Abstract
Septicaemia is an acute inflammatory reaction in the bloodstream to the presence of pathogen-associated molecular patterns. Whole blood stimulation assays capture endotoxin-induced formation of aggregates between platelets and leucocytes using flow cytometry. We wanted to assess extent of spontaneous aggregate formation in whole blood stimulation assays and compare the effects of endotoxin and heat-killed, clinically relevant, bacterial pathogens on aggregate formation and then on adhesion of aggregates to TNFα-stimulated endothelial cells. We found that endotoxin (from Escherichia coli or Salmonella enteritidis) was not a suitable stimulus to provoke platelet-leucocyte aggregates in vitro, as it did not further increase the extent of aggregates formed spontaneously in stasis of hirudin-anticoagulated blood. Specifically, whole blood samples stimulated with or without LPS produced aggregates with a mean surface area of 140.97 and 117.68 μm2, respectively. By contrast, incubation of whole blood with heat-killed Klebsiella pneumoniae or Staphylococcus aureus produced significantly enhanced and complex cellular aggregates (with a mean surface area of 470.61 and 518.39 μm2, respectively) which adhered more frequently to TNFα (and free fatty acid)-stimulated endothelial cells. These were reliably captured by scanning electron microscopy. Adhesion of cellular aggregates could be blocked by incubation of endothelial cells with a commercial P-selectin antibody and an angiopoietin-2 ligand trap. In conclusion, we have developed an in vitro method that models the acute inflammatory reaction in whole blood in the presence of sepsis-relevant bacterial pathogen surfaces.
Highlights
Sepsis is caused by a dysregulated host response to infection with bacterial, viral or fungal pathogens and may lead to life-threatening organ dysfunction, affecting in particular: gut, lung, kidney, heart and brain
Incubation of unstimulated whole blood at 37 °C for 1 h induced a significant increase in platelet–granulocyte aggregate formation compared to baseline (Fig. 1a)
Incubation of whole blood with E. coli 0111: B4 LPS or Salmonella enteritidis LPS for 1 h for both EDTA- and hirudin-anticoagulated samples did not induce an increase in platelet granulocyte aggregate levels beyond that of unstimulated whole blood incubated for 1 h at 37 °C (Fig. 1c)
Summary
Sepsis is caused by a dysregulated host response to infection with bacterial, viral or fungal pathogens and may lead to life-threatening organ dysfunction, affecting in particular: gut, lung, kidney, heart and brain. The lack of biomarkers that trace the extents of the immune response in the course of sepsis (immune activation, over activation and exhaustion during development and progression of sepsis) poses a problem in the design of new treatment approaches [2, 3]. Plateletleucocyte aggregates, whether they are circulating in blood or adhering to the endothelium, are extensively formed in sepsis, play a critical role in sepsis pathophysiology and correlate with severity [4, 5]. Molecular interactions that lead to the formation of these cellular aggregates and their
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