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.

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.

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.

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.

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.

In vitro toxicity assessment of emitted materials collected during the manufacture of water pipe plastic linings.

Objectives: US water infrastructure is in need of widespread repair due to age-related deterioration. Currently, the cured-in-place (CIPP) procedure is the most common method for water pipe repair. This method involves the on-site manufacture of a new polymer composite plastic liner within the damaged pipe. The CIPP process can release materials resulting in occupational and public health concerns. To understand hazards associated with CIPP-related emission exposures, an in vitro toxicity assessment was performed. Materials and Methods: Mouse alveolar epithelial and alveolar macrophage cell lines and condensates collected at 3 worksites utilizing styrene-based resins were utilized for evaluations. All condensate samples were normalized based on the major emission component, styrene. Further, a styrene-only exposure group was used as a control to determine mixture related toxicity. Results: Cytotoxicity differences were observed between worksite samples, with the CIPP worksite 4 sample inducing the most cell death. A proteomic evaluation was performed, which demonstrated styrene-, worksite-, and cell-specific alterations. This examination of protein expression changes determined potential biomarkers of exposure including transglutaminase 2, advillin, collagen type 1, perilipin-2, and others. Pathway analysis of exposure-induced proteomic alterations identified MYC and p53 to be regulators of cellular responses. Protein changes were also related to pathways involved in cell damage, immune response, and cancer. Conclusions: Together these findings demonstrate potential risks associated with the CIPP procedure as well as variations between worksites regarding emissions and toxicity. Our evaluation identified biological pathways that require a future evaluation and also demonstrates that exposure assessment of CIPP worksites should examine multiple chemical components beyond styrene, as many cellular responses were styrene-independent.

Multidimensional Assessment of the Host Response in Mechanically Ventilated Patients with Suspected Pneumonia.

Rationale: The identification of informative elements of the host response to infection may improve the diagnosis and management of bacterial pneumonia. Objectives: To determine whether the absence of alveolar neutrophilia can exclude bacterial pneumonia in critically ill patients with suspected infection and to test whether signatures of bacterial pneumonia can be identified in the alveolar macrophage transcriptome. Methods: We determined the test characteristics of alveolar neutrophilia for the diagnosis of bacterial pneumonia in three cohorts of mechanically ventilated patients. In one cohort, we also isolated macrophages from alveolar lavage fluid and used the transcriptome to identify signatures of bacterial pneumonia. Finally, we developed a humanized mouse model of Pseudomonas aeruginosa pneumonia to determine if pathogen-specific signatures can be identified in human alveolar macrophages. Measurements and Main Results: An alveolar neutrophil percentage less than 50% had a negative predictive value of greater than 90% for bacterial pneumonia in both the retrospective (n = 851) and validation cohorts (n = 76 and n = 79). A transcriptional signature of bacterial pneumonia was present in both resident and recruited macrophages. Gene signatures from both cell types identified patients with bacterial pneumonia with test characteristics similar to alveolar neutrophilia. Conclusions: The absence of alveolar neutrophilia has a high negative predictive value for bacterial pneumonia in critically ill patients with suspected infection. Macrophages can be isolated from alveolar lavage fluid obtained during routine care and used for RNA-Seq analysis. This novel approach may facilitate a longitudinal and multidimensional assessment of the host response to bacterial pneumonia.

Cigarette smoke condensate may disturb immune function with apoptotic cell death by impairing function of organelles in alveolar macrophages.

Considering that cigarette smoke condensate (CSC) is primarily absorbed through the alveoli in the lungs, herein, we used a mouse alveolar macrophage cell line (MH-S cells). After 24 h exposure, CSC decreased dose-dependently cell viability accompanying an increase in intracellular ROS and NO level. CSC structurally or functionally damaged organelles including mitochondria, ER and lysosome and enhanced the expression of proteins related to apoptosis, ER stress and DNA damage accompanying an elevated proportion of annexin V-bound cells. Meanwhile, the expression of certain proteins related to mitochondrial dynamics (OPA1 and DRP1) and autophagy (ATG5) did not overall show significant dose-dependent change in cells exposed to CSC. More importantly, conversion of LC3-I to LC3B-II, a representative marker for autophagy, was also unclear. Considering that intracellular organelles work together in harmony to perform defense mechanism against foreign bodies, we investigated changes in immune response following CSC exposure. The level of IFN-γ and MIP-1α was elevated in CSC-exposed cells, whereas the MCP-1α level decreased. The expression of chemokine receptors (CD195 and CXCR2) and an adhesion molecule (CD54) increased by CSC treatment, the expression of certain antigen presentation-related proteins (MHC class II, CD40, and CD80) were also enhanced. Meanwhile, the expression of CD86, a co-stimulatory molecule for antigen presentation, dose-dependently decreased. In conclusion, we suggest that CSC may induce apoptotic cell death and disturbance in host defense mechanisms by impairing function of cellular components.

