Interleukin-33 treatment reduces secondary injury and improves functional recovery after contusion spinal cord injury.

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Interleukin-33 treatment reduces secondary injury and improves functional recovery after contusion spinal cord injury

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Yuriy Pomeshchik 1, Iurii Kidin 1, Paula Korhonen, Ekaterina Savchenko, Sara Wojciechowski, Katja Kanninen, Jari Koistinaho ⇑, Tarja Malm

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Department of Neurobiology, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland

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Article history: Received 22 April 2014 Received in revised form 11 August 2014 Accepted 12 August 2014 Available online xxxx Keywords: IL-33 SCI Neuroinflammation Astrocytes Cytokines Macrophages

a b s t r a c t Interleukin-33 (IL-33) is a member of the interleukin-1 cytokine family and highly expressed in the naïve mouse brain and spinal cord. Despite the fact that IL-33 is known to be inducible by various inflammatory stimuli, its cellular localization in the central nervous system and role in pathological conditions is controversial. Administration of recombinant IL-33 has been shown to attenuate experimental autoimmune encephalomyelitis progression in one study, yet contradictory reports also exist. Here we investigated for the first time the pattern of IL-33 expression in the contused mouse spinal cord and demonstrated that after spinal cord injury (SCI) IL-33 was up-regulated and exhibited a nuclear localization predominantly in astrocytes. Importantly, we found that treatment with recombinant IL-33 alleviated secondary damage by significantly decreasing tissue loss, demyelination and astrogliosis in the contused mouse spinal cord, resulting in dramatically improved functional recovery. We identified both central and peripheral mechanisms of IL-33 action. In spinal cord, IL-33 treatment reduced the expression of pro-inflammatory tumor necrosis factor-alpha and promoted the activation of anti-inflammatory arginase-1 positive M2 microglia/macrophages, which chronically persisted in the injured spinal cord for up to at least 42 days after the treatment. In addition, IL-33 treatment showed a tendency towards reduced T-cell infiltration into the spinal cord. In the periphery, IL-33 treatment induced a shift towards the Th2 type cytokine profile and reduced the percentage and absolute number of cytotoxic, tumor necrosis factor-alpha expressing CD4+ cells in the spleen. Additionally, IL-33 treatment increased expression of T-regulatory cell marker FoxP3 and reduced expression of M1 marker iNOS in the spleen. Taken together, these results provide the first evidence that IL-33 administration is beneficial after CNS trauma. Treatment with IL33 may offer a novel therapeutic strategy for patients with acute contusion SCI. Ó 2014 Published by Elsevier Inc.

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1. Introduction

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Primary damage after spinal cord injury (SCI) is followed by a wave of secondary inflammatory, apoptotic and degenerative

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Abbreviations: BMS, Basso Mouse Scale; BSA, bovine serum albumin; CBA, cytometric bead assay; CNS, central nervous system; dpi, day post injury; EAE, experimental autoimmune encephalomyelitis; FBS, fetal bovine serum; FoxP3, forkhead box P3; GFAP, glial fibrillary acidic protein; HBSS, Hank’s balanced salt solution; Iba-1, ionized calcium-binding adapter molecule 1; IL, interleukin; IL1RAcP, IL-1 receptor accessory protein; IFN-c, interferon gamma; LFB, Luxol Fast Blue; NF-jB, nuclear factor kappa-B; PFA, paraformaldehyde; SCI, spinal cord injury; Tregs, T regulatory cells; TNF-a, tumor necrosis factor alpha; Th1, type 1 helper T-cell; Th2, type 2 helper T cell. ⇑ Corresponding author. Tel.: +358 403552427; fax: +358 17163030. E-mail address: jari.koistinaho@uef.fi (J. Koistinaho). 1 Equally contributing authors.

changes that greatly increase neurological deficits and complicate the restoration of spinal cord functions (Tator, 1995; Kwon et al., 2004; Rowland et al., 2008; Oyinbo, 2011; Oudega, 2013). Numerous cell types participate in a highly complex inflammatory response after SCI. Although inflammatory cells are known to promote the secondary damage via generation of reactive oxygen species and release of pro-inflammatory mediators, immune cells residing in or invading the spinal cord also participate in clearance of cellular debris and promote regeneration by secreting growth factors and protective cytokines. Despite the controversial role of inflammation in SCI, it is apparent that uncontrolled immune response can damage healthy tissue and aggravate the injury and, therefore, tight regulation is required to maximize functional recovery (Kwon et al., 2004; Rossignol et al., 2007; Rowland et al., 2008; Donnelly and Popovich, 2008; David et al., 2012). Because a growing amount of evidence indicates the dual role of immune

http://dx.doi.org/10.1016/j.bbi.2014.08.002 0889-1591/Ó 2014 Published by Elsevier Inc.

Please cite this article in press as: Pomeshchik, Y., et al. Interleukin-33 treatment reduces secondary injury and improves functional recovery after contusion spinal cord injury. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.08.002

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response in neurodegenerative disorders, including SCI, novel approaches to modulate the immune system towards an anti-inflammatory and regeneration-supporting mode have been suggested as potential therapies (Amor et al., 2013). Macrophages are central players in the innate immune response following injury to the central nervous system (CNS) (David and Kroner, 2011). Exposure of macrophages to type 1 helper T cell (Th1) cytokines, such as interferon gamma (IFN-c) and tumor necrosis factor alpha (TNF-a), leads to their polarization to the M1 subpopulation (the classical pro-inflammatory macrophages), which is associated with cytotoxic processes and correlates with the severity of the disease progression and tissue damage in SCI. In contrast, the ‘‘alternatively activated’’ M2 macrophages are induced by type 2 helper T cell (Th2) cytokines such as interleukin (IL)-4, IL-10 and IL-13. M2 cells demonstrate anti-inflammatory activities, scavenge debris, promote angiogenesis and are involved in tissue remodeling and repair (Gordon, 2003; Mantovani et al., 2004; Schwartz and Yoles, 2006; Kigerl et al., 2009; David and Kroner, 2011; Cassetta et al., 2011; Shechter and Schwartz, 2013). Unfortunately, the microenvironment of the injured spinal cord favors M1 polarization and the appearance of M2 cells remains transient (Kigerl et al., 2009; Schwartz, 2010; David and Kroner, 2011; Shin et al., 2013). Therefore, the polarization of microglia/macrophages to the M2 state may be desirable in situ in the injured spinal cord (David and Kroner, 2011; Guerrero et al., 2012; Jiang et al., 2012b). IL-33 is a member of the IL-1 cytokine family (Schmitz et al., 2005). It is described as a cytokine with two different functions: it acts as an intracellular regulator of gene expression (Carriere et al., 2007; Ali et al., 2011) and as an alarm mediator when released from damaged cells (Cayrol and Girard, 2009; Lamkanfi and Dixit, 2009; Lüthi et al., 2009). Nuclear IL-33 can interact with the transcription factor nuclear factor kappa-B (NF-jB) and dampen its activity in non–IL-33 receptor mediated fashion (Carriere et al., 2007; Ali et al., 2011), whereas extracellular IL33 binds to its receptor consisting of a heterodimer between ST2 and IL-1 receptor accessory protein (IL-1RAcP) (Ali et al., 2007; Chackerian et al., 2007; Liew et al., 2010; Liu et al., 2013). The IL-33 receptor is expressed on a broad range of immune cells, including Th2 cells and macrophages, and can promote Th2 cell expansion and shift the macrophage polarization from M1 to M2 (Xu et al., 1998; Schmitz et al., 2005; Kurowska-Stolarska et al., 2009; Miller et al., 2010). Therefore, increased systemic levels of IL-33 may have a dual function, as its intracellular form can suppress NF-jB-mediated inflammation and its extracellular form stimulates adaptive and innate immune cells in order to clear the initial trigger and repair damaged tissues (Sattler et al., 2013). IL-33 has been reported to be highly expressed in the naïve mouse brain and spinal cord (Schmitz et al., 2005), and to be upregulated by inflammatory stimuli (Hudson et al., 2008; Yasuoka et al., 2011; Christophi et al., 2012; Jiang et al., 2012a; Li et al., 2012; Zhao et al., 2013). However, in pathological conditions, the cellular localization of IL-33 in the CNS remains controversial and has been studied only in experimental autoimmune encephalomyelitis (EAE) (Yasuoka et al., 2011) and bone cancer-induced pain (Zhao et al., 2013) models. Here we show that following SCI, IL-33 is up-regulated and expressed predominantly in spinal cord astrocytes. Administration of recombinant IL-33 in the acute phase of SCI modulated both local and systemic inflammation, resulting in long-term existence of anti-inflammatory M2 microglia/macrophages in the injured spinal cord and leading to reduction of secondary damage and improvement of functional recovery. Therefore, administration of IL-33 may serve as a treatment of patients with acute contusion SCI.

