Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Jun 15.
Published in final edited form as: J Immunol. 2014 May 14;192(12):6009–6019. doi: 10.4049/jimmunol.1303492

mTOR signaling inhibition modulates macrophages/microglia-mediated neuroinflammation and secondary injury via regulatory T cells after focal ischemia

Luokun Xie *, Fen Sun *, Jixian Wang †, XiaoOu Mao ‡, Lin Xie ‡, Shao-Hua Yang *, Dongming Su *, James W Simpkins *,§, David A Greenberg ‡, Kunlin Jin *
PMCID: PMC4128178  NIHMSID: NIHMS587595  PMID: 24829408

Abstract

Signaling by the mammalian target of rapamycin (mTOR) plays an important role in the modulation of both innate and adaptive immune responses. However, the role and underlying mechanism of mTOR signaling in post-stroke neuroinflammation is largely unexplored. Here, we injected rapamycin, an mTOR inhibitor, by the intracerebroventricular route 6 hours after focal ischemic stroke in rats. We found that rapamycin significantly reduced lesion volume and improved behavioral deficits. Notably, infiltration of gamma delta T (γδ T) cells and granulocytes, which are detrimental to the ischemic brain, was profoundly reduced after rapamycin treatment, as was the production of pro-inflammatory cytokines and chemokines by macrophages and microglia. Rapamycin treatment prevented brain macrophage polarization towards the M1 type. In addition, we also found that rapamycin significantly enhanced anti-inflammation activity of regulatory T cells (Tregs), which decreased production of pro-inflammatory cytokines and chemokines by macrophages and microglia. Depletion of Tregs partially elevated macrophage/microglia-induced neuroinflammation after stroke. Our data suggest that rapamycin can attenuate secondary injury and motor deficits after focal ischemia by enhancing the anti-inflammation activity of Tregs to restrain post-stroke neuroinflammation.

INTRODUCTION

Stroke is the fourth leading cause of death and the leading cause of disability in the United States (1). Despite tremendous progress in understanding the pathophysiology of ischemic stroke, translation of this knowledge into effective therapies has largely failed. Systemic thrombolysis with recombinant intravenous tissue plasminogen activator (rtPA) remains the only treatment proven to improve clinical outcome of patients with acute ischemic stroke (2). But because of an increased risk of hemorrhage beyond a few hours post-stroke, only about 1–2% of stroke patients can benefit from rtPA (3, 4).

Molecular and cellular mediators of neuroinflammatory responses play critical roles in the pathophysiology of ischemic stroke, exerting either deleterious effects on the progression of tissue damage or beneficial roles during recovery and repair (5). Therefore, post-ischemic neuroinflammation may provide a novel therapeutic approach in stroke. However, several therapeutic trials targeting neuroinflammatory response have failed to show clinical benefit (6). The cause remains unknown. However, targeting a single cell type or single molecule may not be an adequate clinical strategy. In addition, the biphasic nature of neuroinflammatory effects, which amplify acute ischemic injury but may contribute to long-term tissue repair, complicates anti-inflammatory approaches to stroke therapy.

Mammalian target of rapamycin (mTOR) is a critical regulator of cell growth and metabolism that integrates a variety of signals under physiological and pathological conditions (7, 8). Rapamycin is an FDA-approved immunosuppressant being used to prevent rejection in organ transplantation. Recent data show that mTOR signaling plays an important role in the modulation of both innate and adaptive immune responses (9). In experimental stroke, rapamycin administration 1 hour after focal ischemia ameliorated motor impairment in adult rats (10) and in neonatal rats (11) and improves neuron viability in an in vitro model of stroke (12). However, the mechanisms underlying mTOR-mediated neuroprotection in stroke are unclear. In addition, stroke patients often experience a significant delay between the onset of ischemia and initiation of therapy. So it is important to determine whether rapamycin can protect from ischemic injury when administered at later time points.

In this study, we found that rapamycin administration 6 hours after focal ischemia significantly reduced infarct volume and improved motor function after stroke in rats. In addition, gamma/delta T (γδ T) cells and neutrophil infiltration were decreased, regulatory T cells (Treg) function was increased and pro-inflammatory activity of macrophages and microglia was reduced in the ischemic hemispheres. Tregs from rapamycin-treated brains effectively inhibited pro-inflammatory cytokine and chemokine production by macrophages and microglia. Our data suggest that rapamycin attenuates secondary injury and motor deficits after focal ischemia by modulating post-stroke neuroinflammation.

MATERIALS AND METHODS

Focal cerebral ischemia

Transient focal cerebral ischemia was induced using the suture occlusion technique as previously described (13). Briefly, Male Sprague-Dawley rats weighing 250 to 300 g were anesthetized with 4% isoflurane in 70% N2O/30% O2 using a mask. The neck was incised in the midline, the right external carotid artery (ECA) was carefully exposed and dissected, and a 19-mm long 3–0 monofilament nylon suture was inserted from the ECA into the right internal carotid artery to occlude right MCA at its origin. After 90 minutes, the suture was removed to allow reperfusion, the ECA was ligated and the wound was closed. Sham-operated rats underwent an identical procedure except that the suture was not inserted. Rectal temperature was maintained at 37.0±0.5°C using a heating pad and heating lamp. Regional cerebral blood flow (rCBF) was measured by laser-Doppler flowmetry (Moor Instruments, UK) with the probe positioned over the left hemisphere, 1.5 mm posterior and 3.5 mm lateral to the bregma. After reperfusion for various periods, rats were anesthetized and perfused through the heart with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4). All animal experiments were carried out in accordance with National Institutes of Health guidelines and with the approval of the Institutional Animal Care and Use Committee.

Intracerebroventricular administration of rapamycin

Rats were implanted with an osmotic minipump to the left lateral ventricle 6 hr after MCAO. For neurological behavioral tests and lesion volume measurement, each rat was intracerebroventricularly infused with 0.5µl/hr of either rapamycin (1µM; Calbiochem, La Jolla, CA, USA) or vehicle (artificial cerebrospinal fluid) for 7 days, and then sacrificed 28 days after MCAO. For studying neuroinflammatory response, rats were administrated with either rapamycin or vehicle for 3 days and sacrificed 3 days after treatment.

Lesion volume measurement

Rats were sacrificed 4 weeks after MCAO. 100-µm coronal sections (400-µm apart; 12–16 per rat, 7 rats per group) were stained with crysel violet. Lesion area was measured by a blinded observer as described previously (14). Lesion volumes were expressed as a percentage of the volume of the structures in the control hemispheres,

Neurobehavioral testing

Rats underwent neurobehavioral tests to evaluate functional outcome. The neurobehavioral tests including 1) Beam balance test, 2) Limb placing test, and 3) Elevated body swing test (EBST), was performed according our previously publications (15). Animals were trained prior to surgery and deficits were assessed 1, 3, 7, 14 and 28 days thereafter. The observer was blinded to the experimental condition.

