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Asthma is a common respiratory disease currently affecting more than 300 million worldwide and is characterized by airway inflammation, hyperreactivity, and remodeling. It is a heterogeneous disease consisting of corticosteroid-sensitive T-helper cell type 2-driven eosinophilic and corticosteroid-resistant, T-helper cell type 17-driven neutrophilic phenotypes. One pathway recently described to regulate asthma pathogenesis is cholesterol trafficking. Scavenger receptors, in particular SR-BI (scavenger receptor class B type I), are known to direct cellular cholesterol uptake and efflux. We recently defined SR-BI functions in pulmonary host defense; however, the function of SR-BI in asthma pathogenesis is unknown. To elucidate the role of SR-BI in allergic asthma, SR-BI-sufficient (SR-BI

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.

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.

Hyperpolarized (129)Xe MRI has emerged as a novel means to evaluate pulmonary function via 3D mapping of ventilation, interstitial barrier uptake, and RBC transfer. However, the physiological interpretation of these measurements has yet to be firmly established. Here, we propose a model that uses the three components of (129)Xe gas-exchange MRI to estimate accessible alveolar volume (V(A)), membrane conductance, and capillary blood volume contributions to DL(CO). (129)Xe ventilated volume (VV) was related to V(A) by a scaling factor k(V) = 1.47 with 95% confidence interval [1.42, 1.52], relative (129)Xe barrier uptake (normalized by the healthy reference value) was used to estimate the membrane-specific conductance coefficient k(B) = 10.6 [8.6, 13.6] mL/min/mmHg/L, whereas normalized RBC transfer was used to calculate the capillary blood volume-specific conductance coefficient k(R) = 13.6 [11.4, 16.7] mL/min/mmHg/L. In this way, the barrier and RBC transfer per unit volume determined the transfer coefficient K(CO), which was then multiplied by image-estimated V(A) to obtain DL(CO). The model was built on a cohort of 41 healthy subjects and 101 patients with pulmonary disorders. The resulting (129)Xe-derived DL(CO) correlated strongly (R(2) = 0.75, P < 0.001) with the measured values, a finding that was preserved within each individual disease cohort. The ability to use (129)Xe MRI measures of ventilation, barrier uptake, and RBC transfer to estimate each of the underlying constituents of DL(CO) clarifies the interpretation of these images while enabling their use to monitor these aspects of gas exchange independently and regionally. NEW & NOTEWORTHY The diffusing capacity for carbon monoxide (DL(CO)) is perhaps one of the most comprehensive physiological measures used in pulmonary medicine. Here, we spatially resolve and estimate its key components—accessible alveolar volume, membrane, and capillary blood volume conductances—using hyperpolarized (129)Xe MRI of ventilation, interstitial barrier uptake, and red blood cell transfer. This image-derived DL(CO) correlates strongly with measured values in 142 subjects with a broad range of pulmonary disorders.

BackgroundIncreased exposure to Ozone (O3) is associated with adverse health effects in individuals afflicted with respiratory diseases. Surfactant protein-A (SP-A), encoded bySP-A1andSP-A2, is the largest protein component in pulmonary surfactant and is functionally impaired by O3-oxidation.ObjectiveWe used humanized SP-A2 transgenic mice with allelic variation corresponding to a glutamine (Q) to lysine (K) amino acid substitution at position 223 in the lectin domain to determine the impact of this genetic variation in regards to O3exposure.MethodsMice were exposed to 2ppm O3or Filtered Air (FA) for 3 hours and 24 hrs post-challenge pulmonary function tests and other parameters associated with inflammation were assessed in the bronchoalveolar lavage (BAL) fluid and lung tissue. Additionally, mouse tracheal epithelial cells were cultured and TEER measurements recorded for each genotype to determine baseline epithelial integrity.ResultsCompared to FA, O3exposure led to significantly increased sensitivity to methacholine challenge in all groups of mice. SP-A2 223Q variant mice were significantly protected from O3-induced AHR compared to SP-A-/-and SP-A2 223K mice. Neutrophilia was observed in all genotypes of mice post O3-exposure, however, SP-A2 223Q mice had a significantly lower percentage of neutrophils compared to SP-A-/-mice. Albumin levels in BAL were unchanged in O3-exposed SP-A2 223Q mice compared to their FA controls, while levels were significantly increased in all other genotypes of O3-exposed mice. SP-A 223Q MTECS has significant higher TEER values than all other genotypes, and WT MTECS has significantly higher TEER than the SP-A KO and SP-A 223K MTECS.SignificanceTaken together, our study suggests that expression of a glutamine (Q) as position 223 in SP-A2, as opposed to expression of lysine (K), is more protective in acute exposures to ozone and results in attenuated O3-induced AHR, neutrophilia, and vascular permeability.