Assessment of cytotoxicity and mutagenicity of exfoliated graphene.

Graphene and related materials (GRMs) have unique optical and thermal characteristics and are expected to be adopted for industrial applications. However, there are concerns with respect to their safety to human health. To conduct cytotoxicity and mutagenicity assessments, exfoliated graphene (EGr) dispersed in Tween-20® was diluted in cell culture medium. Rat alveolar macrophage viability significantly decreased after 24 h exposure to 1 and 10 µg/mL EGr. No significant levels of intracellular reactive oxygen species were detected in the 2',7'-dichlorodihydrofluorescin diacetate assay after 24 h of exposure to EGr. The levels of the pro-inflammatory cytokines macrophage inflammatory protein-1α, interleukin (IL)-1ß, IL-18, macrophage chemoattractant protein-1, and tumor necrosis factor α were significantly higher in cells treated with 10 µg/mL EGr for 24 h than in untreated controls. Transmission electron microscopy confirmed that EGr was present in the cytoplasm of the cells. Many genes were upregulated by EGr treatment, and significantly overrepresented gene ontology categories included the biological processes "response to external stimulus", "response to stress", "cell-cell signaling", "biological adhesion", and "cell proliferation". EGr did not induce genetic mutations in E. coli or cause micronucleus induction in mouse bone marrow cells. The results suggest that EGr cytotoxicity should be carefully considered.

Multi-cellular human bronchial models exposed to diesel exhaust particles: assessment of inflammation, oxidative stress and macrophage polarization.

BACKGROUND: Diesel exhaust particles (DEP) are a major component of outdoor air pollution. DEP mediated pulmonary effects are plausibly linked to inflammatory and oxidative stress response in which macrophages (MQ), epithelial cells and their cell-cell interaction plays a crucial role. Therefore, in this study we aimed at studying the cellular crosstalk between airway epithelial cells with MQ and MQ polarization following exposure to aerosolized DEP by assessing inflammation, oxidative stress, and MQ polarization response markers. METHOD: Lung mucosa models including primary bronchial epithelial cells (PBEC) cultured at air-liquid interface (ALI) were co-cultured without (PBEC-ALI) and with MQ (PBEC-ALI/MQ). Cells were exposed to 12.7 µg/cm2 aerosolized DEP using XposeALI®. Control (sham) models were exposed to clean air. Cell viability was assessed. CXCL8 and IL-6 were measured in the basal medium by ELISA. The mRNA expression of inflammatory markers (CXCL8, IL6, TNFα), oxidative stress (NFKB, HMOX1, GPx) and MQ polarization markers (IL10, IL4, IL13, MRC1, MRC2 RETNLA, IL12 andIL23) were measured by qRT-PCR. The surface/mRNA expression of TLR2/TLR4 was detected by FACS and qRT-PCR. RESULTS: In PBEC-ALI exposure to DEP significantly increased the secretion of CXCL8, mRNA expression of inflammatory markers (CXCL8, TNFα) and oxidative stress markers (NFKB, HMOX1, GPx). However, mRNA expressions of these markers (CXCL8, IL6, NFKB, and HMOX1) were reduced in PBEC-ALI/MQ models after DEP exposure. TLR2 and TLR4 mRNA expression increased after DEP exposure in PBEC-ALI. The surface expression of TLR2 and TLR4 on PBEC was significantly reduced in sham-exposed PBEC-ALI/MQ compared to PBEC-ALI. After DEP exposure surface expression of TLR2 was increased on PBEC of PBEC-ALI/MQ, while TLR4 was decreased in both models. DEP exposure resulted in similar expression pattern of TLR2/TLR4 on MQ as in PBEC. In PBEC-ALI/MQ, DEP exposure increased the mRNA expression of anti-inflammatory M2 macrophage markers (IL10, IL4, IL13, MRC1, MRC2). CONCLUSION: The cellular interaction of PBEC with MQ in response to DEP plays a pivotal role for MQ phenotypic alteration towards M2-subtypes, thereby promoting an efficient resolution of the inflammation. Furthermore, this study highlighted the fact that cell-cell interaction using multicellular ALI-models combined with an in vivo-like inhalation exposure system is critical in better mimicking the airway physiology compared with traditional cell culture systems.

