Cell therapy centered on IL-1Ra is neuroprotective in experimental stroke

In this study, we used adult male IL-1Ra KO mice that lacked all isoforms of IL-1Ra and heterozygous IL-1Ra-Tg mice carrying a transgene encoding the secreted form of IL-1Ra (sIL-1Ra) under the control of its endogenous promoter (Table S1). Both mouse strains were kindly donated by Dr. Emmet Hirsh (Columbia University, USA) and have previously been described in detail [32, 36]. Wild-type littermate (LM) mice were used as controls. IL-1Ra-KO mice lacked IL-1Ra mRNA in the brain, and IL-1Ra-Tg mice expressed higher levels of IL-Ra in the brain than LM mice [IL-1Ra: 252 ± 170 pg/mg (Tg), and 8 ± 3 pg/mg (LM), (mean ± SD), n = 14/group]. Both mouse strains were maintained as colonies at the Laboratory of Biomedicine, University of Southern Denmark, Odense, Denmark. In addition, young adult, male and female homozygous C57BL/6-Tg(UBC-GFP)30Scha/J (Stock No. 004353) (GFP-TG) breeding pairs were purchased from the Jackson Laboratory (Bar Harbour, Maine, USA) and transferred to the Department of Biomedicine, University of Aarhus, where they were kept as a colony. IL-1α/β-KO mice [33] were kindly donated by Dr. Yoichiro Iwakura and used for control tissue. All C57BL/6 mice were purchased from Taconic Ltd. (Denmark). The mice were housed under diurnal lighting conditions and given free access to food and water.

This study was performed using postmortem brain tissue from 21 stroke cases. Sex, age, infarcted brain area and the age of the infarct are given in Table S2. Four cases are part of a previous study on surfactant protein-D in human ischemic brain tissue [42]. Control tissues included brain, kidney, pancreas, lung, placenta and tonsil (not shown). The study was approved by the Regional Committee on Health Research Ethics from the Region of Southern Denmark (Project-ID S-20080042) and performed at the Department of Pathology, Odense University Hospital, Denmark.

All studies were conducted using young age-matched male mice. Focal cerebral ischemia was induced by either permanent (p) or transient (t) middle cerebral artery occlusion (MCAo). pMCAo permanent occlusion of the distal part of the left MCA was performed as detailed in Clausen et al. [10]. tMCAo transient occlusion (40 min) of the right MCA was performed using the intraluminal filament placement technique, as detailed in Inacio et al. [35], under 1.5 % isoflurane anesthesia. All studies were conducted as randomized, double-blinded studies, with inclusion of sham-operated mice and unlesioned controls (Ctl). Blood flow and body temperature were monitored using the optical fiber probe (VP10 M200ST, UK) and endoscopic temperature probe (T6a, UK) from Moor Instruments. The study was approved by the Danish Veterinary and Food Administration (J. no. 2011/561-1950). An overview of the different mouse groups used for assessment of the effect of IL-1Ra and IL-1Ra-producing cells on infarct volume is provided in Table S1.

Irradiation BM chimeric mice were generated as detailed in Clausen et al. [10]. All recipient mice were irradiated with a single dose of 9.5 Gy from a ¹³⁷Cs source and BM transplantation into the tail vein was performed within 2 h of whole body irradiation. Recipient mice were allowed to reconstitute for 6 weeks prior to pMCAo. The extent of chimerism was assessed in blood samples taken from C57BL/6 mice reconstituted with BM cells from GFP mice at the time of termination (n = 5). Flow cytometry showed that 91.8 ± 0.9 % (mean ± SD) of the circulating leukocytes were CD11b⁺CD45^high GFP⁺ leukocytes, indicating a successful reconstitution of the mice.

Harvesting and transplantation BM cells were harvested in RPMI medium (Gibco, Life Technologies), and approximately 10⁷ cells were injected into the tail vein of recipient mice 30 min after MCAo [10]. Cell labelling A Vybrant^® CFDA SE Cell (CFSE) Tracer Kit (V12883, Invitrogen) was used for cell labeling and visualization in situ or by flow cytometry. Characterization BM cells harvested from IL-1Ra-Tg and LM mice (n = 5/group) were analyzed with regard to cell numbers and cell size using the Scepter 2.0 automated Cell Counter from Millipore. To assure compatibility with the Scepter correlation range (50 × 10⁵–1.5 × 10⁶ cells/ml), 10 µl of BM cell suspension was first counted using a Bürker-Türk counting chamber. Next, 100 µl of a BM cell suspension from IL-1Ra-Tg and LM mice were counted using the Scepter automated cell counter employing 40-μm Scepter sensors (Millipore). Histograms and overlays were computed utilizing the Scepter™ 2.0 Cell Counter and Software Pro system.

