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1.  Role of MARCO in Innate Immune Tolerance 
Macrophages play a key role in host defense against microbes, in part, through phagocytosis. MARCO (macrophage receptor with collagenous structure) is a scavenger receptor on the cell surface of macrophages that mediates opsonin-independent phagocytosis. The goal of our study is to investigate the role of MARCO in lipopolysaccharide (LPS) or lipotechoic acid (LTA)-induced macrophage tolerance. While it has been established that expression of MARCO and phagocytosis is increased in tolerant macrophages, the transcriptional regulation and biological role of MARCO in tolerant macrophages has not been investigated. Here, we confirm that tolerized mouse bone marrow derived macrophages (BMDM) selectively increase expression of MARCO (both transcript and cell surface receptor) and increase phagocytosis. We found that H3K4me3 dynamic modification of a promoter site of MARCO was increased in tolerized BMDM. Blocking methylation by treatment with 5-Aza-2′-deoxycytidine (5-AZA) resulted in reduced H3K4me3 binding in the promoter of MARCO, decreased expression of MARCO, and impaired phagocytosis in tolerized BMDM. However, 5-AZA had no effect on the inflammatory component of innate immune tolerance. In aggregate, we found that histone methylation was critical to MARCO expression and phagocytosis in tolerized macrophages but did not affect the inflammatory component of innate immune tolerance.
doi:10.4049/jimmunol.1202942
PMCID: PMC3679202  PMID: 23667110
Phagocytosis; MARCO; Macrophage tolerance; Chromatin modification
2.  Adam8 Limits the Development of Allergic Airway Inflammation in Mice 
To determine whether a disintegrin and a metalloproteinase-8 (Adam8) regulates allergic airway inflammation (AAI) and airway hyper-responsiveness (AHR), we compared AAI and AHR in wild type (WT) versus Adam8−/− mice in different genetic backgrounds sensitized and challenged with ovalbumin (OVA) or house dust mite protein extract (HDM). OVA- and HDM-treated Adam8−/− mice had higher lung leukocyte counts, more airway mucus metaplasia, greater lung levels of some TH2 cytokines, and higher methacholine-induced increases in central airway resistance than allergen-treated WT mice. Studies of OVA-treated Adam8 bone marrow chimeric mice confirmed that leukocyte-derived Adam8 predominantly mediated Adam8’s anti-inflammatory activities in murine airways. Airway eosinophils and macrophages both expressed Adam8 in WT mice with AAI. Adam8 limited AAI and AHR in mice by reducing leukocyte survival because: 1) Adam8−/− mice with AAI had fewer apoptotic eosinophils and macrophages in their airways than WT mice with AAI; and 2) Adam8−/− macrophages and eosinophils had reduced rates of apoptosis compared with WT leukocytes when the intrinsic (but not the extrinsic) apoptosis pathway was triggered in the cells in vitro. ADAM8 was robustly expressed by airway granulocytes in lung sections from human asthma patients but, surprisingly, airway macrophages had less ADAM8 staining than airway eosinophils. Thus, ADAM8 has anti-inflammatory activities during AAI in mice by activating the intrinsic apoptosis pathway in myeloid leukocytes. Strategies that increase ADAM8 levels in myeloid leukocytes may have therapeutic efficacy in asthma.
doi:10.4049/jimmunol.1202329
PMCID: PMC3679335  PMID: 23670189
3.  Prenatal Maternal Stress Predicts Childhood Asthma in Girls: Project Ice Storm 
BioMed Research International  2014;2014:201717.
Little is known about how prenatal maternal stress (PNMS) influences risks of asthma in humans. In this small study, we sought to determine whether disaster-related PNMS would predict asthma risk in children. In June 1998, we assessed severity of objective hardship and subjective distress in women pregnant during the January 1998 Quebec Ice Storm. Lifetime asthma symptoms, diagnoses, and corticosteroid utilization were assessed when the children were 12 years old (N = 68). No effects of objective hardship or timing of the exposure were found. However, we found that, in girls only, higher levels of prenatal maternal subjective distress predicted greater lifetime risk of wheezing (OR = 1.11; 90% CI = 1.01–1.23), doctor-diagnosed asthma (OR = 1.09; 90% CI = 1.00–1.19), and lifetime utilization of corticosteroids (OR = 1.12; 90% CI = 1.01–1.25). Other perinatal and current maternal life events were also associated with asthma outcomes. Findings suggest that stress during pregnancy opens a window for fetal programming of immune functioning. A sex-based approach may be useful to examine how prenatal and postnatal environments combine to program the immune system. This small study needs to be replicated with a larger, more representative sample.
