Prak^flox/flox mice were generated by Shanghai Model Organisms Center (China). Lyz2-Cre mice were a gift from Professor Xiaoyu Hu (Tsinghua University). To generate mice with selective Prak deficiency in myeloid cells, Prak^flox/flox mice were crossed with Lyz2-Cre mice, both on C57BL/6 background. Ten to twelve-weeks-old Prak^flox/flox (called “wild-type” here) and Prak^flox/flox Lyz2-Cre (called “knockout” here) mice were littermates and kept in the specific-pathogen-free conditions animal facility of the Peking University Health Science Center. The experimental procedures on use and care of animals were approved by the Ethics Committee of Peking University Health Science Center.

Neutrophils were isolated as described previously (28). Briefly, blood samples were collected from healthy volunteers. Neutrophils were isolated from heparinized venous blood by density gradient centrifugation on PolymorphPrep according to the manufacturer's instructions. The purity of the neutrophils was above 95%. Then neutrophils were washed with PBS and suspended in RPMI 1640 supplemented with 4% heat-inactivated fetal bovine serum (FBS). Neutrophils were pretreated with or without PRAK inhibitor (5 μM) for 1 h, and then stimulated with PMA (100 nM) for 4 h at 37°C and 5% CO₂. The research was approved by the Ethics Committee of Peking University People's Hospital with an approval number of 2016PHB076-01, and performed in accordance with the ethical standards of Declaration of Helsinki.

Briefly, Prak^flox/flox and Prak^flox/flox Lyz2-Cre mice were injected with sterile 4% thioglycollate in the peritoneal cavity (29). After 8–12 h, neutrophils were enriched (75–80% pure as assessed by flow cytometry using Ly6G and CD11b antibodies). Cells from peritoneal lavage fluid were plated and incubated at 37°C and 5% CO₂ for 20 min in RPMI 1640 with 0.5% FBS. The cells were washed once with PBS to remove non-adherent cells. And after more enrichment, the neutrophil purity was consistently >90%. Then, isolated neutrophils were stimulated with PMA (100 nM) 12 h.

The percentage of NET formation was manually quantitated by dividing the number of NET-forming neutrophils (expanded nuclei and releasing DNA fibers) by the total number of cells in 5–8 random microscopic fields and multiplying the values by 100 (30).

Phorbol-myristate-acetate (PMA), lipopolysaccharide (LPS), ionomycin and anti-LC3 antibody were purchased from Sigma-Aldrich. Sytox Green Nucleic Acid Stain, and Dihydroethidium (DHE) were from Life Technologies. The anti-p62 antibody, anti- cleave-caspase3 antibody, phospho- and total mTOR, 70 S6 kinase, 4EBP1, and ERK antibodies were all from Cell Signaling Technology. Horseradish peroxidase-conjugated antibody to rabbit IgG or to mouse IgG was from biodragon-immunotech. The anti-neutrophil elastase antibody and anti-myeloperoxidase antibody were purchased from Abcam. Alexa Fluor 488 goat anti rabbit IgG or mouse IgG and Hoechst 33342 were purchased from Zhongshan Golden Bridge Biotechnology. zVAD-fmk was purchased from Selleck Chemicals. The PRAK inhibitor was synthesized by Wuxi Pharma (China).

NETs were analyzed by immunofluorescence microscopy as previously described (31). Neutrophils were placed on glass coverslips in 24-well-plates in presence or absence of PRAK inhibitor, and stimulated with PMA for 4 h and at 37°C and 5% CO₂. Subsequently, cells were fixed with 4% paraformaldehyde for 2 h and permeabilized with 0.1% Triton X-100 for 5 min at RT. The fixed cells were blocked with blocking buffer (5 mg/ml BSA in PBS) at 37°C for 1 h. Then cells were stained with the primary antibody such as anti-NE, anti-MPO, or anti-LC3 overnight at 4°C. After three washes with PBS, the cells were incubated for another 1 h with secondary goat anti-rabbit IgG or mouse IgG antibody conjugated with Alexa Fluor 488. The DNA was stained with Hoechst 33342 at RT for 5 min. Specimens were captured with confocal microscope.

Neutrophils were placed in a glass-bottomed culture dish for live cell observation and incubated with or without PRAK inhibitor for 1 h in the presence of Hoechst 33342 and Sytox green. Then, the cells were stimulated with PMA at 37°C and 5% CO₂. The fluorescent signals were detected and imaged with a Leica SP5 workstation. Cell morphology was observed using differential interference contrast (DIC). The cells were monitored by autofocus images, and multiple image stacks were captured every 1 min (15, 31).

