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Biochemical and Biophysical Research Communications 472 (2016) 545e550

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier .com/locate/ybbrc

The oxindole Syk inhibitor OXSI-2 blocks nigericin-inducedinflammasome signaling and pyroptosis independent of potassiumefflux

Jordan R. Yaron a, b, Mounica Y. Rao a, Sandhya Gangaraju a, Liqiang Zhang a,Xiangxing Kong a, Fengyu Su a, Yanqing Tian a, Honor L. Glenn a, Deirdre R. Meldrum a, *

a Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, Tempe, AZ, 85287, USAb Biological Design Graduate Program, School of Biological and Health Systems Engineering, Ira A. Fulton Schools of Engineering, Arizona State University,Tempe, AZ, 85287, USA

a r t i c l e i n f o

Article history:Received 11 February 2016Accepted 6 March 2016Available online 9 March 2016

Keywords:InflammasomePyroptosisInflammationPotassiumTyrosine kinase

Abbreviations: NLRP3, NLR family, pyrin domainleukin-1 beta; ROS, reactive oxygen species; mROS, mspecies; LPS, lipopolysaccharide.* Corresponding author. Center for Biosignatures

Biodesign Institute, Arizona State University, 1001877101, Tempe, AZ, 85287-7101, USA.

E-mail address: Deirdre.Meldrum@asu.edu (D.R. M

http://dx.doi.org/10.1016/j.bbrc.2016.03.0210006-291X/© 2016 The Authors. Published by Elsevier

a b s t r a c t

The inflammasome is a caspase-1-activating complex that is implicated in a growing number of acuteand chronic pathologies. Interest has increased in identifying small molecular inhibitors of inflamma-some signaling because of its role in clinically relevant diseases. It was recently reported that the proteintyrosine kinase, Syk, regulates pathogen-induced inflammasome signaling by phosphorylating a mo-lecular switch on the adapter protein ASC. However, several aspects of the role of Syk in inflammasomesignaling and the effects of its inhibition remain unclear. The aim of the present study is to explore indetail the effects of the oxindole Syk inhibitor OXSI-2 on various aspects of nigericin-induced inflam-masome signaling. Our results indicate that OXSI-2 inhibits inflammasome assembly, caspase-1 activa-tion, IL-1b processing and release, mitochondrial ROS generation, and pyroptotic cell death. Using a novellive cell potassium sensor we show that Syk inhibition with OXSI-2 has no effect on potassium effluxkinetics and that blockade of potassium efflux with extracellular potassium alters Syk phosphorylation.The effects of OXSI-2 identified in this study provide context for the role of Syk in inflammasomesignaling and demonstrate its importance in oxidative signaling upstream of inflammasome activationand downstream of ion flux.© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

The innate immune system rapidly responds to a variety ofendogenous and exogenous danger signaling by assembling amulti-protein complex called the inflammasome [1]. The outcomeof inflammasome assembly is activation of the enzyme caspase-1,leading to the processing and release of interleukin-1b (IL-1b)and other pro-inflammatory cytokines, as well as a pro-inflammatory cell death called pyroptosis [2,3].

Recent evidence highlights the importance of post-translational

-containing 3; IL-1b, inter-itochondrial reactive oxygen

Discovery Automation, TheS. McAllister Ave., P.O. Box

eldrum).

Inc. This is an open access article u

signaling in licensing the inflammasome for assembly [4]. Thisrapid licensing is in contrast to canonical models for inflammasomeactivation that depend on a TLR4-dependent priming period fol-lowed by a rapid stimulation period [5,6]. Rapid licensing is bio-logically rational as post-translational signaling occurs morequickly than de novo transcription and translation of effector pro-teins, and enables a more rapid innate immune response to dangersignals. A role for post-translational modifications in regulation ofthe inflammasome is further supported by the discovery of atyrosine phosphorylation site on the inflammasome adapter pro-tein Apoptosis-associated Speck-like protein containing a Caspaserecruitment domain (ASC) that acts as a molecular switch con-trolling inflammasome assembly [7,8]. Phosphorylation of ASC ismediated in large part by spleen tyrosine kinase (Syk), a proteintyrosine kinase that is essential for inflammasome-mediated re-sponses to fungi, mycobacteria and malarial hemozoin [9e11].However, beyond the interaction between Syk and ASC, there islittle understanding of the context of Syk in the inflammasome

