T cell–derived interleukin (IL)-21 promotes brain injury following stroke in mice

IL-21 is closely related to IL-2 and IL-15 and signals through the IL-21 receptor, which is comprised of an IL-21–specific α subunit and a common γ subunit shared with IL-2, IL-7, IL-9, and IL-15. IL-21 is known to regulate immune responses by promoting antibody production, T cell–mediated immunity, and NK cell and CD8⁺ T cell cytotoxicity. Recently, others have shown that stress signals from necrotic tissue can induce rapid IL-21 production from naive T cells (Holm et al., 2009), and co-stimulation with TLR3 ligands during polyclonal T cell activation significantly increases IL-21 secretion that contributes to small intestine localized pediatric celiac disease (van Leeuwen et al., 2013). To test whether IL-21 is up-regulated in brain after ischemic necrosis induced by MCAO and to better understand the cytokines involved in T cell–mediated cerebral I/R injury, we measured changes in inflammatory gene expression in the brain within 24 h after tMCAO in mice using PCR-based gene array analysis. In addition to verifying the up-regulation of several previously reported inflammatory genes, we found that IL-21 was one of the most highly expressed inflammatory genes among those measured (Fig. 1 a). Gene expression levels from arrays were normalized to interquartile spot intensity. Arrays did not differ systemically in gene expression levels before or after normalization (not depicted). This increase in IL-21 gene expression was confirmed by real-time (RT) PCR analysis, which detected a >24-fold relative increase in IL-21 gene expression in the ipsilateral ischemic brain tissues compared with the contralateral hemisphere at 24 h reperfusion (Fig. 1 b). IL-21 was not detectable by this method in healthy brain tissue (Fig. 1 b).

Whether IL-21 contributes to ischemic tissue injury had not been directly studied. However, in the last few years it has become evident that IL-21 expression is associated with acute rejection in mice after kidney, heart, or liver allograft (Baan et al., 2007; Hecker et al., 2009; Xie et al., 2010). Because these models also involve reperfusion of ischemic tissues, these findings support the potential role of IL-21 in I/R injury.

We evaluated the levels of cerebral I/R injury in IL-21–deficient (IL-21 KO) mice. Infarct volumes in IL-21 KO mice were reduced to ∼35% of the infarct volumes observed in congenic C57BL6/J WT mice as early as 24 h after tMCAO, as measured by triphenyltetrazolium chloride (TTC; Fig. 1 c). Similar effects were also seen at 4 d (not depicted) and 7 d after tMCAO (Fig. 1 d), indicating that IL-21 contributes to both immediate and delayed brain injury and suggesting that in the absence of IL-21 tissue repair can occur. Analysis of intracranial vascular anatomy revealed no differences between WT and IL-21 KO mice in the patency of the posterior communicating artery that would account for the observed differences in tissue injury (Fig. 1 g). Nor did we observe differences between WT and IL-21 KO mice in heart rate, or blood pressure before or after tMCAO (Fig. 1 h). WT and IL-21 KO CD4⁺ T cells, CD8⁺ T cells, and CD11b⁺ myeloid cells showed no difference in IL-2, IL-17A, IFN-γ, or TNF production when stimulated in vitro (Fig. 2, g–l). The reduction in infarct volumes corresponded with less weight loss (unpublished data), less spleen atrophy (Fig. 2 c), and improved neurological functioning in IL-21 KO mice compared with WT mice as assessed by both grip strength (Fig. 1 e) and Bederson (Fig. 1 f) scoring 1, 4, and 7 d after tMCAO.