2. Materials and methods

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2.1. Animals and surgical procedure

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Female 10–12 week old C57BL/6J mice were obtained from the National Laboratory Animal Centre, University of Eastern Finland. The mice were housed in groups of three in cages under 12-h light/dark cycle with water and standard rodent chow provided ad libitum. Additional water and powdered food was made available for the first 7 days after SCI. The experimental procedures were approved by the Animal Experiment Committee in State Provincial Office of Southern Finland and conducted according to the national regulation of the usage and welfare of laboratory animals. The mice were anesthetized with 5% isoflurane in 30% O2/70% N2O and maintained in surgical depth anesthesia with 1–1.5% isoflurane delivered through a nose mask during the operation. For the surgery the mice were placed on a controlled heating blanket to maintain body temperature at a constant level of 37 ± 1 °C. A clinically relevant moderate contusion SCI (60 kDynes force) was performed at T10 level using an Infinite Horizons Impactor (Precision Scientific Instrumentation, Lexington, KY) as described in Pomeshchik et al. (2014). The model was chosen as it results in bilateral injury and paralysis, and is also optimal for conclusive evaluation of therapeutic treatments. The mice were kept on 37 °C heating pads for three days after surgery. Analgesia was provided with buprenorfine (TemgesicÒ, Schering-Plough, Belgium) 0.1 mg/kg injected subcutaneously 30 min before surgery and then same dosage every 12 h for 3 days. The bladder was manually expressed two times daily for approximately 2 weeks until mice were able to regain normal bladder function. Mice that underwent laminectomy without impact served as sham controls.

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2.2. IL-33 treatment

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Before intraperitoneal administration, recombinant mouse IL-33 (Mouse IL-33 protein, Biorbyt, San Francisco, CA) was diluted in 0.0025% bovine serum albumin (BSA) in sterile PBS to 5 lg/ml concentration. The mice were randomly divided into IL-33 and vehicle groups. In IL-33 treatment group mice received 1 lg of IL-33 immediately after wound closing, followed by 1 lg at 3 days post injury (dpi) and 0.5 lg at 7 and 10 dpi. The dosing was based on a previous mouse study of atherosclerosis (Miller et al., 2008). Control and sham mice were injected similarly with 0.0025% BSA in PBS.

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2.3. Functional assessment

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Hindlimb motor function recovery was assessed using the Basso Mouse Scale (BMS) (Basso et al., 2006) by two raters blinded to the experimental groups. Each mouse was observed separately for 4 min per session and BMS scores for the right and left hindlimbs were recorded. For further data processing averages of the BMS scores for right and left hindlimbs were used. The BMS is a sensitive, valid and reliable scale allowing assessing the degree of hind-limb functional recovery after SCI. The scale ranges from 0 (no ankle movement) to 9 (complete functional recovery) points and includes the assessment of ankle movement, plantar placement, weight support, stepping, coordination, paw position and trunk stability. Motor function was assessed 24 h after injury, and then weekly for 42 days. The mice with a BMS score higher than one at 24 h after injury were excluded from future evaluation.

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2.4. Blood collection and processing

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Blood was collected from the heart and tail vein 24 h after SCI and mixed with 3.8% trisodium citrate (1:10) as anticoagulant.

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Plasma was separated by centrifugation at 2000g for 15 min, then the upper layer was further centrifuged at 12,000g for 3 min. Plasma was placed at 70 °C until analysis. Cell pellets were immediately used for flow cytometry.

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2.5. Tissue collection

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After terminal anesthesia with tribromoethanol (Avertin, Sigma–Aldrich, St. Louis, MO) the mice were transcardially perfused with heparinized (2500 IU/L) saline. The spleen, inguinal, axillary, brachial lymph nodes, and a 6-mm piece of spinal cord centered by lesion epicenter (or respective area in shams) were dissected, and either snap-frozen in liquid nitrogen and stored at 70 °C for future processing, or used immediately for flow cytometry.

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2.6. Quantitative real-time reverse transcription-PCR (RT-PCR)

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For RNA isolation from spleens and spinal cords TRIzol reagent (Life Technologies, Carlsbad, CA) was used in accordance with the manufacturer’s instructions. The final purity and concentration of total RNA were measured with a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). cDNA was synthesized from 500 ng of total RNA using random hexamer primers (Promega, Madison, WI) as a template and Maxima reverse transcriptase (Thermo Fisher Scientific). The gene expression reactions were performed according to the manufacturer’s instructions for RT-PCR System (StepOnePlus; Life Technologies) using Taqman chemistry and relative expression levels of mRNA encoding genes of interest were measured by using specific assays-on-demand (Life Technologies) target mixes. The expression levels were obtained by normalizing the target gene to ribosomal RNA and are presented as fold change in the expression ± standard error of the mean.

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2.7. Cytokine protein expression

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Frozen spleens were homogenized in buffer containing 10 mM Tris–HCl (pH 8.0), 5 mM EDTA, 150 mM NaCl, 10 % glycerol, 0.1 % Nonidet P-40 and protease inhibitor cocktail (Complete; Roche Applied Science, Mannheim, Germany). From plasma and spleen homogenates the levels of IL-2, IL-4, IL-6, IFN-c, TNF-a, IL-17 and IL-10 proteins were measured by using the cytometric bead assay (CBA) Th1/Th2/Th17 kit (BD Biosciences, Franklin Lakes, NJ) according to manufacturer’s instructions. Data were acquired using FACSCalibur (BD Biosciences, San Jose, CA) and analyzed by FCAP Array software (Soft Flow Inc., St. Louis Park, MN).

