Central nervous system (CNS) injury initializes a reactive tissue response, which is characterized by a cascade of signals that result in inflammatory cytokine release and neuroinflammation. These signals are induced in part by activated microglia that migrates to the injury site in an attempt to remove dead tissue and promote healing. The ability to control the reactive tissue response is of significant importance to a variety of applications, such as neuroprostheses, whose functional lifespan is limited by glial cell activation. A possible strategy to mitigate glial cell activation is to reduce the release of inflammatory cytokines following a brain injury. One pathway that facilitates inflammatory cytokine release is the mitogen-activated protein kinase-activated protein kinase 2 (MK2) pathway, which increases cytokine mRNA synthesis, stability and translation following activation. Therefore, inhibiting MK2-mediated cytokine release can reduce microglial activationfollowing CNS injury. Through in vitro studies, we demonstrate the ability of a cell-penetrating peptide inhibitor of MK2 (MK2i) to reduce MK2-mediated cytokine release in mixed cortical cultures. Immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), and cytotoxicity assays in 7- -10-day-old E17 Sprague-Dawley rat mixed cortical cultures showed that MK2i treatment significantly lowered inflammatory cytokine production and increased cortical cell viability following glial cell activation with tumor necrosis factor- α (TNF- α ) and lipopolysaccharide (LPS). Results suggest that MK2i may reduce damage caused by activated glia in the inflamed CNS.
During the brain’s initial injury response, the blood-brain barrier is broken, resulting in hypoxia, injury, and eventual death of neuronal and glial cells [
One possible way to reduce glial cell activation and neuroinflammation is through the down-regulation of inflammatory cytokines. While there is evidence that overexpression of cytokines, such as IL-1β, IL-6, and TNF-α, leads to a more intense secondary injury response [
One enzyme involved in inflammatory cytokine release is mitogen-activated protein kinase-activated protein kinase 2 (MK2), which plays a direct role in inflammatory cytokine upregulation in the brain [
Protein kinase regulation can be accomplished through the use of substrate-based inhibiting peptides [
Embryonic rat cortical tissue was obtained from E17 Sprague-Dawley rats immediately following removal of embryos from a pregnant rat’s abdomen or was purchased from BrainBits, LLC (Springfield, IL). This tissue was placed in a 50 mL conical tube containing 5 mL of Solution 1 (NaCl 7.24 g/L; KCl 0.4 g/L; NaH2PO4 0.14 g/L; Glucose 2.61 g/L; Hepes 5.96 g/L; MgSO4 0.295 g/L; Bovine Serum Albumin 3 g/L) or 5 mL of Hibernate E (BrainBits, Springfield, IL). Under sterile conditions, 18 µL of trypsin solution (7.5 mg/mL in 0.9% saline) was added and tissue was passed through a 5 mL pipet several times to disassociate tissue. After the conical tube was placed in a 37˚C water bath for 20 minutes, 100 µL of trypsin inhibitor/DNAse solution (2.5 mg/mL trypsin inhibitor, 400 µg/mL DNase in 0.9% saline) was added to the tube and the tissue was pipetted several times with a 5-mL pipet. Tissue was centrifuged at 1000 rpm for 5 minutes at room temperature and supernatant was poured off. Cells were re-suspended in 16 mL of Hibernate E and 100 µL of trypsin inhibitor/DNAse solution and pipetted up and down several times. Cells were filtered through a cell strainer and centrifuged at 1400 rpm for 5 min at room temperature. Supernatant was poured off and cells were re-suspended in media. Primary cells were then plated in poly-D-lysine-coated 96-well plates at a seeding density of 625,000 cells/cm2 and incubated for 7 - 9 days at 37˚C, with media being changed every other day. After 7 - 9 days, separate groups of cortical cells were treated with 5 or 10 ng/mL TNF-α or 50 ng/mL LPS + cell media solution, respectively, for 4, 8, and 24 hours to activate microglia. Separate groups of cortical cells were also treated with a 0.5, 1, or 3 mM MK2i + 5 or 10 ng/mL TNF-α or 50 ng/mL LPS + cell media solution for 4, 8, and 24 hours. Additionally, a separate group of cortical cells received only cell media to determine basal cytokine levels. For a given time point, all treatments were conducted on cortical cells from the same embryonic dissection. After treatments, media was removed and stored at −80˚C.
