSS) and radiographic abnormalities decreased after 12 months of statin therapy (all p < 0.05). The factors associated with response were higher levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody and baseline total cholesterol/high-density lipoprotein cholesterol (TC/HDL) (p = 0.015 and p = 0.035, respectively). The area under the receiver operating characteristic curve (AUROC) of dose of atorvastatin for predicting the response to statin therapy for PAP was 0.859 (95% CI: 0.738-0.979, p < 0.001). The cutoff dose of atorvastatin was 67.5 mg daily with their corresponding specificity (64.3%) and sensitivity (96.2%). No severe side effects were observed during the study. Conclusions: In PAP patients without hypercholesterolemia, statin therapy resulted in improvements in arterial blood gas (ABG) measurement, pulmonary function, and radiographic assessment.

Effect of mycobacterial proteins that target mitochondria on the alveolar macrophages activation during Mycobacterium tuberculosis infection.

Purpose of the study: During the early and progressive (late) stages of murine experimental pulmonary tuberculosis, the differential activation of macrophages contributes to disease development by controlling bacterial growth and immune regulation. Mycobacterial proteins P27 and PE_PGRS33 can target the mitochondria of macrophages. This study aims to evaluate the effect of both proteins on macrophage activation during mycobacterial infection. Materials and methods: We assess both proteins for mitochondrial oxygen consumption, and morphological changes, as well as bactericide activity, production of metabolites, cytokines, and activation markers in infected MQs. The cell line MH-S was used for all the experiments. Results: We show that P27 and PE_PGRS33 proteins modified mitochondrial dynamics, oxygen consumption, bacilli growth, cytokine production, and some genes that contribute to macrophage alternative activation and mycobacterial intracellular survival. Conclusions: Our findings showed that these bacterial proteins partially contribute to promoting M2 differentiation by altering mitochondrial metabolic activity.

PyCoCa:A quantifying tool of carbon content in airway macrophage for assessment the internal dose of particles.

Given the lack of a comprehensive understanding of the complex metabolism and variable exposure environment, carbon particles in macrophages have become a potentially valuable biomarker to assess the exposure level of atmospheric particles, such as black carbon. However, the tedious and subjective quantification method limits the application of carbon particles as a valid biomarker. Aiming to obtain an accurate carbon particles quantification method, the deep learning and binarization algorithm were implemented to develop a quantitative tool for carbon content in airway macrophage (CCAM), named PyCoCa. Two types of macrophages, normal and foamy appearance, were applied for the development of PyCoCa. In comparison with the traditional methods, PyCoCa significantly improves the identification efficiency for over 100 times. Consistency assessment with the gold standard revealed that PyCoCa exhibits outstanding prediction ability with the Interclass Correlation Coefficient (ICC) values of over 0.80. And a proper fresh dye will enhance the performance of PyCoCa (ICC = 0.89). Subsequent sensitivity analysis confirmed an excellent performance regarding accuracy and robustness of PyCoCa under high/low exposure environments (sensitivity > 0.80). Furthermore, a successful application of our quantitative tool in cohort studies indicates that carbon particles induce macrophage foaming and the foaming decrease the carbon particles internalization in reverse. Our present study provides a robust and efficient tool to accurately quantify the carbon particles loading in macrophage for exposure assessment.

Alcohol-Induced Glycolytic Shift in Alveolar Macrophages Is Mediated by Hypoxia-Inducible Factor-1 Alpha.