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2.8. Cell preparation and flow cytometric staining

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Spleen and lymph nodes were crushed through 70 lm and 40 lm cell strainers (BD Biosciences), respectively, and resuspended in 1% fetal bovine serum (FBS) (Life Technologies) in Hank’s balanced salt solution (HBSS) (BioWhittakerÒ Lonza, Basel, Switzerland), centrifuged for 5 min at 400g, and resuspended in 5% FBS in RPMI-1640 (Sigma–Aldrich). Red blood cells were lysed with BD PharmLyse, washed twice with 1 % FBS in HBSS and resuspended in 5% FBS in RPMI-1640. The cells were counted on a Z2 Coulter Counter (3.5–10 lm). All antibodies used for flow cytometry were obtained from eBioscience unless otherwise noted: CD16/32 Fc receptor block (24.G2, BD Biosciences), CD4-FITC (GK1.5 and RM4-5), CD8-PerCP eFluor710 (53–6.7), CD25-APC (PC61.5), Foxp3-PE (FJK-16s), isotype RatIgG2a-PE, TNFa-PE (MP6-XT22), IFNc-PE (XMG 1.2). Extracellular stains were incubated at 4 °C for 45 min and then washed twice with 1 % FBS in HBSS. Staining of T regulatory FoxP3+ cells

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(Tregs) and TNFa and IFNc cytokines was done according to eBioscience’s intracellular staining protocol. Cytokine enumeration was performed on CD4+ isolated cells from the spleen using magnetically labeled CD4 (L3T4) MicroBeads (Miltenyi Biotec, Lund, Sweden) according to manufacturer’s instructions. Purity was confirmed (92.3 ± 0.93%) by flow cytometry. Cytokine stimulation was done as described by Foster et al. (2007). Briefly, 1 106 cells were stimulated for 5 h at 37 °C in 5% CO2 in the presence of Brefeldin A (10 lg/ml, Sigma–Aldrich), Ionomycin (500 ng/ml, Sigma–Aldrich) and phorbol 12-myristate 13-acetate (PMA) (10 ng/ml, Sigma–Aldrich). Samples were acquired on BD FACSCalibur equipped with a single 488 nm argon laser or BD FACSAriaIII equipped with 488 and 633 nm lasers with the standard detector configuration. Post-acquisition data analysis was performed using FCS Express4 (DeNovo, Los Angeles, CA) or Cellquest Pro™ software BD Biosciences).

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2.9. Histology

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Following perfusion with 4% paraformaldehyde (PFA) the spinal cords were post-fixed in 4% PFA at 4 °C for 21 h followed by cryoprotection in 10% sucrose for 24 h and 20% sucrose for the next 24 h. A 6-mm piece of the spinal cord centered on the lesion epicenter (or respective area in shams) was embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek, Zouterwoude, the Netherlands), frozen on liquid-nitrogen-supercooled isopentane and stored at 70 °C for transversal 20 lm cryostat serial sectioning (Leica Microsystems GmH, Wetzlar Germany). All histological studies were performed in a blinded fashion. Luxol Fast Blue (LFB) staining was performed as described elsewhere (Yune et al., 2007). Frozen sections were processed for immunofluorescence staining with primary antibodies against glial fibrillary acidic protein (GFAP), 1:200, (Dako, Glostrup, Denmark); ionized calcium-binding adapter molecule 1 (Iba-1), 1:250, (Wako Pure Chemical Industries, Ltd, Tokyo, Japan); CD3, 1:5000 (AbD Serotec, Kidlington, UK); Arginase-1 (N-20), 1:200, (Santa Cruz Biotechnology, Inc, Heidelberg, Germany); IL-33, 1:200, (R&D Systems, Inc, Minneapolis, MN) or NeuN, 1:200, (Merck Millipore, Billerica, MA). For IL-33 staining permeabilization with 0.4% Triton X-100 (Sigma–Aldrich) for 30 min was performed. For Arginase-1 staining antigen retrieval was performed using 0.3% sodium citrate dehydrate aqueous solution (pH 6) preheated to 92 °C (Sigma–Aldrich). The following day, appropriate Alexa Fluor-conjugated secondary antibodies (all from Life Technologies) were applied and, after washing and air-drying, the sections were mounted with Vectashield mounting media with Dapi (Vector Laboratories, INC. Burlingame, CA) or consequentially processed for double-staining with compatible antibodies. For CD3 staining tyramide-based signal amplification (TSA Biotin System; PerkinElmer, Waltham, MA) was used after the incubation with the secondary antibody according to the manufacturer’s instructions. Spinal cord sections were photographed using a digital camera (Color View 12 or F-view; SoftImaging Systems, Münster, Germany) attached to an Olympus AX70 microscope and the stainings were quantified using ImagePro Plus (Media Cybernetics, Rockville, MD) or ImageJ (Wayne Rasband, National Institutes of Health, Bethesda, MD) software by a researcher blinded to the treatment groups. For assessing the spinal cord tissue sparing, we used the previously described method (López-Vales et al., 2005; Q2 Klopstein et al., 2012) where the total remaining area covered by GFAP is delineated (GFAP-positive tissue) on sections with an interval of 200 lm. The spinal cord section containing the lowest amount of preserved tissue was considered as the lesion epicenter. The myelinated area were analyzed on LFB stained sections with an interval of 200 lm. Astrogliosis was assessed on high-magnification images (40) by calculating the GFAP immunoreactivity in a

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7.5 103 lm2 area in the lateral white matter at the lesion epicenter, as well as 600 and 1200 lm rostrally and caudally, and was expressed as the percentage of GFAP immunoreactivity within the selected area (GFAP-density). Microglial activation was assessed in a 3 104 lm2 area in the lateral white matter at the injury epicenter, 400 and 800 lm rostrally and caudally and expressed as the percentage of the selected area occupied by Iba-1. Arginase-1 immunoreactivity was assessed in images from the injury epicenter, 200, 600 and 1000 lm rostrally and caudally and expressed as a percent occupied by Arginase-1 immunoreactivity within the total area of the section. IL-33 positive cells were manually counted in a 3 104 lm2 area in the lateral white matter at the lesion epicenter, as well as 400 and 800 lm rostrally and caudally and in the same area of the ventral horns at distance 800 lm rostrally and caudally from the injury epicenter, and expressed as number of cell profiles within the selected area. CD3 positive cells were manually counted in 5 sections per spinal cord 200 lm apart within 1 mm around the injury epicenter and expressed as average number of cell profiles per section. For quantification of GFAP, Iba-1 and IL-33 immunoreactivities, images were taken from the left and right sides of the spinal cord and results expressed as averages of the left and right sides at the selected distance. To assess co-localization, the double-stained sections were imaged using a Zeiss LSM 700 confocal microscope (Zeiss Inc., Maple Grove, USA) with an attached digital camera (Color View 12 or F-View; Soft Imaging System, Munster, Germany) running Zen 2009 Image analysis Software (Zeiss Inc., Maple Grove, USA).

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2.10. Magnetic resonance imaging

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Two weeks after injury the mice were anesthetized with isoflurane, positioned in a custom-made animal holder with warm water heating system and imaged using a 9.4 Tesla Varian scanner (Varian Inc., Palo Alto, CA) as previously described in Pomeshchik et al. (2014). Data were post-processed using in-house-built Matlab software (Aedes, http://aedes.uef.fi) by an observer blinded to the treatment and converted to micrometers (lm). A graph of the experimental protocol and timelines of analyses performed is shown as a supplement figure (Suppl. Fig. 1).

