Neutrophil swarming and extracellular trap formation play a significant role in Alum adjuvant activity

J Stephen,D Erben,R A Benson,H E Scales,J M Brewer,P Garside

doi:10.1038/s41541-016-0001-5

Abstract

There are over 6 billion vaccine doses administered each year, most containing aluminium-based adjuvants, yet we still do not have a complete understanding of their mechanisms of action. Recent evidence has identified host DNA and downstream sensing as playing a significant role in aluminium adjuvant (aluminium hydroxide) activity. However, the cellular source of this DNA, how it is sensed by the immune system and the consequences of this for vaccination remains unclear. Here we show that the very early injection site reaction is characterised by inflammatory chemokine production and neutrophil recruitment. Intravital imaging demonstrates that the Alum injection site is a focus of neutrophil swarms and extracellular DNA strands. These strands were confirmed as neutrophil extracellular traps due to their sensitivity to DNAse and absence in mice deficient in peptidylarginine deiminase 4. Further studies in PAD4−/− mice confirmed a significant role for neutrophil extracellular trap formation in the adjuvant activity of Alum. By revealing neutrophils recruited to the site of Alum injection as a source of the DNA that is detected by the immune system this study provides the missing link between Alum injection and the activation of DNA sensors that enhance adjuvant activity, elucidating a key mechanism of action for this important vaccine component.

Highlights

Following its discovery in 1926,1 aluminium hydroxide (Alum) has been unique in its prolonged use as an adjuvant in human vaccines
While a small number of GFP+ neutrophils were observed at the OVA injection site (Fig. 3a; Supplementary Movie 1) treatment with OVA/Alum produced greatly enhanced neutrophil recruitment (Fig. 3b; Supplementary Movie 2) consistent with the flow cytometric analysis (Fig. 1a, b)
The neutrophil identity of the GFP+ cells was confirmed by flow cytometry (FACS) analysis that demonstrated fourfold higher expression of GFP in neutrophils compared with monocytes and macrophages

Summary

INTRODUCTION

Following its discovery in 1926,1 aluminium hydroxide (Alum) has been unique in its prolonged use as an adjuvant in human vaccines. Intravital imaging revealed neutrophil swarming and cell death focussed around Alum, and the presence of DNA strands within the tissue. These strands were subsequently confirmed as neutrophil extracellular traps (NETs) via their sensitivity to DNase treatment. NETs were absent in PAD4deficient mice, which displayed markedly reduced immune responses following Alum injection. These studies demonstrate that the mechanism of neutrophil death plays an important role in the adjuvant activity of Alum, and explain how the cellular reaction at the injection site drives the liberation of host DNA, which subsequently impacts on adjuvant activity

RESULTS

DISCUSSION

METHODS