Serum and Glucocorticoid-Regulated Kinase 1 Regulates Neutrophil Clearance During Inﬂammation Resolution

Published January 15, 2014, doi:10.4049/jimmunol.1300087 The Journal of Immunology

Serum and Glucocorticoid-Regulated Kinase 1 Regulates Neutrophil Clearance during Inﬂammation Resolution

Joseph Burgon,* Anne L. Robertson,† Pranvera Sadiku,† Xingang Wang,‡ Edward Hooper-Greenhill,x Lynne R. Prince,† Paul Walker,{ Emily E. Hoggett,* Jonathan R. Ward,* Stuart N. Farrow,‖ William J. Zuercher,# Philip Jeffrey,x Caroline O. Savage,x Philip W. Ingham,‡ Adam F. Hurlstone,{ Moira K. B. Whyte,*,† and Stephen A. Renshaw*,†

The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid–regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease. The Journal of Immunology, 2014, 192: 000–000.

he inflammatory response is triggered by tissue injury or and are an essential component of the cellular inflammatory re- infection and is essential to protect the body from invading sponse. Their main role is the removal of microbes through phago- T pathogens and to return damaged tissues to homeostasis. cytosis and intracellular killing (2). In addition, neutrophils may Neutrophils are the first cells recruited to sites of inflammation (1) release intracellular contents either as neutrophil extracellular traps (3) or by degranulation of their potentially harmful granule

by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. contents into the inflammatory environment (4). Thus, tight regu- *Medical Research Council Centre for Developmental and Biomedical Genetics, lation of neutrophil function is essential to the maintenance of University of Sheffield, Sheffield S10 2TN, United Kingdom; †Department of Infec- tion and Immunity, University of Sheffield, Sheffield S10 2RX, United Kingdom; health. In a perfectly balanced inflammatory response, there is ‡Institute of Molecular and Cellular Biology, Proteos, Singapore 138673, Singapore; removal of the initiating stimulus combined with the avoidance xImmuno-Inflammation Therapy Area Unit, GlaxoSmithKline Research and Devel- { of tissue damage by successful inflammation resolution. A number opment, Stevenage SG1 2NY, United Kingdom; Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom; ‖Respiratory Therapy Area, of prerequisites must be met for the resolution of inflammation; of GlaxoSmithKline Research and Development, Stevenage SG1 2NY, United Kingdom; which, the safe removal of leukocytes from the inflammatory site and #Department of Chemical Biology, GlaxoSmithKline, Research Triangle Park, NC 27709-3398 is of critical importance (5). An important mechanism of clearance is the apoptosis of neutrophils and their removal by macro- Received for publication January 9, 2013. Accepted for publication December 5, http://classic.jimmunol.org 2013. phage phagocytosis in situ (6). At sites of inflammation, many This work was supported by Medical Research Council Senior Clinical Fellowship signals are produced that suppress neutrophil apoptosis leading Grant G0701932 (to S.A.R.) and Medical Research Council Centre Grant G0700091. to an extended lifespan. Host-derived signals such as ATP (7) or Microscopy studies were supported by Wellcome Trust Grant GR077544AIA to the GM-CSF (8) and pathogen-associated molecular patterns (9) com- Molecular Biology and Biotechnology/Biomedical Science Light Microscopy Facility. bine with physical properties of the inflammatory environment, such M.K.B.W., S.A.R., and J.R.W. conceived the study and designed the experiments with J.B.; S.N.F. and W.J.Z. provided advice on pharmacological inhibition of SGK1 as hypoxia (10), to suppress neutrophil apoptosis, leading to delay

Downloaded from including supply of GSK1558634A and GSK398689A; P.J. and C.O.S. provided or failure of inflammation resolution. This in turn results in tissue advice on pharmacological assessment of SGK1 inhibition; P.W. and A.F.H. provided damage, which is a further stimulus for neutrophil recruitment and the Sgk1 morpholino and the SGK1 gene comparison and background advice; J.B., M.K.B.W., and S.A.R. wrote the manuscript; and J.B., A.L.R., P.S., E.H.-G., L.R.P., survival via damage-associated molecular patterns and underlies and E.E.H. performed the experiments. X.W. and P.W.I. generated the PH-AKT-line many chronic inflammatory diseases (11, 12). Understanding how and provided advice on its use after background advice. these survival signals act at a molecular level within the neutrophil Address correspondence and reprint requests to Dr. Stephen A. Renshaw, Medical Research should identify targets for novel drug therapies for inflammatory Council Centre for Developmental and Biomedical Genetics, University of Sheffield, Western Bank, Sheffield S10 2TN, U.K. E-mail address: s.a.renshaw@sheffield.ac.uk disease. The online version of this article contains supplemental material. GM-CSF is an important host-derived signal influencing neu- Abbreviations used in this article: AUR, aurora kinase; DMOG, dimethyloxaloylgly- trophil lifespan and, upon binding to its receptor, initiates well- cine; dpf, days postfertilization; EGFP, enhanced GFP; hpi, h postinjury; NDRG, characterized intracellular signaling events. The heterodimeric N-myc downstream regulated; Mcl-1, myeloid cell leukemia-1; PDK, phosphoinositide- GM-CSF receptor complex activates at least two distinct signaling dependent kinase; SGK, serum and glucocorticoid–regulated kinase. pathways: activation of class IA PI3Ks (13) and JAK/STAT sig- Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 naling (14). PI3K activation leads to generation of the secondary

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300087 2 SGK1 IN NEUTROPHILIC INFLAMMATION

messenger phosphatidylinositol 3,4,5-triphosphate and downstream according to the manufacturer’s instructions. RT-PCR used Superscript III phosphorylation of AKT (15, 16), which in turn phosphorylates a RT Enzyme (Invitrogen), and PCR used Finnzymes Phusion (New England wide range of cellular substrates with diverse cellular effects (17). Biolabs, Hertfordshire, U.K.) and was carried out in a C1000 Thermal Cycler. Primers used are shown in Supplemental Table I. AKT is seen as a central player in the transduction of cell surface signals to the core apoptotic machinery, and AKT inhibitors have Zebrafish husbandry and assays been shown to delay neutrophil apoptosis (18). However, fMLP Adult fish were maintained on a 14:10-h light/dark cycle at 28˚C in the U.K. receptors also signal via PI3K activation and activate AKT but Home Office–approved facilities. Experiments were performed using the without affecting neutrophil apoptosis (19). Therefore, there is a Tg(mpx:GFP)i114 line, which specifically labels neutrophils with GFP dissociation of apoptosis signaling and AKT activation, suggesting (28), or the Tg(lyz:PHAkt-EGFP) i277, which detects PI3K activation in neutrophils by recruitment of the enhanced GFP (EGFP)-tagged pleckstrin roles for other signaling molecules in addition to PI3K activation homology domain of AKT to phosphatidylinositol 3,4,5-triphosphate in in apoptosis regulation. the membrane (29). Larvae were collected and maintained according to Activation of the JAK/STAT pathway downstream of GM-CSF standard protocols (30). Assays of neutrophil recruitment to tailfin injury stimulation is thought to act predominantly via transcriptional ef- were performed as described previously (28). For analysis of total neu- fects. Traditionally, neutrophils were thought not to be subject to trophil number, larvae were mounted in 1% agarose and imaged using the 32 objective on a Nikon Eclipse TE2000U Inverted Microscope; transcriptional regulation, but there is evidence of considerable neutrophils were counted blind, using ImageJ and Volocity (PerkinElmer, regulation of neutrophil mRNA repertoire by inflammatory stimuli Cambridge, U.K.). Neutrophil apoptosis rates were assessed in PFA-fixed (including GM-CSF) (20, 21), with potential further regulation by larvae, followed by Tyramide Signal Amplification (TSA) staining (TSA- microRNAs (22). In one study, mRNA changes in neutrophils Plus; PerkinElmer) and ApopTag Red In Situ Apoptosis Detection Kit (TUNEL) (Millipore, Herts, U.K.) as described previously (31). were detected by microarray following GM-CSF stimulation (21). One of the most upregulated mRNAs was that of the known Agilent microarray expression analysis antiapoptotic protein serum and glucocorticoid–regulated kinase 1 Six adult fish (from an mpx:GFP background) were macerated using a scalpel (SGK1), which, with GM-CSF stimulation, was upregulated 13.2- and passed through a series of micrometer meshes to further dissociate the fold. This protein had previously been shown to be regulated by cells. Cells were sorted by FACS analysis into those cells showing appro- GM-CSF and also upon treatment with other neutrophil survival priate side scatter (of light) and forward light scatter to be neutrophil-like agents such as LPS and TNF-a (23). SGK1 shares 54% amino acid and further sorted into GFP-positive and -negative sets. GFP-negative cells were used as a control. RNA was extracted from sorted cells using the homology with AKT (24) and is also regulated by phosphorylation mirVana kit as per manufacturer’s instructions (Ambion, Huntingdon, U.K.). downstream of PI3K (25). We hypothesized that SGK1 might be Microarray analysis was performed using a Zebrafish (V3) Gene Expression an important link between extracellular antiapoptotic stimuli and Microarray containing 43663 probes (Agilent Technologies, Amstelveen, the downstream apoptosis machinery in neutrophils. The Netherlands) as described previously (32). Mass spectrometric analysis of drug penetration of larvae Materials and Methods Reagents Three dpf zebrafish samples (20 fish per time point) were sonicated (Soniprep 150; MSE, Sussex, U.K.) as described previously (33). Ho- Reagents used were qVD-OPh (R&D Systems, Abingdon, U.K.), GM-CSF mogenized zebrafish samples were extracted by protein precipitation with (PeproTech, London, U.K.), dimethyloxaloylglycine (DMOG), LPS, ATPgs, the addition of 750 ml acetonitrile containing a structural analog internal