Excessive alcohol use increases the risk of developing respiratory infections partially due to impaired alveolar macrophage (AM) phagocytic capacity. Previously, we showed that chronic ethanol (EtOH) exposure led to mitochondrial derangements and diminished oxidative phosphorylation in AM. Since oxidative phosphorylation is needed to meet the energy demands of phagocytosis, EtOH mediated decreases in oxidative phosphorylation likely contribute to impaired AM phagocytosis. Treatment with the peroxisome proliferator-activated receptor gamma (PPARγ) ligand, pioglitazone (PIO), improved EtOH-mediated decreases in oxidative phosphorylation. In other models, hypoxia-inducible factor-1 alpha (HIF-1α) has been shown to mediate the switch from oxidative phosphorylation to glycolysis; however, the role of HIF-1α in chronic EtOH mediated derangements in AM has not been explored. We hypothesize that AM undergo a metabolic shift from oxidative phosphorylation to a glycolytic phenotype in response to chronic EtOH exposure. Further, we speculate that HIF-1α is a critical mediator of this metabolic switch. To test these hypotheses, primary mouse AM (mAM) were isolated from a mouse model of chronic EtOH consumption and a mouse AM cell line (MH-S) were exposed to EtOH in vitro. Expression of HIF-1α, glucose transporters (Glut1 and 4), and components of the glycolytic pathway (Pfkfb3 and PKM2), were measured by qRT-PCR and western blot. Lactate levels (lactate assay), cell energy phenotype (extracellular flux analyzer), glycolysis stress tests (extracellular flux analyzer), and phagocytic function (fluorescent microscopy) were conducted. EtOH exposure increased expression of HIF-1α, Glut1, Glut4, Pfkfb3, and PKM2 and shifted AM to a glycolytic phenotype. Pharmacological stabilization of HIF-1α via cobalt chloride treatment in vitro mimicked EtOH-induced AM derangements (increased glycolysis and diminished phagocytic capacity). Further, PIO treatment diminished HIF-1α levels and reversed glycolytic shift following EtOH exposure. These studies support a critical role for HIF-1α in mediating the glycolytic shift in energy metabolism of AM during excessive alcohol use.

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-1ß, 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.

Mechanism of chlorogenic acid in alveolar macrophage polarization in Klebsiella pneumoniae-induced pneumonia.

Chlorogenic acid (CA) has been discovered to regulate macrophage polarization in pneumonia. This study aims to analyze the functional mechanism of CA in alveolar macrophage (AM) polarization and provide a theoretical basis for treatment of Klebsiella pneumoniae (Kp)-induced pneumonia. Mice were infected with Kp, and treated with CA and silent information regulator 1 (SIRT1) inhibitor (Selisistat). Mouse survival rate was recorded and bacterial burden was detected. AM polarization and pathologic change of lung tissues were evaluated. Expressions of SIRT1 and HMGB1 and cytokine levels were detected. MH-S cells were infected with Kp to establish the pneumonia cell model, followed by transfection of si-SIRT1 and HMGB1 overexpression vector. The HMGB1 expression in the nucleus and cytoplasm was detected. HMGB1 subcellular localization and HMGB1 acetylation level were detected. Kp led to high death rates, SIRT down-regulation and increases in inflammatory factor level and bacterial burden, and promoted M1 polarization. CA treatment improved mouse survival rate and promoted M2 polarization and SIRT1 expression. SIRT1 decreased HMGB1 acetylation level to inhibit nuclear to the cytoplasm translocation. Silencing SIRT1 or HMGB1 overexpression reversed the effect of CA on Kp-induced pneumonia. Overall, CA activated SIRT1 to inhibit HMGB1 acetylation level and nuclear translocation, thereby promoting M2 polarization in AMs and alleviating Kp-induced pneumonia.

MicroRNA-205-5p targets E2F1 to promote autophagy and inhibit pulmonary fibrosis in silicosis through impairing SKP2-mediated Beclin1 ubiquitination.

Silicosis is an occupational disease characterized by extensive pulmonary fibrosis, and the underlying pathological process remains uncertain. Herein, we explored the molecular mechanism by which microRNA-205-5p (miR-205-5p) affects the autophagy of alveolar macrophages (AMs) and pulmonary fibrosis in mice with silicosis through the E2F transcription factor 1 (E2F1)/S-phase kinase-associated protein 2 (SKP2)/Beclin1 axis. Alveolar macrophages (MH-S cells) were exposed to crystalline silica (CS) to develop an in vitro model, and mice were treated with CS to establish an in vivo model. Decreased Beclin1 and increased SKP2 and E2F1 were identified in mice with silicosis. We silenced or overexpressed miR-205-5p, E2F1, SKP2 and Beclin1 to investigate their potential roles in pulmonary fibrosis in vivo and autophagy in vitro. Recombinant adenovirus mRFP-GFP-LC3 was transduced into the MH-S cells to assay autophagic flow. Knocking down Beclin1 promoted pulmonary fibrosis and suppressed the autophagy. Co-immunoprecipitation and ubiquitination assays suggested that SKP2 induced K48-linked ubiquitination of Beclin1. Furthermore, chromatin immunoprecipitation-PCR revealed the site where E2F1 bound to the SKP2 promoter between 1638 bp and 1645 bp. As shown by dual-luciferase reporter gene assay, the transfection with miR-205-5p mimic inhibited the luciferase activity of the wild-type E2F1 3'untranslated region, suggesting that miR-205-5p targeted E2F1. Additionally, miR-205-5p overexpression increased autophagy and reduced the pulmonary fibrosis, while overexpression of E2F1 or SKP2 or inhibition of Beclin1 could annul this effect. The current study elucidated that miR-205-5p targeted E2F1, thereby inhibiting SKP2-mediated Beclin1 ubiquitination to promote macrophage autophagy and inhibit pulmonary fibrosis in mice with silicosis.

