udp-induced phagocytosis and atp-stimulated chemotactic … · 2019-09-04 · researcharticle...

Research ArticleUDP-Induced Phagocytosis and ATP-StimulatedChemotactic Migration Are Impaired in STIM1minusminus MicrogliaIn Vitro and In Vivo

Hye Min Lim1 Heo Woon1 Jung Woo Han1 Yoshihiro Baba23 Tomohiro Kurosaki23

Min Goo Lee1 and Joo Young Kim1

1Department of Pharmacology and Brain Korea 21 PLUS Project for Medical Science Yonsei University College of MedicineSeoul 120-752 Republic of Korea2Laboratory for Lymphocyte Differentiation WPI Immunology Frontier Research Center (IFReC) Osaka University SuitaOsaka 565-0871 Japan3Laboratory for Lymphocyte Differentiation RIKEN Research Center for Allergy and Immunology 1-7-22 Suehiro-choTsurumi-ku Yokohama Kanagawa 230-0045 Japan

Correspondence should be addressed to Joo Young Kim jooyoungkimyuhsac

Received 30 September 2016 Revised 22 November 2016 Accepted 6 December 2016 Published 15 February 2017

Academic Editor Luc Vallieres

Copyright copy 2017 Hye Min Lim et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

STIM1 is the only currently known intracellular calcium sensor that functions as the calcium influx regulator controlling immunecell activation STIM1 function in immune cell calcium signalling has been studied extensively however its role in microgliainnate immune cells in brain has not been fully understood Here we report that STIM1minusminus murine microglia lost store-operatedcalcium influx and displayed aberrant immunological functions Microglial functions regulated by chronic and global [Ca2+]ichanges were reduced significantly including cytokine releases and opsonin-dependent phagocytosis More dramatically cellularfunctions governed by Ca2+ regulation in local microdomains at the cell periphery such as UDP-induced phagocytosis and ATP-stimulated chemotactic migration were severely reduced in STIM1minusminus microglia Interestingly UDP-induced Orai1 mobilizationto the peripheral region was greatly attenuated in STIM1minusminus microglia Their chemotactic migration defect was reproduced invivo in embryonic brain the aggregated number of STIM1minusminus microglia in LPS- (lipopolysaccharide-) injected lesions was muchsmaller than that in wild-type microglia Furthermore the neuron phagoptosis activities of activated microglia were significantlydiminished in the STIM1minusminus microgliaThese in vitro and in vivo results suggest that STIM1-mediated store-operated calcium entryis important for the regulation of global [Ca2+]i changes which differentiates into active immune state of microglia but it is morecrucial for the regulation of local [Ca2+] microdomains which mediates the acute motility of murine microglia

1 Introduction

Microglia are resident macrophages in the brain that orches-trate inflammatory responses Microglia detect infectionsites foreign material and pathological alterations in thebrain [1 2] Pathological stimuli such as lipopolysaccharide(LPS) activate microglia and induce several morphologicalchanges including phagocytosis to remove cellular debrisand secretion of chemicalmediators such as proinflammatorycytokines which propagate immunological activities [1] LPSa representative immune function inducer ofmicroglia binds

to the TLR4 receptor (toll-like receptor 4) in microgliaand induces cytokine secretion and phagocytosis whichis dependent on a sustained increase of intracellular Ca2+concentration ([Ca2+]i) [3ndash5] LPS also induces a shift in themicroglial gene expression profile which is mainly mediatedby theNFAT (Nuclear factor of activated T-cell) transcriptionfactor [6] Activated microglia have different gene expressionprofiles secrete cytokines such as TNF-alpha and IL-6 andhave the enhanced opsonin-dependent phagocytotic activity[6] Chronic elevation of [Ca2+]i boosts phagocytosis andmigration via optimizing the related gene expressions but

Hindawi Publishing CorporationMediators of InflammationVolume 2017 Article ID 8158514 13 pageshttpdxdoiorg10115520178158514

2 Mediators of Inflammation

UDP-mediated phagocytosis and ATP-induced chemotacticmigration are primarily regulated by local [Ca2+] in focalmicrodomains at the edge of cells such as the phagosomeor focal adhesion spot [7ndash9] Injured neuronal cells leaknucleotides which activate several P2 receptors in microglia[2 10 11] P2X4 and P2X7 the most widely expressed P2Xsubunits on microglia are activated by ATP and form ahomomeric Ca2+-permeable channel [12 13] Microglia alsoexpress P2Y receptors particularly P2Y6 and P2Y12 are acti-vated by UDP then change [Ca2+]i via Gq-coupled signallingpathways and cause Ca2+ influx from the plasma membrane[8 10]

It is well known that Ca2+ influx through store-operatedcalcium entry (SOCE) mediates sustained calcium increasesduring activation of immune functions in microglia [5 14]Recently STIM1 (stromal interaction molecule 1) and STIM2(stromal interaction molecule 2) have been identified asthe only protein family that functions simultaneously as anintracellular calcium storage sensor and as a regulator ofcalcium influx from the extracellular environment STIM1is a type 1 single-spanning membrane protein with an EFhand Ca2+-binding motif which functions as the sensor ofER (Endoplasmic reticulum)-luminal Ca2+ [15 16] Depletionof ER-luminal Ca2+ stores induces a conformational changein STIM1 then the C-terminal transmembrane domainbinds and activates the plasma membrane store-operatedchannel [SOC Orai or transient receptor potential channel(TRPC)] which induce Ca2+ influx [17 18] However Ca2+influx through SOCE in microdomains was not studied inmicroglia

In the present study we investigate the function ofSTIM1-mediated SOCE in pathogen- or purinergic-mediatedmicroglial immune function using STIM1minusminus mice whichhave complete depletion of STIM1 Experiments using pri-mary cultured microglia indicated that innate immune func-tions governed by chronic and global [Ca2+]i changes suchas cytokine release and opsonin-dependent phagocytosiswere reduced significantly in STIM1minusminus microglia Above allfunctions regulated by the local [Ca2+] microdomains at theedge or periphery of cells such asUDP-induced phagocytosisand ATP-stimulated chemotactic migration were essentiallyabolished in STIM1minusminusmicrogliaThemigration impairmentsof STIM1minusminus microglia were also observed in the embryonicbrain in vivo The number of aggregated STIM1minusminus microgliainto the LPS-injected lesion for 24 hours was significantlylower than the number of aggregated WT microglia intothe LPS-injected lesion Furthermore the neuron phago-cytotic activity of microglia had significantly diminishedin STIM1minusminus microglia These in vitro and in vivo resultssuggest that STIM1 is crucial for the regulation of local[Ca2+]microdomains in peripheral region ofmicrogliawhichmediates the process of acute motility of murine microglia

2 Materials and Methods

21 Mice STIM1+minus heteromice (C57BL6 background) weregenerated as previously described [19 20] The mice were

bred and maintained according to the Yonsei Medical Cen-ter animal research requirements and all procedures wereapproved by the Committee on Animal Research at YonseiMedical Center (protocol number 2011-0112) Since STIM1minusminusmice die within 1 day after birth due to defect in thecirculatory system 17 day embryos taken off from pregnantSTIM1+minus females mated with STIM1+minus male mice were usedto purify eachWTand STIM1minusminusmicroglia For themicroglialmigration assays in vivo the brains of the same aged embryoswere used Genomic DNA isolated from the embryo tailwas used for genotyping Genomic DNA for genotypingwas extracted using prepGEM Tissue (PTI0200 ZyGEMHamilton New Zealand)

22 Isolation and Culture of Primary Mouse MicroglialCells Astrocytes and Neurons Primary cultures of mousemicroglia were prepared from the whole brain of an embry-onic day 17 (E17) mouse Each embryo tail was used for geno-type analyses to identify WT and STIM1minusminus microglia Pri-mary mixed cells that had been chemically and mechanicallydissociated of the whole brain were seeded in DMEMF12(1 1) culture medium Cells were cultured at 37∘C in anincubator (Thermo Fisher Scientific Marietta OH USA)humidified with 5 CO2 Cell confluency was achievedafter 3sim4 weeks After 4 weeks the primary mixed cellsof WT and STIM1minusminus were used a mild trypsinization andshaking method [19] The upper cell layer of mixed cells wasremoved in one piece by treatingwith trypsin 025 (1x) solu-tion (SH3004201 Hyclone Laboratories Thermo ScientificLogan UT USA) diluted 1 4 in DMEMF12 (1 1) culturemedium (Dulbeccorsquos modified Eagle medium 11320-033Gibco Life Technologies Carlsbad CA USA) containing10 fetal bovine serum (26140-079 Gibco Life TechnologiesCarlsbad CA USA) and 1 penicillin-streptomycin (15140-122 Gibco Life Technologies Carlsbad CA USA) for 20minat 37∘C The trypsin diluted 1 4 in DMEMF12 was suc-tioned and replaced with 12mL of new culture medium Theremained pure microglial cells were attached to the bottomof the T75 flask and were isolated by shaking the T75 flasksof WT STIM1minusminus at 120 rpm (SLOS-20 Seoulin BioscienceSeoul Korea) at 37∘C for 1 h 12mL of each medium werecollected from each flasks placed a cell strainer with 40120583mpore size (352340 BD Falcon San Jose CA USA) filteredinto a fresh 15-mL conical tube and centrifuged at 4000 rpmat room temperature for 5min Glial cells were grown at ahigh density into 75 T flasks to extract puremicroglia at 7 to 9days in vitro After isolate microglia the remaining cells weremaintained in astrocyte-conditionedmedium and remainingmicroglia were depleted by adding 50mM L-leucine-methylester (LME Sigma Aldrich St Louis MO USA) for 4hours To culture the cortical neuron the culture of corticalneuron was prepared from 1 day postnatal pups (C57BL6strain) as described previously [1] with some modificationsThe cerebella were dissociated in Versene solution (1 5000)and plated at 05 times 106 cellscm2 in 24-well plates coatedwith Laminin (10120583gmL in serum-free DMEM) Cells wereincubated in Dulbeccorsquos modified Eaglersquos medium (DMEM)supplemented with heat-inactivated horse serum (5) fetal

Mediators of Inflammation 3

calf serum (5) 13mM glucose 05mM HEPES buffer25mM KCl and 2mM L -glutamine Cells were maintainedat in a humidified atmosphere of 37∘C 5 CO295 air Toinhibit growth of nonneuronal cells 75120583MAra-C (cytosine-D-arabinoside Sigma Aldrich St Louis MO USA) wastreated to the medium 48 h after plating Cortical neuronalcells were used at 9 days after primary culture in experi-ment to ensure morphological and physiological maturityCells purities were checked with immunostaining of anti-GFAP antibody anti-Iba1 antibody and MAP antibody forglial cells microglia and neuron respectively The numbersof cells were counted with haemocytometer (MarienfeldLauda-Konigshofen Germany)