by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. and the DMSO control (all from Sigma-Aldrich, Poole, U.K.). Three SGK standard. Samples were mixed and centrifuged at 2200 3 g for 20 min. inhibitors were used: GSK650394 (Tocris Bioscience, Bristol, U.K.), Liquid chromatography-tandem mass spectrometry analysis was performed GSK1558634A, and GSK398689A (GlaxoSmithKline, Stevenage, U.K.). using an API 4000 triple quadrupole mass spectrometer (AB Sciex) with an The sgk1 morpholino and standard control morpholino were from Gene- electrospray ionization source operating in positive ion mode. The UHPLC tools (Philomath, OR). system incorporated a Jasco XLC dual pump system with a gradient elution method using acetonitrile +0.1% formic acid (v/v) and water +0.1% formic Purification of peripheral blood neutrophils acid (v/v) mobile phase and a Phenomenex Kinetex analytical column 3 Peripheral venous blood was taken from healthy volunteers in accordance (C18, 2.6 mm, 50 2.1 mm). Samples were analyzed against calibration with the specific approval of the South Sheffield Research Ethics Committee standards prepared in a control zebrafish homogenate matrix over a con- (reference number STH13927), and neutrophils were prepared as described centration range of 1–5000 ng/ml, with an assay LLQ of 1 ng/ml. We previously (26). Negatively selected neutrophil preparations were .98% estimated the volume of a zebrafish larva to be 0.0375 ml. http://classic.jimmunol.org pure. Apoptotic neutrophils were counted by an observer blind to the ex- Statistical analyses perimental condition, from cytospins stained with Quick-Diff (Gentaur, Brussels, Belgium). Data were analyzed (Prism 5.0; GraphPad Software) using either unpaired, two-tailed Student t tests for comparisons between two groups or one-way Protein detection by Western blot analysis ANOVA with Bonferroni posttest adjustment for other data, except where Samples were prepared from ∼5 3 106 neutrophils as previously described indicated in the figure legend. (26) or from 30 pooled 5-d postfertilization (dpf) zebrafish embryos, and Downloaded from Western blotting was performed according to standard protocols (27) using Results a Protean 2 gel assembly (Bio-Rad, Hemel Hempstead, U.K.) and transferred to Hybond-P polyvinylidene difluoride membrane (GE Healthcare Life Sci- Multiple SGK isoforms are expressed in neutrophils and ences, Bucks, U.K.) using a Trans-Blot semidry blotter (Bio-Rad). Abs used regulated by GM-CSF were anti-SGK (number 3272; Cell Signaling Technology, Hertfordshire, U.K.) Kobayashietal.(21)showedGM-CSF stimulation increased at 1:1000, anti–myeloid cell leukemia-1 (Mcl-1) (sc-819; Santa Cruz Bio- technology, Santa Cruz, CA) at 1:1000, anti–phospho-N-myc downstream levels of SGK1 mRNA and prevented time-dependent decreases in regulated (NDRG)-1 (number 3217; Cell Signaling Technology) at 1:1000, SGK1 protein levels. There are, however, three SGK genes present anti-total NDRG-1 (number 5196; Cell Signaling Technology) at 1:1000, or in humans: SGK1, SGK2, and SGK3, each sharing 80% homology anti-actin (A2066; Sigma-Aldrich) at 1:2000 and polyclonal goat anti-rabbit at their catalytic domain (34). In addition, there are four alterna- secondary Ab (DakoCytomation, Cambridge, U.K.) at 1:2000 (1:5000 for anti- actin). Densitometry was performed using ImageJ as described previously (26). tively spliced isoforms of SGK1 and two for both SGK2 and 3 (Supplemental Fig. 1A). We determined the expression profile of PCR analysis of SGK mRNA isoforms and morpholino these different genes and isoforms in ultrapure human neutrophils knockdown (35). Using specific primer pairs in RT-PCRs, we identified full- RNA was extracted from ∼7 3 106 human neutrophils (.96% purity) or length isoforms of SGK1, SGK2, and SGK3 mRNA in human 10–15 zebrafish larvae, using TRIzol reagent (Invitrogen, Paisley, U.K.), neutrophils (Fig. 1A). SGK1 mRNA was detected at higher levels The Journal of Immunology 3

FIGURE 1. Multiple SGK transcripts are present in human neutrophils and are regulated by GM-CSF. Human neutrophils were cultured for the times indicated 6 GM-CSF and lysed for protein or mRNA analysis. (A) RT-PCR analysis of mRNA levels show expression and regulation of multiple SGK genes in human neutrophils. (B) Multiple isoforms of SGK1 are present and regulated in human neutrophils. (C) Western blotting reveals the presence of SGK1 and its regulation by GM-CSF. The example shown is representative of five independent experiments. (D) Densitometry and quantification of Western blots confirm statistically significant regulation of SGK1 expression (t test, *p , 0.05, n = 5). (E) GM-CSF causes a significant reduction in neutrophil apoptosis at 4, 8, and 20 h in the samples used in (D) (**p , 0.01, ***p , 0.005, n = 5).

following GM-CSF stimulation at early time points, whereas of apoptosis. This suggests that levels of SGK1 do not fall as a SGK2 expression was unaltered with GM-CSF stimulation. SGK3 consequence of engagement of neutrophil apoptotic pathways mRNA levels, in contrast to previous reports (34), appeared to be (Supplemental Fig. 2).

by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. increased initially but declined over time. Because GM-CSF regulation was largely confined to SGK1, we examined expression of the SGK1 inhibition blocks GM-CSF–induced human neutrophil different isoforms of this gene. We aligned the sequences of the four survival SGK1 isoforms and identified the important domains and regulatory SGK1 is upregulated at both the mRNA and protein level with regions of the gene (Supplemental Fig. 1B). We found that all GM-CSF treatment. We hypothesized that this upregulation was isoforms varied only at their amino termini, where amino acids functionally important and that the activity of SGK1 contributed to encoded by the first exons varied between isoforms both in length GM-CSF–induced neutrophil survival. To test this hypothesis, we and sequence, with isoform 2 having additional exons. Functional studied the effects on neutrophil apoptosis of the only commer- domains within the protein were maintained between isoforms cially available inhibitor of SGK enzymes, GSK650394. GM- http://classic.jimmunol.org excepting the first 60 aa; these have been identified as playing CSF–induced neutrophil survival was clearly seen at 8 h of cul- a role in the ubiquitination and degradation of SGK1 (36). PCR ture, but with addition of 10 mM GSK650394, this effect was totally analysis shows mRNAs for all SGK1 isoforms are regulated by abrogated (Fig. 2A). GSK650394 also increased constitutive neutro- GM-CSF (Fig. 1B) at early time points, although not at 20 h. phil apoptosis, suggesting that the compound was directly influencing To confirm changes in mRNA expression were paralleled by neutrophil lifespan. To confirm the caspase dependence of the changes at the protein level, we analyzed the change in SGK1 morphological changes of apoptosis observed in these experi-

Downloaded from protein levels over time and following GM-CSF stimulation. We ments, the pan-caspase inhibitor qVD-OPh was shown to prevent found, in agreement with Kobayashi et al. that GM-CSF prevented all cell death (Fig. 2B). the reduction in SGK1 levels over time (Fig. 1C, 1D) in parallel To investigate whether the inhibition of neutrophil survival with increases in neutrophil survival (Fig. 1E). Although the Ab signaling by GSK650394 was specific to GM-CSF survival, we used detects all members of the SGK protein family (SGK1, investigated its effect on a number of other neutrophil survival SGK2, and SGK3), no bands corresponding to full-length SGK2 stimuli including activation of hypoxia-inducible factor signaling or SGK3 were identified. A band was routinely identified corre- (mimicking hypoxia, induced in this study by the chemical DMOG) sponding to full-length SGK1 (isoform 1), which has a predicted (31), LPS (37), and ATPgs (7). All survival factors reduced the size of 49 kDa. To investigate whether the different levels of amount of apoptosis compared with the control, although to a SGK1 detected were due to its decrease over time or merely due to lesser degree than GM-CSF (Fig. 2C). GSK650394 treatment the different numbers of viable neutrophils at the time points abrogated the survival induced by GM-CSF and hypoxia but did analyzed, we used the caspase inhibitor, Q-VD-OPh, to prevent all not affect that induced by LPS and ATPgs (Fig. 2C), indicating neutrophil death (26). Following Q-VD-OPh treatment, SGK1 that SGK1 is important to some, but not all, survival pathways. levels with or without GM-CSF stimulation were unchanged from To further probe the mechanism by which SGK1 inhibition DMSO-treated control levels, despite the almost complete absence might lead to neutrophil apoptosis, we tested whether SGK1 inhibi- 4 SGK1 IN NEUTROPHILIC INFLAMMATION

system, and importantly for these experiments, the zebrafish sgk1 gene is highly similar to the human gene in both amino acid sequence and conservation of key functional domains (Supplemental Fig. 1C). By microarray analysis of FACS-sorted zebrafish neutrophils, sgk1 is present in zebrafish neutrophils at a level 7.84 times higher than in nonneutrophil cells, underlining its importance in this cell type. To investigate whether the chemical inhibitor of human SGK1, GSK650394, could penetrate zebrafish at this larval stage, we incubated 3 dpf larvae with GSK650394 at 10 mM for an hour. We then subjected the larvae to mass spectrometric analysis as described in Materials and Methods. We measured 0.089 nmol compound per larva, or 237 mM, confirming considerable concentration of compound within each larva. To further test whether the compound could successfully inhibit zebrafish Sgk1, we studied its phosphorylation activity in vivo. NDRG1 is known to be phosphorylated solely by SGK1 (39), and thus, its phosphorylation can be used as a read out for SGK1 activity. In larvae treated with GSK650394, there was a dose-dependent reduction in phosphorylated Ndrg1 without any effect on the total amount of Ndrg1 present (Fig. 3A). This indicates that GSK650394 successfully inhibits Sgk1 activity in our assays. Inflammation is induced in 3 dpf zebrafish larvae by transection of the caudal fin (Fig. 3B), and the cellular component of the inflammatory response is quantified by counting the number of neutrophils present at the site of injury. In untreated larvae, recruitment of neutrophils to the site of injury occurs within the first 6 h postinjury (hpi), followed by spontaneous resolution within 24 h. In this model, GSK650394 added during peak inflammation caused specific reduction in neutrophil numbers (Fig. 3C, 3D), without altering numbers of neutrophils elsewhere in the animal (Fig. 3E). To see whether the effects on inflammation resolution were mediated by induction of neutrophil apoptosis in vivo, we dual stained for endogenous neutrophil peroxidase activity and for

