SUMMARY The receptor for advanced glycation endproducts (RAGE) is a pro-inflammatory pattern recognition receptor (PRR) that has been implicated in the pathogenesis of numerous inflammatory diseases. It was discovered in 1992 on endothelial cells and was named for its ability to bind advanced glycation endproducts and promote vascular inflammation in the vessels of patients with diabetes. Further studies revealed that RAGE is most highly expressed in lung tissue and spurred numerous explorations into RAGE’s role in the lung. These studies have found that RAGE is an important mediator in allergic airway inflammation (AAI) and asthma, pulmonary fibrosis, lung cancer, chronic obstructive pulmonary disease (COPD), acute lung injury, pneumonia, cystic fibrosis, and bronchopulmonary dysplasia. RAGE has not yet been targeted in the lungs of paediatric or adult clinical populations, but the development of new ways to inhibit RAGE is setting the stage for the emergence of novel therapeutic agents for patients suffering from these pulmonary conditions.

BackgroundIn myeloid cells the inflammasome plays a crucial role in innate immune defenses against pathogen- and danger-associated patterns such as crystalline silica. Respirable mineral particles impinge upon the lung epithelium causing irreversible damage, sustained inflammation and silicosis. In this study we investigated lung epithelial cells as a target for silica-induced inflammasome activation.MethodsA human bronchial epithelial cell line (BEAS-2B) and primary normal human bronchial epithelial cells (NHBE) were exposed to toxic but nonlethal doses of crystalline silica over time to perform functional characterization of NLRP3, caspase-1, IL-1β, bFGF and HMGB1. Quantitative RT-PCR, caspase-1 enzyme activity assay, Western blot techniques, cytokine-specific ELISA and fibroblast (MRC-5 cells) proliferation assays were performed.ResultsWe were able to show transcriptional and translational upregulation of the components of the NLRP3 intracellular platform, as well as activation of caspase-1. NLRP3 activation led to maturation of pro-IL-1β to secreted IL-1β, and a significant increase in the unconventional release of the alarmins bFGF and HMGB1. Moreover, release of bFGF and HMGB1 was shown to be dependent on particle uptake. Small interfering RNA experiments using siNLRP3 revealed the pivotal role of the inflammasome in diminished release of pro-inflammatory cytokines, danger molecules and growth factors, and fibroblast proliferation.ConclusionOur novel data indicate the presence and functional activation of the NLRP3 inflammasome by crystalline silica in human lung epithelial cells, which prolongs an inflammatory signal and affects fibroblast proliferation, mediating a cadre of lung diseases.

BackgroundPleural fibrosis and malignant mesotheliomas (MM) occur after exposures to pathogenic fibers, yet the mechanisms initiating these diseases are unclear.ResultsWe document priming and activation of the NLRP3 inflammasome in human mesothelial cells by asbestos and erionite that is causally related to release of IL-1β, IL-6, IL-8, and Vascular Endothelial Growth Factor (VEGF). Transcription and release of these proteins are inhibited in vitro using Anakinra, an IL-1 receptor antagonist that reduces these cytokines in a human peritoneal MM mouse xenograft model.ConclusionsThese novel data show that asbestos-induced priming and activation of the NLRP3 inflammasome triggers an autocrine feedback loop modulated via the IL-1 receptor in mesothelial cell type targeted in pleural infection, fibrosis, and carcinogenesis.