Blood gas analysis Venous blood samples [4] collected 30 min after BM injection (n = 6–8/group). were analyzed for pO₂/pCO₂, pH, electrolytes, glucose and lactate. Temperature Rectal temperature was measured on all mice before surgery (0 h), before BM injection (30 min) and 1 and 3 h after pMCAo using a temperature probe with a Model Bat 12 unit (Physiotemp, New Jersey, USA). Body weight All mice were weighed at 0 h and 1, 3 and 5 days after pMCAo.

Grip strength The grip strength test was performed as previously detailed [4, 40] with the principal investigator blinded to the treatment. The highest force score of the front paws was recorded pre- and post-surgically, using the grip strength meter from BIOSEP (BIO-GT-3, France). Hargreaves test Thermal nociception was tested as detailed [5, 49] using the plantar test device (Ugo Basile, Model 37370, Italy). The investigator was blinded to the treatment. Five measurements were registered for the hind paws pre- and post-surgically, the lowest and highest value was excluded and the mean calculated. The mice were allowed to recover for 15 min between trials.

Open field test Locomotor and anxiety-related activities were investigated using the open field test [Box: 45 (W) × 45 (D) × 40 (H) cm] with the investigator blinded to the treatment. This test was conducted as previously detailed [41, 45] over a 10 min period. Movement was recorded automatically, while time to first rear, digging, grooming, jumping, urination and droppings were recorded manually and documented as the number of events.

Fresh (CO ₂ ) frozen tissue Brains were processed into six parallel series of sections (30 μm). Separate series were collected on glass slides and used for infarct size analysis, in situ hybridization (ISH) and immunohistochemistry (IHC) or in Eppendorf tubes and used for qPCR, Western blotting, and ELISA. qPCR The tissue was processed as detailed by Clausen et al. [12]. Flow cytometry The tissue was processed as detailed by Clausen et al. [10]. Paraformaldehyde fixed tissue Brains were fixed and processed into 12 series of sections (16 μm) for histological analysis was performed as detailed by Clausen et al. [10].

Infarct size analysis was performed according to Cavalieri´s principle for infarct volume estimation (IFV) as previously described on one series of sections spanning the infarct [4, 27] with blinded assessment of the outcome.

RNA extraction, cDNA synthesis and qPCR were performed with published primers for IL-1β mRNA and HPRT1 mRNA as described by Clausen et al. [12]. In addition, the following primer sequences were used, all spanning exon–exon junctions with sequence specificity checked using BLAST: IL-1Ra mRNA (sense: AAC CAG CTC ATT GCT GGG TAC TTA; antisense: GCC CAA GAA CAC ACT ATG AAG GTC), IL-1α mRNA (Sense: GTC GGC AAA GAA ATC AAG ATG; antisense GTC TTC GTT TTC ACT GTA ACA G), IL-1R1 mRNA (sense: ACA ACG TGA GCT TCT TCG GAG TA; antisense: GGT CTG TCC CTC TTG TTT TCA TCT ATT), and IL-1R2 mRNA (sense: AGG AAT ACA ACA TCA CTA GGA ATA TTG AAC; antisense: TCA GTC TTG ACC CCA ATG ATG CT). Cytokine mRNA levels were normalized to a pool of non-lesioned brains. Quantitative PCR investigating temporal changes were conducted using C57BL/6 mice with 1, 2, 4, 6, 12 and 24 h survival, including shams and non-lesioned controls (n = 10–12/group).

Alkaline phosphatase (AP)-conjugated oligodeoxynucleotide probe sequences for IL-1β mRNA and GAPDH mRNA were used, as described by Clausen et al. [11, 12]. In addition, the following probes were synthesised: IL-1Ra mRNA (5′TGC CCC CGT GGA TGC CCA AGA ACA C) and IL-1α mRNA (probe 1: 5′GTG CTG ATC TGG GTT GGA TGG TCT CTT C; probe 2: 5′GTT TCT GGC AAC TCC TTC AGC AAC ACG G). Probe specificity was validated as detailed by Clausen et al. [10], with the inclusion of sections from IL-1Ra-KO mice (Figure S1). Temporal profiles for IL-1Ra mRNA, IL-1α mRNA and IL-1β mRNA were obtained from one series of sections from C57BL/6 mice with 30 min, and 1, 4, 6, 12 and 24 h survival, including sections from non-lesioned controls (n = 6/group). IL-1Ra mRNA was also analyzed in one series of sections from mice with 5 days of survival (n = 10/group).