We measured accumulation of monocytes and lymphocytes in the brain after tMCAO in IL-21 KO and WT animals (gating strategy in Fig. 2 a). We did not observe significant differences in the rate of accumulation of monocytes (CD45^highCD11b⁺Ly6c^high), microglia (CD45^intCD11b⁺), B cells (B220⁺), γδ T cells (γδTCR⁺), NK, or NKT cells (NK1.1⁺) in the ischemic brain of WT and IL-21 KO mice after 1, 4, and 7 d of reperfusion (unpublished data). In contrast, as early as 1 d after tMCAO, IL-21 KO mice showed significantly diminished cerebral accumulation of CD4⁺ T cells and CD8⁺ T cells after tMCAO compared with WT mice (Fig. 2 e) and these differences persisted at day 7 (Fig. 2 f). These differences were not reflected in the spleen before or after tMCAO (Fig. 2 b). IL-21 has also been shown to be produced by and modulate the function of regulatory T cells (Peluso et al., 2007; Battaglia et al., 2013), which begin to accumulate in the brain after tMCAO. Thus, we compared the frequency of regulatory CD4 T cells expressing the marker Foxp3 in the brain and spleen 24 h after tMCAO in WT and IL-21 KO mice. IL-21 KO mice exhibited no difference in regulatory T cell abundance in either tissue compared with WT experimental animals. Nor did we observe a difference between WT and IL-21 KO mice in the frequency of lymphocytes producing the antiinflammatory cytokine IL-10 among B cells (B220⁺), CD8⁺ T cells, or CD4⁺ T cells (unpublished data). These data demonstrate that IL-21 deficiency is protective at acute time points after tMCAO and IL-21 levels in the CNS correlate with early infiltration of T cells without affecting regulatory T or B cell accumulation or IL-10 cytokine production during the acute period (day 1–4).

We measured intracellular IL-21 production by various cell populations. IL-21–producing cells were not detected by flow cytometry among cells isolated from healthy brain, but could be detected among mononuclear cells isolated from ischemic brain 24 h after tMCAO. Gating on IL-21⁺ cells revealed that the majority of these cells were CD4⁺ T cells (Fig. 3 a). IL-21–producing CD4⁺ T cells were also detected at low levels among cells isolated from blood, but not spleen, of healthy WT mice, and these levels were unaffected after transient cerebral ischemia (Fig. 2 d), indicating that the increase in IL-21 production was limited to CD4⁺ T cells recovered from the postischemic brain. Next, we adoptively transferred WT or IL-21 KO CD4⁺ T cells into lymphocyte-deficient RAG2 KO mice. Purity of transferred CD4⁺ T cells was confirmed by flow cytometry to be ∼95% (Fig. 3 b). As shown previously (Kleinschnitz et al., 2010), we observed markedly reduced infarcts in RAG KO mice compared with WT mice, and infarct volumes could be restored to WT levels in RAG2 KO by adoptively transferring WT CD4⁺ T cells. Most importantly, RAG2 KO mice that received WT CD4⁺ T cells had significantly larger infarcts than those receiving IL-21 KO CD4⁺ T cells (Fig. 3 c).

We treated WT mice with IL-21 receptor Fc protein (IL-21R.Fc) using a previously described protocol (Jang et al., 2009; McGuire et al., 2011; Spolski et al., 2012). We administered 500 µg of IL-21R.Fc i.p. 1 h before tMCAO. As measured by TTC staining, treated mice showed significantly reduced infarct volumes compared with control-treated mice 24 h after tMCAO (Fig. 4 a). We found a similarly protective effect in mice treated with IL-21R.Fc protein (500 µg i.p.) 2 h after initiation of reperfusion (Fig. 4 a). These differences were associated with decreased locomotor function (decreased resistance to lateral push and increased circling behavior) in control-treated mice compared with those treated with IL-21R.Fc (Fig. 4 b and Video 1). Although we cannot exclude the possibility that IL-21R.Fc exhibits its blocking effect in peripheral immune compartments, using ELISA for human IgG4 we were able to observe that—upon i.p. injection—soluble IL-21R.Fc accumulates in the CNS of mice after tMCAO (Fig. 4 c).

