Omalizumab for treatment of idiopathic angioedema.

BACKGROUND: Omalizumab has been found to improve outcomes in patients with chronic spontaneous urticaria (CSU). Idiopathic angioedema (IAE) is increasingly being recognized as a condition with similar underlying mechanisms as CSU and a form of CSU. We hypothesized that add-on therapy with omalizumab would benefit patients with uncontrolled IAE. OBJECTIVE: To study the safety and efficacy of omalizumab for the treatment of IAE in adults. METHODS: We conducted a randomized, placebo-controlled trial to study the efficacy of omalizumab in adults with 2 or more episodes of angioedema (AE) in the past 6 months for which no clinical or laboratory cause of AE could be found. A total of 10 patients were randomized on a 1:1 basis to receive omalizumab 300 mg subcutaneously or placebo every 4 weeks for 24 weeks with a 12-week follow-up period. The primary endpoint was the change in the Angioedema Activity Score. Secondary endpoints included the Angioedema Quality of Life Questionnaire, the Visual Analogue Scale, and the number of angioedema episodes per month. RESULTS: We observed improvement in the Angioedema Activity Score (-2.93 ln odds; 95% confidence interval [CI], -4.84 to -1.02; P = .003), Visual Analogue Scale (-3.49 ln odds; 95% CI, -6.58 to -0.40; P = .03), Angioedema Quality of Life Questionnaire (-9.43 score; 95% CI, -17.63 to -1.24; P = .03), and number of angioedema episodes per month (-1.93 ln count; 95% CI, -3.23 to -0.63; P = .005) in patients who received omalizumab vs placebo. CONCLUSION: This study provides preliminary prospective evidence that omalizumab improves outcomes in patients with IAE. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02966314. CLINICALTRIALS: gov URL:https://clinicaltrials.gov/ct2/show/NCT02966314?term=omalizumab&cond=Angioedema&draw=2&rank=1.

Orosomucoid-like 3 Supports Rhinovirus Replication in Human Epithelial Cells.

Polymorphism at the 17q21 gene locus and wheezing responses to rhinovirus (RV) early in childhood conspire to increase the risk of developing asthma. However, the mechanisms mediating this gene-environment interaction remain unclear. In this study, we investigated the impact of one of the 17q21-encoded genes, ORMDL3 (orosomucoid-like 3), on RV replication in human epithelial cells. ORMDL3 knockdown inhibited RV-A16 replication in HeLa, BEAS-2B, A549, and NCI-H358 epithelial cell lines and primary nasal and bronchial epithelial cells. Inhibition varied by RV species, as both minor and major group RV-A subtypes RV-B52 and RV-C2 were inhibited but not RV-C15 or RV-C41. ORMDL3 siRNA did not affect expression of the major group RV-A receptor ICAM-1 or initial internalization of RV-A16. The two major outcomes of ORMDL3 activity, SPT (serine palmitoyl-CoA transferase) inhibition and endoplasmic reticulum (ER) stress induction, were further examined: silencing ORMDL3 decreased RV-induced ER stress and IFN-ß mRNA expression. However, pharmacologic induction of ER stress and concomitant increased IFN-ß inhibited RV-A16 replication. Conversely, blockade of ER stress with tauroursodeoxycholic acid augmented replication, pointing to an alternative mechanism for the effect of ORMDL3 knockdown on RV replication. In comparison, the SPT inhibitor myriocin increased RV-A16 but not RV-C15 replication and negated the inhibitory effect of ORMDL3 knockdown. Furthermore, lipidomics analysis revealed opposing regulation of specific sphingolipid species (downstream of SPT) by myriocin and ORMDL3 siRNA, correlating with the effect of these treatments on RV replication. Together, these data revealed a requirement for ORMDL3 in supporting RV replication in epithelial cells via SPT inhibition.

Eosinophil cytolysis on Immunoglobulin G is associated with microtubule formation and suppression of rho-associated protein kinase signalling.

BACKGROUND: 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 integrin-dependent eosinophil cytolysis of IL3-primed blood eosinophils seeded on heat-aggregated immunoglobulin G (HA-IgG). METHODS: Cytoskeletal events and signalling pathways potentially involved in cytolysis were 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.

Characterizing asthma from a drop of blood using neutrophil chemotaxis.

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.

Interaction between allergy and innate immunity: model for eosinophil regulation of epithelial cell interferon expression.

BACKGROUND: Eosinophils in asthmatic airways are associated with risk of exacerbations. The most common cause of asthma exacerbations is viral respiratory infections, particularly human rhinovirus (HRV). OBJECTIVE: To determine the mechanism by which eosinophils may influence virus-induced responses. METHODS: We used an in vitro coculture model of primary human eosinophils and the BEAS-2B epithelial cell line either stimulated with HRV1A infection or polyinosinic-polycytidylic acid (poly[I:C]). The messenger RNA (mRNA) expression of interferon (IFN) ß1 and IFN-λ1 was assessed by quantitative reverse-transcriptase polymerase chain reaction and the protein level of IFN- λ1 by enzyme-linked immunosorbent assay. RESULTS: Both poly(I:C) and HRV1A infection induced BEAS-2B expression of IFN-ß1 and IFN-λ1 mRNA. Coculture of eosinophils resulted in suppression of poly(I:C)-stimulated IFN-ß1 and IFN-λ1 mRNA expression (2.5-fold and 3.6-fold less, respectively). Separation of cells did not block eosinophil regulatory activity. Coculture of eosinophils with HRV1A-infected BEAS-2B cells also suppressed IFN-ß1 and IFN-λ1 mRNA (5.7-fold and 5.0-fold less, respectively) and reduced IFN-λ1 protein secretion (1.6-fold decrease). This corresponded to a 34% increase in the quantity of HRV1A virus RNA on coculture with eosinophils. Recombinant transforming growth factor ß suppressed IFN-λ1 from HRV1A-infected BEAS-2B cells. Coculture of eosinophils and BEAS-2B cells induced transforming growth factor ß secretion, which may mediate suppression of HRV-induced interferon expression. CONCLUSION: Eosinophils suppressed HRV-induced expression of interferons from epithelial cells, resulting in increased quantity of HRV. This represents one mechanism for interaction between allergic inflammation and innate immunity.

Autophagy Protects against Eosinophil Cytolysis and Release of DNA.

The presence of eosinophils in the airway is associated with asthma severity and risk of exacerbations. Eosinophils deposit their damaging products in airway tissue, likely by degranulation and cytolysis. We previously showed that priming blood eosinophils with IL3 strongly increased their cytolysis on aggregated IgG. Conversely, IL5 priming did not result in significant eosinophil cytolysis in the same condition. Therefore, to identify critical events protecting eosinophils from cell cytolysis, we examined the differential intracellular events between IL5- and IL3-primed eosinophils interacting with IgG. We showed that both IL3 and IL5 priming increased the eosinophil adhesion to IgG, phosphorylation of p38, and production of reactive oxygen species (ROS), and decreased the phosphorylation of cofilin. However, autophagic flux as measured by the quantification of SQSTM1-p62 and lipidated-MAP1L3CB over time on IgG, with or without bafilomycin-A1, was higher in IL5-primed compared to IL3-primed eosinophils. In addition, treatment with bafilomycin-A1, an inhibitor of granule acidification and autophagolysosome formation, enhanced eosinophil cytolysis and DNA trap formation in IL5-primed eosinophils. Therefore, this study suggests that increased autophagy in eosinophils protects from cytolysis and the release of DNA, and thus limits the discharge of damaging intracellular eosinophilic contents.

An open microfluidic coculture model of fibroblasts and eosinophils to investigate mechanisms of airway inflammation.

Interactions between fibroblasts and immune cells play an important role in tissue inflammation. Previous studies have found that eosinophils activated with interleukin-3 (IL-3) degranulate on aggregated immunoglobulin G (IgG) and release mediators that activate fibroblasts in the lung. However, these studies were done with eosinophil-conditioned media that have the capacity to investigate only one-way signaling from eosinophils to fibroblasts. Here, we demonstrate a coculture model of primary normal human lung fibroblasts (HLFs) and human blood eosinophils from patients with allergy and asthma using an open microfluidic coculture device. In our device, the two types of cells can communicate via two-way soluble factor signaling in the shared media while being physically separated by a half wall. Initially, we assessed the level of eosinophil degranulation by their release of eosinophil-derived neurotoxin (EDN). Next, we analyzed the inflammation-associated genes and soluble factors using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and multiplex immunoassays, respectively. Our results suggest an induction of a proinflammatory fibroblast phenotype of HLFs following the coculture with degranulating eosinophils, validating our previous findings. Additionally, we present a new result that indicate potential impacts of activated HLFs back on eosinophils. This open microfluidic coculture platform provides unique opportunities to investigate the intercellular signaling between the two cell types and their roles in airway inflammation and remodeling.

Comparison of IL-33 and IL-5 family mediated activation of human eosinophils.

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.