Cell media was replaced with phosphate-buffered 4% formaldehyde solution (pH 7.4) for 5 minutes, followed by three washes using HEPES-buffered Hanks’ saline containing 10 mg/L sodium azide; pH 7.4 (HBHS). A Triton X-100 (Sigma-Aldrich, St. Louis, MO) solution (0.25% by volume in HBHS) was applied for 10 minutes, followed by three washes in HBHS. Blocking solution, consisting of 10% normal goat serum in HBHS (V/V), was then applied for 30 minutes, followed by three washes with HBHS. Microglia, astrocytes, and neurons were labeled using antibodies against ionized calcium binding adaptor molecule 1 (Iba1) (019-19741, Wako Pure Chemical Industries, Osaka, Japan), glial fibrillary acidic protein (GFAP) (AB5541, Millipore, Temecula, CA), and β-3-tubulin (β-3-tub) (MMS-435P, Covance, Princeton, NJ), respectively. Antibodies were applied for 2 hours, at a dilution of 1:400 in HBHS. After two 10 minute washes and one 30 minute wash, secondary antibodies were applied. Alexa fluor 488 goat anti-mouse, Alexa fluor 555 goat anti-chicken, and Alexa fluor 635 goat anti-rabbit (all from Invitrogen) were applied at 1:400, along with Hoechst 33,342 at 1:10,000 (Invitrogen, Eugene, OR), in HBHS for 2 hours. All immunocytochemistry procedures were performed at room temperature. Cells were stored away from light at 4˚C in HBHS prior to imaging.
Cell imaging was accomplished using an Olympus IX81 microscope equipped with Olympus FV1000 laser confocal system, through Olympus 10×/0.40 air and 40×/0.80 water emersion objectives (Olympus America, Center Valley, PA). Image channels were collected sequentially using 488 nm, 543 nm, and 633 nm laser lines (CVI Melles Griot, Carlsbad, CA), along with a tunable MaiTai laser (Spectra-Physics/Newport, Santa Clara, CA) set to 740 nm. Two-photon excitation of Hoechst 33,342 was driven by this 740 nm excitation, and the emission was collected by an external PMT (R3896, Hamamatsu, Bridgewater, NJ) equipped with a 405/40 nm filter (Chroma, Rockingham, VT). Internal detectors collected all other channels. A wide aperture setting (400 µm) was used to capture representative images from the labeled cultures; laser power and PMT voltage settings were held constant. Related image-channel levels were set equal, allowing visual comparison of fluorescent intensity, using Olympus Fluoview V1.7 software. Microglia cell number quantification was accomplished using the CellCount for the public domain program ImageJ (National Institutes of Health, Bethesda, MD), with two 1272 × 1272 µm images counted per treatment. Fluorescent intensity quantification was performed by measuring mean intensity across 800 × 800 pixel (1272 × 1272 µm) raw images (two per treatment) using the analysis function in ImageJ, then averaging the mean intensities within each treatment. Scale bars were added and figure layouts were designed using Photoshop CS2 (Adobe Systems, San Jose, CA). Statistical analysis was conducted using Statistical Analysis Software (SAS, Cary, NC) version 9.2.
IL-6 ELISAs were conducted to quantify changes in inflammatory cytokine production using the Rat IL-6 ELISA Development Kit (Peprotech Inc., Rocky Hill, NJ). 100 µL of 1 µg/mL antigen-affinity purified goat anti-rat IL-6 was added to ELISA microplate wells (NuncMaxisorp, Rochester, NY) and incubated at room temperature overnight. Wells were washed 4 times with 300 µL wash buffer (0.05% Tween-20 in 1x phosphate buffered saline (PBS)). 300 µL block buffer (1% BSA in 1x PBS) was added to each well and incubated for 1 hour at room temperature. Block buffer was aspirated and wells were washed 4 times with wash buffer. 8 ng/mL recombinant IL-6 was then diluted to zero in diluent (0.05% Tween-20, 0.1% BSA in 1x PBS). 100 µL of diluted recombinant IL-6 solutions were added in triplicate into microplate wells. 100 µL of media from all treatments were also added to microplate wells in sextuplicate. Microplates were then incubated at room temperature for 2 hours, aspirated, and washed 4 times with wash buffer. 100 µL of 0.25 µg/mL biotinylated antigen-affinity purified goat anti-rat IL-6 was added to microplate wells and incubated for 2 hours. Microplates were aspirated and washed 4 times with wash buffer. 6 µL Avidin Peroxidase 1:2000 was diluted in 12 mL diluent. 100 µL of this solution was added to wells and incubated for 30 minutes at room temperature, aspirated, and washed 4 times with wash buffer. 100 µL of 2,2’-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) Liquid Substrate Solution (Sigma-Aldrich, St. Louis, MO) was added to each well. Absorbance was then measured using a Spectramax M5 Microplate Reader (Molecular Devices, Sunnyvale, CA). Absorbance values were monitored at 5-minute intervals for 50 minutes.
