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Hyperpolarized xenon 129 MRI and spectroscopy are sensitive to abnormal interstitial barrier uptake and red blood cell transfer in participants with nonspecific interstitial pneumonia.

Ozone (O3) is a criteria air pollutant known to increase the morbidity and mortality of cardiopulmonary diseases. This occurs through a pulmonary inflammatory response characterized by increased recruitment of immune cells into the airspace, pro-inflammatory cytokines, and pro-inflammatory lipid mediators. Recent evidence has demonstrated sex-dependent differences in the O3-induced pulmonary inflammatory response. However, it is unknown if this dimorphic response is evident in pulmonary lipid mediator metabolism. We hypothesized that there are sex-dependent differences in lipid mediator production following acute O3 exposure. Male and female C57BL/6J mice were exposed to 1 part per million O3 for 3 h and were necropsied at 6 or 24 h following exposure. Lung lavage was collected for cell differential and total protein analysis, and lung tissue was collected for mRNA analysis, metabololipidomics, and immunohistochemistry. Compared with males, O3-exposed female mice had increases in airspace neutrophilia, neutrophil chemokine mRNA, pro-inflammatory eicosanoids such as prostaglandin E2, and specialized pro-resolving mediators (SPMs), such as resolvin D5 in lung tissue. Likewise, precursor fatty acids (arachidonic and docosahexaenoic acid; DHA) were increased in female lung tissue following O3 exposure compared with males. Experiments with ovariectomized females revealed that loss of ovarian hormones exacerbates pulmonary inflammation and injury. However, eicosanoid and SPM production were not altered by ovariectomy despite depleted pulmonary DHA concentrations. Taken together, these data indicate that O3 drives an increased pulmonary inflammatory and bioactive lipid mediator response in females. Furthermore, ovariectomy increases susceptibility to O3-induced pulmonary inflammation and injury, as well as decreases pulmonary DHA concentrations.

The lung mucosa functions as a principal barrier between the body and inhaled environmental irritants and pathogens. Precise and targeted surveillance mechanisms are required at this lung-environment interface to maintain homeostasis and preserve gas exchange. This is performed by the innate immune system, a germline-encoded system that regulates initial responses to foreign irritants and pathogens. Environmental pollutants, such as particulate matter (PM), Ozone (O(3)), and other products of combustion (NO(2), SO(3), etc.) both stimulate and disrupt the function of the innate immune system of the lung, leading to the potential for pathologic consequences. PURPOSE OF REVIEW: The purpose of this review is to explore recent discoveries and investigations into the role of the innate immune system in responding to environmental exposures. This focuses on mechanisms by which the normal function of the innate immune system is modified by environmental agents leading to disruptions in respiratory function. RECENT FINDINGS: This is a narrative review of mechanisms of pulmonary innate immunity and the impact of environmental exposures on these responses. Recent findings highlighted in this review are categorized by specific components of innate immunity including epithelial function, macrophages, pattern recognition receptors, and the microbiome. Overall, the review supports broad impacts of environmental exposures to alterations to normal innate immune functions and have important implications for incidence and exacerbations of lung disease. SUMMARY: The innate immune system plays a critical role in maintaining pulmonary homeostasis in response to inhaled air pollutants. As many of these agents are unable to be mitigated, understanding their mechanistic impact is critical to develop future interventions to limit their pathologic consequences.

Defining responses of the structural and immune cells in biologic systems is critically important to understanding disease states and responses to injury. This requires accurate and sensitive methods to define cell types in organ systems. The principal method to delineate the cell populations involved in these processes is flow cytometry. Although researchers increasingly use flow cytometry, technical challenges can affect its accuracy and reproducibility, thus significantly limiting scientific advancements. This challenge is particularly critical to lung immunology, as the lung is readily accessible and therefore used in preclinical and clinical studies to define potential therapeutics. Given the importance of flow cytometry in pulmonary research, the American Thoracic Society convened a working group to highlight issues and technical challenges to the performance of high-quality pulmonary flow cytometry, with a goal of improving its quality and reproducibility.