Ethyl rosmarinate inhibits lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in alveolar macrophages.

In this study, a series of rosmarinic acid and analogs were investigated for their anti-inflammatory potential against LPS-induced alveolar macrophages (MH-S). Our results showed that, among the test compounds, ethyl rosmarinate (3) exhibited the most potent inhibitory effect on NO production in LPS-induced MH-S cells, with low cytotoxicity. Compound 3 exhibited remarkable inhibition of the production of PGE2 in LPS-induced MH-S cells. The inhibitory potency of compound 3 against LPS-induced NO and PGE2 release was approximately two-fold higher than that of dexamethasone. Compound 3 significantly decreased the mRNA and protein expression of iNOS and COX-2 and suppressed p65 expression in the nucleus in LPS-induced MH-S cells. These results suggested that compound 3 inhibited NO and PGE2 production, at least in part, through the down-regulation of NF-κB activation. Analysis of structure-activity relationship revealed that the free carboxylic group did not contribute to inhibitory activity and that the alkyl group of the corresponding alkyl ester analogs produced a strong inhibitory effect. We concluded that compound 3, a structurally modified rosmarinic acid, possessed potent inhibitory activity against lung inflammation, which strongly supported the development of this compound as a novel therapeutic agent for the treatment of macrophage-mediated lung inflammatory diseases, such as COPD.

Physicochemical characterization of spray-dried PLGA/PEG microspheres, and preliminary assessment of biological response.

CONTEXT: The use of spray-drying to prepare blended PLGA:PEG microspheres with lower immune detection. OBJECTIVE: To study physical properties, polymer miscibility and alveolar macrophage response for blended PLGA:PEG microspheres prepared by a laboratory-scale spray-drying process. METHODS: Microspheres were prepared by spray-drying 0-20% w/w ratios of PLGA 65:35 and PEG 3350 in dichloromethane. Particle size and morphology was studied using scanning electron microscopy. Polymer miscibility and residual solvent levels evaluated by thermal analysis (differential scanning calorimetry - DSC and thermogravimetric analysis - TGA). Immunogenicity was assessed in vitro by response of rat alveolar macrophages (NR8383) by the MTT-based cell viability assay and reactive oxygen species (ROS) detection. RESULTS: The spray dried particles were spherical, with a size range of about 2-3 µm and a yield of 16-60%. Highest yield was obtained at 1% PEG concentration. Thermal analysis showed a melting peak at 59 °C (enthalpy: 170.61 J/g) and a degradation-onset of 180 °C for PEG 3350. PLGA 65:35 was amorphous, with a Tg of 43 °C. Blended PLGA:PEG microspheres showed a delayed degradation-onset of 280 °C, and PEG enthalpy-loss corresponding to 15% miscibility of PEG in PLGA. NR8383 viability studies and ROS detection upon exposure to these cells suggested that blended PLGA:PEG microspheres containing 1 and 5% PEG are optimal in controling cell proliferation and activation. CONCLUSION: This research establishes the feasibility of using a spray-drying process to prepare spherical particles (2-3 µm) of molecularly-blended PLGA 65:35 and PEG 3350. A PEG concentration of 1-5% was optimal to maximize process yield, with minimal potential for immune detection.

Proinflammatory Effects of Pyrogenic and Precipitated Amorphous Silica Nanoparticles in Innate Immunity Cells.