Background: Epidemiological studies support the hypothesis that diabetes alters pulmonary responses to air pollutants like ozone (O3). The mechanism(s) underlying these associations and potential links among diabetes, O3, and lung inflammation and remodeling are currently unknown. Objectives: The goal was to determine whether pulmonary responses to repetitive ozone exposures are exacerbated in murine strains that are hyperglycemic and insulin resistant. Methods: Normoglycemic and insulin-sensitive C57BL/6J mice; hyperglycemic, but mildly insulin-resistant, KK mice; and hyperglycemic and markedly insulin-resistant KKAy mice were used for ozone exposure studies. All animals were exposed to filtered air (FA) or repetitive ozone (0.5 ppm O3, 4 h/d, for 13 consecutive weekdays). Tissue analysis was performed 24 h following the final exposure. This analysis included bronchoalveolar lavage (BAL) for cell and fluid analysis, and tissue for pathology, immunohistology, mRNA, and hydroxyproline. Results: Following repetitive O3 exposure, higher bronchoalveolar lavage fluid inflammatory cells were observed in all mice (KKAy>KK>C57BL/6), with a notable influx of neutrophils and eosinophils in KK and KKAy mice. Although the lungs of O3-exposed C57BL/6J and KK mice had minimal centriacinar histological changes without fibrosis, the lungs of O3-exposed KKAy mice contained marked epithelial hyperplasia in proximal alveolar ducts and adjacent alveoli with associated centriacinar fibrosis. Fibrosis in O3-exposed KKAy lungs was confirmed with immunohistochemistry, tissue hydroxyproline content, and tissue mRNA expression of fibrosis-associated genes (Ccl11, Il13, and Mmp12). Immunofluorescence staining and confocal microscopy revealed alterations in the structure and composition of the airway and alveolar epithelium in regions of fibrosis. Discussion: Our results demonstrate that in diabetic animal strains repetitive ambient ozone exposure led to early and exaggerated pulmonary inflammation and remodeling. Changes in distal and interstitial airspaces and the activation of Th2 inflammatory and profibrotic pathways in experimental animals provide a preliminary, mechanistic framework to support the emerging epidemiological associations among air pollution, diabetes, and lung disease. https://doi.org/10.1289/EHP7255

Lung disease causes significant morbidity and mortality, and is exacerbated by environmental injury, for example through lipopolysaccharide (LPS) or ozone (O3). Toll-like receptors (TLRs) orchestrate immune responses to injury by recognizing pathogen- or danger-associated molecular patterns. TLR4, the prototypic receptor for LPS, also mediates inflammation after O3, triggered by endogenous hyaluronan. Regulation of TLR4 signaling is incompletely understood. TLR5, the flagellin receptor, is expressed in alveolar macrophages, and regulates immune responses to environmental injury. Using in vivo animal models of TLR4-mediated inflammations (LPS, O3, hyaluronan), we show that TLR5 impacts the in vivo response to LPS, hyaluronan and O3. We demonstrate that immune cells of human carriers of a dominant negative TLR5 allele have decreased inflammatory response to O3 exposure ex vivo and LPS exposure in vitro. Using primary murine macrophages, we find that TLR5 physically associates with TLR4 and biases TLR4 signaling towards the MyD88 pathway. Our results suggest an updated paradigm for TLR4/TLR5 signaling., eLife digest Immune cells in the lung help guard against infections. On the surface of these cells are proteins called TLR receptors that recognize dangerous molecules or DNA from disease-causing microbes such as bacteria. When the immune cells detect these invaders, the TLR receptors spring into action and trigger an inflammatory response to destroy the microbes. This inflammation usually helps the lung clear infections. But it can also be harmful and damage the lung, for example when inflammation is caused by non-infectious substances such as pollutants in the atmosphere. There are several TLR receptors that each recognize a specific molecule. In 2010, researchers showed that the receptor TLR4 is responsible for causing inflammation in the lung after exposure to pollution. Another receptor called TLR5 also helps activate the immune response in the lung. But it was unclear whether this receptor also plays a role in pollution-linked lung damage. Now, Hussain, Johnson, Sciurba et al. – including one of the researchers involved in the 2010 study – have investigated the role of TLR5 in immune cells from the lungs of humans and mice. The experiments showed that TLR5 works together with TLR4 and helps trigger an inflammatory response to both pollutants and bacteria. Hussain et al. found that people lacking a working TLR5 receptor (which make up 3–10% of the population) are less likely to experience lung inflammation when exposed to pollution or bacterial proteins that activate TLR4. These findings suggest that people without TLR5 may be protected from pollution-induced lung injury. Further research into the role of TLR5 could help develop genetic tests for identifying people who are more sensitive to damage from pollution. This information could then be used to determine the likelihood of a patient experiencing certain lung diseases.

RATIONALE Patients with asthma demonstrate depletion of the endogenous bronchodilator GSNO and upregulation of GSNOR. OBJECTIVES An exploratory proof of concept clinical study of N6022 in mild asthma to determine the potential bronchoprotective effects of GSNOR inhibition. Mechanistic studies aimed to provide translational evidence of effect. METHODS Fourteen mild asthma patients were treated with intravenous N6022 (5 mg) or placebo and observed for 7 days, with repeated assessments of the provocative dose of methacholine causing a 20% fall in FEV1 (methacholine PC20 FEV1), followed by a washout period and crossover treatment and observation. In vitro studies in isolated eosinophils investigated the effect of GSNO and N6022 on apoptosis. MEASUREMENTS AND MAIN RESULTS This was a negative trial as it failed to reach its primary endpoint, which was change from baseline in methacholine PC20 FEV1 at 24 h. However, our exploratory analysis demonstrated significantly more two dose-doubling increases in PC20 FEV1 for N6022 compared with placebo (21% vs 6%, P

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.

The innate immune components that modulate allergic contact hypersensitivity (CHS) responses are poorly defined. Using human skin from contact dermatitis patients and a mouse model of CHS, we find that hapten allergens disrupt the Arginase1 (Arg1) and inducible NO synthase (iNOS) dynamic in monocytes/macrophages (mono/MΦ), which renders those cells ineffectual in suppressing skin inflammation. Mice lacking Arg1 in MΦ develop increased CHS characterized by elevated ear thickening, mono/MΦ−dominated dermal inflammation, and increased iNOS and IL-6 expression compared with control mice. Treatment of Arg1flox/flox; LysMCre+/− mice with a selective NOS inhibitor or knockout of Nos2, encoding iNOS, significantly ameliorates CHS. Our findings suggest a critical role for Arg1 in mono/MΦ in suppressing CHS through dampening Nos2 expression. These results support that increasing Arg1 may be a potential therapeutic avenue in treating allergic contact dermatitis.

Lung disease causes significant morbidity and mortality, and is exacerbated by environmental injury, e.g. through lipopolysaccharide (LPS) or ozone (O3). Toll-like receptors (TLRs) orchestrate immune responses to injury by recognizing pathogen- or danger-associated molecular patterns. TLR4, the prototypic receptor for LPS, also mediates inflammation after O3, triggered by endogenous hyaluronan. Regulation of TLR4 signaling is incompletely understood. TLR5, the flagellin receptor, is expressed in alveolar macrophages, and regulates immune responses to environmental injury. Using in vivo animal models of TLR4-mediated inflammations (LPS, O3, hyaluronan), we show that TLR5 impacts the in vivo response to LPS, hyaluronan and O3. We demonstrate that immune cells of human carriers of a dominant negative TLR5 allele have decreased inflammatory response to O3 exposure ex vivo and LPS exposure in vitro. Using primary murine macrophages, we find that TLR5 physically associates with TLR4 and biases TLR4 signaling towards the MyD88 pathway. Our results suggest an updated paradigm for TLR4/TLR5 signaling.

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.

Objective: Provide information for pharmacists on idiopathic pulmonary fibrosis (IPF) and its treatment. Study Selection and Data Extraction: All articles with data from randomized controlled trials of nintedanib or pirfenidone were reviewed. Data Synthesis: IPF is a progressive and ultimately fatal interstitial lung disease characterized by decline in lung function and worsening dyspnea. It is uncommon and mainly occurs in individuals aged >60 years, particularly men with a history of smoking. Nintedanib and pirfenidone were approved in the United States for the treatment of IPF in 2014 and received conditional recommendations in the 2015 American Thoracic Society/European Respiratory Society/Japanese Respiratory Society/Latin American Thoracic Association treatment guidelines. These drugs slow the progression of IPF by reducing the rate of decline in lung function. Their adverse event profile is characterized mainly by gastrointestinal events, which can be managed through dose adjustment and symptom management. Management of IPF should also include smoking cessation, vaccinations, and supportive care such as patient education, pulmonary rehabilitation, and the use of supplemental oxygen as well as optimizing the management of comorbidities. Relevance to Patient Care and Clinical Practice: This review provides clinical pharmacists with information on the course of IPF, what can be expected of current treatments, and how to help patients manage their drug therapy. Conclusions: IPF is a progressive disease, but treatments are available that can slow the progression of the disease. Clinical pharmacists can play an important role in the care of patients with IPF through patient education, monitoring medication compliance and safety, ensuring drugs for comorbidities are optimized, and preventive strategies such as immunizations.

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.

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.

Assessing the immunotoxicity of xenobiotics by current regulatory testing has revealed compounds that can cause immunosuppression and stimulation. Flow cytometry is a cutting edge technique that can provide data on how toxicants can alter the quality and quantity of the immune response after exposure. Here we describe protocols for how to use flow cytometry to measure the immune response in multiple rodent organs (blood and lymphoid and nonlymphoid) as well as in novel models recently being utilized in the field of toxicology. These methods can be used for current testing and to determine mechanisms by which a xenobiotic can cause immunotoxicity.

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.

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.

Despite recent advances in understanding macrophage activation, little is known regarding how human alveolar macrophages in health calibrate its transcriptional response to canonical TLR4 activation. In this study, we examined the full spectrum of LPS activation and determined whether the transcriptomic profile of human alveolar macrophages is distinguished by a TIR-domain-containing adapter-inducing interferon-β (TRIF)-dominant type I interferon signature. Bronchoalveolar lavage macrophages were obtained from healthy volunteers, stimulated in the presence or absence of ultrapure LPS in vitro, and whole transcriptomic profiling was performed by RNA sequencing (RNA-Seq). LPS induced a robust type I interferon transcriptional response and Ingenuity Pathway Analysis predicted interferon regulatory factor (IRF)7 as the top upstream regulator of 89 known gene targets. Ubiquitin-specific peptidase (USP)-18, a negative regulator of interferon α/β responses, was among the top up-regulated genes in addition to IL10 and USP41, a novel gene with no known biological function but with high sequence homology to USP18. We determined whether IRF-7 and USP-18 can influence downstream macrophage effector cytokine production such as IL-10. We show that IRF-7 siRNA knockdown enhanced LPS-induced IL-10 production in human monocyte-derived macrophages, and USP-18 overexpression attenuated LPS-induced production of IL-10 in RAW264.7 cells. Quantitative PCR confirmed upregulation of USP18, USP41, IL10, and IRF7. An independent cohort confirmed LPS induction of USP41 and IL10 genes. These results suggest that IRF-7 and predicted downstream target USP18, both elements of a type I interferon gene signature identified by RNA-Seq, may serve to fine-tune early cytokine response by calibrating IL-10 production in human alveolar macrophages.

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.