Amandine Vargas

Neutrophil-Derived Exosomes: A New Mechanism Contributing to Airway Smooth Muscle Remodeling.

Neutrophils infiltrate the airways of patients with asthma of all severities, yet their role in the pathogenesis of asthma and their contribution to airway remodeling is largely unknown. We hypothesized that neutrophils modulate airway smooth muscle (ASM) proliferation in asthma by releasing bioactive exosomes. These newly discovered nano-sized vesicles have the capacity to modulate immune responses, cell migration, cell differentiation, and other aspects of cell-to-cell communication. The aim of the study is to determine whether bioactive exosomes are released by neutrophils, and, if so, characterize their proteomic profile and evaluate their capacity to modulate ASM cell proliferation. Exosomes were isolated from equine neutrophil supernatants by differential centrifugation and filtration methods, followed by size-exclusion chromatography. Nanovesicles were characterized using electron microscopy, particle size determination, and proteomic analyses. Exosomes were cocultured with ASM cells and analyzed for exosome internalization by confocal microscopy. ASM proliferation was measured using an impedance-based system. Neutrophils release exosomes that have characteristic size, morphology, and exosomal markers. We identified 271 proteins in exosomes from both LPS and unstimulated neutrophils, and 16 proteins that were differentially expressed, which carried proteins associated with immune response and positive regulation of cell communication. Furthermore, neutrophil-derived exosomes were rapidly internalized by ASM cells and altered their proliferative properties. Upon stimulation of LPS, neutrophil-derived exosomes can enhance the proliferation of ASM cells and could therefore play an important role in the progression of asthma and promoting airway remodeling in severe and corticosteroid-insensitive patients with asthma.

The Interleukin-17 Induced Activation and Increased Survival of Equine Neutrophils Is Insensitive to Glucocorticoids

Background Glucocorticoids (GCs) are the most effective drugs for the treatment of human asthma. However, a subgroup of asthmatic patients with neutrophilic airway inflammation is insensitive to GCs. Interleukin-17 (IL-17), a cytokine upregulated in the airways of a subset of human asthmatic patients, contributes to the recruitment of neutrophils and induces a glucocorticoid resistance in human airway epithelial cells. We hypothesized that IL-17 similarly activates neutrophils and contributes to their persistence in the asthmatic airways in spite of glucocorticoid therapy. Objective To determine whether IL-17 directly activates neutrophils and whether this response is attenuated by GCs. Methods Neutrophils were isolated from the blood of horses and incubated in the presence of recombinant equine IL-17, LPS and dexamethasone. mRNA and protein expression of IL-17 receptors (IL-17RA/IL-17RC) were assessed by qPCR and immunoblot, respectively. Pro-inflammatory cytokine expression, cell viability and apoptosis were determined by qPCR, Trypan Blue exclusion test, and flow cytometry, respectively. Results Equine neutrophils express both IL-17RA and IL-17RC at the mRNA and protein levels. Neutrophil stimulation with IL-17 increases the mRNA expression of IL-8, which is not attenuated by dexamethasone (p = 0.409). Also, neutrophil viability is significantly increased (p<0.0001) by IL-17 in the presence of LPS when compared to LPS alone. Flow cytometry and light microscopy revealed that LPS-induced apoptosis is decreased by IL-17 (p = 0.02 and p = 0.006 respectively). Conclusion These results indicate that IL-17 directly activates equine neutrophils at 24 hours, and that the expression of IL-8 thus induced is not attenuated by GCs. Additionally, IL-17 increases neutrophil viability and decreases apoptosis. These findings suggest an important role of IL-17 in pulmonary persistence of neutrophils in the asthmatic airways.

CONTRIBUTION OF SRF, ELK-1 AND MYOCARDIN TO AIRWAY SMOOTH MUSCLE REMODELING IN HEAVES, AN ASTHMA-LIKE DISEASE OF HORSES

Myocyte hyperplasia and hypertrophy contribute to the increased mass of airway smooth muscle (ASM) in asthma. Serum-response factor (SRF) is a transcription factor that regulates myocyte differentiation in vitro in vascular and intestinal smooth muscles. When SRF is associated with phosphorylated (p)Elk-1, it promotes ASM proliferation while binding to myocardin (MYOCD) leading to the expression of contractile elements in these tissues. The objective of this study was therefore to characterize the expression of SRF, pElk-1, and MYOCD in ASM cells from central and peripheral airways in heaves, a spontaneously occurring asthma-like disease of horses, and in controls. Six horses with heaves and five aged-matched controls kept in the same environment were studied. Nuclear protein expression of SRF, pElk-1, and MYOCD was evaluated in peripheral airways and endobronchial biopsies obtained during disease remission and after 1 and 30 days of naturally occurring antigenic exposure using immunohistochemistry and immunofluorescence techniques. Nuclear expression of SRF (P = 0.03, remission vs. 30 days) and MYOCD (P = 0.05, controls vs. heaves at 30 days) increased in the peripheral airways of horses with heaves during disease exacerbation, while MYOCD (P = 0.04, remission vs. 30 days) decreased in the central airways of control horses. No changes were observed in the expression of pElk-1 protein in either tissue. In conclusion, SRF and its cofactor MYOCD likely contribute to the hypertrophy of peripheral ASM observed in equine asthmatic airways, while the remodeling of the central airways is more static or involves different transcription factors.