Amorphous silica nanoparticles (ASNP) can be synthetized via several processes, 2 of which are the thermal route (to yield pyrogenic silica) and the wet route from a solution containing silicate salts (to obtain precipitated, colloidal, mesoporous silica, or silica gel). Both methods of synthesis lead to ASNP that are applied as food additive (E551). Current food regulation does not require that production methods of additives are indicated on the product label, and, thus, the ASNP are listed without mentioning the production method. Recent results indicate, however, that pyrogenic ASNP are more cytotoxic than ASNP synthesized through the wet route. The present study was aimed at clarifying if 2 representative preparations of ASNP, NM-203 (pyrogenic) and NM-200 (precipitated), of comparable size, specific surface area, surface charge, and hydrodynamic radius in complete growth medium, had different effects on 2 murine macrophage cell lines (MH-S and RAW264.7 cells). Our results show that, when incubated in protein-rich fluids, NM-203 adsorbed on their surface more proteins than NM-200 and, once incubated with macrophages, elicited a greater oxidative stress, assessed from Hmox1 induction and ROS production. Flow cytometry and helium ion microscopy indicated that pyrogenic NM-203 interacted with macrophages more strongly than the precipitated NM-200 and triggered a more evident inflammatory response, evaluated with Nos2 induction, NO production and the secretion of TNF-α, IL-6 and IL-1ß. Moreover, both ASNP synergized macrophage activation by bacterial lipopolysaccharide (LPS), with a higher effect observed for NM-203. In conclusion, the results presented here demonstrate that, compared to precipitated, pyrogenic ASNP exhibit enhanced interaction with serum proteins and cell membrane, and cause a larger oxidative stress and stronger proinflammatory effects in macrophages. Therefore, these 2 nanomaterials should not be considered biologically equivalent.

An in vitro alveolar macrophage assay for the assessment of inflammatory cytokine expression induced by atmospheric particulate matter.

Exposures to air pollution in the form of particulate matter (PM) can result in excess production of reactive oxygen species (ROS) in the respiratory system, potentially causing both localized cellular injury and triggering a systemic inflammatory response. PM-induced inflammation in the lung is modulated in large part by alveolar macrophages and their biochemical signaling, including production of inflammatory cytokines, the primary mechanism via which inflammation is initiated and sustained. We developed a robust, relevant, and flexible method employing a rat alveolar macrophage cell line (NR8383) which can be applied to routine samples of PM from air quality monitoring sites to gain insight into the drivers of PM toxicity that lead to oxidative stress and inflammation. Method performance was characterized using extracts of ambient and vehicular engine exhaust PM samples. Our results indicate that the reproducibility and the sensitivity of the method are satisfactory and comparisons between PM samples can be made with good precision. The average relative percent difference for all genes detected during 10 different exposures was 17.1%. Our analysis demonstrated that 71% of genes had an average signal to noise ratio (SNR) ≥ 3. Our time course study suggests that 4 h may be an optimal in vitro exposure time for observing short-term effects of PM and capturing the initial steps of inflammatory signaling. The 4 h exposure resulted in the detection of 57 genes (out of 84 total), of which 86% had altered expression. Similarities and conserved gene signaling regulation among the PM samples were demonstrated through hierarchical clustering and other analyses. Overlying the core congruent patterns were differentially regulated genes that resulted in distinct sample-specific gene expression "fingerprints." Consistent upregulation of Il1f5 and downregulation of Ccr7 was observed across all samples, while TNFα was upregulated in half of the samples and downregulated in the other half. Overall, this PM-induced cytokine expression assay could be effectively integrated into health studies and air quality monitoring programs to better understand relationships between specific PM components, oxidative stress activity and inflammatory signaling potential.

