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Alveolar macrophages (AMs) in patients with chronic obstructive pulmonary disease (COPD) orchestrate persistent inflammation in the airway. However, subpopulations of AMs participating in chronic inflammation have been poorly characterized. We previously reported that Siglec-1 expression on AMs, which is important for bacteria engulfment, was decreased in COPD. Here, we show that Siglec-1-negative AMs isolated from COPD lung tissues exhibit a proinflammatory phenotype and are associated with poor clinical outcomes in patients with COPD. Using flow cytometry, we segregated three subsets of AMs based on the expression of Siglec-1 and their side scattergram (SSC) and forward scattergram (FSC) properties: Siglec-1+SSChiFSChi, Siglec-1−SSChiFSChi, and Siglec-1−SSCloFSClo subsets. The Siglec-1−SSCloFSClo subset number was increased in COPD. RNA sequencing revealed upregulation of multiple proinflammatory signaling pathways and emphysema-associated matrix metalloproteases in the Siglec-1−SSCloFSClo subset. Gene set enrichment analysis indicated that the Siglec-1−SSCloFSClo subset adopted intermediate phenotypes between monocytes and mature alveolar macrophages. Functionally, these cells produced TNF-α, IL-6, and IL-8 at baseline, and these cytokines were significantly increased in response to viral RNA. The increase in Siglec-1-negative AMs in induced sputum is associated with future exacerbation risk and lung function decline in patients with COPD. Collectively, the novel Siglec-1−SSCloFSClo subset of AMs displays proinflammatory properties, and their emergence in COPD airways may be associated with poor clinical outcomes.NEW & NOTEWORTHY Alveolar macrophages (AMs) in patients with chronic obstructive pulmonary disease (COPD) orchestrate persistent inflammation in the airway. We find that Siglec-1-negative alveolar macrophages have a wide range of proinflammatory landscapes and a protease-expressing phenotype. Moreover, this subset is associated with the pathogenesis of COPD and responds to viral stimuli. [ABSTRACT FROM AUTHOR]

Acute respiratory distress syndrome (ARDS) is characterized by dysregulated inflammation and increased permeability of lung microvascular cells. CD26/dipeptidyl peptidase-4 (DPP4) is a type II membrane protein that is expressed in several cell types and mediates multiple pleiotropic effects. We previously reported that DPP4 inhibition by sitagliptin attenuates lipopolysaccharide (LPS)-induced lung injury in mice. The current study characterized the functional role of CD26/DPP4 expression in LPS-induced lung injury in mice, isolated alveolar macrophages, and cultured lung endothelial cells. In LPS-induced lung injury, inflammatory responses [bronchoalveolar lavage fluid (BALF) neutrophil numbers and several proinflammatory cytokine levels] were attenuated in Dpp4 knockout (Dpp4 KO) mice. However, multiple assays of alveolar capillary permeability were similar between the Dpp4 KO and wild-type mice. TNF-α and IL-6 production was suppressed in alveolar macrophages isolated from Dpp4 KO mice. In contrast, in cultured mouse lung microvascular endothelial cells (MLMVECs), reduction in CD26/DPP4 expression by siRNA resulted in greater ICAM-1 and IL-6 expression after LPS stimulation. Moreover, the LPS-induced vascular monolayer permeability in vitro was higher in MLMVECs treated with Dpp4 siRNA, suggesting that CD26/DPP4 plays a protective role in endothelial barrier function. In summary, this study demonstrated that genetic deficiency of Dpp4 attenuates inflammatory responses but not permeability in LPS-induced lung injury in mice, potentially through differential functional roles of CD26/DPP4 expression in resident cellular components of the lung. CD26/DPP4 may be a potential therapeutic target for ARDS and warrants further exploration to precisely identify the multiple functional effects of CD26/DPP4 in ARDS pathophysiology. NEW & NOTEWORTHY: We aimed to clarify the functional roles of CD26/DPP4 in ARDS pathophysiology using Dpp4-deficient mice and siRNA reduction techniques in cultured lung cells. Our results suggest that CD26/DPP4 expression plays a proinflammatory role in alveolar macrophages while also playing a protective role in the endothelial barrier. Dpp4 genetic deficiency attenuates inflammatory responses but not permeability in LPS-induced lung injury in mice, potentially through differential roles of CD26/DPP4 expression in the resident cellular components of the lung. [ABSTRACT FROM AUTHOR]

Prolonged labor can lead to infection, fetal distress, asphyxia, and life-threatening harm to both the mother and the baby. Surfactant protein A (SP-A) was shown to contribute to the maintenance of pregnancy and timing of term labor. SP-A modulates the stoichiometric expression of the SP-R210L and SP-R210S isoforms of the SP-R210 receptor on alveolar macrophages (AMs). Lack of SP-R210L dysregulates macrophage inflammatory responses. We asked whether SP-A alters normal and inflammation-induced parturition through SP-R210 using SP-A- and SP-R210L-deficient mice. Labor and delivery of time-pregnant mice were monitored in real time using a time-lapse infrared camera. Intrauterine injection with either vehicle or Escherichia coli lipopolysaccharide (LPS) on embryonic (E) day 18.5 post coitus was used to assess the effect of gene disruption in chorioamnionitis-induced labor. We report that either lack of SP-A or disruption of SP-R210L delays parturition by 0.40 and 0.55 days compared with controls, respectively. LPS induced labor at 0.60, 1.01, 0.40, 1.00, and 1.31 days earlier than PBS controls in wild type (WT), SP-A-deficient, littermate controls, heterozygous, and homozygous SP-R210L-deficient mice, respectively. Lack of SP-A reduced litter size in PBS-treated mice, whereas the total number of pups delivered was similar in all LPS-treated mice. The number of live pups, however, was significantly reduced by 50%–70% in SP-A and SP-R210L-deficient mice compared with controls. Differences in gestational length were not associated with intrauterine growth restriction. The present findings support the novel concept that the SP-A/SP-R210 pathway modulates timely labor and delivery and supports fetal lung barrier integrity during fetal-to-neonatal transition in term pregnancy. NEW & NOTEWORTHY: To our knowledge, this study is the first to report that SP-A prevents delay of labor and inflammation-induced stillbirth through the receptor SP-R210L. [ABSTRACT FROM AUTHOR]

Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages (AMs) and bone marrow-derived macrophages (BMDMs) from wild-type (wt) and TREK-1–/– mice, we measured responses to inflammasome priming [using lipopolysaccharide (LPS)] and activation (LPS + ATP). We measured IL-1β, caspase-1, and NLRP3 via ELISA and Western blot. A membrane-permeable potassium indicator was used to measure potassium efflux during ATP exposure, and a fluorescencebased assay was used to assess changes in membrane potential. Inflammasome activation induced by LPS + ATP increased IL-1β secretion in wt AMs, whereas activation was significantly reduced in TREK-1–/– AMs. Priming of BMDMs using LPS was not affected by either genetic deficiency or pharmacological inhibition of TREK-1 with Spadin. Cleavage of caspase-1 following LPS + ATP treatment was significantly reduced in TREK-1–/– BMDMs. The intracellular potassium concentration in LPS-primed wt BMDMs was significant[ly lower compared with TREK-1–/– BMDMs or wt BMDMs treated with Spadin. Conversely, activation of TREK-1 with BL1249 caused a decrease in intracellular potassium in wt BMDMs. Treatment of LPS-primed BMDMs with ATP caused a rapid reduction in intracellular potassium levels, with the largest change observed in TREK-1–/– BMDMs. Intracellular K+ changes were associated with changes in the plasma membrane potential (Em), as evidenced by a more depolarized Em in TREK-1–/– BMDMs compared with wt, and Em hyperpolarization upon TREK-1 channel opening with BL1249. These results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages. NEW & NOTEWORTHY Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages and bone marrow-derived macrophages from wild-type and TREK-1–/– mice, we measured responses to inflammasome priming (using LPS) and activation (LPS + ATP). Our results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages. [ABSTRACT FROM AUTHOR]

Cell-free hemoglobin (CFH) is elevated in the airspace of patients with acute respiratory distress syndrome (ARDS) and is sufficient to cause acute lung injury in a murine model. However, the pathways through which CFH causes lung injury are not well understood. Toll-like receptor 4 (TLR4) is a mediator of inflammation after detection of damage- and pathogen-associated molecular patterns. We hypothesized that TLR4 signaling mediates the proinflammatory effects of CFH in the airspace. After intratracheal CFH, BALBc mice deficient in TLR4 had reduced inflammatory cell influx into the airspace [bronchoalveolar lavage (BAL) cell counts, median TLR4 knockout (KO): 0.8 × 104/mL [IQR 0.4–1.2 × 104/mL], wild-type (WT): 3.0 × 104/mL [2.2–4.0 × 104/mL], P < 0.001] and attenuated lung permeability (BAL protein, TLR4KO: 289 µg/mL [236–320], WT: 488 µg/mL [422–536], P < 0.001). These mice also had attenuated production of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in the airspace. C57Bl/6 mice lacking TLR4 on myeloid cells only (LysM.Cre+/−TLR4fl/fl) had reduced cytokine production in the airspace after CFH, without attenuation of lung permeability. In vitro studies confirm that WT primary murine alveolar macrophages exposed to CFH (0.01–1 mg/mL) had dose-dependent increases in IL-6, IL-1 β, CXC motif chemokine ligand 1 (CXCL-1), TNF-α, and IL-10 (P < 0.001). Murine MH-S alveolar-like macrophages show TLR4-dependent expression of IL-1β, IL-6, and CXCL-1 in response to CFH. Primary alveolar macrophages from mice lacking TLR4 adaptor proteins myeloid differentiation primary response 88 (MyD88) or TIR-domain-containing adapter-inducing interferon-β (TRIF) revealed that MyD88KO macrophages had 71–96% reduction in CFH-dependent proinflammatory cytokine production (P < 0.001), whereas macrophages from TRIFKO mice had variable changes in cytokine responses. These data demonstrate that myeloid TLR4 signaling through MyD88 is a key regulator of airspace inflammation in response to CFH.NEW & NOTEWORTHY Cell-free hemoglobin (CFH) is elevated in the airspace of most patients with acute respiratory distress syndrome and causes severe inflammation. Here, we identify that CFH contributes to macrophage-induced cytokine production via Toll-like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) signaling. These data increase our knowledge of the mechanisms through which CFH contributes to lung injury and may inform development of targeted therapeutics to attenuate inflammation. [ABSTRACT FROM AUTHOR]