nder the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

J.R. Yaron et al. / Biochemical and Biophysical Research Communications 472 (2016) 545e550546

signaling pathway [12,13].Interest has grown in identifying pharmacological inhibitors of

the inflammasome due to the clinical relevance of inflammation[14]. Here, we focus on the Syk inhibitor OXSI-2 [2,3-dihydro-3-[(1-methyl-1H-indol-3-yl)methylene]-2-oxo-1H-indole-5-sulfonamide] because the adenine-mimetic oxindole backbone ishighly flexible for chemical modification and has been useful fordeveloping other small molecule inhibitors, including bloodebrainbarrier-penetrating inhibitors [15e18]. OXSI-2 was discovered in ascreen for bioavailable, cell-permeable Syk inhibitors and wasidentified as the most effective (EC50 ¼ 313 nM; IC50 ¼ 14 nM) incell-based assays among those identified [18]. Indeed, OXSI-2 isalso marketed under the explicit name “Syk Inhibitor” and wasincluded in the initial screen identifying a role of Syk in ASCphosphorylation [7].

Our results indicate that OXSI-2 blocks inflammasome assemblyin response to the bacterial pore-forming toxin nigericin and,accordingly, caspase-1 activation, IL-1b processing and release andpyroptotic cell death. Further, OXSI-2 treatment potently sup-presses mitochondrial reactive oxygen species (mROS) generation.Utilizing a novel live cell potassium sensor we also show that theeffects of OXSI-2 treatment are downstream of nigericin-inducedpotassium efflux and that Syk phosphorylation is affected by po-tassium blockade. These results provide support for an early role forSyk in the inflammasome signaling pathway, downstream of ionflux and upstream of oxidative signaling leading to inflammasomeassembly, pro-inflammatory signaling and pyroptosis.

2. Materials and methods

2.1. Reagents

LPS (from Escherichia coli O111:B4) was purchased from SigmaAldrich (St. Louis, MO, USA). Nigericin was purchased from Inviv-ogen (San Diego, CA, USA) and Cayman (Ann Arbor, MI, USA). OXSI-2 was purchased from Cayman. KS6 intracellular potassium sensorwas developed in-house (Center for Biosignatures DiscoveryAutomation, The Biodesign Institute, Arizona State University,Tempe, AZ, USA) [19]. For complete list of reagents see Supple-mental materials.

2.2. Cell culture

The mouse monocyte/macrophage cell line J774A.1 (ATCC TIB-67™, Manassas, VA, USA) was grown in DMEM supplemented with10% FBS, 100 m/mL Penicillin G and 100 mg/mL Streptomycin Sulfate(all from Gibco, Grand Island, NY, USA). Tissue culture flasks werepassaged every 3e4 days by scraping and cells were counted fordensity and viability with a Countess® Automated Cell Counter (LifeTechnologies) using the Trypan Blue dye exclusion assay.

2.3. Live cell potassium and mROS imaging

For imaging, cells were seeded in an 8-chamber m-Slide (Ibidi,Verona, WI, USA) at a density of 1e2 � 105 per well in 200 mL ofcomplete DMEM. The following day cells were primed for 4 h with1 mg/mL LPS. Inhibitors were added for the last 15 min of priming.Cells were stimulated with 20 mM nigericin after an initial baselinewas taken. Cells were imaged on a Nikon Ti microscope equippedwith a C2si confocal scanner (Nikon Instruments, Melville, NY, USA)and a Tokai Hit stage-top incubator (Tokai Hit Co., Shizuoka, Japan).