IL-21⁺ cells were recently detected in human brain tissue during different neuroinflammatory conditions (Tzartos et al., 2011). Thus, we stained groups of postmortem brain tissue from patients with acute and chronic stroke lesions. In acute infarcts, rare CD4⁺ T cells were found in the necrotic brain parenchyma (Fig. 4 g, ii, arrows), which was predominantly infiltrated by foamy macrophages (Fig. 4 g, i). In contrast, CD4⁺ T cells were consistently found within the Virchow Robins space of vessels bordering acute infarcts (not depicted) and in the subarachnoid space adjacent to meningeal vessels (arrows, Fig. 4 e, i). IL-21 staining was limited almost exclusively to these perivascular spaces. Compared with control stained tissue (Fig. 4 d, i), anti–IL-21 staining labeled cells extensively in the subarachnoid space of deep sulci penetrating the infarcted tissue—showing a similar distribution to CD4⁺ cells in serial sections (arrows, Fig. 4 f). Additionally, double staining with antibodies for CD4 and IL-21 revealed the presence of CD4⁺ IL-21⁺ cells in this perivascular niche (arrow, Fig. 4 g, iii). In summary, CD4⁺ T cells that can secrete IL-21 were detected within the CSF-filled subarachnoid and perivascular spaces during cerebral infarction in humans.

RT-PCR analysis of primary mouse neurons and murine neuronal cell lines (Neuro2A) indicated that IL-21R expression was higher on neuronal cells than on other brain cells, including astrocytes and endothelial cells (Fig. 5, a and c). This is consistent with another report where in situ hybridization detected neuron-restricted IL-21R expression in inflamed human brain tissue (Tzartos et al., 2011). Moreover, treating Neuro2a cells with IL-21 after in vitro oxygen glucose deprivation (OGD) significantly increased cell death as measured by XTT cell viability assay (Fig. 5 b). In subsequent studies we found that treatment of primary neurons with IL-21 up-regulated mRNA levels of the autophagy associated gene ATG6 (Fig. 5 d). These data suggest that IL-21 could directly affect neuronal autophagy during ischemic injury, which has been implicated in neuronal death in infarcted and periinfarcted brain tissue. Thus, we stained WT and IL-21 KO postischemic brain tissues for ATG6. We observed significantly fewer ATG6⁺ cells in infarcted brain tissue of IL-21 KO mice compared with WT, suggesting that IL-21 may contribute to increased cerebral autophagy after stroke (Fig. 5 e).

In conclusion, we implicate IL-21 as a lymphocyte-derived factor with a pronounced effect on brain injury after focal ischemia in mice. We also present data demonstrating that IL-21–producing CD4⁺ T cells are present in the brain of patients with acute stroke. These data warrant investigation of the therapeutic potential of IL-21–modifying treatments in isolation and combination with current anti-thrombotic treatments for ischemic stroke.

C57BL/6 WT mice were obtained from The Jackson Laboratory. IL-21–deficient mice (IL-21^tm1Lex) were purchased from the Mutant Mouse Regional Resources Center. All mice underwent 1 h tMCAO and 24 h reperfusion. All animal procedures used in this study were conducted in strict compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the University of Wisconsin Center for Health Sciences Research Animal Care Committee. All mice (∼25 g) were anesthetized with 5% halothane for induction and 1.0% halothane for maintenance vaporized in N₂O and O₂ (3:2), and all efforts were made to minimize suffering.

Changes in rCBF at the surface of the left cortex were recorded using a blood perfusion monitor (Laserflo BPM2; Vasamedics) with a fiber optic probe (0.7 mm diam). The tip of the probe was fixed with glue on the skull over the core area supplied by the MCA (2 mm posterior and 6 mm lateral from bregma). Changes in rCBF after MCAO were recorded as a percentage of the baseline value. Mice included in these investigations had >80% relative decrease in rCBF during MCAO.