Basic leucine zipper transcription factor ATF-like 2 (BATF2) is a transcription factor that is emerging as an important regulator of the innate immune system. BATF2 is among the top upregulated genes in human alveolar macrophages treated with LPS, but the signaling pathways that induce BATF2 expression in response to Gram-negative stimuli are incompletely understood. In addition, the role of BATF2 in the host response to pulmonary infection with a Gram-negative pathogen like Klebsiella pneumoniae (Kp) is not known. We show that induction of Batf2 gene expression in macrophages in response to Kp in vitro requires TRIF and type I interferon (IFN) signaling, but not MyD88 signaling. Analysis of the impact of BATF2 deficiency on macrophage effector functions in vitro showed that BATF2 does not directly impact macrophage phagocytic uptake and intracellular killing of Kp. However, BATF2 markedly enhanced macrophage proinflammatory gene expression and Kp-induced cytokine responses. In vivo, Batf2 gene expression was elevated in lung tissue of wild-type (WT) mice 24 h after pulmonary Kp infection, and Kp-infected BATF2-deficient (Batf2-/-) mice displayed an increase in bacterial burden in the lung, spleen, and liver compared with WT mice. WT and Batf2-/- mice showed similar recruitment of leukocytes following infection, but in line with in vitro observations, proinflammatory cytokine levels in the alveolar space were reduced in Batf2-/- mice. Altogether, these results suggest that BATF2 enhances proinflammatory cytokine responses in macrophages in response to Kp and contributes to the early host defense against pulmonary Kp infection. NEW & NOTEWORTHY: This study investigates the signaling pathways that mediate induction of BATF2 expression downstream of TLR4 and also the impact of BATF2 on the host defense against pulmonary Kp infection. We demonstrate that Kp-induced upregulation of BATF2 in macrophages requires TRIF and type I IFN signaling. We also show that BATF2 enhances Kp-induced macrophage cytokine responses and that BATF2 contributes to the early host defense against pulmonary Kp infection. [ABSTRACT FROM AUTHOR]

Few new therapeutics exist to target airway inflammation in mild-to-moderate asthma. Alveolar macrophages regulate airway inflammation by producing proresolving eicosanoids. We hypothesized that stimulation of the purinergic receptor P2X7 in macrophages from individuals with asthma produces eicosanoids associated with airway inflammation and resolution, and that these responses are predicted, in part, by P2X7 pore function. Study subjects were recruited in an Institutional Review Board (IRB)- approved study. Alveolar macrophages were recovered from bronchoalveolar lavage fluid following bronchoscopy. Purinergic receptor classification was performed using flow cytometry and fluorescent cell assay. Macrophages were stimulated in vitro and eicosanoids were measured via ELISA or enzyme immunoassay (EIA) in the presence and absence of P2X7-specific agonist [20 (30)-O- (4-Benzoylbenzoyl)adenosine-50-triphosphate tri(triethylammonium) salt (Bz-ATP)] and antagonist (AZD9056). Functional P2X7 pore status was confirmed in a live cell assay using P2X7-specific agonists and antagonists. Alveolar macrophages produced increased quantities of the oxylipins lipoxin A4 (LXA4), resolvin D1 (RvD1), and 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) following stimulation with Bz-ATP compared with vehicle controls, responses that were attenuated in the presence of the P2X7-selective antagonist, AZD9056. LXA4 and RvD1 production was greatest at 1 h, whereas 15(S)-HETE was maximally produced 24 h. Prostaglandin E-2 and resolvin E1 were minimally produced by P2X7 activation, indicating differential signaling pathways involved in eicosanoid production in alveolar macrophages derived from individuals with asthma. The early production of the proresolving eicosanoids, LXA4 and resolvin D1, is regulated by P2X7, whereas generation of the proinflammatory eicosanoid, 15(S)-HETE, is only partially regulated through P2X7 signaling and reaches maximal production after the peak in proresolving eicosanoids. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is characterized by increased collagen accumulation that is progressive and nonresolving. Although fibrosis progression may be regulated by fibroblasts and alveolar macrophage (AM) interactions, this cellular interplay has not been fully elucidated. To study AM-fibroblast interactions, cells were isolated from IPF and normal human lung tissue and cultured independently or together in direct 2-D coculture, direct 3-D coculture, indirect transwell, and in 3-D hydrogels. AM influence on fibroblast function was assessed by gene expression, cytokine/chemokine secretion, and hydrogel contractility. Normal AMs cultured in direct contact with fibroblasts downregulated extracellular matrix (ECM) gene expression whereas IPF AMs had little to no effect. Fibroblast contractility was assessed by encapsulating cocultures in 3-D collagen hydrogels and monitoring gel diameter over time. Both normal and IPF AMs reduced baseline contractility of normal fibroblasts but had little to no effect on IPF fibroblasts. When stimulated with Toll-like receptor (TLR) agonists, IPF AMs increased production of pro-inflammatory cytokines TNFa and IL-1b, compared with normal AMs. TLR ligand stimulation did not alter fibroblast contraction, but stimulation with exogenous TNFa and TGFb did alter contraction. To determine if the observed changes required cell-to-cell contact, AM-conditioned media and transwell systems were utilized. Transwell culture showed decreased ECM gene expression changes compared with direct coculture and conditioned media from AMs did not alter fibroblast contraction regardless of disease state. Taken together, these data indicate that normal fibroblasts are more responsive to AM crosstalk, and that AM influence on fibroblast behavior depends on cell proximity. [ABSTRACT FROM AUTHOR]

Legionella pneumophila is the main etiological agent of Legionnaires' disease, a severe bacterial pneumonia. L. pneumophila is initially engulfed by alveolar macrophages (AMs) and subvert normal cellular functions to establish a replicative vacuole. Cigarette smokers are particularly susceptible to developing Legionnaires' disease and other pulmonary infections; however, little is known about the cellular mechanisms underlying this susceptibility. To investigate this, we used a mouse model of acute cigarette smoke exposure to examine the immune response to cigarette smoke and subsequent L. pneumophila infection. Contrary to previous reports, we show that cigarette smoke exposure alone causes a significant depletion of AMs using enzymatic digestion to extract cells, or via imaging intact lung lobes by light-sheet microscopy. Furthermore, treatment of mice deficient in specific types of cell death with smoke suggests that NLRP3-driven pyroptosis is a contributor to smoke-induced death of AMs. After infection, smoke-exposed mice displayed increased pulmonary L. pneumophila loads and developed more severe disease compared with air-exposed controls. We tested if depletion of AMs was related to this phenotype by directly depleting them with clodronate liposomes and found that this also resulted in increased L. pneumophila loads. In summary, our results showed that cigarette smoke depleted AMs from the lung and that this likely contributed to more severe Legionnaires' disease. Furthermore, the role of AMs in L. pneumophila infection is more nuanced than simply providing a replicative niche, and our studies suggest they play a major role in bacterial clearance. [ABSTRACT FROM AUTHOR]

Although vitamin E acetate (VEA) is suspected to play a causal role in the development of electronic-cigarette, or vaping, product use-associated lung injury (EVALI), the underlying biological mechanisms of pulmonary injury are yet to be determined. In addition, no study has replicated the systemic inflammation observed in humans in a murine EVALI model, nor investigated potential additive toxicity of viral infection in the setting of exposure to vaping products. To identify the mechanisms driving VEA-related lung injury and test the hypothesis that viral infection causes additive lung injury in the presence of aerosolized VEA, we exposed mice to aerosolized VEA for extended times, followed by influenza infection in some experiments. We used mass spectrometry to evaluate the composition of aerosolized VEA condensate and the VEA deposition in murine or human alveolar macrophages. Extended vaping for 28 days versus 15 days did not worsen lung injury but caused systemic inflammation in the murine EVALI model. Vaping plus influenza increased lung water compared with virus alone. Murine alveolar macrophages exposed to vaped VEA hydrolyzed the VEA to vitamin E with evidence of oxidative stress in the alveolar space and systemic circulation. Aerosolized VEA also induced cell death and chemokine release and reduced efferocytotic function in human alveolar macrophages in vitro. These findings provide new insights into the biological mechanisms of VEA toxicity. [ABSTRACT FROM AUTHOR]

Although vitamin E acetate (VEA) is suspected to play a causal role in the development of electronic-cigarette, or vaping, product use-associated lung injury (EVALI), the underlying biological mechanisms of pulmonary injury are yet to be determined. In addition, no study has replicated the systemic inflammation observed in humans in a murine EVALI model, nor investigated potential additive toxicity of viral infection in the setting of exposure to vaping products. To identify the mechanisms driving VEA-related lung injury and test the hypothesis that viral infection causes additive lung injury in the presence of aerosolized VEA, we exposed mice to aerosolized VEA for extended times, followed by influenza infection in some experiments. We used mass spectrometry to evaluate the composition of aerosolized VEA condensate and the VEA deposition in murine or human alveolar macrophages. Extended vaping for 28 days versus 15 days did not worsen lung injury but caused systemic inflammation in the murine EVALI model. Vaping plus influenza increased lung water compared with virus alone. Murine alveolar macrophages exposed to vaped VEA hydrolyzed the VEA to vitamin E with evidence of oxidative stress in the alveolar space and systemic circulation. Aerosolized VEA also induced cell death and chemokine release and reduced efferocytotic function in human alveolar macrophages in vitro. These findings provide new insights into the biological mechanisms of VEA toxicity. [ABSTRACT FROM AUTHOR]

