ROLE OF MHC‐CLASS II ACTIVATION ON STAPHYLOCOCCAL ENTEROTOXINS TYPE A (SEA) AND B (SEB)‐INDUCED NEUTROPHIL DYSFUNCTION

Sepsis is a major cause of death worldwide but its orchestrating components remain incompletely understood. Sepsis by gram‐positive bacteria has a higher mortality rate when compared with sepsis by gram‐negative bacteria. However, experimental models that adequately mimic the signs of sepsis by gram‐positive bacteria are limited. An inefficient neutrophils (NE) mobilization in Staphylococcus aureus ‐induced sepsis is the main cause of death in patients infected with this bacterium. Staphylococcus aureus pathological effects are strong related to the secretion of staphylococcal enterotoxins (SEs). Recently, we have shown that human eosinophils and mice bone marrow granulocytes incubated with SEs exhibit reduced in vitro chemotaxis and adhesion activity. The present study aims to identify the effects of SEA and SEB on the functional properties of human NE as well as the relevance of MHC class II activation for their effects. Blood was collected from healthy volunteers after approval from the local ethics committee (Protocol No 61370616.3.0000.5412). Blood samples were placed on isotonic Percoll solution. After centrifugation, the mononuclear cell layer was removed and the pellet containing erythrocytes and granulocytes was aspirated and subjected to isotonic lysis. After lysis cells (98% NE) were resuspended to 4 × 10 6 cells/ml and submitted to in vitro incubation with SEA or SEB (100 ng/ml; 2 h). Adhesion assays were carried out in 96‐well plates pre‐coated with recombinant human VCAM‐1 and ICAM‐1 for 30 min in the presence of interleukin‐8 (IL‐8). The NE adhesion was calculated by measuring myeloperoxidase (MPO) activity on adherent cells. NE migration assay was performed using a 48‐well microchemotaxis Boyden chamber. In separated assays, neutrophils were incubated for 30 min at 37°C with human anti MHC class II blocking antibody (2 μg/ml) before addition of SEA, SEB or MEM. Both SEA and SEB reduces human neutrophil adhesion in VCAM‐1 (Control: 4.7 ± 0.5; untreated NE + IL‐8: 6.7 ± 0.6; SEA treated NE+IL‐8: 4.9 ± 0.8; SEB treated NE + IL‐8: 3.7 ± 0.6*# optic density/NE × 10 6 cells; ) or ICAM‐1(Control: 4.3 ± 0.5; untreated NE + IL‐8: 6.1 ± 0.6; SEA treated NE+IL‐8: 5.5 ± 1.0; SEB treated NE + IL‐8: 3.7 ± 0.9 optic density/NE × 10 6 cells) coated plates. Similar results were observed for NE chemotactic activity (Control: 18.7 ± 0.8; untreated NE +IL‐8: 52.9 ± 2.7; SEA treated NE+IL‐8: 28.5 ± 1.4; SEB treated NE + IL‐8: 34.2 ± 1.4 NE per high‐power field). Anti‐MHC class II antibody prevented the reductions by SEA or SEB on IL‐8‐induced neutrophil adhesion and chemotaxis in comparison to non‐treated cells. The inhibitory effect of SEA and SEB on human NE in vitro adhesion and chemotaxis via MHC class II activation suggests a role of these toxins on the neutrophil dysfunction associated with the severe sepsis by Staphylococcus aureus . Support or Funding Information Fundação de Amparo à Pesquisa do Estado de São Paulo ‐ FAPESP (2017/25867‐8) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .