NET formation can occur independently of RIPK3 and MLKL signaling

The importance of neutrophil extracellular traps (NETs) in innate immunity is well established but the molecular mechanisms responsible for their formation are still a matter of scientific dispute. Here, we aim to characterize a possible role of the receptor‐interacting protein kinase 3 (RIPK3) and the mixed lineage kinase domain‐like (MLKL) signaling pathway, which are known to cause necroptosis, in NET formation. Using genetic and pharmacological approaches, we investigated whether this programmed form of necrosis is a prerequisite for NET formation. NETs have been defined as extracellular DNA scaffolds associated with the neutrophil granule protein elastase that are capable of killing bacteria. Neither Ripk3‐deficient mouse neutrophils nor human neutrophils in which MLKL had been pharmacologically inactivated, exhibited abnormalities in NET formation upon physiological activation or exposure to low concentrations of PMA. These data indicate that NET formation occurs independently of both RIPK3 and MLKL signaling.


Introduction
Extracellular DNA traps are a part of the innate immune response and are seen with many infectious, allergic and autoimmune diseases. They can be generated by several different leukocytes, including neutrophils, eosinophils, basophils and monocytes, as well as mast cells [1], whereby the neutrophil extracellular traps (NETs) have been most frequently studied. Extracellular DNA traps can bind and kill bacteria [2] and fungi [3] in the extracellular space, but may also contribute to immunopathology [2] or even cause autoimmunity [4,5]. While most investigators agree that NETs contribute significantly to innate immunity, the molec-ular mechanisms responsible for their formation remain unclear and in dispute. Our focus is a simple question: Does the neutrophil need to die in order to provide the extracellular DNA scaffold characteristic for NETs or not?
The type of cell death postulated as being required for NET formation has been called "NETosis" [12][13][14]. Since it has been suggested that a neutrophil nonapoptotic type of death is required for NET formation in inflammatory processes [2,[12][13][14], it seemed possible that the RIPK3-MLKL signaling pathway might regulate this process. The aim of this study was to investigate whether NETosis is actually necroptosis and whether RIPK3 contributes to NET formation. Using genetically deficient mice and pharmacological approaches, we now report that NET formation is a RIPK3-MLKL-independent process.

Ripk3-deficient neutrophils are able to form functional NETs
To investigate whether NETosis is actually necroptosis, we stimulated Ripk3-deficient GM-CSF-primed mouse neutrophils with C5a or LPS, or alternatively, in the absence of priming, with E. coli or phorbol-12-myristate-13-acetate (PMA), all known triggers of NET formation. Ripk3-deficient neutrophils (Fig. 1A) were able to form NETs just as well as neutrophils from wild-type mice ( Fig. 1B and Supporting Information Fig. 1A) and showed no increased spontaneous cell death (Supporting Information Fig. 1B). However, as expected, Ripk3-deficient neutrophils were less susceptible to undergoing necroptosis as compared to wild-type neutrophils (Supporting Information Fig. 1B). In addition, the stimuli used to trigger NET formation did not induce cell death within a time period of 1 h (Supporting Information Fig. 1C and Movie 1). We established the formation of NETs by demonstrating the presence of extracellular DNA fibers associated with elastase (Fig. 1C).
To quantify the dsDNA released by activated neutrophils, we also collected the culture supernatants and measured the amount of released dsDNA using PicoGreen fluorescent dye [15]. We observed no differences in dsDNA release between Ripk3-deficient and wild-type mouse neutrophils, but, as expected, there were statistically significant differences in dsDNA release between control and activated neutrophils derived from both wild-type and RIPK3deficient mouse neutrophils (Fig. 1D). Moreover, Ripk3-deficient neutrophils were able to generate the same amounts of reactive oxygen species (ROS), measured both by fluorescent and luminescent methods ( Fig. 1E and F), which are known to be required for NET formation [16]. In addition, Ripk3-deficient neutrophils exhibited the same extracellular antibacterial killing activity as wild-type neutrophils (Fig. 1G). Taken together, these data clearly demonstrate that NET formation by mouse neutrophils is independent of RIPK3.

Pharmacological inhibition of MLKL does not prevent functional NET formation by human neutrophils
Since mouse and human neutrophils may differ in some functional responses, we also performed experiments in the human system, using a pharmacological approach. To trigger NET formation, we stimulated human neutrophils in the same way as mouse neutrophils, including E. coli ( Fig. 2A). Again, no effect on cell viability upon NET formation was observed in human neu-trophils upon NET formation (Supporting Information Fig. 2). The association between extracellular DNA fibers and elastase was considered as defining for NET formation ( Fig. 2A, right panels). Quantification of dsDNA release was also verified by PicoGreen fluorescent dye (Fig. 2B). Pharmacological inhibition of MLKL by necrosulfonamide (NSA) had no effect on NET formation ( Fig. 2A and B), ROS production ( Fig. 2C) or extracellular antibacterial killing activity (Fig. 2D). In contrast, NSA completely prevented TNF-α/IAP-antagonist, Smac mimetic AT-406-mediated death in caspase-8-deficient Jurkat cells, indicating that the compound was functionally active and able to block necroptosis (Fig. 2E).
Taken together, our data clearly show that NET formation by mouse and human neutrophils can occur independently of both RIPK3 and MLKL signaling, and hence independently of necroptosis [6][7][8][9]. In fact, this study confirms previous work demonstrating that NET formation does not require cell death to occur at all [16]. The idea of a "suicidal NETosis" [17] and the general concept of cell death as a requirement for NET formation both need to be reconsidered.

Confocal laser scanning microscopy and quantification of NET formation
Human neutrophils were resuspended in X-VIVO TM  Neutrophil elastase co-localization with extracellular DNA was analyzed by indirect immunofluorescence as previously described [20]. Briefly, cells were fixed with 4% paraformaldehyde and permeabilized with 0.05% saponin. Monoclonal mouse antibody to human neutrophil elastase (1:1000) or polyclonal goat anti-mouse neutrophil elastase (1:100) served as primary antibodies. Alexa Fluor R 488 conjugated secondary antibody was used at 1:400. In these experiments, cells were stained with 10 μg/mL PI for extracellular DNA detection.
Slides were examined and images acquired by LSM 700 (Carl Zeiss Micro Imaging, Jena, Germany) using 63×/1.40 Oil DIC objective and followed by analysis with IMARIS software as previously reported [16,20].

Quantification of released dsDNA in culture supernatants
Released dsDNA was quantified as previously described [15]. Briefly, 2 × 10 6 neutrophils in 500 μL of X-VIVO TM 15 medium were stimulated as described above. At the end of the incubation time, a low concentration of DNase I (2.5 U/mL; Worthington) was added for an additional 10 min. Reactions were stopped by addition of 2.5 mM EDTA, pH 8.0. Cells were centrifuged at 200 × g for 5 min. One hundred microliters supernatant were transferred to black, glass-bottom 96-well plates (Greiner Bio-One GmbH) and the fluorescent activity of PicoGreen dye bound to dsDNA was excited at 502 nm and the fluorescence emission intensity was measured at 523 nm using a spectrofluorimeter (SpectraMax M2, www.eji-journal.eu Molecular Devices, Biberach an der Riß, Germany), according to the instructions described in the Quant-iT TM PicoGreen R Assay Kit.

Reactive oxygen species measurements
ROS measurements were performed in stimulated mouse and human neutrophils by two different methods. The fluorescent detection of ROS activity was done by flow cytometry as previously described [16,20]. Briefly, 2 × 10 6 /mL neutrophils were resuspended in X-VIVO TM 15 medium and 100 μL of cell suspension was preincubated in the presence and absence of different inhibitors for 30 min, before priming with 25 ng/mL GM-CSF for 20 min. Cells were then stimulated with 10 −8 M C5a or 100 ng/mL LPS. As a control, we also stimulated neutrophils with 25 nM PMA for 15 min in the absence of GM-CSF priming. Dihydrorhodamine 123 (DHR 123) was added to the cells at the final concentration of 1 μM. The reaction was stopped by adding 200 μL of ice-cold PBS, and ROS activity of the samples was measured immediately by flow cytometer (BD FACSCalibur) and quantified using FlowJo software (Ashland, OR, USA). ROS was also quantified using the ROS-Glo TM H 2 O 2 kit (Promega AG), a luminescent assay that can measure H 2 O 2 levels directly in cell cultures. Briefly, 100 μL of 2.5 × 10 6 /mL neutrophils in X-VIVO TM 15 medium were transferred to white 96-well plates (Greiner Bio-One GmbH) and 25 μM of H 2 O 2 substrate was added to the cell suspension. Cells were activated as described above. ROS-Glo detection solution (D-cysteine, Signal Enhancer solution and Luciferin detection solution, provided by the kit) was then added and incubated for 20 min at room temperature, protected from light. Luminescence activity was detected by GloMax R Explorer machine (Promega AG, Dübendorf, Switzerland).

Bacterial killing assay
The bacterial killing assay was performed as previously described [19]. Briefly, a single colony of GFP-E. coli was cultured in Luria broth base (LB) medium at 37°C, shaking at 220 r.p.m. overnight. On the next day, the bacterial culture was diluted 1:100 in LB medium and grown until a mid-logarithmic growth phase (OD 600 = 0.7). Following centrifugation at 1000 × g for 5 min, bacterial pellets were washed twice with 1× Hank's Balanced Salt Solution (HBSS; LuBioScience GmbH) and centrifuged at 100 × g for 5 min to remove any clumped bacteria. Bacteria were opsonized with 10% mouse sera (heat-inactivated) in 1 × HBSS, rotating end-over-end (6 r.p.m.) for 20 min at 37°C and used immediately. Activated neutrophils (1 × 10 7 /mL in RPMI 1640 plus 2% FCS) were mixed with an equal volume of opsonized bacteria (5 × 10 7 /mL) in the presence and absence of 100 U/mL DNase I. The co-cultures of cells and bacteria were rotated endover-end (6 r.p.m.) for 30 min at 37°C. At the end of the incubation period, an equal volume of ice-cold 1 × HBSS was added to each tube to stop the reaction, and cells were pelleted by gentle centrifugation (5 min, 100 × g, 4°C) using a swing-out rotor.
Supernatants containing bacteria were collected, diluted 1:300, plated on agar and grown overnight before counting the colonies. The tubes containing bacteria alone were treated the same way and used as controls.
Functional inhibition of MLKL was controlled by measuring viability in TNF-α treated caspase-8-deficient Jurkat cells (kind gift of Dr. T. Brunner, University of Konstanz, Germany). Briefly, caspase-8-deficient Jurkat cells (1 × 10 6 /mL) were cultured in complete culture medium with 20 ng/mL recombinant human TNF-α, in the presence or absence of 100 nM Smac mimetic AT-406, 20 μM z-VAD, and 5 μM NSA. Cell viability was determined after 24 and 48 h by an ethidium bromide exclusion test as described above.

Statistical analysis
Mean values (± SEM) are provided. To compare groups, one-way ANOVA was applied. P values < 0.05 were considered statistically significant.