José A Cañas

Bio: José A Cañas is an academic researcher from Carlos III Health Institute. The author has contributed to research in topics: Asthma & Medicine. The author has an hindex of 10, co-authored 30 publications receiving 394 citations.

Exosome secretion by eosinophils: A possible role in asthma pathogenesis

Abstract: Background Eosinophils secrete several granules that are involved in the propagation of inflammatory responses in patients with pathologies such as asthma. Objective We hypothesized that some of these granules are exosomes, which, when transferred to the recipient cells, could modulate asthma progression. Methods Eosinophils were purified from peripheral blood and cultured with or without IFN-γ or eotaxin. Multivesicular bodies (MVBs) in eosinophils were studied by using fluorescence microscopy, transmission electron microscopy (TEM), and flow cytometry. Exosome secretion was measured and exosome characterization was performed with TEM, Western blotting, and NanoSight analysis. Results Generation of MVBs in eosinophils was confirmed by using fluorescence microscopy and flow cytometry and corroborated by means of TEM. Having established that eosinophils contain MVBs, our aim was to demonstrate that eosinophils secrete exosomes. To do this, we purified exosomes from culture medium of eosinophils and characterized them. Using Western blot analysis, we demonstrated that eosinophils secreted exosomes and that the discharge of exosomes to extracellular media increases after IFN-γ stimulation. We measured exosome size and quantified exosome production from healthy and asthmatic subjects using nanotracking analysis. We found that exosome production was augmented in asthmatic patients. Conclusion Our findings are the first to demonstrate that eosinophils contain functional MVBs and secrete exosomes and that their secretion is increased in asthmatic patients. Thus exosomes might play an important role in the progression of asthma and eventually be considered a biomarker.

Novel Modulators of Asthma and Allergy: Exosomes and MicroRNAs.

Abstract: Intercellular communication is crucial to the immune system response. In the recent years the discovery of exosomes has changed the way immune response orchestration was understood. Exosomes are able to operate as independent units that act as mediators in both physiological and pathological conditions. These structures contain proteins, lipidic mediators, and nucleic acids, and notoriously include microRNAs (miRNAs). miRNAs are short RNA sequences (around 19–22 nucleotides) with a high phylogenetic conservation and can partially or totally regulate multiple mRNAs, inhibiting protein synthesis. In respiratory diseases such as asthma and allergic sensitization, exosomes released by several cell types and their specific content perform crucial functions in the development and continuation of the pathogenic mechanisms. Released exosomes and miRNAs inside them have been found in different types of clinical samples, such as bronchoalveolar lavage fluids and sputum supernatants, providing new data about the environmental factors and mediators that participate in the inflammatory responses that lead to the exacerbation of asthma. In this review, we summarize our current knowledge of the role of exosomes and miRNAs in asthma and allergic sensitization, paying attention to the functions that both exosomes and miRNAs are described to perform through the literature. We review the effect of exosomes and miRNAs in cells implicated in asthma pathology and the genes and pathways that they modify in them, depicting how their behavior is altered in disease status. We also describe their possible repercussion in asthma diagnosis through their possible role as biomarkers. Therefore, both exosomes and miRNAs can be viewed as potential tools to be added to the arsenal of therapeutics to treat this disease.

Eosinophil-derived exosomes contribute to asthma remodelling by activating structural lung cells.