IL-1β ELISAs were conducted to quantify changes in inflammatory cytokine production using the Rat IL-1β ELISA Development Kit (Peprotech Inc., Rocky Hill, NJ). Methods were identical to those of the IL-6 ELISA assays. Absorbance values were monitored at 5-minute intervals for 25 minutes.
Cytokine assays were conducted to quantify changes in inflammatory cytokine production using Proinflammatory Panel 1 (rat) Kits (Meso Scale Discovery, Rockville, MD). 150 µL of Blocker H was added into each well and shaken at 700 rpm for 1 hour. The 96-well MSD plate was then washed three times with 150 µL of wash buffer (1x PBS + 0.05% Tween 20) per well and 50 µL of calibrator or sample solution was added into each well of the plate. The plate was then sealed with adhesive tape and shaken at 700 rpm for 2 hours at room temperature. After three washes with wash buffer, the detection antibody was then added (25 µL/well) and the plate was again sealed and shaken at 700 rpm for 2 hours. After three more washes with wash buffer, 150 µL of 2× read buffer was added to each well and the plate was analyzed using Sector Imager 2400A (Meso Scale Discovery, Rockville, MD). Both IL-6 and IL-1β were examined in this study and data were analyzed using MSD Discovery Workbench Software.
Live-dead assays were conducted using the Molecular Probe LIVE/DEAD® Viability/Cytotoxicity Kit for Mammalian Cells to quantify cell death in cortical cell cultures following all treatments. A sample size of six was used for each treatment. Prior to experiments, optimal concentration of the live cell dye, Calcein-AM (CA), and the dead cell dye, Ethidium-1 (EthD-1), was determined to be 6 µM. Also, optimal time for dye incubation was 30 minutes.
One-half hour before the 24-hour time point, untreated cultures were killed with a 30-minute 70% ethanol treatment, then washed twice with 250 µL 1x PBS. One group of control dead cells received 100 µL of the 6 µM CA solution to determine the background fluorescence of CA. A second group of control dead cells received 100 µL of the 6 µM EthD-1 solution to determine the maximum fluorescence for EthD-1. Conversely, one group of control live cells received 100 µL of the 6 µM EthD-1 solution to determine the background fluorescence of EthD-1, while a second group of control live cells received 100 µL of the 6 µM CA solution to determine the maximum fluorescence for CA.
At the 24 hour time point, cultures were washed twice with 250 µL 1x PBS. Then, 100 µL of a 6 µM EthD- 1/6 µM CA working solution was added to each treated well. Fluorescence was then measured using a Spectramax M5 Microplate Reader (Molecular Devices). EthD-1 required an excitation wavelength of 530 nm and an emission wavelength of 645 nm. CA required an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
The following equation was used to calculate the ramification index (RI) of individual microglia [
Cell images were obtained as previously described and, using Image J, microglia imaged from each treatment well was carefully traced. Subsequently, Image J was used to calculate cell area and cell perimeter for each tracing. These values were then utilized to obtain an average RI for each treatment group using the above equation.