by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. dsDNA breaks as a marker for apoptosis using TUNEL. These FIGURE 2. Chemical inhibition of SGK1 abrogates neutrophil survival experiments were performed by adding the compound at peak through an increase in apoptosis. Human neutrophils were treated as indicated for 8 h, and percentage apoptosis was scored by cytospin analysis. neutrophil number (6 hpi) and measuring apoptosis at 12 hpi (A) GSK650394 causes an increase in apoptosis rates, and the 10 mM during peak inflammation resolution. Neutrophil numbers were concentration significantly inhibits GM-CSF induced neutrophil survival reduced significantly in these experiments compared with control (t test, *p , 0.05, n = 5). (B) Caspase inhibition prevents all neutrophil (Fig. 3F). Rates of apoptosis were comparable to previous studies death (t test, *p , 0.05, n = 4). (C) Neutrophil survival factors lower (31) and were significantly higher for the GSK650394 group (Fig. apoptosis rates at 8 h, and GSK650394 treatment abrogates survival caused 3G). Interestingly, these effects were specific to inflammatory by GM-CSF and DMOG but not that by LPS and ATPgs(*p , 0.05, n =3). neutrophils, because rates of neutrophil apoptosis were not in- http://classic.jimmunol.org creased with GSK650394 treatment of uninjured larvae (data not tion might cause decreases in the levels of Mcl-1, a key neutro- shown). phil survival protein. By Western blotting, we found that Mcl-1 GSK650394 inhibits the recruitment of neutrophils to the site of levels fell with GSK650394 treatment, suggesting a possible mech- injury through disruption of the direction and speed of anism for enhanced apoptosis (Supplemental Fig. 3). neutrophil chemotaxis Sgk1 inhibition enhances the resolution of inflammation Downloaded from To test whether the SGK1 inhibitor had any effect on other aspects in vivo, in part through an increase in neutrophil apoptosis of neutrophil function in vivo, we assessed the effects of GSK650394 Acceleration of apoptosis in vitro may not always correspond to on recruitment assays in the injured transgenic zebrafish model. acceleration of inflammation resolution in vivo, particularly if Using Volocity software, we tracked neutrophils as they moved antiapoptotic factors predominate at the inflammatory site or large toward the site of tailfin injury (Fig. 4A) and saw a marked re- numbers of apoptotic neutrophils overwhelm clearance mecha- duction in the movement of neutrophils treated with GSK650394. nisms. Therefore, we wished to study the effect of Sgk1 inhibition Further analysis revealed that this was due to a reduction in in a simple in vivo model of neutrophilic inflammation. Our preferred neutrophil speed (Fig. 4B), whereas the meandering index (dis- model for visualizing individual inflammatory cell behaviors in a placement divided by path length) of neutrophils was unaffected genetically and pharmacologically manipulable organism is the (Fig. 4C). We noted that many neutrophils remained in one area, transgenic zebrafish model established in our laboratory (28). extending and retracting lamellipodia without making any direc- This model allows direct visualization of vertebrate neutrophils tional movements. This was reflected in a reduction in the dis- during inflammation in vivo and has proven invaluable in the placement of neutrophils toward the wound in GSK650394-treated identification of unique features of neutrophil behavior (31, 38). larvae (Fig. 4D). In addition, the bearing at which neutrophils By 3 dpf, zebrafish larvae have a fully functioning innate immune moved is significantly different after GSK650394 treatment: The Journal of Immunology 5 by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd.

FIGURE 3. Inhibition of Sgk1 enhances inflammation resolution in a model of tissue injury. (A) Protein extracts (30 mg) from 30 pooled 5-dpf zebrafish embryos exposed for 4 h to GSK650394 at the indicated concentrations were immunoblotted for p-NDRG1 and total NDRG1 showing inhibition of Sgk1 activity by GSK650394. (B–D) Three-dpf mpx:GFP zebrafish larvae were subjected to tailfin transection at the site shown in (B) and treated as indicated at 4 hpi; neutrophil number was counted at 8 hpi. (C) Representative micrographs of the larvae used for (D). (D) GSK650394 significantly reduced the number of neutrophils at the site of injury in a dose-dependent manner (*p , 0.05, **p , 0.01, n = 18 performed as three independent experiments). (E) Total neutrophil counts show no difference between GSK650394 treatment and the control. (F and G) Three-dpf mpx:GFP larvae were injured as above, treated at http://classic.jimmunol.org 6 hpi, and at 12 hpi, neutrophil number was counted or larvae were fixed and subsequently stained with neutrophil markers (TSA) and apoptosis markers (TUNEL) to allow calculation of percentage apoptosis. (F) GSK650394 causes a highly significant reduction in neutrophil number at the site of injury (***p , 0.0001, n = 18 performed as three independent experiments). (G) GSK650394 treatment causes a significant increase in neutrophil apoptosis (**p , 0.005, n = 135 performed as three independent experiments).

Downloaded from control neutrophils preferentially migrate toward the wound but was confirmed by RT-PCR analysis on RNA extracted from mor- GSK650394-treated larvae do not (Fig. 4E). phant larvae, with maintenance of the intron seen as a 385-nt shift in the sgk1 band detected (Fig. 5A). Genetic inhibition of sgk1 has a different anti-inflammatory To isolate and identify the specific stage of the inflammatory phenotype to pharmacological inhibition of Sgk1 response at which Sgk1 inhibition was having its effect, we ana- Pharmacological modulators of neutrophil function are prone to lyzed neutrophil number at the site of injury over the course of an confounding results by off-target effects, and genetic confirmation entire inflammatory response by performing neutrophil counts at of the phenotype is essential to confirm potential drug targets. 2, 4, 6, 8, 12, and 24 hpi. In GSK650394-treated larvae, there was a Efficient knockdown of gene function in zebrafish can be achieved clear inhibition of neutrophil recruitment (Fig. 5B). by injection of morpholino-modified antisense constructs into the To investigate whether reduction in neutrophil recruitment was fertilized egg (40). This can lead to altered expression of the target due to specific inhibition of Sgk1 we repeated this time course using gene for many days. We therefore designed a “morpholino” to alter sgk1 morpholino injected larvae. We found that genetic knockdown the pre-mRNA splicing of the sgk1 gene by targeting the splice site of of sgk1 caused an increase in the resolution of the inflammatory intron 5 and causing intron inclusion, introducing a premature stop response (Fig. 5C), without affecting recruitment or total neutrophil codon and leading to protein truncation. Effective gene knockdown number (Fig. 5D). Additional confirmation of the importance of 6 SGK1 IN NEUTROPHILIC INFLAMMATION

