Cytomegalovirus subverts macrophage identity.

Cytomegaloviruses (CMVs) have co-evolved with their mammalian hosts for millions of years, leading to remarkable host specificity and high infection prevalence. Macrophages, which already populate barrier tissues in the embryo, are the predominant immune cells at potential CMV entry sites. Here we show that, upon CMV infection, macrophages undergo a morphological, immunophenotypic, and metabolic transformation process with features of stemness, altered migration, enhanced invasiveness, and provision of the cell cycle machinery for viral proliferation. This complex process depends on Wnt signaling and the transcription factor ZEB1. In pulmonary infection, mouse CMV primarily targets and reprograms alveolar macrophages, which alters lung physiology and facilitates primary CMV and secondary bacterial infection by attenuating the inflammatory response. Thus, CMV profoundly perturbs macrophage identity beyond established limits of plasticity and rewires specific differentiation processes, allowing viral spread and impairing innate tissue immunity.

Novel insight from the first lung transplant of a COVID-19 patient.

BACKGROUND: To reveal detailed histopathological changes, virus distributions, immunologic properties and multi-omic features caused by SARS-CoV-2 in the explanted lungs from the world's first successful lung transplantation of a COVID-19 patient. MATERIALS AND METHODS: A total of 36 samples were collected from the lungs. Histopathological features and virus distribution were observed by optical microscope and transmission electron microscope (TEM). Immune cells were detected by flow cytometry and immunohistochemistry. Transcriptome and proteome approaches were used to investigate main biological processes involved in COVID-19-associated pulmonary fibrosis. RESULTS: The histopathological changes of the lung tissues were characterized by extensive pulmonary interstitial fibrosis and haemorrhage. Viral particles were observed in the cytoplasm of macrophages. CD3+ CD4- T cells, neutrophils, NK cells, γ/δ T cells and monocytes, but not B cells, were abundant in the lungs. Higher levels of proinflammatory cytokines iNOS, IL-1ß and IL-6 were in the area of mild fibrosis. Multi-omics analyses revealed a total of 126 out of 20,356 significant different transcription and 114 out of 8,493 protein expression in lung samples with mild and severe fibrosis, most of which were related to fibrosis and inflammation. CONCLUSIONS: Our results provide novel insight that the significant neutrophil/ CD3+ CD4- T cell/ macrophage activation leads to cytokine storm and severe fibrosis in the lungs of COVID-19 patient and may contribute to a better understanding of COVID-19 pathogenesis.

Cytotoxicity profiles of multi-walled carbon nanotubes with different physico-chemical properties.

Multi-walled carbon nanotubes (MWCNTs) have industrial applications in the nanotechnology field. The physico-chemical properties of MWCNTs vary greatly depending on MWCNT manufacture and application. It has been pointed out that their needle shape and high durability are important factors that determine the biopersistence of fibers and can lead to inhalation toxicity or cytotoxicity. In this study, we prepared six suspensions of MWCNTs differing in diameter and length, and performed in vitro cell-based assays for 24 h using NR8383 rat alveolar macrophages. Rigid, needle-shaped MWCNTs with a large diameter (>50 µm) penetrated the cytoplasm and decreased cell survival without generating intracellular reactive oxygen species (ROS), significantly up-regulated many genes involved in inflammatory responses, response to oxidative stress and apoptosis, and extracellular matrix degradation. Bent MWCNTs with a small diameter (<20 µm) were phagocytosed in vacuole-like cellular compartments and decreased cell survival along with intracellular ROS generation. Straight, thin MWCNTs with a small diameter (<20 µm) caused a slight intracellular ROS generation but no decrease in cell viability. Some straight, long, and thin MWCNTs were found in the mitochondria and near the nuclei; however, no mutagenesis was observed. The in vitro cell-based assays showed high cytotoxicity of MWCNTs with a large diameter (>50 µm), moderate and low cytotoxicity of MWCNTs with a small diameter (<20 µm). These results suggested that the diameter of MWCNTs considerably contributes to their cytotoxicity.

TNF-α-TNFR signal pathway inhibits autophagy and promotes apoptosis of alveolar macrophages in coal worker's pneumoconiosis.

