Regulation of extracellular chromatin release from neutrophils

Abstract

Neutrophils use intricate mechanisms for capturing and killing invading microorganisms. One mechanism entails the release of relaxed chromatin from the cell. Microbes are trapped by the extracellular chromatin and exposed to high local concentrations of bactericidal compounds. We examine the regulation of chromatin release by testing the contribution of microtubules and the actin cytoskeleton to the deployment of neutrophil extracellular traps (NETs). Incubation of human neutrophils with nocodazole, a tubulin polymerization inhibitor, or cytochalasin D, an inhibitor of actin filamentation, severely diminished the ability of neutrophils to respond to LPS by releasing chromatin from the cells. In addition, pretreatment of neutrophils with M1/70, a monoclonal antibody to the Mac-1 integrin adhesion receptor, drastically reduced the deployment of chromatin into NETs. Analysis of histone deimination, the conversion of arginine to citrulline in 3 of the 4 core histones by peptidylarginine deiminase 4, revealed that the treatments inhibiting NET formation also reduced histone deimination. Our data indicate that NET formation requires functional tubulin and actin filaments and responds to engagement of Mac-1 integrins. Because histone deimination coincides with the release of NETs, we propose that these events represent overlapping mechanisms of neutrophil responses to infections.

Fig. 1

Diverse inflammatory stimuli induce histone deimination and NET release. a Histone deimination was determined by Western blot (CitH3). Neutrophils were incubated in buffer alone (–) or in the presence of TNF (+ = 2 ng/ml; ++ = 8 ng/ml), LPS (+ = 10 ng/ml; ++ = 100 ng/ml), zymosan (Zymo; + = 1 μg/ml; ++ = 10 μg/ml), LTA (+ = 1 μg/ml; ++ = 6 μg/ml) or hydrogen peroxide (H₂O₂; + = 10 μM; ++ = 100 μM). Each stimulus enhanced histone deimination, with higher concentrations of stimuli inducing greater deimination. The samples that were analyzed contained comparable amounts of total histone H3 (TotH3). b Neutrophils retain normal morphology with typical multilobed nuclei following incubation in buffer alone. DNA was visualized with Sytox orange (blue) and the plasma membrane with annexin V (green). c Neutrophils incubated in 100 μM H₂O₂ display NETs. The NET released from the cell at top right (violet arrow) also reacts with antibody to deiminated histone H3 (red), whereas the NET from the cell on the bottom right (blue arrow) does not. Broken lines indicate the footprints of the two cells. In addition to the composite color images, corresponding separate color images are shown for b and c. 1 = Antibody binding; 2 = annexin V; 3 = DNA staining.

Fig. 2

Drugs that depolymerize microtubules or actin filaments inhibit histone deimination and NET release. a Deiminated histone H3 (CitH3) and total H3 (TotH3) were determined by Western blot following incubation of neutrophils in 100 ng/ml LPS. Neutrophils were preincubated in the absence (–) or presence (+) of 10 μM nocodazole (Noc), 10 μM cytochalasin D (CytoD) or 100 μM apocynin (Apo). b Microscopy of cells incubated in 100 ng/ml LPS shows nuclear dissolution and NET release. Antideiminated histone H3 binding is visualized in red. Other colors are as in figure 1. c Cells preincubated in nocodazole show a greatly reduced morphological response to LPS. d Pre-incubation in cytochalasin D reduced NET release in response to LPS and caused accumulation of cells with increased diameter whose interior was filled with homogeneously dispersed chromatin (arrows and inset). e Multiple views of the cell populations shown in b–d versus untreated cells were recorded, analyzed for NET release, and the results were plotted.

Fig. 3

Pretreatment of neutrophils with anti-Mac-1 antibody or secinH3 reduces response to LPS. a Neutrophils were incubated with 10 μg/ml of M1/70, an anti-Mac-1 IgG, or an isotype-matched control IgG, for 30 min prior to addition of 100 ng/ml LPS. Deiminated histone H3 was detected by Western blot (CitH3), indicating that M1/70 reduced deimination. Neutrophil lysates also exhibited a faster migrating immunoreactive band [18]. b Neutrophils incubated without inhibitor (No), with 50 μM Wortmannin (Wort) or 10 μM secinH3 (SecH3) prior to addition of 100 ng/ml LPS. SecinH3 inhibited histone deimination. c Deimination was measured by densitometry of immunoreactive bands and displayed as percent of deimination observed in the absence or presence of M1/70 or SecinH3. d Cells incubated with 100 ng/ml LPS show NETs and histone deimination. Colors represent fluorescence acquired as in figure 1. e Cells treated with M1/70 show a greatly reduced morphological response to LPS than cells in d.