The E.coli strain BL21 was grown to log phase in Luria Bertani medium at 37°C. Neutrophils were pretreated with or without PRAK inhibitor for 1 h and further stimulated with PMA for 3 h at 37°C and 5% CO₂ to induce formation of NETs. To block bacterial phagocytosis, neutrophils were incubated with phagocytosis inhibitor Cytochalasin D before infection of bacteria. To inhibit NET-mediated bacterial killing, neutrophils were incubated with DNase prior to the addition of bacteria. Then cells were incubated with bacteria for 1 h. Bacterial cultures were grown on Luria Bertani agar plates. After a 24-h bacterial culture, bacterial counts were determined (5, 18).

Intracellular ROS was determined using a fluorescent probe, Dihydroethidium (DHE) assay. Neutrophils were pretreated with or without PRAK inhibitor for 1 h and further stimulated with PMA for 30 min. Then, neutrophils were incubated with DHE for 20 min and washed with PBS, followed by measurement of DHE fluorescence with flow cytometry.

Western blot analysis was performed on cells stimulated with PMA for 30 min with or without PRAK inhibitor pretreatment. Neutrophils were lysed in cell lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH7.5), 1 mM EDTA, 1% Na-deoxycholale, 1% Triton-100, 0.1% SDS and 10 mM “cocktail” of protease inhibitors. Protein was separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. Subsequently, the (PVDF) membranes were probed by incubation with the appropriate primary antibodies at a dilution of 1:500 (or as stated otherwise below) overnight at 4°C, followed by incubation with secondary antibody horseradish peroxidase (HRP)-conjugated antibody to rabbit IgG or to mouse IgG (diluted 1:5,000) for 1 h at room temperature. The protein bands were visualized with a SuperSignal WestPico Kit according to the manufacturer's instructions (Thermo Fisher).

Sepsis was induced by cecal ligation and puncture (CLP) as described previously (32). Briefly, the mice were anesthetized with pelltobarbitalum natricum and shaved an incision in the abdomen. The cecum was exposed and ligated distal to the cecum. The cecum was punctured with an 18-g needle, and a small amount of cecum content was extruded. Then the cecum was replaced into the abdominal cavity, and the incision was closed.

The concentrations of mouse IL-6, and TNF-α in the peritoneal lavage fluid after CLP 6 h were measured by ELISA kits (BioLegend) according to the manufacturer's instructions.

Statistical analyses were performed using SPSS 17.0 and GraphPad Prism 7. Data are presented as the mean ± standard deviation (SD). Two-tailed Student's t-test or paired t-test were used for single comparison, and two-way ANOVA was used for multiple comparisons. The survival rates of CLP model were analyzed with Mantel-Cox log-rank test. In all tests, P < 0.05 were considered statistically significant.

As we know, ROS is the main production of NADPH oxidase, which is fundamental for NET formation (17, 18), hence there is a certain possibility that by manipulating ROS production, PRAK may be involved in NETosis. To verify this hypothesis, fresh isolated neutrophils from healthy human blood were treated with or without PRAK inhibitor for 1 h, and then stimulated with PMA for 4 h at 37°C to induce NET formation. Compared to neutrophils stimulated with PMA alone, neutrophils pretreated with PRAK inhibitor showed attenuated levels of NET formation with far less typical DNA filaments and less diffused nuclei (Figure 1A). Based on the morphology (Figures 1A,B) and cell-free DNA (cf-DNA) concentration (Figure S1A), pharmacologic inhibition of PRAK statistically decreased NET formation by about 70% in vitro induction. And PRAK inhibitor could exert this obstructive effect on NET formation with a dose-dependent manner (Figures S1B,C) but was not due to its toxicity (Figure S1D). To further examine PRAK's effect on the dynamic process of NET formation, PMA-stimulated neutrophils pretreated with or without PRAK inhibitor were monitored via live cell imaging. By using the cell-impermeable DNA dye Sytox green and the cell-permeable DNA dye Hoechst 33342, we could clearly estimate the status of nucleus transition and the membrane integrity during NETosis (15, 31). In the presence of PRAK inhibitor, 240 min after PMA stimulation, which was the end point of our observation, rarely neutrophils showed Sytox positive but contained pyknotic nuclei, demonstrating the membranes of these cells were still integral and probably they were not in the process of NETosis (Figure 1C). In parallel, majority of neutrophils stimulated with PMA alone concurrently were stained with Sytox green and showed intracellular chromatin decondensation, indicating disintegration of cell membrane and formation of NETs (Figure 1C). Later, we got the similar results from Prak knockout mice. Neutrophils from WT mice peritoneal cavity fluid can form clearly web-like structure and elevate MPO expression, while neutrophils from Prak knockout mice can hardly form NETosis with the stimulation of PMA (Figures 1D,E). To make a solid judgement, we further examined the translocation of neutrophil elastase (NE) to the nucleus during NETosis, which was the critical step in NET formation (33, 34). We chose to monitor the localization of NE after PMA-stimulation for 2 h with or without PRAK inhibitor pretreatment. In PMA alone group, NE was predominantly found in the nucleus when neutrophils underwent NETosis as reported (Figure 1F; Figure S1E). In contrast, in PRAK inhibitor pretreatment group, NE was excluded from the nucleus (Figure 1F), with < 30% of the total NE in the nucleus 2 h after stimulation (Figure 1G). Taken together, these results suggested that pharmacologic inhibition of PRAK hurt the emigration of the critical enzyme, membrane split and chromatin decondensation and finally hampered NET formation.