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2.11. Statistical analyses

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All data are shown as mean ± standard error of mean. Unpaired t-test was employed for comparing means of two treatment groups. For multiple comparisons one-way ANOVA followed by Tukey’s post hoc test was used. Behavioral data were analyzed using repeated measures two-way ANOVA followed by Bonferroni’s post hoc test. All statistical analyses were performed in GraphPad Prism version 5.03 for Windows software (GraphPad Software, La Jolla, CA, http://www.graphpad.com).

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3. Results 3.1. IL-33 is up-regulated and expressed predominantly in astrocyte nuclei in injured spinal cord To understand the role of IL-33 in SCI we first examined whether the injury affects the expression of the IL-33 gene (Il33) in the spinal cord. RT-PCR revealed more than a 3-fold increase in the Il33 mRNA levels in vehicle-treated SCI mice compared to their sham-operated controls at 24 h after injury (Fig. 1A). IL-33 treatment had no effect on Il33 expression (Fig. 1A). To estimate if IL-33 induction in the injured spinal cord persists for an extended time after SCI we quantified the number of cells

expressing IL-33 in lateral white matter and ventral horns at 42 dpi. The number of IL-33 positive cell profiles was significantly increased in the lesion epicenter and adjacent spinal cord sections of the injured mice treated with vehicle compared to the corresponding sections in the sham mice (Fig. 1B–E). The number of IL-33 positive cell profiles in IL-33-treated mice was not significantly different from the vehicle-treated group (Fig. 1B–E). Double-labelling with GFAP revealed a high incidence of IL-33 within nuclei in astrocytes (Fig. 1F). IL-33 was not observed in NeuN positive neurons or Iba-1 positive microglia/macrophages (Suppl. Fig. 2). We did not observe extranuclear localization of IL-33. The observed pattern of IL-33 expression was similar in all studied mouse groups.

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3.2. IL-33 treatment modulates the expression of IL-33 receptor subunits in injured spinal cord

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To understand whether SCI affects the expression of the IL-33 receptor subunits we assessed the mRNA levels of the two IL-33 receptor subunits, ST2 (Il1rl1) and IL-1RAcP (Il1rap), in the sham and injured spinal cord. Twenty-four hours after SCI, the expression of Il1rap in the vehicle- treated mice was approximately 2-fold higher in comparison to the sham-operated mice (Fig. 2A). IL-33 treatment significantly down-regulated IL-33 Il1rap compared to the vehicle-treated injured mice, yet the levels remained significantly higher compared to the sham-operated controls at this early time point (Fig. 2A) At the same time point observed a trend towards up-regulation of Il1rl1 in the vehicle-treated group, yet this difference was not statistically significant when compared to non-injured mice (Fig. 2B). At 8 dpi the expression of Il1rap was significantly reduced in both vehicle and IL-33-treated mice when compared to the sham-operated controls (Fig. 2C). In contrast, the levels of Il1rl1 mRNA showed a trend towards up-regulation in the vehicletreated group when compared with shams, and become significantly up-regulated upon IL-33 treatment (Fig. 2D).

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3.3. IL-33 treatment promotes functional recovery after SCI

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To evaluate the effect of IL-33 treatment on functional recovery we applied the BMS (Basso et al., 2006). As expected, contusion SCI resulted in complete paralysis of hindlimbs (BMS score 0) 24 h after injury with slow recovery of hindlimb function during the following 42 days (Fig. 3). IL-33 treatment significantly improved motor recovery at 21 dpi when compared to the vehicle treated animals. At 42 dpi the mice treated with IL-33 attained an average BMS score of 3.6 ± 0.26 which corresponds to plantar placement of the paw and occasional plantar stepping, whereas mice treated with vehicle attained an average BMS score of 2.5 ± 0.14, which corresponds to extensive ankle movements or plantar placement of the paw, but not plantar stepping. IL-33 treatment did not affect the lesion volume compared to vehicle-treated injured mice as measured by MRI at 14 dpi, which was consistent with BMS scores at this time point (Suppl. Fig. 3).

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3.4. IL-33 treatment reduces spinal cord tissue loss and demyelination after SCI

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Next we studied whether the functional recovery observed after IL-33 treatment is linked to a reduction of secondary tissue injury at 42 dpi. When the area covered by GFAP immunoreactivity was used as a measure for the preserved spinal cord tissue (LópezVales et al., 2005; Klopstein et al., 2012), IL-33 treatment significantly reduced the spinal cord tissue loss compared to the vehicle treated controls. This effect was significant in regions adjacent to

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Fig. 1. IL-33 expression in the spinal cord after SCI and administration of recombinant IL-33. (A) Il33 mRNA expression was measured in the spinal cord by RT-PCR at 24 h after SCI. (B and C) The number of IL-33 positive cell profiles was quantified in the ventral horns and white matter 42 days after SCI. RVH – ventral horn rostrally, CVH – ventral horn caudally, RWM – white matter rostrally, CWM – white matter caudally. (D) Schematic images showing the area of a spinal cord section taken for quantification of IL-33 immunohistochemistry in studied groups. Scale bars = 250 lm. (E) Representative high-magnification fluorescence micrographs showing IL-33 immunohistochemistry in the white matter (WM) and ventral horns (VH) of sham, vehicle-treated and IL-33 treated mice (as in E). Scale bars = 25 lm. (F) Confocal images showing double-labelling for IL-33 and an astrocyte marker, GFAP. Scale bars = 20 lm. The data are shown as mean ± standard error of mean. n = 8–10. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.

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injury epicenter up to 1 mm rostrally and 0.6 mm caudally to the epicenter (Fig. 4). LFB staining was used to estimate the spinal cord demyelination. IL-33 treated mice showed significantly reduced demyelination at certain levels caudally and rostrally to the lesion epicenter when compared to the vehicle-treated group (Fig. 5). Although this beneficial effect of IL-33 treatment on white matter sparing did not reach significant values at all spinal cord levels studied (especially caudally to the lesion epicenter), the areas of LFB-stained myelin were consistently larger in IL-33-treated mice compared to vehicle-treated mice in all levels that were analyzed. Taken together, the LFP quantification data suggested that IL-33 treatment reduces demyelination in the contused spinal cord.

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3.5. IL-33 treatment reduces astrogliosis after SCI

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We also assessed whether administration of IL-33 affects the SCI-induced activation of astrocytes and microglia. IL-33 treated mice showed significantly reduced astrogliosis at the lesion

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epicenter and in the adjacent sections at 42 dpi (Fig. 6) as measured by the density of GFAP immunoreactivity. However, while SCI induced a 10–15-fold increase in microglial activity compared to sham operated controls, IL-33 treatment did not affect the degree of Iba-1 immunoreactivity (Fig. 7).

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3.6. IL-33 treated mice showed reduced expression levels of Cd3d in the spinal cord at 8 dpi

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To estimate the effect of IL-33 treatment on T cell infiltration into the injured spinal cord we measured the mRNA expression levels of Cd3d and Cd4, representing markers for total T cell population and T-helper cells, respectively, in the spinal cord at 8 dpi. In addition, we analyzed CD3 immunopositive cells in the spinal cord at 42 dpi by immunohistochemistry. At 8 dpi the mRNA expression level of Cd3d was not significantly induced upon SCI, whereas the Cd4 mRNA expression was heavily elevated (Fig. 8B). IL-33 treatment resulted in a significant reduction in the mRNA expression level of Cd3d compared to the vehicle treated group (Fig. 8A). In

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Fig. 2. IL-33 receptor subunit expression profiles in the spinal cord following SCI and IL-33 administration. The relative expression of Il1rap (A and C) and Il1rl1 (B, D, and E) were measured with RT-PCR in the spinal cord (A–D) 24 h (A and B) and 8 days (C and D) after SCI. The data are shown as mean ± standard error of mean. n = 3–6. ⁄p < 0.05, ⁄⁄ p < 0.01, ⁄⁄⁄p < 0.001.

tested whether the treatment with IL-33 can reduce the expression of IFN-c and TNF-a in CD4+ T helper cells in the spleen. At 8 dpi the mice treated with IL-33 had a significantly lower percentage and absolute number of CD4+ T helper cells expressing TNF-a than vehicle-treated or non-injured mice (Fig. 9A and B). Meanwhile, the percentage and absolute number of CD4+ T helper cells expressing IFN-c remained unaltered (Suppl. Fig. 5).

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3.8. IL-33 treatment increases expression of T regulatory cell marker FoxP3 in spleen after SCI

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IL-33 is known to induce the generation and proliferation of suppressive T regulatory cells (Tregs) (Brunner et al., 2011; Turnquist et al., 2011). To investigate if IL-33 is able to expand Treg cell pools in SCI we examined the expression level of transcription factor forkhead box P3 (FoxP3) gene (Foxp3), a marker of Treg cells, in the spleen and spinal cord at 24 h and in the spinal cord 8 dpi after SCI. SCI did not induce the expression of Foxp3 in the spinal cord and spleen. In turn, IL-33 treatment did not induce the expression of Foxp3 in the spinal cord at either studied time point (data not shown). In contrast to the spinal cord, Foxp3 mRNA expression was significantly elevated in the spleen of the group treated with IL-33 when compared to the vehicle-treated mice already 24 h after SCI and the first dose of IL-33 (Fig. 10). However, flow cytometry showed that Foxp3 up-regulation in the spleen at the early time point (24 h) did not result in higher numbers of Treg+ (CD4+/CD25+/FoxP3+) lymphocytes either in spleen or in plasma at 8 dpi (data not shown).

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3.9. IL-33 treatment reduces expression of pro-inflammatory TNF-a in injured spinal cord and modulates the protein levels of pro- and anti-inflammatory cytokines in plasma and spleen after SCI

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3.7. IL-33 treatment suppresses generation of pro-inflammatory T helper cells in spleen after SCI To further evaluate the effect of IL-33 treatment on cellmediated immunity we used flow cytometric analysis to quantify immune cells and their subsets circulating or residing in the spleen and lymph nodes. We analyzed the effect of SCI and IL-33 treatment on CD4 and CD8 lymphocyte populations in the blood, spleen and lymph nodes. At 8 dpi neither SCI nor IL-33 treatment altered the percentage of CD4+ T helper or CD8+ T killer cells in the spleen (Suppl. Fig. 4), blood or lymph nodes (data not shown). Next, we

Next we determined the effect of SCI and IL-33 treatment on expression of inflammatory cytokines in the spinal cord and spleen. Twenty-four hours after SCI the expression levels of TNF-a (Tnf), IL-6 (Il6) and IL-1b (Il1b) genes were significantly up-regulated in the spinal cord (Fig. 11A–C). Administration of IL-33 led to a significant reduction in the expression levels of Tnf compared to the vehicle-treated SCI mice (p < 0.05), although the levels of this cytokine remained elevated compared with sham-operated mice

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Fig. 3. IL-33 treatment improves behavioral recovery after contusion SCI. Hindlimb motor function was assessed by BMS at 1, 7, 14, 21, 35 and 42 days after SCI. IL-33 treated mice show significantly improved recovery compared to vehicle treated controls starting at 21 days post injury. The data are shown as mean ± standard error of mean. n = 10–13. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.

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addition, IL-33 treatment reduced the expression level of Cd4 by approximately 40%, however, this reduction did not reach statistical significance. At 42 dpi the infiltration of CD3 immunopositive cells into the spinal cord was heavily induced upon SCI. IL-33 treatment caused a trend towards reduction in the number of CD3 positive cells when compared to the vehicle (p = 0.13, unpaired t-test between the vehicle and IL-33 treated mice; Fig. 8C and D).


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Fig. 4. IL-33 treatment reduces tissue loss after contusion SCI. (A) Spared tissue in the sham, vehicle and IL-33 treated groups at lesion epicenter and adjacent sections was quantified by delineating the area covered by GFAP immunoreactivity 42 days after SCI. (B) Representative schematic images showing preserved spinal cord tissue in experimental groups at a distance of 600 lm rostrally to the injury epicenter. The data are shown as mean ± standard error of mean. n = 8–10. ⁄p < 0.05, ⁄⁄p < 0.01. Scale bars = 250 lm.

Fig. 5. IL-33 treatment reduces demyelination after contusion SCI. (A) Spared white matter in the sham, vehicle and IL-33 treated groups at lesion epicenter and adjacent sections was quantified as LFB positive area 42 days after SCI. (B) Representative images showing myelin sparing in experimental groups at a distance of 800 lm rostrally to the injury epicenter. The data are shown as mean ± standard error of mean. n = 8–10. ⁄p < 0.05, ⁄⁄p < 0.01. Scale bars = 250 lm.

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(Fig. 11A). IL-33 treatment did not affect the expression levels of Il6 and Il1b mRNA (Fig. 11B and C). The mRNA levels of Il10, Il4 and INFc gene (Infg) were below the detection limit in the spinal cord tissue (data not shown). SCI did not alter expression of Tnf, Infg, Il10, Il6 and Il4 genes in the spleen 24 h after SCI (data not shown). Additionally, we assessed the protein levels of Th1/Th2/Th17 cytokines in the spleen and plasma 24 h after injury and the first dose of IL-33. Although SCI did not affect the level of cytokines in plasma and the spleen at this time point (data not shown), the mice treated with IL-33 had significantly lower levels of IFN-c and IL-17 protein in plasma (Fig. 11D and E) and a higher level of IL-10 protein in the spleen compared to the vehicle-treated SCI mice (Fig. 11F). 3.10. IL-33 treatment induces polarization of microglia/macrophages towards M2 type and prolongs their persistence in the injured spinal cord Polarization of macrophages to the beneficial M2 type has been proposed as a main mechanism of IL-33 action (KurowskaStolarska et al., 2009; Liew et al., 2010; Miller et al., 2010). To study

whether IL-33 administration induces a shift towards M2 microglia/ macrophages in SCI we assessed expression of iNOS gene (Nos2), a marker of M1 cells, as well as Arginase-1 (Arg1) and Ym1 (Chi3l3) genes, markers of M2 cells in the injured spinal cord. Twenty-four hours after SCI we observed a significant increase in the expression of both M1 and M2 microglia/macrophage markers in the injured groups when compared to the non-injured mice (Fig. 12A–C). At this time point we did not detect a difference in M1 or M2 markers between the vehicle and IL-33 treated mice. When we assessed mRNA levels of these markers at 8 dpi we did not detect altered expression of Nos2 (data not shown), whereas Arg1 and Chi3l3 were significantly elevated in the group treated with IL-33 when compared to their vehicle treated controls (Fig. 12D and E). To further investigate the duration of M2 microglia/macrophage persistence in the injured spinal cord we evaluated Arginase-1 protein expression by immunohistochemistry at 42 dpi in the IL-33 and vehicle-treated groups. Arginase-1 immunoreactivity was significantly increased in the group treated with IL-33 when compared to the vehicle-treated group within a 2-mm segment around the injury epicenter (Fig. 13A and B). The maximum staining