Potassium imaging was performed as previously described[19,20]. Briefly, KS6 was diluted 1:1 with 10% w/v Pluronic F127 andadded to priming cells at 1:100 dilution. Final concentration of KS6applied to cells was 5 mM. KS6 was excited at 561 nm and emission

was collected in the TRITC channel.For mROS imaging, MitoSOXwas added 15min after stimulation

at a final concentration of 5 mM, concurrently with 10 mg/mLHoechst 33342 (Life Technologies) and incubated for an additional15 min prior to imaging.

2.4. Caspase-1 FLICA assay

J774A.1 were seeded and primed as described for live cell im-aging. During the last hour of priming cells were loaded with1 � FAM-YVAD-FMK (Caspase-1 FLICA) and 10 mg/mL Hoechst33342 in complete medium. Additional inhibitors were addedduring the last 15e20 min of priming. Cells were stimulated with20 mMnigericin for 30 min, washed 2x with warm DMEM and fixedin 2% formaldehyde for 10 min. Cells were washed 1x with PBS andsubmerged in 200 mL mounting medium (90% glycerol with 10XPBS and 0.1% NaN3). Samples were imaged by laser-scanningconfocal microscopy as a series of 0.5 mm z-stacks. Stacks wereprepared as maximum intensity projections using ImageJ/FIJI(v1.49f, http://www.fiji.sc).

2.5. Lactate dehydrogenase release assay

Cells were seeded in a 96-well tissue culture-treated plate at aconcentration of 105 cells/well. The following day the mediumwasexchanged for 100 mL of either freshmedium ormedium containing1 mg/mL LPS and incubation was continued for 4 h. Inhibitors wereadded during the third hour of incubation. For stimulation, thecomplete medium of each well was exchanged for 100 mL of eitherfresh medium, medium containing 1% Triton X-100 as a maximumrelease control, or the indicated drugs and/or inhibitors andreturned to the incubator for 1 h. Fifty mL of supernatant wassampled for each well. Released lactate dehydrogenase wasmeasured using the CytoTox 96® Non-Radioactive CytotoxicityAssay according to manufacturer's instructions. The developedassay was measured for absorbance at 492 nm on a Biotek SynergyH4 multi-mode plate reader.

2.6. Syk immunoprecipitation

Briefly, supernatants were collected from stimulated cell cul-tures, then combined with cell lysates. The samples were centri-fuged and protein content of the supernatant was quantified byBCA assay. For immunoprecipitation, 1% BSA was added to eachprotein sample and 40 mL of magnetic beads containing Syk captureantibody were added. Samples were rotated overnight at 4 �C. Thenthe beads were washed 3x with cold RIPA by pull-down using amagnetic bead stand and protein was collected by heating thebeads in denaturing Laemmli at 95 �C for 10 min. See Supplementalmaterial for detailed protocol.

2.7. Immunoblotting

For non-immunoprecipitated protein collection, after stimula-tion, supernatants were collected from cells and concentrated with10 mL/mL StrataClean Resin. Cell lysates were collected by directlyadding 100 mL 1 � hot denaturing Laemmli buffer to each well.

Proteins were run on 4e12% Mini-PROTEAN TGX gel thentransferred to 0.2 mm pore nitrocellulose membranes. Membraneswere probed independently with rabbit polyclonal against caspase-1 p10, or goat polyclonal against IL-1b. For immunoprecipitationexperiments, membranes were multiplexed with mouse polyclonalagainst p-Tyr to detect phospho-Syk in Syk-enriched IP eluates andrabbit polyclonal against Syk to detect total Syk. Membranes wereincubated with appropriate secondary antibodies then imaged

J.R. Yaron et al. / Biochemical and Biophysical Research Communications 472 (2016) 545e550 547

using a Li-Cor Odyssey CLx infrared scanner. See Supplementalmaterials for detailed protocol.