PI3K/ NF-κB-dependent TNF-α and HDAC activities facilitate LPS-induced RGS10 suppression in pulmonary macrophages.

Regulator of G-protein signaling 10 (RGS10) is a member of the superfamily of RGS proteins that canonically act as GTPase activating proteins (GAPs). RGS proteins accelerate GTP hydrolysis on the G-protein α subunits and result in termination of signaling pathways downstream of G protein-coupled receptors. Beyond its GAP function, RGS10 has emerged as an anti-inflammatory protein by inhibiting LPS-mediated NF-κB activation and expression of inflammatory cytokines, in particular TNF-α. Although RGS10 is abundantly expressed in resting macrophages, previous studies have shown that RGS10 expression is suppressed in macrophages following Toll-like receptor 4 (TLR4) activation by LPS. However, the molecular mechanism by which LPS induces Rgs10 silencing has not been clearly defined. The goal of the current study was to determine whether LPS silences Rgs10 expression through an NF-κB-mediated proinflammatory mechanism in pulmonary macrophages, a unique type of innate immune cells. We demonstrate that Rgs10 transcript and RGS10 protein levels are suppressed upon LPS treatment in the murine MH-S alveolar macrophage cell line. We show that pharmacological inhibition of PI3K/ NF-κB/p300 (NF-κB co-activator)/TNF-α signaling cascade and the activities of HDAC (1-3) enzymes block LPS-induced silencing of Rgs10 in MH-S cells as well as microglial BV2 cells and BMDMs. Further, loss of RGS10 generated by using CRISPR/Cas9 amplifies NF-κB phosphorylation and inflammatory gene expression following LPS treatment in MH-S cells. Together, our findings strongly provide critical insight into the molecular mechanism underlying RGS10 suppression by LPS in pulmonary macrophages.

Dual antitubercular drug loaded liposomes for macrophage targeting: development, characterisation, ex vivo and in vivo assessment.

AIM: The present study was conducted to formulate and investigate liposomes for the dual drug delivery based on anti-tubercular drug(s) combination i.e. Isoniazid (INH) and Rifampicin (RIF). MATERIALS AND METHODS: Mannosylated and non mannosylated liposomes were prepared by lipid thin film hydration method, using DSPC: Chol at a molar ratio 6:4 while in case of mannosylated liposomes DSPC: Chol: Man-C4-Chol at a molar ratio 6.0:3.5:0.5 were used and extensively characterised. The particle size and zeta potential were recorded to be 1.29 ± 0.24 µm and -9.1 ± 0.11 mV. The drug entrapment (%) was recorded to be 84.7 ± 1.25% for Rifampicin and 31.8 ± 0.12% for Isoniazid. RESULTS: The antitubercular activity studied in Balb/C mice was maximum in the case of mannosylated liposomes. The biodistribution studies also revealed higher drug(s) concentration (accumulation) maintained over a protracted period. CONCLUSIONS: The liposomal preparations are passively as well as actively uptaken by the alveolar macrophages which are the cellular tropics of infection. The mannosylated liposomes appear to be a potential carrier for dual drug delivery and targeted antitubercular therapy.

Infection of Mycobacterium tuberculosis Promotes Both M1/M2 Polarization and MMP Production in Cigarette Smoke-Exposed Macrophages.