Flow cytometry was performed using the FACSCalibur flow cytometer and CellQuest Pro Software (BD Bioscience) [10, 40]. IL-1α⁺ and IL-1β⁺ microglia [CD11b⁺CD45^dim] and leukocytes [CD11⁺CD45^high] were identified as detailed by Clausen et al. [10]. Samples analyzed for IL-1Ra protein were fixed and permeabilized in Cytofix/Cytoperm for 20 min at 4 °C. Cells were washed in 1 ml 1 × PermWash™ buffer (BD Bioscience), blocked using 10 % fetal calf serum (FCS) with 50 μg/ml Syrian Hamster IgG (11.3 mg/ml) (Jackson Immunoresearch) and incubated with anti-IL-1Ra prior to surface marker labeling of microglia and macrophages in mice with 6, 12 and 24 h survival in addition to non-lesioned controls (Ctl) (n = 4–8/group) [10]. BM cells were quantified based on the CFSE signal, and cell numbers were estimated in mice with 6 h survival as described previously [3, 64]. All antibodies used are listed in Table S3.

Mouse brain tissue CD41, IL-1α and IL-1RII were visualized using the streptavidin/horseradish peroxidase (HRP) technique, IL-1β, using the peroxidase labeled “Ready to use” EnVision⁺ polymer (Dakocytomation, Denmark) [10, 12] and IL-1Ra and IL-1RI, using the Vectastain ABC kit (PK4005 and PK6104; Vector Laboratories) on series of sections from mice with 4, 6, 12 and 24 h survival in addition to non-lesioned controls (n = 6/group). IHC double-fluorescence staining This was performed as detailed in Clausen et al. [10] on 16 sections spanning the anterior commissure including areas of both frontal and parietal cortices (n = 5–6 mice/staining). The specificity of staining for IL-1α, IL-1β and IL-1Ra was verified by the absence of staining in IL-1α/β-KO and IL-1Ra-KO brains, and the specificity of all other antibodies was verified by the substitution of the primary antibody with isotype or serum controls (Table S3), which were devoid of signal (data not shown). Human brain tissue Formalin-fixed paraffin-embedded [31] hematoxylin and eosin (HE)-stained tissue samples were evaluated by independent pathologists before serial sections were stained for IL-1Ra, rIgG2a, Iba1 CD45, CD68 and GFAP (Table S3). Slides were stained on the AutostainerPlus platform (Dako, Denmark) as previously described by Hermansen et al. [31]. The PowerVision⁺Poly_HRP IHC detection System (PV6107, Leica Biosystems) was used for the detection of all antibodies [31]. Sections stained with IgG or sections where primary antibodies were omitted were devoid of staining. IHC double staining was performed using the BenchMark fully automated staining instrument and the Vectastain Universal Elite ABC kit (Vector Labs, USA) [14]. Sections were counterstained with Mayer’s hematoxylin (Bie & Berntsen, Herlev, Denmark). All antibodies used are listed in Table S3.

The infarcts were categorized according to age as follows at 1–2 days (n = 8) and 5 to ≥7 days (n = 13). The infarct core, the peri-infarct and normal-appearing tissue were identified in hematoxylin-stained sections. The regions were transferred onto parallel sections stained for IL-1Ra and scored as follows: Score 0: no staining; Score 1: very weak staining; Score 2: weak staining; Score 3: moderate staining; Score 4: strong staining; Score 5: very strong staining. The scoring was performed by two observers blinded to the age of the infarcts.

IL-1Ra (IL-1Ra Kit: MRA00, R&D) and IL-1β (IL-1β Kit: CMC0813, Invitrogen) ELISA were performed on one series of sections and conducted as specified in the ELISA kits. Measurements were normalized to the total protein content of the sample as measured using the method by Bradford [7].

Western blotting was performed on one series of sections as described in Clausen et al. [9] with minor modifications. Western blotting for phosphorylated (p)-SAPK/JNK (Thr183/Tyr185) (1:1000), p-p44/p42 MAPK (ERK1/2)(1:2000) and p-p38 MAPK (Tyr180/Tyr182) (1:1000) (S9910 Kit, Cell Signaling) was performed using 4–12 % gels (Life Technologies) using transcription factor IIB (TFIIB) (Cell Signaling, 1:1000) as a loading control and SeeBlue Plus2 standard (Invitrogen) as an indicator of size. Bands were amplified using a SuperSignal^®Extended duration substrate (34075, Thermo) and visualized by chemiluminescence using the FUSION 7X camera. Densitometry was performed using Image J (version 1.47v) (NIH) following recommendations of the Image J developers. Analysis was performed on two independent gels with n = 2/group and data were normalized to TFIIB and represented as percentages relative to naive LM mice.

Quantitative data are presented as mean ± SD. Comparison between two groups was performed using Mann–Whitney or paired t test. Multiple comparisons were performed using one-way ANOVA and repeated-measures ANOVA, followed by appropriate post hoc tests. Correlations were established using the non-parametric Spearman test. Statistical analysis was performed using the Prism 5 software for Windows (GraphPad). Statistical significance was established for P < 0.05.

To confirm the findings by others suggesting that endogenous IL-1Ra is neuroprotective in experimental stroke [43], we initially subjected IL-1Ra knockout (IL-1Ra-KO) mice, mice overproducing IL-1Ra under the control of the natural promoter (IL-1Ra-Tg) and their respective littermate (LM) mice to permanent middle cerebral artery (MCA) occlusion (pMCAo) (Table S1). Twenty-four hours after pMCAo, IL-1Ra-KO mice developed significantly larger infarcts (Fig. 1a) and IL-1Ra-Tg mice significantly smaller infarcts (Fig. 1b) than their respective LM controls. Additionally, at 24 h after pMCAo, we observed numerous IL-1Ra mRNA expressing (IL-1Ra mRNA⁺) cells, and IL-1Ra immunoreactive (IL-1Ra⁺) cells within the peri-infarct in C57BL/6 mice (Fig. 1c, d). The location of the IL-1Ra-producing cells in the tissue at risk for degeneration is consistent with a neuroprotective role of endogenous IL-1Ra.

To obtain detailed insight into the cellular orchestration of IL-1Ra and IL-1(α/β), we examined the synthesis of all three cytokines in C57BL/6 mice after pMCAo. By the use of quantitative reverse transcription PCR (qPCR), we observed small fluctuations in IL-1Ra mRNA until 6 h (Fig. 2a) and a significant up-regulation at 12 and 24 h after pMCAo, compared to non-lesioned and sham controls (Fig. 2a). Within the peri-infarct, we found multiple process-bearing microglial-like cells with abundant IL-1Ra mRNA (Fig. 2b–d) and protein accumulation (Fig. 2f, g) 6, 12 and 24 h after pMCAo, and round vessel-associated leukocyte-like cells from 4 to 6 h after pMCAo (insert in Fig. 2b, e).

The level of IL-1α mRNA was significantly up-regulated at 6, 12 and 24 h (Fig. 2h), with IL-1α mRNA and protein peaking in microglial-like cells located in the peri-infarct 12 and 24 h after pMCAo (Fig. 2i–m). IL-1β mRNA was increased at 1, 12 and 24 h (Fig. 2n) and showed larger increases than IL-1α mRNA (Fig. 2h). IL-1β mRNA⁺ cells were detected from 30 min after pMCAo and onward, increasing in number until 24 h, at which time IL-1β mRNA⁺ cells also occurred perivascularly (Fig. 2o–q). IL-1β was expressed by microglial-like cells in the peri-infarct at 4 and 24 h and by leukocyte-like cells 24 h after pMCAo (Fig. 2r, s). When performing Spearman correlation analysis, we observed a positive correlation between IL-1Ra and IL-1β mRNA at 1, 2, 6, 12 and 24 h after pMCAo and between IL-1Ra and IL-1α mRNA 24 h after pMCAo (Table S4). No correlation was observed between IL-1α and IL-1β mRNA (Table S4). IHC double-fluorescence staining showed the expression of IL-1Ra, IL-1α and IL-1β in CD11b⁺ cells (Fig. 2t–v), but not in glial fibrillary acidic protein-expressing astrocytes (data not shown), suggesting microglia and/or leukocytes as the major source of these cytokines. In addition, we found IL-1α in CD41⁺ platelets at 4–6 h, but not 24 h, after pMCAo (Fig. 2w, x), which adds to in vitro findings of IL-1α in platelets [62].

