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.
Highlights
Types and pathways[17,18,19]
We found that LPS, but not phorbol myristate acetate (PMA), activates JNK in a dose-dependent fashion, and that JNK activation is responsible for LPS, but not PMA, mediated ROS production and subsequent Nox-dependent suicidal NETosis
To determine the relevance of JNK in NETosis, we examined the effect of LPS on JNK activation in neutrophils
Summary
Types and pathways[17,18,19]. Here we tested whether JNK is an important molecular sensor that initiates NETosis in response to increasing concentrations of LPS- and Gram-negative bacteria. PMA is used often as an agonist to induce Nox-dependent NETosis It activates protein kinase C (PKC), which activates Nox for producing ROS7, 8, 10. The main concept has been that LPS engages TLR4 present on platelets, and subsequently the activated platelets directly or indirectly induce Nox-independent vital NETosis; certain types of LPS can induce Nox-dependent NETosis[20,21,22,23]. To determine the direct and regulatory effects of LPS on NETosis, we induced NETosis with PMA, LPS and two typical Gram-negative bacteria, E. coli and P. aeruginosa. Gram-negative bacteria such as E. coli and P. aeruginosa induce NETosis in a multiplicity of infection (MOI)-dependent manner that can be significantly inhibited by SP600125. JNK acts as a molecular rheostat that uniquely regulates LPS-mediated NETosis by regulating ROS production in neutrophils
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