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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.

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. While 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 (PAMPs) and tissue damage-associated molecular patterns (DAMPs), 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.

Von Willebrand factor (VWF) is classically associated with primary hemostasis and platelet-rich arterial thromboses, but recently has also been implicated in fibrin clotting and venous thrombosis. Direct interaction between fibrin and VWF may mediate these processes, although prior reports are conflicting.We combined two complementary platforms to characterize VWF-fibrin(ogen) interactions and identify their potential physiologic significance.Engineered microvessels were lined with human endothelial cells, cultured under flow, and activated to release VWF and form transluminal VWF fibers. Fibrinogen, fibrin monomers, or polymerizing fibrin were then perfused, and interactions with VWF evaluated. Thrombin and fibrinogen were perfused into living versus paraformeldahyde-fixed microvessels and the pressure drop across microvessels monitored. Separately, protein binding to tethered VWF was assessed on a single-molecule level using total internal reflection fluorescence (TIRF) microscopy.Within microvessels, VWF fibers colocalized with polymerizing fibrin, but not fibrinogen. TIRF microscopy showed no colocalization between VWF and fibrinogen or fibrin monomers in a microfluidic flow chamber across a range of shear rates and protein concentrations. Thrombin-mediated fibrin polymerization within living microvessels triggered endothelial VWF release, increasing the rate and amount of microvessel obstruction compared to fixed vessels with an inert endothelium.We did not identify specific binding between fibrin(ogen) and VWF at a single-molecule level. Despite this, our results suggest that rapid release of endothelial VWF during clotting may provide a physical support for fibrin polymerization and accelerate thrombosis. This interaction may be of fundamental importance for the understanding and treatment of human thrombotic disease.

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.

Versican, a chondroitin sulfate proteoglycan, is an essential component of the extracellular matrix (ECM) in inflammatory lung disease. Versican’s potential as an immunomodulatory molecule makes it a promising therapeutic target for controlling host immune responses in the lungs. To establish changes to versican expression and accumulation during influenza A viral pneumonia, we document the temporal and spatial changes to versican mRNA and protein in concert with pulmonary inflammatory cell infiltration. These studies were performed in the lungs of wild-type C57BL6/J mice on days 3, 6, 9, and 12 post-infection with influenza A virus using immunohistochemistry, in situ hybridization, and quantitative digital pathology. Using duplex in situ hybridization, we demonstrate that type I interferon signaling contributes significantly to versican expression in lung stromal cells. Our findings show that versican is a type I interferon–stimulated gene in pulmonary fibroblasts and pericytes in the context of viral pneumonia. These data also provide a guide for future studies to determine the role of versican in the pulmonary immune response to influenza infection

Subtypes of pulmonary arterial hypertension (PAH) differ in both fundamental disease features and clinical outcomes. Angiogenesis and inflammation represent disease features that may differ across subtypes and are of special interest in connective tissue disease-associated PAH (CTD-PAH). We compared inflammatory and angiogenic biomarker profiles across different etiologies of PAH and related them to clinical outcomes.Participants with idiopathic PAH, CTD-PAH, toxin-associated PAH (tox-PAH), or congenital heart disease-associated PAH (CHD-PAH) were enrolled into a prospective observational cohort. Baseline serum concentrations of 33 biomarkers were related to 3-year mortality, echocardiogram, REVEAL score, and 6-minute walk distance (6MWD). Findings were validated using plasma proteomic data from the UK PAH Cohort Study.One hundred twelve patients were enrolled: 45 idiopathic, 27 CTD-PAH, 20 tox-PAH, and 20 CHD-PAH. Angiogenic and inflammatory biomarkers were distinctly elevated within the CTD-PAH cohort. Six biomarkers were associated with mortality within the entire PAH cohort: interleukin-6 (IL-6, HR:1.6, 95% CI:1.18-2.18), soluble fms-like tyrosine kinase 1 (sFlt-1, HR:1.35, 95% CI:1.02-1.80), placental growth factor (PlGF, HR:1.55, 95% CI:1.07-2.25), interferon gamma-induced protein 10 (IP-10, HR:1.44, 95% CI:1.04-1.99), tumor necrosis factor-beta (TNF-β, HR:1.81, 95% CI:1.11-2.95), and NT-proBNP (HR:2.19, 95% CI:1.52-3.14). Only IL-6 and NT-proBNP remained significant after controlling for multiple comparisons. IL-6, IP-10, and sFlt-1 significantly associated with mortality in CTD-PAH, but not non-CTD-PAH subgroups. In the UK cohort, IP-10, PlGF, TNF-β, and NT-proBNP significantly associated with 5-year survival.Levels of angiogenic and inflammatory biomarkers are elevated in CTD-PAH, compared with other etiologies of PAH, and may correlate with clinical outcomes including mortality.

Secreted phospholipase A(2) (sPLA(2)) enzymes release free fatty acids, including arachidonic acid and generate lysophosphospholipds from phospholipids, including membrane phospholipids from cells and bacteria and surfactant phospholipids. We have shown that an endogenous enzyme sPLA(2) group X (sPLA(2)-X) is elevated in the airways of asthmatics and that mice lacking the sPLA(2)-X gene (Pla2g10) display attenuated airway hyperresponsiveness (AHR), 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 sPLA(2)-X/Pla2g10. In the periphery, a sPLA(2) found in bee venom (bee venom PLA(2), bvPLA(2)) administered with the incomplete antigen ovalbumin (OVA) leads to an antigen specific immune response. In this study, we demonstrate that both bvPLA(2) and murine sPLA(2)-X have adjuvant activity, leading to a type-2 immune response in the lung with features of airway hyperresponsiveness (AHR) and antigen-specific type-2 airway inflammation following peripheral sensitization and subsequent airway challenge with OVA. Further, the adjuvant effects of sPLA(2)-X that result in the type-2 biased OVA-specific adaptive immune response in the lung was dependent upon the catalytic activity of the enzyme, as a catalytically inactive mutant form of sPLA(2)-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 sPLA(2)s play an important role in peripheral sensitization resulting in airway responses to inhaled antigens.

Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in life sciences analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose 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 analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such specimens remain, in comparison, inefficient. To mitigate this bottleneck, we have developed a low-cost user-friendly system, the CryoGrid, that consists of CryoBlock, thermometer/thermocouple, and QR coded CryoTrays to freeze and store frozen tissue fragments, and hand-held CryoCore tool for tissue sampling supported by iPad and Google apps to display tissues, direct coring and share metadata.RNA is one of the most studied analytes. There is a decades-long history of developing methods to isolate and analyze RNA. Still, the throughput of sampling and RNA extraction from tissues has not matched that of the high throughput transcriptome analytical platforms. To address this need, we have integrated the CryoGrid system with PIXUL-based methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. TRIzol is commonly used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. We developed a PIXUL-based TRIzol-free RNA isolation fast protocol that uses a buffer containing proteinase K (PK). Virtually every disease (and often therapeutic agents’ toxicity) is a systemic syndrome but often only one organ is examined. CryoGrid-PIXUL, integrated with either TRIzol or PK buffer RNA isolation protocols, yielded similar RNA profiles in a multiorgan (brain, heart, kidney and liver) mouse model of sepsis. Thus, RNA isolation using the CryoGrid-PIXUL system combined with the PK buffer offers an inexpensive user-friendly workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.

Vascular dysfunction and capillary leak are common in critically ill COVID-19 patients, but identification of endothelial pathways involved in COVID-19 pathogenesis has been limited. Angiopoietin-like 4 (ANGPTL4) is a protein secreted in response to hypoxic and nutrient-poor conditions that has a variety of biological effects including vascular injury and capillary leak.To assess the role of ANGPTL4 in COVID-19-related outcomes.Two hundred twenty-five COVID-19 ICU patients were enrolled from April 2020 to May 2021 in a prospective, multicenter cohort study from three different medical centers, University of Washington, University of Southern California and New York University.Plasma ANGPTL4 was measured on days 1, 7, and 14 after ICU admission. We used previously published tissue proteomic data and lung single nucleus RNA (snRNA) sequencing data from specimens collected from COVID-19 patients to determine the tissues and cells that produce ANGPTL4.Higher plasma ANGPTL4 concentrations were significantly associated with worse hospital mortality (adjusted odds ratio per logANGPTL4 is expressed in pulmonary epithelial cells and fibroblasts and is associated with clinical prognosis in critically ill COVID-19 patients.

Elevated secreted phospholipase A2 (sPLA2) activity in the airways has been implicated in the pathogenesis of asthma and allergic disease for some time. The identity and function of these enzymes in asthma is becoming clear from work in our lab and others. We focused on sPLA2 group X (sPLA2-X) after identifying increased levels of this enzyme in asthma, and that it is responsible for a large portion of sPLA2 activity in the airways and that the levels are strongly associated with features of airway hyperresponsiveness (AHR). In this review, we discuss studies that implicated sPLA2-X in human asthma, and murine models that demonstrate a critical role of this enzyme as a regulator of type-2 inflammation, AHR and production of eicosanoids. We discuss the mechanism by which sPLA2-X acts to regulate eicosanoids in leukocytes, as well as effects that are mediated through the generation of lysophospholipids and through receptor-mediated functions. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau.

BackgroundEosinophils 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.MethodsIn 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.ResultsIntraepithelial 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 betweenIL5expression 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 LTC4along with IL-33 increasedIL13expression in mast cells and altered their protease profile.ConclusionsWe conclude that intraepithelial eosinophils are associated with endogenous AHR and T2 inflammation and may interact with intraepithelial mast cellsviaCysLTs to regulate airway inflammation.

The Adverse Outcome Pathway (AOP) framework is serving as a basis to integrate new data streams in order to enhance the power of predictive toxicology. AOP development for engineered nanomaterials (ENM), including silver nanoparticles (AgNP), is currently lagging behind other chemicals of regulatory interest due to our limited understanding of the mechanism by which underlying genetics or diseases directly modify host response to AgNP exposures. This also highlights the importance of considering the Aggregate Exposure Pathway (AEP) framework, which precedes the AOP framework and outlines source to target site exposure. The AEP and AOP frameworks interface at the target site, where a molecular initiating event (MIE) occurs and is followed by key events (KE) for adverse cellular and organ responses along a biological pathway and ends with the adverse organism response. The primary goal of this study is to use AgNP to interrogate the AEP-AOP framework by organizing and integrating in vitro dose-response data and in vivo exposure-response data from previous studies to evaluate the effects of interactions between host genetic and acquired factors, or gene × environment interactions (G × E), on AgNP toxicity in the respiratory system. Using this framework will help us to identify plausible key event relationships (KER) between MIE and adverse organism responses when KE are not measured using the same assay in order to derive future predictive models, guide research, and support development of tools for making risk-based, regulatory decisions on ENM. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

BackgroundWe 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).MethodsWe purified pericyte-like cells from lung digests using PDGFRβ as a selection marker and expanded them 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 h. 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.ResultsWe observed robust global transcriptional changes in pericyte-like cells following treatment with uninjured and injured BALF at 6 h, but this response persisted for 24 h only after exposure to injured BALF. Functional enrichment analysis of pericytes treated with injured BALF revealed activation of immuno-inflammatory, cell migration and angiogenesis-related pathways, whereas processes associated with tissue development and remodeling were down-regulated. We validated select targets in the inflammatory, angiogenesis-related, and cell migratory pathways using functional biological assays in vitro and in vivo.ConclusionLung 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.

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 A2group 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 PGD2levels 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.

Silver nanoparticles (AgNP) are used in multiple applications but primarily in the manufacturing of antimicrobial products. Previous studies have identified AgNP toxicity in airway epithelial cells, but no

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.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.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.Both strains showed signs of QD-induced acute lung inflammation. However, lung mechanics were impacted by QD exposure in A/J mice only.Our findings suggest that susceptibility to QDs and similar ENM-induced changes in lung function may depend at least in part on genetic background.

Growing evidence suggests that versican is important in the innate immune response to lung infection. Our goal was to understand the regulation of macrophage-derived versican and the role it plays in innate immunity. We first defined the signaling events that regulate versican expression, using bone marrow-derived macrophages (BMDMs) from mice lacking specific Toll-like receptors (TLRs), TLR adaptor molecules, or the type I interferon receptor (IFNAR1). We show that LPS and polyinosinic-polycytidylic acid [poly(I:C)] trigger a signaling cascade involving TLR3 or TLR4, the Trif adaptor, type I interferons, and IFNAR1, leading to increased expression of versican by macrophages and implicating versican as an interferon-stimulated gene. The signaling events regulating versican are distinct from those for hyaluronan synthase 1 (HAS1) and syndecan-4 in macrophages. HAS1 expression requires TLR2 and MyD88. Syndecan-4 requires TLR2, TLR3, or TLR4 and both MyD88 and Trif. Neither HAS1 nor syndecan-4 is dependent on type I interferons. The importance of macrophage-derived versican in lungs was determined with LysM/ Vcan−/−mice. These studies show increased recovery of inflammatory cells in the bronchoalveolar lavage fluid of poly(I:C)-treated LysM/ Vcan−/−mice compared with control mice. IFN-β and IL-10, two important anti-inflammatory molecules, are significantly decreased in both poly(I:C)-treated BMDMs from LysM/ Vcan−/−mice and bronchoalveolar lavage fluid from poly(I:C)-treated LysM/ Vcan−/−mice compared with control mice. In short, type I interferon signaling regulates versican expression, and versican is necessary for type I interferon production. These findings suggest that macrophage-derived versican is an immunomodulatory molecule with anti-inflammatory properties in acute pulmonary inflammation.

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.

Pericytes are perivascular PDGF receptor-β+ (PDGFRβ+) stromal cells required for vasculogenesis and maintenance of microvascular homeostasis in many organs. Because of their unique juxtaposition to microvascular endothelium, lung PDGFRβ+ cells are well situated to detect proinflammatory molecules released following epithelial injury and promote acute inflammatory responses. Thus we hypothesized that these cells represent an unrecognized immune surveillance or injury-sentinel interstitial cell. To evaluate this hypothesis, we isolated PDGFRβ+ cells from murine lung and demonstrated that they have characteristics consistent with a pericyte population (referred to as pericyte-like cells for simplicity hereafter). We showed that pericyte-like cells expressed functional Toll-like receptors and upregulated chemokine expression following exposure to bronchoalveolar lavage fluid (BALF) collected from mice with sterile lung injury. Interestingly, BALF from mice without lung injury also induced chemokine expression in pericyte-like cells, suggesting that pericyte-like cells are primed to sense epithelial injury (permeability changes). Following LPS-induced lung inflammation, increased numbers of pericyte-like cells expressed IL-6, chemokine (C-X-C motif) ligand-1, chemokine (C-C motif) ligand 2/ monocyte chemotactic protein-1, and ICAM-1 in vivo. Sterile lung injury in pericyte-ablated mice was associated with decreased inflammation compared with normal mice. In summary, we found that pericyte-like cells are immune responsive and express diverse chemokines in response to lung injury in vitro and in vivo. Furthermore, pericyte-like cell ablation attenuated inflammation in sterile lung injury, suggesting that these cells play an important functional role in mediating lung inflammatory responses. We propose a model in which pericyte-like cells function as interstitial immune sentinels, detecting proinflammatory molecules released following epithelial barrier damage and participating in recruitment of circulating leukocytes.

Serum amyloid A3 (Saa3) derives mainly from extrahepatic tissue and is not detected in plasma from moderately inflamed obese mice. In contrast, it is present in plasma from mice acutely inflamed by injection of high dose of lipopolysaccharide (LPS). To reconcile these differences, we evaluated whether different acute inflammatory stimuli could affect the presence of Saa3 in plasma. Saa3 appeared dose dependently in plasma after LPS injection. In contrast, only very low levels were detected after sterile inflammation with silver nitrate despite levels of Saa1 and Saa2 being comparable to high dose LPS. Saa3 was not detected in plasma following casein administration. Although most Saa3 was found in HDL, a small amount was not lipoprotein associated. Gene expression and proteomic analysis of liver and adipose tissue suggested that a major source of Saa3 in plasma after injection of LPS was adipose tissue rather than liver. We conclude that Saa3 only appears in plasma after induction of acute inflammation by some but not all inflammatory stimuli. These findings are consistent with the observation that Saa3 is not detectable in plasma in more moderate chronic inflammatory states such as obesity.

Silver nanoparticles (AgNP) are used in multiple applications but primarily in the manufacturing of antimicrobial products. Previous studies have identified AgNP toxicity in airway epithelial cells, but no in vitro studies to date have used organotypic cultures as a high-content in vitro model of the conducting airway to characterize the effects of interactions between host genetic and acquired factors, or gene × phenotype interactions (G×P), on AgNP toxicity. In the present study, we derived organotypic cultures from primary murine tracheal epithelial cells (MTEC) to characterize nominal and dosimetric dose-response relationships for AgNP-induced barrier dysfunction, glutathione (GSH) depletion, reactive oxygen species (ROS) production, lipid peroxidation, and cytotoxicity across two genotypes (A/J and C57BL/6J mice), two phenotypes (“Normal” and “Type 2 [T2]-Skewed”), and two exposures (an acute exposure of 24 h and a subacute exposure of 4 hours, every other day, over 5 days [5×4 h]). We characterized the “T2-Skewed” phenotype as an in vitro model of chronic respiratory diseases, which was marked by increased sensitivity to AgNP-induced barrier dysfunction, GSH depletion, ROS production, lipid peroxidation, and cytotoxicity, suggesting that asthmatics are a sensitive population to AgNP exposures in occupational settings. This also suggests that exposure limits, which should be based upon the most sensitive population, should be derived using in vitro and in vivo models of chronic respiratory diseases. This study highlights the importance of considering dosimetry as well as G×P effects when screening and prioritizing potential respiratory toxicants. Such in vitro studies can be used to inform regulatory policy aimed at special protections for all populations.

Asthma is a heterogeneous syndrome that has been subdivided into physiologic phenotypes and molecular endotypes. The most specific phenotypic manifestation of asthma is indirect airway hyperresponsiveness (AHR), and a prominent molecular endotype is the presence of type 2 inflammation. The underlying basis for type 2 inflammation and its relationship to AHR are incompletely understood. We assessed the expression of type 2 cytokines in the airways of subjects with and without asthma who were extensively characterized for AHR. Using quantitative morphometry of the airway wall, we identified a shift in mast cells from the submucosa to the airway epithelium specifically associated with both type 2 inflammation and indirect AHR. Using ex vivo modeling of primary airway epithelial cells in organotypic coculture with mast cells, we show that epithelial-derived IL-33 uniquely induced type 2 cytokines in mast cells, which regulated the expression of epithelial IL33 in a feed-forward loop. This feed-forward loop was accentuated in epithelial cells derived from subjects with asthma. These results demonstrate that type 2 inflammation and indirect AHR in asthma are related to a shift in mast cell infiltration to the airway epithelium, and that mast cells cooperate with epithelial cells through IL-33 signaling to regulate type 2 inflammation.

The airway epithelium is critical for maintaining innate and adaptive immune responses, and occupational exposures that disrupt its immune homeostasis may initiate and amplify airway inflammation. In our previous study, we demonstrated that silver nanoparticles (AgNP), which are engineered nanomaterials used in multiple applications but primarily in the manufacturing of many antimicrobial products, induce toxicity in organotypic cultures derived from murine tracheal epithelial cells (MTEC), and those differentiated toward a “Type 2 [T2]-Skewed” phenotype experienced an increased sensitivity to AgNP toxicity, suggesting that asthmatics could be a sensitive population to AgNP exposures in occupational settings. However, the mechanistic basis for this genotype × phenotype (G × P) interaction has yet to be defined. In this study, we conducted transcriptional profiling using RNA-sequencing to predict the enrichment of specific canonical pathways and upstream transcriptional regulators to assist in defining a mechanistic basis for G × P effects on AgNP toxicity. Organotypic cultures were derived from MTEC across 2 genetically inbred mouse strains (A/J and C57BL/6J mice), 2 phenotypes (“Normal” and “T2-Skewed”), and 1 AgNP exposure (an acute 24 h exposure) to characterize G × P effects on transcriptional response to AgNP toxicity. The “T2-Skewed” phenotype was marked by increased pro-inflammatory T17 responses to AgNP toxicity, which are significant predictors of neutrophilic/difficult-to-control asthma and suggests that asthmatics could be a sensitive population to AgNP exposures in occupational settings. This study highlights the importance of considering G × P effects when identifying these sensitive populations, whose underlying genetics or diseases could directly modify their response to AgNP exposures.

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.

The extracellular matrix in asthmatic lungs contains abundant low-molecular-weight hyaluronan, and this is known to promote antigen presentation and allergic responses. Conversely, high-molecular-weight hyaluronan (HMW-HA), typical of uninflamed tissues, is known to suppress inflammation. We investigated whether HMW-HA can be adapted to promote tolerance to airway allergens. HMW-HA was thiolated to prevent its catabolism and was tethered to allergens via thiol linkages. This platform, which we call "XHA," delivers antigenic payloads in the context of antiinflammatory costimulation. Allergen/XHA was administered intranasally to mice that had been sensitized previously to these allergens. XHA prevents allergic airway inflammation in mice sensitized previously to either ovalbumin or cockroach proteins. Allergen/XHA treatment reduced inflammatory cell counts, airway hyperresponsiveness, allergen-specific IgE, and T helper type 2 cell cytokine production in comparison with allergen alone. These effects were allergen specific and IL-10 dependent. They were durable for weeks after the last challenge, providing a substantial advantage over the current desensitization protocols. Mechanistically, XHA promoted CD44-dependent inhibition of nuclear factor-κB signaling, diminished dendritic cell maturation, and reduced the induction of allergen-specific CD4 T-helper responses. XHA and other potential strategies that target CD44 are promising alternatives for the treatment of asthma and allergic sinusitis.

Secreted phospholipase A2s (sPLA2s) regulate eicosanoid formation and have been implicated in asthma. Although sPLA2s function as enzymes, some of the sPLA2s bind with high affinity to a C-type lectin receptor, called PLA2R1, which has functions in both cellular signaling and clearance of sPLA2s. We sought to examine the expression of PLA2R1 in the airway epithelium of human subjects with asthma and the function of the murine Pla2r1 gene in a model of asthma. Expression of PLA2R1 in epithelial brushings was assessed in two distinct cohorts of children with asthma by microarray and quantitative PCR, and immunostaining for PLA2R1 was conducted on endobronchial tissue and epithelial brushings from adults with asthma. C57BL/129 mice deficient in Pla2r1 (Pla2r1−/−) were characterized in an ovalbumin (OVA) model of allergic asthma. PLA2R1 was differentially overexpressed in epithelial brushings of children with atopic asthma in both cohorts. Immunostaining for PLA2R1 in endobronchial tissue localized to submuco...

Fibrotic disease is associated with matrix deposition that results in the loss of organ function. Pericytes, the precursors of myofibroblasts, are a source of pathological matrix collagens and may be promising targets for treating fibrogenesis. Here, we have shown that pericytes activate a TLR2/4- and MyD88-dependent proinflammatory program in response to tissue injury. Similarly to classic immune cells, pericytes activate the NLRP3 inflammasome, leading to IL-1β and IL-18 secretion. Released IL-1β signals through pericyte MyD88 to amplify this response. Unexpectedly, we found that MyD88 and its downstream effector kinase IRAK4 intrinsically control pericyte migration and conversion to myofibroblasts. Specific ablation of MyD88 in pericytes or pharmacological inhibition of MyD88 signaling by an IRAK4 inhibitor in vivo protected against kidney injury by profoundly attenuating tissue injury, activation, and differentiation of myofibroblasts. Our data show that in pericytes, MyD88 and IRAK4 are key regulators of 2 major injury responses: inflammatory and fibrogenic. Moreover, these findings suggest that disruption of this MyD88-dependent pathway in pericytes might be a potential therapeutic approach to inhibit fibrogenesis and promote regeneration.

Sepsis-induced multiple organ dysfunction syndrome (MODS) is a major cause of morbidity and mortality in critically ill patients and remains impervious to most therapeutic interventions. We utilized a clinically relevant murine model of systemic inflammatory response syndrome (SIRS) during early MODS induced by ventilator-associated pneumonia to systematically delineate pathways dysregulated in lung, liver, and kidney. We focused on processes commonly activated across at-risk organs and constructed an SIRS-associated network based on connectivity among the gene members of these functionally coherent pathways. Our analyses led to the identification of several putative drivers of early MODS whose expression was regulated by epidermal growth factor receptor. Our unbiased, integrative method is a promising approach to unravel mechanisms in system-wide disorders afflicting multiple compartments such as sepsis-induced MODS, and identify putative therapeutic targets.

Elevated secreted phospholipase A(2) (sPLA(2)) activity in the airways has been implicated in the pathogenesis of asthma and allergic disease for some time. The identity and function of these enzymes in asthma is becoming clear from work in our lab and others. We focused on sPLA(2) group X (sPLA(2)-X) after identifying increased levels of this enzyme in asthma, and that it is responsible for a large portion of sPLA(2) activity in the airways and that the levels are strongly associated with features of airway hyperresponsiveness (AHR). In this review, we discuss studies that implicated sPLA(2)-X in human asthma, and murine models that demonstrate a critical role of this enzyme as a regulator of type-2 inflammation, AHR and production of eicosanoids. We discuss the mechanism by which sPLA(2)-X acts to regulate eicosanoids in leukocytes, as well as effects that are mediated through the generation of lysophospholipids and through receptor-mediated functions.

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.

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.

Phospholipase A2 (PLA2) enzymes regulate the formation of eicosanoids and lysophospholipids that contribute to allergic airway inflammation. Secreted PLA2 group X (sPLA2-X) was recently found to be increased in the airways of asthmatics and is highly expressed in airway epithelial cells and macrophages. In the current study, we show that allergen exposure increases sPLA2-X in humans and in mice, and that global deletion of Pla2g10 results in a marked reduction in airway hyperresponsiveness (AHR), eosinophil and T cell trafficking to the airways, airway occlusion, generation of type-2 cytokines by antigen-stimulated leukocytes, and antigen-specific immunoglobulins. Further, we found that Pla2g10-/- mice had reduced IL-33 levels in BALF, fewer type-2 innate lymphoid cells (ILC2s) in the lung, less IL-33-induced IL-13 expression in mast cells, and a marked reduction in both the number of newly recruited macrophages and the M2 polarization of these macrophages in the lung. These results indicate that sPLA2-X serves as a central regulator of both innate and adaptive immune response to proteolytic allergen.