Frontline Science: TNF‐α and GM‐CSF1 priming augments the role of SOS1/2 in driving activation of Ras, PI3K‐γ, and neutrophil proinflammatory responses

Abstract Circulating neutrophils are, by necessity, quiescent and relatively unresponsive to acute stimuli. In regions of inflammation, mediators can prime neutrophils to react to acute stimuli with stronger proinflammatory, pathogen‐killing responses. In neutrophils G protein‐coupled receptor (GPCR)‐driven proinflammatory responses, such as reactive oxygen species (ROS) formation and accumulation of the key intracellular messenger phosphatidylinositol (3,4,5)‐trisphosphate (PIP3), are highly dependent on PI3K‐γ, a Ras‐GTP, and Gβγ coincidence detector. In unprimed cells, the major GPCR‐triggered activator of Ras is the Ras guanine nucleotide exchange factor (GEF), Ras guanine nucleotide releasing protein 4 (RasGRP4). Although priming is known to increase GPCR–PIP3 signaling, the mechanisms underlying this augmentation remain unclear. We used genetically modified mice to address the role of the 2 RasGEFs, RasGRP4 and son of sevenless (SOS)1/2, in neutrophil priming. We found that following GM‐CSF/TNFα priming, RasGRP4 had only a minor role in the enhanced responses. In contrast, SOS1/2 acquired a substantial role in ROS formation, PIP3 accumulation, and ERK activation in primed cells. These results suggest that SOS1/2 signaling plays a key role in determining the responsiveness of neutrophils in regions of inflammation.


INTRODUCTION
Neutrophils constitute around 60% of the circulating white blood cells in humans. They play a critical role in immune defence against invading pathogens and have a major role in inflammation. They respond quickly by migrating to the sites of inflammation and neutralize potentially injurious agents by phagocytosis and by releasing degradative enzymes, neutrophil extracellular traps (NETs) and reactive oxygen species (ROS). These actions are toxic for pathogens but can also damage local tissue and drive many inflammatory diseases such as rheumatoid arthritis or acute respiratory distress syndrome. It is therefore very important to restrict their activation in time and space. 1 It is believed that "priming" is one of the key mechanisms that bring this Abbreviations: G , heterotrimeric G-protein subunit dimer; GEF, guanine nucleotide exchange factor; GPCR, G protein-coupled receptor; NET, neutrophil extracellular trap; PIP 3 , phosphatidylinositol (3,4,5)-trisphosphate; PLC, phospholipase C; RBD, Ras-binding domain; ROS, reactive oxygen species; RTK, receptor tyrosine kinase; SOS, son of sevenless; RasGRP4, Ras guanine nucleotide releasing protein 4; TAN, tumor-associated neutrophil; WT, wild type Class I PI3Ks are responsible for receptor-stimulated production of the phospholipid phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 , also known as PIP 3 ). Class I PI3Ks are subdivided into class IA (PI3K-, PI3K-, and PI3K-) and IB (PI3K-), based on the properties of their regulatory subunits. The class IA PI3Ks are characteristically activated by receptor tyrosine kinase (RTK)-based mechanisms involving binding of the SH2 domains in their regulatory subunits to phosphotyrosine residues within YXXM recognition sequences. 5 PI3K-is abundant in myeloid cells, particularly in neutrophils. It is a heterodimer comprising of a catalytic subunit, p110-, and a regulatory subunit, p84 or p101. 6 Genetic deletion of p110-revealed that it is required for G protein-coupled receptor (GPCR)-stimulated PIP 3 accumulation, PKB activation, ROS formation, and chemokinesis. [7][8][9] PI3K-is primarily regulated by heterotrimeric G-protein subunit dimer (G ) subunits that bind directly to both the catalytic and regulatory subunits and activate the complex. 10 and changed transcription and expression of many genes, particularly those encoding cytokines or chemokines. 20 A defining feature of the priming process is that it leads to a dramatic increase in responses to a subsequent, and usually different, ligand suggesting that the mechanisms are not based on "additivity" or a common mechanism of action in the process. Given the central role of PI3K-and PIP 3 signaling in neutrophil responses, it seems likely that priming agents would target this pathway to modulate neutrophil responsiveness and there is evidence that supports this concept. Some neutrophil priming agents can stimulate PIP 3 accumulation directly (e.g., GM-CSF and GPCR-ligands, such as fMLP, C5a) 21,22 and hence this creates a complex background in which to answer this question. TNF-, however, cannot increase PIP 3 or GTP-Ras levels in isolated neutrophils, but can prime granulocytes.
The mechanism appeared to be dependent on a TNF--elicited, large increase in PIP 3 accumulation after relatively prolonged (1-2 min) stimulation with the 2 0 agonist. Importantly, priming with TNF-did not increase signaling via the closely related PLC 2/ 3 pathway indicating these events were not driven by increased GPCR signaling. 23,24 The mechanism by which priming elicited an increase in sustained, fMLP-stimulated PIP 3 accumulation is unclear; it appears to involve augmentation of class IA PI3K-/ activity, but is also dependent on PI3K-activity, leading to the idea that there is sequential activation of PI3K-and PI3K-/ . 24 Mixtures of TNF-and GM-CSF are known to be amongst the most powerful priming agents for both human and mouse neutrophils, and in this study, we have aimed to understand the mechanism by which a mixture of TNF-and GM-CSF, which does not significantly increase PIP 3 accumulation in isolation, substantially augments PIP 3 accumulation and ROS formation in response to a subsequent dose of fMLP.

