Interleukin-2-Dependent Allergen-Specific Tissue-Resident Memory Cells Drive Asthma.

Exposure to inhaled allergens generates T helper 2 (Th2) CD4(+) T cells that contribute to episodes of inflammation associated with asthma. Little is known about allergen-specific Th2 memory cells and their contribution to airway inflammation. We generated reagents to understand how endogenous CD4(+) T cells specific for a house dust mite (HDM) allergen form and function. After allergen exposure, HDM-specific memory cells persisted as central memory cells in the lymphoid organs and tissue-resident memory cells in the lung. Experimental blockade of lymphocyte migration demonstrated that lung-resident cells were sufficient to induce airway hyper-responsiveness, which depended upon CD4(+) T cells. Investigation into the differentiation of pathogenic Trm cells revealed that interleukin-2 (IL-2) signaling was required for residency and directed a program of tissue homing migrational cues. These studies thus identify IL-2-dependent resident Th2 memory cells as drivers of lung allergic responses.

Regulation of versican expression in macrophages is mediated by canonical type I interferon signaling via ISGF3.

Growing evidence supports a role for versican as an important component of the inflammatory response, with both pro- and anti-inflammatory roles depending on the specific context of the system or disease under investigation. Our goal is to understand the regulation of macrophage-derived versican and the role it plays in innate immunity. In previous work, we showed that LPS triggers a signaling cascade involving Toll-like receptor (TLR)4, the Trif adaptor, type I interferons, and the type I interferon receptor, leading to increased versican expression by macrophages. In the present study, we used a combination of chromatin immunoprecipitation, siRNA, chemical inhibitors, and mouse model approaches to investigate the regulatory events downstream of the type I interferon receptor to better define the mechanism controlling versican expression. Results indicate that transcriptional regulation by canonical type I interferon signaling via interferon-stimulated gene factor 3 (ISGF3), the heterotrimeric transcription factor complex of Irf9, Stat1, and Stat2, controls versican expression in macrophages exposed to LPS. This pathway is not dependent on MAPK signaling, which has been shown to regulate versican expression in other cell types. The stability of versican mRNA may also contribute to prolonged versican expression in macrophages. These findings strongly support a role for macrophage-derived versican as a type I interferon-stimulated gene and further our understanding of versican's role in regulating inflammation.NEW & NOTEWORTHY We report the novel finding that versican expression is regulated by the interferon-stimulated gene factor 3 (ISGF3) arm of canonical type I Ifn signaling in LPS-stimulated macrophages. This pathway is distinct from mechanisms that control versican expression in other cell types. This suggests that macrophage-derived versican may play a role in limiting a potentially excessive inflammatory response. The detailed understanding of how versican expression is regulated in different cells could lead to unique approaches for enhancing its anti-inflammatory properties.

Distinct Epithelial-Innate Immune Cell Transcriptional Circuits Underlie Airway Hyperresponsiveness in Asthma.

Rationale: Indirect airway hyperresponsiveness (AHR) is a highly specific feature of asthma, but the underlying mechanisms responsible for driving indirect AHR remain incompletely understood. Objectives: To identify differences in gene expression in epithelial brushings obtained from individuals with asthma who were characterized for indirect AHR in the form of exercise-induced bronchoconstriction (EIB). Methods: RNA-sequencing analysis was performed on epithelial brushings obtained from individuals with asthma with EIB (n = 11) and without EIB (n = 9). Differentially expressed genes (DEGs) between the groups were correlated with measures of airway physiology, sputum inflammatory markers, and airway wall immunopathology. On the basis of these relationships, we examined the effects of primary airway epithelial cells (AECs) and specific epithelial cell-derived cytokines on both mast cells (MCs) and eosinophils (EOS). Measurements and Main Results: We identified 120 DEGs in individuals with and without EIB. Network analyses suggested critical roles for IL-33-, IL-18-, and IFN-γ-related signaling among these DEGs. IL1RL1 expression was positively correlated with the density of MCs in the epithelial compartment, and IL1RL1, IL18R1, and IFNG were positively correlated with the density of intraepithelial EOS. Subsequent ex vivo modeling demonstrated that AECs promote sustained type 2 (T2) inflammation in MCs and enhance IL-33-induced T2 gene expression. Furthermore, EOS increase the expression of IFNG and IL13 in response to both IL-18 and IL-33 as well as exposure to AECs. Conclusions: Circuits involving epithelial interactions with MCs and EOS are closely associated with indirect AHR. Ex vivo modeling indicates that epithelial-dependent regulation of these innate cells may be critical in indirect AHR and modulating T2 and non-T2 inflammation in asthma.

Rhinovirus infection of the airway epithelium enhances mast cell immune responses via epithelial-derived interferons.

BACKGROUND: Mast cells (MCs) within the airway epithelium in asthma are closely related to airway dysfunction, but cross talk between airway epithelial cells (AECs) and MCs in asthma remains incompletely understood. Human rhinovirus (RV) infections are key triggers for asthma progression, and AECs from individuals with asthma may have dysregulated antiviral responses. OBJECTIVE: We utilized primary AECs in an ex vivo coculture model system to examine cross talk between AECs and MCs after epithelial rhinovirus infection. METHODS: Primary AECs were obtained from 11 children with asthma and 10 healthy children, differentiated at air-liquid interface, and cultured in the presence of laboratory of allergic diseases 2 (LAD2) MCs. AECs were infected with rhinovirus serogroup A 16 (RV16) for 48 hours. RNA isolated from both AECs and MCs underwent RNA sequencing. Direct effects of epithelial-derived interferons on LAD2 MCs were examined by real-time quantitative PCR. RESULTS: MCs increased expression of proinflammatory and antiviral genes in AECs. AECs demonstrated a robust antiviral response after RV16 infection that resulted in significant changes in MC gene expression, including upregulation of genes involved in antiviral responses, leukocyte activation, and type 2 inflammation. Subsequent ex vivo modeling demonstrated that IFN-ß induces MC type 2 gene expression. The effects of AEC donor phenotype were small relative to the effects of viral infection and the presence of MCs. CONCLUSIONS: There is significant cross talk between AECs and MCs, which are present in the epithelium in asthma. Epithelial-derived interferons not only play a role in viral suppression but also further alter MC immune responses including specific type 2 genes.

CryoGrid-PIXUL-RNA: high throughput RNA isolation platform for tissue transcript analysis.

BACKGROUND: Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in tissue multiomics analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose 96-well microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream multiomics analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such biospecimens remain, in comparison, inefficient. RESULTS: To mitigate this tissue interrogation bottleneck, we have developed a low-cost user-friendly system, CryoGrid, to catalog, cryostore and sample tissue fragments. TRIzol is widely used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. Columns are also commonly used to extract RNA but they involve many steps, are prone to human errors, and are also expensive. Both TRIzol and column protocols use test tubes. We developed a microplate PIXUL-based TRIzol-free and column-free RNA isolation protocol that uses a buffer containing proteinase K (PK buffer). We have integrated the CryoGrid system with PIXUL-based PK buffer, TRIzol, and PureLink column methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. CryoGrid-PIXUL, when integrated with either PK buffer, TRIzol or PureLink column RNA isolation protocols, yielded similar transcript profiles in frozen organs (brain, heart, kidney and liver) from a mouse model of sepsis. CONCLUSIONS: RNA isolation using the CryoGrid-PIXUL system combined with the 96-well microplate PK buffer method offers an inexpensive user-friendly high throughput workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.

Lung pericytes as mediators of inflammation.

Pericytes are microvascular mural cells that directly contact endothelial cells. They have long been recognized for their roles in vascular development and homeostasis, but more recently have been identified as key mediators of the host response to injury. In this context, pericytes possess a surprising degree of cellular plasticity, behaving dynamically when activated and potentially participating in a range of divergent host responses to injury. Although there has been much interest in the role of pericytes in fibrosis and tissue repair, their involvement in the initial inflammatory process has been understudied and is increasingly appreciated. Pericytes mediate inflammation through leukocyte trafficking and cytokine signaling, respond to pathogen-associated molecular patterns and tissue damage-associated molecular patterns, and may drive vascular inflammation during human SARS-CoV-2 infection. In this review, we highlight the inflammatory phenotype of activated pericytes during organ injury, with an emphasis on novel findings relevant to pulmonary pathophysiology.

Multicompartmental analysis of the murine pulmonary immune response by spectral flow cytometry.

Studies of pulmonary inflammation require unique considerations due to the complex structure and composition of the lungs. The lungs have multiple compartments and diverse immune cell populations, with inherently high autofluorescence, and are involved in the host response to pulmonary pathogens. We describe a protocol that accounts for these factors through a novel combination of methodologies-in vivo compartmental analysis and spectral flow cytometry with a broad panel of antibodies. In vivo compartmental analysis enables the precise localization of immune cells within the marginated vasculature, lung interstitium, nonlavageable airways, and lavageable airways of the lungs, as well as the pulmonary lymph nodes. Spectral flow cytometry with a broad panel of antibodies supports an unbiased exploratory approach to investigating diverse immune cell populations during pulmonary inflammation. Most importantly, spectral flow uses cellular autofluorescence to aid in the resolution and identification of immune cell populations. This methodology enables the acquisition of high-quality data compatible with informed gating and dimensionality reduction algorithms. In addition, our protocol emphasizes considerations for compartmentalization of the inflammatory response, spectral flow panel design, and autofluorescence spectra analysis. These methodologies are critical for increasing the rigor of pulmonary research. We apply this protocol for the precise characterization and localization of leukocytes in the pulmonary host response to influenza A virus in C57BL/6J mice. In particular, we demonstrate that this protocol improves the quantification and localization of alveolar macrophages within the airways. The methodology is modifiable and expandable to allow for further characterization of leukocyte populations of special interest.NEW & NOTEWORTHY We describe a novel combination of methodologies that incorporates dual in vivo compartmental analysis using intravascular and intratracheal CD45 labeling, a broad panel of antibodies for identifying lymphoid and nonlymphoid cells, and spectral flow cytometry that uses cellular autofluorescence to aid in resolving and identifying immune cell populations. This methodology allows precise localization of immune cells in the lavageable airways, nonlavageable airways, interstitial lung tissue, and marginated in the lung vasculature.

Update on the Features and Measurements of Experimental Acute Lung Injury in Animals: An Official American Thoracic Society Workshop Report.

Advancements in methods, technology, and our understanding of the pathobiology of lung injury have created the need to update the definition of experimental acute lung injury (ALI). We queried 50 participants with expertise in ALI and acute respiratory distress syndrome using a Delphi method composed of a series of electronic surveys and a virtual workshop. We propose that ALI presents as a "multidimensional entity" characterized by four "domains" that reflect the key pathophysiologic features and underlying biology of human acute respiratory distress syndrome. These domains are 1) histological evidence of tissue injury, 2) alteration of the alveolar-capillary barrier, 3) presence of an inflammatory response, and 4) physiologic dysfunction. For each domain, we present "relevant measurements," defined as those proposed by at least 30% of respondents. We propose that experimental ALI encompasses a continuum of models ranging from those focusing on gaining specific mechanistic insights to those primarily concerned with preclinical testing of novel therapeutics or interventions. We suggest that mechanistic studies may justifiably focus on a single domain of lung injury, but models must document alterations of at least three of the four domains to qualify as "experimental ALI." Finally, we propose that a time criterion defining "acute" in ALI remains relevant, but the actual time may vary based on the specific model and the aspect of injury being modeled. The continuum concept of ALI increases the flexibility and applicability of the definition to multiple models while increasing the likelihood of translating preclinical findings to critically ill patients.

