Characterization of immortalized MARCO and SR-AI/II-deficient murine alveolar macrophage cell lines

<p>Abstract</p> <p>Background</p> <p>Alveolar macrophages (AM) avidly bind and ingest unopsonized inhaled particles and bacteria through class A scavenger receptors (SRAs) MARCO and SR-AI/II. Studies to characterize the function of these SRAs have used AMs from MARCO or SR-AI/II null mice, but this approach is limited by the relatively low yield of AMs. Moreover, studies using both MARCO and SR-AI/II-deficient (MS^-/-) mice have not been reported yet. Hence, we sought to develop continuous cell lines from primary alveolar macrophages from MS^-/-mice.</p> <p>Results</p> <p>We used <it>in vitro </it>infection of the primary AMs with the J2 retrovirus carrying the <it>v-raf </it>and <it>v-myc </it>oncogenes. Following initial isolation in media supplemented with murine macrophage colony-stimulating factor (M-CSF), we subcloned three AM cell lines, designated ZK-1, ZK-2 and ZK-6. These cell lines grow well in RPMI-1640-10% FBS in the absence of M-CSF. These adherent but trypsin-sensitive cell lines have a doubling time of approximately 14 hours, exhibit typical macrophage morphology, and express macrophage-associated cell surface Mac-1 (CD11b) and F4/80 antigens. The cell lines show robust Fc-receptor dependent phagocytosis of opsonized red blood cells. Similar to freshly isolated AMs from MS^-/-mice, the cell lines exhibit decreased phagocytosis of unopsonized titanium dioxide (TiO₂), fluorescent latex beads and bacteria (<it>Staphylococcus aureus</it>) compared with the primary AMs from wild type (WT) C57BL/6 mice.</p> <p>Conclusion</p> <p>Our results indicated that three contiguous murine alveolar macrophage cell lines with MS^-/-(ZK1, ZK2 and ZK6) were established successfully. These cell lines demonstrated macrophage morphology and functional activity. Interestingly, similar to freshly isolated AMs from MS^-/-mice, the cell lines have a reduced, but not absent, ability to bind and ingest particles, with an altered pattern of blockade by scavenger receptor inhibitors. These cell lines will facilitate <it>in vitro </it>studies to further define MARCO and SR-AI/II function, and may also be useful to identify other novel scavenger-type macrophage receptors and for additional studies of particle toxicology.</p

Link between Epigenomic Alterations and Genome-Wide Aberrant Transcriptional Response to Allergen in Dendritic Cells Conveying Maternal Asthma Risk

We investigated the link between epigenome-wide methylation aberrations at birth and genomic transcriptional changes upon allergen sensitization that occur in the neonatal dendritic cells (DC) due to maternal asthma. We previously demonstrated that neonates of asthmatic mothers are born with a functional skew in splenic DCs that can be seen even in allergen-naïve pups and can convey allergy responses to normal recipients. However, minimal-to-no transcriptional or phenotypic changes were found to explain this alteration. Here we provide in-depth analysis of genome-wide DNA methylation profiles and RNA transcriptional (microarray) profiles before and after allergen sensitization. We identified differentially methylated and differentially expressed loci and performed manually-curated matching of methylation status of the key regulatory sequences (promoters and CpG islands) to expression of their respective transcripts before and after sensitization. We found that while allergen-naive DCs from asthma-at-risk neonates have minimal transcriptional change compared to controls, the methylation changes are extensive. The substantial transcriptional change only becomes evident upon allergen sensitization, when it occurs in multiple genes with the pre-existing epigenetic alterations. We demonstrate that maternal asthma leads to both hyper- and hypomethylation in neonatal DCs, and that both types of events at various loci significantly overlap with transcriptional responses to allergen. Pathway analysis indicates that approximately 1/2 of differentially expressed and differentially methylated genes directly interact in known networks involved in allergy and asthma processes. We conclude that congenital epigenetic changes in DCs are strongly linked to altered transcriptional responses to allergen and to early-life asthma origin. The findings are consistent with the emerging paradigm that asthma is a disease with underlying epigenetic changes

Risk for Asthma in Offspring of Asthmatic Mothers versus Fathers: A Meta-Analysis