Abstract: SummaryBackground Eosinophils, a central factor in asthma pathogenesis, have the ability to secrete exosomes. However, the precise role played by exosomes in the biological processes leading up to asthma has not been fully defined. Objective We hypothesized that exosomes released by eosinophils contribute to asthma pathogenesis by activating structural lung cells. Methods Eosinophils from asthmatic patients and healthy volunteers were purified from peripheral blood, and exosomes were isolated from eosinophils of asthmatic and healthy individuals. All experiments were performed with eosinophil-derived exosomes from healthy and asthmatic subjects. Epithelial damage was evaluated using primary small airway epithelial cell lines through 2 types of apoptosis assays, that is, flow cytometry and TUNEL assay with confocal microscopy. Additionally, the epithelial repair was analysed by performing wound healing assays with epithelial cells. Functional studies such as proliferation and inhibition-proliferation assays were carried out in primary bronchial smooth muscle cell lines. Also, gene expression analysis of pro-inflammatory molecules was evaluated by real-time PCR on epithelial and muscle cells. Lastly, protein expression of epithelial and muscle cell signalling factors was estimated by Western blot. Results Asthmatic eosinophil-derived exosomes induced an increase in epithelial cell apoptosis at 24 hour and 48 hour, impeding wound closure. In addition, muscle cell proliferation was increased at 72 hours after exosome addition and was linked with higher phosphorylation of ERK1/2. We also found higher expression of several genes when both cell types were cultured in the presence of exosomes from asthmatics: CCR3 and VEGFA in muscle cells, and CCL26, TNF and POSTN in epithelial cells. Healthy eosinophil-derived exosomes did not exert any effect over these cell types. Conclusions and Clinical Relevance Eosinophil-derived exosomes from asthmatic patients participate actively in the development of the pathological features of asthma via structural lung cells.

Exosomes from eosinophils autoregulate and promote eosinophil functions.

Abstract: Eosinophils are able to secrete exosomes that have an undefined role in asthma pathogenesis. We hypothesized that exosomes released by eosinophils autoregulate and promote eosinophil function. Eosinophils of patients with asthma (n = 58) and healthy volunteers (n = 16) were purified from peripheral blood, and exosomes were isolated and quantified from eosinophils of the asthmatic and healthy populations. Apoptosis, adhesion, adhesion molecules expression, and migration assays were performed with eosinophils in the presence or absence of exosomes from healthy and asthmatic individuals. Reactive oxygen species (ROS) were evaluated by flow cytometry with an intracellular fluorescent probe and nitric oxide (NO) and a colorimetric kit. In addition, exosomal proteins were analyzed by mass spectrometry. Eosinophil-derived exosomes induced an increase in NO and ROS production on eosinophils. Moreover, exosomes could act as a chemotactic factor on eosinophils, and they produced an increase in cell adhesion, giving rise to a specific augmentation of adhesion molecules, such as ICAM-1 and integrin α2. Protein content between exosomes from healthy and asthmatic individuals seems to be similar in both groups. In conclusion, we found that exosomes from the eosinophils of patients with asthma could modify several specific eosinophil functions related to asthma pathogenesis and that they could contribute fundamentally to the development and maintenance of asthma.

Asthma diagnosis using integrated analysis of eosinophil microRNAs.

Abstract: Background Asthma is a syndrome characterized by airway inflammation and obstruction. Due to its heterogeneity, the difficulties in asthma diagnosis and treatment make the discovery of new biomarkers a focus of research. So, we determined the differential miRNA expression of eosinophils between healthy and asthmatic patients and to establish a differentially expressed miRNA profile detectable in sera for use as biomarker. Methods MicroRNAs from peripheral eosinophils from healthy and asthmatic subjects were isolated and analyzed by next-generation sequencing and confirmed by quantitative PCR in 29 asthmatics and 10 healthy individuals. The levels of serum miRNAs were performed by quantitative PCR in 138 asthmatics and 39 healthy subjects. Regression analysis and Random Forest models were performed. Results We found a set of miRNAs whose expression differs between eosinophils from asthmatics and healthy subjects. These miRNAs can classify asthmatics into two clusters that differed in the number of eosinophils and periostin concentration in serum. Some of these miRNAs were also confirmed in sera, as miR-185-5p which discriminates asthmatics from healthy subjects. Together with other two miRNAs, miR-185-5p allowed us to create a logistic regression model to discriminate better both conditions and a Random Forest model that can even sort the asthmatics into intermittent, mild persistent, moderate persistent, and severe persistent asthma. Conclusion Our data show that miRNAs profile in eosinophils can be used as asthma diagnosis biomarker in serum and that this profile is able to rank asthma severity.

Inflammation and tumor progression: signaling pathways and targeted intervention.