OBJECTIVE: Exposure to coal dust causes the development of coal worker's pneumoconiosis (CWP), which is associated with accumulating macrophages in the lower respiratory tract. This study was performed to investigate the effect of tumor necrosis factor-α (TNF-α)-tumor necrosis factor receptor (TNFR) signal pathway on autophagy and apoptosis of alveolar macrophages (AMs) in CWP. METHODS: AMs from controls exposed to coal dust and CWP patients were collected, in which expressions of TNF-α and TNFR1 were determined. Autophagy was observed by transmission electron microscopy, and apoptosis by light microscope and using terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. AMs in CWP patients were treated with TNF-α or anti-TNF-α antibody. Besides, expressions of autophagy marker proteins, apoptosis-related factors, FAS, caspase-8, and receptor-interacting serine-threonine-protein kinase 3 (RIPK3) were determined by western Blot. Activities of caspase-3 and caspase-8 were determined by a fluorescence kit. Flow cytometry was applied to measure the expression of TNFR1 on the surface of the AM. RESULTS: TNF-α expression and TNFR1 expression on the surface of AM, as well as autophagy and apoptotic index were significantly increased in AMs of CWP patients. In response to the treatment of TNF-α, TNF-α expression and TNFR1 expression on the surface of AM as well as LC3I expression were increased, autophagy was decreased, and LC3, LC3II, Beclin1 and B-cell lymphoma 2 expressions decreased, whereas FAS expression and activity and expression of caspase-3 and caspase-8 increased, and apoptotic index increased. Moreover, the situations were reversed with the treatment of anti-TNF-α antibody. CONCLUSION: TNF-α-TNFR signal pathway was involved in the occurrence and development of CWP by activating FAS-caspase-8 and thus inhibiting autophagy while promoting apoptosis of AM.

Bismuth Ferrite Second Harmonic Nanoparticles for Pulmonary Macrophage Tracking.

Recently, second harmonic generation (SHG) nanomaterials have been generated that are efficiently employed in the classical (NIR) and extended (NIR-II) near infrared windows using a multiphoton microscope. The aim was to test bismuth ferrite harmonic nanoparticles (BFO-HNPs) for their ability to monitor pulmonary macrophages in mice. BFO-loaded MH-S macrophages are given intratracheally to healthy mice or BFO-HNPs are intranasally instilled in mice with allergic airway inflammation and lung sections of up to 100 µM are prepared. Using a two-photon-laser scanning microscope, it is shown that bright BFO-HNPs signals are detected from superficially localized cells as well as from deep within the lung tissue. BFO-HNPs are identified with an excellent signal-to-noise ratio and virtually no background signal. The SHG from the nanocrystals can be distinguished from the endogenous collagen-derived SHG around the blood vessels and bronchial structures. BFO-HNPs are primarily taken up by M2 alveolar macrophages in vivo. This SHG imaging approach provides novel information about the interaction of macrophages with cells and the extracellular matrix in lung disease as it is capable of visualizing and tracking NP-loaded cells at high resolution in thick tissues with minimal background fluorescence.

Pulmonary and pleural toxicity of potassium octatitanate fibers, rutile titanium dioxide nanoparticles, and MWCNT-7 in male Fischer 344 rats.

Potassium octatitanate (K2O·8TiO2, POT) fibers are used as an alternative to asbestos. Their shape and biopersistence suggest that they are possibly carcinogenic. However, inhalation studies have shown that respired POT fibers have little carcinogenic potential. We conducted a short-term study in which we administered POT fibers, and anatase and rutile titanium dioxide nanoparticles (a-nTiO2, r-nTiO2) to rats using intra-tracheal intra-pulmonary spraying (TIPS). We found that similarly to other materials, POT fibers were more toxic than non-fibrous nanoparticles of the same chemical composition, indicating that the titanium dioxide composition of POT fibers does not appear to account for their lack of carcinogenicity. The present report describes the results of the 3-week and 52-week interim killing of our current 2-year study of POT fibers, with MWCNT-7 as a positive control and r-nTiO2 as a non-fibrous titanium dioxide control. Male F344 rats were administered 0.5 ml vehicle, 62.5 µg/ml and 125 µg/ml r-nTiO2 and POT fibers, and 125 µg/ml MWCNT-7 by TIPS every other day for 2 weeks (eight doses: total doses of 0.25 and 0.50 mg/rat). At 1 year, POT and MWCNT-7 fibers induced significant increases in alveolar macrophage number, granulation tissue in the lung, bronchiolo-alveolar cell hyperplasia and thickening of the alveolar wall, and pulmonary 8-OHdG levels. The 0.5 mg POT- and the MWCNT-7-treated groups also had increased visceral and parietal pleura thickness, increased mesothelial cell PCNA labeling indices, and a few areas of visceral mesothelial cell hyperplasia. In contrast, in the r-nTiO2-treated groups, none of the measured parameters were different from the controls.