23 Ca2+ InfluxMeasurements in STIM1minusminusMice Ca2+ influxwasmeasured by recording [Ca2+]i with Fura-2-AM labelling(F1201 Invitrogen Molecular Probes Eugene Oregon USA)as described previously [17] Briefly WT and STIM1minusminusmicroglia grown on glass cover slips were loaded with cell-permeable Fura-2-AM and trapped Fura-2 fluorescence wasmeasured with a spectrofluorometer (Photon TechnologyInternational Birmingham NJ USA) Cells were perfusedwith a solution containing 150mM NaCl 5mM KCl 1mMMgCl2 10mM glucose 10mM HEPES (pH 74 adjusted withNaOH) containing either 1-2mM CaCl2 or 05mM EGTA(to chelate any Ca2+ in the solution) The osmolality of allsolutions was adjusted to 310 Osm with the major salt TheFura-2 ratio was recorded using dual-excitation wavelengthsat 340 and 380 nm and emission wavelengths above 510 nmwere monitored Cells were treated with 25 120583Mcyclopiazonicacid (CPAC1530 SigmaAldrich St LouisMOUSA) whichinhibits the endoplasmic reticulum (ER) Ca2+-ATPase Cellswere treated with 100 120583M uridine 51015840-(trihydrogen diphos-phate) sodium salt (UDP U4125 Sigma Aldrich St LouisMO USA) or 50 120583M adenosine 51015840-triphosphate di(tris) salthydrate (ATP A9062 Sigma Aldrich St Louis MO USA)

24 Immunocytochemistry Primary cultured WT andSTIM1minusminus microglia were seeded on glass cover slips into24-well plates at 5 times 104 cellswell and incubated at 37∘C inan incubator enriched with 5 CO2 atmosphere overnightCells were treated with or without 1 120583M thapsigargin (TGT9033 Sigma Aldrich St Louis MO USA) for 5min fixedwith 4 paraformaldehyde in phosphate-buffered saline(PBS) (19943 1 LT Affymetrix Cleveland Ohio USA)at room temperature for 10min and then rinsed threetimes with PBS Cells were permeabilized by incubatingin permeabilization solution [01 Triton X-100 1 horseserum and 1 bovine serum albumin (BSA) in PBS pH74] at room temperature for 10min and then rinsed threetimes with PBS To avoid nonspecific antibody bindingcells were treated with blocking solution (5 horse serum1 BSA 01 gelatin and 0001 sodium azide in PBSpH 74) at room temperature for 30min Immunostainingwas performed in blocking solution using 1 100 dilutionof anti-Orai1 (D-15) (sc-74776 Santa Cruz BiotechnologySanta Cruz CA USA) or 1 100 dilution of anti-STIM1(610954 BD Biosciences 1 100) as primary antibodies and

the appropriate secondary antibodies tagged with AlexaFluor-FITC or Alexa Fluor-rhodamine Nuclei were stainedwith 5 120583gmL 410158406-diamidino-2-phenylindole (DAPI D3571Invitrogen Molecular Probes Eugene Oregon USA) Thecoverslips were rinsed mounted with fluorescent mountingmedium (S3023 Dako Glostrup Denmark) and cells werevisualised on a confocal microscope (LSM 710 controlledwith Zen software Carl Zeiss Jena Germany)

25 ELISA Assay WT and STIM1minusminus microglia were seededinto 6-well plates at 2 times 106 cellswell and incubated at 37∘CovernightThen cells were treated with or without 100 ngmLLPS and incubated at 37∘C in an incubator enriched with 5CO2 atmosphere for 24 h The next day supernatants wereassayed for levels of TNF-120572 and IL-6 using an ELISA kit(430901 and 431301 resp BioLegend San Diego CA USA)according to the manufacturerrsquos instructions Cytokine levelsin LPS-activated microglia were analysed for 30min (at 5-min intervals) using the kinetic method and a VersaMaxspectrophotometer plate reader (Molecular Devices Sunny-vale CA USA) at 650 nm

26 Phagocytosis Assay WT and STIM1minusminus microglia wereseeded into 12-well plates at 3 times 105 cellswell and incubatedat 37∘C overnight Then cells were treated with or without100 ngmL lipopolysaccharides from Escherichia coli 0111B4(L4391 Sigma Aldrich St Louis MO USA) and incubated at37∘C in an incubator enriched with 5 CO2 atmosphere for24 h LPS-activated microglia were treated with BioParticlesFluorescent Particles (conjugated with Alexa Fluor 488 E-13231 Invitrogen Molecular Probes Eugene Oregon USA)and incubated at 37∘C in an incubator enriched with 5CO2 atmosphere for 2 h and then cells were rinsed threetimes with PBS To quench the particle fluorescence cellswere treated with 04 trypan blue stain (15250-061 GibcoLife Technologies Carlsbad CA USA) at room temperaturefor 2min and then cells were rinsed three times with PBSCells were fixed with 4 paraformaldehyde in PBS at roomtemperature for 10min and then rinsed three times withPBS Nuclei were stained with 5 120583gmL DAPI Cells werevisualised on an inverted fluorescence microscope (IX73-F22PH Olympus) and fluorescence intensity was quantifiedwith MetaMorph software (Molecular Devices SunnyvaleCA USA) Flow cytometry experiments were conductedas follows WT and STIM1minusminus microglia were seeded into12-well plates at 3 times 105 cellswell and incubated at 37∘Covernight Then cells were treated with or without 100120583MUDP and incubated at 37∘C in an incubator enriched with 5CO2 atmosphere for 20min UDP-activated microglia weretreated with BioParticles Fluorescent Particles as describedabove Cells were rinsed three times with PBS treated with005 trypsin-EDTA (25300-062 Gibco Life TechnologiesCarlsbad CA USA) at 37∘C for 10min in a CO2-enrichedincubator and then cells were harvested by centrifugationat 2000 rpm at 4∘C for 5min Supernatant was removed500120583L ice-cold serum-free medium was added and cellswere resuspended Cells were held on ice and analysed by flowcytometry using BD FACSVerse (BD Biosciences FranklinLakes NJ USA) and a total gated cell number of 10000 cells


Flow cytometry data were analysed with FlowJo software(Treestar Ashland OR USA)

27 In Vitro Chemotaxis Assay Analysis of microglial migra-tion was performed in triplicate using chemotaxis chambers(101-8 Neuro Probe Gaithersburg MD USA) as previouslydescribed [13 21] Polycarbonate filters with 8-120583mpores werecoated with 10 120583gmL fibronectin (F0895 Sigma AldrichSt Louis MO USA) in PBS at room temperature for1 h The lower chambers were filled with 29 120583L of serum-free DMEM-F12 media containing 50120583M ATP The dryfibronectin-coated filter was installed to separate the lowerchambers from the upper chambersThenWT and STIM1minusminusmicroglia were seeded into the upper chambers at 4 times 104cellswell in 20120583L serum-free DMEM-F12 and chamberswere incubated at 37∘C in an incubator enriched with 5CO2 atmosphere for 15 30 60 and 90min After incubationthe chemotaxis chambers were disassembled and filters wereremoved Microglial cells in the lower chambers were stainedwith tomato lectin (conjugated with DyLight 594 1 200dilution Vector Labs Carlsbad CA USA) nuclei werestained with 5 120583gmL DAPI at room temperature for 10minand then cells were rinsed three times with PBS Cells thathad migrated to the lower chambers were visualised andcounted using an inverted fluorescent microscope (IX73-F22PH Olympus Tokyo Japan) The total number of cellsstained withDAPI was quantified usingMetaMorph software(Molecular Devices Sunnyvale CA USA)

28 Microinjection of Embryonic Mouse Brain In Vivo Weused microinjection to target specific regions of embryonicmouse brain in vivo as previously described [22] Nanoliter2000 microinjector and capillary glass (35 inches) (WPISarasota FL USA) were used as injection tools Timedpregnant STIM1+minus hetero female mice carrying embryonicday 17 (E17) embryos were anesthetised with Isoflurane andplaced on a heating pad Embryonic mice in utero wereinjected with 30 nL of LPS mixed with 4 methylene blue(66720 Sigma Aldrich St Louis MO USA) The finalconcentration was 12 ng of LPS per one embryo in uteroThe mother was sutured and recovered on a warming padfor 1 h and then moved back to her home cage After24 h the mother was reanesthetized with Isoflurane and theabdominal incision was reopened to approach the embryosAfter the embryos were taken out of the utero the motherwas inevitably sacrificed Embryonic whole brains were fixedwith 10 formalin at 4∘C overnight Then the tissue wastransferred to 3M sucrose in PBS solution and incubatedat 4∘C until the tissue sinks to the bottom of the tube (acouple of hours) This sucrose-infiltration is necessary fortissue cryopreservation The fixed embryo brain tissue wasused for cryosectioning 10 120583M thick slices and embryo tailswere used for genotype analyses Sections of embryonic WTand STIM1minusminus brains were stained in blocking solutions withanti-Iba1 (1 200 dilution 019-19741 Wako Osaka Japan) anantibody specific for microglia and a secondary antibodylabelled with Alexa Fluor 488 (Invitrogen Molecular ProbesEugene Oregon USA) and then rinsed three times withPBS Nuclei were stained with 5 120583gmL DAPI After rinsing

coverslips were mounted with fluorescent mountingmedium(S3023 Dako Glostrup Denmark) and visualised on aconfocal microscope (LSM 710 controlled with Zen softwareCarl Zeiss Jena Germany)Three brains ofWTand STIM1minusminusembryonic mice were used for the experiments The brainslices were illustrated using a tile scan mode to obtain thelarger images of the entire brain Among the larger imagesthe images which showed the injected legion with methyleneblue were selected for counting the aggregated microglia andthe highest number of aggregating microglia were used forsummaryThe total numbers of cells in lesion and counterpartareas were measured by computer vision using MetaMorphsoftware

29 Microglial Phagoptosis of Neurons Normal neurons werecultured in the bottomdish of transwell plates to induce dam-age by secreted molecules from the LPS-activated microglialocalized in the upper transwell dishes Using this coculturesystem for 48 hours the neuron became damaged accordingto Neher et al [23 24] WT and STIM1minusminus microglia weretreated with LPS for 24 hours to full activation ActivatedWT and STIM1minusminus microglia were treated directly in contactwith the damaged neuron to allow phagoptosis for six hoursThe same number of microglia in the same number ofdamaged neurons were used and no activated microgliatreated neurons were used as mocks After six hours allcells were fixed with 37 formaldehyde in the PBS thenimmunostaining was performed as above Cells were alsostained with DAPI (for nuclei blue) tomato lectin (formicroglia Rhodamine-red) and MAP (for neuron FITC-green) respectively The phagocytosis activity was relativelyanalysed with the portion of the fully enlarged microglia cellnumber (gt35 120583m2) from the total number of the activatedmicroglia (gt25 120583m2) The relative neuron survival rate fromthe microglial phagoptosis was summarized by calculatingeach MAP (FITC) staining intensity from the remainingneurons in the five randomly selected images from the fieldsNo microglia treated only neurons were used for mocks tocompare their phagoptosis activities