Cortical cells were grown for 1 week in 30 mm Petri dishes, followed by 24-hour MK2i treatment. Media was removed and cells were rinsed two times with ice-cold Tris-Buffered Saline (TBS). 100 µL of lysis buffer (0.54 g/mL 9M Urea, 0.04 g/mL CHAPS, 10 µL Sigma Phosphatase Inhibitor Cocktail 1/mL) was added to dishes and cells were scraped to a corner of the dish using a cell scraper. Cells were added to a chilled, 1.5 mL LoBind protein tube (Sigma Aldrich, St. Louis, MO) and frozen at −80˚C. Thawed cells were vortexed using the Disruptor Genie (Scientific Industries #SI D236, Bohemia, NY) for three hours at 4˚C, then centrifuged at 20, 160 Relative Centrifugal Force (RCF) for 20 minutes at 4˚C. Supernatant was collected into a LoBind tube and frozen at −80˚C. The bicinchoninic assay (BCA) Protein Assay Kit (Pierce #PI23225) was used to run a total protein assay on thawed supernatant. Protein standards were made using bovine serum albumin (BSA). 25 µL of each standard and sample were pipetted in triplicate into a 96-well microplate. 200 µL of working reagent was added to each well and mixed thoroughly on a plate shaker for 30 seconds. The microplate was then covered, incubated at 37˚C for 30 minutes, and cooled at room temperature. The absorbance of each well was then measured at 562 nm using a Spectramax M5 Microplate Reader (Molecular Devices, Sunnyvale, CA).
Following the protein assay, 15 µg of total protein was added to aliquot tubes and diluted with 50 µL Beta Mercaptoethanol (Bio-Rad #161-0710, Hercules, CA) and 950 µL Laemmli Sample Buffer. Tubes were heated for 5 minutes at 100˚C, vortexed, and centrifuged. 2 µL of the solution was then loaded into Tris-hydrochloride (Tris-HCl) Bio-Rad gel lanes, and the gel was run. Polyvinylidenefluoride (PVDF) membranes (Bio-Rad #162- 0174) were soaked in methanol for 1 - 2 minutes in preparation for gel transfer. Gels were then transferred to the PVDF membrane using a Trans-Blot SD System (Bio-Rad #1703848) and placed into a blocking buffer solution (Odyssey, #927-40000) in a cold room overnight on a rocking platform (VWR, Rocking Platform Model 200). The membrane was washed twice with TBS, with each wash preceding a 15-minute incubation on the rocking platform.
Primary antibodies for Beta Actin (Abcam-ab8226, Cambridge, England) and MK2 (Abcam-ab32567) were added to a 5 mL solution of TBS, 0.05% Tween-20, and 0.25% BSA. The solution and membrane were placed in a plastic pouch, sealed and incubated on a rocking platform overnight at 4˚C. The membrane was removed and rinsed four times with a 1× TBS +0.05% Tween-20 solution. Secondary antibodies were then added to a 5 mL solution of TBS, 0.05% Tween-20, and 0.25% BSA. The solution and membrane were placed in a plastic pouch, sealed and incubated in the dark on a rocking platform for 1 hour at room temperature. The membrane was removed, rinsed four times with a 1× TBS +0.05% Tween-20 solution, and washed with 1× TBS for 15 minutes on a rocking platform to remove residual Tween-20. The membrane was then incubated in stripping buffer (15 g glycine, 1 g SDS, 10 mL Tween-20) for 10 - 15 minutes, rinsed with 1× TBS +0.05% Tween-20 for 5 minutes, and rinsed with TBS. Protein-antibody complexes were visualized using an Odyssey Imaging System. Complexes were quantified by measuring the intensity of each blot and normalizing MK2 protein expression to the β-actin control.
Data analysis for all experiments was conducted with Statistical Analysis Software (SAS, Cary, NC) version 9.2. First, Dunnett’s method for multiple pairwise comparisons was used to determine the effect of TNF-α and LPS treatments on IL-6 concentration (pg/mL) compared to untreated cells (control). Control sample means were compared to sample means from 5 ng/mL and 10 ng/mL TNF-α and 50 ng/mL LPS treatments. No significant difference in IL-6 concentration was shown when comparing untreated controls to 5 or 10 ng/mL TNF-α treatments and therefore, 5 or 10 ng/mL TNF-α treatments were used as two controls for two separate sets of experiments. However, a significant difference in IL-6 concentration was shown when comparing untreated controls to 50 ng/mL LPS treatments and therefore, untreated cells were used as a control in those experiments. Dunnett’s method for multiple pairwise comparisons was then used to compare the effect of TNF-α + MK2i treatment on IL-6 concentration to the TNF-α controls. Sample means from 5 ng/mL TNF-α treatment were compared to sample means following 5 ng/mL + 0.5 mM MK2i, 5 ng/mL + 1 mM MK2i, and 5 ng/mL + 3 mM MK2i treatment. Similarly, sample means from 10 ng/mL TNF-α treatment were compared to sample means following 10 ng/mL + 0.5 mM MK2i, 10 ng/mL + 1 mM MK2i, and 10 ng/mL + 3 mM MK2i treatment. Dunnett’s method for multiple pairwise comparisons was also used to compare the effect of LPS + MK2i treatment on IL-6 concentration to the untreated controls. Sample means from untreated controls and 50 ng/mL LPS treat- ment were compared to sample means following 50 ng/mL + 0.5 mM MK2i and 50 ng/mL + 1 mM MK2i treatment. IL-1β ELISA analysis was identical to IL-6 ELISA analysis.