Inhaled corticosteroids modulate the (+)insert smooth muscle myosin heavy chain in the equine asthmatic airways

Rationale Overexpression of the (+)insert smooth muscle myosin heavy chain (SMMHC) isoform could contribute to airway bronchospasm by increasing the velocity of contraction. Whether the (+)insert isoform is present in the small airways and its expression is reversible in asthma are unknown. Objectives To determine the anatomical location and the expression kinetics of the (+)insert SMMHC isoform in airways of horses with heaves and to evaluate its modulation in response to disease status. Methods We evaluated the (+)insert SMMHC isoform in the airways of horses with heaves during disease exacerbation and remission, and in controls. The expression kinetics of the SMMHC (+)insert was then assessed at multiple time points in two studies: first, in horses with heaves treated for a 1-year period with antigen avoidance alone, inhaled corticosteroids alone or both; second, in horses with heaves before and after a 30-day natural antigen exposure. Gene expression analysis was assessed by quantitative PCR and protein expression was confirmed by targeted mass spectrometry. Measurements and main results The (+)insert SMMHC isoform was significantly increased in central and peripheral airways, but not in the trachea of heaves-affected horses in clinical exacerbation when compared horses with heaves in remission and controls. Both corticosteroid administration and antigen avoidance led to a significant reduction of the (+)insert expression in the airways. The (+)insert SMMHC isoform was not significantly increased in airways after 1 month of antigenic re-exposure. Conclusions The (+)insert SMMHC expression is increased throughout the bronchial tree in horses with heaves and reversible by corticosteroids administration and antigen avoidance.

Fluticasone/salmeterol reduces remodelling and neutrophilic inflammation in severe equine asthma

Asthmatic airways are inflamed and undergo remodelling. Inhaled corticosteroids and long-acting β2-agonist combinations are more effective than inhaled corticosteroid monotherapy in controlling disease exacerbations, but their effect on airway remodelling and inflammation remains ill-defined. This study evaluates the contribution of inhaled fluticasone and salmeterol, alone or combined, to the reversal of bronchial remodelling and inflammation. Severely asthmatic horses (6 horses/group) were treated with fluticasone, salmeterol, fluticasone/salmeterol, or with antigen avoidance for 12 weeks. Lung function, central and peripheral airway remodelling, and bronchoalveolar inflammation were assessed. Fluticasone/salmeterol and fluticasone monotherapy decreased peripheral airway smooth muscle remodelling after 12 weeks (p = 0.007 and p = 0.02, respectively). On average, a 30% decrease was observed with both treatments. In central airways, fluticasone/salmeterol reversed extracellular matrix remodelling after 12 weeks, both within the lamina propria (decreased thickness, p = 0.005) and within the smooth muscle layer (p = 0.004). Only fluticasone/salmeterol decreased bronchoalveolar neutrophilia (p = 0.03) to the same extent as antigen avoidance already after 8 weeks. In conclusion, this study shows that fluticasone/salmeterol combination decreases extracellular matrix remodelling in central airways and intraluminal neutrophilia. Fluticasone/salmeterol and fluticasone monotherapy equally reverse peripheral airway smooth muscle remodelling.

Characterization of Circulating Low-Density Neutrophils Intrinsic Properties in Healthy and Asthmatic Horses

Low-density neutrophils (LDNs) are a subset of neutrophils first described in the bloodstream upon pathological conditions, and recently, in the blood of healthy humans. LDNs may have an enhanced pro-inflammatory (low-density granulocytes, LDGs) or an immunosuppressive (Granulocytic myeloid-derived suppressor cells, G-MDSCs) profile. Whether these characteristics are specific to LDNs or related to disease states is unknown. Thus, we sought to investigate the properties of LDNs in both health and disease states, and to compare them to those of autologous normal-density neutrophils (NDNs). We studied 8 horses with severe equine asthma and 11 healthy animals. LDNs were smaller and contained more N-formylmethionine-leucyl-phenylalanine receptors than NDNs, but the myeloperoxidase content was similar in both cell populations. They also had an increased capacity to produce neutrophil extracellular traps, and were more sensitive to activation by phorbol-12-myristate-13-acetate. This profile is suggestive of LDGs. These characteristics were similar in both healthy and diseased animals, indicating that these are intrinsic properties of LDNs. Furthermore, these results suggest that LDNs represent a population of primed and predominantly mature cells. This study is the first to characterize LDNs in health, and to compare their properties with those of NDNs and of animals with a naturally occurring disease.