Since IL-1(α/β) and IL-1Ra are known to impact on infarct development, we additionally analyzed the mRNA expression of IL-1R1 and -1R2 by qPCR. We found small fluctuations in IL-R1 mRNA until 6 h and small increases at 12 and 24 h after pMCAo compared to non-lesioned and sham controls (Figure S2a, c). IL-1R1 immunoreactivity was associated with vascular cells at 6 h and glial-like cells at 24 h after pMCAo (Figure S2b). In comparison, IL-1R2 mRNA was significantly increased at 6, 12 and 24 h compared to non-lesioned and sham controls (Figure S2c), significantly correlating to the ischemia-induced increase in IL-1α mRNA (Table S4) and the occurrence of IL-1R2⁺ leukocyte-like cells in the peri-infarct (Figure S2d).

A quantitative assessment of microglial versus leukocyte-derived IL-1Ra, IL-1α and IL-1β was performed by flow cytometry separating CD11b⁺ cells into CD11b⁺CD45^dim microglia and CD11b⁺CD45^high leukocytes (Fig. 3a) [10, 40, 58]. As previously shown [10, 40], the number of recruited CD11b⁺CD45^high leukocytes gradually increased from 6 to 24 h after pMCAo in C57BL/6 mice, whereas the number of CD11b⁺CD45^dim microglia overall remained constant (Fig. 3a). IL-1Ra, IL-1α and IL-1β were expressed by both CD11b⁺CD45^dim microglia (Fig. 3b) and CD11b⁺CD45^high leukocytes (Fig. 3c), however, with significant differences in baseline and temporal expression profiles (Figs. 3b, S3a–c). Interestingly, there were significantly higher numbers of IL-1Ra⁺ compared to IL-1α⁺ and IL-1β⁺ microglia in non-lesioned control mice and at 6 h after pMCAo, but not at 12 h, when both IL-1α⁺ and IL-1β⁺ microglia had increased compared to controls (Fig. 3b). By 24 h, the proportion of IL-1Ra⁺ microglia reached 34 ± 11 % [mean ± SD, (n = 4)] of the gated CD11b⁺CD45^dim population, while IL-1α⁺ reached 9 ± 1 % [mean ± SD, (n = 4)] and IL-1β⁺ reached 18 ± 5 % [mean ± SD, (n = 4)].

The number of IL-1β⁺ microglia exceeded the number of IL-1α⁺ microglia after pMCAo at all times of observation (Fig. 3b). Surprisingly, few CD11b⁺CD45^high leukocytes were IL-1Ra⁺ apart from 6 h after pMCAo, when 16 ± 7 % (n = 4) were IL-1Ra⁺ (Fig. 3c). By 24 h, less than 0.3 ± 0.05 % (n = 4) of the leukocytes were either IL-1Ra⁺ or IL-1α⁺, whereas 17 ± 9 % (n = 4) expressed IL-1β (Fig. 3c). A comparison of the number of microglia and leukocytes expressing IL-1Ra, IL-1α and IL-1β showed that microglia was the major source of IL-1Ra and IL-1α, whereas both microglia and leukocytes were a source for IL-1β 24 h after pMCAo (Fig. 3b, c; Table S5). These findings were confirmed by the use of GFP irradiation BM chimeric mice (Figure S3a–c) showing that the majority of the IL-1Ra⁺ and IL-1α⁺ cells were GFP⁻ microglia (Figure S3a, b), while IL-1β was expressed by both GFP⁻ microglia and GFP⁺ leukocytes (Figure S3c). These results clearly show microglia as the major source of neuroprotective IL-1Ra after pMCAo, and additionally suggest that increasing IL-1Ra production in recruited cells could be of relevance in stroke therapy.

Based on our previous findings showing that IL-1β and TNF are expressed by largely different subsets of microglia [10], we assessed the proportion of CD11b⁺CD45^dim microglia co-expressing IL-1Ra, IL-1α and/or IL-1β 24 h after pMCAo. We found IL-Ra and IL-1β to be produced by segregated subsets of CD11b⁺CD45^dim microglia with no co-expression (Fig. 3d, e). Approximately, 5 % of the gated IL-1Ra⁺ microglia co-expressed IL-1α (5.1 ± 1.9 % (n = 4)), while significantly higher proportions, approximately 50 % of the gated IL-1α⁺ cells co-expressed IL-1Ra (50.4 ± 6.5 % (n = 4)) (Figure S3d). Further, we found that approximately 8 % of the gated IL-1β⁺ microglia co-expressed IL-1α (8.0 ± 4.1 % (n = 4)) (Fig. 3e), with higher proportions, and approximately 17 % of the gated IL-1α⁺ microglia co-expressing IL-1β (17.3 ± 6.1 % (n = 4)) (Figs. 3e, S3d). Double-immunofluorescence staining confirmed the absence of co-expression between IL-1Ra and IL-1β 24 h after pMCAo, showing spatially segregated cells producing either IL-1Ra or IL-1β (Fig. 3f, i). We confirmed the co-expression of IL-1Ra and IL-1α (Fig. 3g, j) and IL-1α and IL-1β; the latter however was only observed in a few number of cells 24 h after pMCAo (Fig. 3h, k). Taken together, these results demonstrate a functional diversity among microglia at the site of injury.

Based on the observation that IL-1Ra is expressed by a small subset of infiltrating CD11b⁺CD45^high leukocytes, we next tested whether increasing IL-1Ra production in infiltrating leukocytes would be neuroprotective. As previously done [10, 40], we used a BM chimeric approach, comparing infarct development in whole body-irradiated recipient C57BL/6 mice (B6*) mice reconstituted with BM cells from either IL-1Ra-Tg, IL-1Ra-KO or B6 mice (Table S1). We observed no difference in infarct size between KO-B6* BM chimeric mice and B6–B6* BM chimeric mice (Fig. 4a), which indicates that leukocytes under normal conditions fail to produce sufficient IL-1Ra to influence infarct development. In comparison, the Tg–B6* BM chimeras developed significantly smaller infarcts compared to the B6–B6* BM chimeric mice, 24 h after pMCAo (Fig. 4a), suggesting that increasing the level of leukocyte-produced IL-1Ra is neuroprotective.

To track IL-1Ra synthesizing BM-derived leukocytes in the ischemic brain, we also subjected IL-1Ra-KO mice reconstituted with BM cells from IL-1Ra-Tg mice (Tg–KO*) to pMCAo. ISH of sections from these mice showed multiple IL-1Ra mRNA⁺ cells in the peri-infarct of irradiated IL-1Ra-KO mice reconstituted with BM cells from IL-1Ra-Tg mice (Tg–KO*), compared to no cells in IL-1Ra-KO mice, 24 h after pMCAo (Fig. 4b, c). By analyzing RNA and protein isolated from parallel sections, we, as expected, observed a robust expression of IL-1Ra mRNA and protein in Tg–KO* mice, and not in IL-1Ra-KO mice, 24 h after pMCAo (Fig. 4d, e). Interestingly, the increase in IL-1Ra synthesis was associated with a lower ischemia-induced increase in IL-1β mRNA levels in Tg–KO* compared to IL-1Ra-KO mice (qPCR, IL-1Ra-KO: 96.6 ± 67.5, n = 12; Tg–KO*: 16.6 ± 11.3, n = 14; P < 0.001; Mann–Whitney test).

In combination, these results provide first evidence that increasing IL-1Ra expression by infiltrating leukocytes, controlled by endogenous kinetics can promote and support neuroprotective signaling pathways in the ischemic cortex.

Having shown that leukocyte-derived IL-1Ra can be neuroprotective using irradiation BM chimeric mice, we next tested the ability of BM cells to infiltrate the ischemic cortex when injected post-stroke. First, we evaluated the ability of BM cells to infiltrate the developing infarct, by injecting GFP⁺ BM cells into the tail vein of C57BL/6 mice 30 min after pMCAo. We identified GFP⁺ cells as soon as 2 h after pMCAo, increasing in numbers at 4 and 6 h (n = 4/group), while no cells were found in the cortex of non-lesioned controls (Fig. 5a). Next, we analyzed BM samples isolated from IL-1Ra-Tg and LM mice, and showed that samples, as expected, were similar regarding the BM profiles and the unit volume (cells/ml), but that BM cells from IL-1Ra-Tg mice showed higher expression of IL-1Ra mRNA than BM cells from sibling donors (Fig. 5b).

Prior to the therapeutic injections of BM cells obtained from either IL-1Ra-Tg or LM mice, we labeled them with the fluorescent marker CFSE (carboxyfluorescein diacetate succinimidyl ester), allowing the cellular detection by flow cytometry 6 h after pMCAo (Fig. 5c). We found that approximately 6 % of the recruited CD11b⁺CD45^high cells in both LM–LM and Tg–LM mice were CFSE⁺ BM cells, and that the numbers of recruited CFSE⁻ CD11b⁺CD45^high leukocytes were comparable 6 h after pMCAo (Fig. 5d). We analyzed the mean fluorescence intensity (MFI) of IL-1Ra in the CFSE⁺ BM recruits uncovering significantly higher IL-1Ra synthesis in BM cells originating from IL-1Ra-Tg (Tg) compared to LM mice 6 h after pMCAo (Fig. 5e).