210 Statistical Analyses Data are presented as the meansplusmn standard error of the mean Statistical analysis was per-formed with Studentrsquos 119905-test or with analysis of variance(ANOVA) followed by Tukeyrsquos multiple comparison testsusing the GraphPad Prism software package (version 50) asappropriate 119901 lt 005 was considered statistically significant

3 Results31 LPS-Stimulated Phagocytosis and Cytokine Secretion inWT and STIM1minusminusMicroglia Thebasal characteristics ofWTand STIM1minusminusmicrogliawere confirmedbefore using them forfurther experiments (Supplementary Figures 1sim3 in Supple-mentary Material available online at httpsdoiorg10115520178158514) LPS is a potent immune activator of microgliawhich binds and activates TLR4 receptor signalling [25]STIM1 effect on microglial immune function phagocyticactivity and cytokine secretion was compared in WT andSTIM1minusminus microglia To compare phagocytosis activity in


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Figure 1 Lipopolysaccharide-induced phagocytosis and cytokine secretion inwild-type and STIM1minusminusmicroglia (a b) Representative imagesof image-based phagocytosis measurement using phagocytic engulfment of FITC bead in WT and STIM1minusminus microglia Summary of fiveindependent phagocytosis measurements Seven images were used for phagocytosis measurements and then depicted as a bar graph lowastlowastlowast119901 le00001 in LPS-induced fold-induction ofWT lowast119901 le 0005 inWTmock 119899 = 7 (c d) Comparisons of LPS-induced cytokine secretion activityTNF-120572 secretion (c) and IL-6 secretion (d) were compared inWT and STIM1minusminus microglia 119901 le 00001 119899 = 4 where 119899 is independent ELISAexperiment ns no significant difference fromWTmock

WT and STIM1minusminus microglia resting and LPS-stimulated(100 ngmL for 24 h) phagocytotic activities were tested withengulfment of E coli opsonised FITC-labelled bioparticlesMicroglial cells were treated with bioparticle beads for 2 hThen free beads and attached beads on the cell surface werequenched by trypan blue and cells were visualised usingfluorescence microscopy (Figures 1(a) and 1(b)) STIM1minusminusmicroglia under resting-state conditions showed a slightreduction in phagocytotic activity InWTcells LPS treatment

for 24 h increased phagocytic activity by approximately 25-fold but STIM1minusminus microglia had smaller increases in phago-cytic activity These results indicate that the LPS-stimulatedgreater phagocytotic activity was more obviously found inWT microglia Next we compared LPS-induced secretion ofTNF-120572 and IL-6 cytokines (Figures 1(c) and 1(d))The resultsshow that STIM1minusminus microglia secreted less than or equal tohalf of the amount of TNF-120572 and IL-6 as that secreted byWTmicroglia These data indicate that STIM1 depletion reduces


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Figure 2 Comparison of UDP-induced Ca2+ signalling and phagocytosis in wild-type and STIM1minusminus microglia (a) Representative curves ofUDP-induced [Ca2+]i mobilization inWT and STIM1minusminusmicroglia 100 120583MUDP induced ER-Ca2+ efflux whilst store depletion induced Ca2+influx (b) Relative UDP-induced Ca2+ transient release from ER lowastlowastlowast119901 le 00001 119899 = 15 (c) Comparison of UDP-induced SOCE activitydetermined by the slope of Ca2+ influx lowastlowastlowast119901 le 00001 119899 = 15 (d) Representative flow cytometry images of the phagocytosis measurementsFITC intensities of phagocytic bead engulfment with or without 100 120583MUDPwere measured at 20min Grey region indicates the populationof bead-engulfedmicroglia (e) Summary of FACS from three independent phagocytosis experiments Geometricmean represents the relativephagocytosis activity in each set lowastlowast119901 le 0001 in comparisons of bead engulfment in WT microglia

phagocytotic activity and cytokine section in the microgliaunder LPS-stimulation conditions

32 UDP-Induced Phagocytosis Is Impaired in STIM1minusminusMicroglia Phagocytosis of microglia is activated by UDPthrough P2Y6 metabotropic receptor that is coupled to theGq-protein [10] RT-PCR analysis indicated that microgliaexpresses P2X3 P2X4 and P2X7 receptors and all types ofP2Y receptors (Supplementary Figure 4) In particular theexpression of P2Y receptors were somewhat increased inSTIM1minusminus microglia which implies that the compensatory

up-regulation of P2Y receptors might exist to cope with thereduced Ca2+ influxes in STIM1minusminus microglia We measuredUDP-induced [Ca2+]i in WT and STIM1minusminus microglia (Fig-ure 2(a)) In STIM1minusminus microglia 100 120583M UDP induced atransient increase in [Ca2+]i but no sustained Ca2+ influx(Figures 2(a)ndash2(c)) This result indicates that UDP-inducedcalcium influx is almost disappeared in STIM1minusminus microgliaTo more precisely test the microglial phagocytosis activitywe tested the phagocytotic engulfment of E coli opsonisedFITC-labelled bioparticles using FACS (Figures 2(d) and


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Figure 3 Chemotactic migration in wild-type and STIM1minusminus microglia (a) Treatment with 50 120583M ATP-induced [Ca2+]i changes in 2mMCa2+ media (b) Time-dependent migration toward 50 120583MATP determined in chemotaxis chambers Cells migrating toward ATP were fixedstained with DAPI and counted after 15 30 60 and 90min The numbers of migrating cells at each time point were determined from sixindependent experiments and are depicted graphically lowast119901 le 005 lowastlowast119901 le 0001 and lowastlowastlowast119901 le 00001 versus the corresponding control (119899 = 6where 119899 is the number of independent experiments)

2(e)) This method enabled discrimination of the UDP-induced phagocytosis peak from the basal phagocytosispeak After phagocytotic bead engulfment for 20min withor without UDP microglia were detached by trypsin andcollected cells were counted by FACS Figure 2(d) shows therepresentative FACS images of WT and STIM1minusminus microgliaPhagocytosis activity of WT and STIM1minusminus microglia after20min of UDP treatment is presented in Figure 2(e) UDP-induced phagocytosis in STIM1minusminus microglia was severelydefected compared to that in WT These data indicate thatSTIM1 is critically important in UDP-induced phagocytosis

33 Chemotactic Migration Is Severely Defective in STIM1minusminusMicroglia ATP is a chemoattractant of microglia and ATPconcentrations of approximately 50ndash100120583M activate severalpurinergic receptors [12 26] First we monitored [Ca2+]i bymeasuring Fura-2-AM fluorescence under the same condi-tions that were used for chemotactic migration experimentsSTIM1minusminusmicroglia had a reduced Ca2+ evocation signal thanWT microglia (Figure 3(a)) Next chemotactic migration toATP in chemotaxis chambers inWT and STIM1minusminus microgliawas compared The number of cells migrating into theATP chamber after 15 30 60 and 90min were countedafter DAPI staining WT microglial migration was time-dependent and increased with longer incubation times Bycontrast STIM1minusminus microglia did not show a time-dependentincrease in migration and the number of cells migrating intothe ATP chamber was one-third of that for WT microgliaThese results clearly indicate that STIM1minusminus microglia have

defective chemotactic migration to ATP (Figure 3(b)) Thesedata show that STIM1 is crucial for microglial chemotacticmigration activity

34 PeripheralCentral Localization of Orai1 Is Controlledby STIM1 upon UDP Stimulation We supposed that Orai1is a microdomain indicator of [Ca2+]i since Ca2+ influxesthrough Orai1 which can change [Ca2+]i underneath theplasma membrane and build a microdomain of [Ca2+]i Totest whether the peripheral localization of Orai1 was con-trolled by STIM1 we compared localization of Orai1 underUDP treatment which induce acute phagocytic movementin WT and STIM1minusminus microglia [27] At the resting stateOrai1 was merely found at the cell periphery After 40min ofUDP treatment Orai1 clusters in the peripheral region weresignificantly increased and some of them were colocalizedwith STIM1 clusters in WT microglia (Figure 4(a)) Suchclusters of Orai1 in peripheral regions were not observedin STIM1minusminus microglia (Figure 4(b)) These results suggestedthat STIM1 regulates Orai1 localization upon UDP treatedconditions and it might imply that STIM1 controls [Ca2+]microdomains in the cell periphery that requires for localCa2+ signalling for phagocytic or chemotactic migrationmovements

35 Microglial Aggregation in LPS-Injected Lesions Was Abol-ished in STIM1minusminus Embryonic Brain To test the in vivobehaviour ofmicroglia we injected 30 nL LPS plusmethyleneblue mix into brains of 17-day-old embryos impregnated


Orai1

1120583m

Orai1

1120583m

STIM1

1120583m

STIM1

1120583m

Orai1STIM1

1120583m

Orai1STIM1

1120583m

Orai1STIM1DAPI

5120583m

Orai1STIM1DAPI

5120583m 0

100

200

Inte

nsity

5 10 15 200

Inte

nsity

5 10 15 20 25 3000

50100150200250

Orai1DAPI

DIC

Distance (120583m)

Distance (120583m)

Mock

UDP40min

WT

WT

250

150

50

(a)

Orai1DAPI Orai1

Orai1DAPI Orai1

1120583m

1120583m

5120583m

5120583m

0

100

200

Inte

nsity

0

100

200

Inte

nsity

5 10 15 20 250

5 10 15 20 250

Orai1DAPI

DIC

Distance (120583m)

Distance (120583m)

Mock

UDP40min

STIM

1minusminus

STIM

1minusminus

150

250

250

150

50

50

(b)

Mock UDP Mock UDP

lowast

0

10

20

30

40

50

Perip

hery

loca

lized

Ora

i1 (

of t

otal

Ora

i1)

STIM1minusminusWT

(c)

Figure 4 Retarded peripheral localization of Orai1 in STIM1minusminus microglia upon UDP stimulation UDP induces movement of Orai1 (green)and STIM1 (red) in WT microglia (a) and STIM1minusminus microglia (b) respectively Treatment with 100 120583M UDP for 40min increased Orai1localization at the cell surface throughout the cell border in WT(a) and STIM1minusminus microglia (b) Insets were magnified following separatedthree (a) or one image (b) Orai1-FITC-intensity profiles in red lined regions were shown in each of the rightmost images The cell border isindicated by dotted lines Clustered Orai1 in peripheral regions of WTmicroglia were indicated by white arrows and the absence of Orai1 inperipheral regions of STIM1minusminus microglia was indicated by black arrows (c) Intensity of surface Orai1 in cell periphery was compared withthe UDP condition Intensity of surface Orai1 in cell periphery was determined by the intensity of ROI with 065120583m away from the cell edgesusing MetaMorph software

in STIM1+minus female mice mated with STIM1+minus male miceMethylene blue was used to label the injection site in the tis-sue slices At 24 h after injection each embryo brain was har-vested and their genotypes were checked WT and STIM1minusminusbrains were sectioned and the sections containing lesionslabelled with methylene blue were selected and stained withIba1 to visualisemicroglia Two sets of representativeWT andSTIM1minusminus brain tissue sections at different magnifications arepresented in Figure 5(a) InWTbrain Iba1-labelledmicrogliaaggregated at the LPS-methylene blue injected lesion but the

counterpart region of the noninjected hemisphere displayednormal ramified microglia High-magnification images ofboth hemispheres clearly show their distinct morphologiesas ramified resting state and amoeboid activated state InSTIM1minusminus brain tissue the LPS-methylene blue injected lesionwas not crowded with microglia and the microglia localizedin the lesion showed similar amoeboid shapes as those ofWTWe calculated the total microglial cell number in the whole-brain slices and did not find any significant difference in thecell numbers as shown in mocks in STIM1minusminus brain tissue

Mediators of Inflammation 9W

T

Set 1 Set 2

50120583M

B

10120583M

C

50120583M

D

10120583M

E

50120583M

G

10120583M

H

10120583M

J

50120583M

I

500120583M

No injection

LPS + MB

F

50120583M

L

10120583M

M

50120583M

N

10120583M

O

No injection

LPS + MB 500120583M

K

10120583M

R

50120583M

Q

50120583M

S

10120583M

T

No injection

LPS + MB 500120583M

P

500120583M

No injection

LPS + MB

A

STIM

1minusminus

(a)

ns

ns

Iba1

posit

ive c

ells

Non

inje

cted

Inje

cted

Non

inje

cted

Inje

cted

lowast

lowastlowast

WT STIM1minusminus

0

100

200

300

(b)

Figure 5 In vivo behaviour of microglia in lipopolysaccharide-injected brains of wild-type and STIM1minusminus embryonic mice (a) Twoexperimental sets representing microglial aggregation at the site of injection of LPS and methylene blue (MB) in WT and STIM1minusminus micebrain No LPS injection (upper right hemisphere) and LPS-injected (lower left hemisphere) are indicated in low-magnification images ofimmunostaining with Iba1 antibody (A F K and P bar = 500120583m) White boxed regions are twofold magnifications ((B D G I L N Qand S bar = 50 120583m) and (C E H J M O R and T bar = 10 120583m)) Merged images of green Iba1-FITC and grey-DIC are shown in BndashE GndashJLndashO and QndashT microglia are shown in green and methylene blue particles are shown in grey Resting ramified microglia (C H M and R)were identified in the noninjected hemisphere and the stimulated amoeboid shapes of microglia engulfing methylene blue particles (E J Oand T) were identified only in the LPS + MB injected region (b) Comparison of aggregated microglial numbers at the LPS injection siteThe numbers of microglia at the LPS + MB injection hemisphere and noninjected hemisphere were counted lowast119901 le 005 lowastlowast119901 le 001 ns nosignificant difference fromWTmock Data are representative of three experiments


These results also were observed when the number of cellsafter primary culture of each mouse was counted HoweverSTIM1minusminus brain slices had definitely reduced numbers ofIba1 positive microglia aggregated in LPS-injected lesions(Figure 5(b)) which indicates that STIM1minusminus microglia havea defect in migration activity toward the legionThese in vivodata indicate that STIM1minusminus microglia have severe defects inchemotactic migration activity in brain environment

36 Neuron Phagocytosis Activity Is Attenuated in STIM1minusminusMicroglia To compare themicroglial phagocytosis activitieswe tested the phagocytic activity of WT and STIM1minusminusmicroglia in the condition of direct contact with damagedcortical neurons Since the LPS treatment without microgliadoes not cause damage of neuron [24] we applied thecoculture systems using WT microglia with 100 ngmL LPStreatment on transwell inserts and the primary culturedcortical WT neurons on the bottom of the plate for two daysto prepare for the phosphatidylserine- (PS-) exposed WTneuron [24] After two days the transwell insert was removedand the medium was also washed out and preactivated(100 ngmL LPS for 24 h) WT or STIM1minusminus microglia weredirectly added to PS-exposed damaged neuronal cells [23]After 6 h the cortical neuron and microglia cells werefixed and cells were indicated with corresponding antibodies(Figures 6(a) and 6(b)) We analysed each size of tomatolectin positive microglia cells and counted the fully enlargedmicroglia engulfing neurons The total number of fullyenlarged microglia (gt35 120583m2) out of the total number ofactivated microglia (gt25 120583m2) was significantly higher inWT microglia (Figure 6(c)) These data indicated that LPS-activated WT microglia had higher neuron phagocytosisactivities than STIM1minusminus microglia Furthermore the neu-ronal survival rate from phagoptosis by STIM1minusminus microgliawasmuch higher than that ofWTmicroglia (Figure 6(d)) Allof the data suggests that STIM1 is important in phagoptosisactivities of microglia

4 Discussion

One of the most crucial signalling pathways during immunecell differentiation is sustained calcium elevation in thecytosol which is mediated primarily by SOCE [14] Thesignificance of SOCE was demonstrated in studies usingpharmacological inhibitors and depletion of extracellularcalcium with calcium chelators but it has not been fullyevaluated inmicroglial cells under completely blocked condi-tions until STIM1 was identified [15 20] Complete depletionof SOCE was necessary to determine whether the completeSOCE depletion abolished immune response activation ofimmune responses which is mediated by sustained [Ca2+]iincrease in response to several agonists A complete depletionof SOCE was achieved in purified lymphocytes of STIM1minusminusmice including mast cells T cells platelets neutrophils andmicroglia [20 28ndash32] Our previous using siRNA for STIM1was not able to completely deplete all STIM1 protein sothe effect of residual STIM1 in activated immune functionof microglia could not be ruled out [33] In this study we

evaluate the in vitro and in vivo effects of STIM1-regulatedSOCE onmicroglial immune functions including phagocyto-sis cytokine secretion and chemotactic migration Not onlythe Ca2+ signal which changes the profile or efficiency of geneexpression but also the Ca2+ signal which acutely regulatesthe function such as UDP-induced phagocytosis and ATP-induced chemotaxis was significantly decreased in STIM1depletion condition

STIM2 was less effective to activate SOCE than STIM1 inT cells [14 28] and microglia [32] Michaelis et al recentlyreported that STIM2minusminus microglia displayed SOCE activationby store depletion and nucleotide stimulation comparedwiththe complete absence of SOCE in STIM1minusminus microglia [32]Western blot analyses and [Ca2+]i measurements indicatedthat STIM1 protein expressionwas greater than that of STIM2(Supplementary Figure 1B) SOCE was essentially abolishedafter CPA-induced Ca2+ depletion from ER in STIM1minusminusmicroglia (Supplementary Figures 2A and 2D) in spite ofthe presence of STIM2 (Supplementary Figure 1B) Theseresults indicate that STIM1 is the major regulator of SOCEin microglia

Important roles of STIM1 in differentiation of activemicroglial status were proved by attenuated immune func-tion of activated microglia treated with LPS LPS-inducedactivated microglia showed increased phagocytosis activityand cytokine secretion of TNFminus120572 and IL-6 but those weresignificantly reduced in STIM1minusminus microglia The sustainedhigh [Ca2+]i via Ca

2+ influx causes the change of Ca2+ con-centration of nucleus which yields the change of gene expres-sion for fully differentiation [14] These reduced chronicimmune function of STIM1minusminus microglia might be caused bythe reduced Ca2+ influx and lower [Ca2+]i status which abol-ishes the increase of nuclear Ca2+ concentration to changethe gene expression for fully differentiation Moreover thecrucial role of STIM1 in acute movement was proved byUDP-induced phagocytosis and ATP-stimulated chemotaxisThe UDP-induced phagocytosis is relatively faster functionwhich demands the cytoskeletal movement governed bylocal Ca2+ signal in the cell periphery [10] The markedlyimpaired UDP-induced phagocytosis in STIM1minusminus microgliaseems to be caused by local increased Ca2+ concentrationright after the influx Similarly ATP-stimulated microglialchemotaxis and migration were greatly reduced in STIM1minusminusmicroglia subjected to chemotaxis assays Time-dependentATP-stimulated cell migration was essentially abolished inSTIM1minusminus microglia (Figure 3(b)) These defects might berelated with the retarded movement of Orai1 in STIM1minusminusmicroglia (Figure 4) Orai1 pores can constitute a [Ca2+]microdomain underneath the plasmamembrane through theCa2+ influx Attenuated movements of Orai1 in STIM1minusminusmicroglia implied that STIM1 is critical for the organizationof [Ca2+] microdomains in microglia

This in vitro result was confirmed in in vivo studiesThe cell migration to the injured site was severely impairedin the brain environment (Figure 5) and damaged neuronphagoptosis was much more retarded in STIM1minusminus microglia(Figure 6) Injured cells release nucleotides such as ATP


WT neuronWT microglia

WT neuron WT microgliaWT microglia

WTmicroglia

200120583m 20120583m

(a)

WT neuron

200120583m 20120583m

WT neuronSTIM1minusminus microglia

STIM1minusminus microgliaSTIM1minusminus microglia

STIM1minusminus

microglia

(b)

25120583m2 lt lt 35120583m2

gt 35120583m2

+ activated microgliafor 6hr

WT STIM1minusminus

lowastlowastlowast

0

20

40

60

80

100

120

( o

f tot

al ac

tivat

ed m

icro

glia

)Re

lativ

e act

ivat

ed m

icro

glia

size

(c)

Rela

tive s

urvi

val n

euro

n

Mock


lowast

lowastlowastlowast

WT STIM1minusminus0

25

50

75

100

125(

of o

nly

neur

on M

AP2

posit

ive c

ells)

(d)

Figure 6 Decreased neuron phagoptosis activity of STIM1minusminus microglia (a) Neuron phagoptosis by WT microglia was shown byimmunostaining of neuron and microglia after damaging neuron and LPS-activated WT microglia (bar = 200120583m) Normal neurons werestained with MAP specific antibodies (green) and WT microglia were stained with tomato lectins (red) The second image shows enlargedmicroglia indicated via arrowsThe third image shows highlymagnified images of the activatedmicroglia engulfing neurons soMAP stainingwas found inside of themicroglia stained with tomato lectin (b) Similar image to the above but used LPS-activated STIM1minusminusmicroglia whichcoexisted with neurons The second image shows some enlarged STIM1minusminus microglia indicated with arrows The third image shows highlymagnified images showing the activatedmicroglia near the neurons (c)The relative size of the activatedWTand STIM1minusminusmicroglia indicatedvia bar graph 25 120583m2 was determined as the minimum size of activated microglia and the number of cells bigger than 35 120583m2 was assignedas the ldquofully enlarged microglia engulfing neuronrdquo which is significantly small in STIM1minusminus microglia population lowastlowastlowast119901 le 0001 119899 ge 100119899 = cell number from 3 independent experiments (d) The relative neuron survival rate was analysed using MAP2 staining intensity of theremaining neuron after 6 hr phagoptosis in which images were delivered from five randomly selected fields of leftmost images Each of thephagoptosis activities was compared with the total MAP2 intensity of the only neuron which was without phagoptosis by activated microglialowastlowastlowast119901 le 0001 lowast119901 le 005 119899 ge 15 and 119899 = 5 images from 3 independent experiments


which is a known chemotactic attractant and an activatorof purinergic receptors such as P2X4 and P2Y12 and rapidcell migration is essential for microglial recruitment to thesite of brain lesions [12 13] Although the severe impairmentof cell migration in STIM1minusminus embryonic brain is not onlydue to defective chemotaxis of STIM1minusminus microglia sincethe environment of STIM1minusminus brains is also differs fromthat of WT brains in our experimental conditions How-ever defective chemotaxis of STIM1minusminus microglia in vitroand retarded phagocytosis activity toward damaged neuronsstrongly suggest that STIM1 is essential for regulating chemo-tactic migration and phagocytosis activated by chemical cueor damaged neurons

Cell migration is coordinated by Ca2+ signalling inpart through local Ca2+ signalling in the cell leading edge[9] Previous reports have identified STIM1 localization inpodosome [34] phagosome [34] and cell-matrix adhesionat the front of migrating cells [9] Recent work showedthat receptor tyrosine kinase signalling and phospholipaseC signalling are restricted to the front of the migratingendothelial leader cells which triggers local Ca2+ pulseslocal depletion of ER-Ca2+ and local STIM1 activation thesecascades orchestrate pulsatile retraction and adhesion of theleading cell front [9] Another study reported that STIM1localization is juxtaposed to ER and phagosomes whichgenerate Ca2+ hotspots that boost phagocytosis [7] Ourresults are in agreement with these previous studies thatSTIM1-regulated local Ca2+ influx in the leading focal adhe-sion microdomain is important for the function mediatedby local cytoskeletal rearrangement such as UDP-inducedrapid phagocytosis and ATP-induced chemotactic migrationNotably STIM1 depletion had a stronger adverse effect on therate of UDP-induced rapid phagocytosis and ATP-inducedchemotacticmigration (Figures 2(d) 2(e) and 3(b)) than thaton activation of LPS-induced phagocytotic activity (Figures1(a) and 1(b)) and LPS-induced cytokine secretion (Figures1(c) and 1(d)) These data might imply that the role of STIM1in local Ca2+ microdomains is more critical than the role ofSTIM1 in [Ca2+]i change through the cells

A recent study indicated that STIM1 inhibition in lym-phocytes had beneficial effects for anaphylactic responses[20] ischemic brain infarction [30] and autoimmune disease[31] The current model of neuronal degeneration considersthat inflamed microglia have detrimental effects that leadto neuronal loss [2] The retarded LPS-induced activationof STIM1minusminus microglia implied that inhibition of STIM1 canattenuate the magnitude of flammability of microglia Ourresult showed that STIM1minusminusmicroglia has reduced phagopto-sis activity toward damaged neurons (Figure 6)These resultssuggest that STIM1 can be used as a new therapeutic target toprevent the excessive immunological microglial function andthereby delay the progression of neurodegenerative diseases

In conclusion we found that STIM1minusminus microglia showedreduced cytokine secretion and opsonin-dependent phago-cytosis in LPS condition More importantly UDP-inducedphagocytosis and ATP-stimulated chemotactic migrationwere severely impaired in STIM1minusminus microglia in vitro and

in vivo These results suggest that STIM1-mediated store-operated calcium entry is also important for the regulation ofglobal [Ca2+]i changes but it is indispensable for regulatinglocal [Ca2+] microdomains in murine microglia

Abbreviations

SOCE Store-operated calcium entry[Ca2+]i Intracellular Ca

2+ concentrationSOC Store-operated channelSTIM1 Stromal interaction molecule 1STIM2 Stromal interaction molecule 2UDP Uridine diphosphateATP Adenosine triphosphateLPS LipopolysaccharideCPA Cyclopiazonic acidTG ThapsigarginTNF-120572 Tumour necrosis factor-120572IL-6 Interleukin-6siRNA Small interfering RNAWT Wild-typeMB Methylene blueTLR4 Toll-like receptor 4NFAT Nuclear factor of activated T cellsER Endoplasmic reticulum

Competing Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Joo Young Kim conceived and designed the experimentsHye Min Lim performed most of the experiments HeoWoon confirmed most of experiment result Jung Woo Hanhelped to maintain the mice strain and participated inprimary culture of microglia Joo Young Kim Hye MinLim and Heo Woon analysed the data Yoshihiro Baba andTomohiro Kurosaki produced and provided the STIM1minusminusmice Min Goo Lee participated in design of the experimentsand contributed to reagents materials and instruments JooYoung Kim wrote the manuscripts All authors read andapproved the final manuscript Hye Min Lim and HeoWoonequally contributed to this work

Acknowledgments

This work was supported by grants from the NationalResearch Foundation of Korea (NRF-2009-0090035 NRF-2015R1D1A1A02062027 and MSIP-2013R1A3A2042197) anda faculty research grant from the Yonsei University College ofMedicine for 2015 (6-2015-0048)

References

[1] A Nimmerjahn F Kirchhoff and F Helmchen ldquoRestingmicroglial cells are highly dynamic surveillants of brainparenchyma in vivordquo Science vol 308 no 5726 pp 1314ndash13182005


[2] CKGlass K Saijo BWinnerMCMarchetto and FHGageldquoMechanisms underlying inflammation in neurodegenerationrdquoCell vol 140 no 6 pp 918ndash934 2010

[3] E C Toescu T Moller H Kettenmann and A VerkhratskyldquoLong-term activation of capacitative Ca2+ entry in mousemicroglial cellsrdquo Neuroscience vol 86 no 3 pp 925ndash935 1998

[4] A Hoffmann O Kann C Ohlemeyer U-K Hanisch andH Kettenmann ldquoElevation of basal intracellular calcium asa central element in the activation of brain macrophages(microglia) suppression of receptor-evoked calcium signalingand control of release functionrdquoThe Journal ofNeuroscience vol23 no 11 pp 4410ndash4419 2003

[5] K Farber and H Kettenmann ldquoFunctional role of calciumsignals for microglial functionrdquoGlia vol 54 no 7 pp 656ndash6652006

[6] K Nagamoto-Combs and C K Combs ldquoMicroglial phenotypeis regulated by activity of the transcription factor NFAT(nuclear factor of activatedT cells)rdquo Journal of Neuroscience vol30 no 28 pp 9641ndash9646 2010

[7] P Nunes D Cornut V Bochet et al ldquoSTIM1 juxtaposes ER tophagosomes generating Ca2+ hotspots that boost phagocyto-sisrdquo Current Biology vol 22 no 21 pp 1990ndash1997 2012

[8] C Barcia C M Ros V Annese et al ldquoROCKCdc42-mediatedmicroglial motility and gliapse formation lead to phagocyto-sis of degenerating dopaminergic neurons in vivordquo ScientificReports vol 2 article 809 2012

[9] F-C Tsai A Seki H W Yang et al ldquoA polarized Ca2+diacylglycerol and STIM1 signalling system regulates directedcell migrationrdquo Nature Cell Biology vol 16 no 2 pp 133ndash1442014

[10] S Koizumi Y Shigemoto-Mogami K Nasu-Tada et al ldquoUDPacting at P2Y6 receptors is a mediator of microglial phagocyto-sisrdquo Nature vol 446 no 7139 pp 1091ndash1095 2007

[11] E Czirr and T Wyss-Coray ldquoThe immunology of neurodegen-erationrdquo Journal of Clinical Investigation vol 122 no 4 pp 1156ndash1163 2012

[12] D Davalos J Grutzendler G Yang et al ldquoATP mediatesrapid microglial response to local brain injury in vivordquo NatureNeuroscience vol 8 no 6 pp 752ndash758 2005

[13] K Ohsawa Y Irino Y Nakamura C Akazawa K Inoue andS Kohsaka ldquoInvolvement of P2X4 and P2Y12 receptors in ATP-inducedmicroglial chemotaxisrdquoGlia vol 55 no 6 pp 604ndash6162007

[14] M Oh-hora and A Rao ldquoCalcium signaling in lymphocytesrdquoCurrent Opinion in Immunology vol 20 no 3 pp 250ndash2582008

[15] J Liou M L Kim W D Heo et al ldquoSTIM is a Ca2+ sensoressential for Ca2+-store- depletion-triggered Ca2+ influxrdquo Cur-rent Biology vol 15 no 13 pp 1235ndash1241 2005

[16] G N Huang W Zeng J Y Kim et al ldquoSTIM1 carboxyl-terminus activates native SOC 119868119888119903119886119888 and TRPC1 channelsrdquoNature Cell Biology vol 8 no 9 pp 1003ndash1010 2006

[17] D K Heo W Y Chung H W Park J P Yuan M G Lee andJ Y Kim ldquoOpposite regulatory effects of TRPC1 and TRPC5 onneurite outgrowth in PC12 cellsrdquo Cellular Signalling vol 24 no4 pp 899ndash906 2012

[18] P F Worley W Zeng G N Huang et al ldquoTRPC channelsas STIM1-regulated store-operated channelsrdquo Cell Calcium vol42 no 2 pp 205ndash211 2007

[19] T T Tamashiro C L Dalgard and K R Byrnes ldquoPrimarymicroglia isolation frommixed glial cell cultures of neonatal rat

brain tissuerdquo Journal of Visualized Experiments no 66 ArticleID e3814 2012

[20] Y Baba K Nishida Y Fujii T Hirano M Hikida and TKurosaki ldquoEssential function for the calcium sensor STIM1in mast cell activation and anaphylactic responsesrdquo NatureImmunology vol 9 no 1 pp 81ndash88 2008

[21] E K De Jong A H De Haas N Brouwer et al ldquoExpressionof CXCL4 in microglia in vitro and in vivo and its possiblesignaling through CXCR3rdquo Journal of Neurochemistry vol 105no 5 pp 1726ndash1736 2008