A real-time assay for neutrophil chemotaxis

Neutrophils are the predominant cells during acute phases of inflammation, and it is now recognized that these leukocytes play an important role in modulation of the immune response. Directed migration of these cells to the sites of injury, known as chemotaxis, is driven by chemoattractants present at the endothelial cell surface or in the extracellular matrix (ECM). Since uncontrolled or excessive neutrophil chemotaxis is involved in pathological conditions such as atherosclerosis and severe asthma, studying the chemical cues triggering neutrophil migration is essential for understanding the biology of these cells and developing new anti-inflammatory therapies. Although several methods have been developed to evaluate neutrophil chemotaxis, these techniques are generally labor-intensive or alter the native form of these cells and their physiological response. Here we report a rapid, non-invasive, impedance-based, and label-free assay for real-time assessment of neutrophil chemotaxis.

Evaluation of contractile phenotype in airway smooth muscle cells isolated from endobronchial biopsy and tissue specimens from horses

OBJECTIVE To develop a method to maintain the initial phenotype of airway smooth muscle (ASM) cells isolated from equine endobronchial biopsy specimens in long-term cell culture. SAMPLE Endobronchial tissue specimens (8 to 10/horse) collected from the lungs of previously healthy horses at necropsy (n = 12) and endobronchial biopsy specimens collected from standing, sedated, heaves-affected horses in clinical remission of the disease (5) and control horses (4). PROCEDURES A sampling protocol was developed to recover and maintain a contractile phenotype in ASM cells from endobronchial specimens from freshly harvested equine lungs and from healthy and heaves-affected horses. Immunologic techniques were used to evaluate the contractile phenotype of ASM cells in culture. RESULTS Characteristic ASM cells were successfully cultured from endobronchial tissue or biopsy specimens from both healthy and heaves-affected horses, and their contractile phenotype was maintained for up to 7 passages. Moreover, the capacity of cells at the seventh passage to contract in a collagen gel in response to methacholine was maintained. CONCLUSIONS AND CLINICAL RELEVANCE ASM cells isolated from equine endobronchial tissue and biopsy specimens were able to maintain a contractile phenotype in long-term cell cultures, suggesting they could be used for tissue engineering and in vitro studies of equine ASM cells.

Glucocorticosteroids administration is associated with increased regulatory T cells in equine asthmatic lungs

Recurrent inflammation in severe equine asthma causes a remodeling of the airways leading to incompletely reversible airway obstruction. Despite the improvement of clinical signs and lung function with glucocorticoids (GC), inflammation, translated by an increased percentage of neutrophils, persists in the airways. Regulatory T cells (Treg) have been shown to have anti-inflammatory properties and play an important role in balancing the immune response by suppressing effector lymphocyte activity. However, interactions between Treg, neutrophils and glucocorticosteroids in vivo are unclear, particularly in asthma. Furthermore, the effects of GC on Treg in the airway of asthmatic horses have not been investigated. We hypothesized that horses with severe asthma display a decreased population of pulmonary Treg when compared to heathy controls, and that treatment with GC lead to an increased pulmonary Treg cell population only in affected horses. Using lung function measurements and flow cytometry with surface antigens CD4 and FoxP3, we investigated Treg in airway luminal cells obtained by bronchoalveolar lavage fluid (BALF) from 6 asthmatic horses in exacerbation of the disease and 6 aged-match controls, kept in the same environment, before and following a 2-week treatment with dexamethasone. Results showed that the number of Treg increases only in the lungs of asthmatic horses following GC therapy, despite continued presence of increased numbers of neutrophils. Our results support the complexity of the interaction between Treg, neutrophils and GC.

SRSF6 is upregulated in asthmatic horses and involved in the MYH11 SMB expression

Smooth muscle has a central role in bronchospasm‐induced airway obstruction in asthma. Alternative mRNA splicing of the smooth muscle myosin heavy chain (myh11) gene produces four different isoforms, one of which (SMB) is characterized by the inclusion of the exon5b, which doubles the smooth muscle cells contraction velocity. Deciphering the regulation of the expression levels of the SMB isoform would represent a major step for the understanding of the triggers and pathways leading to airway smooth muscle contraction in asthma. Our objective was therefore, to study the splicing regulation mechanisms of the exon5b in airway smooth muscle cells. Bioinformatics analysis was performed to identify the cis‐regulatory elements present in the exon5b using HSF finder 3 tool. The expression of the corresponding serine/arginine rich protein (SR) genes thus identified was evaluated by quantitative RT‐PCR (qPCR). SRSF1, SRSF6, and hnRNPA1 cis‐acting elements were identified by in silico analysis of the exon5b sequence as splicing regulator candidates. QPCR analyses showed that SRSF1 and SRSF6 are upregulated in ASM cells from asthmatic horses in exacerbation (n = 5) compared to controls (n = 5). The inhibition of the identified splicing factors by small interfering RNA allowed identifying the regulation of the SMB isoform by SRSF6. Our results implicate for the first time the upregulation of SRSF6 and SRSF1 in the asthmatic ASM cells and indicate that SRSF6 induces the exon5b inclusion. This study provides an important first step for the understanding of the triggers and pathways leading to ASM hypercontraction and identifies a possible new target for asthma.