JNK Activation Turns on LPS- and Gram-Negative Bacteria-Induced NADPH Oxidase-Dependent Suicidal NETosis

Abstract

Neutrophils cast neutrophil extracellular traps (NETs) to ensnare microbial pathogens. Nevertheless, the molecular rheostats that regulate NETosis in response to bacteria are not clearly established. We hypothesized that stress-activated protein kinase or c-Jun N-terminal Kinase (SAPK/JNK) is a molecular switch that turns on NETosis in response to increasing concentrations of lipopolysaccharide (LPS)- and Gram-negative bacteria. Here we show that Escherichia coli LPS (0111:B4; 10–25 μg/ml), but not phorbol myristate acetate (PMA), activates JNK in human neutrophils in a dose-dependent manner. JNK inhibitors SP600125 and TCSJNK6o, and a TLR4 inhibitor TAK242 suppress reactive oxygen species production and NETosis in LPS-, but not PMA-treated neutrophils. Diphenyleneiodonium suppresses LPS-induced NETosis, confirming that endotoxin induces NADPH oxidase-dependent NETosis. Immunoblots, Sytox Green assays, and confocal microscopy of cleaved caspase-3 and nuclear morphology show that JNK inhibition does not induce apoptosis in LPS-stimulated neutrophils. JNK inhibition also suppresses NETosis induced by two typical Gram-negative bacteria, E. coli and Pseudomonas aeruginosa. Therefore, we propose that neutrophils use a TLR4-dependent, JNK-mediated molecular sensing mechanism to initiate NADPH oxidase-dependent suicidal NETosis in response to increasing concentrations of LPS, and Gram-negative bacteria. The LPS-TLR4-JNK activation axis determines the fate of these cells: to be or not to be NETotic neutrophils.

Neutrophil Extracellular Traps Induce Human Th17 Cells: Effect of Psoriasis-Associated TRAF3IP2 Genotype

“…We utilized 25 nM PMA as an alternative stimulus for NETosis and were interested to find that PMA-induced NETs were completely ineffective for induction of Th17 cells (Supplementary Figure S2c), despite PMA being a strong stimulus for NETosis ( Supplementary Figures S2a, S2b). Recent studies (Khan et al, 2017) have shown that LPS and PMA utilize different signaling pathways to induce NETosis, with LPS triggering a Toll-like receptor 4edependent activation of nicotinamide adenine dinucleotide phosphate oxidase to effect NETosis (Khan et al, 2017). While both sets of experiments utilized E. coli 0111:B4 LPS, we used a much lower concentration (5 ng/ml vs. 100e25,000 ng/ml).…”

“…Quantitation of DNA released from NETs revealed that treatment with 5 ng/ml LPS for 4 hours did not significantly increase NETosis (Supplementary Figure S2b). We also generated NETs using phorbol myristate acetate (PMA), another frequently used stimulus for NET formation (Khan et al, 2017). Interestingly, while 25 nM PMA was highly effective in inducing NETosis (Supplementary Figure S2b), the resulting NETs were completely ineffective in inducing Th17 cells (Supplementary Figure S2c).…”

Identification of a novel HIF-1α-αMβ2Integrin-NETosis axis in fibrotic interstitial lung disease

“…Having found an effect on NETosis, we next examined integrin activation and neutrophil adhesion, which are also implicated in NET release 34,35 . We measured neutrophil adhesion to primary human endothelial cells in the absence or presence of PMA (a general integrin activator) or lipopolysaccharide (LPS) (to mimic infectious stimuli), stimuli that activate NETosis via distinct pathways 36 . Hypoxia increased both unstimulated (23.6±4.0% vs 2.7±1.6%, p<0.05) and LPS-stimulated (35.7±4.8% vs 11.3±1.4%, p<0.05) adhesion to resting endothelium, whilst PMA-stimulated adhesion, which was already high, was unaffected (fig.…”

Mechanisms and disease relevance of neutrophil extracellular trap formation

“…Immune complexes were found to drive NET formation through FcγRIIIb signalling, and opsonization of S. aureus with IgA induces the release of NETs through engagement of FcαR . NET formation requires the activity of mitogen‐activated protein kinases (MAPKs) . Recently, a number of other kinases have been linked to the formation of NETs in response to micro‐organisms, immune complexes and MSU crystals, such as spleen tyrosine kinase (SYK), phosphoinositide 3‐kinase (PI3K), IL‐1 receptor‐associated kinase (IRAK), PKC and AKT .…”

“…60 NET formation requires the activity of mitogen-activated protein kinases (MAPKs). 61,62 Recently, a number of other kinases have been linked to the formation of NETs in response to micro-organisms, immune complexes and MSU crystals, such as spleen tyrosine kinase (SYK), phosphoinositide 3-kinase (PI3K), IL-1 receptorassociated kinase (IRAK), PKC and AKT. 18,55,59,[63][64][65][66] In line with the involvement of PI3K in NET release, studies have identified a role for autophagy, which also requires this signalling intermediate, in the formation of NETs.…”