Isolation of immune cells from brains

Isolation of brain immune cells was performed following a well-established method (16) with a few modifications at day 3 post-ischemia. Briefly, rat cerebral hemispheres were cut into about 1mm3 pieces before digestion with digestion buffer for 45 min at 37°C. Digestion buffer consists of RPMI 1640 medium supplemented with 10% fetal bovine serum, 1mg/ml collagenase type IV (Sigma-Aldrich), 50µg/ml DNase I (Sigma-Aldrich), 5mM CaCl2 (Fisher Scientific). Digested tissues were then gently pressed through 40-µm cell strainers to prepare homogenized tissue suspension. Tissue suspension was then mixed with 4 volumes of 30% Percoll (GE Healthcare). The mixture was loaded onto 2 ml of 37% Percoll, which was above 2 ml of 70% Percoll, followed by centrifugation at 500g for 20 min. Cells in the interface between 37% Percoll and 70% Percoll were collected, washed with PBS, and resuspended in PBS for further use..

Flow Cytometry analysis and cell sorting

For immune cell staining, the following anti-rat antibodies were used: Alexa Fluor® 647 anti-TCRαβ (R73), FITC anti-TCRγδ (V65), APC-Cy7 anti-CD4 (W3/25), PE anti-CD8 (G28), PE anti-CD25 (OX-39), PE/Cy7 anti-CD45 (OX-1), PE anti-CD11b/c (OX-42), and FITC anti-RTIB (MHC-II, OX-6) (Biolegend); Alexa Fluor® 647 anti-CD163 (ED2, Abdserotec); biotinylated anti-granulocyte (RP-1, BD Pharmingen), and biotinylated or PE anti-CD3 (eBioG4.18, eBioscience). Stained cells were analyzed on a BD LSR-II flow cytometer. Dead cells and debris were excluded by staining with propidium iodide (PI, eBioscience). For Treg detection, Alexa Fluor 488 anti-mouse/rat/human Foxp3 (150D) was purchased from Biolegend. For cell sorting, stained cells were sorted on a BD InFlux Cell Sorter. For intracellular staining, cells were stained with antibodies against surface antigens, followed by fixation, permeabilization and staining with PE-Cy7 anti-IL-17A (eBio17B7, eBioscience) according to BD intracellular cytokine staining manual.

In vitro cell culture

On day 3 after ischemia/reperfusion (I/R), anti-granulocyte− CD11b/c+ myeloid cells (mainly macrophages and microglia) were sorted from vehicle-treated ischemic brain hemispheres by flow cytometry. TCRαβ+CD4+CD25hi Treg-enriched cells were sorted from either vehicle-treated or rapamycin-treated ischemic brain hemispheres by flow cytometry. Treg-enriched cells from 2~3 ischemic brains of each group were pooled. 1 × 104 myeloid cells and 1 × 104 Treg-enriched cells were co-cultured in 96-well V-bottom microplates at 37°C in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. After 24 h co-culture, cells were centrifuged at 1000g for 5 min and were incubated in PBS containing 1 mM EDTA and biotinylated anti-CD3 monoclonal Ab for 15 min on ice. CD3+ cells (Tregs) were depleted with Dynabeads® biotin binder (invitrogen) following the manufacture’s protocol. Unbound cells were collected and subject to Q-RTPCR. To check the efficiency of Treg depletion, unbound cells were incubated with FITC anti-CD5 monoclonal Ab (HIS47, eBioscience) and subject to flow cytometry analysis.

To explore the direct effect of rapamycin on myeloid cells, anti-granulocyte− CD11b/c+ myeloid cells were isolated from vehicle-treated ischemic brain hemispheres as described above on day 3 after I/R. Cell density was adjusted to 1 × 106/ml in supplemented RPMI 1640. Fifty microliters of cell suspension was added into each well of a 96-well microplate and rapamycin was added at a final concentration of 10 nM. Cells were cultured at 37°C for 24 h before collection for RNA extraction and Q-RTPCR.

γδ T cell migration assay

On day 3 after I/R, spleens from vehicle-treated rats were isolated and single splenocyte suspension was prepared by mechanical dissociation in 40-µm cell strainers. Red blood cells were lysed with BD RBC lysis buffer. Splenocytes were incubated with FITC anti-TCRγδ Ab for 15 min on ice. TCRγδ+ cells were sorted by flow cytometry. CD45hi CD11b/c+ macrophages and CD45lo CD11b/c+ microglia were sorted from vehicle-treated and rapamycin-treated ischemic brain hemispheres by flow cytometry, respectively. All cells were resuspended in supplemented RPMI 1640. 2 × 105 macropahges or microglia were seeded into each well of a 96-well transwell plate (Corning). 1.25 × 104 γδ T cells were seeded into each well of the insert. The cells were cultured at 37°C for 18 h. All cells in the lower wells (not the insert wells) were then incubated in 1 mM EDTA-PBS for 10 min and collected. Cells were incubated with PE anti-CD3 Ab for 15 min on ice. The number of CD3+ cells was enumerated by flow cytometry.

Depletion of regulatory T cells in vivo

To deplete Tregs in vivo, anti-rat CD25 monoclonal antibody (OX-39, AbD Serotec) was used according to previous literatures with modifications (17, 18). Briefly, 2.5 mg of Ab in PBS was intraperitoneally injected into each rat once a day for 2 days prior to I/R. Peripheral blood was collected through tail vein at indicated time points to determine the efficiency of Treg depletion by flow cytometry analysis. Rats receiving PBS were used as vehicle control.

Quantitative RT-PCR (Q-RTPCR

Total RNAs were reversely transcribed to cDNAs using SuperScript® III First-Strand Synthesis System (Invitrogen) according to the manufactures’ instructions. Q-RTPCR was performed using Fast SYBR® Green Master Mix (Invitrogen) on a 7300 Real-Time PCR System (Invitrogen). Data was analyzed with 7300 system software. Primer sequences for each gene were shown in Table I.

Table I.

Primer sequences for Q-RTPCR

Genes Primers (Forward and reverse)
Il10 5’-TAAAAGCAAGGCAGTGGAGC-3’
5’-GATGCCGGGTGGTTCAATTT-3’
Tgfb1 5’-GTCAACTGTGGAGCAACACG-3’
5’-TTCCGTCTCCTTGGTTCAGC-3’
Ebi3 5’-TTCTAGCCTTTGTGGCGGAA-3’
5’-AGCGAAGTCGGTACTTGAGAG-3’
Tnfα 5’-TCGGTCCCAACAAGGAGGAG-3’
5’-GGGCTTGTCACTCGAGTTTTG-3’
Il1b 5’-TGTCTGACCCATGTGAGCTG-3’
5’-GCCACAGGGATTTTGTCGTT-3’
Il6 5’-ACTTCACAAGTCGGAGGCTT-3’
5’-TTCTGACAGTGCATCATCGCT-3’
Inos 5’-TTCCTCAGGCTTGGGTCTTGT-3’
5’-GGCAAGCCATGTCTGTGACTT-3’
Il23a 5’-CTCTGTAACTGCCTGCTTAGTC-3’
5’-GCTTTTGTCACAGGTCGGTAT-3’
Il12b 5’-TCCAGACACATCAGACCAAGCA-3'
5’-AGTGGAGACACCAGCAAAACC-3’
Ikzf2 5’ATAACGTCAGGTGACAATGAGCTT-3’
5’-CTTCATCACTCTGCATTTCCAGC-3’
Nrp1 5’-AGCTACTGGGCTGTGAAGTA-3’
5’-CTGGTCATCGTCACACTCG-3’
Fgf2 5’-CCGGTACCTGGCTATGAAGG-3’
5’-TTCCGTGACCGGTAAGTGTT-3’
Cxcl12 5’-CTGCCGATTCTTTGAGAGCCA-3’
5’-GGGCTGTTGTGCTTACTTGTTT-3’
Cxcl2 5’-ACATCCAGAGCTTGACGGTG-3’
5’-CAGGTCAGTTAGCCTTGCCT-3’
Ccl2 5’-TAGCATCCACGTGCTGTCTC-3’
5’-CAGCCGACTCATTGGGATCA-3’
Ccl3 5’-CTCAGCACCATGAAGGTCTCC-3’
5’-CGTCCATAGGAGAAGCAGCAG-3’
Gapdh 5’-GATGGTGAAGGTCGGTGTGA-3’
5’-TGAACTTGCCGTGGGTAGAG-3’
Open in a new tab