RationaleMineral particles in the lung cause inflammation and silicosis. In myeloid and bronchial epithelial cells the inflammasome plays a role in responses to crystalline silica. Thioredoxin (TRX) and its inhibitory protein TRX-interacting protein link oxidative stress with inflammasome activation. We investigated inflammasome activation by crystalline silica polymorphs and modulation by TRX in vitro, as well as its localization and the importance of silica surface reactivity in rats.MethodsWe exposed bronchial epithelial cells and differentiated macrophages to silica polymorphs quartz and cristobalite and measured caspase-1 activity as well as the release of IL-1β, bFGF and HMGB1; including after TRX overexpression or treatment with recombinant TRX. Rats were intratracheally instilled with vehicle control, Dörentruper quartz (DQ12) or DQ12 coated with polyvinylpyridine N-oxide. At days 3, 7, 28, 90, 180 and 360 five animals per treatment group were sacrificed. Hallmarks of silicosis were assessed with Haematoxylin-eosin and Sirius Red stainings. Caspase-1 activity in the bronchoalveolar lavage and caspase-1 and IL-1β localization in lung tissue were determined using Western blot and immunohistochemistry (IHC).ResultsSilica polymorphs triggered secretion of IL-1β, bFGF and HMGB1 in a surface reactivity dependent manner. Inflammasome readouts linked with caspase-1 enzymatic activity were attenuated by TRX overexpression or treatment. At day 3 and 7 increased caspase-1 activity was detected in BALF of the DQ12 group and increased levels of caspase-1 and IL-1β were observed with IHC in the DQ12 group compared to controls. DQ12 exposure revealed silicotic nodules at 180 and 360 days. Particle surface modification markedly attenuated the grade of inflammation and lymphocyte influx and attenuated the level of inflammasome activation, indicating that the development of silicosis and inflammasome activation is determined by crystalline silica surface reactivity.ConclusionOur novel data indicate the pivotal role of surface reactivity of crystalline silica to activate the inflammasome in cultures of both epithelial cells and macrophages. Inhibitory capacity of the antioxidant TRX to inflammasome activation was evidenced. DQ12 quartz exposure induced acute and chronic functional activation of the inflammasome in the heterogeneous cell populations of the lung in associated with its crystalline surface reactivity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-014-0058-0) contains supplementary material, which is available to authorized users.

BackgroundExposure to respirable crystalline silica particles, as opposed to amorphous silica, is associated with lung inflammation, pulmonary fibrosis (silicosis), and potentially with lung cancer. We used Affymetrix/GeneSifter microarray analysis to determine whether gene expression profiles differed in a human bronchial epithelial cell line (BEAS 2B) exposed to cristobalite vs. amorphous silica particles at non-toxic and equal surface areas (75 and 150 × 106μm2/cm2). Bio-Plex analysis was also used to determine profiles of secreted cytokines and chemokines in response to both particles. Finally, primary human bronchial epithelial cells (NHBE) were used to comparatively assess silica particle-induced alterations in gene expression.ResultsMicroarray analysis at 24 hours in BEAS 2B revealed 333 and 631 significant alterations in gene expression induced by cristobalite at low (75) and high (150 × 106μm2/cm2) amounts, respectively (p < 0.05/cut off ≥ 2.0-fold change). Exposure to amorphous silica micro-particles at high amounts (150 × 106μm2/cm2) induced 108 significant gene changes. Bio-Plex analysis of 27 human cytokines and chemokines revealed 9 secreted mediators (p < 0.05) induced by crystalline silica, but none were induced by amorphous silica. QRT-PCR revealed that cristobalite selectively up-regulated stress-related genes and cytokines (FOS, ATF3, IL6 and IL8) early and over time (2, 4, 8, and 24 h). Patterns of gene expression in NHBE cells were similar overall to BEAS 2B cells. At 75 × 106μm2/cm2, there were 339 significant alterations in gene expression induced by cristobalite and 42 by amorphous silica. Comparison of genes in response to cristobalite (75 × 106μm2/cm2) revealed 60 common, significant gene alterations in NHBE and BEAS 2B cells.ConclusionsCristobalite silica, as compared to synthetic amorphous silica particles at equal surface area concentrations, had comparable effects on the viability of human bronchial epithelial cells. However, effects on gene expression, as well as secretion of cytokines and chemokines, drastically differed, as the crystalline silica induced more intense responses. Our studies indicate that toxicological testing of particulates by surveying viability and/or metabolic activity is insufficient to predict their pathogenicity. Moreover, they show that acute responses of the lung epithelium, including up-regulation of genes linked to inflammation, oxidative stress, and proliferation, as well as secretion of inflammatory and proliferative mediators, can be indicative of pathologic potential using either immortalized lines (BEAS 2B) or primary cells (NHBE). Assessment of the degree and magnitude of these responses in vitro are suggested as predictive in determining the pathogenicity of potentially harmful particulates.

