Opposition between PKC isoforms regulates histone deimination and neutrophil extracellular chromatin release

Abstract

In response to inflammation, neutrophils deiminate histones and externalize chromatin. Neutrophil extracellular traps (NETs) are an innate immune defense mechanism, yet NETs also may aggravate chronic inflammatory and autoimmune disorders. Activation of peptidylarginine deiminase 4 (PAD4) is associated with NET release (NETosis) but the precise mechanisms of PAD4 regulation are unknown. We observed that, in human neutrophils, calcium ionophore induced histone deimination, whereas phorbol myristate acetate (PMA), an activator of protein kinase C (PKC), suppressed ionophore-induced deimination. Conversely, low doses of chelerythrine and sanguinarine, two inhibitors of PKC, reversed PMA inhibition and enhanced ionophore-stimulated deimination. In addition, a peptide inhibitor of PKCα superinduced ionophore activation of PAD4, thus identifying PKCα as the PMA-induced inhibitor of PAD4. At higher doses, chelerythrine, sanguinarine, and structurally unrelated PKC inhibitors blocked histone deimination, suggesting that a different PKC isoform activates histone deimination. We identify PKCζ as activator of PAD4 because a specific peptide inhibitor of this PKC isoform suppressed histone deimination. Confocal microscopy confirmed that, in the presence of PMA, NETosis proceeds without detectable histone deimination, and that ionophore cooperates with PMA to induce more extensive NET release. Broad inhibition of PKC by chelerythrine or specific inhibition of PKCζ suppressed NETosis. Our observations thus reveal an intricate antagonism between PKC isoforms in the regulation of histone deimination, identify a dominant role for PKCα in the repression of histone deimination, and assign essential functions to PKCζ in the activation of PAD4 and the execution of NETosis. The precise balance between opposing PKC isoforms in the regulation of NETosis affirms the idea that NET release underlies specific and vitally important evolutionary selection pressures.

Keywords: NETosis; PAD4; deimination; inflammation; protein kinase C.

FIGURE 1

Phorbol myristate acetate (PMA) suppresses histone deimination, whereas ionophore or ionomycin stimulate deimination. Purified neutrophils were incubated in buffers containing calcium (Ca), A23187 ionophore (Io) or ionomycin (Im), and/or PMA for 2 h. Cells were lysed and analyzed by western blotting to detect deiminated histone H3 (dH3) vs. total H3 (tH3). The presence of stimuli is indicated by a plus sign. Panels (A,B) show blots of lysates from cells incubated with the indicated stimuli or calcium alone. Panel (A) shows results of a blot detecting dH3, whereas panel (B) shows antibody binding to modified citrullines. Migration of H3 and H4 histones and position of molecular weight markers are indicated. Asterisk indicates a protein of approximately 50 kD that is deiminated in response to ionophore treatment. Substitution of PBS for HBSS did not qualitatively alter the results of the treatments (C, top), nor did substitution of ionomycin for ionophore (C, bottom). The highest levels of dH3 were observed after ionophore or ionomycin treatment. Inclusion of PMA reduced histone deimination and PMA alone failed to stimulate deimination. Microscopy of unstimulated neutrophils showed cells with multilobed nuclei (D), whereas PMA treatment induced nuclear swelling, rupture, and NET release (E). In these pictures, DNA is displayed in blue and binding of antibodies to deiminated histone H3 in green. Diffuse NETs are indicated by arrow heads here and in (G). Ionophore induced histone deimination in cytoplasm and decondensed nuclei (F). A combination of PMA and ionophore led to enhanced NETosis with little to no detectable histone deimination (G). Purified NET DNA was solubilized by micrococcal nuclease digestion and quantified by fluorescence (H). MPO released by nuclease treatment was measured in solution by the TMB method (I). All experiments were performed at least five times with consistent results.

FIGURE 2

Conditions in which PKC inhibitors enhance histone deimination. (A) Histone deimination that is repressed by PMA can be derepressed by 5 μM chelerythrine (Chel) or 1 μM sanguinarine (Sang) but higher concentrations of these inhibitors lead to renewed repression. Levels of deiminated histone H3 (dH3) vs. total H3 (tH3) were estimated by western blotting. (B) A narrow range of inhibitor concentrations also superinduced histone deimination in response to ionophore (Io). (C) Pseudosubstrate peptide inhibitor for PKCα/β enhanced histone deimination that was stimulated by ionophore. The vertical white lines separate lanes from the same gel that were taken with identical autoradiographic exposure but were arranged differently in the original gel. The presence of stimuli is indicated by a plus sign.

FIGURE 3

Histone deimination induced by ionophore is only partially suppressed by calphostin C or bisindolylmaleimide 1 (BIM1) but is fully suppressed by a specific peptide inhibitor of PKCζ. Calphostin C (Calp) partially suppressed histone deimination (A), as did addition of 100 nM BIM1 (B). These two compounds do not effectively block atypical PKC isoforms. In contrast, ionophore-induced histone deimination was completely suppressed by a peptide inhibitor of PKCζ but not by an inhibitor of PKCτ (C). The vertical white lines separate lanes from the same gel that were taken with identical autoradiographic exposure but were arranged differently in the original gel. The presence of stimuli is indicated by a plus sign. Addition of LPS in PBS (100 ng/ml) enhanced histone deimination, and a peptide inhibitor of PKCζ but not of PKCτ or α/β suppressed the elevated level of deimination (D).

FIGURE 4

Effect of PKC inhibitors on NETosis. Neutrophils were incubated with A23187 ionophore in the presence of 5 μM chelerythrine (A), or 20 μM chelerythrine (B). Antibody binding to deiminated histone H3 (green) and the DNA contained in cells or released as NETs (blue) was observed in 5 μM chelerythrine, whereas 20 μM inhibitor precluded deimination and most other morphologic features of NETosis. Cells incubated with PKCα/β pseudosubstrate peptide inhibitor deiminated histone H3 and proceeded to NET release (C). The PKCζ inhibitor peptide suppressed histone deimination and NET release but earlier stages of NETosis, such as a partial heterochromatin decondensation and nuclear de lobulation, were observed (D). As in Figure 1E, untreated neutrophils showed typical multilobed nuclei without apparent evidence of NETosis (E).