FIGURE 4. GSK650394 inhibits neutrophil recruitment through disrupted chemotaxis. Three-dpf mpx:GFP larvae were pretreated for 2 h and injured, and a time-lapse image sequence was taken for 1 h. Neutrophil chemotaxis was analyzed using Volocity software. (A) Representative images of treated larvae with neutrophil tracks superimposed. (B) Neutrophil speed is signiﬁcantly reduced with GSK650394 treatment (**p , 0.005, n = 14 performed as three independent experiments). (C) Neutrophil meandering index is unaffected. (D) GSK650394 treated neutrophils show a lack of movement toward the site of injury, as represented by positive values (**p , 0.005, n = 14 performed as three independent experiments). (E) Analysis of the bearing at which neutrophils move demonstrates that directional movement of neutrophils toward the wound is removed with GSK650394 treatment (**p , 0.005, ***p , 0.001, n = 14 performed as three independent experiments).

Sgk1 levels on resolution of inﬂammation was obtained by sep- in response to pharmacological treatments (29). This assay gives a arating larvae into groups showing the highest and lowest rates numerical value of the cell polarization as indicated by PHAkt- of inﬂammation resolution. The group having fewer neutrophils had EGFP distribution within neutrophils. Using this system, we were

by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. better knockdown of sgk1 (Fig. 5E), confirming its importance in able to show that both PI3K inhibitors and GSK650394 reduced regulating inflammation resolution. the polarity index (Fig. 6D, 6E) whereas genetic manipulation of Sgk1 did not (Fig. 6F). GSK650394 inhibits neutrophil recruitment by off-target This phenotype demonstrates the complexity of understanding effects, in part through the inhibition of PI3K off-target pharmacological inhibition, with only part of the effect of Detailed analysis of neutrophil recruitment behavior in sgk1 mor- pharmacological Sgk inhibition likely to act via the PI3K pathway phants revealed no difference in the morphant neutrophils’ speed while underscoring the importance of genetic confirmation for all of movement (Fig. 5F) or their ability to follow the chemotactic inhibitor studies. gradient (Fig. 5G–I), suggesting GSK650394 might be acting Highly specific SGK1 inhibitors block GM-CSF induced http://classic.jimmunol.org on a target other than Sgk1 to block chemotaxis. The profound effect on neutrophil recruitment and differences in the shape of neutrophil survival and also lower neutrophil number at an GSK650394-treated neutrophils (data not shown) led us to hy- in vivo site of injury pothesize that these effects might relate to off-target inhibition of We have shown that sgk1 genetic inhibition removes neutrophils the PI3K pathway. This was possible as GSK650394 was known from a site of inflammation; however, for this to be useful in to inhibit .30 enzymes, other than SGK1, with a pIC50 of .6 a clinical situation, SGK1 inhibition would have to be achieved

