Stephane Esnault - Academia.edu (original) (raw)

Papers by Stephane Esnault

Metabolites

Asthma is a complex syndrome associated with episodic decompensations provoked by aeroallergen ex... more Asthma is a complex syndrome associated with episodic decompensations provoked by aeroallergen exposures. The underlying pathophysiological states driving exacerbations are latent in the resting state and do not adequately inform biomarker-driven therapy. A better understanding of the pathophysiological pathways driving allergic exacerbations is needed. We hypothesized that disease-associated pathways could be identified in humans by unbiased metabolomics of bronchoalveolar fluid (BALF) during the peak inflammatory response provoked by a bronchial allergen challenge. We analyzed BALF metabolites in samples from 12 volunteers who underwent segmental bronchial antigen provocation (SBP-Ag). Metabolites were quantified using liquid chromatography-tandem mass spectrometry (LC–MS/MS) followed by pathway analysis and correlation with airway inflammation. SBP-Ag induced statistically significant changes in 549 features that mapped to 72 uniquely identified metabolites. From these features, ...

Genes (131) down regulated by >1.5 fold in both fibroblasts lines (L20 and L21) cultured with ... more Genes (131) down regulated by >1.5 fold in both fibroblasts lines (L20 and L21) cultured with IL3IgG eosinophil conditioned media (average of 2 eosinophil donors), compared to HLF cultured in medium only, and cultured with rhIL-3 plus HA-IgG. (PDF 88Â kb)

IL-3-pre-activated eosinophils strongly adhere to coated HA-IgG and ultimately release free granu... more IL-3-pre-activated eosinophils strongly adhere to coated HA-IgG and ultimately release free granule proteins. Human blood eosinophils were activated with IL-3 or IL-5 for 20 h and were then added on heat-aggregated (HA) human serum IgG. Photomicroscopy of IL-3 and IL-5-pre-activated eosinophils on HA-IgG was performed at 2 h, 4 h and at 6 h, using a digital camera from Olympus. (PDF 223 kb)

Biological Psychiatry, 2020

Journal of Proteome Research, 2018

Purified human eosinophils treated for 18-24 h with IL-3 adopt a unique activated phenotype marke... more Purified human eosinophils treated for 18-24 h with IL-3 adopt a unique activated phenotype marked by increased reactivity to aggregated immunoglobulin-G (IgG). To characterize this phenotype, we quantified protein abundance and phosphorylation by multi-plexed isobaric labeling combined with high-resolution mass spectrometry. Purified blood eosinophils of five individuals were treated with IL-3 or no cytokine for 20 hours, and comparative data were obtained on abundance of 5385 proteins and phosphorylation at 7330 sites. The 1150 proteins that were significantly up-regulated (q<0.05, pair-wise t test with Benjamini-Hoachberg correction) by IL-3 included the IL3RA and CSF2RB subunits of the IL-3 receptor, the low-affinity receptor for IgG (FCGR2B), 96 proteins involved in protein translation, and 55 proteins involved in cytoskeleton organization. Among the 703 proteins that decreased were 78 mitochondrial proteins. Dynamic regulation of protein phosphorylation was detected at 4218 sites. These included multiple serines in CSF2RB; Y694 of STAT5, a key site of activating phosphorylation downstream of IL3RA/CSF2RB; and multiple sites in RPS6KA1, RPS6, and EIF4B, which are responsible for translational initiation. We conclude that IL-3 up-regulates overall protein synthesis and targets specific proteins for up-regulation, including its own receptor.

IEEE Transactions on Plasma Science, 2005

A hollow-cathode microplasma was used to modify the lumenal surface of small-diameter polyethylen... more A hollow-cathode microplasma was used to modify the lumenal surface of small-diameter polyethylene (PE). We make use of two microplasma diagnostics to monitor the plasma properties during the treatment process. A microwave cavity was used to measure the density of the microplasma. Emitted light from the microplasma was fed into a monochromator at various positions along the PE tube to assess uniformity of the microplasma. Effectiveness of plasma treatments were evaluated using the capillary-rise method at various positions along the tubing. We show a correlation between the properties of the inner surface of the PE tubing and the light emitted from the plasma. A Poly(ethylene oxide) (PEO) surfactant was immobilized to the lumenal surface of the PE tubing using the microplasma discharge. An in vitro blood-circulation loop was constructed to test the hematocompatibility of the PE tubes. After blood exposure, scanning electron microscope images were taken to assess the density of adhering platelets along the length of the tubes. The plasma-treated tubing showed fewer blood adherents than the untreated tubing. By suitably controlling the pressure drop along the tube, the uniformity of the microplasma treatment along the tubing can be optimized.

Clinical Immunology, 2014

Semaphorin 7A (Sema7A) plays a major role in TGF-β1-induced lung fibrosis. Based on the accumulat... more Semaphorin 7A (Sema7A) plays a major role in TGF-β1-induced lung fibrosis. Based on the accumulating evidence that eosinophils contribute to fibrosis/remodeling in the airway, we hypothesized that airway eosinophils may be a significant source of sema7A. In vivo, sema7A was expressed on the surface of circulating eosinophils and upregulated on bronchoalveolar lavage eosinophils obtained after segmental bronchoprovocation with allergen. Based on mRNA levels in unfractionated and isolated bronchoalveolar cells, eosinophils are the predominant source of sema7A. In vitro, among the members of the IL-5-family cytokines, sema7A protein on the surface of blood eosinophils was increased more by IL-3 than by GM-CSF or IL-5. Cytokineinduced expression of cell surface sema7A required translation of newly synthetized protein. Finally, a recombinant sema7A induced alpha-smooth muscle actin production in human bronchial fibroblasts. Semaphorin 7A is a potentially important modulator of eosinophil profibrotic functions in the airway remodeling of patients with chronic asthma.

Clinical & Experimental Allergy, 2021

Background: In asthma, IL-6 is a potential cause of enhanced inflammation, tissue damage and airw... more Background: In asthma, IL-6 is a potential cause of enhanced inflammation, tissue damage and airway dysfunction. IL-6 signaling is regulated by its receptor, which is composed of two proteins, IL-6R and GP130. In addition to their membrane form, these two proteins may be found as extracellular soluble forms. The interaction of IL-6 with soluble IL-6R (sIL-6R) can trigger IL-6 trans-signaling in cells lacking IL-6R. Conversely, the soluble form of GP130 (sGP130) competes with its membrane form to inhibit IL-6 trans-signaling. Objectives: We aimed to analyze IL-6 trans-signaling proteins in the airways of subjects after an allergen challenge. Methods: We used a model of segmental bronchoprovocation with an allergen (SBP-Ag) in human subjects with allergy. Before and 48h after SBP-Ag, bronchoalveolar lavages (BAL) allowed for the analysis of proteins in BAL fluids (BALF) by ELISA, and membrane proteins on the surface of BAL cells by flow cytometry. In addition, we performed RNA-sequencing (RNAseq) and used proteomics data to further inform on the expression of the IL-6R subunits by

Primer sequences used for real-time PCR. (DOCX 15Â kb)

Abstract: The rising worldwide prevalence of asthma has intensified interest in the natural histo... more Abstract: The rising worldwide prevalence of asthma has intensified interest in the natural history of asthma. An improved understanding of the genetic, environmental, and developmental factors contributing to the inception and exacerbation of asthma will be crucial to efforts to devise effective preventive and therapeutic interventions. There is increasing evidence that the complex interplay of early life respiratory viral infections and allergic sensitization is important in the development of asthma. Major causes of asthma exacerbations are respiratory viral infections and aeroallergen exposure, which may have interactive co-morbid effects. This review describes the potential role of thymic stromal lymphopoietin (TSLP) as a connection between the innate immune response to respiratory viral infections and the type-2 adaptive immune response in the development and exacerbation of asthma.

Journal of Allergy and Clinical Immunology

Mechanical forces have long been recognized as fundamental drivers in biological processes, such ... more Mechanical forces have long been recognized as fundamental drivers in biological processes, such as embryogenesis, tissue formation and disease regulation. The collagen gel contraction (CGC) assay has served as a classic tool in the field of mechanobiology to study cell-induced contraction of extracellular matrix (ECM), which plays an important role in inflammation and wound healing. In a conventional CGC assay, cell-laden collagen is loaded into a cell culture vessel (typically a well plate) and forms a disk-shaped gel adhering to the bottom of the vessel. The decrement in diameter or surface area of the gel is used as a parameter to quantify the degree of cell contractility. In this study, we developed a microscale CGC assay with an engineered well plate insert that uses surface tension forces to load and manipulate small volumes (14 µL) of cell-laden collagen. The system is easily operated with two pipetting steps and the microscale device moves dynamically as a result of cellula...

Frontiers in Medicine

We have recently reported that, unlike IL-5 and GM-CSF, IL-3 induces increased translation of a s... more We have recently reported that, unlike IL-5 and GM-CSF, IL-3 induces increased translation of a subset of mRNAs. In addition, we have demonstrated that Pin1 controls the activity of mRNA binding proteins, leading to enhanced mRNA stability, GM-CSF protein production and prolonged eosinophil (EOS) survival. In this review, discussion will include an overview of cap-dependent protein translation and its regulation by intracellular signaling pathways. We will address the more general process of mRNA post-transcriptional regulation, especially regarding mRNA binding proteins, which are critical effectors of protein translation. Furthermore, we will focus on (1) the roles of IL-3-driven sustained signaling on enhanced protein translation in EOS, (2) the mechanisms regulating mRNA binding proteins activity in EOS, and (3) the potential targeting of IL-3 signaling and the signaling leading to mRNA binding activity changes to identify therapeutic targets to treat EOS-associated diseases.

Journal of Leukocyte Biology

Eosinophils and basophils are a vital component of allergic inflammation in asthma and other dise... more Eosinophils and basophils are a vital component of allergic inflammation in asthma and other diseases. Both granulocytes express the 3 common bc-chain signaling cytokine receptors for IL-5, GM-CSF, and IL-3. Therefore, the identification of the specific signaling pathways induced by IL-3 could contribute to new therapeutic options that will dampen the functions of these inflammatory cells. Recently, Kӓmpfer et al. [1] published an important study in the Journal of Leukocyte Biology, demonstrating long-term IL-3R-mediated signaling in basophils and eosinophils. Also recently, our group has reported very similar long-term IL-3 effects on eosinophils [2], but our work was not mentioned by Kӓmpfer et al. [1]. Therefore, I found it important to discuss further the similarities and differences in IL-3 signaling observed in basophils and eosinophils. This Letter will help answer some of the questions or uncertainties raised by the Kӓmpfer et al. manuscript [1], which primarily focuses on basophils. In agreement between Kӓmpfer and colleagues’ work [1] and our study [2], IL-3-induced, prolonged intracellular events required continuous presence of IL-3 and IL-3R signaling. Furthermore, Kӓmpfer et al. [1] found that IL-3 was unique among the common b-chain signaling cytokines to prolong signaling for .48 h and to induce the production of key basophil-specific proteins. Phosphorylation of STAT5 was the most remarkably prolonged event in IL-3-activated basophils. To some extent, STAT3, protein kinase B, ERK, p38, and the ribosomal S6 protein were all continuously phosphorylated exclusively by IL-3 [1]. Interestingly, Pim1 (a phosphorylated STAT5-induced protein), retinaldehyde dehydrogenase 2, and granzyme B protein products were induced by IL-3 at 18 h, whereas IL-5 and GM-CSF had no effect on these proteins. Similarly with basophils, the prolonged phosphorylation of STAT5 and production of Pim1 were observed in IL-3-activated eosinophils, whereas little effect was seen following stimulation with GM-CSF or IL-5 [1]. In agreement with Kӓmpfer et al. [1], our group has recently reported that IL-3 was more potent than GM-CSF or IL-5 in maintaining intracellular signaling in eosinophils [2]. STAT5 phosphorylation was not analyzed in our study, but phosphorylation of both p90S6 kinase and ribosomal S6 proteins was found to be maintained for at least 2 d after addition of IL-3 [2]. Therefore, as for basophils, IL-3 is unique in its ability to prolong ribosomal S6 phosphorylation in eosinophils. The mechanism responsible for the prolonged S6 phosphorylation in IL-3-activated basophils was not pursued in the Kӓmpfer et al. study [1]; however, given our study [2], p90S6 kinase may be the distinguishing factor. Similarly to the Kӓmpfer and coworkers’ basophil study [1], we also examined ERK activation [2], which was required upstream of S6 phosphorylation. Although we did not analyze the continuous phosphorylation of ERK in activated eosinophils, as was shown in the Kӓmpfer et al. study [1], we did find that inhibition of ERK activation, 3 h after addition of IL-3, blocked long-term p90S6 kinase and S6 phosphorylations, indicating that ERK must be activated for at least 3 h in IL-3activated eosinophils. Furthermore, among the proteins solely induced by IL-3 in basophils, granzyme B is quickly and strongly up-regulated at the mRNA level [3]. This can account for the IL-3-induced upregulation of granzyme B protein in basophils, as observed by Kӓmpfer et al. [1]. In eosinophils, however, IL-3 is unique compared with IL-5 and GM-CSF to induce an increased translation rate of semaphorin-7A without changing its mRNA level. Whereas transcriptional and post-transcriptional analyses of the IL-3-induced basophil proteins were clearly beyond the scope of the Kӓmpfer et al. study [1], the potential role of S6 phosphorylation on protein translation rate in basophils should have been discussed in their paper. Finally, Kӓmpfer and coworkers’ work [1] supports the premise that in basophils, the continuous intracellular IL-3 signaling versus the ephemeral signaling induced by IL-5 or GMCSF is probably a result of the dynamic of their respective receptors. However, it is important to note the differences and commonalities between basophils and eosinophils regarding the regulation of these receptors. In basophils, IL-3, GM-CSF, or IL-5 maintain the high basal level of IL-3Ra over time, whereas IL5Ra and GM-CSFRa are quickly removed from the cell surface. On the contrary, in eosinophils, the basal level of IL-3Ra is low and is increased over time (after 4 h) by IL-3, GM-CSF, or IL-5. Interestingly, whereas these cytokines result in the loss of surface IL-5Ra, GM-CSFRa is generally unaffected by the cytokine treatments [2, 4, 5]. The different dynamics of GM-CSFRa in activated basophils versus eosinophils may explain why GM-CSF

Journal of Visualized Experiments, 2016

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing ... more Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (ΔS) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (α) or a truncated transactivation domain with 7 unique residues (β). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (Sα, Sβ, ΔSα and ΔSβ) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of cosplicing at other tandem splicing sites.

A hollow-cathode microplasma modified the lumenal surface of small diameter (0.28 and 0.8 mm) up ... more A hollow-cathode microplasma modified the lumenal surface of small diameter (0.28 and 0.8 mm) up to 1 meter long polyethylene (PE) tubing. PE glycol was grafted to the lumenal surface using O2 plasma followed by Ar plasma. Feedstock gases and reaction products drift along the tubing which may cause nonuniform treatment. Emitted light from the plasma was fed into a monochromator at various positions to assess uniformity. Effectiveness was evaluated using capillary-rise, which is related to contact angle. Uniformity of the atomic surface composition along the length of the inner surface of the PE tubing was analyzed by XPS and transmission FTIR. To test hematocompatibility, a flow loop circulated heparinized human blood for various times at 37 C. Plasma-treated and untreated tubing were then examined with a scanning SEM to assess the morphology of adhering platelets. By modifying plasma parameters, uniformity along the tubing and proximity to the peristaltic pump can be optimized.

Eosinophils function contributes to human allergic and autoimmune diseases, many of which current... more Eosinophils function contributes to human allergic and autoimmune diseases, many of which currently lack curative treatment. Development of more effective treatments for eosinophil-related diseases requires expanded understanding of eosinophil signaling and biology. Cell signaling requires integration of extracellular signals with intracellular responses, and is organized in part by cholesterol rich membrane microdomains (CRMMs), commonly referred to as lipid rafts. Formation of these organizational membrane domains is in turn dependent upon the amount of available cholesterol, which can fluctuate widely with a variety of disease states. We tested the hypothesis that manipulating membrane cholesterol content in primary human peripheral blood eosinophils (PBEos) would selectively alter signaling pathways that depend upon membrane-anchored signaling proteins localized within CRMMs (e.g., mitogen activated protein kinase [MAPK] pathway), while not affecting pathways that signal through...