Next, Dunnett’s method for multiple pairwise comparisons was used to determine the effect of TNF-α treatment on the percentage of dead cortical cells (%Dead) compared to untreated cells (control). Control sample means were compared to sample means from 5 ng/mL and 10 ng/mL TNF-α treatments. No significant difference in %Dead was shown when comparing untreated controls to 5 or 10 ng/mL TNF-α treatment. Therefore, 5 or 10 ng/mL TNF-α treatments were used as two controls for two separate sets of experiments. Dunnett’s method for multiple pairwise comparisons was then used to compare the effect of TNF-α + MK2i treatment on %Dead to the TNF-α controls. Sample means from 5 ng/mL TNF-α treatment were compared with sample means following 5 ng/mL + 0.5 mM MK2i, 5 ng/mL + 1 mM MK2i, and 5 ng/mL + 3 mM MK2i treatment. Similarly, sample means from 10 ng/mL TNF-α treatment were compared to sample means following 10 ng/mL + 0.5 mM MK2i, 10 ng/mL + 1 mM MK2i, and 10 ng/mL + 3 mM MK2i treatment.
Next, Dunnett’s method for multiple pairwise comparisons was used to determine the effect of TNF-α treatment on microglia cell number compared to untreated cells (control). Control sample means were compared to sample means from 5 ng/mL and 10 ng/mL TNF-α treatments. No significant difference in microglia cell number was shown following treatment with 5 or 10 ng/mL TNF-α. Therefore, 5 or 10 ng/mL TNF-α treatments were used as two controls for two separate sets of experiments. Dunnett’s method for multiple pairwise comparisons was then used to compare the effect of TNF-α + MK2i treatment on microglia cell number to the TNF-α controls. Sample cell number means from 5 ng/mL TNF-α treatment were compared to sample means following 5 ng/mL + 0.5 mM MK2i, 5 ng/mL + 1 mM MK2i, and 5 ng/mL + 3 mM MK2i treatment. Similarly, sample means from 10 ng/mL TNF-α treatment were compared to sample means following 10 ng/mL + 0.5 mM MK2i, 10 ng/mL + 1 mM MK2i, and 10 ng/mL + 3 mM MK2i treatment. Β3-tubulin, GFAP, and Iba1 fluorescence analysis was identical to Iba1 cell number analysis.
Immunocytochemical images confirm the presence of microglia (Iba1), neurons (β-3-tub), and astrocytes (GFAP) in 1-week old mixed cortical cultures (
Dunnett’s method was used to compare the effect of TNF-α + MK2i treatment on IL-6 concentration to TNF-α treatmentalone. For the 5 and 10 ng/mL TNF-α controls, analysis showed that mean IL-6 concentrations were significantly lowered when TNF-α treatment was combined with 0.5, 1, or 3 mM MK2i for 4, 8, or 24 hours (Figures 2(a)-(c)).
Dunnett’s method was also used to compare the effect of TNF-α + MK2i treatment on IL-1β concentration to
TNF-α treatmentalone (Figures 2(d)-(f)). For the 5 ng/mL TNF-α controls, mean IL-1β concentrations were significantly lowered when TNF-α treatment was combined with 0.5, 1, or 3 mM MK2i for 4, 8, and 24 hours. For the 10 ng/mL TNF-α controls, mean IL-1β concentrations were significantly lowered when TNF-α treatment was combined with 0.5, 1, and 3 mM MK2i for 4 and 8 hours. After 24 hours, mean IL-1β concentrations were also lowered when TNF-α treatment was combined with 0.5, 1, and 3 mM MK2i; however, only 3 mM MK2i produced a statistically significant decrease. It should be noted that the concentrations of TNF-α used in these experiments were chosen for their ability to upregulate inflammatory cytokine expression as lower concentrations of TNF-α showed no significant effects (data not shown).
Dunnett’s method was used to compare the effect of LPS + MK2i treatment on IL-6 and IL-1β concentrations to untreated controls and LPS treatment. Results were similar to TNF-α stimulated cultures as LPS combined with 0.5 and 1 mM MK2i lowered IL-6 and IL-1β concentrations compared to LPS treatment alone; however, only a select number of specific time points and concentrations showed a statistically significant decrease (data not shown). The ability of MK2i to downregulate cytokine concentration following LPS stimulation is consistent with our previously published work [
Dunnett’s method was used to compare the effect of TNF-α + MK2i treatment on the percentage of dead cortical
cells to TNF-α treatment. In
Untreated microglia displayed an amoeboid or round morphology with high Iba1 intensity in the center of the
cell (
To further quantify these microglia observations, the Ramification Index (RI) was calculated and used to compare TNF-α + MK2i treatments to untreated controls and TNF-α only treatments. 5 and 10 ng/mL TNF-α treatments significantly lowered RI compared to untreated controls, indicating a more branched appearance (
Treatments of 5 and 10 ng/mL TNF-α resulted in increased neuronal cell death while treatments of TNF-α + 0.5 or 1 mM MK2i suppressed the neuronal cell death (
Analysis of image data showed no significant differences in Iba1, β3-tubulin, or GFAP fluorescence intensity following 24-hour TNF-α + MK2i treatments (data not shown). There was also no significant change in microglia cell number following 24-hour TNF-α + MK2i treatments (data not shown). These results confirm a similar concentration of cell-type specific proteins within each culture following various treatments.
Western blot analysis determined the effect of the MK2i peptide on MK2 protein expression. Comparison of control blots to treatment blots showed no significant reduction in MK2 expression following 24 hour 0.5 mM MK2i treatment (
MK2i is a novel type of cell-penetrating peptide (CPP). CPPs can be utilized to deliver bioactive molecules into cells. Frankel and Pabo’s discovery of the TAT HIV protein’s ability to be rapidly taken up by cells launched a
great interest into the therapeutic use of TAT to deliver compounds across the cell membrane [
matory cytokine production without causing neuronal cell damage after microglial activation in vitro. The images of microglia, probed with Iba-1, and the calculated ramification index show that MK2i suppresses microglial spreading induced by treatment with TNF-a. This is consistent with the understanding that non-activated embryonic microglia are naturally amoeboid in shape and branch out when they mature or become activated [
While the use of MK2i focuses on inhibiting inflammatory cytokine release from microglia, there are other mechanisms that may play a role in the neuronal cell death and glial cell activation following a brain injury. The release of the interferon-γ cytokine from injured neurons introduces β-Amyloid, which induces microglia nitric oxide production. This cascade of reactions inhibits respiratory enzymes, oxidizes the SH group of proteins, and enhances DNA injury, resulting in neuronal cell death [
Activated microglia also expresses a variety of chemokines that play a role in migration and communication [
Microglia and astrocytes also have Toll-like receptors (TLRs) that recognize host molecules from damaged tissues [
Through in vitro studies, we demonstrate the ability of a novel cell-penetrating peptide (MK2i) to down-regulate, but not completely eliminate MK2-mediated cytokine expression following glial cell activationin mixed cortical cell cultures. MK2i was also shown to increase cell viability, reduce microglial activation and elicit a possible neural protective effect. Given the critical role of cytokine production in chronic neuroinflammation and glial scar formation, these results demonstrate the potential of MK2i as a therapeutic to reduce glial activation following CNS trauma, such as that which occurs upon implantation of neuroprosthetic devices.
The authors would like to thank the Purdue Research Foundation, the Indiana Spinal Cord and Brain Injury Research Board (grant #00014975), and the National Science Foundation (NSF CCLI-0728668) for their financial support, as well as the Weldon School of Biomedical Engineering and the Purdue Graduate School.