Characterisation of a New Human Alveolar Macrophage-Like Cell Line (Daisy).

PURPOSE: There is currently no true macrophage cell line and in vitro experiments requiring these cells currently require mitogenic stimulation of a macrophage precursor cell line (THP-1) or ex vivo maturation of circulating primary monocytes. In this study, we characterise a human macrophage cell line, derived from THP-1 cells, and compare its phenotype to the THP-1 cells. METHODS: THP-1 cells with and without mitogenic stimulation were compared to the newly derived macrophage-like cell line (Daisy) using microscopy, flow cytometry, phagocytosis assays, antigen binding assays and gene microarrays. RESULTS: We show that the cell line grows predominantly in an adherent monolayer. A panel of antibodies were chosen to investigate the cell surface phenotype of these cells using flow cytometry. Daisy cells expressed more CD11c, CD80, CD163, CD169 and CD206, but less CD14 and CD11b compared with mitogen-stimulated THP-1 cells. Unlike stimulated THP-1 cells which were barely able to bind immune complexes, Daisy cells showed large amounts of immune complex binding. Finally, although not statistically significant, the phagocytic ability of Daisy cells was greater than mitogen-stimulated THP-1 cells, suggesting that the cell line is more similar to mature macrophages. CONCLUSIONS: The observed phenotype suggests that Daisy cells are a good model of human macrophages with a phenotype similar to human alveolar macrophages.

Graphene quantum dots in alveolar macrophage: uptake-exocytosis, accumulation in nuclei, nuclear responses and DNA cleavage.

BACKGROUND: Given the tremendous potential for graphene quantum dots (QDs) in biomedical applications, a thorough understanding of the interaction of these materials with macrophages is essential because macrophages are one of the most important barriers against exogenous particles. Although the cytotoxicity and cellular uptake of graphene QDs were reported in previous studies, the interaction between nuclei and the internalized graphene QDs is not well understood. We thus systematically studied the nuclear uptake and related nuclear response associated with aminated graphene QDs (AG-QDs) exposure. RESULTS: AG-QDs showed modest 24-h inhibition to rat alveolar macrophages (NR8383), with a minimum inhibitory concentration (MIC) of 200 µg/mL. Early apoptosis was significantly increased by AG-QDs (100 and 200 µg/mL) exposure and played a major role in cell death. The internalization of AG-QDs was mainly via energy-dependent endocytosis, phagocytosis and caveolae-mediated endocytosis. After a 48-h clearance period, more than half of the internalized AG-QDs remained in the cellular cytoplasm and nucleus. Moreover, AG-QDs were effectively accumulated in nucleus and were likely regulated by two nuclear pore complexes genes (Kapß2 and Nup98). AG-QDs were shown to alter the morphology, area, viability and nuclear components of exposed cells. Significant cleavage and cross-linking of DNA chains after AG-QDs exposure were confirmed by atomic force microscopy investigation. Molecular docking simulations showed that H-bonding and π-π stacking were the dominant forces mediating the interactions between AG-QDs and DNA, and were the important mechanisms resulting in DNA chain cleavage. In addition, the generation of reactive oxygen species (ROS) (e.g., •OH), and the up-regulation of caspase genes also contributed to DNA cleavage. CONCLUSIONS: AG-QDs were internalized by macrophages and accumulated in nuclei, which further resulted in nuclear damage and DNA cleavage. It is demonstrated that oxidative damage, direct contact via H-bonding and π-π stacking, and the up-regulation of caspase genes are the primary mechanisms for the observed DNA cleavage by AG-QDs.

Morphometric Characterization of Rat and Human Alveolar Macrophage Cell Models and their Response to Amiodarone using High Content Image Analysis.