WT and IL-21 KO mice were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). After thoracotomy was performed, a cannula was introduced into the ascending aorta through the left ventricle. Transcardial perfusion fixation was performed with 2 ml saline and 2 ml of 3.7% formaldehyde. Carbon lampblack (C198-500; Thermo Fisher Scientific) in an equal volume of 20% gelatin in ddH₂O (1 ml) was injected through the cannula. The brains were removed and fixed in 4% PFA overnight at 4°C. Posterior communicating arteries (PComA) connect vertebrobasilar arterial system to the Circle of Willis and internal carotid arteries, and its development affects brain sensitivity to ischemia among different mouse strains (Barone et al., 1993). Development of PComA in both hemispheres was examined and graded on a scale of 0–3, as reported previously (Majid et al., 2000). 0, no connection between anterior and posterior circulation; 1, anastomosis in capillary phase (present but poorly developed); 2, small truncal PComA; 3, truncal PComA.

Focal cerebral ischemia in mice was induced by occlusion of the left MCA, as described previously (Longa et al., 1989). Operators performing surgeries were masked to experimental groups. In brief, the left common carotid artery was exposed, and the occipital artery branches of the external carotid artery (ECA) were isolated and coagulated. After coagulation of the superior thyroid artery, the ECA was dissected distally and coagulated along with the terminal lingual and maxillary artery branches. The internal carotid artery (ICA) was isolated, and the extracranial branch of the ICA was then dissected and ligated. A standardized polyamide resin glue-coated 6.0 nylon monofilament (3021910; Doccol Corp) was introduced into the ECA lumen, and then advanced ∼9–9.5 mm in the ICA lumen to block MCA blood flow. During the entire procedure, mouse body temperature was kept between 37 and 38°C with a heating pad. The suture was withdrawn 60 min after occlusion. The incision was closed, and the mice underwent recovery.

After 24 h reperfusion, mice were sacrificed and brains were removed and frozen at −80°C for 5 min. 2-mm coronal slices were made with a rodent brain matrix (Ted Pella, Inc.). The sections were stained for 20 min at 37°C with 2% TTC (Sigma-Aldrich). Infarction volume was calculated with the method reported by Swanson et al. (1990) to compensate for brain swelling in the ischemic hemisphere. In brief, the sections were scanned, and the infarction area in each section was calculated by subtracting the noninfarct area of the ipsilateral side from the area of the contralateral side with National Institutes of Health image analysis software, ImageJ. Infarction areas on each section were summed and multiplied by section thickness to give the total infarction volume.

Ipsilateral brain hemispheres were dissected and stored in RNAlater (QIAGEN) at 4°C until further use. Total RNA was extracted and purified with RNeasy Protect Mini kit (QIAGEN) according to the manufacturer’s instructions. Purified RNA samples were analyzed by GeArray S Serious Mouse Autoimmune and Inflammatory Gene array (SuperArray; Bioscience Corporation). Results from GeArray were filtered for genes with spot intensities higher than the mean local background of the bottom 75% of nonbleeding spots. For RT-PCR, 1 µg total RNA from each sample was reverse transcribed using SuperScript II first strand cDNA synthesis kit (Invitrogen). RT-PCR was performed on a Smart Cycler (Model SC 100–1; Cepheid) using IL-21 TaqMan gene expression assay (Mm00517640_m1; Applied Biosystems), RT² qPCR Primer assay for mouse Becn1 (PPM32434A; SABiosciences), or RT² qPCR Primer Assay for Mouse IL21r (PPM03762A; SABiosciences). The data were normalized to an internal reference gene, GAPDH.

Brains were removed from perfused animals, weighed, minced, transferred to Medicon inserts, and ground in a MediMachine (BD) for 20–30 s. The cell suspension was washed with HBSS, and cells were resuspended in 70% Percoll (Pharmacia) and overlaid with 30% Percoll. The gradient was centrifuged at 2,250 g for 30 min at 4°C without brake. The interface was removed and washed once for further analysis. CD11b-positive and -negative fractions were isolated using Imag anti-CD11b magnetic particles (BD), following the manufacturer’s protocol. A total of 10⁶ cells were incubated for 30 min on ice with saturating concentrations of labeled antibodies with 40 µg/ml unlabeled 2.4G2 mAb to block binding to Fc receptors, and then washed 3 times with 1% BSA in PBS.