Asthma is a generalized term that describes a scope of distinct pathologic phenotypes of variable severity, which share a common complication of reversible airflow obstruction. Asthma is estimated to affect almost 400 million people worldwide, and nearly ten percent of asthmatics have what is considered "severe" disease. The majority of moderate to severe asthmatics present with a "type 2-high" (T2-hi) phenotypic signature, which pathologically is driven by the type 2 cytokines Interleukin-(IL)-4, IL-5, and IL-13. However, "type 2-low" (T2-lo) phenotypic signatures are often associated with more severe, steroid-refractory neutrophilic asthma. A wide range of clinical and experimental studies have found that the receptor for advanced glycation endproducts (RAGE) plays a significant role in the pathogenesis of asthma and allergic airway disease (AAD). Current experimental data indicates that RAGE is a critical mediator of the type 2 inflammatory reactions which drive the development of T2-hi AAD. However, clinical studies demonstrate that increased RAGE ligands and signaling strongly correlate with asthma severity, especially in severe neutrophilic asthma. This review presents an overview of the current understandings of RAGE in asthma pathogenesis, its role as a biomarker of disease, and future implications for mechanistic studies, and potential therapeutic intervention strategies.

Respiratory syncytial virus (RSV) remains the most common cause of lower respiratory tract infections in children worldwide. Development of a vaccine has been hindered by the risk of developing enhanced respiratory disease (ERD) upon natural exposure to the virus. Generation of higher quality neutralizing antibodies with stabilized pre-fusion F protein antigens has been proposed as a strategy to prevent ERD. We sought to test whether there was evidence of ERD in naïve BALB/c mice immunized with an unadjuvanted, stabilized pre-fusion F protein, and challenged with RSV line 19. We further sought to determine the extent to which formulation with a Th2-biased (alum) or a more Th1/Th2-balanced (Advax-SM) adjuvant influenced cellular responses and lung pathology. When exposed to RSV, mice immunized with pre-fusion F protein alone (PreF) exhibited increased airway eosinophilia and mucus accumulation. This was further exacerbated by formulation of PreF with Alum (aluminum hydroxide). Conversely, formulation of PreF with a Th1/Th2-balanced adjuvant, Advax-SM, not only suppressed RSV viral replication, but also inhibited airway eosinophilia and mucus accumulation. This was associated with lower numbers of lung innate lymphocyte cells (ILC2s) and CD4+ T cells producing IL-5+ or IL-13+ and increased IFNγ+ CD4+ and CD8+ T cells, in addition to RSV F-specific CD8+ T cells. These data suggest that in the absence of preimmunity, stabilized PreF antigens may still be associated with aberrant Th2 responses that induce lung Eichinger et al. Protection With Th1/Th2-Balanced Prefusion Adjuvant pathology in response to RSV infection, and can be prevented by formulation with more Th1/Th2-balanced adjuvants that enhance CD4+ and CD8+ IFNγ+ T cell responses. This may support the use of stabilized PreF antigens with Th1/Th2-balanced adjuvants like, Advax-SM, as safer alternatives to alum in RSV vaccine candidates.

Background: Asthma is estimated to effect more than 300 million persons worldwide, leading to nearly 250,000 deaths annually. The majority of patients with mild-to-severe asthma have what is deemed ''type-2 high'' asthma, which is driven by the prototypical type 2 cytokines IL-4, IL-5, and IL-13. Studies have indicated that the receptor for advanced glycation end products (RAGE) is a critical molecule in the pathogenesis of experimental asthma/allergic airway inflammation. More specifically, RAGE expressed on stromal cells, rather than hematopoietic cells, is critical to induction of asthma/allergic airway inflammation by driving type 2 inflammatory responses. However, the role of RAGE in directly mediating type 2 cytokine signaling has never been investigated. Objective: The goal of this study was to test the hypothesis that RAGE mediates type 2 cytokine-induced signal transduction, airway inflammation, and mucus metaplasia in the lungs. Methods: Wild-type (WT) and RAGE knockout (RAGE 2/2) mice, were intranasally administered rIL-5/rIL-13 or rIL-4 alone, and signal transducer and activator of transcription 6 (STAT6) signaling, airway inflammation, and mucus metaplasia were assessed. A RAGE small-molecule antagonist was used to determine the effects of pharmacologically inhibiting RAGE on type 2 cytokine-induced effects. Results: Administration of type 2 cytokines induced pronounced airway inflammation and mucus metaplasia in WT mice, which was nearly completely abrogated in RAGE 2/2 mice. In addition, treatment with a RAGE-specific antagonist diminished the effects of type 2 cytokines in WT mice and in primary human bronchial epithelial cell cultures. Genetic ablation or pharmacologic inhibition of RAGE blocks the effects of IL-13 and IL-4 by inhibiting sustained STAT6 activation and downstream target gene expression in mice and in human bronchial epithelial cells. Conclusions: This study is the first to indicate that RAGE is a critical component of type 2 cytokine signal transduction mechanisms, which is a driving force behind type 2-high asthma.