PURPOSE: Progress to the clinic may be delayed or prevented when vacuolated or "foamy" alveolar macrophages are observed during non-clinical inhalation toxicology assessment. The first step in developing methods to study this response in vitro is to characterize macrophage cell lines and their response to drug exposures. METHODS: Human (U937) and rat (NR8383) cell lines and primary rat alveolar macrophages obtained by bronchoalveolar lavage were characterized using high content fluorescence imaging analysis quantification of cell viability, morphometry, and phospholipid and neutral lipid accumulation. RESULTS: Cell health, morphology and lipid content were comparable (p < 0.05) for both cell lines and the primary macrophages in terms of vacuole number, size and lipid content. Responses to amiodarone, a known inducer of phospholipidosis, required analysis of shifts in cell population profiles (the proportion of cells with elevated vacuolation or lipid content) rather than average population data which was insensitive to the changes observed. CONCLUSIONS: A high content image analysis assay was developed and used to provide detailed morphological characterization of rat and human alveolar-like macrophages and their response to a phospholipidosis-inducing agent. This provides a basis for development of assays to predict or understand macrophage vacuolation following inhaled drug exposure.

Structure of Rat Lungs after Administration of Magnetomicelles Based on the Carbon-Coated Iron Nanoparticles.

We studied the effects of single administration of a suspension of magnetomicelles based on carbon-coated iron nanoparticles on the structure of rat lungs within 40 days. Histological analysis revealed a complex of hemodynamic alterations in the lungs. Described changes persisted in the lung stroma from day 1 until day 40, but their intensity decreased by the end of the experiment. Using immunohistochemical Perls reaction we identified cells morphologically corresponding to alveolar and interstitial lung macrophages. The number of Perls+ cells decreased by day 40 of the experiment. Ultrastructural analysis showed endocytosis of modified iron nanoparticles and their accumulation in intracellular digestionorganelles (endo- and lysosomes) of mononuclear phagocyte system cells. Accumulation of magnetomicelles in the lungs was not associated with damage to pneumocytes, macrophages, and blood-air barrier.

The sst1 resistance locus regulates evasion of type I interferon signaling by Chlamydia pneumoniae as a disease tolerance mechanism.

The sst1, "supersusceptibility to tuberculosis," locus has previously been shown to be a genetic determinant of host resistance to infection with the intracellular pathogen, Mycobacterium tuberculosis. Chlamydia pneumoniae is an obligate intracellular bacterium associated with community acquired pneumonia, and chronic infection with C. pneumoniae has been linked to asthma and atherosclerosis. C. pneumoniae is a highly adapted pathogen that can productively infect macrophages and inhibit host cell apoptosis. Here we examined the role of sst1 in regulating the host response to infection with C. pneumoniae. Although mice carrying the sst1 susceptible (sst1(S) ) locus were not impaired in their ability to clear the acute infection, they were dramatically less tolerant of the induced immune response, displaying higher clinical scores, more severe lung inflammation, exaggerated macrophage and neutrophil influx, and the development of fibrosis compared to wild type mice. This correlated with increased activated caspase-3 in the lungs of infected sst1(S) mice. Infection of sst1(S) macrophages with C. pneumoniae resulted in a shift in the secreted cytokine profile towards enhanced production of interferon-ß and interleukin-10, and induced apoptotic cell death, which was dependent on secretion of interferon-ß. Intriguingly macrophages from the sst1(S) mice failed to support normal chlamydial growth, resulting in arrested development and failure of the organism to complete its infectious cycle. We conclude that the sst1 locus regulates a shared macrophage-mediated innate defense mechanism against diverse intracellular bacterial pathogens. Its susceptibility allele leads to upregulation of type I interferon pathway, which, in the context of C. pneumoniae, results in decreased tolerance, but not resistance, to the infection. Further dissection of the relationship between type I interferons and host tolerance during infection with intracellular pathogens may provide identification of biomarkers and novel therapeutic targets.

Differential bioreactivity of neutral, cationic and anionic polystyrene nanoparticles with cells from the human alveolar compartment: robust response of alveolar type 1 epithelial cells.

