Transforming Growth Factor-β-Activated Kinase 1 Is Required for Human FcγRIIIb-Induced Neutrophil Extracellular Trap Formation

Abstract

Neutrophils (PMNs) are the most abundant leukocytes in the blood. PMN migrates from the circulation to sites of infection where they are responsible for antimicrobial functions. PMN uses phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds, such as Phorbol 12-myristate 13-acetate (PMA), are efficient inducers of NETs. Antigen–antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. Direct cross-linking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of extracellular signal-regulated kinase (ERK) and nuclear factor Elk-1, and the transforming growth factor-β-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies, and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-β-induced TAK1 phosphorylation. A MEK (ERK kinase)-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis.

Pathogenic Entamoeba histolytica, but not Entamoeba dispar, induce neutrophil extracellular trap (NET) formation

“…It is also equally possible that the amoeba lectin binds to a neutrophil glycoprotein that in turn functions as the receptor inducing NETosis. So far, only 2 receptors on the human neutrophil have been described as bona fide inducers of NET release, the IgA receptor FcαR and the IgG receptor FcγRIIIb . In the case of human protozoan parasites as well as many other pathogens, no distinct neutrophil receptor for PAMPs has been identified .…”

“…So far, only 2 receptors on the human neutrophil have been described as bona fide inducers of NET release, the IgA receptor Fc R 46 and the IgG receptor Fc RIIIb. 37,38 In the case of human protozoan parasites as well as many other pathogens, no distinct neutrophil receptor for PAMPs has been identified. 29 Independently of what the actual receptor on the neutrophil is for inducing NET formation, our data strongly indicate that recognition of the parasite and induction of NETosis involve a carbohydrate with an axial HO-group at carbon 4 of a hexose, such as the one present in Gal and GalNAc (Fig.…”

“…NET formation was quantified by detecting DNA release spectrophotometrically with the DNA‐binding dye SYTOX ® Green as previously described . Briefly, neutrophils were resuspended at 1 × 10 6 cell/ml in RPMI‐1640 medium (Gibco ® ), containing 500 nM SYTOX ® Green.…”

“…NET formation was quantified by detecting DNA release spectrophotometrically with the DNA-binding dye SYTOX R Green as previously described. 32,37,38 Briefly, neutrophils were resuspended at 1 × In selected experiments, neutrophils were incubated at 37 • C for 20 min with various concentrations of different saccharides, before stimulation with the amoebas.…”

“…NET are strongly induced by PMA, a potent activator of protein kinase C. 45 Ab Fc receptors, Fc RI 46 and Fc RIIIb, 37,38 are also strong activators of NET formation. In addition, various protozoan parasites including Leishmania, 47 Toxoplasma gondii, 48 Trypanosoma cruzi, 49 and E. histolytica trophozoites 31,32 have been shown to induce NET formation.…”