Rationale: Interstitial macrophages (IMs) and airspace macrophages (AMs) play critical roles in lung homeostasis and host defense, and are central to the pathogenesis of a number of lung diseases. However, the absolute numbers of macrophages and the precise anatomic locations they occupy in the healthy human lung have not been quantified. Objectives: To determine the precise number and anatomic location of human pulmonary macrophages in nondiseased lungs and to quantify how this is altered in chronic cigarette smokers. Methods: Whole right upper lobes from 12 human donors without pulmonary disease (6 smokers and 6 nonsmokers) were evaluated using design-based stereology. CD206 (cluster of differentiation 206)-positive/CD43(+) AMs and CD206(+)/CD43(−) IMs were counted in five distinct anatomical locations using the optical disector probe. Measurements and Main Results: An average of 2.1 × 10(9) IMs and 1.4 × 10(9) AMs were estimated per right upper lobe. Of the AMs, 95% were contained in diffusing airspaces and 5% in airways. Of the IMs, 78% were located within the alveolar septa, 14% around small vessels, and 7% around the airways. The local density of IMs was greater in the alveolar septa than in the connective tissue surrounding the airways or vessels. The total number and density of IMs was 36% to 56% greater in the lungs of cigarette smokers versus nonsmokers. Conclusions: The precise locations occupied by pulmonary macrophages were defined in nondiseased human lungs from smokers and nonsmokers. IM density was greatest in the alveolar septa. Lungs from chronic smokers had increased IM numbers and overall density, supporting a role for IMs in smoking-related disease.

Exposure to ozone (O(3)) induces lung injury, pulmonary inflammation, and alters lipid metabolism. During tissue inflammation, specialized pro-resolving lipid mediators (SPMs) facilitate the resolution of inflammation. SPMs regulate the pulmonary immune response during infection and allergic asthma; however, the role of SPMs in O(3)-induced pulmonary injury and inflammation is unknown. We hypothesize that O(3) exposure induces pulmonary inflammation by reducing SPMs. To evaluate this, male C57Bl/6J mice were exposed to filtered air (FA) or 1 ppm O(3) for 3 h and necropsied 24 h after exposure. Pulmonary injury/inflammation was determined by bronchoalveolar lavage (BAL) differentials, protein, and lung tissue cytokine expression. SPMs were quantified by liquid chromatography tandem mass spectrometry and SPM receptors leukotriene B(4) receptor 1 (BLT-1), formyl peptide receptor 2 (ALX/FPR2), chemokine-like receptor 1 (ChemR23), and SPM-generating enzyme (5-LOX and 12/15-LOX) expression were measured by real time PCR. 24 h post-O(3) exposure, BAL PMNs and protein content were significantly increased compared to FA controls. O(3)-induced lung inflammation was associated with significant decreases in pulmonary SPM precursors (14-HDHA, 17-HDHA), the SPM PDX, and in pulmonary ALX/FPR2, ChemR23, and 12/15-LOX expression. Exogenous administration of 14-HDHA, 17-HDHA, and PDX 1 h prior to O(3) exposure rescued pulmonary SPM precursors/SPMs, decreased proinflammatory cytokine and chemokine expression, and decreased BAL macrophages and PMNs. Taken together, these data indicate that O(3)-mediated SPM reductions may drive O(3)-induced pulmonary inflammation.

Elevated ambient temperatures and extreme weather events have increased the incidence of wildfires world-wide resulting in increased wood smoke particle (WSP). Epidemiologic data suggests that WSP exposure associates with exacerbations of respiratory diseases, and with increased respiratory viral infections. To assess the impact of WSP exposure on host response to viral pneumonia, we performed WSP exposures in rodents followed by infection with mouse adapted influenza (HINI-PR8). C57BL/6 male mice aged 6–8 weeks were challenged with WSP or PBS by oropharyngeal aspiration in acute (single dose) or sub-acute exposures (day 1, 3, 5, 7 and 10). Additional groups underwent sub-acute exposure followed by infection by influenza or heat-inactivated (HI) virus. Following exposures/infection, bronchoalveolar lavage (BAL) was performed to assess for total cell counts/differentials, total protein, protein carbonyls and hyaluronan. Lung tissue was assessed for viral counts by real time PCR. When compared to PBS, acute WSP exposure associated with an increase in airspace macrophages. Alternatively, sub-acute exposure resulted in a dose dependent increase in airspace neutrophils. Sub-acute WSP exposure followed by influenza infection was associated with improved respiratory viral outcomes including reduced weight loss and increased blood oxygen saturation, and decreased protein carbonyls and viral titers. Flow cytometry demonstrated dynamic changes in pulmonary macrophage and T cell subsets based on challenge with WSP and influenza. This data suggests that sub-acute WSP exposure can improve host response to acute influenza infection.