BACKGROUND: Engineered nanoparticles (NP) are being developed for inhaled drug delivery. This route is non-invasive and the major target; alveolar epithelium provides a large surface area for drug administration and absorption, without first pass metabolism. Understanding the interaction between NPs and target cells is crucial for safe and effective NP-based drug delivery. We explored the differential effect of neutral, cationic and anionic polystyrene latex NPs on the target cells of the human alveolus, using primary human alveolar macrophages (MAC) and primary human alveolar type 2 (AT2) epithelial cells and a unique human alveolar epithelial type I-like cell (TT1). We hypothesized that the bioreactivity of the NPs would relate to their surface chemistry, charge and size as well as the functional role of their interacting cells in vivo. METHODS: Amine- (ANP) and carboxyl- surface modified (CNP) and unmodified (UNP) polystyrene NPs, 50 and 100 nm in diameter, were studied. Cells were exposed to 1-100 µg/ml (1.25-125 µg/cm(2); 0 µg/ml control) NP for 4 and 24 h at 37 °C with or without the antioxidant, N-acetyl cysteine (NAC). Cells were assessed for cell viability, reactive oxygen species (ROS), oxidised glutathione (GSSG/GSH ratio), mitochondrial integrity, cell morphology and particle uptake (using electron microscopy and laser scanning confocal microscopy). RESULTS: ANP-induced cell death occurred in all cell types, inducing increased oxidative stress, mitochondrial disruption and release of cytochrome C, indicating apoptotic cell death. UNP and CNP exhibited little cytotoxicity or mitochondrial damage, although they induced ROS in AT2 and MACs. Addition of NAC reduced epithelial cell ROS, but not MAC ROS, for up to 4 h. TT1 and MAC cells internalised all NP formats, whereas only a small fraction of AT2 cells internalized ANP (not UNP or CNP). TT1 cells were the most resistant to the effects of UNP and CNP. CONCLUSION: ANP induced marked oxidative damage and cell death via apoptosis in all cell types, while UNP and CNP exhibited low cytotoxicity via oxidative stress. MAC and TT1 cell models show strong particle-internalization compared to the AT2 cell model, reflecting their cell function in vivo. The 50 nm NPs induced a higher bioreactivity in epithelial cells, whereas the 100 nm NPs show a stronger effect on phagocytic cells.

Autophagy in pulmonary macrophages mediates lung inflammatory injury via NLRP3 inflammasome activation during mechanical ventilation.

The inflammatory response is a primary mechanism in the pathogenesis of ventilator-induced lung injury. Autophagy is an essential, homeostatic process by which cells break down their own components. We explored the role of autophagy in the mechanisms of mechanical ventilation-induced lung inflammatory injury. Mice were subjected to low (7 ml/kg) or high (28 ml/kg) tidal volume ventilation for 2 h. Bone marrow-derived macrophages transfected with a scrambled or autophagy-related protein 5 small interfering RNA were administered to alveolar macrophage-depleted mice via a jugular venous cannula 30 min before the start of the ventilation protocol. In some experiments, mice were ventilated in the absence and presence of autophagy inhibitors 3-methyladenine (15 mg/kg ip) or trichostatin A (1 mg/kg ip). Mechanical ventilation with a high tidal volume caused rapid (within minutes) activation of autophagy in the lung. Conventional transmission electron microscopic examination of lung sections showed that mechanical ventilation-induced autophagy activation mainly occurred in lung macrophages. Autophagy activation in the lungs during mechanical ventilation was dramatically attenuated in alveolar macrophage-depleted mice. Selective silencing of autophagy-related protein 5 in lung macrophages abolished mechanical ventilation-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and lung inflammatory injury. Pharmacological inhibition of autophagy also significantly attenuated the inflammatory responses caused by lung hyperinflation. The activation of autophagy in macrophages mediates early lung inflammation during mechanical ventilation via NLRP3 inflammasome signaling. Inhibition of autophagy activation in lung macrophages may therefore provide a novel and promising strategy for the prevention and treatment of ventilator-induced lung injury.

Autophagy plays an essential role in the clearance of Pseudomonas aeruginosa by alveolar macrophages.

Intracellular bacteria have been shown to cause autophagy, which impacts infectious outcomes, whereas extracellular bacteria have not been reported to activate autophagy. Here, we demonstrate that Pseudomonas aeruginosa, a Gram-negative extracellular bacterium, activates autophagy with considerably increased LC3 punctation in both an alveolar macrophage cell line (MH-S) and primary alveolar macrophages. Using the LC3 Gly120 mutant, we successfully demonstrated a hallmark of autophagy, conjugation of LC3 to phosphatidylethanolamine (PE). The accumulation of typical autophagosomes with double membranes was identified morphologically by transmission electron microscopy (TEM). Furthermore, the increase of PE-conjugated LC3 was indeed induced by infection rather than inhibition of lysosome degradation. P. aeruginosa induced autophagy through the classical beclin-1-Atg7-Atg5 pathway as determined by specific siRNA analysis. Rapamycin and IFN-γ (autophagy inducers) augmented bacterial clearance, whereas beclin-1 and Atg5 knockdown reduced intracellular bacteria. Thus, P. aeruginosa-induced autophagy represents a host protective mechanism, providing new insight into the pathogenesis of this infection.

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.

Foamy macrophage responses in the rat lung following exposure to inhaled pharmaceuticals: a simple, pragmatic approach for inhaled drug development.

Successes in the field of respiratory medicines are largely limited to three main target classes: ß2 -adrenergic receptor agonists, muscarinic antagonists and corticosteroids. A significant factor in attrition during the development of respiratory medicines is the induction of foamy macrophage responses, particularly, in rats. The term foamy macrophage describes a vacuolated cytoplasmic appearance, seen by light microscopy, which is ultrastructurally characterized by the presence of lysosomal lamellar bodies, neutral lipid droplets or drug particles. We propose a simple classification, based light-heartedly on the theme 'the good, the bad and the ugly', which allows important distinctions to be made between phenotypes, aetiologies and adversity. Foamy macrophages induced in rat lungs by exposure to inhaled ß2 -agonists, antimuscarinics and corticosteroids are simple aggregates of uniform cells without other associated pathologies. In contrast, macrophage reactions induced by some other inhaled drug classes are more complex, associated with neutrophilic or lymphocytic infiltrations with/without damage to the adjacent alveolar walls. Foamy macrophage responses induced by inhaled drugs may be ascribed to either phagocytosis of poorly soluble drug particles, or to pharmacology. Both corticosteroids and ß2 -agonists increase surfactant synthesis whereas muscarinic antagonists may decrease surfactant breakdown, due to inhibition of phospholipase C, both of which lead to phagocytosis of excess surfactant. Simple foamy macrophage responses are considered non-adverse, whereas ones that are more complex are designated as adverse. The development of foamy macrophage responses has led to confusion in interpretation and we hope this review helps clarify what is in fact a relatively simple, predictable, easily interpretable, commonly induced change.

NOD2 enhances the innate response of alveolar macrophages to Mycobacterium tuberculosis in humans.

A role for the nucleotide-binding oligomerization domain 2 (NOD2) receptor in pulmonary innate immune responses has recently been explored. In the present study, we investigated the role that NOD2 plays in human alveolar macrophage innate responses and determined its involvement in the response to infection with virulent Mycobacterium tuberculosis. Our results showed that NOD2 was expressed in human alveolar macrophages, and significant amounts of IL-1ß, IL-6, and TNF-α were produced upon ligand recognition with muramyldipeptide (MDP). NOD2 ligation induced the transcription and protein expression of the antimicrobial peptide LL37 and the autophagy enzyme IRGM in alveolar macrophages, demonstrating a novel function for this receptor in these cells. MDP treatment of alveolar macrophages improved the intracellular growth control of virulent M. tuberculosis; this was associated with a significant release of TNF-α and IL-6 and overexpression of bactericidal LL37. In addition, the autophagy proteins IRGM, LC3 and ATG16L1 were recruited to the bacteria-containing autophagosome after treatment with MDP. In conclusion, our results suggest that NOD2 can modulate the innate immune response of alveolar macrophages and play a role in the initial control of respiratory M. tuberculosis infections.

Immuno-phenotypic and functional characterization of rabbit pulmonary intravascular macrophages.

Pulmonary intravascular macrophages (PIMs) are present in species such as cattle, sheep and horse and promote acute lung inflammation (ALI). Rabbits are often used as a model of ALI but there is controversy about the presence of PIMs in these species. Rabbits were treated with 10 mg/kg of gadolinium chloride intravenously (GC; n = 6) or saline (n = 6) followed by euthanasia at 48 h post-treatment to determine the presence of PIMs. In a subsequent study, rabbits were pre-treated with GC or 0.9 % saline followed by 100 µg/kg of E. coli lipopolysaccharide intravenously 48 h later. Rabbits were euthanized 24 h post-LPS treatment. Light and electron microscopy showed that PIMs attached to the capillary endothelium and were positive for RAM-11 anti-macrophage antibody. While GC treatment induced apoptotic PIMs, there was no difference in the PIM number between control and GC-treated rabbits. Rabbits administered with LPS were 3.5 times more likely to die before the end of the 24-h period than those pre-treated with GC. Lung heterophil accumulation and IL-1ß, TNFα and IL-6 mRNA expression were significantly higher in rabbits administered with LPS compared to those administered with GC before the LPS injection. PIMs from the LPS-treated rabbits were positive for TNFα. Lung, BAL and serum IL-8 and MCP-1 expression was not different between LPS rabbits with or without pre-treatment with GC. We conclude that rabbit lungs contain PIMs and that their depletion reduces endotoxin-induced lung inflammation. The presence of PIMs in rabbit lungs may need to be considered while using rabbit to model acute lung injury.

Computerized image analysis of iron-stained macrophages.

Analysis of iron levels in single cells is critical to understand the consequences of impaired regulation of iron homeostasis. Here we establish a method to analyze intracellular iron deposits by computerized image analysis of Prussian blue-stained alveolar macrophages as a test system. We efficiently detected small differences in macrophage steady-state iron levels in Hfe (-/-) mice as well as inflammation-induced iron sequestration upon lipopolysaccharide instillation. In conclusion, computerized image analysis of single cells is a robust and reproducible tool suitable for iron measurements in small sample sets with limited cell yield.

Cellular uptake and localization of inhaled gold nanoparticles in lungs of mice with chronic obstructive pulmonary disease.

BACKGROUND: Inhalative nanocarriers for local or systemic therapy are promising. Gold nanoparticles (AuNP) have been widely considered as candidate material. Knowledge about their interaction with the lungs is required, foremost their uptake by surface macrophages and epithelial cells. METHODS: Scnn1b-Tg and Wt mice inhaled a 21-nm AuNP aerosol for 2 h. Immediately (0 h) or 24 h thereafter, bronchoalveolar lavage (BAL) macrophages and whole lungs were prepared for stereological analysis of AuNP by electron microscopy. RESULTS: AuNP were mainly found as singlets or small agglomerates of ≤ 100 nm diameter, at the epithelial surface and within lung-surface structures. Macrophages contained also large AuNP agglomerates (> 100 nm). At 0 h after aerosol inhalation, 69.2±4.9% AuNP were luminal, i.e. attached to the epithelial surface and 24.0±5.9% in macrophages in Scnn1b-Tg mice. In Wt mice, 35.3±32.2% AuNP were on the epithelium and 58.3±41.4% in macrophages. The percentage of luminal AuNP decreased from 0 h to 24 h in both groups. At 24 h, 15.5±4.8% AuNP were luminal, 21.4±14.2% within epithelial cells and 63.0±18.9% in macrophages in Scnn1b-Tg mice. In Wt mice, 9.5±5.0% AuNP were luminal, 2.2±1.6% within epithelial cells and 82.8±0.2% in macrophages. BAL-macrophage analysis revealed enhanced AuNP uptake in Wt animals at 0 h and in Scnn1b-Tg mice at 24 h, confirming less efficient macrophage uptake and delayed clearance of AuNP in Scnn1b-Tg mice. CONCLUSIONS: Inhaled AuNP rapidly bound to the alveolar epithelium in both Wt and Scnn1b-Tg mice. Scnn1b-Tg mice showed less efficient AuNP uptake by surface macrophages and concomitant higher particle internalization by alveolar type I epithelial cells compared to Wt mice. This likely promotes AuNP depth translocation in Scnn1b-Tg mice, including enhanced epithelial targeting. These results suggest AuNP nanocarrier delivery as successful strategy for therapeutic targeting of alveolar epithelial cells and macrophages in COPD.