Hereditary pulmonary alveolar proteinosis (hPAP) is a rare disorder caused by recessive mutations in GM-CSF receptor subunit a/b genes (CSF2RA/CSF2RB, respectively) characterized by impaired GM-CSF-dependent surfactant clearance by alveolar macrophages (AMs) resulting in alveolar surfactant accumulation and hypoxemic respiratory failure. Because hPAP is caused by CSF2RA mutations in most patients, we created an animal model of hPAP caused by Csf2ra gene disruption (Csf2ra-/- mice) and evaluated the effects on AMs and lungs. Macrophages from Csf2ra-/- mice were unable to bind and clear GM-CSF, did not exhibit GM-CSF signaling, and had functional defects in phagocytosis, cholesterol clearance, and surfactant clearance. Csf2ra-/- mice developed a time-dependent, progressive lung disease similar to hPAP in children caused by CSF2RA mutations with respect to the clinical, physiological, histopathological, biochemical abnormalities, biomarkers of PAP lung disease, and clinical course. In contrast, Csf2ra+/- mice had functionally normal AMs and no lung disease. Pulmonary macrophage transplantation (PMT) without myeloablation resulted in long-term engraftment, restoration of GM-CSF responsiveness to AMs, and a safe and durable treatment effect that lasted for the duration of the experiment (6 mo). Results demonstrate that homozygous (but not heterozygous) Csf2ra gene ablation caused hPAP identical to hPAP in children with CSF2RA mutations, identified AMs as the cellular site of hPAP pathogenesis in Csf2ra-/- mice, and have implications for preclinical studies supporting the translation of PMT as therapy of hPAP in humans. [ABSTRACT FROM AUTHOR]

Mechanical ventilation is a life-sustaining therapy for patients with respiratory failure but can cause further lung damage known as ventilator-induced lung injury (VILI). However, the intrinsic molecular mechanisms underlying recovery of VILI remain unknown. Phagocytosis of apoptotic cells (also known as efferocytosis) is a key mechanism orchestrating successful resolution of inflammation. Here we show the positive regulation of macrophage Toll-like receptor (TLR) 4 in efferocytosis and resolution of VILI. Mice were depleted of alveolar macrophages and then subjected to injurious ventilation (tidal volume, 20 mL/kg) for 4 h. On day 1 after mechanical ventilation, Tlr4+/+ or Tlr4-/- bone marrow-derived macrophages (BMDMs) were intratracheally administered to alveolar macrophage-depleted mice. We observed that mice depleted of alveolar macrophages exhibited defective resolution of neutrophilic inflammation, exuded protein, lung edema, and lung tissue injury after ventilation, whereas these delayed responses were reversed by administration of Tlr4+/+ BMDMs. Importantly, these proresolving effects by Tlr4+/+ BMDMs were abolished in mice receiving Tlr4-/- BMDMs. The number of macrophages containing apoptotic cells or bodies in bronchoalveolar lavage fluid was much less in mice receiving Tlr4-/- BMDMs than that in those receiving Tlr4+/+ BMDMs. Macrophage TLR4 deletion facilitated a disintegrin and metalloprotease 17 maturation and enhanced Mer cleavage in response to mechanical ventilation. Heat shock protein 70 dramatically increased Mer tyrosine kinase surface expression, phagocytosis of apoptotic neutrophils, and rescued the inflammatory phenotype in alveolar macrophage-depleted mice receiving Tlr4+/+ BMDMs, but not Tlr4-/- BMDMs. Our results suggest that macrophage TLR4 promotes resolution of VILI via modulation of Mer-mediated efferocytosis. [ABSTRACT FROM AUTHOR]

Increased lung vascular permeability and neutrophilic inflammation are hallmarks of acute lung injury. Alveolar macrophages (AMΦ), the predominant sentinel cell type in the airspace, die in massive numbers while fending off pathogens. Recent studies indicate that the AMΦ pool is replenished by airspace-recruited monocytes, but the mechanisms instructing the conversion of recruited monocytes into reparative AMΦ remain elusive. Cyclic AMP (cAMP) is a vascular barrier protective and immunosuppressive second messenger in the lung. Here, we subjected mice expressing GFP under the control of the Lysozyme-M promoter (LysM-GFP mice) to the LPS model of rapidly resolving lung injury to address the impact of mechanisms determining cAMP levels in AMΦ and regulation of mobilization of the reparative AMΦ-pool. RNA-seq analysis of flow-sorted MΦ identified phosphodiesterase 4b (PDE4b) as the top LPS-responsive cAMP-regulating gene. We observed that PDE4b expression markedly increased at the time of peak injury (4 h) and then decreased to below the basal level during the resolution phase (24 h). Activation of transcription factor NFATc2 was required for the transcription of PDE4b in MΦ. Inhibition of PDE4 activity at the time of peak injury, using intratracheal rolipram, increased cAMP levels, augmented the reparative AMΦ pool, and resolved lung injury. This response was not seen following conditional depletion of monocytes, thus establishing airspace-recruited PDE4b-sensitive monocytes as the source of reparative AMΦ. Interestingly, adoptive transfer of rolipram-educated AMΦ into injured mice resolved lung edema. We propose suppression of PDE4b as an effective approach to promote reparative AMΦ generation from monocytes for lung repair. [ABSTRACT FROM AUTHOR]

Patients with cystic fibrosis (CF) have defective macrophage phagocytosis and efferocytosis. Several reports demonstrate that neutrophil elastase (NE), a major inflammatory protease in the CF airway, impairs macrophage phagocytic function. To date, NE-impaired macrophage phagocytic function has been attributed to cleavage of cell surface receptors or opsonins. We applied an unbiased proteomic approach to identify other potential macrophage targets of NE protease activity that may regulate phagocytic function. Using the murine macrophage cell line, RAW 264.7, human blood monocyte-derived macrophages, and primary alveolar macrophages from Cftr-null and wild-type littermate mice, we demonstrated that NE exposure blocked phagocytosis of Escherichia coli bio-particles. We performed liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteomic analysis of the conditioned media from RAW264.7 treated either with active NE or inactive (boiled) NE as a control. Out of 840 proteins identified in the conditioned media, active NE upregulated 142 proteins and downregulated 211 proteins. NE released not only cell surface proteins into the media but also cytoskeletal, mitochondrial, cytosolic, and nuclear proteins that were detected in the conditioned media. At least 32 proteins were associated with the process of phagocytosis including 11 phagocytic receptors [including lipoprotein receptor-related protein 1 (LRP1)], 7 proteins associated with phagocytic cup formation, and 14 proteins involved in phagocytic maturation (including calpain-2) and phagolysosome formation. NE had a broad effect on the proteome required for regulation of all stages of phagocytosis and phagolysosome formation. Furthermore, the NE sheddome/secretome included proteins from other macrophage cellular domains, suggesting that NE may globally regulate macrophage structure and function. [ABSTRACT FROM AUTHOR]

Electronic (e-) cigarettes are growing in popularity despite uncertainties regarding their long-term health implications. The link between cigarette smoking and initiation of chronic lung disease took decades to unpick so in vitro studies mimicking e-cigarette exposure aim to detect early indicators of harm. In response to e-cigarette exposure, alveolar macrophages adopt a proinflammatory phenotype of increased secretion of proinflammatory cytokines, reduction in phagocytosis, and efferocytosis and reactive oxygen species generation. These effects are largely driven by free radical exposure, changes in PI3K/Akt signaling pathways, nicotine-induced reduction in phagocytosis receptors, and impaired lipid homeostasis leading to a foam-like lipidladen phenotype. Neutrophils exhibit disrupted chemotaxis and transmigration to chemokines, reduced phagocytosis and bacterial killing, and an increase in protease secretion without corresponding antiproteases in response to e-cigarette exposure. This is driven by an altered ability to respond and to polarize toward chemoattractants, an activation of the p38 MAPK signaling pathway and inability to assemble NADPH oxidase. E-cigarettes induce lung epithelial cells to display decreased ciliary beat frequency and ion channel conductance as well as changes in chemokine secretion and surface protein expression. Changes in gene expression, mitochondrial function, and signaling pathways have been demonstrated in lung epithelial cells to explain these changes. Many functional outputs of alveolar macrophages, neutrophils, and lung epithelial cells have not been fully explored in the context of e-cigarette exposure and the underlying driving mechanisms are poorly understood. This review discusses current evidence surrounding the effects of e-cigarettes on alveolar macrophages, neutrophils, and lung epithelial cells with particular focus on the cellular mechanisms of change. [ABSTRACT FROM AUTHOR]

Microbial metabolites produced by the gut microbiome, e.g. short-chain fatty acids (SCFA), have been found to influence lung physiology and injury responses. However, how lung immune activity is regulated by SCFA is unknown. We examined fresh human lung tissue and observed the presence of SCFA with interindividual variability. In vitro, SCFA were capable of modifying the metabolic programming in LPS-exposed alveolar macrophages (AM). We hypothesized that lung immune tone could be defined by baseline detection of lung intracellular IL-1b. Therefore, we interrogated naïve mouse lungs with intact gut microbiota for IL-1b mRNA expression and localized its presence within alveolar spaces, specifically within AM subsets. We established that metabolically active gut microbiota, which produce SCFA, can transmit LPS and SCFA to the lung and thereby could create primed lung immunometabolic tone. To understand how murine lung cells sensed and upregulated IL-1b in response to gut microbiome-derived factors, we determined that, in vitro, AM and alveolar type II (AT2) cells expressed SCFA receptors, free fatty acid receptor 2 (FFAR2), free fatty acid receptor 3 (FFAR3), and IL-1b but with distinct expression patterns and different responses to LPS. Finally, we observed that IL-1b, FFAR2, and FFAR3 were expressed in isolated human AM and AT2 cells ex vivo, but in fresh human lung sections in situ, only AM expressed IL-1b at rest and after LPS challenge. Together, this translational study using mouse and human lung tissue and cells point to an important role for the gut microbiome and their SCFA in establishing and regulating lung immune tone. [ABSTRACT FROM AUTHOR]

Macrophage activation is a key contributing factor for excessive inflammatory responses of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Geranylgeranyl diphosphate synthase (GGPPS) plays a key role in the development of inflammatory diseases. Our group previously showed that GGPPS in alveolar epithelium have deleterious effects on acute lung injury induced by LPS or mechanical ventilation. Herein, we examined the role of GGPPS in modulating macrophage activation in ALI/ ARDS. We found significant increased GGPPS expression in alveolar macrophages in patients with ARDS compared with healthy volunteers and in ALI mice induced by LPS. GGPPS-floxed control (GGPPSfl/fl) and myeloid-selective knockout (GGPPSfl/flLysMcre) mice were then generated. Interestingly, using an LPS-induced ALI mouse model, we showed that myeloid-specific GGPPS knockout significantly increased mortality, aggravated lung injury, and increased the accumulation of inflammatory cells, total protein, and inflammatory cytokines in BALF. In vitro, GGPPS deficiency upregulated the production of LPS-induced IL-6, IL-1b, and TNF-a in alveolar macrophages, bone marrow-derived macrophages (BMDMs), and THP-1 cells. Mechanistically, GGPPS knockout increased phosphorylation and nuclear translocation of NF-κB p65 induced by LPS. In addition, GGPPS deficiency increased the level of GTP-Rac1, which was responsible for NF-κB activation. In conclusion, decreased expression of GGPPS in macrophages aggravates lung injury and inflammation in ARDS, at least partly by regulating Rac1-dependent NF-κB signaling. GGPPS in macrophages may represent a novel therapeutic target in ARDS. [ABSTRACT FROM AUTHOR]

Few patients with bacteremia from a nonpulmonary source develop acute respiratory distress syndrome (ARDS). However, the mechanisms that protect the lung from injury in bacteremia have not been identified. We simulated bacteremia by adding Streptococcus pneumoniae to the perfusate of the ex vivo perfused human lung model. In contrast to a pneumonia model in which bacteria were instilled into the distal air spaces of one lobe, injection of high doses of S. pneumoniae into the perfusate was not associated with alveolar epithelial injury as demonstrated by low protein permeability of the alveolar epithelium, intact alveolar fluid clearance, and the absence of alveolar edema. Unexpectedly, the ex vivo human lung rapidly cleared large quantities of S. pneumoniae even though the perfusate had very few intravascular phagocytes and lacked immunoglobulins or complement. The bacteria were cleared in part by the small number of neutrophils in the perfusate, alveolar macrophages in the airspaces, and probably by interstitial pathways. Together, these findings identify one mechanism by which the lung and the alveolar epithelium are protected from injury in bacteremia. [ABSTRACT FROM AUTHOR]

Acute lung injury is a major complication of hemorrhagic shock and the required resuscitation with large volumes of crystalloid fluids and blood products. We previously identified a role of macrophagederived chemokine (CCL22/MDC) pulmonary inflammation following hemorrhage and resuscitation. However, further details regarding the induction of CCL22/MDC and its precise role in pulmonary inflammation after trauma remain unknown. In the current study we used in vitro experiments with a murine alveolar macrophage cell line, as well as an in vivo mouse model of hemorrhage and resuscitation, to identify key regulators in CCL22/MDC production. We show that trauma induces expression of IFNγ, which leads to production of CCL22/MDC through a signaling mechanism involving p38 MAPK, NF-κB, JAK, and STAT-1. IFNγ also activates TNFα production by alveolar macrophages, potentiating CCL22/MDC production via an autocrine mechanism. Neutralization of IFNγ or TNFα with specific antibodies reduced histological signs of pulmonary injury after hemorrhage and reduced inflammatory cell infiltration into the lungs. [ABSTRACT FROM AUTHOR]

Ghrelin has proven to be protective against sepsis-induced acute lung injury (ALI) via anti-inflammatory effects. However, its mechanisms remain poorly understood. Alveolar macrophages (AMs) play a key role in mediating inflammatory responses during sepsis-induced ALI by secretion of cytokines and chemokines. This study was undertaken to investigate whether ghrelin suppresses inflammatory effects of AMs and therefore may help to attenuate sepsis-induced ALI. A sepsis model in rats was achieved using cecal ligation and puncture. Ghrelin treatment markedly improved histopathological changes in the lungs and reduced pulmonary inflammation in septic rats. NF-κB translocation and p-Akt and inducible nitric oxide synthase (iNOS) activities in AMs from septic rats were suppressed by ghrelin. In vitro data indicated that ghrelin decreased the levels of LPS-induced IL-1β, TNF-α, and IL-6, NF-κB translocation, and iNOS and Akt activities of AMs. Furthermore, the NF-κB/iNOS pathway or Akt signaling was positively correlated with LPS-induced inflammatory production of AMs in vitro. In conclusion, ghrelin exerts a protective role against sepsis-induced ALI probably by reducing the production of inflammatory cytokines from AMs via inhibition of the NF-κB/iNOS pathway or Akt signaling. [ABSTRACT FROM AUTHOR]

Smoking alters pulmonary reverse lipid transport and leads to intracellular lipid accumulation in alveolar macrophages. We investigated whether stimulating reverse lipid transport with an agonist of the liver X receptor (LXR) would help alveolar macrophages limit lipid accumulation and dampen lung inflammation in response to cigarette smoke. Mice were exposed to cigarette smoke and treated intraperitoneally with the LXR agonist T0901317. Expression of lipid capture and lipid export genes was assessed in lung tissue and alveolar macrophages. Pulmonary inflammation was assessed in the bronchoalveolar lavage (BAL). Finally, cholesterol efflux capacity and pulmonary surfactant levels were determined. In room air-exposed mice, T0901317 increased the expression of lipid export genes in macrophages and the whole lung and increased cholesterol efflux capacity without inducing inflammation or affecting the pulmonary surfactant. However, cigarette smoke-exposed mice treated with T0901317 showed a marked increase in BAL neutrophils, IL-1α, C-C motif chemokine ligand 2, and granulocyte-colony-stimulating factor levels. T0901317 treatment in cigarette smoke-exposed mice failed to increase the ability of alveolar macrophages to export cholesterol and markedly exacerbated IL-1α release. Finally, T0901317 led to pulmonary surfactant depletion only in cigarette smoke-exposed mice. This study shows that hyperactivation of LXR and the associated lipid capture/export mechanisms only have minor pulmonary effects on the normal lung. However, in the context of cigarette smoke exposure, where the pulmonary surfactant is constantly oxidized, hyperactivation of LXR has dramatic adverse effects, once again showing the central role of lipid homeostasis in the pulmonary response to cigarette smoke exposure. [ABSTRACT FROM AUTHOR]

Neuropilins are multifunctional receptors that play important roles in immune regulation. Neuropilin-2 (NRP2) is expressed in the lungs, but whether it regulates airway immune responses is unknown. Here, we report that Nrp2 is weakly expressed by alveolar macrophages (AMs) in the steady state but is dramatically upregulated following in vivo lipopolysaccharide (LPS) inhalation. Ex vivo treatment of human AMs with LPS also increased NRP2 mRNA expression and cell-surface display of NRP2 protein. LPS-induced Nrp2 expression in AMs was dependent upon the myeloid differentiation primary response 88 signaling pathway and the transcription factor NF-κB. In addition to upregulating display of NRP2 on the cell membrane, inhaled LPS also triggered AMs to release soluble NRP2 into the airways. Finally, myeloid-specific ablation of NRP2 resulted in increased expression of the chemokine (C-C motif) ligand 2 (Ccl2) in the lungs and prolonged leukocyte infiltration in the airways following LPS inhalation. These findings suggest that NRP2 expression by AMs regulates LPS-induced inflammatory cell recruitment to the airways and reveal a novel role for NRP2 during innate immune responses in the lungs. [ABSTRACT FROM AUTHOR]

Surfactant protein C (SPC), a key component of pulmonary surfactant, also plays a role in regulating inflammation. SPC deficiency in patients and mouse models is associated with increased inflammation and delayed repair, but the key drivers of SPC-regulated inflammation in response to injury are largely unknown. This study focuses on a new mechanism of SPC as an anti-inflammatory molecule using SPC-TK/SPC-KO (surfactant protein C-thymidine kinase/surfactant protein C knockout) mice, which represent a novel sterile injury model that mimics clinical acute respiratory distress syndrome (ARDS). SPC-TK mice express the inducible suicide gene thymidine kinase from by the SPC promoter, which targets alveolar type 2 (AT2) cells for depletion in response to ganciclovir (GCV). We compared GCV-induced injury and repair in SPC-TK mice that have normal endogenous SPC expression with SPC-TK/SPC-KO mice lacking SPC expression. In contrast to SPC-TK mice, SPC-TK/SPC-KO mice treated with GCV exhibited more severe inflammation, resulting in over 90% mortality; there was only 8% mortality of SPC-TK animals. SPC-TK/SPC-KO mice had highly elevated inflammatory cytokines and granulocyte infiltration in the bronchoalveolar lavage (BAL) fluid. Consistent with a proinflammatory phenotype, immunofluorescence revealed increased phosphorylated signal transduction and activation of transcription 3 (pSTAT3), suggesting enhanced Janus kinase (JAK)/STAT activation in inflammatory and AT2 cells of SPC-TK/SPC-KO mice. The level of suppressor of cytokine signaling 3, an anti-inflammatory mediator that decreases pSTAT3 signaling, was significantly decreased in the BAL fluid of SPC-TK/SPC-KO mice. Hyperactivation of pSTAT3 and inflammation were rescued by AZD1480, a JAK1/2 inhibitor. Our findings showing a novel role for SPC in regulating inflammation via JAK/STAT may have clinical applications. [ABSTRACT FROM AUTHOR]

To advance our understanding of the pathobiology of sarcoidosis, we developed a multiwall carbon nanotube (MWCNT)-based murine model that shows marked histological and inflammatory signal similarities to this disease. In this study, we compared the alveolar macrophage transcriptional signatures of our animal model with human sarcoidosis to identify overlapping molecular programs. Whole genome microarrays were used to assess gene expression of alveolar macrophages in six MWCNT-exposed and six control animals. The results were compared with the transcriptional profiles of alveolar immune cells in 15 sarcoidosis patients and 12 healthy humans. Rigorous statistical methods were used to identify differentially expressed genes. To better elucidate activated pathways, integrated network and gene set enrichment analysis (GSEA) was performed. We identified over 1,000 differentially expressed between control and MWCNT mice. Gene ontology functional analysis showed overrepresentation of processes primarily involved in immunity and inflammation in MCWNT mice. Applying GSEA to both mouse and human samples revealed upregulation of 92 gene sets in MWCNT mice and 142 gene sets in sarcoidosis patients. Commonly activated pathways in both MWCNT mice and sarcoidosis included adaptive immunity, T-cell signaling, IL-12/IL-17 signaling, and oxidative phosphorylation. Differences in gene set enrichment between MWCNT mice and sarcoidosis patients were also observed. We applied network analysis to differentially expressed genes common between the MWCNT model and sarcoidosis to identify key drivers of disease. In conclusion, an integrated network and transcriptomics approach revealed substantial functional similarities between a murine model and human sarcoidosis particularly with respect to activation of immune-specific pathways. [ABSTRACT FROM AUTHOR]

There is no therapeutic intervention proven to prevent acute respiratory distress syndrome (ARDS). Novel mechanistic insights into the pathophysiology of ARDS are therefore required. Platelets are implicated in regulating many of the pathogenic processes that occur during ARDS; however, the mechanisms remain elusive. The platelet receptor CLEC-2 has been shown to regulate vascular integrity at sites of acute inflammation. Therefore the purpose of this study was to establish the role of CLEC-2 and its ligand podoplanin in a mouse model of ARDS. Platelet-specific CLEC-2-deficient, as well as alveolar epithelial type I cell (AECI)-specific or hematopoietic-specific podoplanin deficient, mice were established using cre-loxP strategies. Combining these with intratracheal (IT) instillations of lipopolysaccharide (LPS), we demonstrate that arterial oxygen saturation decline in response to IT-LPS in platelet-specific CLEC-2-deficient mice is significantly augmented. An increase in bronchoalveolar lavage (BAL) neutrophils and protein was also observed 48 h post-IT-LPS, with significant increases in pro-inflammatory chemokines detected in BAL of platelet-specific CLEC-2-deficient animals. Deletion of podoplanin from hematopoietic cells but not AECIs also reduces lung function and increases pro-inflammatory chemokine expression following IT-LPS. Furthermore, we demonstrate that following IT-LPS, platelets are present in BAL in aggregates with neutrophils, which allows for CLEC-2 interaction with podoplanin expressed on BAL inflammatory alveolar macrophages. Taken together, these data suggest that the platelet CLEC-2-podoplanin signaling axis regulates the severity of lung inflammation in mice and is a possible novel target for therapeutic intervention in patients at risk of developing ARDS. [ABSTRACT FROM AUTHOR]

Metabolic reprogramming has been intrinsically linked to macrophage activation. Alveolar macrophages are known to play an important role in the pathogenesis of pulmonary fibrosis. However, systematic characterization of expression profile in these cells is still lacking. Furthermore, main metabolic programs and their regulation of cellular phenotype are completely unknown. In this study, we comprehensively analyzed the expression profile and main metabolic programs in alveolar macrophages from mice with or without experimental pulmonary fibrosis. We found that alveolar macrophages from both bleomycin and active TGF-β1-induced fi- brotic mouse lungs demonstrated a primarily profibrotic M2-like profile that was distinct from the well-defined M1 or any of the M2 subtypes. More importantly, we found that fibrotic lung alveolar macrophages assumed augmented glycolysis, which was likely attributed to enhanced expression of multiple key glycolytic mediators. We also found that fatty acid oxidation was upregulated in these cells. However, the profibrotic M2-like profile of fibrotic lung alveolar macrophages was not dependent on fatty acid oxidation and synthesis or lipolysis, but instead on glycolysis, in contrast to the typical IL-4-induced macrophages M(IL-4). Additionally, glutaminolysis, a key metabolic program that has been implicated in numerous pathologies, was not required for the profibrotic M2-like phenotype of these macrophages. In summary, our study identifies a unique expression and metabolic profile in alveolar macrophages from fibrotic lungs and suggests glycolytic inhibition as an effective antifibrotic strategy in treating lung fibrosis. [ABSTRACT FROM AUTHOR]

Downregulation of the alveolar macrophage (AM) receptor with collagenous structure (MARCO) leads to susceptibility to postinfluenza bacterial pneumonia, a major cause of morbidity and mortality. We sought to determine whether immunomodulation of MARCO could improve host defense and resistance to secondary bacterial pneumonia. RNAseq analysis identified a striking increase in MARCO expression between days 9 and 11 after influenza infection and indicated important roles for Akt and Nrf2 in MARCO recovery. In vitro, primary human AM-like monocyte-derived macrophages (AMMDMs) and THP-1 macrophages were treated with IFNγ to model influenza effects. Activators of Nrf2 (sulforaphane) or Akt (SC79) caused increased MARCO expression and a MARCO-dependent improvement in phagocytosis in IFNγ-treated cells and improved survival in mice with postinfluenza pneumococcal pneumonia. Transcription factor analysis also indicated a role for transcription factor E-box (TFEB) in MARCO recovery. Overexpression of TFEB in THP-1 cells led to marked increases in MARCO. The ability of Akt activation to increase MARCO expression in IFNγ-treated AMMDMs was abrogated in TFEB-knockdown cells, indicating Akt increases MARCO expression through TFEB. Increasing MARCO expression by targeting Nrf2 signaling or the Akt-TFEB-MARCO pathway are promising strategies to improve bacterial clearance and survival in postinfluenza bacterial pneumonia. [ABSTRACT FROM AUTHOR]

Lung injury can release intracellular actin into the alveolar milieu and is also associated with increased susceptibility to secondary infections. We investigated the effect of free (extracellular) actin on lung macrophage host defense functions. Western blot analysis demonstrated free actin release into the lung lavage fluids of mouse models of ozone injury, influenza infection, and secondary pneumococcal pneumonia and in samples from patients following burn and inhalation injury. Using levels comparable with those observed in lung injury, we found that free actin markedly inhibited murine lung macrophage binding and uptake in vitro of S. pneumoniae, S. aureus, and E. coli, (e.g., S. pneumoniae, mean %inhibition, actin vs. vehicle: 85 ± 0.3 (SD); n = 22, P < .001). Similar effects were observed on the ability of primary human macrophages to bind and ingest fluorescent S. aureus (75% inhibition). Plasma gelsolin (pGSN), a protein that functions to bind and cleave actin, restored bacterial binding and uptake by both murine and human macrophages. Scavenger receptor inhibitors reduced binding of fluorescent actin by murine macrophages [fluorescence index (x10-3) after incubation with vehicle, actin, or actin _ polyinosinic acid, respectively: 0.8 ± 0.7, 101.7 ± 50.7, or 52.7 ± 16.9; n = 5-6, P < 0.05]. In addition, actin binding was reduced in a MARCO/SRAI/II-deficient cell line and by normal AMs obtained from MARCO-/- mice. After release from injured cells during lung injury, free actin likely contributes to impaired host defense by blocking scavenger receptor binding of bacteria. This mechanism for increased risk of secondary infections after lung injury or inflammation may represent another target for therapeutic intervention with pGSN. [ABSTRACT FROM AUTHOR]

Keratinocyte growth factor (KGF) is an epithelial mitogen that has been reported to protect the lungs from a variety of toxic and infectious insults. In prior studies we found that recombinant human KGF accelerates clearance of bacteria from the murine lung by augmenting the function of alveolar macrophages (AM). In this study we tested the hypothesis that endogenous KGF plays a role in the maintenance of innate pulmonary defense against gram-negative bacterial infections. KGF-deficient mice exhibited delayed clearance of Escherichia coli from the lungs, attenuated phagocytosis by AM, and decreased antimicrobial activity in bronchoalveolar lavage (BAL) fluid, due in part to reductions in levels of surfactant protein A, surfactant protein D, and lysozyme. These immune deficits were accompanied by lower alveolar type II epithelial cell counts and reduced alveolar type II epithelial cell expression of collectin and lysozyme genes on a per cell basis. No significant between-group differences were detected in selected inflammatory cytokines or BAL inflammatory cell populations at baseline or after bacterial challenge in the wild-type and KGF-deficient mice. A single intranasal dose of recombinant human KGF reversed defects in bacterial clearance, AM function, and BAL fluid antimicrobial activity. We conclude that KGF supports alveolar innate immune defense through maintenance of alveolar antimicrobial protein levels and functions of AM. Together these data demonstrate a role for endogenous KGF in maintenance of normal pulmonary innate immune function. [ABSTRACT FROM AUTHOR]