Inhalational exposure to crystalline silica is linked to several debilitating systemic autoimmune diseases characterized by a prominent humoral immune component, but the mechanisms by which silica induces autoantibodies is poorly understood. To better understand how silica lung exposure breaks B cell tolerance and unleashes autoreactive B cells, we exposed both wildtype mice of healthy C57BL/6 and lupus-prone BXSB, MRL, and NZB strains and mice carrying an autoantibody transgene on each of these backgrounds to instilled silica or vehicle and monitored lung injury, autoimmunity, and B cell fate. Silica exposure induced lung damage and pulmonary lymphoid aggregates in all strains, including in genetically diverse backgrounds and in autoantibody transgenic models. In wildtype mice strain differences were observed in specificity of autoantibodies and site of enhanced autoantibody production, consistent with genetic modulation of the autoimmune response to silica. The unique autoantibody transgene reporter system permitted the in vivo fate of autoreactive B cells and tolerance mechanisms to be tracked directly, and demonstrated the presence of transgenic B cells and antibody in pulmonary lymphoid aggregates and bronchoalveolar lavage fluid, respectively, as well as in spleen and serum. Nonetheless, B cell enumeration and transgenic antibody quantitation indicated that B cell deletion and anergy were intact in the different genetic backgrounds. Thus, silica exposure sufficient to induce substantial lung immunopathology did not overtly disrupt central B cell tolerance, even when superimposed on autoimmune genetic susceptibility. This suggests that silica exposure subverts tolerance at alternative checkpoints, such as regulatory cells or follicle entry, or requires additional interactions or co-exposures to induce loss of tolerance. This possibility is supported by results of differentiation assays that demonstrated transgenic autoantibodies in supernatants of Toll-like receptor (TLR)7/TLR9-stimulated splenocytes harvested from silica-exposed, but not vehicle-exposed, C57BL/6 mice. This suggests that lung injury induced by silica exposure has systemic effects that subtly alter autoreactive B cell regulation, possibly modulating B cell anergy, and that can be unmasked by superimposed exposure to TLR ligands or other immunostimulants.

Radiation–induced pulmonary fibrosis (RTPF) is a progressive, serious condition in many subjects treated for thoracic malignancies or after accidental nuclear exposure. No biomarker exists for identifying the irradiated subjects most susceptible to pulmonary fibrosis (PF). Previously, we determined that gastrin-releasing peptide (GRP) was elevated within days after birth in newborns exposed to hyperoxia who later developed chronic lung disease. The goal of the current study was to test whether radiation (RT) exposure triggers GRP release in mice and whether this contributes to RTPF in vivo. We determined urine GRP levels and lung GRP immunostaining in mice 0 to 24 after post-thoracic RT (15 Gy). Urine GRP levels were significantly elevated between 24 hours post-RT; GRP-blocking monoclonal antibody 2A11, given minutes post-RT, abrogated urine GRP levels by 6 to 12 hours and also altered phosphoprotein signaling pathways at 24 hours post-RT. Strong extracellular GRP immunostaining was observed in lung at 6 hours post-RT. Mice given one dose of GRP monoclonal antibody 2A11 24 hours post-RT had significantly reduced myofibroblast accumulation and collagen deposition 15 weeks later, indicating protection against lung fibrosis. Therefore, elevation of urine GRP could be predictive of RTPF development. In addition, transient GRP blockade could mitigate PF in normal lung after therapeutic or accidental RT exposure.