Single-cell suspensions from various tissues were cultured at 37°C in 10% FBS in RPMI 1640 media supplemented with GolgiStop (BD) in the presence of either phorbol myristate acetate (50 ng/ml) and ionomycin (1 µg/ml) for 5 h. After surface staining with antibodies against CD4, NK1.1, and γδTCR, cell suspensions were fixed and permeabilized by Cytofix/Cytoperm solution (BD), followed by staining with anti–IL-21 antibodies. Fluorochrome-labeled antibodies against CD45, CD11b, Ly6c, B220, CD4, CD8a, NK1.1, IFN-γ, and appropriate isotype controls were purchased from BD. Fluorochrome-labeled antibody against IL-21 and γδTCR was purchased from eBioscience. Cell staining was acquired on a FACSCalibur or LSRII (BD) and analyzed with FlowJo (Tree Star) software version 5.4.5.

Neuromuscular coordination was assessed by grip strength test, as previously described (Kleinschnitz et al., 2010). For this test, mice were placed on a horizontal string midway between two supports. Mice were scored from 0 to 5 as follows: 0, falls off within 2 s; 1, hangs on with forepaw(s); 2, hangs on with forepaws and moves laterally on string; 3, hangs onto string with forepaws and hindpaw(s); 4, hangs onto string with forepaws, hindpaw(s) and tail; 5, escape to supports. Mice were allowed to rest between trials. Scores for each mouse were determined by averaging 5–10 trials (each lasting ∼15 s). Global neurological deficit was determined by a modified Bederson scoring system: 0, no deficit; 1, forelimb flexion; 2, unidirectional circling after being lifted by tail; 3, spontaneous unidirectional circling; 4, longitudinal rolling upon being lifted by tail; 5, spontaneous longitudinal rolling.

Chinese hamster ovary cell line (Korn et al., 2007) expressing the extracellular domain (aa 20–236) of mouse IL-21R fused to the fragment crystallizable (Fc) portion of human IgG4 (IL-21R.Fc) were maintained in UltraCHO (BioWhittaker). IL-21R.Ig was purified from the culture supernatant by passage through a protein G–Sepharose column and concentrated by ultrafiltration. Concentration was determined spectrophotometrically. Purity and molecular weight were confirmed by sodium dodecyl-sulfate PAGE and human-IgG4 ELISA (eBioscience) following the manufacturer’s instructions. The IL-21R.Fc reagent was tested in vitro for its ability to suppress IL-21–induced T cell proliferation.