Defining the versican interactome in lung health and disease.

The extracellular matrix (ECM) imparts critical mechanical and biochemical information to cells in the lungs. Proteoglycans are essential constituents of the ECM and play a crucial role in controlling numerous biological processes, including regulating cellular phenotype and function. Versican, a chondroitin sulfate proteoglycan required for embryonic development, is almost absent from mature, healthy lungs and is reexpressed and accumulates in acute and chronic lung disease. Studies using genetically engineered mice show that the versican-enriched matrix can be pro- or anti-inflammatory depending on the cellular source or disease process studied. The mechanisms whereby versican develops a contextual ECM remain largely unknown. The primary goal of this review is to provide an overview of the interaction of versican with its many binding partners, the "versican interactome," and how through these interactions, versican is an integrator of complex extracellular information. Hopefully, the information provided in this review will be used to develop future studies to determine how versican and its binding partners can develop contextual ECMs that control select biological processes. Although this review focuses on versican and the lungs, what is described can be extended to other proteoglycans, tissues, and organs.

Effect of lung pericyte-like cell ablation on the bleomycin model of injury and repair.

We previously showed that pericyte-like cells derived from the FoxD1-lineage contribute to myofibroblasts following bleomycin-induced lung injury. However, their functional significance in lung fibrosis remains unknown. In this study, we used a model of lung pericyte-like cell ablation to test the hypothesis that pericyte-like cell ablation attenuates lung fibrosis in bleomycin-induced lung injury. Lung fibrosis was induced by intratracheal instillation of bleomycin. To ablate pericyte-like cells in the lung, diphtheria toxin (DT) was administered to Foxd1-Cre;Rosa26-iDTR mice at two different phases of bleomycin-induced lung injury. For early ablation, we coadministered bleomycin with DT and harvested mice at days 7 and 21. To test the effect of ablation after acute injury, we delivered DT 7 days after bleomycin administration. We assessed fibrosis by lung hydroxyproline content and semiquantitative analysis of picrosirius red staining. We performed bronchoalveolar lavage to determine cell count and differential. We also interrogated mRNA expression of fibrosis-related genes in whole lung RNA. Compared with DT-insensitive littermates where pericyte-like cells were not ablated, DT-sensitive animals exhibited no difference in fibrosis at day 21 both in the early and late pericyte ablation models. However, early ablation of pericytes reduced acute lung inflammation, as indicated by decreased inflammatory cells. Our data confirm a role for pericytes in regulating pulmonary inflammation in early lung injury.

Location of eosinophils in the airway wall is critical for specific features of airway hyperresponsiveness and T2 inflammation in asthma.

BACKGROUND: Eosinophils are implicated as effector cells in asthma, but the functional implications of the precise location of eosinophils in the airway wall is poorly understood. We aimed to quantify eosinophils in the different compartments of the airway wall and associate these findings with clinical features of asthma and markers of airway inflammation. METHODS: In this cross-sectional study, we utilised design-based stereology to accurately partition the numerical density of eosinophils in both the epithelial compartment and the subepithelial space (airway wall area below the basal lamina including the submucosa) in individuals with and without asthma and related these findings to airway hyperresponsiveness (AHR) and features of airway inflammation. RESULTS: Intraepithelial eosinophils were linked to the presence of asthma and endogenous AHR, the type that is most specific for asthma. In contrast, both intraepithelial and subepithelial eosinophils were associated with type 2 (T2) inflammation, with the strongest association between IL5 expression and intraepithelial eosinophils. Eosinophil infiltration of the airway wall was linked to a specific mast cell phenotype that has been described in asthma. We found that interleukin (IL)-33 and IL-5 additively increased cysteinyl leukotriene (CysLT) production by eosinophils and that the CysLT LTC4 along with IL-33 increased IL13 expression in mast cells and altered their protease profile. CONCLUSIONS: We conclude that intraepithelial eosinophils are associated with endogenous AHR and T2 inflammation and may interact with intraepithelial mast cells via CysLTs to regulate airway inflammation.

Pericyte-like cells undergo transcriptional reprogramming and distinct functional adaptations in acute lung injury.

We previously reported on the role of pericyte-like cells as functional sentinel immune cells in lung injury. However, much about the biological role of pericytes in lung injury remains unknown. Lung pericyte-like cells are well-positioned to sense disruption to the epithelial barrier and coordinate local inflammatory responses due to their anatomic niche within the alveoli. In this report, we characterized transcriptional responses and functional changes in pericyte-like cells following activation by alveolar components from injured and uninjured lungs in a mouse model of acute lung injury (ALI). Purified pericyte-like cells from lung digests using PDGFRß as a selection marker were expanded in culture as previously described (1). We induced sterile acute lung injury in mice with recombinant human Fas ligand (rhFasL) instillation followed by mechanical ventilation (1). We then collected bronchoalveolar lavage fluid (BALF) from injured and uninjured mice. Purified pericyte-like cells in culture were exposed to growth media only (control), BALF from uninjured mice, and BALF from injured mice for 6 and 24 hours. RNA collected from these treatment conditions were processed for RNAseq. Targets of interest identified by pathway analysis were validated using in vitro and in vivo assays. We observed robust global transcriptional changes in pericyte-like cells following treatment with uninjured and injured BALF at 6 hours, but this response persisted for 24 hours only after exposure to injured BALF. Functional enrichment analysis of pericytes treated with injured BALF revealed the activation of pro-inflammatory, cell migration, and angiogenesis-related pathways, whereas processes associated with tissue development and cell differentiation were down-regulated. We validated select upregulated targets in the inflammatory, angiogenic, and cell migratory pathways using functional biological assays in vitro and in vivo. We conclude that lung pericyte-like cells are highly responsive to alveolar compartment content from both uninjured and injured lungs, but injured BALF elicits a more sustained response. The inflammatory, angiogenic, and migratory changes exhibited by activated pericyte-like cells underscore the phenotypic plasticity of these specialized stromal cells in the setting of acute lung injury.

Secreted Phospholipase A2 Group X Acts as an Adjuvant for Type 2 Inflammation, Leading to an Allergen-Specific Immune Response in the Lung.

Secreted phospholipase A2 (sPLA2) enzymes release free fatty acids, including arachidonic acid, and generate lysophospholipids from phospholipids, including membrane phospholipids from cells and bacteria and surfactant phospholipids. We have shown that an endogenous enzyme sPLA2 group X (sPLA2-X) is elevated in the airways of asthmatics and that mice lacking the sPLA2-X gene (Pla2g10) display attenuated airway hyperresponsiveness, innate and adaptive immune responses, and type 2 cytokine production in a model of airway sensitization and challenge using a complete allergen that induces endogenous adjuvant activity. This complete allergen also induces the expression of sPLA2-X/Pla2g10 In the periphery, an sPLA2 found in bee venom (bee venom PLA2) administered with the incomplete Ag OVA leads to an Ag-specific immune response. In this study, we demonstrate that both bee venom PLA2 and murine sPLA2-X have adjuvant activity, leading to a type 2 immune response in the lung with features of airway hyperresponsiveness and Ag-specific type 2 airway inflammation following peripheral sensitization and subsequent airway challenge with OVA. Further, the adjuvant effects of sPLA2-X that result in the type 2-biased OVA-specific adaptive immune response in the lung were dependent upon the catalytic activity of the enzyme, as a catalytically inactive mutant form of sPLA2-X does not elicit the adaptive component of the immune response, although other components of the immune response were induced by the inactive enzyme, suggesting receptor-mediated effects. Our results demonstrate that exogenous and endogenous sPLA2s play an important role in peripheral sensitization, resulting in airway responses to inhaled Ags.

Exercise-induced alterations in phospholipid hydrolysis, airway surfactant, and eicosanoids and their role in airway hyperresponsiveness in asthma.

The mechanisms responsible for driving endogenous airway hyperresponsiveness (AHR) in the form of exercise-induced bronchoconstriction (EIB) are not fully understood. We examined alterations in airway phospholipid hydrolysis, surfactant degradation, and lipid mediator release in relation to AHR severity and changes induced by exercise challenge. Paired induced sputum (n = 18) and bronchoalveolar lavage (BAL) fluid (n = 11) were obtained before and after exercise challenge in asthmatic subjects. Samples were analyzed for phospholipid structure, surfactant function, and levels of eicosanoids and secreted phospholipase A2 group 10 (sPLA2-X). A primary epithelial cell culture model was used to model effects of osmotic stress on sPLA2-X. Exercise challenge resulted in increased surfactant degradation, phospholipase activity, and eicosanoid production in sputum samples of all patients. Subjects with EIB had higher levels of surfactant degradation and phospholipase activity in BAL fluid. Higher basal sputum levels of cysteinyl leukotrienes (CysLTs) and prostaglandin D2 (PGD2) were associated with direct AHR, and both the postexercise and absolute change in CysLTs and PGD2 levels were associated with EIB severity. Surfactant function either was abnormal at baseline or became abnormal after exercise challenge. Baseline levels of sPLA2-X in sputum and the absolute change in amount of sPLA2-X with exercise were positively correlated with EIB severity. Osmotic stress ex vivo resulted in movement of water and release of sPLA2-X to the apical surface. In summary, exercise challenge promotes changes in phospholipid structure and eicosanoid release in asthma, providing two mechanisms that promote bronchoconstriction, particularly in individuals with EIB who have higher basal levels of phospholipid turnover.

Quantum dots and mouse strain influence house dust mite-induced allergic airway disease.

Quantum dot nanoparticles (QDs) are engineered nanomaterials (ENMs) that have utility in many industries due to unique optical properties not available in small molecules or bulk materials. QD-induced acute lung inflammation and toxicity in rodent models raise concerns about potential human health risks. Recent studies have also shown that some ENMs can exacerbate allergic airway disease (AAD). In this study, C57BL/6J and A/J mice were exposed to saline, house dust mite (HDM), or a combination of HDM and QDs on day 1 of the sensitization protocol. Mice were then challenged on days 8, 9 and 10 with HDM or saline only. Significant differences in cellular and molecular markers of AAD induced by both HDM and HDM + QD were observed between C57BL/6J and A/J mice. Among A/J mice, HDM + QD co-exposure, but not HDM exposure alone, significantly increased levels of bronchoalveolar lavage fluid (BALF). IL-33 compared to saline controls. BALF total protein levels in both mouse strains were also only significantly increased by HDM + QD co-exposure. In addition, A/J mice had significantly more lung type 2 innate lymphoid cells (ILC2s) cells than C57BL/6J mice. A/J lung ILC2s were inversely correlated with lung glutathione and MHC-IIhigh resident macrophages, and positively correlated with MHC-IIlow resident macrophages. The results from this study suggest that 1) QDs influence HDM-induced AAD by potentiating and/or enhancing select cytokine production; 2) that genetic background modulates the impact of QDs on HDM sensitization; and 3) that potential ILC2 contributions to HDM induced AAD are also likely to be modulated by genetic background.

The Effects of Gene × Environment Interactions on Silver Nanoparticle Toxicity in the Respiratory System.

Silver nanoparticles (AgNP) are used in multiple applications but primarily in the manufacturing of antimicrobial products. AgNP toxicity in the respiratory system is well characterized, but few in vitro or in vivo studies have evaluated the effects of interactions between host genetic and acquired factors or gene × environment interactions (G × E) on AgNP toxicity in the respiratory system. The primary goal of this article is to review host genetic and acquired factors identified across in vitro and in vivo studies and prioritize those necessary for defining exposure limits to protect all populations. The impact of these exposures and the work being done to address the current limited protections are also discussed. Future research on G × E effects on AgNP toxicity is warranted and will assist with informing regulatory or recommended exposure limits that enforce special protections for all populations to AgNP exposures in occupational settings.

Matrix Metalloproteinase-28 Is a Key Contributor to Emphysema Pathogenesis.

Chronic obstructive pulmonary disease (COPD) comprises chronic bronchitis and emphysema, and is a leading cause of morbidity and mortality. Because tissue destruction is the prominent characteristic of emphysema, extracellular proteinases, particularly those with elastolytic ability, are often considered to be key drivers in this disease. Several human and mouse studies have implicated roles for matrix metalloproteinases (MMPs), particularly macrophage-derived proteinases, in COPD pathogenesis. MMP-28 is expressed by the pulmonary epithelium and macrophage, and we have found that it regulates macrophage recruitment and polarization. We hypothesized that MMP-28 has contributory roles in emphysema via alteration of macrophage numbers and activation. Because of the established association of emphysema pathogenesis to macrophage influx, we evaluated the inflammatory changes and lung histology of Mmp28-/- mice exposed to 3 and 6 months of cigarette smoke. At earlier time points, we found altered macrophage polarization in the smoke-exposed Mmp28-/- lung consistent with other published findings that MMP-28 regulates macrophage activation. At both 3 and 6 months, Mmp28-/- mice had blunted inflammatory responses more closely resembling nonsmoked mice, with a reduction in neutrophil recruitment and CXCL1 chemokine expression. By 6 months, Mmp28-/- mice were protected from emphysema. These results highlight a previously unrecognized role for MMP-28 in promoting chronic lung inflammation and tissue remodeling induced by cigarette smoke and highlight another potential target to modulate COPD.

Genetic determinants of susceptibility to silver nanoparticle-induced acute lung inflammation in mice.

Silver nanoparticles (AgNPs) are employed in a variety of consumer products; however, in vivo rodent studies indicate that AgNPs can cause lung inflammation and toxicity in a strain- and particle type-dependent manner, but mechanisms of susceptibility remain unclear. The aim of this study was to assess the variation in AgNP-induced lung inflammation and toxicity across multiple inbred mouse strains and to use genome-wide association (GWA) mapping to identify potential candidate susceptibility genes. Mice received doses of 0.25 mg/kg of either 20-nm citrate-coated AgNPs or citrate buffer using oropharyngeal aspiration. Neutrophils in bronchoalveolar lavage fluid (BALF) served as markers of inflammation. We found significant strain- and treatment-dependent variation in neutrophils in BALF. GWA mapping identified 10 significant single-nucleotide polymorphisms (false discovery rate, 15%) in 4 quantitative trait loci on mouse chromosomes 1, 4, 15, and 18, and Nedd4l (neural precursor cell expressed developmentally downregulated gene 4-like; chromosome 18), Ano6 (anocatmin 6; chromosome 15), and Rnf220 (Ring finger protein 220; chromosome 4) were considered candidate genes. Quantitative RT-PCR revealed significant inverse associations between mRNA levels of these genes and neutrophil influx. Nedd4l, Ano6, and Rnf220 are candidate susceptibility genes for AgNP-induced lung inflammation that warrant additional exploration in future studies.-Scoville, D. K., Botta, D., Galdanes, K., Schmuck, S. C., White, C. C., Stapleton, P. L., Bammler, T. K., MacDonald, J. W., Altemeier, W. A., Hernandez, M., Kleeberger, S. R., Chen, L.-C., Gordon, T., Kavanagh, T. J. Genetic determinants of susceptibility to silver nanoparticle-induced acute lung inflammation in mice.

Neutrophil extracellular traps (NETs) are increased in the alveolar spaces of patients with ventilator-associated pneumonia.

BACKGROUND: Neutrophils release neutrophil extracellular traps (NETs) in response to invading pathogens. Although NETs play an important role in host defense against microbial pathogens, they have also been shown to play a contributing mechanistic role in pathologic inflammation in the absence of infection. Although a role for NETs in bacterial pneumonia and acute respiratory distress syndrome (ARDS) is emerging, a comprehensive evaluation of NETs in the alveolar space of critically ill patients has yet to be reported. In this study, we evaluated whether markers of NET formation in mechanically ventilated patients are associated with ventilator-associated pneumonia (VAP). METHODS: We collected bronchoalveolar lavage fluid from 100 critically ill patients undergoing bronchoscopy for clinically suspected VAP. Subjects were categorized by the absence or presence of VAP and further stratified by ARDS status. NETs (myeloperoxidase (MPO)-DNA complexes) and the NET-associated markers peroxidase activity and cell-free DNA were analyzed by enzyme-linked immunosorbent assay and colorimetric assays, respectively. Quantitative polymerase chain reaction of nuclear and mitochondrial DNA was used to determine the origin of the extruded DNA. Interleukin (IL)-8 and calprotectin were assayed as measures of alveolar inflammation and neutrophil activation. Correlations between NETs and markers of neutrophil activation were determined using Spearman's correlation. We tested for associations with VAP and bacterial burden by logistic and linear regression, respectively, using log10-transformed NETs. RESULTS: MPO-DNA concentrations were highly correlated with other measures of NET formation in the alveolar space, including cell-free DNA and peroxidase activity (r = 0.95 and r = 0.87, p < 0.0001, respectively). Alveolar concentrations of MPO-DNA were higher in subjects with VAP and ARDS compared with those with ARDS alone (p < 0.0001), and higher MPO-DNA was associated with increased odds of VAP (odds ratio 3.03, p < 0.0001). In addition, NET concentrations were associated with bacterial burden (p < 0.0001) and local alveolar inflammation as measured by IL-8 (r = 0.89, p < 0.0001). CONCLUSIONS: Alveolar NETs measured by MPO-DNA complex are associated with VAP, and markers of NETosis are associated with local inflammation and bacterial burden in the lung. These results suggest that NETs contribute to inflammatory responses involved in the pathogenesis of VAP.

Quantum dot induced acute changes in lung mechanics are mouse strain dependent.

INTRODUCTION: Concerns have been raised regarding occupational exposure to engineered nanomaterials (ENMs). Potential impacts on lung function from inhalation exposures are of concern as the lung is a sensitive ENM target in animals. Epidemiological data suggest that occupational exposure to ENMs may impact respiratory and cardiovascular health. Quantum dots (QDs) are ENMs with outstanding semiconductor and fluorescent properties with uses in biomedicine and electronics. QDs are known to induce inflammation and cytotoxicity in rodents and high dose exposures impact lung function 2 weeks after exposure. However, effects of mouse strain and the temporality of QD effects on lung function at more occupationally relevant doses have not been well-established. OBJECTIVE: We evaluated the impact of QD exposure on respiratory mechanics in C57BL/6J and A/J mice. Previous work found a greater initial inflammatory response to QD exposure in A/J mice compared to C57BL/6J mice. Thus, we hypothesized that A/J mice would be more sensitive to QD-induced effects on lung mechanics. METHODS: C57BL/6J and A/J mice were exposed to 6 µg/kg Cd equivalents of amphiphilic polymer-coated Cd/Se core, ZnS shell QDs via oropharyngeal aspiration. Lung mechanics were measured using forced oscillation, and inflammation was characterized by neutrophils and cytokines in bronchoalveolar lavage fluid. RESULTS: Both strains showed signs of QD-induced acute lung inflammation. However, lung mechanics were impacted by QD exposure in A/J mice only. CONCLUSIONS: Our findings suggest that susceptibility to QDs and similar ENM-induced changes in lung function may depend at least in part on genetic background.