Sex differences clearly exist in incidence, susceptibility, and severity of airway disease and in pulmonary responses to air pollutants such as ozone (O3). Prior rodent O3 exposure studies demonstrate sex-related differences in the expression of lung inflammatory mediators and signaling. However, whether or not sex modifies O3-induced airway physiologic responses remains less explored. To address this, we exposed 8- to 10-week-old male and female C57BL/6 mice to either 1 or 2 ppm O3 or filtered air (FA) for 3 h. At 12, 24, 48, and 72 h following exposure, we assessed airway hyperresponsiveness to methacholine (MCh), bronchoalveolar lavage fluid cellularity, cytokines and total protein/albumin, serum progesterone, and whole lung immune cells by flow cytometry. Male mice generated consistent airway hyperresponsiveness to MCh at all time points following exposure. Alternatively, females had less consistent airway physiologic responses to MCh, which were more variable between individual experiments and did not correlate with serum progesterone levels. Bronchoalveolar lavage fluid total cells peaked at 12 h and were persistently elevated through 72 h. At 48 h, bronchoalveolar lavage cells were greater in females versus males. Bronchoalveolar lavage fluid cytokines and total protein/albumin increased following O3 exposure without sex differences. Flow cytometry of whole lung tissue identified dynamic O3-induced immune cell changes also independent of sex. Our results indicate sex differences in acute O3-induced airway physiology responses and airspace influx without significant difference in other injury and inflammation measures. This study highlights the importance of considering sex as a biological variable in acute O3-induced airway physiology responses.

Previous studies of the lung microbiome have focused on characterizing the community and attempts to understand the role of community membership concerning disease or exposures such as cigarette smoke. However, we still lack an understanding of two critical aspects of the lung microbiome: the origin of the community members and their fate. As we continue to better understand how the lung microbiome influences human health, it is essential to determine how the environment shapes the lung microbiome membership. Using a pig model, we explored the relationship that the surrounding environment has on the resident lung bacteria by collecting environmental samples (soil, air, water, feed) to compare with lung samples (swab, lavage, and tissue). Results suggest that airborne bacteria make up the highest portion of the lung microbiome. Furthermore, bacteria from samples taken from the bronchioles can be correctly identified by which farm they originated, whereas those from alveolar samples are indistinguishable. The findings suggest that while the environment may shape the microbiome of the bronchioles, a distinct community exists within the alveoli. Our findings expand upon the current understanding of the lung microbiome and provide a model of how microbial communities within the lung relate to their surrounding environment.

Clear identification of specific cell populations by flow cytometry is important to understand functional roles. A well-defined flow cytometry panel for myeloid cells in human bronchoalveolar lavage (BAL) and lung tissue is currently lacking. The objective of this study was to develop a flow cytometry-based panel for human BAL and lung tissue. We obtained and performed flow cytometry/sorting on human BAL cells and lung tissue. Confocal images were obtained from lung tissue using antibodies for cluster of differentiation (CD)206, CD169, and E cadherin. We defined a multicolor flow panel for human BAL and lung tissue that identifies major leukocyte populations. These include macrophage (CD206(+)) subsets and other CD206(-) leukocytes. The CD206(-) cells include: (1) three monocyte (CD14(+)) subsets, (2) CD11c(+) dendritic cells (CD14(-), CD11c(+), HLA-DR(+)), (3) plasmacytoid dendritic cells (CD14(-), CD11c(-), HLA-DR(+), CD123(+)), and (4) other granulocytes (neutrophils, mast cells, eosinophils, and basophils). Using this panel on human lung tissue, we defined two populations of pulmonary macrophages: CD169(+) and CD169(-) macrophages. In lung tissue, CD169(-) macrophages were a prominent cell type. Using confocal microscopy, CD169(+) macrophages were located in the alveolar space/airway, defining them as alveolar macrophages. In contrast, CD169(-) macrophages were associated with airway/alveolar epithelium, consistent with interstitial-associated macrophages. We defined a flow cytometry panel in human BAL and lung tissue that allows identification of multiple immune cell types and delineates alveolar from interstitial-associated macrophages. This study has important implications for defining myeloid cells in human lung samples.

