Neutrophils produce high amounts of ROS which act
Neutrophils produce high amounts of ROS, which act as antimicrobial agents, signaling molecules or initiators during PMA-induced NET formation. Of the intravenous anesthetics used in this study, propofol was the best at decreasing PMA-induced ROS levels (Fig. 3A and B). Our previous study also demonstrated that propofol is the most effective anesthetic at inhibiting total ROS production in Staphylococcus aureus-infected RAW264.7 Thiomyristoyl mg . In addition to propofol, midazolam and ketamine have been demonstrated to suppress ROS production in phagocytes [12,14,25], and Yang et al.  reported that propofol significantly reduced superoxide generation in fMLF-activated human neutrophils. Conversely, our results indicated that propofol reduced the total amount of ROS (Fig. 3A and B) but not the superoxide (Fig. 3C) produced in response to PMA. This discrepancy may be due to the different stimulators used because our results are supported by those of Davidson et al. . Inhibitors of NADPH oxidase and MPO reduce PMA-induced NET release, indicating that superoxide and MPO-derived HOCl are involved in NET release . In our study, propofol specifically decreased the level of HOCl (Fig. 3D), which was related to the reduction in total ROS production stimulated by PMA (Fig. 3A and B). This propofol-driven reduction in the HOCl level may further mediate the decrease in NET formation observed with the MPO inhibitor aminopyrine (Fig. 4B). However, the decreased HOCl level induced by propofol treatment was not directly mediated by inhibiting the activity of MPO (Fig. 4A) and may instead be due to the HOCl-scavenging activity of propofol .
Two main studies have reported related pathways involved in PMA-induced NET formation [10,11]. Hakkim et al.  showed that RAF, MEK and ERK inhibitors can each block ROS production and that phosphorylation of ERK was not prevented by DPI treatment, indicating that the RAF-MEK-ERK pathway is upstream of NADPH oxidase. In contrast, Keshari et al.  found that the ERK inhibitor U0126 failed to reduce PMA-induced superoxide generation and DPI that inhibited ERK phosphorylation, suggesting that ERK is downstream of NADPH oxidase. This discrepancy may be because of the neutrophil diversity among individuals or the detection method and concentrations of the different inhibitors used . In addition, Hakkim et al.  did not detect superoxide and did not clearly describe the method used to measure total ROS. In the present study, we found that the level of PMA-induced superoxide was not influenced by the ERK inhibitor PD98059 (Fig. 3C), which is consistent with the study by Keshari et al. . Furthermore, we demonstrated that propofol inhibited PMA-induced NET formation by decreasing the level of p-ERK (Figs. 2B and 5B) but not that of superoxide (Fig. 3C). These data indicate that propofol has the potential to inhibit NET formation in vivo. Further investigation of NET formation in sepsis patients with propofol sedation would be interesting.
Conclusions By comparing the effects of four intravenous anesthetics, propofol was found to be the most effective at inhibiting PMA-induced NET formation. This inhibition was mediated by two pathways: reduced ERK phosphorylation and HOCl levels (Fig. 6). The two pathways are independent because ERK and MPO inhibitors did not reduce the levels of the product of the other pathway. In the present study, we define a new immunomodulatory function of intravenous anesthetics, particularly propofol, against NET formation.
Acknowledgments This work was supported by the Ditmanson Medical Foundation of Chia-Yi Christian Hospital under grant number R106-33.
Main Text A classical mechanism in organismal development is the formation of morphogen gradients. Morphogen gradients result from localized secretion and long-range diffusion of signaling molecules, which generate a graded concentration profile along the tissue. Classical works on EGFR-ERK signaling suggested that this signaling pathway exhibits such a morphogen gradient behavior (Gabay et al., 1997, Schweitzer and Shilo, 1997). However, in recent years this picture is challenged by several works showing a wave-like propagation dynamics of ERK activity (Aoki et al., 2017, de la Cova et al., 2017, Lim et al., 2015). In this issue of Developmental Cell, Ogura et al. (2018) provide new evidence that EGFR-ERK signaling in the Drosophila trachea propagates from cell to cell through a relay mechanism rather than passive diffusion. This relay mechanism is essential for the proper invagination of the tracheal placode.