Neutrophils take the advantage of the sticky web-like characteristic of NETs to disarm and kill a variety of microbes in extracellular (5, 35), therefore NETs are featured as another killing tool of neutrophils, apart from phagocytosis (18, 36). To verify whether PRAK regulation of NETosis did influence NET-mediated extracellular bacterial killing, we performed bacterial killing assays as previously described (5, 18). PMA-stimulated neutrophils were incubated with Cytochalasin D, which blocked phagocytosis without affecting NETs and then infected with E.coli. As shown in Figure 2A; Figure S2A, there were large quantities of bacteria left in PRAK inhibitor pretreatment group and hence the percentage of bacterial killing was significantly decreased compared to untreated neutrophils. Moreover, the treatment with various concentrations of PRAK inhibitor resulted in a reduction of NET-mediated extracellular bacterial killing in a dose-dependent manner (Figure 2B; Figure S2B). Since our hypothesis suggested that PRAK dysfunction would specially manipulate NET formation and the other functions of neutrophils such as phagocytosis were supposed not to be affected. To verify it, the effect of PRAK inhibitor on the phagocytic function of neutrophils was then examined. The neutrophils were treated with DNase before infection with E.coli, which dismantled NETosis but did not affect phagocytosis. As expected, there was no significant difference in phagocytosis between the PRAK inhibitor pretreatment or not pretreatment groups of cells (Figures 2C,D). This result suggested that PRAK regulated NET-mediated extracellular microbial killing, but had hardly any effect on phagocytic function of neutrophils.

Series of studies by other groups have demonstrated that NETosis would be interfered if caspase-dependent apoptosis was overactive, and vice versa (15, 37). Therefore, there was a possibility that the effect of PRAK dysfunction on NET formation was on account of its influence on the extent of apoptosis. In fact, the results on caspase-3 expression in PMA plus PRAK inhibitor-pretreated neutrophils revealed a much higher activation of apoptosis (Figure 3A). As we know, mTOR-autophagy pathway was usually in charge of cell apoptosis, we next sought whether the enhanced apoptosis and reduced NETosis by PRAK inhibitor were caused by mTOR signaling or autophagy accumulation. The data showed that PRAK inhibitor-pretreated neutrophils with PMA exhibited clearly higher production of the LC3 fluorescence with a stronger punctate staining pattern than untreated neutrophils (Figures 3B,C). Later, the autophagy formation in response to PMA stimulation was further confirmed by immunoblotting. Although PRAK inhibitor alone would not induce the expression of LC3-II, it would sharply upregulate LC3-II expression upon PMA stimulation (Figure 3D). Similarly, the p62 protein level, as another autophagy activation indicator, was significantly decreased in PRAK inhibitor-pretreated neutrophils compared to that in untreated neutrophils (Figure 3D). As we know, mTOR signal was a master negative regulator of autophagy formation (23, 38), we next examined to verify the enhanced autophagy activation by PRAK inhibitor was determined by the deactivation mTOR. As Figure 3E shown, treatment of neutrophils with PRAK inhibitor resulted in a marked suppression of the phosphorylation of mTOR induced by PMA stimulation (Figure 3E). Additionally, the phosphorylation of 4EBP1 and 70S6K, which are the direct downstream of mTORC1, were decreased in PRAK inhibitor- pretreated neutrophils (Figure 3E). And this regulation on mTOR signal by PRAK inhibitor was specific, since other signaling such as ERK was not varied (Figure S3A) (39). Briefly, these results illustrated that the attenuation of NET formation of neutrophils by PRAK dysfunction may be associated with elevated caspase-dependent apoptosis, owing to over-activity of autophagy formation caused by the enhanced mTOR repression.

As we shown above, PRAK inhibitor would promote apoptosis and suppress NETosis upon PMA stimulation. To further examine whether the altered NET formation of neutrophils by PRAK inhibitor was actually resulted from its facilitation of apoptosis, we used zVAD-fmk, a pan-caspase inhibitor, to forcedly cut down the apoptosis of neutrophils after PMA stimulation. Indeed, as Figure 4A shown, zVAD-fmk could clearly restrain the activity of caspase 3. Furthermore, the addition of zVAD-fmk could partially rescue the decreased MPO expression and enhance the chromatin decondensation in PRAK inhibitor-pretreated neutrophils during PMA-induced NETosis (Figures 4B,C). In consistent, the percentage of nuclear NE was also increased in the zVAD-fmk-treated group (Figures 4D,E). Hence, these data suggested that the impaired NET formation by PRAK inhibitor could be, at least, partially rescued by reduced apoptosis.

Our previous research has shown that PRAK could regulate intracellular ROS generation to reduce oxidative damage (data not published). As shown by flow cytometry analysis, PRAK inhibitor-pretreated neutrophils showed increased production of ROS in response to PMA stimulation compared to the control cells (Figure 5A). Due to the possibility that excessive ROS was the main reason for high caspases expression and apoptosis (40), we tested the apoptosis and NETosis of neutrophils in PRAK inhibitor pretreatment after rectifying their ROS accumulation in a hypoxic environment (5% O₂). Compared to the neutrophils stimulated with PMA under normoxic PO₂, neutrophils showed a mild decreased intracellular ROS content in response to PMA stimulation under hypoxic condition but it can surely reduce the elevated ROS caused by PRAK inhibitor (Figure 5B). From our data, hypoxic environment (5% O₂) could rescue the release of the web-like structures composed of decondensed chromatin and MPO in PRAK inhibitor-pretreated neutrophils during PMA-induced NETosis (Figures 5C,D). Furthermore, as Figures 5E,F shown, reduced ROS production in 5% O₂ could restrain the over-activity of autophagy in PRAK inhibitor-pretreated neutrophils during PMA stimulation and may partially rescue the apoptosis. Consistently, the attenuation of NET-mediated bacterial killing and NE translocation of neutrophils by PRAK dysfunction could be also rescued by treatment with lower ROS in hypoxia (Figure S4). In addition, PRAK inhibitor could also inhibit LPS-stimulated NOX-dependent NETs (Figures S5A,B). Since ROS was firmly related to NADPH oxidase and theoretically ROS level will not interfere with NOX-independent NETs, to examine whether PRAK specifically affects the formation of NOX-dependent NETs, we also examined the effect of PRAK inhibitor on ionomycin-induced NETs. As expected, the suppressive effect of PRAK inhibitor on NOX-independent NETs was not observed (Figures S5C,D). Briefly, these data above suggested that the rectification of ROS and autophagy may rescue the defective neutrophil NETosis brought by PRAK inhibitor.

To investigate whether PRAK would influence the NETosis and killing function of neutrophils during bacterial infection, the mouse CLP-induced sepsis model was used to measure the bacterial cleaning of neutrophils (41). As Figure 6A shown, Mantel-Cox log-rank survival analysis for the WT and Prak knockout mice after puncture in the caecum illustrated that the Prak knockout mice had a lower overall survival capacity (Figure 6A). Prak knockout mice had died very fast and all mice in the KO group died within 4 days, while 25% mice (2 in 8) in the WT group still survived for 7 days (Figure 6A). Moreover, we analyzed NET formation in the neutrophils from septic mice. 6 h after CLP, neutrophils were isolated and purified from the mouse peritoneal lavage fluid and stimulated with PMA for 12 h. Due to the bacterial infection, WT neutrophils were pretty active and produced a huge amount of NETosis upon PMA stimulation with large nuclear areas and plenty of released DNA fibers (Figures 6B,C). In comparison, barely NET formation was observed in neutrophils from Prak knockout mice (Figures 6B,C). Likewise, the serum concentrations of cf-DNA (NETs) were higher in WT mice than that in KO mice (Figure S6A). To further assess the effects of NETs capacity on impeding bacterial spreading during sepsis, the bacterial load was examined in the peritoneal cavity. Compared to WT mice, amount of bacterial load was observed in the peritoneal cavity of knockout mice and could not be cleaned up by these Prak knockout neutrophils (Figure 6D). Notably, PRAK deficiency specifically harmed the NETosis in neutrophils but did not affect their other functions, such as cytokine secretion (Figure 6E). Overall, these results showed that PRAK was essential for neutrophil NET formation in bacterial killing following CLP-induced sepsis.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.