Inhibition of airway inflammation by the roots of Angelica decursiva and its constituent, columbianadin.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots of Angelica decursiva Fr. Et Sav (Umbelliferae) have been frequently used in traditional medicine as anti-inflammatory, antitussive, analgesic agents and expectorant, especially for treating cough, asthma, bronchitis and upper respiratory tract infections. To establish the scientific rationale for the clinical use of Angelica decursiva and to identify new agents for treating inflammatory lung disorders, pharmacological evaluation of the roots of Angelica decursiva and the isolated constituents was performed. METHODS: In vitro study was carried out using two lung cells, lung epithelial cells (A549) and alveolar macrophages (MH-S). The inflammatory markers such as IL-6 and nitric oxide (NO) for each cell line were examined. For in vivo study, a mouse model of lipopolysaccharide (LPS)-induced acute lung injury was used and the effects on lung inflammation were established by measuring the cell numbers in bronchoalveolar lavage fluid (BALF) and by histological observation. RESULTS: Water and 70% ethanol extracts of the roots of Angelica decursiva showed considerable inhibitory activity against LPS-induced lung inflammation in mice following oral administration at a dose of 400 mg/kg. Five coumarin derivatives including columbianadin, umbelliferone, umbelliferone 6-carboxylic acid, nodakenin and nodakenetin were isolated. Among the isolated compounds, columbianadin was found to possess strong inhibitory activity against the inflammatory response of IL-1ß-treated A549 cells and LPS-treated MH-S cells. Columbianadin was found to inhibit NO production by down-regulation of inducible NO synthase. Moreover, columbianadin was also proved to possess significant inhibitory activity against LPS-induced lung inflammation following oral administration at a dose of 20-60 mg/kg. CONCLUSIONS: The roots of Angelica decursiva were proved to be effective in the treatment of lung inflammation. Columbianadin can be a potential new agent for treating inflammatory lung disorders.

Magnetite- and maghemite-induced different toxicity in murine alveolar macrophage cells.

The unique properties of nanoparticles and biological systems are important factors affecting the biological response following nanoparticle exposure. Iron oxide nanoparticles are classified mainly as magnetite (M-FeNPs) and maghemite (NM-FeNPs). In our previous study, NM-FeNPs induced autophagic cell death in RAW264.7, a murine peritoneal macrophage cell line, which has excellent lysosomal activity. In this study, we compared the toxicity of M-FeNPs and NM-FeNPs in MH-S, a murine alveolar macrophage cell line, which has relatively low lysosomal activity. At 24 h post-exposure, M-FeNPs decreased cell viability and ATP production, and elevated the levels of reactive oxygen species, nitric oxide, and pro-inflammatory cytokines to a higher extent than NM-FeNPs. Damage of mitochondria and the endoplasmic reticulum and the down-regulation of mitochondrial function and transcription-related genes were also higher in cells exposed to M-FeNPs than in cells exposed to NM-FeNPs (50 µg/ml). In addition, cells exposed to M-FeNPs (50 µg/ml) showed an increase in the number of autophagosome-like vacuoles, whereas cells exposed to NM-FeNPs formed large vacuoles in the cytosol. However, an autophagy-related molecular response was not induced by exposure to either FeNPs, unlike the results seen in our previous study with RAW264.7 cells. We suggest that M-FeNPs induced higher toxicity compared to NM-FeNPs in MH-S cells, and lysosomal activity plays an important role in determining cell death pathway.

Glutathione attenuates ethanol-induced alveolar macrophage oxidative stress and dysfunction by downregulating NADPH oxidases.

Chronic alcohol abuse increases lung oxidative stress and susceptibility to respiratory infections by impairing alveolar macrophage (AM) function. NADPH oxidases (Nox) are major sources of reactive oxygen species in AMs. We hypothesized that treatment with the critical antioxidant glutathione (GSH) attenuates chronic alcohol-induced oxidative stress by downregulating Noxes and restores AM phagocytic function. Bronchoalveolar lavage (BAL) fluid and AMs were isolated from male C57BL/6J mice (8-10 wk) treated ± ethanol in drinking water (20% wt/vol, 12 wk) ± orally gavaged GSH in methylcellulose vehicle (300 mg x kg(-1) x day(-1), during week 12). MH-S cells, a mouse AM cell line, were treated ± ethanol (0.08%, 3 days) ± GSH (500 µM, 3 days or last 1 day of ethanol). BAL and AMs were also isolated from ethanol-fed and control mice ± inoculated airway Klebsiella pneumoniae (200 colony-forming units, 28 h) ± orally gavaged GSH (300 mg/kg, 24 h). GSH levels (HPLC), Nox mRNA (quantitative RT-PCR) and protein levels (Western blot and immunostaining), oxidative stress (2',7'-dichlorofluorescein-diacetate and Amplex Red), and phagocytosis (Staphylococcus aureus internalization) were measured. Chronic alcohol decreased GSH levels, increased Nox expression and activity, enhanced oxidative stress, impaired phagocytic function in AMs in vivo and in vitro, and exacerbated K. pneumonia-induced oxidative stress. Although how oral GSH restored GSH pools in ethanol-fed mice is unknown, oral GSH treatments abrogated the detrimental effects of chronic alcohol exposure and improved AM function. These studies provide GSH as a novel therapeutic approach for attenuating alcohol-induced derangements in AM Nox expression, oxidative stress, dysfunction, and risk for pneumonia.

Reduced in vitro toxicity of fine particulate matter collected during the 2008 Summer Olympic Games in Beijing: the roles of chemical and biological components.

Beijing has implemented systematic air pollution control legislation to reduce particulate emissions and improve air quality during the 2008 Summer Olympics, but whether the toxicity of fine fraction of particles (PM(2.5)) would be changed remains unclear. In present study we compared in vitro biological responses of PM(2.5) collected before and during the Olympics and tried to reveal possible correlations between its chemical components and toxicological mechanism(s). We measured cytotoxicity, cytokines/chemokines, and related gene expressions in murine alveolar macrophages, MH-S, after treated with 20 PM(2.5) samples. Significant, dose-dependent effects on cell viability, cytokine/chemokine release and mRNA expressions were observed. The cytotoxicity caused at equal mass concentration of PM(2.5) was notably reduced (p<0.05) by control measures, and significant association was found for viability and elemental zinc in PM(2.5). Endotoxin content in PM(2.5) correlated with all of the eight detected cytokines/chemokines; elemental and organic carbon correlated with four; arsenic and chromium correlated with six and three, respectively; iron and barium showed associations with two; nickel, magnesium, potassium, and calcium showed associations with one. PM(2.5) toxicity in Beijing was substantially dependent on its chemical components, and lowering the levels of specific components in PM(2.5) during the 2008 Olympics resulted in reduced biological responses.

Lectins offer new perspectives in the development of macrophage-targeted therapies for COPD/emphysema.

We have previously shown that the defective ability of alveolar macrophages (AM) to phagocytose apoptotic cells ('efferocytosis') in chronic obstructive pulmonary disease/emphysema (COPD) could be therapeutically improved using the C-type lectin, mannose binding lectin (MBL), although the exact mechanisms underlying this effect are unknown. An S-type lectin, galectin-3, is also known to regulate macrophage phenotype and function, via interaction with its receptor CD98. We hypothesized that defective expression of galectin/CD98 would be associated with defective efferocytosis in COPD and that mechanisms would include effects on cytoskeletal remodeling and macrophage phenotype and glutathione (GSH) availability. Galectin-3 was measured by ELISA in BAL from controls, smokers and current/ex-smokers with COPD. CD98 was measured on AM using flow cytometry. We assessed the effects of galectin-3 on efferocytosis, CD98, GSH, actin polymerisation, rac activation, and the involvement of PI3K (using ß-actin probing and wortmannin inhibition) in vitro using human AM and/or MH-S macrophage cell line. Significant decreases in BAL galectin-3 and AM CD98 were observed in BAL from both current- and ex-smoker COPD subjects vs controls. Galectin 3 increased efferocytosis via an increase in active GTP bound Rac1. This was confirmed with ß-actin probing and the role of PI3K was confirmed using wortmannin inhibition. The increased efferocytosis was associated with increases in available glutathione and expression of CD98. We provide evidence for a role of airway lectins in the failed efferocytosis in COPD, supporting their further investigation as potential macrophage-targeted therapies.

Nanostructured calcium silicate hydrate seeds accelerate concrete hardening: a combined assessment of benefits and risks.

Nanotechnology creates new possibilities to control and improve material properties for civil infrastructure. Special focus in this area is put on Portland cement and gypsum. Together their annual production is by far larger than for any other material worldwide. Nanomodification of these materials can be done during the few hours between dissolution and hardening, especially by nucleation of the re-crystallization with suitable colloids. Here we report first results in homogeneous seeding of the precipitation of calcium silicate hydrates within a real Portland cement composition. The occupational safety during the production phase and during mixing of concrete paste is addressed in detail by in vivo testing. We perform 5-day inhalation with 21-day recovery in rats and analyze organ-specific toxicity and 71 endpoints from bronchoalveolar lavage (BALF) and blood. In BALF parameters, no test-related changes were observed, indicating the generally low toxicity of the test material. Some mild lesions were observed in larynx level. In the lungs, all animals of the 50 mg/m³ concentration group revealed a minimal to mild increase in alveolar macrophages, which recovered back to control level.