2.8. ELISA

Cells were seeded in 96-well plates at a concentration of105 cells/well and incubated overnight. Cells were primed for 4 hwith 1 mg/mL LPS and subsequently stimulated for 60 min with20 mM nigericin in 100 mL medium. Where indicated, inhibitorswere added 15e20 min prior to nigericin stimulation. Supernatantswere collected (100 mL per condition) and released IL-1b wasevaluated with ELISA using the R&D Systems DuoSet kit accordingto the manufacturer's protocol. Developed plates were read on aBiotek Synergy H4 mutli-mode plate reader.

2.9. Statistical analysis

Data were analyzed in GraphPad Prism version 6.05 (GraphPad,La Jolla, CA, USA) using one-way ANOVAwith a Tukey's post-hoc orFischer's LSD comparison. Results were considered significant ifp < 0.05.

3. Results

3.1. OXSI-2 blocks nigericin-induced inflammasome assembly

We first explored the effect of Syk inhibition with OXSI-2 onnigericin-induced NLRP3 inflammasome assembly [21]. Activatedcaspase-1 can be fluorescently tagged at the explicit site of acti-vation (i.e., in the inflammasome itself) by pre-exposure toCaspase-1 FLICA, a FAM-conjugated irreversible inhibitor forcaspase-1 containing the YVAD caspase-1 recognition sequence[22]. Confocal imaging showed that LPS-primed, nigericin-treated

Fig. 1. Syk activity is required for nigericin-induced inflammasome assembly. J774A.1 mand stimulated with 20 mM nigericin for 30 min. Where indicated, cells were treated with 13perinuclear caspase-1 specks classical for inflammasome assembly. Bar graph indicates meaone-way ANOVA with Tukey's post-hoc comparison. Blue fluorescence is Hoechst 33342 an

J774A.1 assemble the inflammasome as indicated by single, peri-nuclear specks of FLICA-labeled caspase-1 (Fig. 1). As expected,treatment with 130 mM extracelluar KCl to inhibit Kþ efflux, in-hibits the assembly of the inflammasome. Importantly, OXSI-2 re-sults in significant suppression of caspase-1 specks.

3.2. OXSI-2 suppresses pro-inflammatory protein processing, mROSgeneration and pyroptotic cell death

Immunoblot analysis reveals a robust suppression of caspase-1and IL-1b processing and release by both Syk inhibition andextracellular potassium blockade as indicated by a reduction incaspase-1 p10 and IL-1b p17 fragments in concentrated superna-tants of stimulated J774A.1 cells (Fig. 2a). In agreement with theimmunoblot results, quantitative detection of processed andreleased IL-1b by ELISA shows robust inhibition upon treatmentwith extracellular KCl and OXSI-2 (Fig. 2b).

Mitochondrial destabilization and oxidative signaling have beenimplicated in triggering inflammasome assembly [20,23e25].Mitochondrial ROS generation was visualized by live cell imagingwith the reactive oxygen probe MitoSOX (Fig. 2c). MitoSOX oxida-tion was suppressed by treatment with extracellular KCl or OXSI-2demonstrating that this process is dependent on both potassiumefflux and Syk activity.

Inflammasome assembly can result in pro-inflammatory celldeath by caspase-1-dependent pyroptosis [3]. Pyroptosis wasmeasured by release of lactate dehydrogenase into the medium.Pyroptosis in J774A.1 requires both LPS priming and nigericinstimulation and, importantly, was blocked by both extracellular KCland treatment with OXSI-2 (Fig. 2d).

ouse macrophage cells were left untreated, primed for 4 h with 1 mg/mL LPS or primed0 mM KCl or 2 mM OXSI-2 for 15e20 min prior to addition of nigericin. Arrows indicaten and standard error of at least 3 fields from 2 independent experiments evaluated byd green fluorescence is caspase-1 FLICA. Scale bar represents 25 mm.

