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EP.016
Human neutrophil function is rapidly impaired by complement C5a in a clinically relevant model of bacteraemia

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Human neutrophil function is rapidly impaired by complement C5a in a clinically relevant model of bacteraemia

Alex JT Wood,1 Arlette Vassallo,1 Klaus Okkenhaug,2 A John Simpson,3 Jonathon Scott,3 Charlotte Summers,1 Edwin R Chilvers,1 Andrew Conway-Morris.1,2

1.Department of Medicine, University of Cambridge, Cambridge, UK
2.Signalling Programme, Babraham Institute, Cambridge, UK
3.Institute of Cellular Medicine, Newcastle University, Newcastle, UK
 

Key messages

•We have refined the assessment of multiple neutrophil functions in small samples of minimally-manipulated human blood.
•The anaphylatoxin C5a rapidly induces a prolonged defect in human neutrophil phagocytosis, even after a short ‘pulse’ of C5a exposure.
•Our new assay sheds light on important differences between isolated and whole blood neutrophils which may be relevant to drug therapies.
 
 

Previously, we have demonstrated that the complement protein C5a (present at high concentrations in plasma from critically ill patients) impairs phagocytosis of yeast particles by neutrophils and is associated with nosocomial infections.1-3 In this study, we investigated the underlying mechanism, duration and preventability of C5a-induced neutrophil dysfunction in a clinically relevant in vitro model. 

 

Figure 1: pHrodo Bioparticles are a sensitive measure of phagocytosis in neutrophils. Neutrophils were incubated with S. aureus green bioparticles for 30 minutes. White arrows indicate intracellular S. aureus Bioparticles (green). Imaging by confocal microscopy at X63 objective.

 

Methods

A new flow cytometric assay was developed to assess neutrophil functions using small (<2 mL) volumes of blood. Healthy human samples were exposed to C5a or control, with addition of pH-sensitive Staphylococcus aureus bioparticles (pHrodo™ Life Technologies, Paisley, UK) and the reactive oxygen species (ROS) indicator DHR-123 (Sigma, Gillingham, UK). 

 

Figure 2: The anticoagulant Argatroban allows assessment of multiple neutrophil functions in small samples of whole blood. 50 uL of healthy donor whole blood samples (n=4-6) were incubated with pHrodo and DHR-123 for 1 hour in the presence of soluble neutrophil priming agents. Phagocytosis and ROS production were quantified by flow cytometry.

 

Figure 3: C5a induces long-term impairment of phagocytosis.

S. aureus bioparticles were incubated with PMNs in the presence of C5a or PBS, with removal of C5a/PBS (A; n = 4) or continuous exposure from different time points (B; n = 5) before measurement of phagocytosis. 

 

Figure 4: C5a-induced phagocytic impairment can be prevented by PI3-kinase inhibition in purified neutrophils but not in whole blood neutrophils.

Purified human neutrophils (A; n = 9) or human whole blood samples (B; n = 7) were incubated with isoform-selective small molecule inhibitors of PI3-kinase enzymes prior to addition of S. aureus bioparticles and measurement of phagocytosis.

 

Results summary

•We have developed a model of bacteraemia which allows rapid interrogation of multiple neutrophil functions in small blood samples without purification or lysis steps.
•The effect of C5a occurs after even a brief exposure (30 mins), and lasts for up to 7 hours after C5a has been washed off (Figure 3A).
•The temporal relationship between C5a exposure and phagocytic target exposure is crucial; C5a exposure after target exposure does not impair phagocytosis (Figure 3B).
•PI3-kinase inhibition appears to prevent C5a-induced phagocytic impairment in purified neutrophils, but not in whole blood (Figure 4). This finding may influence the efficacy of locally- or systemically-administered therapies. 

References

1.Conway Morris A et al. Blood. 2011;117:5178-88
2.Conway Morris A et al. Br J Anaesth, 2013;111:778-87.
3.Conway Morris A et al. Am J Respir Crit Care Med. 2009;180:19-28.

 

 
 

 

 

 

 

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