Many human epidemiologic studies demonstrate that maternal asthma confers greater risk of asthma to offspring than does paternal disease. However, a handful have shown the opposite. Given this disparity, a meta-analysis is necessary to determine the veracity and magnitude of the "maternal effect."We screened the medical literature from 1966 to 2009 and performed a meta-analysis to compare the effect of maternal asthma vs. paternal asthma on offspring asthma susceptibility. Aggregating data from 33 studies, the odds ratio for asthma in children of asthmatic mothers compared with non-asthmatic mothers was significantly increased at 3.04 (95% confidence interval: 2.59-3.56). The corresponding odds ratio for asthma in children of asthmatic fathers was increased at 2.44 (2.14-2.79). When comparing the odds ratios, maternal asthma conferred greater risk of disease than did paternal asthma (3.04 vs. 2.44, p = 0.037). When analyzing the studies in which asthma was diagnosed by a physician the odds ratios were attenuated and no significant differences were observed (2.85 vs. 2.48, N = 18, p = 0.37). Similarly, no significant differences were observed between maternal and paternal odds ratios when analyzing the studies in which the patient population was 5 years or older (3.15 vs. 2.60, p = 0.14). However, in all cases the trend remained the same, that maternal asthma was a greater risk factor for asthma than paternal.The results show that maternal asthma increases offspring disease risk to a greater extent than paternal disease

Alveolar Macrophage Interaction With Air Pollution Particulates

We applied flow cytometric analysis to characterize the in vitro response of alveolar macrophages (AM) to air pollution particulates. Normal hamster AM were incubated with varying concentrations of residual oil fly ash (ROFA) or concentrated ambient air particulates (CAP). We found a dose-dependent increase in AM-associated right angle light scatter (RAS) after uptake of ROFA (e.g., mean channel number 149.4 ± 6.5, 102.5 ± 4.1, 75.8 ± 3.5, and 61.0 ± 4.6 at 200, 100, 50, and 25 mg/ml, respectively) or CAP. A role for scavenger-type receptors (SR) in AM uptake of components of ROFA and CAP was identified by marked inhibition of RAS increases in AM pretreated with the specific SR inhibitor polyinosinic acid. We combined measurement of particle uptake (RAS) with flow cytometric analysis of intracellular oxidation of dichlorofluorescin. Both ROFA and CAP caused a dose-related intracellular oxidant stress within AM, comparable to that seen with phorbol myristate acetate (PMA) (e.g., fold increase over control, 6.6 ± 0.4, 3.6 ± 0.4, 4.6 ± 0.5, 200 mg/ml ROFA, 100 mg/ml ROFA, and $10^{-7}$ M PMA, respectively). We conclude that flow cytometry of RAS increases provides a useful relative measurement of AM uptake of complex particulates within ROFA and CAP. Both ROFA and CAP cause substantial intracellular oxidant stress within AM, which may contribute to subsequent cell activation and production of proinflammatory mediators

HSV-1 exploits the innate immune scavenger receptor MARCO to enhance epithelial adsorption and infection

HSV-1 is an important epithelial pathogen and has the potential for significant morbidity in humans. Here we demonstrate that a cell surface scavenger receptor, macrophage receptor with collagenous structure (MARCO), previously thought to enhance antiviral defense by enabling nucleic acid recognition, is usurped by HSV-1 and functions together with heparan sulfate proteoglycans to mediate adsorption to epithelial cells. Ligands of MARCO dramatically inhibit HSV-1 adsorption and infection of human keratinocytes and protect mice against infection. HSV-1 glycoprotein C (gC) closely co-localizes with MARCO at the cell surface, and gC binds directly to purified MARCO with high affinity. Increasing MARCO expression enhances HSV-1 infection while MARCO-/- mice have reduced susceptibility to infection by HSV-1. These findings demonstrate that HSV-1 binds to MARCO to enhance its capacity for disease, and suggests a new therapeutic target to alter pathogenicity of HSV-1 in skin infection

Signaling pathways required for macrophage scavenger receptor-mediated phagocytosis: analysis by scanning cytometry

Background: Scavenger receptors are important components of the innate immune system in the lung, allowing alveolar macrophages to bind and phagocytose numerous unopsonized targets. Mice with genetic deletions of scavenger receptors, such as SR-A and MARCO, are susceptible to infection or inflammation from inhaled pathogens or dusts. However, the signaling pathways required for scavenger receptor-mediated phagocytosis of unopsonized particles have not been characterized. Methods: We developed a scanning cytometry-based high-throughput assay of macrophage phagocytosis that quantitates bound and internalized unopsonized latex beads. This assay allowed the testing of a panel of signaling inhibitors which have previously been shown to target opsonin-dependent phagocytosis for their effect on unopsonized bead uptake by human in vitro-derived alveolar macrophage-like cells. The non-selective scavenger receptor inhibitor poly(I) and the actin destabilizer cytochalasin D were used to validate the assay and caused near complete abrogation of bead binding and internalization, respectively. Results: Microtubule destabilization using nocodazole dramatically inhibited bead internalization. Internalization was also significantly reduced by inhibitors of tyrosine kinases (genistein and herbimycin A), protein kinase C (staurosporine, chelerythrine chloride and Gö 6976), phosphoinositide-3 kinase (LY294002 and wortmannin), and the JNK and ERK pathways. In contrast, inhibition of phospholipase C by U-73122 had no effect. Conclusion: These data indicate the utility of scanning cytometry for the analysis of phagocytosis and that phagocytosis of unopsonized particles has both shared and distinct features when compared to opsonin-mediated phagocytosis

The relative resistance of children to sepsis mortality: from pathways to drug candidates

Attempts to develop drugs that address sepsis based on leads developed in animal models have failed. We sought to identify leads based on human data by exploiting a natural experiment: the relative resistance of children to mortality from severe infections and sepsis. Using public datasets, we identified key differences in pathway activity (Pathprint) in blood transcriptome profiles of septic adults and children. To find drugs that could promote beneficial (child) pathways or inhibit harmful (adult) ones, we built an in silico pathway drug network (PDN) using expression correlation between drug, disease, and pathway gene signatures across 58,475 microarrays. Specific pathway clusters from children or adults were assessed for correlation with drug-based signatures. Validation by literature curation and by direct testing in an endotoxemia model of murine sepsis of the most correlated drug candidates demonstrated that the Pathprint-PDN methodology is more effective at generating positive drug leads than gene-level methods (e.g., CMap). Pathway-centric Pathprint-PDN is a powerful new way to identify drug candidates for intervention against sepsis and provides direct insight into pathways that may determine survival

Heterogeneity in Macrophage Phagocytosis of Staphylococcus aureus Strains: High-Throughput Scanning Cytometry-Based Analysis

Alveolar macrophages (AMs) can phagocytose unopsonized pathogens such as S. aureus via innate immune receptors, such as scavenger receptors (SRs). Cytoskeletal events and signaling pathways involved in phagocytosis of unopsonized bacteria likely govern the fate of ingested pathogens, but are poorly characterized. We have developed a high-throughput scanning cytometry-based assay to quantify phagocytosis of S. aureus by adherent human blood-derived AM-like macrophages in a 96-well microplate format. Differential fluorescent labeling of internalized vs. bound bacteria or beads allowed automated image analysis of collapsed confocal stack images acquired by scanning cytometry, and quantification of total particles bound and percent of particles internalized. We compared the effects of the classic SR blocker polyinosinic acid, the cytoskeletal inhibitors cytochalasin D and nocodazole, and the signaling inhibitors staurosporine, Gö 6976, JNK Inhibitor I and KN-93, on phagocytosis of a panel of live unopsonized S. aureus strains, (Wood, Seattle 1945 (ATCC 25923), and RN6390), as well as a commercial killed Wood strain, heat-killed Wood strain and latex beads. Our results revealed failure of the SR inhibitor polyinosinic acid to block binding of any live S. aureus strains, suggesting that SR-mediated uptake of a commercial killed fluorescent bacterial particle does not accurately model interaction with viable bacteria. We also observed heterogeneity in the effects of cytoskeletal and signaling inhibitors on internalization of different S. aureus strains. The data suggest that uptake of unopsonized live S. aureus by human macrophages is not mediated by SRs, and that the cellular mechanical and signaling processes that mediate S. aureus phagocytosis vary. The findings also demonstrate the potential utility of high-throughput scanning cytometry techniques to study phagocytosis of S. aureus and other organisms in greater detail

Manipulation of environmental oxygen modifies reactive oxygen and nitrogen species generation during myogenesis

Regulated changes in reactive oxygen and nitrogen species (RONS) activities are important in maintaining the normal sequence and development of myogenesis. Both excessive formation and reduction in RONS have been shown to affect muscle differentiation in a negative way. Cultured cells are typically grown in 20% O2 but this is not an appropriate physiological concentration for a number of cell types, including skeletal muscle. The aim was to examine the generation of RONS in cultured skeletal muscle cells under a physiological oxygen concentration condition (6% O2) and determine the effect on muscle myogenesis. Primary mouse satellite cells were grown in 20% or 6% O2 environments and RONS activity was measured at different stages of myogenesis by real-time fluorescent microscopy using fluorescent probes with different specificities i.e. dihydroethidium (DHE), 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA) and 5-(and-6)-chloromethyl-2′,7′ -dichlorodihydrofluorescein diacetate (CM-DCFH-DA). Data demonstrate that satellite cell proliferation increased when cells were grown in 6% O2 compared with 20% O2. Myoblasts grown in 20% O2 showed an increase in DCF fluorescence and DHE oxidation compared with myoblasts grown at 6% O2. Myotubes grown in 20% O2 also showed an increase in DCF and DAF-FM fluorescence and DHE oxidation compared with myotubes grown in 6% O2. The catalase and MnSOD contents were also increased in myoblasts and myotubes that were maintained in 20% O2 compared with myoblasts and myotubes grown in 6% O2. These data indicate that intracellular RONS activities in myoblasts and myotubes at rest are influenced by changes in environmental oxygen concentration and that the increased ROS may influence myogenesis in a negative manner