Animal-Free Human Whole Blood Sepsis Model to Study Changes in Innate Immunity

Abstract

Studying innate immunity in humans is crucial for understanding its role in the pathophysiology of systemic inflammation, particularly in the complex setting of sepsis. Therefore, we standardized a step-by-step process from the venipuncture to the transfer in a human model system, while closely monitoring the inflammatory response for up to three hours. We designed an animal-free, human whole blood sepsis model using a commercially available, simple to use, tubing system. First, we analyzed routine clinical parameters, including cell count and blood gas analysis. Second, we demonstrated that extracellular activation markers (e.g., CD11b and CD62l) as well as intracellular metabolic (intracellular pH) and functional (generation of radical oxygen species) features remained stable after incubation in the whole blood model. Third, we mimicked systemic inflammation during early sepsis by exposure of whole blood to pathogen-associated molecular patterns. Stimulation with lipopolysaccharide revealed the capability of the model system to evoke a sepsis-like inflammatory phenotype of innate immunity. In summary, the presented model serves as a convenient, economic, and reliable platform to study innate immunity in human whole blood, which may yield clinically important insights.

Activation of Neutrophil Granulocytes by Platelet-Activating Factor Is Impaired During Experimental Sepsis

“…To translate the findings to a more clinically relevant setting, an ex vivo model of human endotoxemia was used analyzing the modulation of the PAF-induced response in systemic inflammation. As previously reported ( 36 ), sole contact with the tubing system of the model did not promote neutrophil activation (data not shown). However, the exposure to LPS (100 ng/ml) resulted in an activated neutrophil phenotype as reflected by of the upregulation of the surface expression of CD11b and downregulation of CD62L.…”

“…For example, in murine sepsis, an increase in neutrophil cell size ( 14 ) and pH i were reported, with the latter verified in patients with sepsis ( 15 ). Both alterations have been confirmed in an ex vivo model of lipopolysaccharide (LPS)-induced inflammation ( 36 ). In this context, it is tempting to speculate that a shift in baseline levels of neutrophil parameters during systemic inflammation affects the response under additional stimulation by inflammatory mediators such as PAF, however, this remains to be further elucidated.…”

“…Platelet–neutrophil complex (PNC) formation, defined as a neutrophil with at least one platelet in direct proximity, was assessed as described previously by light microscopy and flow cytometry ( 36 , 42 ). For blood smears, whole blood was diluted 1:1 with phosphate-buffered saline containing calcium and magnesium (PBS) and incubated for 15 min at 37°C with or without 1 μM PAF on a spinning wheel (Snijders Labs, Tilburg, Netherlands) at 3 rpm.…”

“…An animal-free human whole blood model of endotoxemia was used to investigate the effect of LPS exposure on the PAF-induced response of neutrophils, which was described previously in detail ( 36 ). In brief, 0.5 IU/ml heparin (B. Braun Melsungen AG, Melsungen, Germany) and 100 ng/ml LPS (#L2630, Merck) or PBS (control) were added to 9 ml blood, which was transferred into a Cortiva BioActive Surface (Medtronic, Meerbusch, Germany) coated tubing system using a coated connector (Medtronic) to interlink both ends.…”

“…Following 1 h of rotation (3 rpm) on a spinning wheel (Snijders Labs) at 37°C, the blood was transferred to citrate anti-coagulated monovettes (Sarstedt). Neutrophil activation markers were analyzed directly in whole blood as described previously ( 36 ) with or without exposure to PAF (1 μM, 15 min, 37°C). The remaining whole blood was processed to isolate and to analyze neutrophils as described above.…”