Pulmonary tuberculosis (PTB) is a risk factor for COPD. Our previous study revealed more severe emphysema in COPD patients (mostly smokers) with prior tuberculosis. However, the mechanisms of interactions between cigarette smoke (CS) and Mycobacterium tuberculosis (Mtb) are unknown. In this study, we found that the frequencies of both M1 and M2 macrophages, and levels of MMP9 and MMP12 in bronchoalveolar lavage were increased in PTB patients with smoking. Between-group analysis showed that the frequency of M1 macrophages was higher in non-smoker PTB patients while more M2 macrophages were found in smokers without PTB, as compared to the non-smoker healthy controls. Bacille Calmette-Guérin (BCG) infection in CS extract (CSE)-incubated MH-S cells further enhanced secretion of M1-related (iNOS, IFN-γ and TNF-α) and M2-related (TGF-ß and IL-10) cytokines, reactive oxygen species (ROS) production and cellular apoptosis, concomitantly with up-regulation of MMP9 and MMP12, but not TIMP1. Moreover, BCG infection in acutely CS-exposed mice promoted macrophage polarization toward both M1 and M2 phenotypes, along with increased lung inflammatory infiltration. MMP9 and MMP12, but not TIMP1, were further up-regulated in lung tissues and BAL fluid after BCG infection in this model. Taken together, Mtb Infection promoted CS-exposed macrophages to polarize toward both M1 and M2 phenotypes, along with enhanced production of MMP9 and MMP12. These findings provide insights into the mechanistic interplay between CS exposure and tuberculosis in the pathogenesis of COPD.

Organic dust-induced lung injury and repair: Bi-directional regulation by TNFα and IL-10.

Exposure to organic dust increases chronic airway inflammatory disorders. Effective treatment strategies are lacking. It has been reported that hog barn dust extracts (HDE) induce TNFα through protein kinase C (PKC) activation and that lung inflammation is enhanced in scavenger receptor A (SRA/CD204) knockout (KO) mice following HDE. Because interleukin (IL)-10 production can limit excessive inflammation, it was hypothesized here that HDE-induced IL-10 would require CD204 to effect inflammatory responses. C57BL/6 wild-type (WT), SRA KO, and IL-10 KO mice were intranasally challenged daily for 8 days with HDE and subsequently rested for 3 days with/without recombinant IL-10 (rIL-10) treatment. Primary peritoneal macrophages (PM) and murine alveolar macrophages (MH-S cells) were treated in vitro with HDE, SRA ligand (fucoidan), rIL-10, and/or PKC isoform inhibitors. HDE induced in vivo lung IL-10 in WT, but not SRA KO mice, and similar trends were demonstrated in isolated PM from same treated mice. Lung lymphocyte aggregates and neutrophils were elevated in in vivo HDE-treated SRA and IL-10 KO mice after a 3-d recovery, and treatment during recovery with rIL-10 abrogated these responses. In vitro rIL-10 treatment reduced HDE-stimulated TNFα release in MH-S and WT PM. In SRA KO macrophages, there was reduced IL-10 and PKC zeta (ζ) activity and increased TNFα following in vitro HDE stimulation. Similarly, blocking SRA (24 hr fucoidan pre-treatment) resulted in enhanced HDE-stimulated macrophage TNFα and decreased IL-10 and PKCζ activation. PKCζ inhibitors blocked HDE-stimulated IL-10, but not TNFα. Collectively, HDE stimulates IL-10 by an SRA- and PKCζ-dependent mechanism to regulate TNFα. Enhancing resolution of dust-mediated lung inflammation through targeting IL-10 and/or SRA may represent new approaches to therapeutic interventions.

miR-26a-5p mediates TLR signaling pathway by targeting CTGF in LPS-induced alveolar macrophage.

To explore the regulation mechanism of miR-26a-5p and connective tissue growth factor (CTGF) in lipopolysaccharide (LPS)-induced alveolar macrophages, which is a severe pneumonia cell model. MH-S cells were grouped into Normal group, Model group, negative control (NC) group, miR-26a-5p mimic group, oe-CTGF group, miR-26a-5p mimic + oe-CTGF group. The expression level of miR-26a-5p, CTGF and Toll-like receptor (TLR) signaling related molecules (TLR2, TLR4 and nuclear factor-κB p65) were detected by qRT-PCR and WB, respectively. The cell viability and apoptosis rate were detected by methyl thiazolyl tetrazolium (MTT) and flow cytometry, respectively. Compared with the Normal group, the expression level of miR-26a-5p was significantly decreased, while CTGF protein level was significantly increased in the Model group. Compared with the Model group, MH-S cells with miR-26a-5p overexpression showed enhanced cell viability, decreased apoptosis rate, declined expression level of TLR signaling related molecules and reduced level of tumor necrosis factor-α (TNF-α), interleukin (IL) 6 (IL-6) and IL-1ß, while those with CTGF overexpression had an opposite phenotype. In conclusion, miR-26a-5p can inhibit the expression of CTGF and mediate TLR signaling pathway to inhibit the cell apoptosis and reduce the expression of proinflammatory cytokines in alveolar macrophages which is a cell model of severe pneumonia.

In vitro toxicity assessment of respirable solid surface composite sawing particles.

Solid surface composites (SSCs) are a class of popular construction materials composed of aluminum trihydrate and acrylic polymers. Previous investigations have demonstrated that sawing SSC releases substantial airborne dusts, with a number-based geometric mean diameter of 1.05 µm. We reported that in mice, aspiration exposure to airborne SSC dusts induced symptoms of pulmonary inflammation at 24-h postexposure: neutrophilic influx, alveolitis, and increased lactate dehydrogenase (LDH) and pro-inflammatory cytokine levels in lavage fluid. The particles appeared to be poorly cleared, with 81% remaining at 14-day postexposure. The objective of this study was to determine the toxicity specifically of respirable particles on a model of human alveolar macrophages (THP-1). The relative toxicities of subfractions (0.07, 0.66, 1.58, 5.0, and 13.42 µm diameter) of the airborne particles were also determined. THP-1 macrophages were exposed for 24 h to respirable particles from sawing SSC (0, 12.5, 25, 50, or 100 µg/ml) or size-specific fractions (100 µg/ml). Exposure to respirable SSC particles induced THP-1 macrophage toxicity in a dose-dependent manner. Viability was decreased by 15% and 19% after exposure to 50 and 100 µg/ml SSC, respectively, which correlated with increased cell culture supernatant LDH activity by 40% and 70% when compared to control. Reactive oxygen species (ROS) production and inflammatory cytokines were increased in a dose-dependent manner. A size-dependent cytotoxic effect was observed in the cells exposed to subfractions of SSC particles. SSC particles of 0.07, 0.66, and 1.58 µm diameter killed 36%, 17%, and 22% of cells, respectively. These results indicate a potential for cytotoxicity of respirable SSC particles and a relationship between particle size and toxicity, with the smallest fractions appearing to exhibit the greatest toxicity.

α-Ketoglutarate Modulates Macrophage Polarization Through Regulation of PPARγ Transcription and mTORC1/p70S6K Pathway to Ameliorate ALI/ARDS.

As tissue-resident cells in the lung, alveolar macrophages display remarkable heterogeneity and play a crucial role in the development and control of septic acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Recent evidence suggests that α-ketoglutarate (α-KG) plays an important role in alternative activation of macrophage (M2) through metabolic and epigenetic reprogramming, and thus possesses anti-inflammatory properties. However, the underlying mechanisms of α-KG's effect on alveolar macrophage polarization and the potential effects of α-KG in ALI/ARDS remain unclear. Here, we examined the effects and mechanisms of α-KG on alveolar macrophage polarization, and investigated the possible effects of α-KG on lipopolysaccharide (LPS)-induced ALI/ARDS in a mouse model. We found that α-KG inhibited M1 macrophage polarization and promoted IL-4-induced M2 macrophage polarization in MH-S cells (a murine alveolar macrophage cell line). Further experiments showed that α-KG down-regulated the expression of M1-polarized marker genes and inhibited the activities of mammalian target of rapamycin complex 1 (mTORC1)/p70 ribosomal protein S6 kinase (p70S6K) signaling pathway in M1-polarized MH-S cells. Moreover, our results showed that α-KG promoted IL-4-induced M2 polarization of MH-S cells by augmenting nuclear translocation of peroxisome proliferator-activated receptor γ (PPARγ) and increasing expression of relevant fatty acid metabolic genes. Finally, using an LPS-induced ALI/ARDS mouse model, we found that α-KG ameliorated the LPS-induced inflammation and lung pathological damage, as well as α-KG pretreated mice had better clinical scores compared with the LPS group. These findings reveal new mechanisms of α-KG in regulating macrophage polarization which may provide novel strategies for the prevention and treatment of inflammatory diseases, including sepsis and septic ALI/ARDS.

Cigarette smoke-induced HMGB1 translocation and release contribute to migration and NF-κB activation through inducing autophagy in lung macrophages.

High-mobility group box 1 (HMGB1) shows pro-inflammatory activity in various inflammatory diseases and has been found up-regulated in chronic obstructive pulmonary disease (COPD). Lung macrophages play an important role in airway inflammation and lung destruction in COPD, yet whether HMGB1 is involved in cigarette smoke (CS)-induced lung macrophage dysfunction is unknown. We sought to evaluate the intracellular localization and release of HMGB1 in lung macrophages from COPD patients and CS-exposed mice, and to investigate the role of HMGB1 in regulating autophagy in CS extract (CSE)-treated lung macrophages (MH-S cells). Our results showed that HMGB1 was highly expressed in lung tissues and sera of COPD patients and CS-exposed mice, along with predominantly cytoplasmic exporting from nuclei in lung macrophages. In vitro experiments revealed that CSE promoted the expression, nucleocytoplasmic translocation and release of HMGB1 partly via the nicotinic acetylcholine receptor (nAChR). Blockade of HMGB1 with chicken anti-HMGB1 polyclonal antibody (anti-HMGB1) or glycyrrhizin (Gly) attenuated the increase of LC3B-II and Beclin1, migration and p65 phosphorylation, suggesting the involvement of HMGB1 in autophagy, migration and NF-κB activation of lung macrophages. Hydroxychloroquine (CQ), an autophagy inhibitor, enhanced the increase of LC3B-II but not Beclin1 in CSE or rHMGB1-treated MH-S cells, and inhibition of autophagy by CQ and 3-methyladenine (3-MA) abrogated the migration and p65 phosphorylation of CSE-treated cells. These results indicate that CS-induced HMGB1 translocation and release contribute to migration and NF-κB activation through inducing autophagy in lung macrophages, providing novel evidence for HMGB1 as a potential target of intervention in COPD.

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure.

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

Down-regulation of long non-coding RNA SNHG14 protects against acute lung injury induced by lipopolysaccharide through microRNA-34c-3p-dependent inhibition of WISP1.

BACKGROUND: Accumulating evidence has shown the important roles of long non-coding RNAs (lncRNAs) in acute lung injury (ALI). This study aimed to investigate the potential role of lncRNA small nucleolar RNA host gene 14 (SNHG14) in lipopolysaccharides (LPS)-induced ALI. METHODS: Expression of SNHG14, microRNA-34c-3p (miR-34c-3p) and Wnt1 inducible signaling pathway protein 1 (WISP1) in LPS-exposed mouse alveolar macrophages (MH-S) and lung tissues from mice with LPS-induced ALI was determined by reverse transcription quantitative polymerase chain reaction. The interactions among SNHG14, miR-34c-3p and WISP1 were analyzed by dual-luciferase reporter and RIP assays. Using gain-of-function or loss-of-function approaches, the contents of proinflammatory proteins were determined and MH-S cell viability was assessed to evaluate the in vitro functions of SNHG14, miR-34c-3p and WISP1, and wet/dry weight ratio and proinflammatory proteins in lung tissues were determined to assess their in vivo effects. RESULTS: SNHG14 and WISP1 expression was increased, while miR-34c-3p was decreased in ALI models. SNHG14 bound to miR-34c-3p, resulting in impaired miR-34c-3p-dependent down-regulation of WISP1. Both SNHG14 silencing and miR-34c-3p over-expression reduced the levels of proinflammatory proteins IL-18, IL-1ß, TNF-α and IL-6 and inhibited MH-S cell viability. SNHG14 silencing or miR-34c-3p over-expression decreased the wet/dry weight ratio in lung tissues from ALI mice. The reductions induced by SNHG14 silencing or miR-34c-3p over-expression were rescued by WISP1 over-expression. CONCLUSION: This study demonstrated that lncRNA SNHG14 silencing alleviated inflammation in LPS-induced ALI through miR-34c-3p-mediated inhibition of WISP1. Our findings suggest that lncRNA SNHG14 may serve as a therapeutic target for ALI.