The most severe complication of influenza is viral pneumonia, which can lead to the acute respiratory distress syndrome. Alveolar epithelial cells (AECs) are the first cells that influenza virus encounters upon entering the alveolus. Infected epithelial cells produce cytokines that attract and activate neutrophils and macrophages, which in turn induce damage to the epithelial-endothelial barrier. Hepatocyte growth factor (HGF)/c-Met and transforming growth factor-α (TGF-α)/epidermal growth factor receptor (EGFR) are well known to regulate repair of damaged alveolar epithelium by stimulating cell migration and proliferation. Recently, TGF-α/EGFR signaling has also been shown to regulate innate immune responses in bronchial epithelial cells. However, little is known about whether HGF/c-Met signaling alters the innate immune responses and whether the innate immune responses in AECs are regulated by HGF/c-Met and TGF-α/EGFR. We hypothesized that HGF/c-Met and TGF-α/EGFR would regulate innate immune responses to influenza A virus infection in human AECs. We found that recombinant human HGF (rhHGF) and rhTGF-α stimulated primary human AECs to secrete IL-8 and granulocyte macrophage colony-stimulating factor (GM-CSF) strongly and IL-6 and monocyte chemotactic protein 1 moderately. Influenza infection stimulated the secretion of IL-8 and GM-CSF by AECs plated on rat-tail collagen through EGFR activation likely by TGF-α released from AECs and through c-Met activated by HGF secreted from lung fibroblasts. HGF secretion by fibroblasts was stimulated by AEC production of prostaglandin E2 during influenza infection. We conclude that HGF/c-Met and TGF-α/EGFR signaling enhances the innate immune responses by human AECs during influenza infections. [ABSTRACT FROM AUTHOR]

Alveolar macrophage (AM) immune function depends on the activation of the transcription factor PU.1 by granulocyte macrophage colony-stimulating factor. We have determined that chronic alcohol ingestion dampens PU.1 signaling via an unknown zinc-dependent mechanism; specifically, although PU.1 is not known to be a zinc-dependent transcription factor, zinc treatment reversed alcohol-mediated dampening of PU.1 signaling. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a zinc-dependent basic leucine zipper protein essential for antioxidant defenses, is also impaired by chronic alcohol ingestion and enhanced by zinc treatment. We hypothesized that the response of PU.1 to zinc treatment may result from the action of Nrf2 on PU.1. We first performed Nrf2/PU.1 protein coimmunoprecipitation on a rat AM cell line (NR8383) and found no evidence of protein-protein interactions. We then found evidence of increased Nrf2 binding to the PU.1 promoter region by chromatin immunoprecipitation. We next activated Nrf2 using either sulforaphane or an overexpression vector and inhibited Nrf2 with silencing RNA to determine whether Nrf2 could actively regulate PU.1. Nrf2 activation increased protein expression of both factors as well as gene expression of their respective downstream effectors, NAD(P)H dehydrogenase[quinone] 1 (NQO1) and cluster of differentiation antigen-14 (CD14). In contrast, Nrf2 silencing decreased the expression of both proteins, as well as gene expression of their effectors. Activating and inhibiting Nrf2 in primary rat AMs resulted in similar effects. Taken together, these findings suggest that Nrf2 regulates the expression and activity of PU.1 and that antioxidant response and immune activation are coordinately regulated within the AM. [ABSTRACT FROM AUTHOR]

Hematopoietic stem cell transplantation (HSCT) is complicated by pulmonary infections that manifest posttransplantation. Despite engraftment, susceptibility to infections persists long after reconstitution. Previous work using a murine bone marrow transplant (BMT) model implicated increased cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in promoting impaired alveolar macrophage (AM) responses. However, mechanisms driving COX-2 overexpression remained elusive. Previously, transforming growth factor-β (TGF-β) signaling after BMT was shown to promote hypomethylation of the COX-2 gene. Here, we provide mechanistic insight into how this occurs and show that TGF-β induces microRNA (miR)-29b while decreasing DNA methyltransferases (DNMT)1, DNMT3a, and DNMT3b in AMs after BMT. De novo DNMT3a and DNMT3b were decreased upon transient transfection of miR-29b, resulting in decreased methylation of the COX-2 promoter and induction of COX-2. As a consequence, miR-29b-driven upregulation of COX-2 promoted AM dysfunction, and transfection of BMT AMs with a miR-29b inhibitor rescued the bacterial-killing defect. MiR-29b-mediated defects in BMT AMs were dependent on increased levels of PGE2, as miR-29b-transfected AMs treated with a novel E prostanoid receptor 2 antagonist abrogated the impaired bacterial killing. We also demonstrate that patients that have undergone HSCT exhibit increased miR-29b; thus these studies highlight miR-29b in driving defective AM responses and identify this miRNA as a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Excessive neutrophil activation is a major component in septic lung injury. Neutrophil-derived DNA may form extracellular traps in response to bacterial invasions. The aim of the present study was to investigate the potential role of neutrophil extracellular traps (NETs) in septic lung injury. Male C57BL/6 mice were treated with recombinant human (rh)DNAse (5 mg/kg) after cecal ligation and puncture (CLP). Extracellular DNA was stained by Sytox green, and NET formation was quantified by confocal microscopy and cell-free DNA in plasma, peritoneal cavity, and lung. Blood, peritoneal fluid, and lung tissue were harvested for analysis of neutrophil infiltration, NET levels, tissue injury, as well as CXC chemokine and cytokine formation. We observed that CLP caused increased formation of NETs in plasma, peritoneal cavity, and lung. Administration of rhDNAse not only eliminated NET formation in plasma, peritoneal cavity, and bronchoalveolar space but also reduced lung edema and tissue damage 24 h after CLP induction. Moreover, treatment with rhDNAse decreased CLP-induced formation of CXC chemokines, IL-6, and high-mobility group box 1 (HMGB1) in plasma, as well as CXC chemokines and IL-6 in the lung. In vitro, we found that neutrophil-derived NETs had the capacity to stimulate secretion of CXCL2, TNF-, and HMGB1 from alveolar macrophages. Taken together, our findings show that NETs regulate pulmonary infiltration of neutrophils and tissue injury via formation of proinflammatory compounds in abdominal sepsis. Thus we conclude that NETs exert a proinflammatory role in septic lung injury. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae (Kp) is a Gram-negative bacterium that can cause serious infections in humans. Autophagy-related gene 7 (Atg7) has been implicated in certain bacterial infections; however, the role of Atg7 in macrophage-mediated immunity against Kp infection has not been elucidated. Here we showed that Atg7 expression was significantly increased in murine alveolar macrophages (MH-S) upon Kp infection, indicating that Atg7 participated in host defense. Knocking down Atg7 with small-interfering RNA increased bacterial burdens in MH-S cells. Using cell biology assays and whole animal imaging analysis, we found that compared with wild-type mice atg7 knockout (KO) mice exhibited increased susceptibility to Kp infection, with decreased survival rates, decreased bacterial clearance, and intensified lung injury. Moreover, Kp infection induced excessive proinflammatory cytokines and superoxide in the lung of atg7 KO mice. Similarly, silencing Atg7 in MH-S cells markedly increased expression levels of proinflammatory cytokines. Collectively, these findings reveal that Atg7 offers critical resistance to Kp infection by modulating both systemic and local production of proinflammatory cytokines. [ABSTRACT FROM AUTHOR]

Apoptosis of alveolar macrophages and their subsequent clearance by neighboring phagocytes are necessary steps in the resolution of acute pulmonary inflammation. We have recently identified that activation of the Fas death receptor on the cell surface of macrophages drives macrophage apoptosis. However, the source of the cognate ligand for Fas (FasL) responsible for induction of alveolar macrophage apoptosis is not defined. Given their known role in the resolution of inflammation and ability to induce macrophage apoptosis ex vivo, we hypothesized that T lymphocytes represented a critical source of FasL. To address this hypothesis, C57BL/6J and lymphocyte-deficient (Rag- 1-/-) mice were exposed to intratracheal lipopolysaccharide to induce pulmonary inflammation. Furthermore, utilizing mice expressing nonfunctional FasL, we adoptively transferred donor lymphocytes into inflamed lymphocyte-deficient mice to characterize the effect of lymphocyte-derived FasL on alveolar macrophage apoptosis in the resolution of inflammation. Herein, evidence is presented that lymphocytes expressing FasL enhance alveolar macrophage apoptosis during the resolution of LPS-induced inflammation. Moreover, lymphocyte induction of alveolar macrophage apoptosis results in contraction of the alveolar macrophage pool, which occurs in a FasLdependent manner. Specifically, FasL-expressing CD8+ T lymphocytes potently induce alveolar macrophage apoptosis and contraction of the alveolar macrophage pool. Together, these studies identify a novel role for CD8+ T lymphocytes in the resolution of acute pulmonary inflammation. [ABSTRACT FROM AUTHOR]

The article discusses a study on the role of glutathione (GSH) levels in ethanol-induced alveolar macrophage (AM) oxidative stress and dysfunction by downregulating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Topics covered include the effects of chronic alcohol consumption on the development of respiratory ailments including acute respiratory distress syndrome and pneumonia, the role of reactive oxygen species (ROS), and the effect of oxidative stress on ROS generation.

Female sex predisposes individuals to poorer outcomes during respiratory disorders like cystic fibrosis and influenza-associated pneumonia. A common link between these disorders is dysregulation of alveolar fluid clearance via disruption of epithelial sodium channel (ENaC) activity. Recent evidence suggests that female sex hormones directly regulate expression and activity of alveolar ENaC. In our study, we identified the mechanism by which estradiol (E2) or progesterone (P4) independently regulates alveolar ENaC. Using cell-attached patch clamp, we measured ENaC single-channel activity in a rat alveolar cell line (L2) in response to overnight exposure to either E2 or P4. In contrast to P4, E2 increased ENaC channel activity (NPo) through an increase in channel open probability (Po) and an increased number of patches with observable channel activity. Apical plasma membrane abundance of the ENaC -subunit (ENaC) more than doubled in response to E2 as determined by cell surface biotinylation. ENaC membrane abundance was approximately threefold greater in lungs from female rats in proestrus, when serum E2 is greatest, compared with diestrus, when it is lowest. Our results also revealed a significant role for the G protein-coupled estrogen receptor (Gper) to mediate E2's effects on ENaC. Overall, our results demonstrate that E2 signaling through Gper selectively activates alveolar ENaC through an effect on channel gating and channel density, the latter via greater trafficking of channels to the plasma membrane. The results presented herein implicate E2-mediated regulation of alveolar sodium channels in the sex differences observed in the pathogenesis of several pulmonary diseases. [ABSTRACT FROM AUTHOR]

Mechanical ventilation induces pulmonary apoptosis and inhibits alveolar development in preterm infants, but the molecular basis for the apoptotic injury is unknown. The objective was to determine the signaling mechanism(s) of ventilation (stretch)-induced apoptosis in newborn rat lung. Seven-day-old rats were ventilated with room air for 24 h using moderate tidal volumes (8.5 ml/kg). Isolated fetal rat lung epithelial and fibroblast cells were subjected to continuous cyclic stretch (5, 10, or 17% elongation) for up to 12 h. Prolonged ventilation significantly increased the number of apoptotic alveolar type II cells (i.e., terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling and anti-cleaved caspase-3 immunochemistry) and was associated with increased expression of the apoptotic mediator Fas ligand (FasL). Fetal lung epithelial cells, but not fibroblasts, subjected to maximal (i.e., 17%, but not lesser elongation) cyclic stretch exhibited increased apoptosis (i.e., nuclear fragmentation and DNA laddering), which appeared to be mediated via the extrinsic pathway (increased expression of FasL and cleaved caspase-3, -7, and -8). The intrinsic pathway appeared not to be involved [minimal mitochondrial membrane depolarization (JC-1 flow analysis) and no activation of caspase-9]. Universal caspases inhibition and neutralization of FasL abrogated the stretch-induced apoptosis. Prolonged mechanical ventilation induces apoptosis of alveolar type II cells in newborn rats and the mechanism appears to involve activation of the extrinsic death pathway via the FasL/Fas system. [ABSTRACT FROM AUTHOR]

Acrolein, an α,β unsaturated electrophile, is an environmental pollutant released in ambient air from diesel exhausts and cooking oils. This study examines the role of acrolein in altering mitochondrial function and metabolism in lung-specific cells. RLE-6TN, H441, and primary alveolar type II (pAT2) cells were exposed to acrolein for 4 h, and its effect on mitochondrial oxygen consumption rates was studied by XF Extracellular Flux analysis. Low-dose acrolein exposure decreased mitochondrial respiration in a dose-dependent manner because of alteration in the metabolism of glucose in all the three cell types. Acrolein inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, leading to decreased substrate availability for mitochondrial respiration in RLE-6TN, H441, and pAT2 cells; the reduced GAPDH activity was compensated in pAT2 cells by an increase in the activity of glucose-6-phosphate dehydrogenase, the regulatory control of the pentose phosphate pathway. The decrease in pyruvate from glucose metabolism resulted in utilization of alternative sources to support mitochondrial energy production: palmitate-BSA complex increased mitochondrial respiration in RLE-6TN and pAT2 cells. The presence of palmitate in alveolar cells for surfactant biosynthesis may prove to be the alternative fuel source for mitochondrial respiration. Accordingly, a decrease in phosphatidylcholine levels and an increase in phospholipase A2 activity were found in the alveolar cells after acrolein exposure. These findings have implications for understanding the decrease in surfactant levels frequently observed in pathophysiological situations with altered lung function following exposure to environmental toxicants. [ABSTRACT FROM AUTHOR]

Active Na+ transport mediated by epithelial Na+ channel (ENaC) is vital for fetal lung fluid reabsorption at birth and pulmonary edema resolution. Previously, we demonstrated that ENaC expression and activity are downregulated in alveolar epithelial cells by cycloheximide (Chx) and Pseudomonas aeruginosa. The regulatory mechanisms of ENaC mRNA expression by Chx and lipopolysaccharide (LPS) from P. aeruginosa were further studied in the present work. Both agents decreased ENaC mRNA expression to 50% of control values after 4 h. Chx repressed ENaC expression in a dose-dependent manner independently of protein synthesis. Although extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogenactivated protein kinase (MAPK) pathways were activated by the two treatments, their mechanisms of ENaC mRNA modulation were different. First, activation of the signaling pathways was sustained by Chx but only transiently by LPS. Second, ERK1/2 or p38 MAPK inhibition attenuated the effects of Chx on ENaC mRNA, whereas suppression of both signaling pathways was necessary to alleviate the outcome of LPS on ENaC mRNA. The molecular mechanisms involved in the decrease of ENaC expression were investigated in both conditions. LPS, but not Chx, significantly reduced ENaC promoter activity via the ERK1/2 and p38 MAPK pathways. These results suggest that LPS attenuates ENaC mRNA expression via diminution of transcription, whereas Chx could trigger some posttranscriptional mechanisms. Although LPS and Chx downregulate ENaC mRNA expression similarly and with similar signaling pathways, the mechanisms modulating ENaC expression are different depending on the nature of the cellular stress. [ABSTRACT FROM AUTHOR]

Organophosphorus pesticides (OPs) are implicated in human asthma. We previously demonstrated that, at concentrations that do not inhibit acetylcholinesterase activity, the OP parathion causes airway hyperreactivity in guinea pigs as a result of functional loss of inhibitory M2 muscarinic receptors on parasympathetic nerves. Because macrophages are associated with asthma, we investigated whether macrophages mediate parathion-induced M2 receptor dysfunction and airway hyperreactivity. Airway physiology was measured in guinea pigs 24 h after a subcutaneous injection of parathion. Pretreatment with liposome-encapsulated clodronate induced alveolar macrophage apoptosis and prevented parathion-induced airway hyperreactivity in response to electrical stimulation of the vagus nerves. As determined by qPCR, TNF-α and IL-1β mRNA levels were increased in alveolar macrophages isolated from parathion-treated guinea pigs. Parathion treatment of alveolar macrophages ex vivo did not significantly increase IL-1β and TNF-α mRNA but did significantly increase TNF-α protein release. Consistent with these data, pretreatment with the TNF-α inhibitor etanercept but not the IL-1β receptor inhibitor anakinra prevented parathion-induced airway hyperreactivity and protected M2 receptor function. These data suggest a novel mechanism of OP-induced airway hyperreactivity in which low-level parathion activates macrophages to release TNF-α-causing M2 receptor dysfunction and airway hyperreactivity. These observations have important implications regarding therapeutic approaches for treating respiratory disease associated with OP exposures. [ABSTRACT FROM AUTHOR]

Interleukin-8 (IL-8/ CXCL8) is an important neutrophil chemoattractant known to be elevated in the airways of cigarette smokers and in patients with chronic obstructive pulmonary disease (COPD). We examined the acute effect of aqueous cigarette smoke extract (CSE) on IL-8 expression in primary human pulmonary cells, in particular in normal human bronchial smooth muscle cells (HBSMCs). IL-8 mRNA levels increased upon CSE exposure in a concentration- and time-dependent manner, and such an effect was accompanied by IL-8 secretion. CSE-evoked elevation of IL-8 mRNA was mimicked by its component acrolein. Both CSE and acrolein induced p38 mitogen-activated protein kinase (MAPK) phosphorylation, accompanied by the phosphorylation of MAPK-activated kinase 2 (MK2), a known downstream substrate of the p38 MAPK, both in HBSMCs and in human airway epithelial cells. Furthermore, pharmacological inhibition of p38 MAPK or MK2 strongly accelerated the decay of IL-8 mRNA levels upon stimulation with CSE or acrolein and subsequent blockade of mRNA neosynthesis with actinomycin D in pulmonary structural cells (HBSMCs and airways epithelial cells) as well as in human alveolar macrophages. Conversely, pharmacological inhibition of ERK1/2 signaling inhibited CSE-induced steady-state levels of IL-8 mRNA without affecting mRNA stability, thus suggesting inhibition at the transcriptional level. In sum, p38 MAPK/MK2 signaling is an important posttranscriptional mechanism underlying upregulation of IL-8 mRNA levels elicited by CSE and acrolein. Given the pivotal role of IL-8 in neutrophil chemotaxis and activation, our results shed light on the mechanisms through which cigarette smoke can initiate inflammation in the lung. [ABSTRACT FROM AUTHOR]

Alveolar macrophages (AMs) are exposed to frequent challenges from inhaled particulates and microbes and function as a first line of defense with a highly regulated immune response because of their unique biology as prototypic alternatively activated macrophages. Lung collectins, particularly surfactant protein A (SP-A), contribute to this activation state by fine-tuning the macrophage inflammatory response. During short-term (10 min-2 h) exposure, SP-A's regulation of human macrophage responses occurs through decreased activity of kinases required for proinflammatory cytokine production. However, AMs are continuously exposed to surfactant, and the biochemical pathways underlying long-term reduction of proinflammatory cytokine activity are not known. We investigated the molecular mechanism(s) underlying SP-A- and surfactant lipid-mediated suppression of proinflammatory cytokine production in response to Toll-like receptor (TLR) 4 (TLR4) activation over longer time periods. We found that exposure of human macrophages to SP-A for 6-24 h upregulates expression of IL-1 receptor-associated kinase M (IRAK-M), a negative regulator of TLR-mediated NF-κB activation. Exposure to Survanta, a natural bovine lung extract lacking SP-A, also enhances IRAK-M expression, but at lower magnitude and for a shorter duration than SP-A. Surfactant-mediated upregulation of IRAK-M in macrophages suppresses TLR4-mediated TNF-α and IL-6 production in response to LPS, and IRAK-M knockdown by small interfering RNA reverses this suppression. In contrast to TNF-α and IL-6, the surfactant components upregulate LPS-mediated immunoregulatory IL-10 production, an effect reversed by IRAK-M knockdown. In conclusion, these data identify an important signaling regulator in human macrophages that is used by surfactant to control the long-term alveolar inflammatory response, i.e., enhanced IRAK-M activity. [ABSTRACT FROM AUTHOR]

