Low levels of microRNA-21 in neutrophil-derived exosomes may contribute to airway smooth muscle hyperproliferation in horses with severe asthma.

OBJECTIVE: Neutrophilic inflammation is associated with the degree of airway obstruction in severe equine asthma (SEA), but the contribution of these leukocytes to bronchial remodeling remains ill defined. Neutrophils could cause structural alterations of the airways by the release of exosomes, a type of cell-derived nanoparticles that can modify the biology of local and distant cells. Neutrophil-derived exosomes have been shown to increase airway smooth muscle (ASM) cell proliferation in humans and horses. Therefore, this study aimed to identify neutrophil exosomal microRNAs (miRs) implicated in the regulation of ASM biology in SEA. ANIMALS: 6 horses with SEA and 6 healthy controls. METHODS: The expression of selected miRs in exosomes from peripheral neutrophils was studied by quantitative PCR. The effects of miR-21 transfection in ASM cells were evaluated by gene expression analysis and proliferation studies. RESULTS: The miR-21 was downregulated in neutrophil exosomes from SEA horses, and it attenuated the proliferation of ASM cells stimulated with lipopolysaccharide. CLINICAL RELEVANCE: The lower level of miR-21 in neutrophil-derived exosomes could contribute to ASM hyperproliferation, which could, in turn, promote the thickening of the bronchial wall in SEA.

Recellularization of Bronchial Extracellular Matrix With Primary Bronchial Smooth Muscle Cells.

Severe asthma is associated with an increased airway smooth muscle (ASM) mass and altered composition of the extracellular matrix (ECM). Studies have indicated that ECM-ASM cell interactions contribute to this remodeling and its limited reversibility with current therapy. Three-dimensional matrices allow the study of complex cellular responses to different stimuli in an almost natural environment. Our goal was to obtain acellular bronchial matrices and then develop a recellularization protocol with ASM cells. We studied equine bronchi as horses spontaneously develop a human asthma-like disease. The bronchi were decellularized using Triton/Sodium Deoxycholate. The obtained scaffolds retained their anatomical and histological properties. Using immunohistochemistry and a semi-quantitative score to compare native bronchi to scaffolds revealed no significant variation for matrixial proteins. DNA quantification and electrophoresis revealed that most DNA was 29.6 ng/mg of tissue ± 5.6, with remaining fragments of less than 100 bp. Primary ASM cells were seeded on the scaffolds. Histological analysis of the recellularizations showed that ASM cells migrated and proliferated primarily in the decellularized smooth muscle matrix, suggesting a chemotactic effect of the scaffolds. This is the first report of primary ASM cells preferentially repopulating the smooth muscle matrix layer in bronchial matrices. This protocol is now being used to study the molecular interactions occurring between the asthmatic ECMs and ASM to identify effectors of asthmatic bronchial remodeling.

An Integrative miRNA-mRNA Expression Analysis Reveals Striking Transcriptomic Similarities between Severe Equine Asthma and Specific Asthma Endotypes in Humans.

Severe equine asthma is an incurable obstructive respiratory condition affecting 10-15% of horses in temperate climates. Upon exposure to airborne antigens from hay feeding, affected horses show neutrophilic airway inflammation and bronchoconstriction, leading to increased respiratory effort. The resulting implications range from welfare concerns to economic impacts on equestrian sports and horse breeding. Immunological and pathophysiological characteristics of severe equine asthma show important parallels with allergic and severe neutrophilic human asthma. Our study aimed at investigating regulatory networks underlying the pathophysiology of the disease by profiling miRNA and mRNA expression in lung tissue samples from asthmatic horses compared with healthy controls. We sequenced small RNAs and mRNAs from lungs of seven asthmatic horses in exacerbation, five affected horses in remission, and eight healthy control horses. Our comprehensive differential expression analyses, combined with the miRNA-mRNA negative correlation approach, revealed a strong similarity on the transcriptomic level between severe equine asthma and severe neutrophilic asthma in humans, potentially through affecting Th17 cell differentiation. This study also showed that several dysregulated miRNAs and mRNAs are involved in airway remodeling. These results present a starting point for a better transcriptomic understanding of severe equine asthma and its similarities to asthma in humans.

Use of a biopolymer delivery system to investigate the influence of interleukin-4 on recruitment of neutrophils in equids.

OBJECTIVE: To use a biopolymer delivery system to investigate the ability of interleukin (IL)-4 to recruit neutrophils into subcutaneous tissues of equids. ANIMALS: 16 horses and 2 ponies. PROCEDURES: Animals were assigned to 3 experiments (6/experiment). Effects of recombinant equine (Req) IL-4 (100, 250, or 500 ng/site) versus a positive control (ReqIL-8; 100 ng, 250 ng, or 1 µg/site) and a negative control (Dulbecco PBSS or culture medium) on neutrophil chemotaxis were assessed after SC injection into the neck with an injectable biopolymer used as the vehicle. Tissue samples including the biopolymer plug were collected by biopsy at various time points from 3 hours to 7 days after injection. Neutrophil infiltration was evaluated by histologic scoring (experiments 1, 2, and 3) or flow cytometry (experiment 3). RESULTS: Histologic neutrophil infiltration scores did not differ significantly among treatments at most evaluated time points. On flow cytometric analysis, log-transformed neutrophil counts in biopsy specimens were significantly greater for the ReqIL-8 treatment (1 µg/site) than the negative control treatment at 3 but not 6 hours after injection; results did not differ between ReqIL-4 and control treatments at either time point. Negative control treatments induced an inflammatory response in most equids in all experiments. CONCLUSIONS AND CLINICAL RELEVANCE: Flow cytometry was a more reliable method to estimate neutrophil migration than histologic score analysis. The ReqIL-4 treatment did not induce a detectable neutrophil response, compared with the negative control treatment in this study. Evidence of inflammation in negative control samples suggested the biopolymer is not a suitable vehicle for use in equids.

Effects of a propriety oiled mixed hay feeding system on lung function, neutrophilic airway inflammation and oxidative stress in severe asthmatic horses.

BACKGROUND: Hay feeding is considered the main triggering factor for airway obstruction and inflammation in severe equine asthma (SEA). Finding alternate strategies allowing hay feeding while controlling clinical signs of SEA is of importance. The Nutri-Foin Système is believed to decrease inhaled dust by incorporating soybean oil to mechanically processed hay. OBJECTIVES: We compared airflow obstruction and airway inflammation in horses with SEA fed oiled hay or alfalfa pellet regimen. STUDY DESIGN: Controlled trial in asthmatic research horses. METHODS: Twelve horses in exacerbation of SEA from a research herd were studied. Horses were fed either oiled treated hay (n = 6) or alfalfa pelleted hay (n = 6) for 3 months while being stabled. Lung function, bronchoalveolar lavage fluid cytology and serum antioxidant enzyme kinetics were sequentially evaluated. RESULTS: Pelleted hay and the hay treated with the Nutri-Foin Système similarly improved lung function, airway neutrophilia and serum antioxidant enzyme kinetics over time. MAIN LIMITATIONS: The small number of horses in each group. CONCLUSIONS: We conclude from this study that Nutri-Foin Système is an appropriate alternative to pelleted hay for the control of the airway obstruction in horses with SEA.

Endogenous retrovirus-encoded Syncytin-2 contributes to exosome-mediated immunosuppression of T cells†.

Modulation of the activation status of immune cell populations during pregnancy depends on placental villous cytotrophoblast (VCT) cells and the syncytiotrophoblast (STB). Failure in the establishment of this immunoregulatory function leads to pregnancy complications. Our laboratory has been studying Syncytin-2 (Syn-2), an endogenous retroviral protein expressed in placenta and on the surface of placental exosomes. This protein plays an important role not only in STB formation through its fusogenic properties, but also through its immunosuppressive domain (ISD). Considering that Syn-2 expression is importantly reduced in preeclamptic placentas, we were interested in addressing its possible immunoregulatory effects on T cells. Activated Jurkat T cells and peripheral blood mononuclear cells (PBMCs) were treated with monomeric or dimerized version of a control or a Syn-2 ISD peptide. Change in phosphorylation levels of ERK1/2 MAP kinases was selectively noted in Jurkat cells treated with the dimerized ISD peptide. Upon incubation with the dimerized Syn-2 ISD peptide, significant reduction in Th1 cytokine production was further demonstrated by ELISA and Human Th1/Th2 Panel Multi-Analyte Flow Assay. To determine if exosome-associated Syn-2 could also be immunosuppressive placental exosomes were incubated with activated Jurkat and PBMCs. Quantification of Th1 cytokines in the supernatants revealed severe reduction in T cell activation. Interestingly, exosomes from Syn-2-silenced VCT incubated with PBMCs were less suppressive when compared with exosome derived from VCT transfected with control small interfering RNA (siRNA). Our results suggest that Syn-2 is an important immune regulator both locally and systemically, via its association with placental exosomes.

Galectin-1 interacts with the human endogenous retroviral envelope protein syncytin-2 and potentiates trophoblast fusion in humans.

Syncytin (Syn)-2 is an important fusogenic protein that contributes to the formation of the placental syncytiotrophoblast. Galectin (Gal)-1, a soluble lectin, is also involved in trophoblast cell fusion and modulates the interaction of certain retroviral envelopes with their cellular receptor. This study aimed to investigate the association between Syn-2 and Gal-1 during human trophoblast cell fusion. This association was evaluated in vitro on primary villous cytotrophoblasts (vCTBs) and cell lines using recombinant Gal-1 and Syn-2-pseudotyped viruses. Using lactose, a Gal antagonist, and Gal-1-specific small interfering RNA (siRNA) transfections, we confirmed the implication of Gal-1 in vCTBs and BeWo cell fusion, although RT-PCR and ELISA analyses suggested that Gal-1 alone did not induce syncytialization. Infection assays showed a specific and significant effect of Gal-1 on the infectivity of Syn-2-pseudotyped viruses that depended on the expression of major facilitator superfamily domain-containing 2A (MFSD2a). Moreover, Gal-3, another placental Gal, did not modulate the infectivity of Syn-2-positive viruses, strengthening the specific association between Gal-1 and Syn-2. Interestingly, Gal-1 significantly reduced the infectivity of Syn-1-pseudotyped viruses, suggesting the opposite effects of Gal-1 on Syn-1 and -2. Finally, coimmunoprecipitation experiments showed a glycan-dependent interaction between Syn-2-bearing virions and Gal-1. We conclude that Gal-1 specifically interacts with Syn-2 and possibly regulates Syn-2/MFSD2a interaction during syncytialization of trophoblastic cells.-Toudic, C., Vargas, A., Xiao, Y., St-Pierre, G., Bannert, N., Lafond, J., Rassart, É., Sato, S., Barbeau, B. Galectin-1 interacts with the human endogenous retroviral envelope protein syncytin-2 and potentiates trophoblast fusion in humans.

Randomised study of the immunomodulatory effects of azithromycin in severely asthmatic horses.

Neutrophilic inflammation is believed to contribute to the airway obstruction and remodelling in equine asthma. Azithromycin, an antibiotic with immunomodulatory properties, reduces pulmonary neutrophilia and hyper-responsiveness in human asthmatics and decreases airway remodelling in rodent models of asthma. It was therefore hypothesised that azithromycin would improve lung function, mucus accumulation and central airway remodelling by decreasing luminal neutrophilia in severe equine asthma. The effects of a 10-day treatment with either azithromycin or ceftiofur, an antimicrobial without immune-modulating activity, were assessed using a blind, randomised, crossover design with six severe asthmatic horses in clinical exacerbation. Lung function, tracheal mucus accumulation, tracheal wash bacteriology, bronchial remodelling, airway neutrophilia and mRNA expression of proinflammatory cytokines (interleukin (IL)-8, IL-17A, IL-1ß, tumour necrosis factor-α) in bronchoalveolar lavage fluid were evaluated. Azithromycin decreased the expression of IL-8 (P=0.03, one-tailed) and IL-1ß (P=0.047, one-tailed) but failed to improve the other variables evaluated. Ceftiofur had no effect on any parameter. The reduction of neutrophilic chemoattractants (IL-8, IL-1ß) justifies further efforts to investigate the effects of a prolonged treatment with macrolides on airway neutrophilia and remodelling. The lack of efficacy of ceftiofur suggests that severe equine asthma should not be treated with antibiotics at first-line therapy.

MicroRNA-221 is overexpressed in the equine asthmatic airway smooth muscle and modulates smooth muscle cell proliferation.

Airway wall remodeling, including hyperplasia and hypertrophy of smooth muscle (ASM) cells leading to an increased smooth muscle mass, is considered central to asthma. However, molecular pathways responsible for ASM remodeling remain poorly understood. MicroRNAs (miRNAs) have emerged as key regulators of inflammatory and repair processes affecting the lungs and can downregulate protein expression by inhibiting target mRNA translation. We therefore hypothesized that miRNAs are involved in ASM remodeling in asthma by modulating ASM proliferation. We have analyzed the expression of miRNAs in bronchial smooth muscle from asthmatic horses during disease exacerbation and remission and from controls. Their involvement in ASM cell proliferation was then studied. Our results shown that miR-26a, miR-133, and miR-221 were upregulated in ASM from horses with asthma exacerbation compared with asthma remission and controls. MiR-221 induced cell hyperproliferation and reduced the expression of contractile gene markers in ASM cells. These changes were associated with the decreased mRNA expression of cell cycle regulatory genes (p53, p21, and p27). In conclusion, we demonstrated for the first time an upregulation of miR-221 in asthmatic airway smooth muscle and confirm the involvement of miR-221 in ASM cell proliferation by regulation of the cell cycle arrest genes. Targeting miR-221 network genes may represent a novel approach for the treatment of ASM remodeling in asthma.

Investigation of blood biomarkers for the diagnosis of mild to moderate asthma in horses.

BACKGROUND: Asthma in horses is associated with nonspecific respiratory clinical signs and may be manifested only as exercise intolerance. Its diagnosis relies on bronchoalveolar lavage fluid (BALF) cytology in the presence of compatible clinical signs. The identification of blood biomarkers for this condition would facilitate diagnosis in the field, because there are regional areas where BAL is not routinely performed in clinical practice. OBJECTIVE: Identification of blood biomarkers for the diagnosis of asthma in horses. ANIMALS: Fourteen horses with asthma with increased neutrophil numbers in BALF (neutrophilic asthma), 9 healthy control horses, and 10 horses with other pathologic conditions (pathologic controls). METHODS: Physical examination, clinical score, hematology, and BALF cytology (in a subset of horses) were performed. Serum concentrations of surfactant protein D (SP-D), haptoglobin, and secretoglobin (SCGB) were measured using commercial ELISA assays. RESULTS: Serum concentration of SP-D > 43 ng/mL, serum concentration of haptoglobin >5730 ng/mL, and serum concentration of SCGB <19 ng/mL allowed differentiation of horses with neutrophilic asthma from horses of the control groups (healthy and pathologic) with sensitivity of 55, 95, and 75%, and specificity of 67, 28, and 60%, respectively. Specificity of 100% and sensitivity of 45% were obtained with the combination of SP-D, haptoglobin, and SCGB at the serum concentrations indicated above. Specificity of 95% and sensitivity of 45% were obtained with the combination of SP-D and SCGB serum concentrations. CONCLUSIONS AND CLINICAL IMPORTANCE: Haptoglobin, SCGB, and SP-D may be diagnostic aids in horses with clinical signs of lower airway disease and neutrophilic pulmonary inflammation.

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.

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.

Neutrophil extracellular traps are downregulated by glucocorticosteroids in lungs in an equine model of asthma.

BACKGROUND: Severe neutrophilic asthma is poorly responsive to glucocorticosteroids (GC). Neutrophil extracellular traps (NETs) within the lungs have been associated with the severity of airway obstruction and inflammation in asthma, and were found to be unaffected by GC in vitro. As IL-17 is overexpressed in neutrophilic asthma and contributes to steroid insensitivity in different cell types, we hypothesized that NETs formation in asthmatic airways would be resistant to GC through an IL-17 mediated pathway. METHODS: Six neutrophilic severe asthmatic horses and six healthy controls were studied while being treated with dexamethasone. Lung function, bronchoalveolar lavage fluid (BALF) cytology and NETs formation, as well as the expression of CD11b and CD13 by blood and airway neutrophils were evaluated. The expression of IL-17 and its role in NETs formation were also studied. RESULTS: Airway neutrophils from asthmatic horses, as opposed to blood neutrophils, enhanced NETs formation, which was then decreased by GC. GC also tended to decrease the expression of CD11b in blood neutrophils, but not in airway neutrophils. IL-17 mRNA was increased in BALF cells of asthmatic horses and was unaffected by GC. However, both GC and IL-17 inhibited NETs formation in vitro. CONCLUSION: GC decreased NETs formation in vitro and also in vivo in the lungs of asthmatic horses. However, airway neutrophil activation during asthmatic inflammation was otherwise relatively insensitive to GC. The contribution of IL-17 to these responses requires further study.

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.

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

Asthmatic airways are inflamed and undergo remodelling. Inhaled corticosteroids and long-acting ß2-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.

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.

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts.

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

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.

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.

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.