[22] S Davidson H Truong Y Nakagawa and G J Giesler Jr ldquoAmicroinjection technique for targeting regions of embryonicand neonatal mouse brain in vivordquo Brain Research vol 1307 pp43ndash52 2010

[23] J J Neher U Neniskyte J-W Zhao A Bal-Price A MTolkovsky and G C Brown ldquoInhibition of microglial phago-cytosis is sufficient to prevent inflammatory neuronal deathrdquoJournal of Immunology vol 186 no 8 pp 4973ndash4983 2011

[24] M Fricker M J Oliva-Martın and G C Brown ldquoPrimaryphagocytosis of viable neurons by microglia activated with LPSor A120573 is dependent on calreticulinLRP phagocytic signallingrdquoJournal of Neuroinflammation vol 9 article no 196 2012

[25] L A J OrsquoNeill and A G Bowie ldquoThe family of five TIR-domain-containing adaptors in Toll-like receptor signallingrdquoNature Reviews Immunology vol 7 no 5 pp 353ndash364 2007

[26] D Ferrari C Stroh and K Schulze-Osthoff ldquoP2X7P2Zpurinoreceptor-mediated activation of transcription factorNFAT in microglial cellsrdquo Journal of Biological Chemistry vol274 no 19 pp 13205ndash13210 1999

[27] V H Perry J A R Nicoll and C Holmes ldquoMicroglia inneurodegenerative diseaserdquo Nature Reviews Neurology vol 6no 4 pp 193ndash201 2010

[28] M Oh-hora M Yamashita P G Hogan et al ldquoDual functionsfor the endoplasmic reticulum calcium sensors STIM1 andSTIM2 in T cell activation and tolerancerdquo Nature Immunologyvol 9 no 4 pp 432ndash443 2008

[29] H Zhang R A Clemens F Liu et al ldquoSTIM1 calcium sensoris required for activation of the phagocyte oxidase duringinflammation and host defenserdquo Blood vol 123 no 14 pp2238ndash2249 2014

[30] D Varga-Szabo A Braun C Kleinschnitz et al ldquoThe calciumsensor STIM1 is an essentialmediator of arterial thrombosis andischemic brain infarctionrdquo Journal of Experimental Medicinevol 205 no 7 pp 1583ndash1591 2008

[31] A Braun J E Gessner D Varga-Szabo et al ldquoSTIM1 is essentialfor Fc120574receptor activation and autoimmune inflammationrdquoBlood vol 113 no 5 pp 1097ndash1104 2009

[32] M Michaelis B Nieswandt D Stegner J Eilers and R KraftldquoSTIM1 STIM2 and orai1 regulate store-operated calciumentryand purinergic activation of microgliardquo GLIA vol 63 no 4 pp652ndash663 2015

[33] D K Heo H M Lim J H Nam M G Lee and J Y KimldquoRegulation of phagocytosis and cytokine secretion by store-operated calcium entry in primary isolated murine microgliardquoCellular Signalling vol 27 no 1 pp 177ndash186 2015

[34] T A Siddiqui S Lively C Vincent and L C SchlichterldquoRegulation of podosome formation microglial migration andinvasion byCa2+-signalingmolecules expressed in podosomesrdquoJournal of Neuroinflammation vol 9 article 250 2012


UDP-mediated phagocytosis and ATP-induced chemotacticmigration are primarily regulated by local [Ca2+] in focalmicrodomains at the edge of cells such as the phagosomeor focal adhesion spot [7ndash9] Injured neuronal cells leaknucleotides which activate several P2 receptors in microglia[2 10 11] P2X4 and P2X7 the most widely expressed P2Xsubunits on microglia are activated by ATP and form ahomomeric Ca2+-permeable channel [12 13] Microglia alsoexpress P2Y receptors particularly P2Y6 and P2Y12 are acti-vated by UDP then change [Ca2+]i via Gq-coupled signallingpathways and cause Ca2+ influx from the plasma membrane[8 10]

It is well known that Ca2+ influx through store-operatedcalcium entry (SOCE) mediates sustained calcium increasesduring activation of immune functions in microglia [5 14]Recently STIM1 (stromal interaction molecule 1) and STIM2(stromal interaction molecule 2) have been identified asthe only protein family that functions simultaneously as anintracellular calcium storage sensor and as a regulator ofcalcium influx from the extracellular environment STIM1is a type 1 single-spanning membrane protein with an EFhand Ca2+-binding motif which functions as the sensor ofER (Endoplasmic reticulum)-luminal Ca2+ [15 16] Depletionof ER-luminal Ca2+ stores induces a conformational changein STIM1 then the C-terminal transmembrane domainbinds and activates the plasma membrane store-operatedchannel [SOC Orai or transient receptor potential channel(TRPC)] which induce Ca2+ influx [17 18] However Ca2+influx through SOCE in microdomains was not studied inmicroglia

In the present study we investigate the function ofSTIM1-mediated SOCE in pathogen- or purinergic-mediatedmicroglial immune function using STIM1minusminus mice whichhave complete depletion of STIM1 Experiments using pri-mary cultured microglia indicated that innate immune func-tions governed by chronic and global [Ca2+]i changes suchas cytokine release and opsonin-dependent phagocytosiswere reduced significantly in STIM1minusminus microglia Above allfunctions regulated by the local [Ca2+] microdomains at theedge or periphery of cells such asUDP-induced phagocytosisand ATP-stimulated chemotactic migration were essentiallyabolished in STIM1minusminusmicrogliaThemigration impairmentsof STIM1minusminus microglia were also observed in the embryonicbrain in vivo The number of aggregated STIM1minusminus microgliainto the LPS-injected lesion for 24 hours was significantlylower than the number of aggregated WT microglia intothe LPS-injected lesion Furthermore the neuron phago-cytotic activity of microglia had significantly diminishedin STIM1minusminus microglia These in vitro and in vivo resultssuggest that STIM1 is crucial for the regulation of local[Ca2+]microdomains in peripheral region ofmicrogliawhichmediates the process of acute motility of murine microglia

2 Materials and Methods

21 Mice STIM1+minus heteromice (C57BL6 background) weregenerated as previously described [19 20] The mice were

bred and maintained according to the Yonsei Medical Cen-ter animal research requirements and all procedures wereapproved by the Committee on Animal Research at YonseiMedical Center (protocol number 2011-0112) Since STIM1minusminusmice die within 1 day after birth due to defect in thecirculatory system 17 day embryos taken off from pregnantSTIM1+minus females mated with STIM1+minus male mice were usedto purify eachWTand STIM1minusminusmicroglia For themicroglialmigration assays in vivo the brains of the same aged embryoswere used Genomic DNA isolated from the embryo tailwas used for genotyping Genomic DNA for genotypingwas extracted using prepGEM Tissue (PTI0200 ZyGEMHamilton New Zealand)

22 Isolation and Culture of Primary Mouse MicroglialCells Astrocytes and Neurons Primary cultures of mousemicroglia were prepared from the whole brain of an embry-onic day 17 (E17) mouse Each embryo tail was used for geno-type analyses to identify WT and STIM1minusminus microglia Pri-mary mixed cells that had been chemically and mechanicallydissociated of the whole brain were seeded in DMEMF12(1 1) culture medium Cells were cultured at 37∘C in anincubator (Thermo Fisher Scientific Marietta OH USA)humidified with 5 CO2 Cell confluency was achievedafter 3sim4 weeks After 4 weeks the primary mixed cellsof WT and STIM1minusminus were used a mild trypsinization andshaking method [19] The upper cell layer of mixed cells wasremoved in one piece by treatingwith trypsin 025 (1x) solu-tion (SH3004201 Hyclone Laboratories Thermo ScientificLogan UT USA) diluted 1 4 in DMEMF12 (1 1) culturemedium (Dulbeccorsquos modified Eagle medium 11320-033Gibco Life Technologies Carlsbad CA USA) containing10 fetal bovine serum (26140-079 Gibco Life TechnologiesCarlsbad CA USA) and 1 penicillin-streptomycin (15140-122 Gibco Life Technologies Carlsbad CA USA) for 20minat 37∘C The trypsin diluted 1 4 in DMEMF12 was suc-tioned and replaced with 12mL of new culture medium Theremained pure microglial cells were attached to the bottomof the T75 flask and were isolated by shaking the T75 flasksof WT STIM1minusminus at 120 rpm (SLOS-20 Seoulin BioscienceSeoul Korea) at 37∘C for 1 h 12mL of each medium werecollected from each flasks placed a cell strainer with 40120583mpore size (352340 BD Falcon San Jose CA USA) filteredinto a fresh 15-mL conical tube and centrifuged at 4000 rpmat room temperature for 5min Glial cells were grown at ahigh density into 75 T flasks to extract puremicroglia at 7 to 9days in vitro After isolate microglia the remaining cells weremaintained in astrocyte-conditionedmedium and remainingmicroglia were depleted by adding 50mM L-leucine-methylester (LME Sigma Aldrich St Louis MO USA) for 4hours To culture the cortical neuron the culture of corticalneuron was prepared from 1 day postnatal pups (C57BL6strain) as described previously [1] with some modificationsThe cerebella were dissociated in Versene solution (1 5000)and plated at 05 times 106 cellscm2 in 24-well plates coatedwith Laminin (10120583gmL in serum-free DMEM) Cells wereincubated in Dulbeccorsquos modified Eaglersquos medium (DMEM)supplemented with heat-inactivated horse serum (5) fetal

















WT mock



50120583m50120583m

50120583m 50120583m

(a)

lowast


WT STIM1minusminus

LPSMock LPSMock0

1

2

3

Relat

ive p

hago

cyto

sis ac

tivity

(fol

d of

WT

con)


ns

WT

LPSMock LPSMock

STIM1minusminus

0

200

400

600

800

120572(p

gm

L)Se

cret

ion

of T

NF-

(c)


STIM1minusminus

ns0

100

200

300

400

500

LPSMock LPSMock

WT

6(p

gm

L)Se

cret

ion

of IL

-

(d)





WTSTIM1minusminus

100120583M UDP2mM Ca2+

0

100

200

300

400

500

1000

Time (Sec)200 300

[Ca2+] i

(nM

)

(a)

WT STIM1minusminus

lowastlowastlowast

000

025

050

075

100

Relat

ive U

DP-

indu

ced

Ca2+

peak

(WT

STIM

1minusminus

)

(b)

WT STIM1minusminus

lowastlowastlowast

000

025

050

075

100

Rela

tiveC

a2+

influ

x (W

T STIM

1minusminus

)

(c)

B

Bead +UDP

FITC-A

FITC-intensity

FITC-A

A

BeadFITC-intensity

FITC-A

D

FITC-intensity

FITC-A

C

FITC-intensity

FITC-A

FITC-intensity

FITC-A


WT

376285

105104103102101100105104103102101100105104103102101100

105104103102101100105104103102101100105104103102101100

296269729

226

0

10

20

30

Cou

nt

0

10

20

30

40

Cou

nt

0

20

40

60

Cou

nt

0

20

40

60

Cou

nt

0

20

40

60

80

Cou

nt

0

10

20

30

Cou

nt

STIM

1minusminus

(d)