Local pulmonary expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically important for defense of the pulmonary alveolar space. It is required for surfactant homeostasis and pulmonary innate immune responses and is protective against lung injury and aberrant repair. Alveolar epithelial cells (AEC) are a major source of GM-CSF; however, the control of homeostatic expression of GM-CSF is incompletely characterized. Increasing evidence suggests considerable plasticity of expression of AEC phenotypic characteristics. We tested the hypothesis that this plasticity extends to regulation of expression of GM-CSF using 1) MLE-12 cells (a commonly used murine cell line expressing some features of normal type II AEC, 2) primary murine AEC incubated under standard conditions [resulting in rapid spreading and loss of surfactant protein C (SP-C) expression with induction of the putative type I cell marker (T1α)], or 3) primary murine AEC on a hyaluronic acid/collagen matrix in defined medium, resulting in preservation of SP-C expression. AEC in standard cultures constitutively express abundant GM-CSF, with further induction in response to IL-1β but little response to TNF-α. In contrast, primary cells cultured to preserve SP-C expression and MLE-12 cells both express little GM-CSF constitutively, with significant induction in response to TNF-α and limited response to IL-1β. We conclude that constitutive and cytokine-induced expression of GM-CSF by AEC varies in concert with other cellular phenotypic characteristics. These changes may have important implications both for the maintenance of normal pulmonary homeostasis and for the process of repair following lung injury. [ABSTRACT FROM AUTHOR]

Cigarette smoke is the main cause of chronic obstructive pulmonary disease (COPD), where it can contribute to the observed airway inflammation. PGE2 is produced within human airways, and both pro- and anti-inflammatory activities have been reported. We quantitated PGE2 concentrations in induced sputum supematants from different groups of subjects and conelated the obtained values to neutrophil infiltration as well as to the expression of cyclooxygenase-2 (COX-2). Cigarette smoke extract (CSE) was used to evaluate the effect of smoking on COX-2 and PGE2 receptor expression as well as on PGE2 release in neutrophils and alveolar macrophages (AM) obtained from normal donors. The effects of PGE2 and of PGE receptor agonists and antagonists were evaluated on the adhesion of neutrophil to a human bronchial epithelial cell line (16HBE). PGE2 levels, COX-2 expression, and neutrophil infiltration were significantly higher in normal smokers and COPD smokers (P < 0.000 1) compared with controls and COPD former smokers. Induced sputum supernatant caused neutrophil adhesion to 16HBE that was significantly reduced, in COPD smokers only, by PGE2 immunoprecipitation. In vitro experiments confirmed that CSE increased PGE2 release and COX-2 and POE2 receptor expression in neutrophils and AM; PGE2 enhanced the adhesion of neutrophils to 16HBE, and a specific E-prostanoid 4 (EP4) receptor antagonist blunted its effect. These results suggest that CSE promote the induction of COX-2 and contributes to the proinflammatory effects of PGE2 in the airways of COPD subjects. [ABSTRACT FROM AUTHOR]

A significant role for alveolar macrophages (AM) in the pathophysiology of sepsis-induced acute lung injury (ALl) has been shown; however, the mechanisms behind AM-related lung injury remain relatively uncertain. We examined the role of AM nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in pulmonary endothelial cell septic injury. NADPH oxidase is one of the major sources of cellular reactive oxygen species and has been implicated in endothelial injury in ALL. Pulmonary microvascular endothelial cells (PMVEC) mono-layers were grown on Transwell inserts and incubated with wild-type and NADPH oxidase-deficient AM in the presence or absence of cytomix (equimolar TNF-α, IL-β, and IFN-γ). Injury to the mono layers was assessed by trans-PMVEC Evans blue (EB)-labeled albumin flux. We found AM under cytomix stimulation caused significant EB-albumin flux across the PMVEC monolayers, and this effect was attenuated by the genetic deletion of AM NADPH oxidase. The pharmacological inhibition of AM NADPH oxidase with apocynin and PR-39 also significantly reduced AM-dependent PMVEC injury. In the AM-PMVEC cocultures, we also assessed PMVEC injury through measurement of protein oxidation and lipid peroxidation. AM were shown to cause a significant increase in these markers of PMVEC injury, which was also attenuated by the inhibition of NADPH oxidase or through the use of NADPH oxidase-deficient AM. PMVEC NADPH oxidase was shown not to significantly contribute to PMVEC injury in our studies. From our findings we have concluded that AM NADPH oxidase is crucial for the septic increase in pulmonary vascular permeability. [ABSTRACT FROM AUTHOR]

Yamashita C, Forbes A, Tessolini JM, Yao L-J, Lewis JF, Veidhuizen RA. Protective effects of elevated endogenous surfactant pools to injurious mechanical ventilation. Am J Physiol Lung Cell Mo! Physiol 294: L724-L732, 2008. First published January 25, 2008; doi: 10.11 52/ajplung.00389.2007.-Depletion of alveolar macrophages (AM) leads to an increase in endogenous surfactant that lasts several days beyond the repletion of AM. Furthermore, impairment to the endogenous pulmonary surfactant system contributes to ventilation- induced lung injury. The objective of the current study was to determine whether increased endogenous surfactant pools induced via AM depletion was protective against ventilation-induced lung injury. Adult rats were intratracheally instilled with either control or dichlo- romethylene diphosphonic acid (DMDP) containing liposomes to deplete AMs and thereby increase endogenous surfactant pools. Either 3 or 7 days following instillation, rats were exposed to 2 h of injurious ventilation using either an ex vivo or in vivo ventilation protocol and were compared with nonventilated controls. The measured outcomes were oxygenation, lung compliance, lavage protein, and inflammatory cytokine concentrations. Compared with controls, the DMDP-treated animals had significantly reduced AM numbers and increased surfac- tant pools 3 days after instillation. Seven days after instillation, AM numbers had returned to normal, but surfactant pools were still elevated. DMDP-treated animals at both time points exhibited pro- tection against ventilation-induced lung injury, which included supe- rior physiological parameters, lower protein leakage, and lower in- flammatory mediator release into the air space, compared with ani- mals not receiving DMDP. It is concluded that DMDP-liposome administration protects against ventilation-induced lung injury. This effect appears to be due to the presence of elevated endogenous surfactant pools. [ABSTRACT FROM AUTHOR]

Surfactant protein D (SP-D), a member of the collectin superfamily, modulates pulmonary inflammatory responses and innate immunity. Disruption of the SP-D gene in mice induces peribronchiolar inflammation, accumulation of large, foamy macrophages, increased bronchoalveolar lavage (BAL) phospholipid, and pulmonary emphysema. We hypothesized that absence of SP-D aggravates hyperoxia-induced injury. To test this, SP-D-deficient (SP-D-/-) and wild-type (SP-D+/+) mice were exposed to 80% or 21% oxygen. Paradoxically, during 14 days of hyperoxia, SP-D-/- mice had 100% survival vs. 30% in SP-D+/+. The survival advantage in SP-D-/- mice was accompanied by lower histopathological injury scores at days 5 and 14, although total BAL cells (8.2 ± 1.4 × 105 in SP-D-/- vs. 4.04 ± 0.25 × 105 in SP-D+/+ mice) and neutrophils (1.2 ± 0.4 × 105 vs. 0.03 ± 0.02 × 105 in SP-D+/+ and SP-D+/+, respectively) were increased. In addition, BAL protein and lung-to-body weight ratios were similarly elevated in both groups after 3, 5, and 14 days of continuous exposure. Biochemically, in contrast to SP-D+/+, SP-D-/- mice had higher levels of surfactant phospholipid and SP-B at baseline and 5 days after hyperoxia accompanied by a preservation of surfactant biophysical activity. From a multiplex assay of nine cytokines, we found elevated levels of IL-13 in BAL fluid of normoxic SP-D-/- mice compared with SP-D+/+. We conclude that the resistance of SP-D-deficient mice to hyperoxia reflects homeostatic changes in the SP-D-/- phenotype involving both phospholipid and SP-B-mediated induced resistance of surfactant to inactivation as well as changes in the immunomodulatory BAL cytokine profile. [ABSTRACT FROM AUTHOR]

Mice gene targeted for ATP-binding cassette transporter A1 (ABCA1; Abca1-/-) have been shown to have low-serum high-density lipoprotein and abnormal lung morphology. We examined alterations in the structure and function of lungs from -/- mice (DBA1/J). Electron microscopy of the diseased mouse lung revealed areas of focal disease confirming previous results (47). Lipid analysis of the lung tissue of -/- mice showed a 1.2- and 1.4-fold elevation in total phospholipid (PL) and saturated phosphatidylcholine, respectively, and a marked 50% enrichment in total cholesterol content predominately due to a 17.5-fold increase in cholesteryl ester compared with wild type (WT). Lung surfactant in the -/- mice was characterized by alveolar proteinosis (161%), a slight increase in total PL (124%), and a marked increase in free cholesterol (155%) compared with WT. Alveolar macrophages were enriched in cholesterol (4.8-fold) due to elevations in free cholesterol (2.4-fold) and in cholesteryl ester (14.8-fold) compared with WT macrophages. More PL mass was cleared from the alveolar space of -/- mice lungs, measured using intratracheal installation of ³H-PL liposomes. Compared with WT mice, the Abca1-/- mice demonstrated respiratory distress with rapid, shallow breathing. Thus the lungs of mice lacking ABCA1 protein demonstrated abnormal morphology and physiology, with alveolar proteinosis and cholesterol enrichment of tissue, surfactant, and macrophages. The results indicate that the activity of ABCA1 is important for the maintenance of normal lung lipid composition, structure, and function. [ABSTRACT FROM AUTHOR]