Alum triggers infiltration of human neutrophils ex vivo and causes lysosomal destabilization and mitochondrial membrane potential‐dependent NET‐formation

Abstract

Aluminium salts have been used in vaccines for decades. However, the mechanisms underlying their adjuvant effect are still unclear. Neutrophils, the first immune cells at the injection site, can release cellular DNA together with granular material, so‐called neutrophil extracellular traps (NETs). In mice, NETs apparently play a role in aluminium hydroxide (alum)‐adjuvant immune response to vaccines. Although no experimental data exist, this effect is assumed to be operative also in humans. As a first step to verify this knowledge in humans, we demonstrate that the injection of alum particles into human skin biopsies ex vivo leads to similar tissue infiltration of neutrophils and NET‐formation. Moreover, we characterized the mechanism leading to alum‐induced NET‐release in human neutrophils as rapid, NADPH oxidase‐independent process involving charge, phagocytosis, phagolysosomal rupture, Ca2+‐flux, hyperpolarization of the mitochondrial membrane, and mitochondrial ROS. Extracellular flow and inhibition experiments suggested that no additional energy from oxidative phosphorylation or glycolysis is required for NET‐release. This study suggests a so far unappreciated role for neutrophils in the initial phase of immune responses to alum‐containing vaccines in humans and provides novel insights into bioenergetic requirements of NET‐formation.

Adjuvants and Vaccines Used in Allergen-Specific Immunotherapy Induce Neutrophil Extracellular Traps

“…Two different pathways of NET induction exist, a NADPH-oxidase 2 (NOX2)- dependent pathway involving cytosolic (c)ROS as induced by PMA or lipopolysaccharide [ 34 , 35 ], and a NOX2-independent pathway involving mitochondrial (m)ROS as induced, e.g., by ionomycin or alum [ 18 , 35 , 36 ]. Production of NOX2-dependent cROS in neutrophils was measured by flow cytometry using the cell-permeant fluorescent probe CM-H 2 DCFDA, and NOX2-independent mROS were assessed with MitoSox Red using a plate reader.…”

“…Neutrophils were isolated from heparinized blood by Ficoll gradients, erythrocyte sedimentation and lysis of erythrocytes as described [ 18 ]. Obtained cell populations were >99% according to trypan blue staining and consisted of 92.4% ± 5.1 (mean ± SD; n = 14) CD16 + CD66b + neutrophils as evaluated flow cytometry.…”

“…First, 6 × 10 6 neutrophils were seeded into 6 mL HBSS in 6-well plates and NET formation was induced by incubation of the cells with 5 μM ionomycin for 4 h at 37 °C and 5% CO 2 . Since alum creates immense technological problems in isolating pure NETs, and we have demonstrated similar mechanisms in NET induction by alum and ionomycin [ 18 ], the latter was used as the NET-inducer for both procedures. NET formation was controlled by light microscopy.…”

“…Then, cells were stimulated with ionomycin, alum, MPLA and different vaccines at indicated concentrations. mROS-induced fluorescence was measured as described [ 18 ]. To detect the production of cytosolic ROS (cROS), chloromethyl-2’7’-dichlorodihydrofluorescein diacetate (CM-H 2 CFDA) (Molecular probes, Life Technologies, Carlsbad, CA) was used; 1 × 10 6 neutrophils were incubated for 30 min in 1 mL of 1 μM CM-H 2 CFDA in HBSS supplemented with 25 ng/mL GM-CSF at 37 °C and 5% CO 2 in Falcon tubes.…”

“…NETs are 3-dimensional web-like structures composed of decondensed chromatin and mitochondrial DNA that are complexed with toxic granular material such as myeloperoxidase (MPO), neutrophil elastase (NE) or the cationic cathelicidin peptide LL-37, which can keep microbes from growing and spreading [ 16 , 17 ]. We have recently demonstrated that injection of alum particles induces tissue infiltration by neutrophils and NET formation in a human ex vivo skin model [ 18 ].…”