Relat

ive p

hago

cyto

sis ac

tivity

(fold

of W

T U

DP)

A C D

ns

lowastlowast

lowastlowast

WT STIM1minusminus

+UDPBead+UDPBead

B00

05

10

15

20

(e)






[Ca2+] i

(nM

)50120583M ATP

WT

50 75 100250

Time (Sec)

0

100

200

300

400

500

STIM1minusminus

(a)

WTSTIM1minusminus

50120583M ATP

lowastlowast


lowast

0

200

400

600

800

1000

Mig

ratin

g ce

ll nu

mbe

r (ou

t of4

times10

4ce

lls)


(b)








Orai1

1120583m

Orai1

1120583m

STIM1

1120583m

STIM1

1120583m

Orai1STIM1

1120583m

Orai1STIM1

1120583m

Orai1STIM1DAPI

5120583m

Orai1STIM1DAPI

5120583m 0

100

200

Inte

nsity

5 10 15 200

Inte

nsity

5 10 15 20 25 3000

50100150200250

Orai1DAPI

DIC

Distance (120583m)

Distance (120583m)

Mock

UDP40min

WT

WT

250

150

50

(a)

Orai1DAPI Orai1

Orai1DAPI Orai1

1120583m

1120583m

5120583m

5120583m

0

100

200

Inte

nsity

0

100

200

Inte

nsity

5 10 15 20 250

5 10 15 20 250

Orai1DAPI

DIC

Distance (120583m)

Distance (120583m)

Mock

UDP40min

STIM

1minusminus

STIM

1minusminus

150

250

250

150

50

50

(b)

Mock UDP Mock UDP

lowast

0

10

20

30

40

50

Perip

hery

loca

lized

Ora

i1 (

of t

otal

Ora

i1)

STIM1minusminusWT

(c)





T

Set 1 Set 2

50120583M

B

10120583M

C

50120583M

D

10120583M

E

50120583M

G

10120583M

H

10120583M

J

50120583M

I

500120583M

No injection

LPS + MB

F

50120583M

L

10120583M

M

50120583M

N

10120583M

O

No injection

LPS + MB 500120583M

K

10120583M

R

50120583M

Q

50120583M

S

10120583M

T

No injection

LPS + MB 500120583M

P

500120583M

No injection

LPS + MB

A

STIM

1minusminus

(a)

ns

ns

Iba1

posit

ive c

ells

Non

inje

cted

Inje

cted

Non

inje

cted

Inje

cted

lowast

lowastlowast

WT STIM1minusminus

0

100

200

300

(b)





4 Discussion










WTmicroglia

200120583m 20120583m

(a)

WT neuron

200120583m 20120583m



STIM1minusminus

microglia

(b)

25120583m2 lt lt 35120583m2

gt 35120583m2


WT STIM1minusminus

lowastlowastlowast

0

20

40

60

80

100

120

( o

f tot

al ac

tivat

ed m

icro

glia

)Re

lativ

e act

ivat

ed m

icro

glia

size

(c)

Rela

tive s

urvi

val n

euro

n

Mock


lowast

lowastlowastlowast

WT STIM1minusminus0

25

50

75

100

125(

of o

nly

neur

on M

AP2

posit

ive c

ells)

(d)








Abbreviations



Competing Interests




Acknowledgments


References





















































WT mock



50120583m50120583m

50120583m 50120583m

(a)

lowast


WT STIM1minusminus

LPSMock LPSMock0

1

2

3

Relat

ive p

hago

cyto

sis ac

tivity

(fol

d of

WT

con)


ns

WT

LPSMock LPSMock

STIM1minusminus

0

200

400

600

800

120572(p

gm

L)Se

cret

ion

of T

NF-

(c)


STIM1minusminus

ns0

100

200

300

400

500

LPSMock LPSMock

WT

6(p

gm

L)Se

cret

ion

of IL

-

(d)





WTSTIM1minusminus

100120583M UDP2mM Ca2+

0

100

200

300

400

500

1000

Time (Sec)200 300

[Ca2+] i

(nM

)

(a)

WT STIM1minusminus

lowastlowastlowast

000

025

050

075

100

Relat

ive U

DP-

indu

ced

Ca2+

peak

(WT

STIM

1minusminus

)

(b)

WT STIM1minusminus

lowastlowastlowast

000

025

050

075

100

Rela

tiveC

a2+

influ

x (W

T STIM

1minusminus

)

(c)

B

Bead +UDP

FITC-A

FITC-intensity

FITC-A

A

BeadFITC-intensity

FITC-A

D

FITC-intensity

FITC-A

C

FITC-intensity

FITC-A

FITC-intensity

FITC-A


WT

376285

105104103102101100105104103102101100105104103102101100

105104103102101100105104103102101100105104103102101100

296269729

226

0

10

20

30

Cou

nt

0

10

20

30

40

Cou

nt

0

20

40

60

Cou

nt

0

20

40

60

Cou

nt

0

20

40

60

80

Cou

nt

0

10

20

30

Cou

nt

STIM

1minusminus

(d)

Relat

ive p

hago

cyto

sis ac

tivity

(fold

of W

T U

DP)

A C D

ns

lowastlowast

lowastlowast

WT STIM1minusminus

+UDPBead+UDPBead

B00

05

10

15

20

(e)






[Ca2+] i

(nM

)50120583M ATP

WT

50 75 100250

Time (Sec)

0

100

200

300

400

500

STIM1minusminus

(a)

WTSTIM1minusminus

50120583M ATP

lowastlowast


lowast

0

200

400

600

800

1000

Mig

ratin

g ce

ll nu

mbe

r (ou

t of4

times10

4ce

lls)


(b)








Orai1

1120583m

Orai1

1120583m

STIM1

1120583m

STIM1

1120583m

Orai1STIM1

1120583m

Orai1STIM1

1120583m

Orai1STIM1DAPI

5120583m

Orai1STIM1DAPI

5120583m 0

100

200

Inte

nsity

5 10 15 200

Inte

nsity

5 10 15 20 25 3000

50100150200250

Orai1DAPI

DIC

Distance (120583m)

Distance (120583m)

Mock

UDP40min

WT

WT

250

150

50

(a)

Orai1DAPI Orai1

Orai1DAPI Orai1

1120583m

1120583m

5120583m

5120583m

0

100

200

Inte

nsity

0

100

200

Inte

nsity

5 10 15 20 250

5 10 15 20 250

Orai1DAPI

DIC

Distance (120583m)

Distance (120583m)

Mock

UDP40min

STIM

1minusminus

STIM

1minusminus

150

250

250

150

50

50

(b)

Mock UDP Mock UDP

lowast

0

10

20

30

40

50

Perip

hery

loca

lized

Ora

i1 (

of t

otal

Ora

i1)

STIM1minusminusWT

(c)





T

Set 1 Set 2

50120583M

B

10120583M

C

50120583M

D

10120583M

E

50120583M

G

10120583M

H

10120583M

J

50120583M

I

500120583M

No injection

LPS + MB

F

50120583M

L

10120583M

M

50120583M

N

10120583M

O

No injection

LPS + MB 500120583M

K

10120583M

R

50120583M

Q

50120583M

S

10120583M

T

No injection

LPS + MB 500120583M

P

500120583M

No injection

LPS + MB

A

STIM

1minusminus

(a)

ns

ns

Iba1

posit

ive c

ells

Non

inje

cted

Inje

cted

Non

inje

cted

Inje

cted

lowast

lowastlowast

WT STIM1minusminus

0

100

200

300

(b)





4 Discussion










WTmicroglia

200120583m 20120583m

(a)

WT neuron

200120583m 20120583m



STIM1minusminus

microglia

(b)

25120583m2 lt lt 35120583m2

gt 35120583m2


WT STIM1minusminus

lowastlowastlowast

0

20

40

60

80

100

120

( o

f tot

al ac

tivat

ed m

icro

glia

)Re

lativ

e act

ivat

ed m

icro

glia

size

(c)

Rela

tive s

urvi

val n

euro

n

Mock


lowast

lowastlowastlowast

WT STIM1minusminus0

25

50

75

100

125(

of o

nly

neur

on M

AP2

posit

ive c

ells)

(d)








Abbreviations



Competing Interests




Acknowledgments


References






































WTSTIM1minusminus

100120583M UDP2mM Ca2+

0

100

200

300

400

500

1000

Time (Sec)200 300

[Ca2+] i

(nM

)

(a)

WT STIM1minusminus

lowastlowastlowast

000

025

050

075

100

Relat

ive U

DP-

indu

ced

Ca2+

peak

(WT

STIM

1minusminus

)

(b)

WT STIM1minusminus

lowastlowastlowast

000

025

050

075

100

Rela

tiveC

a2+

influ

x (W

T STIM

1minusminus

)

(c)

B

Bead +UDP

FITC-A

FITC-intensity

FITC-A

A

BeadFITC-intensity

FITC-A

D

FITC-intensity

FITC-A

C

FITC-intensity

FITC-A

FITC-intensity

FITC-A


WT

376285

105104103102101100105104103102101100105104103102101100

105104103102101100105104103102101100105104103102101100

296269729

226

0

10

20

30

Cou

nt

0

10

20

30

40

Cou

nt

0

20

40

60

Cou

nt

0

20

40

60

Cou

nt

0

20

40

60

80

Cou

nt

0

10

20

30

Cou

nt

STIM

1minusminus

(d)

Relat

ive p

hago

cyto

sis ac

tivity

(fold

of W

T U

DP)

A C D

ns

lowastlowast

lowastlowast

WT STIM1minusminus

+UDPBead+UDPBead

B00

05

10

15

20

(e)






[Ca2+] i

(nM

)50120583M ATP

WT

50 75 100250

Time (Sec)

0

100

200

300

400

500

STIM1minusminus

(a)

WTSTIM1minusminus

50120583M ATP

lowastlowast


lowast

0

200

400

600

800

1000

Mig

ratin

g ce

ll nu

mbe

r (ou

t of4

times10

4ce

lls)


(b)








Orai1

1120583m

Orai1

1120583m

STIM1

1120583m

STIM1

1120583m

Orai1STIM1

1120583m

Orai1STIM1

1120583m

Orai1STIM1DAPI

5120583m

Orai1STIM1DAPI

5120583m 0

100

200

Inte

nsity

5 10 15 200

Inte

nsity

5 10 15 20 25 3000

50100150200250

Orai1DAPI

DIC

Distance (120583m)

Distance (120583m)

Mock

UDP40min

WT

WT

250

150

50

(a)