Because the major source of IL-1Ra in the ischemic brain is microglia, supplemented by few recruited leukocytes (Fig. 3b, c), we asked whether the CFSE⁺ BM recruits could impact IL-1Ra synthesis by these cells. Flow cytometry analysis showed that the number of IL-1Ra⁺CD11b⁺CD45^high leukocytes was comparable in Tg–LM, LM–LM and LM mice 6 h after pMCAo, as well as in their non-lesioned control mice (Fig. 5f, g). In contrast, we observed a significant increase in the number of IL-1Ra⁺CD11b⁺CD45^dim microglia in both Tg–LM and LM–LM mice compared to non-treated LM mice 6 h after pMCAo (Fig. 5h, i). The finding of significantly higher numbers of IL-1Ra⁺CD11b⁺CD45^dim microglia in Tg–LM compared to LM–LM mice stressed the source-benefit of the cells, revealing BM from IL-1Ra-Tg mice as potent inducers of IL-1Ra synthesis by microglia after stroke (Fig. 5h, i). These data were supported by ELISA data showing significantly more IL-1Ra protein in Tg–LM mice compared to LM mice 6 h after pMCAo (Tg–LM: 10.7 ± 6.5, LM–LM: 2.7 ± 2.6; LM: 2.7 ± 3.2 pg/mg; n = 5 mice/group). Thus, microglia are highly plastic cells which can be induced to increase their production of neuroprotective IL-1Ra.

Since mitogen-activated protein kinase (MAPK) signaling pathways play an important role in the early inflammatory response after pMCAo (25), the effect of the BM treatment on MAPKs signaling pathways, including extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) as well as p38, was explored in the tissue from the same mice (Figure S4a–c). The level of phosphorylated (p) JNK was elevated by 60–80 % 6 h after pMCAo in mice treated with IL-1Ra-overproducing BM cells, compared to pMCAo LM mice (Figure S4b). These findings suggest that the increased availability of IL-1Ra in the BM-treated mice modulate the ischemia-induced activation of the JNK pathway.

As an increase in IL-1Ra production by BM cells or microglia could impact the infarct development, thereby changing the neurological outcome, we finally investigated the therapeutic effect of IL-1Ra-overproducing BM cells in mice subjected to either pMCAo or tMCAo (Fig. 6). BM cells harvested from either LM mice or IL-1Ra-Tg mice (Tg) were injected into the tail veins of LM recipients 30 min after MCAo (Table S1). We first investigated the effect of IL-1Ra-overproducing BM cells in mice subjected to pMCAo with 24 h or 5 days survival (Table S1). We observed a significant reduction in infarct size in Tg–LM compared to LM–LM mice 24 h after pMCAo (survival rate/group >95 %) (Fig. 6a). LM–LM mice showed similar infarct sizes as non-treated LM mice 24 h after pMCAo (compared to Fig. 1b). Encouraged by the prominent effect of IL-1Ra⁺ BM cells, we allowed mice to survive for 5 days to assess any physiological and/or behavioral improvements of this treatment strategy. By day 5, we observed a significant reduction in infarct volume in Tg–LM mice compared to LM–LM mice (survival rate/group >95 %), with no difference between LM–LM-treated mice and LM mice (Fig. 6b). The protective effect of IL-1Ra was not a result of decreased body temperature, as rectal temperature recordings (up to 3 h) showed no differences between treatment groups (Figure S5a). Additionally, blood gas parameters (pO₂/pCO₂, pH, electrolytes, glucose/lactate) recorded 30 min after BM injections (i.e., 1 h after pMCAo) showed no difference among various experimental groups (LM, LM–LM and Tg–LM mice) or when compared to non-lesioned mice (Figure S5b-i). When performing in situ hybridization, we observed IL-1Ra mRNA⁺ cells in the peri-infarct in none of the ten LM mice, one out of ten LM–LM and in two out of ten Tg–LM mice 5 days after pMCAo (Figure S6a, shown for Tg–LM). These results matched qPCR results obtained on RNA isolated from parallel sections, showing elevated levels of IL-1Ra mRNA in Tg–LM compared to both LM–LM and LM 5 days after pMCAo (Figure S6b). These results suggest the continued presence of infiltrating IL-1Ra mRNA⁺ BM cells, and/or a prolonged expression of IL-1Ra mRNA by host cells in BM-grafted mice, 5 days after pMCAo.

We next tested the neuroprotective effect of IL-1Ra-overproducing BM cells, in the model of tMCAo (Table S1), which gives rise to a large lesion comprising both cortical and subcortical areas (Fig. 6c). We observed a significant reduction in infarct size in Tg–LM mice and LM–LM mice compared to LM controls, 24 h after tMCAo (Fig. 6c) (survival rate/group >95 %), thereby supporting the findings in mice subjected to pMCAo, additionally showing an infarct-reducing effect of LM BM cells 24 h after tMCAo.

To address a possible mechanism responsible for the beneficial effect of increased IL-1Ra, we quantified IL-1β mRNA and protein in parallel sections by qPCR and ELISA (Fig. 6d–f). The ischemia-induced increase in IL-1β mRNA was significantly smaller in Tg–LM mice compared to LM–LM mice 24 h after pMCAo (Fig. 6d). Consistent with this observation, we also observed less IL-1β protein in Tg–LM mice compared to LM–LM mice 24 h after pMCAo (Fig. 6d). By day 5, both IL-1β mRNA and protein in LM, LM–LM and Tg–LM were back at baseline levels (Fig. 6e), as measured in non-lesioned B6 mice (see Fig. 2c for mRNA) and non-lesioned LM mice [IL-1β: 137 ± 72 pg/mg (mean ± SD), n = 3]. Similar to the pMCAo mice, we also observed a smaller ischemia-induced increase in IL-1β mRNA and protein in Tg–LM mice compared to LM mice 24 h after tMCAo (Fig. 6f), collectively suggesting an effect of increased IL-1Ra production on IL-1β expression after both pMCAo and tMCAo.

We used a battery of behavioral tests to assess the functional recovery in mice subjected to 5 days pMCAo or mice subjected to tMCAo. As the MCA supplies cortical areas controlling the contralateral front and hind limb, we measured the neuromuscular function of the left and right forelimbs (Fig. 6g–i). We detected a significant contralateral weakness in the right (R) front paw of LM mice 1, 2 and 5 days after pMCAo compared to normal baseline strength (Fig. 6g). A weakness first observed on day 2 and 5 after pMCAo in LM–LM mice was accordingly not observed in Tg–LM mice (Fig. 6g). By day 5, both LM and LM–LM mice showed pronounced grip asymmetry in the left and right forelimbs, whereas the performance of Tg–LM mice was comparable in the two forelimbs (Fig. 6h). A similar grip strength outcome was observed in LM–LM and Tg–LM mice 24 h after tMCAo (Fig. 6i). In addition, performing the Hargreaves and open field test on mice subjected to tMCAo, we observed an improved functional recovery in Tg–LM-treated mice (Table S6). Hargreaves test revealed similar nociception in the left and right hind paw prior to surgery with LM and LM–LM mice showing a significantly lower sensitivity compared to Tg–LM mice 24 h after tMCAo (Table S6). Using the open field test, we observed that the Tg–LM mice exhibited a more explorative activity, revealing that LM–LM and Tg–LM mice traveled longer (cm) and faster (cm/s) than LM mice 24 h after tMCAo. However, only Tg–LM mice showed reduced anxiety-related behavior spending more time in the center of the maze (Table S7).

Despite evidence that IL-1Ra is an important neuroprotective cytokine, its presence in the human brain after a stroke has not yet been investigated. Here, we show that IL-1Ra⁺ cells are present both in the peri-infarct (Fig. 7a) and the infarct core (Fig. 7b) 24 h after stroke onset (Fig. 7a–e). Scoring of IL-1Ra expression in three distinct regions, e.g., infarct core, peri-infarct and normal-appearing tissue, showed higher IL-1Ra expression in the peri-infarct compared to infarct core both 1–2 days (P < 0.04) and 5 to ≥7 days (P < 0.03) post-stroke (Fig. 7f). At ≥7 days, there was a marked increase of IL-1Ra⁺ cells (Table S2). Regions of infarct core, peri-infarct and normal-appearing tissue were identified based on parallel series of HE-stained sections. Iba1, CD45, CD68 and GFAP were used to distinguish the topography of IL-1Ra⁺ cells (Figure S7). IHC double staining showed IL-1Ra and CD45 co-expressing microglia 24 h post-stroke (Fig. 7e). These results provide novel insight into the production of IL-1Ra in the human brain and emphasize the translational relevance of our experimental results.