Accurate and reproducible assessments of experimental lung injury and inflammation are critical for basic and translational research. In particular, investigators use various methods for BAL and euthanasia; however, the impact of these methods on assessments of injury and inflammation is unknown. To define potential effects, we compared methods of lavage and euthanasia in uninjured mice and after a mild lung injury model (ozone). C57BL/6J male mice (8–10 weeks old) underwent BAL after euthanasia with ketamine/xylazine, carbon dioxide (CO(2)), or isoflurane. BAL methods included 800 μl of isotonic solution instilled and withdrawn three times, and one or three passive fills and drainage to 20 cm H(2)O. Parallel experiments were performed 24 hours after 3 hours of ozone (O(3)) exposure at 2 ppm. BAL total cell counts/differentials and total protein/albumin were determined. Lung histology was evaluated for lung inflammation or injury. BAL cells were cultured and stimulated with PBS, PMA, or LPS for 4 hours and supernatants were evaluated for cytokine content. In uninjured mice, we observed differences due to the lavage and euthanasia methods used. The lavage method increased total cells and total protein/albumin in uninjured and O(3)-exposed mice, with the 800-μl instillation having the highest values. Isoflurane increased total BAL cells, whereas CO(2) euthanasia increased the total protein/albumin levels in uninjured mice. These effects limited our ability to detect differences in BAL injury measures after O(3) exposure. In conclusion, the method used for lavage and euthanasia affects measures of lung inflammation/injury and should be considered a variable in model assessments.

Multiparameter flow cytometry of human lungs allows for characterization, isolation, and examination of human pulmonary immune cell composition, phenotype, and function. Here we describe an approach to process lung tissues and then utilize a base antibody panel to define all of the major immune cell types in a single staining condition. This base antibody panel can also be used to identify major immune cell types in human blood and bronchoalveolar lavage (BAL) fluid.

Lung disease is a leading cause of morbidity and mortality worldwide. Innate immune responses in the lung play a central role in the pathogenesis of lung disease and the maintenance of lung health, and thus it is crucial to understand factors that regulate them. Hyaluronan is ubiquitous in the lung, and its expression is increased following lung injury and in disease states. Furthermore, hyaladherins like inter-α-inhibitor, tumor necrosis factor-stimulated gene 6, pentraxin 3 and versican are also induced and help form a dynamic hyaluronan matrix in injured lung. This review synthesizes present knowledge about the interactions of hyaluronan and its associated hyaladherins with the lung immune system, and the implications of these interactions for lung biology and disease.

Although the antibody-based recognition of cell-surface markers has been widely used for the identification of immune cells, overlap in the expression of markers by different cell types and the inconsistent use of antibody panels have resulted in a lack of clearly defined signatures for myeloid cell subsets. We developed a 10-fluorochrome flow cytometry panel for the identification and quantitation of myeloid cells in the lungs, including pulmonary monocytes, myeloid dendritic cells, alveolar and interstitial macrophages, and neutrophils. After the initial sorting of viable CD45(+) leukocytes, we detected three leukocyte subpopulations based on CD68 expression: CD68(-), CD68(low), and CD68(hi). Further characterization of the CD68(hi) population revealed CD45(+)/CD68(hi)/F4/80(+)/CD11b(-)/CD11c(+)/Gr1(-) alveolar macrophages and CD45(+)/CD68(hi)/F4/80(-)/CD11c(+)/Gr1(-)/CD103(+)/major histocompatibility complex (MHC) class II(hi) dendritic cells. The CD68(low) population contained primarily CD45(+)/CD68(low)/F4/80(+)/CD11b(+)/CD11c(+)/Gr1(-)/CD14(low) interstitial macrophages and CD45(+)/CD68(low)/F4/80(+)/CD11b(+)/CD11c(-)/Gr1(low)/CD14(hi) monocytes, whereas the CD68(-) population contained neutrophils (CD45(+)/CD68(-)/F4/80(-)/CD11b(+)/Gr1(hi)). The validity of cellular signatures was confirmed by a morphological analysis of FACS-sorted cells, functional studies, and the depletion of specific macrophage subpopulations using liposomal clodronate. We believe our approach provides an accurate and reproducible method for the isolation, quantification, and characterization of myeloid cell subsets in the lungs, which may be useful for studying the roles of myeloid cells during various pathological processes.

Primary cilia (PC) are solitary cellular organelles that play critical roles in development, homeostasis, and disease pathogenesis by modulating key signaling pathways such as Sonic Hedgehog and calcium flux. The antenna-like shape of PC enables them also to facilitate sensing of extracellular and mechanical stimuli into the cell, and a critical role for PC has been described for mesenchymal cells such as chondrocytes. However, nothing is known about the role of PC in airway smooth muscle cells (ASMCs) in the context of airway remodeling. We hypothesized that PC on ASMCs mediate cell contraction and are thus integral in the remodeling process. We found that PC are expressed on ASMCs in asthmatic lungs. Using pharmacological and genetic methods, we demonstrated that PC are necessary for ASMC contraction in a collagen gel three-dimensional model both in the absence of external stimulus and in response to the extracellular component hyaluronan. Mechanistically, we demonstrate that the effect of PC on ASMC contraction is, to a small extent, due to their effect on Sonic Hedgehog signaling and, to a larger extent, due to their effect on calcium influx and membrane depolarization. In conclusion, PC are necessary for the development of airway remodeling by mediating calcium flux and Sonic Hedgehog signaling.

Chronic obstructive pulmonary disease (COPD) is the fourth major cause of death in the United States and the only disease in the top ten causes of death with a rising incidence in the United States [1]. COPD is a slowly progressive disease characterized by airflow limitation, which is largely irreversible [2]. The development of pulmonary emphysema is a frequent observation in patients with COPD. The incidence of emphysema is reaching worldwide epidemic proportions and predicted to displace stroke as the third major worldwide cause of mortality by 2030. The pathologic feature of pulmonary emphysema is alveolar destruction with the loss of lung functional units. The development of emphysema is accompanied by accumulation of inflammatory cells such as macrophages and neutrophils in the airways and lung parenchyma. The molecular pathogenesis of emphysema includes both protease-antiprotease imbalance and oxidant stress [3–5]. Tobacco smoke is a dominant risk factor for the development of emphysema [6], which has been shown to induce oxidative stress and up-regulate genes responsible for protection from oxidant injury [7]. However, only 15–20% of smokers develop clinically recognized emphysema and approximately 25% of patients with emphysema are lifelong non-smokers [8]. These observations suggest both that host factors contribute to disease susceptibility and that additional environmental exposures are likely to contribute to disease pathogenesis [9]. There are currently no therapies available to slow the rate of decline of lung function in patients with emphysema. Understanding the fundamental mechanisms that contribute to disease pathogenesis could provide insight into novel therapeutic approaches to this common and devastating disease. The lungs are continuously exposed to environmental toxicants, which may lead to enhanced oxidant stress produced either by phagocytes or other cell types within the lung. Normally, the lungs can tolerate the stresses imposed by the ambient environment through the presence of well-developed antioxidant systems [10]. However, when the balance shifts in favor of oxidants, from either an excess of oxidants and/or depletion of its antioxidant responses, oxidative stress can occur. Previous studies have reported that markers of oxidative stress (8-isoprostanes) are elevated in the breath and serum of patients with COPD [11]. Numerous studies have demonstrated that the susceptibility of the lung to oxidative injury depends largely on the up-regulation of protective antioxidant systems [12]. An important transcriptional regulator of antioxidant pathways, Nuclear factor-erythroid factor 2 (Nrf2), has been demonstrated to be essential in cigarette smoke-induced emphysema in mice [13–14]. Nrf2-deficient mice have also shown exacerbated elastase-induced emphysema when compared with control mice [15]. These studies support the potential importance of oxidant stress in the pathobiology of pulmonary emphysema. Nrf2 has an essential protective role in the lungs against emphysema through the activation of antioxidant response elements (ARE) and induced transcription of potentially thousands of ARE-dependent genes. Studies demonstrate that Nrf2 exerts its protective effects through transcriptional activation of anti-proteases, as well as, antioxidants in alveolar macrophages [15]. Many studies have utilized downstream activity of AREs, such as NAD(P)H:quinone oxioreductase 1 (NQO1), as a readout for Nrf2 activity. However, the functional role of individual ARE-dependent genes in the pathogenesis of emphysema remains largely unexplored. NQO1 is a flavoprotein that catalyzes a two electron reduction of quinones, and requires NADH or NADPH as a cofactor. NQO1 may exert either anti-oxidant or pro-oxidant properties depending on the quinone substrate. NQO1 is highly induced by Nrf2 nuclear translocation and binding to AREs. In this setting, NQO1 may act as an antioxidant enzyme by regenerating antioxidant forms of ubiquinone and vitamin E quinone. The role of Nrf2 in human emphysema is supported, in part, by reduced levels of NQO1 in lung macrophages obtained from patients with emphysema [16] and the level of NQO1 from whole lung is inversely associated with severity of COPD [17]. These studies support that reduced Nrf2 activity is associated with emphysema, but do not address the functional role of NQO1 in disease progression. Based on the putative antioxidant potential of NQO1 and the association with reduced levels in human emphysema, we hypothesized that NQO1 is protective in the development of emphysema through attenuation of oxidant stress. In the present study, we demonstrate in vivo a critical protective role of NQO1 in oxidant-induced emphysematous lung disease utilizing mouse models. These novel observations highlight a direct functional role of NQO1 in the pathogenesis of emphysema and identify a potential novel therapeutic target for COPD.

The chemokine, CXCL10, and its cognate receptor, CXCR3, are important mediators of the pathobiology of lung fibrosis. Macrophages are a known source of CXCL10, but their specific source in the lung is poorly defined due to incomplete characterization of macrophage subpopulations. We recently developed a novel flow cytometric approach that discriminates resident alveolar macrophages from recruited exudative macrophages (ExMacs) after infectious lung injury. We hypothesized that ExMacs are present after noninfectious lung injury with bleomycin, and are a source of CXCL10. We found that ExMacs are recruited to the lung after injury, peaking at Day 7, then maintained through Day 28. ExMac recruitment was significantly reduced, but not abolished, in CCR2 null mice. ExMacs, but not alveolar macrophages, produce CXCL10, both constitutively and after stimulation with hyaluronan (HA) fragments. Interestingly, ExMac stimulation with LPS resulted in complete suppression of CXCL10. In contrast, ExMacs produced TNF-α and CXCL2/MIP-2 (Macrophage Inflammatory Protein-2) after stimulation with both HA and LPS. ExMacs were present in CXCR3 null mice after bleomycin, but produced minimal CXCL10. This impairment was overcome by administration of exogenous IFN-γ or IFN-γ with HA. Collectively, these data suggest that ExMacs are recruited and maintained in the lung after noninfectious lung injury, are a source of a variety of cytokines, but importantly, are essential for the production of antifibrotic CXCL10. Understanding the contribution of ExMacs to the pathobiology of lung injury and repair could lead to new treatment options for fibrosing lung diseases.

Pulmonary fibrosis (PF), a devastating disease affecting >105 Americans, can have environmental, autoimmune, or idiopathic causes. In lung, pulmonary neuroendocrine cells secrete GRP in response to...

The present study demonstrates that acute ozone exposure of healthy human subjects enhances lung immune responses to subsequent bacterial stimuli. This highlights common air pollutant exposures as modifiers of the intensity of pulmonary immune activation.

Inhalation of ambient ozone alters populations of lung macrophages. However, the impact of altered lung macrophage populations on the pathobiology of ozone is poorly understood. We hypothesized that subpopulations of macrophages modulate the response to ozone. We exposed C57BL/6 mice to ozone (2 ppm × 3 h) or filtered air. At 24 h after exposure, the lungs were harvested and digested and the cells underwent flow cytometry. Analysis revealed a novel macrophage subset present in ozone-exposed mice, which were distinct from resident alveolar macrophages and identified by enhanced Gr-1+ expression [Gr-1 macrophages (Gr-1 Macs)]. Further analysis showed that Gr-1+ Macs exhibited high expression of MARCO, CX3CR1, and NAD(P)H:quinone oxioreductase 1. Gr-1+ Macs were present in the absence of CCR2, suggesting that they were not derived from a CCR2-dependent circulating intermediate. Using PKH26-PCL to label resident phagocytic cells, we demonstrated that Gr-1 Macs were derived from resident lung cells. This new subset was diminished in the absence of CX3CR1. Interestingly, CX3CR1-null mice exhibited enhanced responses to ozone, including increased airway hyperresponsiveness, exacerbated neutrophil influx, accumulation of 8-isoprostanes and protein carbonyls, and increased expression of cytokines (CXCL2, IL-1β, IL-6, CCL2, and TNF-α). Our results identify a novel subset of lung macrophages, which are derived from a resident intermediate, are dependent upon CX3CR1, and appear to protect the host from the biological response to ozone.