Influence of autologous extracellular traps (ETs) on immune synapses in human leukocyte cultures challenged with heterologous ovalbumin antigen (OVA)

Abstract

Extracellular traps (ETs) produced by immune cells constitute a defense mechanism against pathogenic microorganisms and they are structures composed by chromatin, histones, granular proteins, including myeloperoxidase (MPO) and other molecules such as MHC-II and B7 costimulators. In addition, they have been implicated in tissue damage, thrombosis, autoimmunity, cancer and COVID-19 pathogenesis. Here we proposed to isolate ETs generated in vitro from total human leukocytes extracted from peripheral blood stimulated with lipopolysaccharide (LPS) and study their influence on immune synapses (ISs) generation in autologous cultures challenged with ovalbumin heterologous antigen (OVA).

Autologous leukocyte cultures from healthy human blood samples anticoagulated with heparin were performed, (n = 10 donated by IHH, UNC) with informed consent (Ethics Committee, HNC, FCM) stimulated with LPS for ETs generation and subsequent isolation. Controls: samples from paired cultures. Antigen-presenting cells (APCs) and lymphocytes molecules were labeled by immunofluorescence in macrophage-lymphocyte rosettes (MRL) phenomenon of multiple ISs. Cells labeled quantification was performed with FIJI software and MLRs. Statistical treatment: t test for paired samples.

After 24 hours of culture, a lower percentage MLR formation was observed in cocultures with ETs generated (p<0.05). At 96 hours of culture with isolated ETs and OVA addition, a higher percentage MLRs formation was observed (p<0.05). A lower percentage MPO and HLA-DR positive labeled cells was observed with OVA addition at 24 hours of culture (p<0.05). In cocultures at 24 hours, a higher percentage MPO positive cells with OVA and ETs generated was manifested (p<0.05).

Besides the ETs, the stimulation with LPS to obtain them can have influence about free MPO production in culture medium, which can be internalized by cells and contribute to positivity observed. With OVA addition and isolated ETs, an increase in ISs was observed, although ETs can hinder IS, it is known they carry important molecules for antigen presentation, but they can also interfere with the cell-cell contact required for IS.