Paraffin-embedded postmortem brain tissue sections from individuals with acute and chronic stroke lesions were obtained from the Neuropathology Laboratory of the University of Wisconsin Department of Pathology. After rehydration and deparaffinization, sections underwent heat-induced antigen retrieval in 10 mM sodium citrate, pH 6.0, for surface antigens or Tris-EDTA (10 mM/1 mM) with 0.05% Tween-20 for intracellular antigens. Sections were blocked for 30 min with secondary serum (10% in Tris-buffered saline) and then stained with primary antibodies, 0.5% chicken anti–IL-21 (Lifespan Biosciences) or prediluted mouse anti-CD4 ([1F6]; ab17131; Abcam) for 1–2 h at 37°C or overnight at 4°C. Normal primary sera (5–10%) were used for negative control. After several washes, secondary antibodies (biotin-labeled goat anti–chicken or biotin-labeled goat anti–mouse; Vector Laboratories) were applied to sections and incubated for 2 h at room temperature. Staining was developed using the VECTASTAIN ABC-HRP kit (Vector Laboratories) with diaminobenzidine substrate (BD) or streptavidin-alkaline phosphatase with Fast Red substrate (Laboratory Vision), following the manufacturer’s instructions. Slides were lightly counterstained with hematoxylin, rinsed with running tap water, and mounted. For frozen mouse sections WT and IL-21 KO mice underwent 1-h tMCAO and 1–7-d reperfusion. Brains were perfused with PBS and 3% formalin, embedded in OCT, and cut into 8-µm frozen sections for immunohistochemistry. Frozen sections were thawed for 10 min at room temperature and blocked with 5% goat serum solution in PBS for 15 min. Sections were stained with rabbit polyclonal antibodies for ATG6 for 1 h at room temperature, followed by phycoerythrin-labeled goat anti–rabbit IgG (Santa Cruz Biotechnology, Inc.). Images were acquired on a BX40 microscope equipped with a Q-Color 3 camera using Q-Capture software (Olympus). Digital images were processed and analyzed using Photoshop CS4 software (Adobe). Color balance, brightness, and contrast settings were manipulated to generate final images. All changes were applied equally to entire image.

Primary neuronal cultures derived from embryonic day 14–18 mouse cortices were grown to 80% confluency in neural basal media supplemented with B27 (2%) and penicillin/streptomycin (1%), as previously described (Kintner et al., 2010). Astrocytic and microglial contamination was excluded based on the absence of GFAP⁺ and CD11b⁺ cells when stained by immunocytochemistry. For OGD, media was replaced with neural basal media with or without glucose and placed in a hypoxic chamber or under normoxic conditions for 2 h at 37°C. Afterward, cells were lysed and mRNA isolated using RNeasy mini kit (QIAGEN). For XTT viability assay, Neuro2A underwent OGD and were treated with dose curve of IL-21 immediately after return to normoxic media and incubated for 4 h at 37°C. XTT labeling mixture (50 µl per well; Roche) was added according to manufacturer’s instructions, and at18 h fluorescence was read on a GENious Microplate reader (Tecan). Cell number was calculated using a standard curve of known untreated cells kept under normoxic conditions.

All surgeries were performed in a blinded manner by a third party and measurements masked where possible. Infarct volume measurements from TTC stained sections were averaged from two to three independent blinded observers. Based on power calculations, n = 3–10 sex- and age-matched mice were used for each experiment and group assignment was randomized. Among animals receiving MCAO procedure, 86.5% of WT mice, 93.5% of IL-21KO mice, and 100% of RAG2KO mice were included in analysis. Mice were excluded due to premature death (13.5% of WT mice, 3.2% of IL-21 KO mice) or vessel variation (3.2% of IL-21KO mice). Results are given as means ±1 SD. Multiple comparisons were made using one-way ANOVA. Where appropriate, two-tailed Student’s t test analysis was used for comparing measures made between two groups. For comparison of RT-PCR data, nonparametric Mann-Whitney rank sum analysis was used. P-values <0.05 were considered significant.

Video 1 shows groups of WT C57BL6 mice treated with 500 µg IL-21R.Fc or PBS control via i.p. injection.

We thank Satoshi Kinoshita for expert histopathology services, Guoqing Song for assisting in the surgical procedures, Dr. Wenda Gao for providing reagents and protocols for the purification of IL-21R.Fc protein, and members of our laboratory for helpful discussions and constructive criticisms of this work. We also thank Khen Macvilay and Sinarack Macvilay for their expertise provided for cytofluorimetry and immunohistochemistry studies and Samuel (Joe) Ollar for assisting in the OGD procedure.

This work was supported by awards from the American Heart Association (pre-doctoral fellowship #12PRE12060020 to B.D.S. Clarkson) and the National Institutes of Health (NS037570 and NS076946 to ZF, AI048087 to M.S. Salamat, and AI068730 to J.D. Lambris).

The authors have no competing financial interests.