Fig. 2. Syk and potassium efflux are critical for inflammasome signaling and pyroptosis. (a) Immunoblot analysis of caspase-1 and IL-1b in the cell lysates and concentratedsupernatants. Where indicated, cells were treated with 1 mg/mL LPS, inhibited with 130 mM KCl or 2 mM OXSI-2 and stimulated with nigericin. Caspase-1 p45 and p10 and proIL-1band bioactive IL-1b are indicated by arrows (b) Quantitative ELISA of IL-1b in the supernatants of cells treated as in (a). Bars represent mean and standard error. Statistics werecalculated by one-way ANOVA with Tukey's post-hoc comparison. Results represent at least two independent experiments. (c) J774A.1 cells were untreated, primed with LPS, orprimed and then treated with 20 mM nigericin. Where indicated, cells were treated with 130 mM KCl or 2 mM OXSI-2 for 15e20 min prior to nigericin addition. Cells were stainedwith 5 mM MitoSOX and then imaged by confocal microscopy. Results are representative of two independent experiments. Scale bar represents 25 mm. (d) J774A.1 cells wereuntreated, primed with LPS, or primed and then treated with 20 mM nigericin. Where indicated, cells were treated with 130 mM KCl or 2 mM OXSI-2. Released LDH was measured incell-free supernatants. Bars represent mean and standard error of two independent experiments evaluated by one-way ANOVA with Tukey's post-hoc comparison.

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3.3. Syk activity is dispensable for, and downstream of, nigericin-induced potassium efflux

Because both extracellular potassium blockade and OXSI-2treatment inhibit inflammasome assembly, we sought to deter-mine whether OXSI-2 treatment would modulate nigericin-induced potassium efflux. We recently developed a novel intra-cellular potassium sensor, KS6, for real-time imaging of potassiumdynamics in live cells and have applied it to inflammasomesignaling [19,20]. KS6 responses revealed no difference in the ki-netics of potassium efflux induced by nigericin treatment in LPS-primed cells with or without OXSI-2 treatment (Fig. 3a).

Quantitative, multiplexed immunoblots of immunoprecipitatedSyk, probed for total Syk and phospho-tyrosine residues, indicatedthat blockade of potassium efflux with extracellular KCl results in asignificant alteration of phospho-Syk levels in LPS-primed, niger-icin-stimulated cells (Fig. 3b). In agreement with other reports, aslow loss of phospho-Syk signal after the initial stimulus wasobserved [7]. However, addition of extracellular KCl resulted in arapid and dramatic dephosphorylation of Syk following stimula-tion. Control experiments were performed on unprimed cells todetermine whether simply the addition of KCl could drive this Sykdephosphorylation (Fig. 3c). There was no observable differencebetween treatment with nigericin or KCl alone, or the combinationof nigericin and KCl. These data suggest a requirement for TLR4-dependent priming with LPS in order to produce conditions

wherein Syk phosphorylation is sensitive to potassium efflux.Taken together, these results indicate that Syk phosphorylation isdownstream of potassium efflux in inflammasome signaling.

4. Discussion

How inflammasome assembly is initiated by structurally andfunctionally diverse stimuli is not well understood and presents achallenge for intervening with inflammasome signaling [1,14,26].Specific upstream proteins are attractive for pharmacologic inhi-bition and a number of new targets including Syk have beenrecently identified [7,8,27e31].

Here we investigated the functional effects of pharmacologicSyk inhibition on canonical, nigericin-induced inflammasomesignaling. We report that OXSI-2 treatment potently inhibitsinflammasome complex assembly, caspase-1 activation and IL-1bprocessing and release in the J774A.1 mouse macrophage cell line.

To further elucidate the mechanism by which Syk regulates theinflammasome, we explored effects of OXSI-2 treatment on thegeneration of mitochondrial ROS, since oxidative signaling has beenimplicated in triggering NLRP3 inflammasome assembly andpyroptosis [32,33]. Use of the mROS probe MitoSOX revealed thatactivated cells displayed substantial mROS production. Thisresponse was inhibited by both KCl blockade and OXSI-2 treatment,placing potassium and Syk activity upstream of mitochondrialdysfunction. These results contrast with the work of Hara et al. who