Orai1DAPI Orai1

Orai1DAPI Orai1

1120583m

1120583m

5120583m

5120583m

0

100

200

Inte

nsity

0

100

200

Inte

nsity

5 10 15 20 250

5 10 15 20 250

Orai1DAPI

DIC

Distance (120583m)

Distance (120583m)

Mock

UDP40min

STIM

1minusminus

STIM

1minusminus

150

250

250

150

50

50

(b)

Mock UDP Mock UDP

lowast

0

10

20

30

40

50

Perip

hery

loca

lized

Ora

i1 (

of t

otal

Ora

i1)

STIM1minusminusWT

(c)





T

Set 1 Set 2

50120583M

B

10120583M

C

50120583M

D

10120583M

E

50120583M

G

10120583M

H

10120583M

J

50120583M

I

500120583M

No injection

LPS + MB

F

50120583M

L

10120583M

M

50120583M

N

10120583M

O

No injection

LPS + MB 500120583M

K

10120583M

R

50120583M

Q

50120583M

S

10120583M

T

No injection

LPS + MB 500120583M

P

500120583M

No injection

LPS + MB

A

STIM

1minusminus

(a)

ns

ns

Iba1

posit

ive c

ells

Non

inje

cted

Inje

cted

Non

inje

cted

Inje

cted

lowast

lowastlowast

WT STIM1minusminus

0

100

200

300

(b)





4 Discussion










WTmicroglia

200120583m 20120583m

(a)

WT neuron

200120583m 20120583m



STIM1minusminus

microglia

(b)

25120583m2 lt lt 35120583m2

gt 35120583m2


WT STIM1minusminus

lowastlowastlowast

0

20

40

60

80

100

120

( o

f tot

al ac

tivat

ed m

icro

glia

)Re

lativ

e act

ivat

ed m

icro

glia

size

(c)

Rela

tive s

urvi

val n

euro

n

Mock


lowast

lowastlowastlowast

WT STIM1minusminus0

25

50

75

100

125(

of o

nly

neur

on M

AP2

posit

ive c

ells)

(d)








Abbreviations



Competing Interests




Acknowledgments


References






































[Ca2+] i

(nM

)50120583M ATP

WT

50 75 100250

Time (Sec)

0

100

200

300

400

500

STIM1minusminus

(a)

WTSTIM1minusminus

50120583M ATP

lowastlowast


lowast

0

200

400

600

800

1000

Mig

ratin

g ce

ll nu

mbe

r (ou

t of4

times10

4ce

lls)


(b)








Orai1

1120583m

Orai1

1120583m

STIM1

1120583m

STIM1

1120583m

Orai1STIM1

1120583m

Orai1STIM1

1120583m

Orai1STIM1DAPI

5120583m

Orai1STIM1DAPI

5120583m 0

100

200

Inte

nsity

5 10 15 200

Inte

nsity

5 10 15 20 25 3000

50100150200250

Orai1DAPI

DIC

Distance (120583m)

Distance (120583m)

Mock

UDP40min

WT

WT

250

150

50

(a)

Orai1DAPI Orai1

Orai1DAPI Orai1

1120583m

1120583m

5120583m

5120583m

0

100

200

Inte

nsity

0

100

200

Inte

nsity

5 10 15 20 250

5 10 15 20 250

Orai1DAPI

DIC

Distance (120583m)

Distance (120583m)

Mock

UDP40min

STIM

1minusminus

STIM

1minusminus

150

250

250

150

50

50

(b)

Mock UDP Mock UDP

lowast

0

10

20

30

40

50

Perip

hery

loca

lized

Ora

i1 (

of t

otal

Ora

i1)

STIM1minusminusWT

(c)





T

Set 1 Set 2

50120583M

B

10120583M

C

50120583M

D

10120583M

E

50120583M

G

10120583M

H

10120583M

J

50120583M

I

500120583M

No injection

LPS + MB

F

50120583M

L

10120583M

M

50120583M

N

10120583M

O

No injection

LPS + MB 500120583M

K

10120583M

R

50120583M

Q

50120583M

S

10120583M

T

No injection

LPS + MB 500120583M

P

500120583M

No injection

LPS + MB

A

STIM

1minusminus

(a)

ns

ns

Iba1

posit

ive c

ells

Non

inje

cted

Inje

cted

Non

inje

cted

Inje

cted

lowast

lowastlowast

WT STIM1minusminus

0

100

200

300

(b)





4 Discussion










WTmicroglia

200120583m 20120583m

(a)

WT neuron

200120583m 20120583m



STIM1minusminus

microglia

(b)

25120583m2 lt lt 35120583m2

gt 35120583m2


WT STIM1minusminus

lowastlowastlowast

0

20

40

60

80

100

120

( o

f tot

al ac

tivat

ed m

icro

glia

)Re

lativ

e act

ivat

ed m

icro

glia

size

(c)

Rela

tive s

urvi

val n

euro

n

Mock


lowast

lowastlowastlowast

WT STIM1minusminus0

25

50

75

100

125(

of o

nly

neur

on M

AP2

posit

ive c

ells)

(d)








Abbreviations



Competing Interests




Acknowledgments


References






































Orai1

1120583m

Orai1

1120583m

STIM1

1120583m

STIM1

1120583m

Orai1STIM1

1120583m

Orai1STIM1

1120583m

Orai1STIM1DAPI

5120583m

Orai1STIM1DAPI

5120583m 0

100

200

Inte

nsity

5 10 15 200

Inte

nsity

5 10 15 20 25 3000

50100150200250

Orai1DAPI

DIC

Distance (120583m)

Distance (120583m)

Mock

UDP40min

WT

WT

250

150

50

(a)

Orai1DAPI Orai1

Orai1DAPI Orai1

1120583m

1120583m

5120583m

5120583m

0

100

200

Inte

nsity

0

100

200

Inte

nsity

5 10 15 20 250

5 10 15 20 250

Orai1DAPI

DIC

Distance (120583m)

Distance (120583m)

Mock

UDP40min

STIM

1minusminus

STIM

1minusminus

150

250

250

150

50

50

(b)

Mock UDP Mock UDP

lowast

0

10

20

30

40

50

Perip

hery

loca

lized

Ora

i1 (

of t

otal

Ora

i1)

STIM1minusminusWT

(c)





T

Set 1 Set 2

50120583M

B

10120583M

C

50120583M

D

10120583M

E

50120583M

G

10120583M

H

10120583M

J

50120583M

I

500120583M

No injection

LPS + MB

F

50120583M

L

10120583M

M

50120583M

N

10120583M

O

No injection

LPS + MB 500120583M

K

10120583M

R

50120583M

Q

50120583M

S

10120583M

T

No injection

LPS + MB 500120583M

P

500120583M

No injection

LPS + MB

A

STIM

1minusminus

(a)

ns

ns

Iba1

posit

ive c

ells

Non

inje

cted

Inje

cted

Non

inje

cted

Inje

cted

lowast

lowastlowast

WT STIM1minusminus

0

100

200

300

(b)





4 Discussion










WTmicroglia

200120583m 20120583m

(a)

WT neuron

200120583m 20120583m



STIM1minusminus

microglia

(b)

25120583m2 lt lt 35120583m2

gt 35120583m2


WT STIM1minusminus

lowastlowastlowast

0

20

40

60

80

100

120

( o

f tot

al ac

tivat

ed m

icro

glia

)Re

lativ

e act

ivat

ed m

icro

glia

size

(c)

Rela

tive s

urvi

val n

euro

n

Mock


lowast

lowastlowastlowast

WT STIM1minusminus0

25

50

75

100

125(

of o

nly

neur

on M

AP2

posit

ive c

ells)

(d)








Abbreviations



Competing Interests




Acknowledgments


References






































T

Set 1 Set 2

50120583M

B

10120583M

C

50120583M

D

10120583M

E

50120583M

G

10120583M

H

10120583M

J

50120583M

I

500120583M

No injection

LPS + MB

F

50120583M

L

10120583M

M

50120583M

N

10120583M

O

No injection

LPS + MB 500120583M

K

10120583M

R

50120583M

Q

50120583M

S

10120583M

T

No injection

LPS + MB 500120583M

P

500120583M

No injection

LPS + MB

A

STIM

1minusminus

(a)

ns

ns

Iba1

posit

ive c

ells

Non

inje

cted

Inje

cted

Non

inje

cted

Inje

cted

lowast

lowastlowast

WT STIM1minusminus

0

100

200

300

(b)





4 Discussion










WTmicroglia

200120583m 20120583m

(a)

WT neuron

200120583m 20120583m



STIM1minusminus

microglia

(b)

25120583m2 lt lt 35120583m2

gt 35120583m2


WT STIM1minusminus

lowastlowastlowast

0

20

40

60

80

100

120

( o

f tot

al ac

tivat

ed m

icro

glia

)Re

lativ

e act

ivat

ed m

icro

glia

size

(c)

Rela

tive s

urvi

val n

euro

n

Mock


lowast

lowastlowastlowast

WT STIM1minusminus0

25

50

75

100

125(

of o

nly

neur

on M

AP2

posit

ive c

ells)

(d)








Abbreviations



Competing Interests




Acknowledgments


References








































4 Discussion










WTmicroglia

200120583m 20120583m

(a)

WT neuron

200120583m 20120583m



STIM1minusminus

microglia

(b)

25120583m2 lt lt 35120583m2

gt 35120583m2


WT STIM1minusminus

lowastlowastlowast

0

20

40

60

80

100

120

( o

f tot

al ac

tivat

ed m

icro

glia

)Re

lativ

e act

ivat

ed m

icro

glia

size

(c)

Rela

tive s

urvi

val n

euro

n

Mock


lowast

lowastlowastlowast

WT STIM1minusminus0

25

50

75

100

125(

of o

nly

neur

on M

AP2

posit

ive c

ells)

(d)








Abbreviations



Competing Interests




Acknowledgments


References








































WTmicroglia

200120583m 20120583m

(a)

WT neuron

200120583m 20120583m



STIM1minusminus

microglia

(b)

25120583m2 lt lt 35120583m2

gt 35120583m2


WT STIM1minusminus

lowastlowastlowast

0

20

40

60

80

100

120

( o

f tot

al ac

tivat

ed m

icro

glia

)Re

lativ

e act

ivat

ed m

icro

glia

size

(c)

Rela

tive s

urvi

val n

euro

n

Mock


lowast

lowastlowastlowast

WT STIM1minusminus0

25

50

75

100

125(

of o

nly

neur

on M

AP2

posit

ive c

ells)

(d)








Abbreviations



Competing Interests




Acknowledgments


References











































Abbreviations



Competing Interests




Acknowledgments


References





































udp-induced phagocytosis and atp-stimulated chemotactic … · 2019-09-04 · researcharticle...

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