Transforming Growth Factor-β1 Drives Neutrophil Chemokinesis, Not Chemotaxis

Louise E Crowley,1,2 Ellen Jenkins,1 Kirsty C McGee,1,3 Theodore S Smith,1 Jon Hazeldine,1 Robert A Stockley,1 David R Thickett,1– 3 Dhruv Parekh,1– 3,* Aaron Scott1,31Department of Inflammation and Ageing, School of Infection, Inflammation and Immunology, College of Medicine and Health, University of Birmingham, Birmingham, UK; 2University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK; 3National Institute for Health and Care Research (NIHR) Birmingham Biomedical Research Centre, Birmingham, UK*These authors contributed equally to this workCorrespondence: Aaron Scott, Department of Inflammation and Ageing, University of Birmingham Research Laboratories, Queen Elizabeth Hospital, Birmingham, B15 2WB, UK, Email a.scott@bham.ac.ukPurpose: Idiopathic pulmonary fibrosis (IPF) is a devastating condition that is characterized by dysregulated repair responses to alveolar epithelial damage and progressive remodeling of the lung interstitium. Neutrophils are critical responders to tissue injury, but their role in organ fibrosis pathogenesis remains unclear. Neutrophil numbers are increased in bronchoalveolar spaces of IPF patients, and neutrophil activation markers are elevated in fibrotic lung tissue. However, the key mediators driving neutrophil movement in fibrotic lung diseases are unknown. Transforming growth factor–beta 1 (TGFβ 1) is a critical pro-fibrotic cytokine, but its effect on neutrophil movement remains unclear. This mechanistic, exploratory study aimed to determine whether TGFβ 1 alters neutrophil migration in a direction-dependent (chemotaxis) or direction-independent (chemokinesis) manner and to assess whether TGFβ 1 exposure is accompanied by functional changes consistent with neutrophil priming.Methods: This study utilized human neutrophils from healthy donors to model aspects of a TGFβ 1-rich environment. Neutrophil movement was assessed using a transwell migration assay and by real-time tracking of human neutrophils within an Insall chamber. Extracellular release of superoxide anions and nucleic acid were assessed by lucigenin-amplified chemiluminescence, and fluorometry, respectively. Expression of cell surface markers, TGFβ receptor 1 and pSmad2/3 were assessed using flow cytometry and Western blot.Results: This study revealed that TGFβ 1 drives neutrophil chemokinesis, but not chemotaxis, and that pre-treatment with TGFβ 1 amplified selected movement responses to the established chemoattractants, interleukin-8 and N-Formylmethionine-leucyl-phenylalanine. TGFβ 1 also increased total superoxide anion production and nucleic acid release, but did not alter cell surface expression of adhesion (CD11b and CD62L) or degranulation (CD66b) markers.Conclusion: Together, these data suggest that TGFβ 1 promotes neutrophil chemokinesis, rather than chemotaxis, and that this may reflect transition into a priming-like state with potential relevance to neutrophil behavior in conditions such as fibrotic lung disease.Keywords: chemokines, cytokines, lung, fibrosis