ROS generation is crucial for NETs
ROS generation is crucial for NETs release upon both physiological or pathogenic stimuli [16,30]. In our study, we further verified this conclusion by demonstrating that SRA stimulation by Poly I enhanced intracellular ROS levels while scavenging of ROS by anti-oxidant agents effectively abolished the NETs release. Therefore, our data suggest a possible link between SRA stimulation and NETs associated ROS production. Indeed, recent studies presented similar findings that oxidized LDL, a non-specific ligand of scavenger receptors, induced ROS production in neutrophils [31,32]. NOX2 is regarded as a key factor that catalyzes ROS generation and mediates canonical NETs formation. Moreover, ERK is implicated as a major kinase that mediates NOX2 activation [16,17]. By using chemical inhibitors that either blocks the activity of NOX2 or suppressed ERK activation, we verified that the SRA elicited NETs were induced by NOX2 and mediated by ERK activation. Thus we first characterize a NOX2 dependent canonical pathway involved in NETs formation due to SRA stimulation.
Recently, NOX-independent NETs formation has been observed by stimuli like calcium influx and uric Cy7 NHS ester (non-sulfonated) [19,33]. In this situation, mtROS is generated to trigger NETs release. Our current work demonstrated that SRA activation also induced mtROS dependent NETs formation, in addition to the NOX2 dependent pathway. Although mtROS generation is regarded as the bypass production of the respiratory chain, the upstream modulation of the NETs-triggering mtROS is not well understood . Here we discovered a new mechanism of SRA dependent and NETs triggering mtROS though ERK dependent upregulation of ROMO1 in neutrophils. ROMO1 is known as an important mitochondrial protein that residents in the inner membrane and alters cellular redox state by inducing mtROS production . For a decade since its first discovery in 2006, ROMO1 has been mainly studied in the context of tumor invasion  and apoptosis  by means of inducing mtROS generation. However, its functions in neutrophils remain largely unknown. In fact, we found that ROMO1expression positively correlated with the generation of mtROS in neutrophils (Figs. S2 and S3). Moreover, SRA stimulation by Poly Indirectly increased the ROMO1 expression in neutrophils while ERK activation was required for the upregulation of ROMO1. It is noteworthy that ROMO1 could induce MMP loss and form a nonselective cationic channel in mitochondria, both contributing to mtROS production in tumor cells [35,36]. Our study also demonstrated that Poly I downregulated MMP in neutrophils while inhibiting the channel activity of ROMO1 markedly suppressed NETs release. Therefore, we further characterize the involvement of ROMO1 in mediating mtROS generation and NETs formation upon SRA stimulation, in addition to the abovementioned NOX2 dependent pathway.
Conflicts of interest
Acknowledgements This work was supported by grants from Frontier and Applied Basic Research Program of Chongqing (cstc2016jcyjA0105) and Scientific Innovation Program of Southwest Hospital (2017MPRC-10, SWH2016JCYB-07) to Xin Liu.
Introduction The drug apomorphine is of interest in terms of drug conditioning in that this drug can have pronounced but opposite effects upon dopamine neurotransmission depending upon dose level. In the low dose range in rats (<0.1 mg/kg), apomorphine can induce a profound inhibition of movement presumably by a preferential stimulation of dopamine auto-receptors and thereby inhibiting dopamine activity in the brain [, , ]. At higher dose levels (>0.5 mg/kg) apomorphine increasingly stimulates dopamine post-synaptic receptors and is a potent behavioral stimulant, manifested as hyper-locomotion [, , ]. In line with the importance of the role of dopamine in the modulation of stimulus-response (S-R) associations, it has been reliably reported that the repeated pairing of a high dose of apomorphine with a specific environment induces context dependent sensitization and conditioning of hyper-locomotion elicited by apomorphine [, , , , , ]. Interestingly, both low and high dose apomorphine treatments have behavioral inhibitory/excitatory effects when given post-trial [, , ]. Post-trial treatment effects have been long known to influence the consolidation  or re-consolidation memory process [17,18].