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Fig. 6. IL-33 treatment reduces astrogliosis after contusion SCI. (A) Astrogliosis in the sham, vehicle and IL-33 treated groups at lesion epicenter and adjacent sections was assessed by quantifying the density of GFAP immunoreactivity in highmagnification images 42 days after SCI. (B) Schematic images showing the area of spinal cord section taken for assessment of astrogliosis in experimental groups. Scale bars = 250 lm. (C) Representative high-magnification fluorescence micrographs showing the degree of astrogliosis at the distance of 600 lm rostrally to the injury epicenter. Scale bars = 25 lm. The data are shown as mean ± standard error of mean. n = 8–10. ⁄p < 0.05, ⁄⁄⁄p < 0.001.

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intensity was detected at the lesion epicenter where it was approximately 35-fold higher compared to the vehicle-treated group (Fig. 13A and B). Confocal imaging confirmed that most, if not all, Arginase-1 protein expression was colocalized with Iba-1 positive macrophages/microglia (Fig. 13C and D). We did not detect any Arginase-1 immunoreactivity in the control non-injured mice (Fig. 13B).

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Additionally, we assessed expression of M1 and M2 markers, iNOS and Arginase-1 respectively, in the spleen 42 days after SCI. IL-33 treatment significantly downregulated mRNA expression of iNOS gene Nos2 when compared with vehicle-treated and noninjured controls (Fig. 12F) and showed about 33% increase in Q3 Arg1 expression when compared with vehicle-treated group (p = 0,297, unpaired t-test between the vehicle and IL-33 treated mice; Fig. 12G).

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4. Discussion

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IL-33 is a cytokine with pleiotropic activities in innate and adaptive immune responses in host defense and disease (Liu et al., 2013). Recently the production and function of IL-33 in the CNS have drawn increasing attention (Han et al., 2011), however, the role of IL-33 in traumatic CNS injuries is still unknown. We therefore investigated the expression of IL-33 and its receptors

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Fig. 7. Microglial activation remains unchanged upon IL-33 treatment. Microgliosis in the sham, vehicle and IL-33 treated groups at lesion epicenter and adjacent sections was assessed by Iba-1 immunohistochemistry 42 days after SCI. (B) Schematic images showing the area of spinal cord section taken for assessment of microgliosis in experimental groups. Scale bars = 250 lm. (C) Representative high-magnification fluorescence micrographs showing the degree of microgliosis at the distance of 800 lm caudally to the injury epicenter. Scale bars = 25 lm. The data are shown as mean ± standard error of mean. n = 8–10. ⁄⁄⁄p < 0.001.

following contusion SCI and assessed the effect of IL-33 treatment in SCI. While the brain and spinal cord are known to be the organs with the highest IL-33 expression in the body (Schmitz et al., 2005), and inducibility of IL-33 expression by various inflammatory stimuli is well established (Hudson et al., 2008; Yasuoka et al., 2011; Christophi et al., 2012; Jiang et al., 2012a; Li et al., 2012; Zhao et al., 2013), the cellular localization of IL-33 in the CNS has been controversial. IL-33 has been shown to be up-regulated primarily in spinal cord astrocytes in the EAE (Yasuoka et al., 2011) and bone cancer-induced pain (Zhao et al., 2013) mouse models, as well as in human brains from patients with multiple sclerosis (Christophi et al., 2012). However, in a more recent study utilizing the mouse model of EAE the levels of IL-33 were increased in both neurons and astrocytes (Jiang et al., 2012a). The expression and cellular localization of IL-33 in other neurological diseases have not been examined. To our knowledge, this is the first study to describe the pattern of IL-33 expression in the mouse spinal cord after contusion SCI. Importantly, we report that IL-33 expression was increased in the injured spinal cord 24 h after trauma, and that the number of IL-33 expressing cells remained elevated for up to 42 dpi. Remarkably, almost all the detected IL33 was colocalized with the astrocytic marker GFAP and was restricted to astrocyte nuclei. Our findings support the observations that astrocytes are the main IL-33 expressing cells in the CNS (Hudson et al., 2008; Yasuoka et al., 2011; Christophi et al., 2012; Zhao et al., 2013). As the intracellular IL-33 immunoreactivity is thought to primarily reflect its role as transcriptional repressor of NF-jB activity (Carriere et al., 2007; Ali et al., 2011), the


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Fig. 8. IL-33 treated mice show a tendency towards reduced T-cell infiltration into the spinal cord at 42 dpi. The relative expression of Cd3d (A) and Cd4 (B) were measured with RT-PCR in the spinal cord 8 days after SCI. The data are shown as mean ± standard error of mean. n = 3–5. ⁄p < 0.05, ⁄⁄p < 0.01. (C) The number of CD3 positive cell profiles was quantified in the spinal cord 42 days after SCI. (D) Representative fluorescence images from the lesion epicenter (0 lm) showing CD3 immunohistochemistry in the spinal cord 42 days after SCI. Scale bars = 100 lm. The data are shown as mean ± standard error of mean. n = 8–10. ⁄⁄⁄p < 0.001.

Fig. 9. IL-33 treatment reduces the percentage and absolute number of CD4+ T helper cells expressing TNF-a in the spleen. (A) Percentage and number of CD4+ T helper cells expressing TNF-a were measured by flow cytometry 8 days after SCI. Bars represent the percentage of CD4+ T helper cells expressing cytokine; inside bars are the absolute numbers of TNF-a expressing CD4+ T helper cells in the spleen (106). (B) Representative plots show the percentage of CD4+ T cells expressing TNF-a from all gated CD4+ cells. Data were first gated on live, debris-free cell populations from FSC-H vs SSC-H scatter plot then CD4+ vs SSC-H. Cytokine positivity was gated from unstimulated sample controls. The data are shown as mean ± standard error of mean. n = 4–5. ⁄p < 0.05.


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Fig. 10. IL-33 treatment increases expression of Treg cells (A) marker FoxP3 in spleen. The relative expression of Foxp3 were measured with RT-PCR 24 h after SCI. The data are shown as mean ± standard error of mean. n = 3–6. ⁄p < 0.05, ⁄⁄p < 0.01.