Downloaded from (data not shown). To further investigate this hypothesis, the effect using a chemical inhibitor. We have seen that GSK650394 has off- of PI3K inhibition on neutrophil recruitment was studied in the target effects and would not fulfill this role; we therefore used two zebrafish model. PI3K inhibition reduces the number of neutrophils potent SGK1 inhibitors from a chemical template distinct from recruited to a site of injury in a similar manner to GSK650394 (Fig. that of GSK650394: GSK1558634A and GSK398689A. These 6A). A more detailed investigation of neutrophil chemotaxis shows compounds have a pIC50 of 8.2 and 8.8, respectively, with limited that this reduction in recruitment is due to an inhibition of neutro- off-target effects. GSK1558634A inhibits only two other enzymes phil speed (Fig. 6B), similar to that seen with SGKgk inhibition. with a pIC50 . 6; these are aurora kinase (AUR)A, AURB; However, in contrast to findings with GSK650394-treated larvae, GSK398689A additionally inhibits YAK3 (GSK internal data). the bearing at which neutrophils moved was unaffected (Fig. 6C). Treatment of human neutrophils with either of these new inhibitors Furthermore, using a PHAkt-EGFP line (29) to indicate the sub- removes the survival response to GM-CSF in a dose-dependent cellular localization of phosophoinositides produced by PI3K manner, with total abrogation by 100 mM (Fig. 7A–D). Impor- activity, we investigated whether there were changes in PI3K tantly, these compounds had no effect upon constitutive apoptosis activity in response to GSK650394 or Sgk1 knockdown. To quantify of human neutrophils. PI3K activity in individual cells in vivo, we used an assay we had Furthermore, zebrafish larvae treated with either GSK1558634A previously developed to quantify PI3K activity in neutrophils or GSK398689A after transection of the caudal fin show a signif- The Journal of Immunology 7 by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. FIGURE 5. sgk1 inhibition enhances inflammation resolution without affecting neutrophil recruitment. Three-dpf mpx:GFP larvae were injured and treated as indicated, and neutrophil counts were performed at the time points shown. (A, C–E) One to 2 nl sgk1 morpholino was injected to larvae at the one cell stage. Knockdown disrupts the splicing of sgk1 premRNA causing intron 5 to be retained; this can be detected as a 385-bp band shift by PCR. (A)RT- PCR showing sgk1 knockdown from 1 to 3 dpf with addition of the sgk1 morpholino. (B) GSK650394 causes a highly significant inhibition of neutrophil recruitment to the site of injury (**p , 0.01, ***p , 0.005, n = 18 performed as three independent experiments). (C) The sgk1 morpholino increases resolution of the inflammatory response with a significant lowering of neutrophil number from 8 hpi (*p , 0.05, **p , 0.01, ***p , 0.005, n =34 performed as three independent experiments). (D) Total neutrophil counts show no difference between the sgk1 morpholino and the control. (E) RT-PCR analysis of larvae separated after the morpholino time course into those with high and low neutrophil number at the site of injury show that larvae with fewer neutrophils show more complete morpholino knockdown. (F–I) mpx:GFP larvae were injected with 1–2 nl 0.25 mM sgk1 morpholino at the one cell http://classic.jimmunol.org stage and injured at 3 dpf, and a time-lapse video was taken for 1 h. Neutrophil chemotaxis was analyzed using Volocity. Neutrophils from the sgk1 morphants did not show any significant difference from the controls in their speed (F), meandering (G), or displacement toward the wound (H)(n =11 performed as three independent experiments). (I) There is no difference in the bearing at which neutrophils move between the control and sgk1 morpholino– injected larvae (*p , 0.05, **p , 0.005, n = 11 performed as three independent experiments).

Downloaded from icant and dose dependent reduction in the number of neutrophils at new transcription and regulation of protein and mRNA stability, the site of injury (Fig. 7E, 7F). The ability of two additional, more but we have shown by highly effective caspase inhibition that the specific, inhibitors of SGK1 to replicate both the inhibition of changes in SGK1 protein level are independent of levels of apo- GM-CSF–induced neutrophil survival and the rapid resolution of ptosis. Use of the only commercially available SGK inhibitor, the inflammatory response in vivo suggests that this occurs through GSK650394, together with newer more selective compounds and a inhibition of the SGK1 protein, not through off-target actions, morpholino against sgk1, has shown it to play an important role in adding support for a key role for SGK1 in the regulation of neu- maintaining neutrophils at a site of injury. Sgk inhibition leads to trophil lifespan during inflammation resolution. rapid resolution of the inflammatory response without affecting neutrophil recruitment. SGK1 is therefore an important compo- Discussion nent of the pathways that link certain survival stimuli with the Previous work using human neutrophils has shown increased neutrophil apoptotic machinery. In other cell types, SGK1 is known amounts of SGK1 mRNA and protein following GM-CSF stim- to act downstream of PI3K and is activated by phosphoinositide- ulation (21); we have now produced genetic and pharmacological dependent kinase (PDK)1 through phosphorylation of Thr256 and by data showing the importance of SGK1 in controlling neutrophil PDK2 via Ser422 (25, 41). We do not yet know completely how, lifespan in vitro and in vivo. We have not distinguished between once activated, SGK1 plays its antiapoptotic role; in other cell 8 SGK1 IN NEUTROPHILIC INFLAMMATION

FIGURE 6. PI3K inhibition attenuates neutrophil recruitment through a reduction in speed. Three-dpf mpx:GFP larvae were pretreated for 2 h and injured, and a time-lapse image sequence was taken for 1 h. Neutrophil chemotaxis was analyzed using Volocity. (A) Both GSK650394 and LY294002 reduce the number of neutrophils recruited to a site of injury (***p , 0.0001, n = 12 performed as two independent experiments). (B) Neutrophil speed is by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. significantly reduced with LY294002 treatment (***p , 0.0001, n = 15 performed as two independent experiments). (C) The bearing at which neutrophils migrate is not affected by PI3K inhibition (***p , 0.0001, **p , 0.05, n = 15 performed as two independent experiments). (D–F) Three-dpf PHAkt-EGFP larvae were pretreated for 2 h as indicated and subsequently injured. Neutrophils were imaged, and their polarity index calculated. (D) Representative images of treated neutrophils and fluorescence intensity per pixel graphs. (E) GSK650394 and LY294002 significantly reduce the polarity index of neutrophils (***p , 0.005, n = 22 performed on three separate occasions). (F) Injection of the sgk1 morpholino has no effect upon the polarity index of neutrophils (n = 26 performed on three separate occasions).