doi:10.1155/2014/201717
PMCID: PMC4034394  PMID: 24895550
4.  Pro-fibrotic Activities for Matrix Metalloproteinase-8 During Bleomycin-mediated Lung Injury1 
Matrix metalloproteinase-8 (MMP-8) is a potent interstitial collagenase thought to be expressed mainly by PMNs. To determine whether Mmp-8 regulates lung inflammatory or fibrotic responses to bleomycin, we delivered bleomycin by the intratracheal (IT) route to wild type (WT) vs. Mmp-8−/− mice and quantified Mmp-8 expression, and inflammation and fibrosis in the lung samples. Mmp-8 steady-state mRNA and protein levels increase in whole lung and bronchoalveolar lavage samples when WT mice are treated with bleomycin. Activated murine lung fibroblasts express Mmp-8 in vitro. MMP-8 expression is increased in leukocytes in the lungs of patients with idiopathic pulmonary fibrosis compared with control lung samples. Compared with bleomycin-treated WT mice, bleomycin-treated Mmp-8−/− mice have greater lung inflammation, but reduced lung fibrosis. While bleomycin-treated Mmp-8−/− and WT mice have similar lung levels of several pro- and anti-fibrotic mediators (Tgf-β, Il-13, JE, and Ifn-γ), Mmp-8−/− mice have higher lung levels of Ip-10 and Mip-1α. Genetically deleting either Ip-10 or Mip-1α in Mmp-8−/− mice abrogates their lung inflammatory response to bleomycin but reconstitutes their lung fibrotic response to bleomycin. Studies of bleomycin-treated Mmp-8 bone marrow-chimeric mice show that both leukocytes and lung parenchymal cells are sources of pro-fibrotic Mmp-8 during bleomycin-mediated lung fibrosis. Thus, during bleomycin-mediated lung injury, Mmp-8 dampens the lung acute inflammatory response but promotes lung fibrosis by reducing lung levels of Ip-10 and Mip-1α. These data indicate therapeutic strategies to reduce lung levels of MMP-8 may limit fibroproliferative responses to injury in the human lung.
doi:10.4049/jimmunol.1201043
PMCID: PMC3639121  PMID: 23487425
MMP-8; fibrosis; inflammation; IP-10; interferon-gamma; mice; bleomycin
5.  Targeted Deletion of Nrf2 Impairs Lung Development and Oxidant Injury in Neonatal Mice 
Antioxidants & Redox Signaling  2012;17(8):1066-1082.
Abstract
Aims: Nrf2 is an essential transcription factor for protection against oxidant disorders. However, its role in organ development and neonatal disease has received little attention. Therapeutically administered oxygen has been considered to contribute to bronchopulmonary dysplasia (BPD) in prematurity. The current study was performed to determine Nrf2-mediated molecular events during saccular-to-alveolar lung maturation, and the role of Nrf2 in the pathogenesis of hyperoxic lung injury using newborn Nrf2-deficient (Nrf2−/−) and wild-type (Nrf2+/+) mice. Results: Pulmonary basal expression of cell cycle, redox balance, and lipid/carbohydrate metabolism genes was lower while lymphocyte immunity genes were more highly expressed in Nrf2−/− neonates than in Nrf2+/+ neonates. Hyperoxia-induced phenotypes, including mortality, arrest of saccular-to-alveolar transition, and lung edema, and inflammation accompanying DNA damage and tissue oxidation were significantly more severe in Nrf2−/− neonates than in Nrf2+/+ neonates. During lung injury pathogenesis, Nrf2 orchestrated expression of lung genes involved in organ injury and morphology, cellular growth/proliferation, vasculature development, immune response, and cell–cell interaction. Bioinformatic identification of Nrf2 binding motifs and augmented hyperoxia-induced inflammation in genetically deficient neonates supported Gpx2 and Marco as Nrf2 effectors. Innovation: This investigation used lung transcriptomics and gene targeted mice to identify novel molecular events during saccular-to-alveolar stage transition and to elucidate Nrf2 downstream mechanisms in protection from hyperoxia-induced injury in neonate mouse lungs. Conclusion: Nrf2 deficiency augmented lung injury and arrest of alveolarization caused by hyperoxia during the newborn period. Results suggest a therapeutic potential of specific Nrf2 activators for oxidative stress-associated neonatal disorders including BPD. Antioxid. Redox Signal. 00, 000–000.
doi:10.1089/ars.2011.4288
PMCID: PMC3423869  PMID: 22400915
6.  Link between Epigenomic Alterations and Genome-Wide Aberrant Transcriptional Response to Allergen in Dendritic Cells Conveying Maternal Asthma Risk 
PLoS ONE  2013;8(8):e70387.
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.
doi:10.1371/journal.pone.0070387
PMCID: PMC3741290  PMID: 23950928
7.  TRIF Mediates Toll-Like Receptor 2-Dependent Inflammatory Responses to Borrelia burgdorferi 
Infection and Immunity  2013;81(2):402-410.
TRIF is an adaptor molecule important in transducing signals from intracellularly signaling Toll-like receptor 3 (TLR3) and TLR4. Recently, TLR2 was found to signal from intracellular compartments. Using a synthetic ligand for TLR2/1 heterodimers, as well as Borrelia burgdorferi, which is a strong activator of TLR2/1, we found that TLR2 signaling can utilize TRIF. Unlike TRIF signaling by other TLRs, TLR2-mediated TRIF signaling is dependent on the presence of another adaptor molecule, MyD88. However, unlike MyD88 deficiency, TRIF deficiency does not result in diminished control of infection with B. burgdorferi in a murine model of disease. This appears to be due to the effects of MyD88 on phagocytosis via scavenger receptors, such as MARCO, which are not affected by the loss of TRIF. In mice, TRIF deficiency did have an effect on the production of inflammatory cytokines, suggesting that regulation of inflammatory cytokines and control of bacterial growth may be uncoupled, in part through transduction of TLR2 signaling through TRIF.
doi:10.1128/IAI.00890-12
PMCID: PMC3553797  PMID: 23166161
8.  HSV-1 exploits the innate immune scavenger receptor MARCO to enhance epithelial adsorption and infection 
Nature communications  2013;4:1963.
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.
doi:10.1038/ncomms2963
PMCID: PMC3681428  PMID: 23739639
9.  Role of the Adiponectin Binding Protein, T-Cadherin (cdh13), in Pulmonary Responses to Subacute Ozone 
PLoS ONE  2013;8(6):e65829.
Adiponectin, an adipose derived hormone with pleiotropic functions, binds to several proteins, including T-cadherin. We have previously reported that adiponectin deficient (Adipo−/−) mice have increased IL-17A-dependent neutrophil accumulation in their lungs after subacute exposure to ozone (0.3 ppm for 72 hrs). The purpose of this study was to determine whether this anti-inflammatory effect of adiponectin required adiponectin binding to T-cadherin. Wildtype, Adipo−/−, T-cadherin deficient (T-cad−/−), and bideficient (Adipo−/−/T-cad−/−) mice were exposed to subacute ozone or air. Compared to wildtype mice, ozone-induced increases in pulmonary IL-17A mRNA expression were augmented in T-cad−/− and Adipo−/− mice. Compared to T-cad−/− mice, there was no further increase in IL-17A in Adipo−/−/T-cad−/− mice, indicating that adiponectin binding to T-cadherin is required for suppression of ozone-induced IL-17A expression. Similar results were obtained for pulmonary mRNA expression of saa3, an acute phase protein capable of inducing IL-17A expression. Comparison of lung histological sections across genotypes also indicated that adiponectin attenuation of ozone-induced inflammatory lesions at bronchiolar branch points required T-cadherin. BAL neutrophils and G-CSF were augmented in T-cad−/− mice and further augmented in Adipo−/−/T-cad−/− mice. Taken together with previous observations indicating that augmentation of these moieties in ozone exposed Adipo−/− mice is partially IL-17A dependent, the results indicate that effects of T-cadherin deficiency on BAL neutrophils and G-CSF are likely secondary to changes in IL-17A, but that adiponectin also acts via T-cadherin independent pathways. Our results indicate that T-cadherin is required for the ability of adiponectin to suppress some but not all aspects of ozone-induced pulmonary inflammation.
doi:10.1371/journal.pone.0065829
PMCID: PMC3675043  PMID: 23755285
10.  MARCO Regulates Early Inflammatory Responses against Influenza 
Lung macrophages use the scavenger receptor MARCO to bind and ingest bacteria, particulate matter, and post cellular debris. We investigated the role of MARCO in influenza A virus (IAV) pneumonia. In contrast to higher susceptibility to bacterial infection, MARCO−/− mice had lower morbidity and mortality from influenza pneumonia than wild-type (WT) mice. The early course of influenza in MARCO−/− lungs was marked by an enhanced but transient neutrophilic inflammatory response and significantly lower viral replication compared with the WT mice. At later time points, no significant differences in lung histopathology or absolute numbers of T lymphocyte influx were evident. Uptake of IAV by WT and MARCO−/− bronchoalveolar lavage macrophages in vitro was similar. By LPS coadministration, we demonstrated that rapid neutrophil and monocyte influx during the onset of influenza suppressed viral replication, indicating a protective role of early inflammation. We hypothesized that the presence of increased basal proinflammatory post cellular debris in the absence of scavenging function lowered the inflammatory response threshold to IAV in MARCO−/− mice. Indeed, MARCO−/− mice showed increased accumulation of proinflammatory oxidized lipoproteins in the bronchoalveolar lavage early in the infection process, which are the potential mediators of the observed enhanced inflammation. These results indicate that MARCO suppresses a protective early inflammatory response to influenza, which modulates viral clearance and delays recovery.
doi:10.1165/rcmb.2010-0349OC
PMCID: PMC3262690  PMID: 21562316
inflammation; scavenger receptors; leukocytes; chemokines; pathology; oxidized lipoproteins
11.  MicroRNA-181b regulates NF-κB–mediated vascular inflammation 
The Journal of Clinical Investigation  2012;122(6):1973-1990.
EC activation and dysfunction have been linked to a variety of vascular inflammatory disease states. The function of microRNAs (miRNAs) in vascular EC activation and inflammation remains poorly understood. Herein, we report that microRNA-181b (miR-181b) serves as a potent regulator of downstream NF-κB signaling in the vascular endothelium by targeting importin-α3, a protein that is required for nuclear translocation of NF-κB. Overexpression of miR-181b inhibited importin-α3 expression and an enriched set of NF-κB–responsive genes such as adhesion molecules VCAM-1 and E-selectin in ECs in vitro and in vivo. In addition, treatment of mice with proinflammatory stimuli reduced miR-181b expression. Rescue of miR-181b levels by systemic administration of miR-181b “mimics” reduced downstream NF-κB signaling and leukocyte influx in the vascular endothelium and decreased lung injury and mortality in endotoxemic mice. In contrast, miR-181b inhibition exacerbated endotoxin-induced NF-κB activity, leukocyte influx, and lung injury. Finally, we observed that critically ill patients with sepsis had reduced levels of miR-181b compared with control intensive care unit (ICU) subjects. Collectively, these findings demonstrate that miR-181b regulates NF-κB–mediated EC activation and vascular inflammation in response to proinflammatory stimuli and that rescue of miR-181b expression could provide a new target for antiinflammatory therapy and critical illness.
doi:10.1172/JCI61495
PMCID: PMC3366408  PMID: 22622040
12.  Allergy Risk Is Mediated by Dendritic Cells with Congenital Epigenetic Changes 
One factor predisposing toward allergic responses is a maternal history of allergy. In a mouse model of maternal transmission of asthma risk, offspring of asthmatic, but not normal, mothers show increased allergic susceptibility, recreating epidemiologic observations in humans. Dendritic cells (DCs) capture and process antigens, and can skew immune responses toward a pro-allergic T helper 2 phenotype. Genome-wide analysis shows that neonates of allergic mothers are born with substantial changes in DNA methylation in their splenic CD11c+ DCs, findings observed without any contact with allergens. We demonstrate that these DCs from allergen-naive neonates born to asthmatic mothers, but not DCs from offspring of normal mothers, confer increased allergic susceptibility to multiple allergens when adoptively transferred into normal recipient mice, manifesting as increased airway responsiveness and allergic inflammation. Other immune splenocytes, including macrophages and CD4+ T cells, did not transfer the effect. The “asthma-susceptible” DCs also show enhanced allergen-presentation activity in vitro. Our findings suggest that maternal allergy results in an altered epigenetic profile in neonatal DCs that is independent of encounters with allergens and is linked to pro-allergic function.
doi:10.1165/rcmb.2009-0400OC
PMCID: PMC3095930  PMID: 20118218
dendritic cells; allergy; asthma; epigenetics; DNA methylation
13.  Genome-Wide RNAi Screen in IFN-γ-Treated Human Macrophages Identifies Genes Mediating Resistance to the Intracellular Pathogen Francisella tularensis 
PLoS ONE  2012;7(2):e31752.
Interferon-gamma (IFN-γ) inhibits intracellular replication of Francisella tularensis in human monocyte-derived macrophages (HMDM) and in mice, but the mechanisms of this protective effect are poorly characterized. We used genome-wide RNA interference (RNAi) screening in the human macrophage cell line THP-1 to identify genes that mediate the beneficial effects of IFN-γ on F. tularensis infection. A primary screen identified ∼200 replicated candidate genes. These were prioritized according to mRNA expression in IFN-γ-primed and F. tularensis-challenged macrophages. A panel of 20 top hits was further assessed by re-testing using individual shRNAs or siRNAs in THP-1 cells, HMDMs and primary human lung macrophages. Six of eight validated genes tested were also found to confer resistance to Listeria monocytogenes infection, suggesting a broadly shared host gene program for intracellular pathogens. The F. tularensis-validated hits included ‘druggable’ targets such as TNFRSF9, which encodes CD137. Treating HMDM with a blocking antibody to CD137 confirmed a beneficial role of CD137 in macrophage clearance of F. tularensis. These studies reveal a number of important mediators of IFN-γ activated host defense against intracellular pathogens, and implicate CD137 as a potential therapeutic target and regulator of macrophage interactions with Francisella tularensis.
doi:10.1371/journal.pone.0031752
PMCID: PMC3281001  PMID: 22359626
14.  Scavenger receptors and β-glucan receptors participate in the recognition of yeasts by murine macrophages 
Inflammation Research  2011;61(2):113-126.
Objectives
Numerous receptors have been implicated in recognition of pathogenic fungi by macrophages, including the β-glucan receptor dectin-1. The role of scavenger receptors (SRs) in anti-fungal immunity is not well characterized.
Methods
We studied uptake of unopsonized Saccharomycetes cerevisiae (zymosan) and live Candida albicans yeasts as well as zymosan-stimulated H2O2 production in J774 macrophage-like cells and peritoneal exudate macrophages (PEMs). The role of different receptors was assessed with the use of competitive ligands, transfected cells and receptor-deficient macrophages.
Results
The uptake of zymosan by untreated J774 cells was mediated approximately half by SRs and half by a β-glucan receptor which was distinct from dectin-1 and not linked to stimulation of H2O2 production. Ligands of β-glucan receptors and of SRs also inhibited uptake of C. albicans by macrophages (J774 cells and PEMs). In macrophages pretreated with a CpG motif-containing oligodeoxynucleotide (CpG-ODN) the relative contribution of SRs to yeast uptake increased and that of β-glucan receptors decreased. Whereas the class A SR MARCO participated in the uptake of both zymosan and C. albicans by CpG-ODN-pretreated, but not untreated macrophages, the related receptor SR-A/CD204 was involved in the uptake of zymosan, but not of C. albicans. The reduction of zymosan-stimulated H2O2 production observed in DS-pretreated J774 cells and in class A SRs-deficient PEMs suggest that class A SRs mediate part of this process.
Conclusions
Our results revealed that SRs belong to a redundant system of receptors for yeasts. Binding of yeasts to different receptors in resting versus CpG-ODN-pre-exposed macrophages may differentially affect polarization of adaptive immune responses.
doi:10.1007/s00011-011-0395-5
PMCID: PMC3265724  PMID: 22116297
MARCO; CD204; Oxidative burst
15.  Risk for Asthma in Offspring of Asthmatic Mothers versus Fathers: A Meta-Analysis 
PLoS ONE  2010;5(4):e10134.
Background
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.”
Methodology/Principal Findings
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.
Conclusions/Significance
The results show that maternal asthma increases offspring disease risk to a greater extent than paternal disease.
doi:10.1371/journal.pone.0010134
PMCID: PMC2853568  PMID: 20405032
16.  Heterogeneity in Macrophage Phagocytosis of Staphylococcus aureus Strains: High-Throughput Scanning Cytometry-Based Analysis 
PLoS ONE  2009;4(7):e6209.
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.
doi:10.1371/journal.pone.0006209
PMCID: PMC2703801  PMID: 19593389
17.  Transplacental Passage of Interleukins 4 and 13? 
PLoS ONE  2009;4(3):e4660.
The mechanisms by which prenatal events affect development of adult disease are incompletely characterized. Based on findings in a murine model of maternal transmission of asthma risk, we sought to test the role of the pro-asthmatic cytokines interleukin IL-4 and -13. To assess transplacental passage of functional cytokines, we assayed phosphorylation of STAT-6, a marker of IL-4 and -13 signaling via heterodimeric receptor complexes which require an IL-4 receptor alpha subunit. IL-4 receptor alpha−/− females were mated to wild-type males, and pregnant females were injected with supraphysiologic doses of IL-4 or 13. One hour after injection, the receptor heterozygotic embryos were harvested and tissue nuclear proteins extracts assayed for phosphorylation of STAT-6 by Western blot. While direct injection of embryos produced a robust positive control, no phosphorylation was seen after maternal injection with either IL-4 or -13, indicating that neither crossed the placenta in detectable amounts. The data demonstrate a useful approach to assay for transplacental passage of functional maternal molecules, and indicate that molecules other than IL-4 and IL-13 may mediate transplacental effects in maternal transmission of asthma risk.
doi:10.1371/journal.pone.0004660
PMCID: PMC2645503  PMID: 19252738
18.  Pulmonary Exposure to Particles during Pregnancy Causes Increased Neonatal Asthma Susceptibility 
Maternal immune responses can promote allergy development in offspring, as shown in a model of increased susceptibility to asthma in babies of ovalbumin (OVA)-sensitized and -challenged mother mice. We investigated whether inflammatory responses to air pollution particles (diesel exhaust particles, DEP) or control “inert” titanium dioxide (TiO2) particles are enhanced during pregnancy and whether exposure to particles can cause increased neonatal susceptibility to asthma. Pregnant BALB/c mice (or nonpregnant controls) received particle suspensions intranasally at Day 14 of pregnancy. Lung inflammatory responses were evaluated 48 hours after exposure. Offspring of particle- or buffer-treated mothers were sensitized and aerosolized with OVA, followed by assays of airway hyperresponsiveness (AHR) and allergic inflammation (AI). Nonpregnant females had the expected minimal response to “inert” TiO2. In contrast, pregnant mice showed robust and persistent acute inflammation after both TiO2 and DEP. Genomic profiling identified genes differentially expressed in pregnant lungs exposed to TiO2. Neonates of mothers exposed to TiO2 (and DEP, but not PBS) developed AHR and AI, indicating that pregnancy exposure to both “inert” TiO2 and DEP caused increased asthma susceptibility in offspring. We conclude that (1) pregnancy enhances lung inflammatory responses to otherwise relatively innocuous inert particles; and (2) exposures of nonallergic pregnant females to inert or toxic environmental air particles can cause increased allergic susceptibility in offspring.
doi:10.1165/rcmb.2007-0124OC
PMCID: PMC2176127  PMID: 17656681
maternal asthma; environmental particles; titanuim dioxide; diesel exhaust particles; susceptibility
19.  Air pollution particles diminish bacterial clearance in the primed lungs of mice 
Epidemiological studies reveal increased incidence of lung infection when air pollution particle levels are increased. We postulate that one risk factor for bacterial pneumonia, prior viral infection, can prime the lung for greater deleterious effects of particles via the γ-interferon (IFN-γ) characteristic of successful host anti-viral responses. To test this postulate, we developed a mouse model in which mice were treated with γ-interferon aerosol, followed by exposure to concentrated ambient particles (CAPs) collected from urban air. The mice were then infected with Streptococcus pneumoniae and the effect of these treatments on the lung innate immune response was evaluated. The combination of IFN-γ priming and CAPs exposure enhanced lung inflammation, manifest as increased polymorphonuclear granulocyte (PMN) recruitment to the lung, and elevated expression of pro-inflammatory cytokine mRNAs. Combined priming and CAPs exposure resulted in impaired pulmonary bacterial clearance, as well as increased oxidant production and diminished bacterial uptake by alveolar macrophages (AMs) and PMNs. The data suggest that priming and CAPs exposure lead to an inflamed alveolar milieu where oxidant stress causes loss of antibacterial functions in AMs and recruited PMNs. The model reported here will allow further analysis of priming and CAPs exposure on lung sensitivity to infection.
doi:10.1016/j.taap.2007.04.014
PMCID: PMC2075081  PMID: 17561223
γ-interferon priming; air pollution particles; Streptococcus pneumoniae; AMs; PMNs
20.  Signaling pathways required for macrophage scavenger receptor-mediated phagocytosis: analysis by scanning cytometry 
Respiratory Research  2008;9(1):59.
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.
doi:10.1186/1465-9921-9-59
PMCID: PMC2527561  PMID: 18687123
21.  Characterization of immortalized MARCO and SR-AI/II-deficient murine alveolar macrophage cell lines 
Background
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.
Results
We used in vitro infection of the primary AMs with the J2 retrovirus carrying the v-raf and v-myc 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 (Staphylococcus aureus) compared with the primary AMs from wild type (WT) C57BL/6 mice.
Conclusion
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 in vitro 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.
doi:10.1186/1743-8977-5-7
PMCID: PMC2427050  PMID: 18452625
22.  Effect of Concentrated Ambient Particles on Macrophage Phagocytosis and Killing of Streptococcus pneumoniae 
Particulate air pollution is linked to increased pneumonia epidemiologically and diminished lung bacterial clearance experimentally. We investigated the effect of concentrated ambient particles (CAPs, ⩽ PM2.5) on the interaction of murine primary alveolar macrophages (AMs) and the murine macrophage cell line, J774 A.1, with Streptococcus pneumoniae. We found that CAPs increased binding of bacteria by both primary AMs and J774 cells (66.7 ± 10.6% and 58.9 ± 4.0%, respectively, n = 4). In contrast to bacterial binding, CAPs decreased internalization in both AMs and J774 (55.4 ± 8.5% and 54.7 ± 5.1%, respectively, n = 4). The rate of killing of internalized bacteria was similar, but CAPs caused a decrease in the absolute number of bacteria killed by macrophages, mainly due to decreased internalization. Additional analyses showed that soluble components of CAPs mediated the enhanced binding and decreased internalization of S. pneumoniae. Chelation of iron in soluble CAPs substantially reversed, while addition of iron as ferric ammonium citrate restored inhibition of phagocytosis of S. pneumoniae in vitro. The results identify phagocytic internalization as a specific target for toxic effects of air pollution particles on AMs.
doi:10.1165/rcmb.2006-0293OC
PMCID: PMC1899326  PMID: 17079778
concentrated ambient particles; macrophages; Streptococcus pneumoniae; phagocytosis; killing
23.  Alveolar macrophage cytokine response to air pollution particles: oxidant mechanisms 
Toxicology and applied pharmacology  2006;218(3):256-264.
Alveolar macrophages (AMs) primed with LPS and treated with concentrated ambient air particles (CAPs) showed enhanced release of tumor necrosis factor (TNF) and provide an in vitro model for the amplified effects of air pollution particles seen in people with preexisting lung disease. To investigate the mechanism(s) by which CAPs mediate TNF release in primed rat AMs, we first tested the effect of a panel of antioxidants. N-acetyl cysteine (20mM), dimethyl thiourea (20 mM) and catalase (5 uM) significantly inhibited TNF release by primed AMs incubated with CAPs. Conversely, when LPS-primed AMs were treated with CAPs in the presence of exogenous oxidants (H2O2 generated by glucose oxidase, 10 uM/hr), TNF release and cell toxicity was significantly increased. The soluble fraction of CAPs suspensions caused most of the increased bioactivity in the presence of exogenous H2O2. The metal chelator deferoxamine (DFO) strongly inhibited the interaction of the soluble fraction with H2O2 but had no effect on the bioactivity of the insoluble CAPs fraction. We conclude that CAPs can mediate their effects in primed AMs by acting on oxidant-sensitive cytokine release in at least two distinct ways. In the primed cell, insoluble components of PM mediate enhanced TNF production that is H2O2-dependent (catalase-sensitive) yet independent of iron (DFO-insensitive). In the presence of exogenous H2O2 released by AMs, PMNs, or other lung cells within an inflamed alveolar milieu, soluble iron released from air particles can also mediate cytokine release and cell toxicity.
doi:10.1016/j.taap.2006.11.033
PMCID: PMC1852536  PMID: 17222881
alveolar macrophage; cytokines; air pollution; particles; oxidants
24.  Ipr1 gene mediates innate immunity to tuberculosis 
Nature  2005;434(7034):767-772.
An estimated 8 million people are infected each year with the pathogen, Mycobacterium tuberculosis, and over 2 million die annually1. Yet only about 10% of those infected develop tuberculosis. Genetic variation within host populations is known to play a significant role in humans and animals 2,3, but the nature of genetic control of host resistance to tuberculosis remains poorly understood. Previously we mapped a new genetic locus on mouse chromosome 1, designated sst1 (for supersusceptibility to tuberculosis1)4. Here we demonstrate in sst1 congenic mouse strains that this locus mediates innate immunity, and identify a candidate gene, Intracellular Pathogen Resistance 1 (Ipr1), within the sst1 locus. The Ipr1 gene is upregulated in the sst1 resistant macrophages upon activation and infection, but is not expressed in the sst1 susceptible macrophages. Expression of the Ipr1 transgene in the sst1 susceptible macrophages limits multiplication not only of MTB but also Listeria monocytogenes and switches a cell death pathway of the infected macrophages from necrosis to apoptosis. Our data suggest that the Ipr1 gene product may play a novel role in integrating signals generated by intracellular pathogens with mechanisms controlling innate immunity, cell death and pathogenesis.
doi:10.1038/nature03419
PMCID: PMC1388092  PMID: 15815631
Bacterial; Lung; Inflammation; Innate Immunity; Tuberculosis; Rodent
25.  The Macrophage Scavenger Receptor SR-AI/II and Lung Defense against Pneumococci and Particles 
The class A macrophage scavenger receptor SR-AI/II is implicated as a pattern recognition receptor for innate immunity, but its functional role in lung defense has not been studied. We used mice genetically deficient in SR-AI/II and their wild-type C57BL/6 counterparts to investigate the contribution of this receptor to defense against pneumococcal infection and inhaled particles. SR-AI/II deficiency caused impaired phagocytosis of fluorescent bacteria in vivo, diminished clearance of live bacteria from the lungs, and substantially increased pneumonic inflammation. Survival studies also showed increased mortality in SR-AI/II–deficient mice with pneumococcal lung infection. Similarly, after challenge of the airways with TiO2 particles, SR-AI/II–deficient mice showed increased proinflammatory cytokine levels in lung lavage fluid and a more pronounced neutrophilic inflammation. The data indicate that the lung macrophage class A scavenger receptor SR-AI/II contributes to innate defense against bacteria and inhaled particles.
doi:10.1165/rcmb.2006-0128OC
PMCID: PMC2643266  PMID: 16675784
environmental particles; lung; macrophages; scavenger receptors

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