Abstract: Cancer development and its response to therapy are regulated by inflammation, which either promotes or suppresses tumor progression, potentially displaying opposing effects on therapeutic outcomes. Chronic inflammation facilitates tumor progression and treatment resistance, whereas induction of acute inflammatory reactions often stimulates the maturation of dendritic cells (DCs) and antigen presentation, leading to anti-tumor immune responses. In addition, multiple signaling pathways, such as nuclear factor kappa B (NF-kB), Janus kinase/signal transducers and activators of transcription (JAK-STAT), toll-like receptor (TLR) pathways, cGAS/STING, and mitogen-activated protein kinase (MAPK); inflammatory factors, including cytokines (e.g., interleukin (IL), interferon (IFN), and tumor necrosis factor (TNF)-α), chemokines (e.g., C-C motif chemokine ligands (CCLs) and C-X-C motif chemokine ligands (CXCLs)), growth factors (e.g., vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β), and inflammasome; as well as inflammatory metabolites including prostaglandins, leukotrienes, thromboxane, and specialized proresolving mediators (SPM), have been identified as pivotal regulators of the initiation and resolution of inflammation. Nowadays, local irradiation, recombinant cytokines, neutralizing antibodies, small-molecule inhibitors, DC vaccines, oncolytic viruses, TLR agonists, and SPM have been developed to specifically modulate inflammation in cancer therapy, with some of these factors already undergoing clinical trials. Herein, we discuss the initiation and resolution of inflammation, the crosstalk between tumor development and inflammatory processes. We also highlight potential targets for harnessing inflammation in the treatment of cancer.

Mepolizumab for Severe Eosinophilic Asthma (DREAM): A Multicentre, Double-Blind, Placebo-Controlled Trial

Abstract: ID Pavord, S Korn, P Howarth. Lancet. 2012;380(9842):651–659 To elucidate the efficacy, safety, and patient characteristics of responsiveness to mepolizumab (a humanized monoclonal antibody against interleukin 5). Previous small, proof-of-concept studies in subjects with severe, eosinophilic asthma revealed that mepolizumab decreased exacerbation rates. From 81 multinational centers, 621 pa-tients were enrolled. Major inclusion criteria included: age 12 to 74 years, asthma diagnosis with objective measures, ≥2 asthma exacerbations requiring oral corticosteroids in the last year, refractory asthma as defined by the American …

T H 2 cell development and function

Abstract: T helper 2 (TH2) cells orchestrate protective type 2 immune responses, such as those that target helminths and facilitate tissue repair, but also contribute to chronic inflammatory diseases, such as asthma and allergy. Here, we review recent insights into how diverse molecular signals from cellular sources, including dendritic cells, innate lymphoid cells and the epithelium, are integrated by T cells to guide the transcriptional and epigenetic changes necessary for TH2 cell differentiation. Our improved understanding of these pathways has opened new avenues for therapeutically targeting TH2 cells in asthma and allergy. The advent of comprehensive single-cell transcriptomics along with improvements in single-cell proteomics and the generation of novel in vivo cell fate mapping techniques promise to expand our understanding of T cell diversity and offer new insight into disease-related heterogeneity and plasticity of TH cell responses.

Extracellular vesicles, exosomes and shedding vesicles in regenerative medicine – a new paradigm for tissue repair

Abstract: Tissue regeneration by stem cells is driven by the paracrine activity of shedding vesicles and exosomes, which deliver specific cargoes to the recipient cells. Proteins, RNA, cytokines and subsequent gene expression, orchestrate the regeneration process by improving the microenvironment to promote cell survival, controlling inflammation, repairing injury and enhancing the healing process. The action of microRNA is widely accepted as an essential driver of the regenerative process through its impact on multiple downstream biological pathways, and its ability to regulate the host immune response. Here, we present an overview of the recent potential uses of exosomes for regenerative medicine and tissue engineering. We also highlight the differences in composition between shedding vesicles and exosomes that depend on the various types of stem cells from which they are derived. The conditions that affect the production of exosomes in different cell types are deliberated. This review also presents the current status of candidate exosomal microRNAs for potential therapeutic use in regenerative medicine, and in applications involving widely studied organs and tissues such as heart, lung, cartilage and bone.