types, it regulates this process through inactivation of the FOXO3a given the different effects of treatment with the more specific in- http://classic.jimmunol.org transcription factor (42) and upregulation of NF-kBsignaling hibitors, is that the recruitment phenotype is an off-target effect of (43), both of which are important in neutrophils (44, 45). Inter- GSK650394. Over 30 enzymes are known to be inhibited by estingly, SGK1 phosphorylates and inactivates glycogen synthase GSK650394 with a pIC50 . 6, including activin receptor-like kinase kinase-3 (25), which is known to phosphorylate the key neutrophil 5, AURB, calcium/calmodulin-dependent protein kinase kinase 1 antiapoptotic protein Mcl-1 leading to its destabilization and deg- and2,cyclindependentkinase-2, IL-2–inducible T cell kinase, radation (46), a key upstream event in neutrophil apoptosis (26). We JNK1, JNK3, and the class III PI3K, and vacuolar protein sorting Downloaded from now show that inhibition of Mcl-1 turnover in human neutrophils 34, and it is likely that additional kinases that have not been is a likely final mechanism for the antiapoptotic action of SGK1. included in these kinase profiling studies are also inhibited by Chemical inhibition of Sgk enzymes with GSK650394 accel- GSK650394 (S. Farrow and W. Zuercher, unpublished data). erates inflammation resolution and reduces neutrophil recruitment. Thus, there is considerable scope for significant off-target effects Genetic ablation of sgk1 alone has no effect on neutrophil recruit- at the dose used. To overcome this limitation of the pharmaco- ment. Studies of PI3K inhibition in vivo suggest that GSK650394, logical approach, we used new inhibitors, GSK1558634A and but not the genetic strategy, may act upstream of PI3K to modulate GSK398689A. These compounds have a pIC50 of 8.2 and 8.8, neutrophil recruitment, suggesting that inhibition of neutrophil re- respectively, inhibiting only three and four other enzymes with cruitment is an off-target effect. It remains possible that the lack of a pIC50 . 6. The fact that both compounds show the same pro- effect on neutrophil recruitment with sgk1 morpholino could be due resolution phenotype as GSK650394 strongly supports the role of to incomplete knockdown of sgk1, as shown by the maintenance of SGK1 in the regulation of neutrophil responses to survival signals. a wild-type sgk1 band in our PCRs (Fig. 5A); the small amounts of The effects on chemotaxis may relate to inefficient AKT2 recruit- wild-type sgk1 present may be sufficient for neutrophil recruitment following changes in PI3Kg activation, because knockout of ment, but not their maintenance, at a site of injury. More probable, either gene causes frustrated chemotaxis (47, 48). Interestingly, the The Journal of Immunology 9

FIGURE 7. Highly speciﬁc SGK1 inhibitors removes GM-CSF induced neutrophil survival. (A–D) Human neutrophils were treated as incubated for A C

by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. 8 h, and percentage apoptosis was scored by cytospin analysis. ( and ) GSK1558634A causes a dose-dependent inhibition of GM-CSF–induced neutrophil survival (**p , 0.01, n =4).(B and D) GSK398689A causes an inhibition of GM-CSF–induced neutrophil survival (*p , 0.05, n =5). (E and F) Three-dpf mpx:GFP zebrafish larvae were subjected to tailfin transection and treated as indicated at 6 hpi; neutrophil number was counted at 12 hpi. Both GSK1558634A (E) and GSK398689A (F) significantly reduce the number of neutrophils present at the site of injury in a dose-dependent manner (*p , 0.05, **p , 0.01, ***p , 0.005, n = 4).

morphology of PI3Kg knockout neutrophils is very similar to that inﬂuencing apoptosis, for example, by fMLP (19). Thus, SGK1 seen in our time-lapse videos of GSK650394-treated neutrophils in might in some circumstances act to transduce apoptotic signals

http://classic.jimmunol.org the extending and retracting of lamellopodia. downstream of PI3K, in parallel to AKT. The use of the zebrafish model of inflammation has allowed us to The upregulation of SGK1 by survival signals (21, 23) links to dissect the cellular consequences of SGK inhibition in ways that a clinically important increase in SGK1 expression seen in a would not be possible in mammalian systems. The ability to number of inflammatory or fibrotic diseases during which the dissociate effects of SGK inhibition on recruitment and neutrophil inflammatory response is involved, including Crohn’s disease (50) removal allows the pathway to be understood in detail. Although and glomerulonephritis (51). Prolonged neutrophil lifespan has murine models might allow the identification of reduced neutrophil been implicated in the extensive host tissue damage seen in these Downloaded from numbers, dissociating therapeutic differences in recruitment and conditions, further underlining the therapeutic potential of SGK1 clearance is more challenging but critically important. There is an inhibition. Many current anti-inflammatory treatments block important therapeutic difference between removing an unwanted neutrophil recruitment to the site of injury but fail to remove neu- neutrophil and preventing recruitment of all neutrophils to sites of trophils already there. We propose that SGK1 represents a possible potential infection. target for anti-inflammatory therapeutics specifically targeting the SGK1, like AKT, is activated downstream of PI3K by PDK1/2 resolution phase of inflammation while still allowing neutrophils at similar residues (15, 25, 41), and most protein targets of to mount a response against infection, without the extended sur- SGK1 are also phosphorylated by other protein kinases such as vival and degranulation associated with disease. AKT (49). However, it has previously been noted that some stress stimuli, which activated SGK1, did not result in AKT phosphor- Disclosures ylation, showing separate rather than redundant roles for these two C.S., E.H.-G., W.Z., and S.F. are employees of GlaxoSmithKline, which has protein kinases (50). Although AKT activation is thought to delay financial interest in inhibitors of SGK1 and other targets involved in inflam- neutrophil apoptosis (18), AKT can also be activated without mation resolution. 10 SGK1 IN NEUTROPHILIC INFLAMMATION

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