AB0083 SIGNALING OF PSGL-1 IN HUMAN NEUTROPHILS MIGHT CONTROL NET GENERATION AND APOPTOSIS AND MIGHT BE ALTERED IN SLE PATIENTS
Authors: A. Muñoz-Callejas, E. González Sánchez, E. F. Vicente-Rabaneda, J. Garcia Perez, S. Castañeda, A. Urzainqui
Journal: Annals of the Rheumatic Diseases (2021)

Background: Neutrophil extracellular traps (NETs) are extracellular chromatin fibers decorated with neutrophil antimicrobial proteins and histones, which are formed by neutrophils to trap invading pathogens and facilitate their removal. Excessive presence of NETs has been reported in autoimmune diseases like Systemic Lupus Erythematosus (SLE) and has been related with disease pathogenesis [1]. Our previous work showed that P-selectin KO mice develop an autoimmune syndrome similar to human lupus and that patients with cutaneous lupus have reduced expression of P-selectin in skin vessels [2]. Although it has been reported that P-selectin might induce NET generation in mouse neutrophils [3], there are no studies performed with human neutrophils. Objectives: 1)To analyze the contribution of PSGL-1/P-Selectin interaction to control the generation of NETs by human neutrophils from healthy donors and patients with SLE. 2)To study the implication of PSGL-1/P-Selectin interaction in the control of other types of neutrophil death. Methods: Human neutrophils were isolated from healthy donors and patients with SLE. After incubation in vitro with BSA or P-Selectin in rolling-like conditions (60 rpm shaking at 37 0 C) for 10 min, neutrophils were left to adhere for 1 hour. Then, NETs were labeled with Sytox Green and quantified by fluorimetry and by flow cytometry (FACSCanto II, BD Biosciences). For quantification of necrosis and apoptosis, cells were labeled with propidium iodide and annexing V and analyzed by flow cytometry. Data were analyzed with two-sided Student, two-sided Mann-Whitney U or two-sided Wilcoxon signed rank test using GraphPad Prism software (Version 8.0.1, La Jolla, CA). Results were considered significant at P&lt;.05. Results: Our results show that rolling on BSA, compared to adhesion, induces NET generation and apoptosis of neutrophils isolated from blood of healthy donors. Rolling on P-selectin reduces apoptosis of neutrophils and increase the percentage of NET events, although reducing NET intensity. Comparing to healthy donors, neutrophils from active SLE patients generate NETs with higher intensity in adhesion and do not respond to P-Selectin. Conclusion: The interaction of PSGL-1 with P-Selectin preserves neutrophil death from apoptosis and controls NET generation. Our results suggest that this control might be altered in patients with active SLE. References: [1]Fousert, E., Toes, R., &amp; Desai, J. (2020). Neutrophil extracellular traps (NETs) take the central stage in driving autoimmune responses. Cells , 9 (4), 915. [2]González-Tajuelo, R., Silván, J., Pérez-Frías, A., De La Fuente-fernández, M., Tejedor, R., Espartero-Santos, M. &amp; Urzainqui, A. (2017). P-Selectin preserves immune tolerance in mice and is reduced in human cutaneous lupus. Scientific reports , 7 (1), 1-12. [3]Etulain, J., Martinod, K., Wong, S. L., Cifuni, S. M., Schattner, M., &amp; Wagner, D. D. (2015). P-selectin promotes neutrophil extracellular trap formation in mice. Blood , 126 (2), 242-246. Disclosure of Interests: None declared