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intracellular up-regulation of IL-33 we observed after SCI may be a compensatory mechanism to inhibit an excessive inflammatory response. It is important to note that treating mice with recombinant IL-33 did not increase Il33 mRNA levels or the number of IL-33 expressing cells in the spinal cord any further, suggesting that supplementary IL-33 does not further amplify the intracellular levels of this cytokine and therefore does not affect its intracellular function. The extracellular signaling of IL-33 depends on its binding to the primary receptor ST2 and subsequent recruitment of IL-1RAcP (Schmitz et al., 2005; Ali et al., 2007; Chackerian et al., 2007; Liew et al., 2010; Liu et al., 2013). Neurons express only IL-1RAcP, whereas astrocytes express both ST2 and IL-1RAcP subunits (Andre et al., 2005; Yasuoka et al., 2011). Initially microglia were proposed to express only IL-1RAcP (Andre et al., 2005), although recently the expression of ST2 by microglia has also been reported (Yasuoka et al., 2011). These findings suggest that microglia and astrocytes may be the primary responders to IL-33 (Han et al., 2011) in the CNS. In the present study, the expression levels of IL-33 receptor subunits were differently regulated upon mouse contusion SCI. The expression of IL-1RAcP was transiently up-regulated in the acute phase of SCI but declined by 8 dpi. In line with

the observations reported on the model of bone cancer-induced pain (Zhao et al., 2013) we did not find the levels of ST2 to be significantly increased in SCI. These data are in contrast to reports on a mouse model of EAE where ST2 expression is markedly elevated upon disease development (Jiang et al., 2012a; Li et al., 2012). Moreover, we found that administration of recombinant IL-33 induced an early but transient reduction of IL-1RAcP and later a transition towards ST2 up-regulation in the SCI. The observed alternate response of IL-33 receptor subunits may be explained by the fact that ST2 subunit specifically recognizes IL-33, whereas IL-1RAcP is shared by IL-33 and other IL-1 family members such as IL-1a and IL-1b (Chackerian et al., 2007; Liu et al., 2013). Adaptive immune cells, such as T and B lymphocytes, are activated upon SCI and known to promote tissue damage (Gonzalez et al., 2003; Jones et al., 2005). Chronic T cell activation can induce pathological fibrosis and scarring (Wynn, 2004). In the present study we observed that a significant spinal cord injury induced up-regulation of the mRNA level of T-helper CD4 surface marker at 8 days after SCI and increased the number of CD3 immunopositive T cells at 42 days after SCI which corresponds to the time of the second peak in the T cell infiltration into the injured mouse Q4 spinal cord (Sroga et al., 2003; Kigerl et al., 2006). IL-33 treatment was capable of reducing the expression level of Cd3d and induced a trend towards reduction of the number of T cells at 42 dpi. Our results are well in line with the previously reported capacity of IL-33 to reduce T cell infiltration in vivo (Turnquist et al., 2011). On the other hand, activated T cells can also serve as a source of neurotrophic factors and cytokines activating macrophages to ameliorate neurotoxicity, produce growth factors and remove inhibitors of axonal growth after SCI (Schwartz and Kipnis, 2001; Q5 Hauben et al., 2000, 2001; Schwartz, 2005). M1 macrophages are believed to be responsible for promoting secondary injury and impairing recovery from SCI (Kigerl et al., 2009; David and Kroner, 2011). They are also known to play a role in axonal ‘‘dieback’’ response and regeneration failure within the CNS (Horn et al., 2008; Busch et al., 2009). In contrast, M2 cells are involved in SCI recovery (Schwartz, 2010; David and Kroner, 2011; Guerrero et al., 2012) and are required for oligodendrocyte differentiation and remyelination (Miron et al., 2013). M2 macrophages

Fig. 11. Cytokine gene expression and protein profiles in the spinal cord and spleen following SCI and IL-33 administration. The relative expression of Tnf (A), Il6 (B) and Il1b (C) were measured with RT-PCR in the spinal cord, protein levels of IFN-c (D), IL-17 (E) were measured by CBA in the plasma and protein levels of IL-10 were measured by CBA in the spleen (F) 24 h after SCI. The data are shown as mean ± standard error of mean. n = 10–13 for CBA, n = 6 for RT-PCR. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.


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Fig. 12. Altered expression of M1 and M2 macrophage markers in the spinal cord (A–E) and spleen (F and G) following SCI and IL-33 administration. The relative expression of Nos2 (A and F), Arg1 (B, D and G) and Chi3l3 (C and E) were measured with RT-PCR in the spinal cord 24 h (A–C) and 8 days (D and E) after SCI and in the spleen 42 days after SCI (F and G). The data are shown as mean ± standard error of mean. n = 3–7. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.

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typically disappear within 3 to 7 days after SCI (Kigerl et al., 2009) and, therefore, modification of the SCI microenvironment to increase the number of M2 macrophages is believed to have a beneficial effect. We observed that recombinant IL-33 induced a dramatic increase in the expression of Arginase-1 and Ym-1, markers of M2 cells, in injured spinal cord at 8 dpi, and a remarkable elevation in the number of Arginase-1-positive M2 microglia/ macrophages in injured spinal cord for at least up to 42 dpi. Therefore, even a relatively short-lasting treatment with recombinant IL33 during the acute SCI phase was sufficient to induce a long-term presence of alternatively activated Arginase-1 positive M2 microglia/macrophages around the injured tissue. Our results are in line with a previous study showing that IL-33 treatment induces a shift towards M2 type macrophage activation in the lymph nodes and spleen of a mouse model of EAE (Jiang et al., 2012a). Indeed, at 42 dpi in the group treated with IL-33 we observed significant reduction of M1 marker iNOS expression and tendency towards up-regulation of Arginase-1 expression in the spleen. Because monocytes infiltrating the spinal cord at 7 dpi predominantly originate from the splenic reservoir (Blomster et al., 2013), we hypothesize that recombinant IL-33 treatment may target the infiltrating macrophages, especially those originating from the spleen in our study set up. However, we cannot rule out the possibility that also endogenous microglia contribute to detected M2 polarization. Although IL-33 may exhibit pro-inflammatory properties and induce secretion of TNF-a, IL-1b and IL-6 (Hudson et al., 2008; Espinassous et al., 2009; Enoksson et al., 2011; Yasuoka et al., 2011; Milovanovic et al., 2012; Kempuraj et al., 2013) under certain conditions, we demonstrated that recombinant IL-33

significantly decreased TNF-a induction in injured spinal cord already at 24 h after its administration. The discrepancy between our results and previous reports may be due to the differences in conditions and models used in the studies, as IL-33 either promotes or reduces inflammation depending on the tissue environment, disease state, and the model used (Han et al., 2011; Zhao et al., 2013). The reduction of TNF-a we observed in the injured spinal cord after IL-33 treatment may modulate the SCI microenvironment in favor of M2 polarization. TNF-a itself also plays a detrimental role in the development and persistence of SCI (Yune et al., 2003; Chen et al., 2011) by being able to induce not only neuronal, but also oligodendroglial cell apoptosis (Inukai et al., 2009) and by leading to massive demyelination (Shuman et al., 1997; Esposito and Cuzzocrea, 2011). Therefore, reduction of TNF-a levels by IL-33 treatment might prevent spinal cord tissue loss and demyelination, as was detected at 42 days after injury. Additionally, at 42 dpi the mice treated with IL-33 exhibited reduced astrogliosis, which is likely to be a consequence of the reduced secondary tissue damage in IL-33 treated group at this late stage. Although IL-33 has been reported to activate microglia in vitro (Yasuoka et al., 2011) we did not observe activation of microglia after administration of recombinant IL-33 to the injured mice. In parallel with the reduced TNF-a expression in the spinal cord after IL-33 treatment we also observed a reduction of the Th1/Th17 cytokines IFN-c and IL-17 in plasma, and elevation of the Th2 cytokine IL-10 in the spleen, suggesting a transition of the immune response towards a Th2 response in the periphery. This view is supported by the fact that recombinant IL-33 decreased the percentage and absolute number of CD4+ T helper cells expressing


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Fig. 13. IL-33 treatment induces Arginase-1 immunoreactivity in the injured spinal cord 42 days after injury. (A) Immunoreactivity in the vehicle and IL-33 treated groups at lesion epicenter and adjacent sections was assessed by Arginase-1 immunostaining. Sham mice were devoid of Arginase-1 positive cells (B) Representative images from the lesion epicenter (0 lm) showing increased arginase-1 immunoreactivity after IL-33 treatment. Scale bars = 250 lm. (C and D) Confocal images showing double labelling for Arginase-1 and macrophages/microglia marker Iba-1 in the group treated with IL-33. Scale bars = 20 lm. The data are shown as mean ± standard error of mean. n = 8–10. ⁄ p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.

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TNF-a in the spleen. Despite the fact that we did not detect evidence of SCI itself altering the cytokine levels in plasma and the spleen, the effect observed after IL-33 treatment in the periphery may also play an important role in M2 polarization. For instance, IL-10, which we found to be elevated by IL-33 treatment in the spleen after SCI, is one of the cytokines mainly responsible for M2 polarization (Deng et al., 2012). Although IL-33 has been shown to increase the levels of IL-4 in multiple tissues (Schmitz et al., 2005), we did not detect any effect of IL-33 treatment on IL-4 levels in plasma, the spleen or spinal cord at 24 h after SCI. We cannot rule out the possibility that the time point chosen in this study was not optimal for demonstrating IL-4 up-regulation. Even though the exact mechanism by which recombinant IL-33 modifies the cytokine profile in the spinal cord or the periphery are uncertain, we propose that recombinant IL-33 induced a transition from Th1 towards Th2 response after SCI, thereby causing polarization of macrophages towards the beneficial M2 subtype, thereby leading to improved functional recovery. The transcription factor Foxp3 is specifically expressed in naturally arising CD4+ regulatory T cells and programs their development and function (Fontenot et al., 2003; Hori et al., 2003; Walsh and Kipnis, 2011). Because the spleen is a main organ in which T regulatory cells reside (Wei et al., 2006), we determined the splenic expression of FoxP3 upon SCI and IL-33 treatment. Twenty-four

hours after SCI IL-33 treatment significantly elevated the splenic expression of FoxP3, when compared to the vehicle treated SCI mice. This is in line with the observations that IL-33 induces an expansion of the Treg pool in vivo (Brunner et al., 2011; Turnquist et al., 2011). However, we did not detect an increase in the number of CD4+/CD25+/FoxP3+ cells either in the spleen or plasma at 8 dpi as analyzed by flow cytometry. Furthermore, we did not observe that IL-33 treatment affects FoxP3 expression in the spinal cord at this later time point, indicating either a transient nature of Treg cell induction after the treatment or the presence of some unknown mechanisms suppressing Treg expansion upon SCI. The data presented here demonstrate for the first time that acute SCI is associated with increased IL-33 expression and that the IL-33 levels remain elevated also during the subchronic SCI stage with a predominant localization in astrocytes’ nuclei. Administration of recombinant IL-33 after contusion SCI results in improved and long-lasting motor recovery associated with reduced secondary tissue damage. Already the first dose of IL-33 reduced expression of cytotoxic TNF-a in the injured spinal cord. Furthermore, polarization of microglia/macrophages to the antiinflammatory M2 type permanently persisting in the injured spinal cord together with a tendency towards reduced T cell infiltration upon IL-33 treatment as well as a shift towards the Th2 type cytokine profile in the periphery are identified as mechanisms of


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beneficial IL-33 action in SCI. Even though we observed positive effects of the IL-33 therapy when the treatment was initiated immediately after injury, leaving the outcome of delayed administration of IL-33 to be investigated, our data strongly suggest that IL-33 represents a potential therapeutic approach in acute contusion SCI.

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Conflict of interest

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The authors have no conflicts of interest. Acknowledgments We thank Mrs. Mirka Tikkanen for technical assistance with immunohistochemical stainings, Dr. Piia Valonen for technical Q6 assistance with MRI data analysis, Drs. Merja Jaronen and Sarka Lehtonen for technical help with confocal imaging. This work Q7 was supported by Academy of Finland, Finnish Cultural Foundation and Tekes Funding Agency.

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Appendix A. Supplementary data

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbi.2014.08.002.

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High-dose ascorbic acid administration improves functional recovery in rats with spinal cord contusion injury.

Silencing ephrinB3 improves functional recovery following spinal cord injury.

Tamoxifen Administration Immediately or 24 Hours after Spinal Cord Injury Improves Locomotor Recovery and Reduces Secondary Damage in Female Rats.

Neurological and functional recovery after thoracic spinal cord injury.

The immunomodulator decoy receptor 3 improves locomotor functional recovery after spinal cord injury.

Overexpressing neuroglobin improves functional recovery by inhibiting neuronal apoptosis after spinal cord injury.

Fenbendazole improves pathological and functional recovery following traumatic spinal cord injury.

An acute growth factor treatment that preserves function after spinal cord contusion injury.

Ecto-domain phosphorylation promotes functional recovery from spinal cord injury.

MicroRNA-155 Deficiency Suppresses Th17 Cell Differentiation and Improves Locomotor Recovery after Spinal Cord Injury.

Fgf2 improves functional recovery-decreasing gliosis and increasing radial glia and neural progenitor cells after spinal cord injury.

A Coral-Derived Compound Improves Functional Recovery after Spinal Cord Injury through Its Antiapoptotic and Anti-Inflammatory Effects.

The experimental contusion injury of the spinal cord in sheep.

Locomotor training improves premotoneuronal control after chronic spinal cord injury.

Physical exercise improves arterial stiffness after spinal cord injury.

Plasticity for recovery after partial spinal cord injury – hierarchical organization.

Hyaluronan tetrasaccharide exerts neuroprotective effect and promotes functional recovery after acute spinal cord injury in rats.

Sodium hyaluronate-CNTF gelatinous particles promote axonal growth, neurogenesis and functional recovery after spinal cord injury.

Combinatory repair strategy to promote axon regeneration and functional recovery after chronic spinal cord injury.

E2F4 Promotes Neuronal Regeneration and Functional Recovery after Spinal Cord Injury in Zebrafish.

Nanomedicine strategies for treatment of secondary spinal cord injury.

Delayed Imatinib Treatment for Acute Spinal Cord Injury: Functional Recovery and Serum Biomarkers.

Inflammogenesis of Secondary Spinal Cord Injury.

Curcumin improves the integrity of blood-spinal cord barrier after compressive spinal cord injury in rats.