Western blot

Western blot was performed using the protocol as previously described (19). The primary antibodies were anti-Phosph-4EBP1 (Thr37/46), anti-4EBP1 (Cell Signaling) and anti-actin (Santa Cruz Technology, CA). Membranes were developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and the optical density was analyzed using a Biospetrum 500 imaging system (UVP, LLC).

Statistical analyses

Quantitative data were expressed as mean ± SEM from the indicated number of experiments. Behavioral data were analyzed by two-way analysis of variance (ANOVA) with repeated measures, followed by post hoc multiple comparison tests (Fisher PLSD or Student’s paired t test with the Bonferroni correction). Lesion volume data were analyzed by one-way ANOVA followed by Fisher PLSD post hoc tests. For quantifying immune cellularity and cytokine expression, Student’s t test or one-way ANOVA were used for comparison of mean between the groups. P values < 0.05 were considered significant.

RESULTS

Rapamycin reduces lesion volume and improves motor deficits after MCAO

To assess the roleefficacy of rapamycin when administered more than 1 hr post-stroke, which corresponds more closely to the clinical situationafter focal ischemia, rapamycin or vehicle was administrated beginning 6 hr, which corresponds more closely to the clinical setting, after MCAO for 7 consecutive days, and rats were euthanized 4 weeks after MCAO to measure lesion volume (Fig. 1A). As shown in Fig. 1B & 1C, lesion volume was significantly decreased in the rapamycin- compared with vehicle-treated group. Next, we asked whether blocking mTOR signaling could improve the neurological deficits after MCAO. As shown in Fig. 1D, there was a significant difference in the motor performance as observed in beam balance test, limb placing test and EBST between rapamycin- and vehicle-treated groups, consistent with the effect of rapamycin in functional outcome in rats after I/R.

Figure 1. Rapamycin treatment reduced lesion volume and improved motor deficits after MCAO.

Figure 1

Open in a new tab

(A) The scheme of experimental design. (B) Crystal violet-stained coronal brain sections from rapamycin- and vehicle-treated ischemic rats. (C) Volume loss in vehicle- and rapamycin-treated rats (N=7 per group). (D) Neurobehavioral tests (sham group: n=6~7; MCAO group: n=10~11). Left: Beam-walking test scores, expressed as the mean numbers of forelimb slip steps when traversing an elevated narrow beam; middle: Limb-placing test scores, expressed as a score derived from the number of correct limb placements; right: Elevated body swing test scores, expressed as a percentage of turns to the contralesional (impaired) side. Veh, vehicle; Rapa, rapamycin. *, p<0.05 compared to vehicle-treated rats.

Rapamycin reduces γδ T cell and granulocyte infiltration

Expression of pro-inflammatory cytokines and chemokines such as TNF-α, IL-1β, CCL2 and CCL3 is induced as early as 1~2 h after ischemia and are increased for up to 2~5 days (20–25). Importantly, the temporal profile of chemokines such as CCL2 and CCL3 is in line with that of leukocyte accumulation in the ischemic brain parenchyma (26). Leukocyte accumulation is the character of inflammation. Previous studies have indicated that leukocyte accumulation including T cells, B cells, granulocytes and monocytes starts on day 1 and peaks on day 3 after onset of ischemia (27, 28). Thus, post-ischemic neuroinflammation appears to culminate at day 3 after I/R. So we chose this time point to study the neuroinflamamtion, hoping to observe significant changes and easily isolate recruited leukocytes. To investigate the mechanism by which rapamycin protected from MCAO-induced damage, we firstly determined whether rapamycin inhibited inflammatory cell infiltration in the ischemic brain. Total immune cells recovered from rapamycin-treated brains 3 days after ischemia were significantly less than those from vehicle-treated brain (Fig 2A). We then investigated distinct leukocyte populations. Leukocytes were carefully gated based on CD45 expression and their specific surface markers (Supplemental Fig. 1A). In the ipsilateral (ischemic) hemispheres, there was no significant difference in αβ T cell number between rapamycin- and vehicle-treated groups (Fig. 2B), although a trend of decrease in CD4+ T cells occurred. In contrast, infiltration of γδ T cells and granulocytes in the ipsilateral hemisphere was profoundly inhibited in rapamycin-treated rats compared to vehicle-treated rats (Fig. 2C & 2E). Previous study has shown the pivotal role of γδ T cell-derived IL-17 in the progression of post-ischemic neuroinflammation in mice (28). Thus, we determined IL-17A expression in infiltrated γδ T cells to check if rapamycin could influence IL-17A expression. To our surprise, neither IL-17A protein nor IL-17A mRNA was significantly elevated in infiltrated γδ T cells, and rapamycin had no significant effect on IL-17A expression in γδ T cells (Fig. 2D and Supplemental Fig. 1B). These data suggests unlike mouse MCAO model, IL-17 might not be an important factor for γδ T cell-induced neuroinflammation in rat ischemia.

Figure 2. Rapamycin treatment reduced inflammatory cell infiltration after MCAO.

Figure 2

Open in a new tab

(A) The number of total immune cells recovered from ischemic brains. (B) αβ T cell number in ischemic brains. (C) γδ T cell number in ischemic brains. Left: representative contour plots of brain γδ T cells. Right: statistical analysis of γδ T cell infiltrates. (D) Flow cytometry analysis of IL-17A expression in infiltrated T cells. αβ T, αβ T cells; Ctrl γδ T, γδ T cells in brains of vehicle-treated rats; Rapa γδ T, γδ T cells in brains of rapamycin-treated rats. This is a representative of three independent experiments. (E) Granulocyte number in ischemic brains. Left: representative contour plots of brain granulocytes. Right: statistical analysis of granulocyte infiltrates. Numbers in the plots are the frequencies of each population in total recovered immune cells. Ctrl, vehicle control; Rapa, Rapamycin-treated; Contra, contralateral side; Ipsi, ipsilateral side. N=8 rats per group. **, p<0.01; ***, p<0.001.

Rapamycin inhibits production of pro-inflammatory cytokines and chemokines by macrophages and microglia

The reduction of immune cells in the ipsilateral hemisphere suggests that recruitment of γδ T cells and granulocytes by post-ischemic neuroinflammation is restrained by rapamycin treatment. Macrophages and microglia play critical roles in the initiation of post-ischemic neuroinflammation, including recruitment of blood leukocytes. Therefore, we tested macrophages/microglia-mediated inflammation by detecting production of cytokines and chemokines in these cells. The amount of CD45hiCD11b+ macrophages and CD45loCD11b+ microglia was significantly increased in ipsilateral hemispheres in comparison with the contralateral side, but rapamycin treatment did not alter their number, suggesting rapamycin has no effect on the accumulation of macrophages and microglia after stroke. (Fig. 3A). I/R robustly induced expression of inflammatory cytokines and chemokines in macrophages and microglia in ischemic hemispheres (Supplemental Fig. 2A). Note that induction of IL23a (p19) and IL12b (p40) was not as profound as induction of other cytokines (Supplemental Fig. 2A), suggesting that expression of IL-23 might be relatively low in our model. This could explain why we did not observe significant IL-17 expression in γδ T cells. Compared with vehicle treatment, rapamycin treatment inhibited expression of TNF-α, IL-1β, IL-6, iNOS and IL12b (p40) in macrophages while IL23a (p19) expression was not affected (Fig. 3B), suggesting that rapamycin down-regulates pro-inflammatory activity of macrophages. In microglia, only IL-1β expression was significantly down-regulated by rapamycin treatment, although there was a trend of decrease in TNF-α and iNOS expression (Fig 3B). To our surprise, rapamycin strongly increased CXCL2 mRNA level in macrophages and microglia. In addition, rapamycin significantly reduced CCL2 and CCL3 level in macrophages and microglia (Fig. 3B). The reduction of CCL2 and CCL3 levels could be a reason of reduced leukocyte infiltration in the ischemic brains. To test our hypothesis that rapamycin weakens macrophages/micgroglia-induced chemoattraction of γδ T cells, we performed in vitro migration assay by culturing splenic γδ T cells with post-ischemic brain macrophages or microglia in the transwell plates. Both macrophages and microglia effectively induced migration of γδ T cells 18 h after culture. Compared with control groups, macrophages and microglia isolated from rapamycin-treated brains induced less γδ T cell migration, suggesting their ability to recruit γδ T cells is indeed weakened (Fig. 3C).

Figure 3. Rapamycin treatment inhibited pro-inflammatory activity of macrophages and microglia.

Figure 3

Open in a new tab

(A) Macrophages and microglia in ischemic brains. Left: representative dot plots of flow cytometry analysis showing brain macrophages and microglia. Total isolated cells were gated according to CD45 and CD11b/c expression. Numbers in the plots are the frequencies of each population in total recovered immune cells. Right: the number of macrophages and microglia in ischemic brains with or without rapamycin treatment, respectively. Contra, contralateral side; Ipsi, ipsilateral side. ***, p<0.001. (B) Q-RTPCR for mRNA levels of cytokines and chemokines in brain macrophages and microglia after MCAO. N=6 rats per group. Ctrl, vehicle control; Rapa, Rapamycin-treated rats. *, p<0.05; **, p<0.01; ***, p<0.001. (C) In vitro migration of γδ T cells induced by macrophages and microglia. Left: representative contour plots of flow cytometry analysis showing the gating strategy. γδ T cells were firstly gated based on FSC and SSC, then were further gated by CD3 expression. Right: the number of γδ T cells migrating to the lower wells. Medium, random migration of γδ T cells without macrophages or microglia in the lower wells. Macrophages, migration of γδ T cells induced by macrophages. Microglia, migration of γδ T cells induced by microglia. N=3 per group. ##, p<0.01 compared with medium only; ###, p<0.001 compared with medium only; *, p<0.05 compared with migration induced by macrophages or microglia isolated from vehicle-treated rats.

The changes in cytokine and chemokine expression could be due to direct or indirect effect of rapamycin on macrophages and microglia. To clarify this, we isolated macrophages and microglia from ischemic rat brains without rapamycin injection. These cells were cultured in vitro in the presence or absence of rapamycin for 24 h and mRNA levels of cytokines and chemokines were tested by Q-RTPCR. Rapamycin directly enhanced expression of IL-1β, iNOS, CCL3 and IL23a, but did not affect expression of TNFα, IL-6, CXCL2 and IL12b in macrophages (Supplemental Fig. 2B). In microglia, expression of IL-1β and iNOS were also promoted by rapamycin (Supplemental Fig. 2B). Our data is generally consistent with previous publications which demonstrated that rapamycin enhances pro-inflammatory activity of macrophages (29, 30). Thus, in vivo decrease of pro-inflammatory mediators in macrophages and microglia is unlikely due to the direct effect of rapamycin. It is more likely that rapamycin acts on other cell types first and in turn those affected cell types induce less inflammatory response of macrophages and microglia. Interestingly, rapamycin directly inhibited CCL2 expression in both macrophages and microglia (Supplemental Fig. 2B), which is consistent with the in vivo data. CCL2 has been shown to be critical for γδ T cell recruitment in other disorders (31, 32). Hence, rapamycin might directly inhibit CCL2 production in macrophages and microglia, so as to reduce γδ T cell accumulation in ischemic brains.

The expression of anti-inflammatory TGF-β1 and IL-10 was not altered in macrophages and microglia (Supplemental Fig. 3A & 3B). Expression of FGF-2 was up-regulated in microglia from rapamycin-treated brains, suggesting microglia might promote brain recovery.

Rapamycin treatment favors brain macrophage polarization towards M2 type

Polarization of macrophages between M1 and M2 type is associated with pro- and anti-inflammatory activity, respectively. Above data suggests that polarization of macrophages and microglia might be changed after rapamycin treatment. Our flow cytometry analysis showed that four subpopulations of macrophages—RTIB−CD163−RTIBhiCD163−RTIB+CD163+RTIB−CD163+—were present in contralateral hemispheres (Supplemental Fig. 4). According to published M1 and M2 phenotypes (33, 34), we designated them M0, M1, M1/2 and M2, respectively. However, only very few microglia in contralateral hemispheres expressed RTIB or CD163 (Supplemental Fig. 4). In ipsilateral hemispheres, rapamycin treatment increased the frequency and number of M2 macrophages, compared with vehicle control (Fig. 4A & 4B), suggesting rapamycin treatment favors M2 polarization of macrophages. To our surprise, microglia barely expressed RTIB and CD163 in both contralateral and ipsilateral hemispheres (Fig. 4C & Supplemental Fig. 4), suggesting that these surface markers are not suitable for distinguishing microglial polarization.

Figure 4. Rapamycin treatment enhanced brain macrophage polarization into type M2.

Figure 4

Open in a new tab

(A) Representative dot plots of polarization of brain macrophages and microglia. Macrophages and microglia were gated as in Figure 3A. Then each population was further divided according to MHC-II and CD163 expression. Numbers are the frequency of each population in their parental population. (B) Statistical analysis of frequency (left) and cell number (right) of each macrophage subtype. (C) Statistical analysis of frequency (left) and cell number (right) of each microglia subtype. Ctrl, vehicle control; Rapa, Rapamycin-treated. All data are from ipsilatreal hemispheres. N=6~8 per group. *, p<0.05; **, p<0.01 compared with control group.

Rapamycin enhances anti-inflammatory activity of Tregs

It has been shown that rapamycin enriched the population of Tregs (35–37). Tregs are able to protect ischemic brain damage (38). So we asked whether rapamycin could alter Treg number and/or activity in ischemic brains. Treg number in rapamycin-treated brains was comparable to that in vehicle-treated brains. However, the proportion of Tregs in total recovered immune cells was significantly increased (Fig. 5A). Tregs in rapamycin-treated brains expressed higher Foxp3 and CD25 (Fig. 5B), suggesting they have higher regulatory activity than Tregs in the vehicle control brains. To determine the anti-inflammatory activity of Tregs, we sorted TCRαβ+CD4+CD25hi Treg-enriched cells for Q-RTPCR. Messenger RNA levels of IL-10 in Tregs were not significantly changed, while mRNA levels of TGF-β1 and Ebi3 were elevated in Tregs in rapamycin-treated brains (Fig. 5C). Thus, rapamycin treatment enhanced Treg anti-inflammatory activity. Q-RTPCR revealed that infiltrated Tregs expressed similar levels of helios and neuropilin-1 to those in splenic counterparts (Fig. 5D). Splenic Tregs contain mainly natural Tregs (39). Hence, infiltrated Tregs are mainly natural Tregs as well. This result is consistent with our expectation based on the temporal process of immune response, since adaptive immune reaction has not fully engaged on day 3 after antigen exposure.

Figure 5. Rapamycin administration enhanced Treg proportion and activity after MCAO.

Figure 5

Open in a new tab

(A) Foxp3+ Treg number and proportion in ischemic brains. Left: representative dot plots of Foxp3+ cells gated on CD3+CD4+ T cells in ischemic brains. Right: statistical analysis of Treg number and frequency. N=5 per group. (B) Rapamcin treatment enhanced expression of Foxp3 and CD25 in infiltrating Tregs. Left: representative histograms of Foxp3 and CD25 in Foxp3+ T cells. Dotted curve, isotype control; shaded curve, vehicle-treated rats; solid curve, rapamycin-treated rats. Right: statistical analysis of the mean fluorescence intensity of Foxp3 and CD25. N=5 per group. (C) Anti-inflammatory cytokine production in Tregs. Ctrl, vehicle control; Rapa, rapamycin-treated rats. Data were from ipsilatreal hemispheres. N=5~6 per group. *, p<0.05. **, p<0.01. (D) Expression of helios and neuropilin-1 in Tregs detected by Q-RTPCR. Spleen, splenic Tregs; Ctrl, Tregs from vehicle control rat brains; Rapa, Tregs from rapamycin-treated rat brains. N=3 per group.

Tregs in rapamycin-treated brains inhibit pro-inflammatory activity of macrophages/microglia

Then we tested whether Tregs in rapamycin-treated brains more potently inhibit macrophages/microglia-mediated inflammation. Because rapamycin treatment induced a similar cytokine/chemokine expression change in macrophages and microglia (Fig. 3B), we used macrophages/microglia mixture for the study. Ischemic macrophages/microglia were co-cultured with Tregs isolated form ischemic brains. Tregs, which are CD5+were then effectively depleted with magnetic beads (Fig. 6A). Tregs from vehicle-treated brains (V-Tregs) moderately inhibited cytokine/chemokine expression except for TNF-α and CCL2, while Tregs from rapamycin-treated brains (R-Tregs) more robustly inhibited almost all cytokines/chemokines in comparison with V-Tregs (Fig. 6B). To confirm that rapamycin inhibited mTOR signaling, brain immune cells were isolated to measure mTORC1 activity by Western blot. Ischemic injury significantly up-regulated both 4EBP1 and phosphorylated 4EBP1. Rapamycin treatment did not alter 4EBP1 protein levels, but significantly reduced 4EBP1 phosphorylation (Fig. 6C). Thus, rapamycin down-regulated mTORC1 signaling in infiltrating leukocytes.

Figure 6. Tregs in rapamycin-treated rat brains potently inhibited inflammatory response of macrophages/microglia.

Figure 6

Open in a new tab

(A) Magnetic cell sorting effectively depleted Tregs from the co-culture. PI− live cells were detected for expression of CD5. (B) Cytokine/chemokine production in machophages/microglia after co-culture with Tregs. Ctrl: vehicle control; Rapa: Rapamycin-treated rats. N=5 per group. V-Tregs, Tregs from vehicle-treated brains; R-Tregs, Tregs from rapamycin-treated brains. (C) Phosphorylation of 4EBP1 in immune cells in ischemic brains. This is a representative of three independent experiments. I/R, ischemia/perfusion; Rapa, rapamycin-treated. N=4 per group. *, p<0.05; **, p<0.01; ***, p<0.001.

Depletion of Tregs partially elevates inflammatory response of macrophage/microglia in rapamycin-treated ischemic brains

To further explore whether Tregs are critical for rapamycin-induced alleviation of neuroinflammatory response in macrophage/microglia, intraperitoneal injection of anti-CD25 antibody was applied to deplete peripheral Tregs before MCAO was performed (Fig. 7A). Peripheral blood was drawn to determine the efficiency of Treg depletion. Three days and five days after the initial Ab injection, over 50% and 60% of CD4+Foxp3+ Tregs in the blood were depleted respectively (Fig. 7B). MCAO and rapamycin injection was performed two days after the initial Ab injection. On day 3 after MCAO, brain immune cells were evaluated. Consistent with the reduction of peripheral Tregs, infiltrated Tregs in ischemic brains were also decreased by 60% after Ab treatment (Fig. 7C). In comparison with rats receiving rapamycin and PBS, Treg depletion caused a trend of increase in total immune cell number (Fig. 7D). In comparison with control rats (I/R without additional treatment), the total immune cell number after Treg depletion was relatively lower but it was not statistically significant (Fig. 7D). Treg depletion induced significant increase of γδ T cells and granulocytes in ischemic brains, compared with rats receiving rapamycin and PBS (Fig. 7E). However, their numbers were still less than the amount of γδ T cells and granulocytes in ischemic brains of control rats, suggesting Treg depletion only partially increases the infiltration of γδ T cells and granulocytes. The numbers of infiltrated αβ T cells, macrophages and microglia were not significantly altered by Treg depletion (Fig. 7E &7F).

Figure 7. Depletion of Tregs partially promotes inflammatory response of macrophage/microglia in rapamycin-treated ischemic brains.

Figure 7

Open in a new tab

(A) The scheme of experimental design. Anti-CD25 Ab was injected once a day for 2 days before MCAO and rapamycin treatment. On day 5 after the initial injection of Ab, rats were sacrificed for testing neuroinflammation. (B) Depletion of CD4+Foxp3+ T cells in the blood on day 3 and day 5 after Ab injection. CD3+CD4+ cells were gated for detecting Foxp3+ cells. The numbers in the plots are the frequencies of Foxp3+ cells in CD4+ T cells. This is a representative of two independent experiments. V, PBS (Vehicle) injection; Ab, Ab injection. (C) Decrease of infiltrated Tregs in ischemic brains after Treg depletion. CD3+ cells were gated for detecting CD4+Foxp3+ cells. Left, representative dot plot of infiltrated Tregs after MCAO. Right, number of infiltrated Tregs in ipsilateral hemispheres. Rapa+V, PBS injection before MCAO and rapamycin treatment. Rapa+Ab, Ab injection before MCAO and rapamycin treatment. N=4 per group. *, p<0.05. (D~F) The number of total immune cells (D), αβ T cells, γδ T cells, granulocytes (E), macrophages and microglia (F) in the ipsilateral hemispheres. Ctrl, control group (MCAO without additional treatment). Rapa+V, Vehicle injection before MCAO and rapamycin treatment. Rapa+Ab, Ab injection before MCAO and rapamycin treatment. N=4 per group. *, p<0.05 compared with control group. **, p<0.01 compared with control group. #, p<0.05 compared with Rapa+V group. (G) Cytokine and chemokine expression in infiltrated CD11b/c+ myeloid cells. N=4 per group. *, p<0.05 compared with control group. **, p<0.01 compared with control group. ***, p<0.001 compared with control group. #, p<0.05 compared with Rapa+V group. ##, p<0.01 compared with Rapa+V group. (H) Treg depletion did not affect macrophage phenotype. This is a representative of two independent experiments. The figure in each quadrant is the frequency of each subpopulation of macrophages.

We then tested the cytokine and chemokine production by macrophages/microglia in ischemic brains after Treg depletion. As shown in Fig. 7G, in comparison with administration of rapamycin and PBS, Treg depletion caused significant increases in the mRNA levels of IL-6, iNOS, CCL2 and IL-12b (p40). However, compared with untreated control group, mRNA levels of IL-6 and IL-12b after Treg depletion were still lower. Only iNOS and CCL2 production were relatively close to the control level. There was a trend of increase in the TNF-α mRNA level after Treg depletion but it was statistically insignificant. Production of IL-1β and CCL3 was almost not changed. Our data suggests Tregs indeed play a role in rapamycin-induced restraint of neuroinflammatory response of macrophages/microglia. However, the effect of Tregs was not as profound as we expected. Some other cellular components might have contributed to the efficacy of rapamycin. Phenotypic polarization of macrophages was not altered after Treg depletion in comparison with vehicle-treated group (Fig. 7H).

DISCUSSION

The results presented here reveal that rapamycin administration 6 hr after focal cerebral ischemia significantly reduces lesion volume and improves motor deficits, implying a longer therapeutic window of opportunity for ischemic stroke treatment. In addition, rapamycin is able to modulate post-stroke neuroinflammatory responses by reducing deleterious and enhancing protective actions of immune cells.

Post-stroke neuroinflammation plays critical roles in the pathophysiology of ischemic stroke, which is characterized by peripheral leukocyte influx into the cerebral parenchyma, activation of endogenous microglia, and release of pro-inflammatory mediators (5, 40). These mediators lead to secondary injury of potentially salvageable tissue within the penumbra regions after ischemic stroke. Granulocytes are generally the first leukocyte subtype recruited to the ischemic brain and may potentiate injury by secreting deleterious neuroinflammatory mediators (41). T lymphocytes also influence the ischemic lesion independently of antigen-specificity and co-stimulatory molecules (42), though there are conflicting data (43). The impact of T cell subsets on secondary infarct progression has been disclosed in recent years. γδ T cells have pivotal roles in the evolution of brain infarction and accompanying neurological deficits (16). In contrast, Tregs prevent secondary infarct growth (38). However, their protective role still needs further confirmation since controversies emerge (44, 45).

In our study, administration of rapamycin profoundly reduced the number of γδ T cells in ischemic brains, suggesting rapamycin might protect brains from γδ T cell-mediated damage. However, we could not detect either IL-17 protein or IL-17 mRNA in infiltrated γδ T cells. It might be possible that rat γδ T cells respond differently from their mouse counterparts, using some mediators other than IL-17 to cause the brain damage. It has been shown that γδ T cells also express TNF-α during the development of EAE (46). The cytokine profile of infiltrated γδ T cells is still unclear and needs further investigation.

Interestingly, Treg number in repamycin-treated brains was not significantly altered, but the Treg proportion in brain immune cells was higher. Thus, it is possible that Tregs can function more efficiently to inhibit inflammatory cells in treated brains. TGF-β and IL-10, which can be produced by Tregs, are shown to be neuroprotective (47–49). Our data showed that although IL-10 level was not enhanced in Tregs, the levels of IL-35 and TGF-β in Tregs were significantly increased by rapamycin. Hence, increased Treg anti-inflammatory activity may contribute to alleviating brain damage. However, it remains unclear whether rapamycin directly or indirectly enhances Treg anti-inflammatory activity in ischemic brains. It has been reported that mTOR signaling negatively regulates Treg commitment, expansion and function, while rapamycin increases Treg number and enhances Treg activity both in vitro and in vivo (50–57). The effect of rapamycin on Tregs is associated with stabilization of Foxp3 (51, 54), which is the Treg master regulator controlling Treg development and function. Indeed, we found higher Foxp3 level in Tregs in rapamycin-treated brains, suggesting the expression/stabilization of Foxp3 is enhanced by rapamycin treatment. CD25 expression was also higher on Tregs after rapamycin treatment, possibly due to direct binding of Foxp3 to the promoter region of Il2ra. The elevated expression of Ebi3, which is the subunit of IL-35, can also be attributed to higher Foxp3 level, since Ebi3 is a downstream target of Foxp3 (58). However, the higher Foxp3 level in rapamycin-treated Tregs does not explain the unchanged IL-10 and increased TGF-β, since there is no convincing proof showing Foxp3 directly regulates expression of these cytokines. Thus, rapamycin might regulate their expression independently of direct effect of Foxp3. The higher CD25 expression might reflect higher level of IL-2R on the surface of Tregs in rapamycin-treated brains, thus making these Tregs possess higher affinity for IL-2, which induces more IL-10 expression (59–61). However, rapamycin itself might inhibit IL-10 mRNA and protein in Tregs, as in macrophages (62). Hence, the unchanged IL-10 level might reflect a balance between the effects of IL-2/IL-2R signaling and mTOR inhibition. Researchers have observed rapamycin-induced TGF-β production by lymphocytes and infiltrated Tregs in previous studies (63, 64). Hence, rapamycin might directly increase TGF-β production in Tregs through unknown molecular mechanisms. In addition, Tregs might also be modulated by an indirect effect of rapamycin. It has been reported that rapamycin-treated endothelial cells and dendritic cells may promote Treg activity in ischemic brains (65, 66). Hence, rapamycin-treated endothelial or dendritic cells could induce Tregs to produce immunosuppressive cytokines to restrain the inflammation. However, further investigations are in demand to test our hypothesis.

Previous studies have documented the proinflammatory (M1) and anti-inflammatory (M2) macrophages in the post-ischemic brains (67, 68). Modulation of microglia and macrophage polarization towards the beneficial M2 type would restrain neuroinflammation and favor functional recovery (34). Our work indicated that rapamycin treatment phenotypically inhibited M1 polarization in brain macrophages. Correspondingly, pro-inflammatory cytokine and chemokine production in macrophages were inhibited. However, M2 type-related anti-inflammatory cytokine production was not increased, suggesting the effects of rapamycin on macrophage polarization are more complicated than expected. It is also possible that phenotypical polarization starts earlier than functional polarization, or restraint of M1 cytokine production is prior to elevation of M2 cytokine production. In microglia, both phenotypical and functional polarization was not as significant as in macrophages, suggesting the effects of rapamycin treatment on microglia could be weak or require longer time. Macrophages and microglia showed similar changes in chemokine levels after rapamycin treatment. The profound elevation of CXCL12 was unexpected but could be involved in the migration of neural stem/progenitor cells to the lesion site. Decreased production of other chemokines could explain reduced infiltration of granulocytes and γδ T cells. However, inhibited inflammatory response of macrophages and microglia cannot be attributed to the direct effect of rapamycin, since both our data and previous research demonstrated that rapamycin induces macrophages polarization towards the pro-inflammatory M1 type (30). Thus, the inhibited M1 polarization of macrophages and microglia should be mediated by agents other than rapamycin itself. Interestingly, our data show that the functional change in macrophages/microglia is at least partially due to their interaction with Tregs. Tregs isolated from rapamycin-treated brains more potently inhibited pro-inflammatory cytokine and chemokine production, consistent with their promoted anti-inflammatory activity. So we concluded although rapamycin directly induces M1 polarization of macrophages and microglia, it also strongly enhances Treg activity, which in turn restrains the inflammatory response of macrophages and microglia.

To determine whether Treg is a major target of rapamycin, we depleted Tregs with anti-CD25 Ab before stroke and rapamycin injection. Although Treg depletion was successful, it just partially enhanced inflammatory response in rapamycin-treated brains. It appears that Tregs indeed but limitedly contributed to rapamycin-induced anti-inflammation effect. Other cell types might also play roles in this process. Note that rapamycin was injected 6 h after I/R, early before the recruitment of peripheral immune cells. We speculated that rapamycin could inhibit the initiation of neuroinflammation when leukocytes are still outside the brain parenchyma. Both damaged/stressed neurons and reactive astrocytes quickly initiate post-ischemic inflammation, through producing pro-inflammatory mediators and educating microglia (43, 69, 70). Rapamycin protects neurons after stroke (12, 71) and inhibits reactive astrocytes (72, 73), thus probably preventing the onset of acute neuroinflammation. Hence, the neuroinflammation might have been alleviated even before Tregs entered the brain parenchyma. Decrease of neuroinflammation on day 3 after stroke might be the outcome of both restrained initiation and inhibited progression of neuroinflamamtion. Tregs might be just involved in the progression phase. This could explain why Treg depletion could not completely abolish the effect of rapamycin. However, careful studies, especially on the initiation of neuroinflammation shortly after I/R, will be needed to test our hypothesis in future.

In our study we injected rapamycin into the lateral ventricles, hoping that it functions mainly in the ischemic brains. Based on former studies, peripheral administration of rapamycin also shows neuroprotective effect after stroke (10, 71). Rapamycin can cross the blood brain barrier into the brain parenchyma even in the steady state (74). Thus, peripheral administration of rapamycin might inhibit leukocyte activation in the periphery, prevent neuron death and restrain astrocyte reaction in the brain, exerting similar effects to intracerebroventricular injection. However, peripheral administration might severely interfere with functions of the immune system and other vital organs/tissues, which might not be good for patients.

Taken together, our research suggests intraventricular administration of rapamycin after ischemic stroke restrains pro-inflammatory activity of macrophages and microglia through Tregs. These studies may have implications for novel therapeutic interventions targeting post-ischemic neuroinflammation in stroke.

Supplementary Material

1

ACKNOWLEDGMENTS

Flow cytometry cell sorting was performed by Xiangle Sun in the Flow Cytometry and Laser Capture Microdissection Core Facility at The University of North Texas Health Science Center.

This work was supported by US Public Health Service Grants NS57186 and AG21980 to KJ, and NS054687 and NS054651 to SY.

Abbreviations

MCAO

middle cerebral artery occlusion

IL

interleukin

iNOS

inducible NO synthase

FGF

fibroblast growth factor

mTOR

mammalian target of rapamycin

mTORC

mTOR complex

TGF-β

tumor growth factor beta

TNF-α

tumor necrosis factor alpha

Treg

regulatory T cell

Footnotes

COMPETING INTERESTS STATEMENT

The authors declare no competing financial interests.

REFERENCES

  • 1.Go AS, Mozaffarian D, Roger VL, Benjamin EJ, Berry JD, Blaha MJ, Dai S, Ford ES, Fox CS, Franco S, Fullerton HJ, Gillespie C, Hailpern SM, Heit JA, Howard VJ, Huffman MD, Judd SE, Kissela BM, Kittner SJ, Lackland DT, Lichtman JH, Lisabeth LD, Mackey RH, Magid DJ, Marcus GM, Marelli A, Matchar DB, McGuire DK, Mohler ER, 3rd, Moy CS, Mussolino ME, Neumar RW, Nichol G, Pandey DK, Paynter NP, Reeves MJ, Sorlie PD, Stein J, Towfighi A, Turan TN, Virani SS, Wong ND, Woo D, Turner MB. Heart disease and stroke statistics--2014 update: a report from the American Heart Association. Circulation. 2014;129:e28–e292. doi: 10.1161/01.cir.0000441139.02102.80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Brott T, Bogousslavsky J. Treatment of acute ischemic stroke. N Engl J Med. 2000;343:710–722. doi: 10.1056/NEJM200009073431007. [DOI] [PubMed] [Google Scholar]
  • 3.Wang Y, Zhang Z, Chow N, Davis TP, Griffin JH, Chopp M, Zlokovic BV. An activated protein C analog with reduced anticoagulant activity extends the therapeutic window of tissue plasminogen activator for ischemic stroke in rodents. Stroke; a journal of cerebral circulation. 2012;43:2444–2449. doi: 10.1161/STROKEAHA.112.658997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Wechsler LR, Jovin TG. Intravenous recombinant tissue-type plasminogen activator in the extended time window and the US Food and Drug Administration: confused about the time. Stroke; a journal of cerebral circulation. 2012;43:2517–2519. doi: 10.1161/STROKEAHA.112.670554. [DOI] [PubMed] [Google Scholar]
  • 5.Jin R, Yang G, Li G. Inflammatory mechanisms in ischemic stroke: role of inflammatory cells. J Leukoc Biol. 2010;87:779–789. doi: 10.1189/jlb.1109766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Sughrue ME, Mehra A, Connolly ES, Jr, D'Ambrosio AL. Anti-adhesion molecule strategies as potential neuroprotective agents in cerebral ischemia: a critical review of the literature. Inflamm Res. 2004;53:497–508. doi: 10.1007/s00011-004-1282-0. [DOI] [PubMed] [Google Scholar]
  • 7.Wiederrecht GJ, Sabers CJ, Brunn GJ, Martin MM, Dumont FJ, Abraham RT. Mechanism of action of rapamycin: new insights into the regulation of G1-phase progression in eukaryotic cells. Progress in cell cycle research. 1995;1:53–71. doi: 10.1007/978-1-4615-1809-9_5. [DOI] [PubMed] [Google Scholar]
  • 8.Laplante M, Sabatini DM. mTOR signaling at a glance. Journal of cell science. 2009;122:3589–3594. doi: 10.1242/jcs.051011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Thomson AW, Turnquist HR, Raimondi G. Immunoregulatory functions of mTOR inhibition. Nature reviews. Immunology. 2009;9:324–337. doi: 10.1038/nri2546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Chauhan A, Sharma U, Jagannathan NR, Reeta KH, Gupta YK. Rapamycin protects against middle cerebral artery occlusion induced focal cerebral ischemia in rats. Behav Brain Res. 2011;225:603–609. doi: 10.1016/j.bbr.2011.08.035. [DOI] [PubMed] [Google Scholar]
  • 11.Carloni S, Girelli S, Scopa C, Buonocore G, Longini M, Balduini W. Activation of autophagy and Akt/CREB signaling play an equivalent role in the neuroprotective effect of rapamycin in neonatal hypoxia-ischemia. Autophagy. 2010;6:366–377. doi: 10.4161/auto.6.3.11261. [DOI] [PubMed] [Google Scholar]
  • 12.Fletcher L, Evans TM, Watts LT, Jimenez DF, Digicaylioglu M. Rapamycin treatment improves neuron viability in an in vitro model of stroke. PLoS One. 2013;8:e68281. doi: 10.1371/journal.pone.0068281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Jin K, Minami M, Lan JQ, Mao XO, Batteur S, Simon RP, Greenberg DA. Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat. Proc Natl Acad Sci U S A. 2001;98:4710–4715. doi: 10.1073/pnas.081011098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C, Sharp FR. A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab. 1990;10:290–293. doi: 10.1038/jcbfm.1990.47. [DOI] [PubMed] [Google Scholar]
  • 15.Sun F, Xie L, Mao X, Hill J, Greenberg DA, Jin K. Effect of a contralateral lesion on neurological recovery from stroke in rats. Restor Neurol Neurosci. 2012 doi: 10.3233/RNN-2012-120254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Shichita T, Sugiyama Y, Ooboshi H, Sugimori H, Nakagawa R, Takada I, Iwaki T, Okada Y, Iida M, Cua DJ, Iwakura Y, Yoshimura A. Pivotal role of cerebral interleukin-17-producing gammadeltaT cells in the delayed phase of ischemic brain injury. Nature medicine. 2009;15:946–950. doi: 10.1038/nm.1999. [DOI] [PubMed] [Google Scholar]
  • 17.Ghiringhelli F, Larmonier N, Schmitt E, Parcellier A, Cathelin D, Garrido C, Chauffert B, Solary E, Bonnotte B, Martin F. CD4+CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative. Eur J Immunol. 2004;34:336–344. doi: 10.1002/eji.200324181. [DOI] [PubMed] [Google Scholar]
  • 18.Ghiringhelli F, Puig PE, Roux S, Parcellier A, Schmitt E, Solary E, Kroemer G, Martin F, Chauffert B, Zitvogel L. Tumor cells convert immature myeloid dendritic cells into TGF-beta-secreting cells inducing CD4+CD25+ regulatory T cell proliferation. J Exp Med. 2005;202:919–929. doi: 10.1084/jem.20050463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Hill JJ, Jin K, Mao XO, Xie L, Greenberg DA. Intracerebral chondroitinase ABC and heparan sulfate proteoglycan glypican improve outcome from chronic stroke in rats. Proc Natl Acad Sci U S A. 2012;109:9155–9160. doi: 10.1073/pnas.1205697109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Wang X, Yue TL, Barone FC, White RF, Gagnon RC, Feuerstein GZ. Concomitant cortical expression of TNF-alpha and IL-1 beta mRNAs follows early response gene expression in transient focal ischemia. Mol Chem Neuropathol. 1994;23:103–114. doi: 10.1007/BF02815404. [DOI] [PubMed] [Google Scholar]
  • 21.Liu T, McDonnell PC, Young PR, White RF, Siren AL, Hallenbeck JM, Barone FC, Feurestein GZ. Interleukin-1 beta mRNA expression in ischemic rat cortex. Stroke. 1993;24:1746–1750. doi: 10.1161/01.str.24.11.1746. discussion 1750-1741. [DOI] [PubMed] [Google Scholar]
  • 22.Wiessner C, Gehrmann J, Lindholm D, Topper R, Kreutzberg GW, Hossmann KA. Expression of transforming growth factor-beta 1 and interleukin-1 beta mRNA in rat brain following transient forebrain ischemia. Acta Neuropathol. 1993;86:439–446. doi: 10.1007/BF00228578. [DOI] [PubMed] [Google Scholar]
  • 23.Liu T, Clark RK, McDonnell PC, Young PR, White RF, Barone FC, Feuerstein GZ. Tumor necrosis factor-alpha expression in ischemic neurons. Stroke. 1994;25:1481–1488. doi: 10.1161/01.str.25.7.1481. [DOI] [PubMed] [Google Scholar]
  • 24.Wang X, Yue TL, Barone FC, Feuerstein GZ. Monocyte chemoattractant protein-1 messenger RNA expression in rat ischemic cortex. Stroke. 1995;26:661–665. doi: 10.1161/01.str.26.4.661. discussion 665-666. [DOI] [PubMed] [Google Scholar]
  • 25.Kim JS, Gautam SC, Chopp M, Zaloga C, Jones ML, Ward PA, Welch KM. Expression of monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 after focal cerebral ischemia in the rat. J Neuroimmunol. 1995;56:127–134. doi: 10.1016/0165-5728(94)00138-e. [DOI] [PubMed] [Google Scholar]
  • 26.Pantoni L, Sarti C, Inzitari D. Cytokines and cell adhesion molecules in cerebral ischemia: experimental bases and therapeutic perspectives. Arterioscler Thromb Vasc Biol. 1998;18:503–513. doi: 10.1161/01.atv.18.4.503. [DOI] [PubMed] [Google Scholar]
  • 27.Gelderblom M, Leypoldt F, Steinbach K, Behrens D, Choe CU, Siler DA, Arumugam TV, Orthey E, Gerloff C, Tolosa E, Magnus T. Temporal and spatial dynamics of cerebral immune cell accumulation in stroke. Stroke. 2009;40:1849–1857. doi: 10.1161/STROKEAHA.108.534503. [DOI] [PubMed] [Google Scholar]
  • 28.Shichita T, Sugiyama Y, Ooboshi H, Sugimori H, Nakagawa R, Takada I, Iwaki T, Okada Y, Iida M, Cua DJ, Iwakura Y, Yoshimura A. Pivotal role of cerebral interleukin-17-producing gammadeltaT cells in the delayed phase of ischemic brain injury. Nat Med. 2009;15:946–950. doi: 10.1038/nm.1999. [DOI] [PubMed] [Google Scholar]
  • 29.Baker AK, Wang R, Mackman N, Luyendyk JP. Rapamycin enhances LPS induction of tissue factor and tumor necrosis factor-alpha expression in macrophages by reducing IL-10 expression. Mol Immunol. 2009;46:2249–2255. doi: 10.1016/j.molimm.2009.04.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Mercalli A, Calavita I, Dugnani E, Citro A, Cantarelli E, Nano R, Melzi R, Maffi P, Secchi A, Sordi V, Piemonti L. Rapamycin unbalances the polarization of human macrophages to M1. Immunology. 2013;140:179–190. doi: 10.1111/imm.12126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Penido C, Costa MF, Souza MC, Costa KA, Candea AL, