Effects of Eosinophilopoietins and C-C Chemokines on Chemotaxis, Adhesion, and ROS Production of Blood Eosinophil Subtypes in Asthma Patients.

Purpose: Two distinct blood eosinophil subtypes have recently been described as inflammatory-like (iEOS-like) and resident-like (rEOS-like) eosinophils. Eosinophilopoietins - interleukin (IL)-3, IL-5, and GM-CSF, as well as C-C chemokines - CCL11, CCL24, and CCL26 (eotaxin-1, 2, 3), CCL5 (RANTES), are key regulators of eosinophil activity. However, our understanding of the effects of these cytokines on blood eosinophil subtype chemotaxis, adhesion, and reactive oxygen species (ROS) production in asthma is limited. Patients and Methods: The study enrolled 13 patients with non-severe allergic asthma (AA), 12 patients with severe eosinophilic asthma (SEA), and 10 healthy subjects (HS). Additionally, AA patients underwent bronchial allergen challenge with D. pteronyssinus, and all scheduled tests were repeated 24 hours later. Ex vivo blood eosinophils were purified using Ficoll gradient centrifugation and magnetic separation, subtyped by CD62L-based magnetic separation, isolating iEOS-like (CD62L-) and rEOS-like (CD62L+) cells. In vitro stimulation of the isolated eosinophils was performed using recombinant human cytokines and chemokines (IL-3, IL-5, GM-CSF; CCL5, 11, 24, 26). Eosinophil chemotaxis was evaluated using Transwell migration assay, adhesion to airway smooth muscle cells (ASMC) by eosinophil peroxidase activity assay, and ROS production measured using a dihydrorhodamine-123 fluorescent probe and flow cytometer. Results: In all groups, IL-3, IL-5, and GM-CSF enhanced iEOS-like cells' chemotaxis, ROS production, and adhesion to ASMC, with no impact on rEOS-like cell chemotaxis, and only GM-CSF promoted rEOS-like cell adhesion. SEA patients' rEOS-like cells' ROS production following activation with eosinophilopoietins was higher compared to the AA group. Allergen-activated AA patients' blood eosinophil subtypes' ROS production was further upregulated by IL-3, IL-5, and GM-CSF, p < 0.05, but these cytokines had differential effects on chemotaxis and adhesion to ASMC. In AA and SEA, CCL5, 11, 24 and 26 promoted chemotaxis of blood eosinophil subtypes, p < 0.05. Lastly, CCL26 had a higher effect on iEOS-like cells in AA. Conclusion: Eosinophilopoietins and C-C chemokines increase chemotaxis, adhesion, and ROS production of blood eosinophil subtypes, notably in asthma patients. GM-CSF and CCL26 are the most potent promoters of eosinophil chemotaxis and adhesion, particularly of iEOS-like cells.