Asthma is a chronic inflammatory disorder that affects more than 300 million people worldwide. Asthma management would benefit from additional tools that establish biomarkers to identify phenotypes of asthma. We present a microfluidic solution that discriminates asthma from allergic rhinitis based on a patient's neutrophil chemotactic function. The handheld diagnostic device sorts neutrophils from whole blood within 5 min, and generates a gradient of chemoattractant in the microchannels by placing a lid with chemoattractant onto the base of the device. This technology was used in a clinical setting to assay 34 asthmatic (n = 23) and nonasthmatic, allergic rhinitis (n = 11) patients to establish domains for asthma diagnosis based on neutrophil chemotaxis. We determined that neutrophils from asthmatic patients migrate significantly more slowly toward the chemoattractant compared with nonasthmatic patients (P = 0.002). Analysis of the receiver operator characteristics of the patient data revealed that using a chemotaxis velocity of 1.55 μm/min for asthma yields a diagnostic sensitivity and specificity of 96% and 73%, respectively. This study identifies neutrophil chemotaxis velocity as a potential biomarker for asthma, and we demonstrate a microfluidic technology that was used in a clinical setting to perform these measurements.diagnostics | microfluidics | KOALA | passive pumping

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Esnault and Leet have participated equally to the work.Abbreviations: CD32, receptor for Fc fragment of IgG, low affinity II (FCGRII); DAPI, 4′,6-diamidino-2-phenylindole; DPI, diphenyleneiodonium; EDN, eosinophil-derived neurotoxin;HA-IgG, heat-aggregated immunoglobulin G; MAP4, microtubule-associated protein 4; MAPK, mitogen-activated protein kinase; MT, microtubule; NADPH, dihydronicotinamide-adenine dinucleotide phosphate; PFA, paraformaldehyde; PI3K, phosphatidylinositol 3'-kinase; ROCK, Rho-associated, coiled-coil containing protein kinase; ROS, reactive oxygen species; SBP-Ag, segmental bronchoprovocation with an allergen. AbstractBackground: The presence of eosinophils in the airway is associated with asthma severity and risk of exacerbations. Cell-free eosinophil granules are found in tissues in eosinophilic diseases, including asthma. This suggests that eosinophils have lysed and released cellular content, likely harming tissues.Objective: The present study explores the mechanism of CD32-and αMß2 integrindependent eosinophil cytolysis of IL3-primed blood eosinophils seeded on heat-aggregated immunoglobulin G (HA-IgG). Methods: Cytoskeletal events and signalling pathways potentially involved in cytolysiswere assessed using inhibitors. The level of activation of the identified events and pathways involved in cytolysis was measured. In addition, the links between these identified pathways and changes in degranulation (exocytosis) and adhesion were analysed.Results: Cytolysis of IL3-primed eosinophils was dependent on the production of reactive oxygen species (ROS) and downstream phosphorylation of p-38 MAPK. In addition, formation of microtubule (MT) arrays was necessary for cytolysis and was accompanied by changes in MT dynamics as measured by phosphorylation status of stathmin and microtubule-associated protein 4 (MAP4), the latter of which was regulated by ROS production. Reduced ROCK signalling preceded cytolysis, which was associated with eosinophil adhesion and reduced migration. Conclusion and Clinical Relevance:In this CD32-and αMß2 integrin-dependent adhesion model, lysing eosinophils exhibit reduced migration and ROCK signalling, as well as both MT dynamic changes and p-38 phosphorylation downstream of ROS production. We propose that interfering with these pathways would modulate eosinophil cytolysis and subsequent eosinophil-driven tissue damage. K E Y W O R D Sadhesion, cytolysis, degranulation, Eosinophil, IL3, immunoglobulin G, microtubule, priming | 199 STEPHANE ET Al.

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23 24 25 2 26 ABSTRACT 27 28 Eosinophils are the prominent inflammatory cell involved in allergic asthma, atopic dermatitis, 29 eosinophilic esophagitis, and hypereosinophilic syndrome and are found in high numbers in local tissue 30 and/or circulating blood of affected patients. There is recent interest in a family of alarmins, including 31 TSLP, IL-25 and IL-33, that are epithelial-derived and released upon stimulation of epithelial cells. Several 32 genome wide association studies have found SNPs in genes encoding IL-33 to be risk factors for asthma. 33 In two studies examining the direct role of IL-33 in eosinophils, there were differences in eosinophil 34 responses. We sought to further characterize activation of eosinophils with IL-33 compared to activation 35 by other cytokines and chemokines. We assessed IL-33 stimulated adhesion, degranulation, chemotaxis 36 and cell surface protein expression in comparison to IL-3, IL-5, and eotaxin-1 on human eosinophils. Our 37 results demonstrate that IL-33 can produce as potent or more eosinophil activation than IL-3, IL-5 and 38 eotaxin-1. Thus, when considering specific cytokine targeting strategies, IL-33 will be important to 39 consider for modulating eosinophil function. 40 41 42 111 Mg 2 + and washed with 3 times with HBSS. Cells, 1x10 4 eosinophils, were added to each well along with 112 the respective cytokines (1ng/ml final concentration). After 30 min at 37°C, wells were washed 3x with 113 HBSS to remove non adherent cells. 100ul of HBSS was added to sample wells and 100ul with 1x10 4114 eosinophils were added to untreated wells to provide "Total" eosinophils for comparison. Eosinophil

Eosinophils are the prominent inflammatory cell involved in allergic asthma, atopic dermatitis, eosinophilic esophagitis, and hypereosinophilic syndrome and are found in high numbers in local tissue and/or circulating blood of affected patients. There is recent interest in a family of alarmins, including TSLP, IL-25 and IL-33, that are epithelial-derived and released upon stimulation of epithelial cells. Several genome wide association studies have found SNPs in genes encoding IL-33 to be risk factors for asthma. In two studies examining the direct role of IL-33 in eosinophils, there were differences in eosinophil responses. We sought to further characterize activation of eosinophils with IL-33 compared to activation by other cytokines and chemokines. We assessed IL-33 stimulated adhesion, degranulation, chemotaxis and cell surface protein expression in comparison to IL-3, IL-5, and eotaxin-1 on human eosinophils. Our results demonstrate that IL-33 can produce as potent eosinophil activation as IL-3, IL-5 and eotaxin-1. Thus, when considering specific cytokine targeting strategies, IL-33 will be important to consider for modulating eosinophil function.