Reagents and antibodies
All materials used were of the lowest endotoxin level available and were purchased from Sigma (UK) unless stated otherwise. The antibodies used for Western blots were commercially available: anti-SOS1

Purification of mouse neutrophils
Murine neutrophils were isolated at room temperature from bone marrow of femurs and tibias using Percoll gradients (55% and 62%).

Immunoblots
The protein extracts were separated via SDS-PAGE in 10% polyacrylamide gels and were transferred overnight onto PDVF membranes.
After blocking and incubating with relevant primary and secondary, HRP-labeled, antibodies, the membranes were incubated with ECL reagents (GE Healthcare) and exposed to light-sensitive film. Protein levels were quantified by 2D densitometry using Aida Image Analyzer software v3.27.

ROS production
It was measured by chemiluminescence using a luminol-based assay in

Ras activation assays
To measure the activation of Ras, neutrophils were stimulated while in suspension (4 × 10 6 per condition) then rapidly diluted with cold PBS, sedimented by brief centrifugation (total time ∼10 s), aspirated and solubilized into ice-cold lysis buffer. The lysates were centrifuged (13,500 × g, 10 min, 4 • C) and the supernatants mixed with 4× SDS-PAGE sample buffer. Ras pull-down assays were performed using GST-Raf-RBD as previously described. 16

Statistics
Neutrophils were isolated from at least 2 mice (of the same genotype) and pooled prior to conducting experiments. Depending on the number of comparisons, two-tailed t-tests or ANOVAs followed by Holm-Sidak's multiple comparisons tests were used. When departure from normality was observed, data were log transformed prior to the analyses. Differences were considered significant P-value < 0.05.

PLC 2/ 3, PI3K-, and Ras-activation of PI3Kare required for fMLP-stimulated ROS formation in both unprimed and primed mouse neutrophils
Incubating freshly isolated mouse neutrophils at 37 • C for 1 h in the absence of priming agents leads to a 2-3-fold reduction, compared to freshly prepared cells, in the amount of ROS they produce in response to fMLP (Fig. 1). The reason for this decline in responsiveness is unclear but has been observed previously. 24 Quite surprisingly, there was no parallel reduction in fMLP-stimulated PIP 3 accumulation (see Fig. 2D). When mouse neutrophils were primed for 1 h with GM-CSF and TNF-(2 ng/ml and 500 U/ml), there was about a 10-fold increase, compared to mock-primed cells, in the amount of ROS generated in response to fMLP; however, priming had no effect on ROS production in the absence of fMLP (Fig. 1). Treatment with either TNF-or GM-CSF alone also primed fMLP-stimulated, but not basal, ROS formation, by relatively smaller extents (Fig. 1A).
Mouse neutrophils lacking both PLC 2 and PLC 3 failed to produce trophils isolated from p110 +/+ and p110 -/mice. Data are presented as peak ROS levels with 100% representing the peak response obtained with 10 µM fMLP in p110-+/+ neutrophils primed with 2 ng/ml GM-CSF and 500 U/ml TNF-and are presented as mean ± SEM of 3-8 independent experiments. (D) ROS production from neutrophils isolated from p110-+/+ and p110 DASAA/DASAA mice. Data are presented as peak ROS level with 100% representing the peak response obtained with 10 µM fMLP in p110-+/+ neutrophils primed with 2 ng/ml GM-CSF and 500 U/ml TNF-and are presented as mean ± SEM 3-8 independent experiments performed in duplicate, except for the 500 U/ml TNF-results, which are means ± range of n = 2. Significance of the differences was estimated using unpaired ANNOVA test. *P ≤ 0.050020 vs. WT mice, **P ≤ 0.01 vs. WT mice, ***P ≤ 0.0005 vs. WT mice, ****P ≤ 0.0001 vs. WT mice ROS in response to fMLP, either in the absence (confirming previous results) or presence of priming agents (Fig. 1B). These results are consistent with the idea that PLC 2/ 3-dependent changes in cytosolic free Ca 2+ and DAG/PKC are required for GPCR-stimulated ROS formation 7 and that priming does not change this dependency. Mouse neutrophils lacking p110-produced substantially less ROS in response to fMLP in both unprimed or TNF--and GM-CSF-primed conditions (Fig. 1C), confirming previous work 24 and consistent with the central role of PI3K-in GPCR stimulation of ROS formation. [7][8][9] Mouse neutrophils expressing an endogenous, Ras-insensitive version of p110-(p110 DASAA/DASAA ) had a similar, but weaker, phenotype compared to p110 -/neutrophils (Fig. 1D).
This result demonstrates that a Ras input to PI3K-is needed to allow normal fMLP-stimulated ROS formation in both unprimed 15 and primed neutrophils.

SOS1/2 become the functionally dominant, fMLP-sensitive RasGEFs in TNF--and GM-CSF-primed neutrophils
We fed SOS2 −/− x SOS1 LoxP/LoxP x ERT2-Cre mice a diet containing tamoxifen (see the Material and Methods Section), yielding "SOS1/2-DKO" mice, prepared neutrophils, lysed them, and immunoblotted for SOS1 and 2 (Fig. 3A). This revealed the neutrophils lacked detectable The low recovery neutrophils number has severely restricted the experiments we could conduct; thus, direct assays of Ras activation were impractical. It has been shown previously, however, that in fibroblasts isolated from the same mouse strains, loss of SOS1/2 resulted in an almost complete blockade of EGF or PDGF-stimulated Ras activation. 28 In unprimed cells, fMLP-stimulated ERK activation (Fig. 3A) and ROS formation (Fig. 3C)

CONCLUDING REMARKS
Our results build on work published by other labs identifying roles for a Ras-sensitive-network controlling important neutrophil functions such as ROS formation, NET release, 29 and tissue infiltration (in acute pancreatitis 30 ) and in disease processes, such as autoimmune vasculitis (via antineutrophil cytoplasmic antibodies-ANCA 31 ). Our results provide a molecular framework with which to understand how Ras signaling orchestrates ROS formation in basal and primed neutrophils.
The results presented above confirm that in neutrophils, the primary class I PI3K required for a wide variety of GPCRs to stimulate ROS formation is PI3K-and that Ras-family GTPases play a key role in its activation. 15,24 These conclusions contrast with studies of macrophage-like bone marrow-derived cells that show that PI3Kis the key player in Ras signaling networks constitutively activated by mutations in PTPN11 (Shp2) common in juvenile myelomonocytic leukaemia. 32 Collectively, our results indicate that the level of Ras activation achieved in primed cells via SOS is sufficient to support enhanced class I PI3K activation, ERK activation, and ROS formation, without a requirement for RasGRP4. This may be a result of the ability of GTPbound Ras to stimulate SOS proteins, and hence reinforce activation of Ras through a "feedforward loop" 18