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

Omar Rafael Alemán,Eileen Uribe-Querol,Ricarda Cortes-Vieyra,Nancy Mora,Carlos Rosales

doi:10.3389/fimmu.2016.00277

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.

Highlights

Neutrophils are innate immune cells that migrate from the circulation to sites of inflammation or infection
In order to confirm that the mAb 3G8 (IgG1) was targeting FcγRIIIb, neutrophils were stimulated by the isotypic control antibody TS2/16 (IgG1) that binds to β1 integrins, and by the mAb IV.3 (IgG2b) that binds FcγRIIa
Neither mAb IV.3 nor mAb TS2/16 induced neutrophil extracellular traps (NETs) formation (Figure 1), strengthening the point that FcγRIIIb is the receptor responsible for induction of NETosis. These data indicated that cross-linking FcγRIIIb is an efficient stimulus for NET formation with a faster response than the one induced by Phorbol 12-myristate 13-acetate (PMA)

Summary

Introduction

Neutrophils are innate immune cells that migrate from the circulation to sites of inflammation or infection. Neutrophils are considered the first line of defense since they are the first cells to appear at the affected site, and they display important antimicrobial functions [1]. Neutrophils use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes [2, 3]. NET fibers are composed of chromatin covered with histones [4] and antimicrobial proteins derived from the neutrophil granules, such as the bactericidal/permeabilityincreasing protein (BPI), elastase, myeloperoxidase, lactoferrin, and metalloprotease 9 [2, 5]. NETs prevent further spread of pathogens because they function as a physical barrier where microorganisms get trapped and because they bring antimicrobial proteins in close proximity of pathogens. NETs can kill microorganisms extracellularly and independently of phagocytosis [6]

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Results

Conclusion