Fig. 3. Syk activity is dispensable for, and downstream of, nigericin-induced potassium efflux. (a) LPS-primed J774A.1 macrophages were loaded with KS6 potassium sensor andstimulated with 20 mM nigericin before continuous imaging. Where indicated, cells were treated with 2 mM OXSI-2 for 15e20 min prior to nigericin treatment. Red box indicatesselected region expanded in kymograph panels. Traces indicated mean and standard deviation of KS6 signal change for 5 cells in a representative field from each condition. Scale barrepresents 25 mm. Results are representative of at least two independent experiments. (b) J774A.1 mouse macrophages were left untreated or primed for 4 h with 1 mg/mL LPSbefore treatment with 20 mMnigericin for the indicated time. 130 mM KCl was added to the mediumwhere indicated. Immunoprecipitation was performed on combined lysates andsupernatants. Multiplexed infrared immunoblots were performed with total Syk and phospho-tyrosine (pY) antibodies. The ratios of pY signal to total Syk were calculated andnormalized to untreated controls (bar graph). (c) J774A.1 cells were left untreated or directly treated with 20 mM nigericin, 130 mM KCl or a combination of both for 15 min andprocessed as described in (b). Bar graphs indicate mean of two independent experiments and error bars indicate standard deviations. P-values were calculated using one-wayANOVA with a Fischer's LSD multiple comparison test. IP control indicates total Syk-conjugated Protein A Dynabeads left unexposed to collected protein.

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reported that inhibition of Syk kinase did not suppress nigericin-induced MitoSOX oxidation [7]. This is likely the result of meth-odological differences. In the Hara et al. study, macrophages wereincubated in nigericin and MitoSOX simultaneously for 20 min. LPSpriming induces TLR4-dependent mROS generation and thusMitoSOX fluorescence will increase as soon as it is added to cells[34]. In the current study, MitoSOX was added after a period ofnigericin treatment resulting in a lower background signal from theLPS-induced mROS generation alone. We recommend thisapproach in order to avoid limitations of the MitoSOX sensor.

A terminal outcome of inflammasome assembly is a caspase-1-dependent, pro-inflammatory cell death called pyroptosis [2,3].Despite recent interest in the role of Syk in inflammasomesignaling, it remained unclear whether Syk was involved insignaling events resulting in pyroptotic cell death [13]. Here, wedemonstrate that treatment with OXSI-2 blocks pyroptosis and

prevents the release of lactate dehydrogenase into the supernatant,a conventional read-out of pyroptosis [35].

Potassium ion flux is a driving mechanism in inflammasomesignaling [20,36,37]. Here, we show that Syk inhibitionwith OXSI-2blocks the outcomes of nigericin-induced inflammasome assembly,but does not inhibit potassium efflux as measured in real-time by alive cell probe [19,20]. We explored this further by blocking po-tassium efflux with extracellular potassium. We find that blockingpotassium efflux results in rapid dephosphorylation of Syk in par-allel with inhibition of downstream inflammasome signaling. Ourresults suggest that Syk is downstream of, and dispensable for,potassium efflux. We hypothesize that potassium efflux may pro-mote Syk activation. Further experiments using site mutagenesis ofSyk phosphorylation sites may be helpful in revealing the details ofthis relationship.

The finding that OXSI-2 inhibits inflammasome assembly, pro-

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inflammatory cytokine secretion and pyroptotic cell death ispromising for modulating innate immune system-driven inflam-matory processes. Indeed, there are numerous Syk inhibitorscurrently in clinical and pre-clinical trials for treating inflammatoryand autoimmune pathologies [38e41]. The novel finding that po-tassium blockade affects Syk phosphorylation may point to a newavenue for modulating Syk-dependent inflammatory pathologiesby targeting channels and processes that regulate ion homeostasis.

Acknowledgments

Support for this work was provided by the Microscale LifeSciences Center, an NIH NHGRI Center of Excellence in GenomicScience (5P50 HG002360 to D.R.M.), and the NIH Common FundLINCS (Library of Integrated Network-Based Cellular Signatures)program (U01CA164250 to D.R.M.).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.bbrc.2016.03.021.

Conflict of interest

The authors state no conflict of interest.

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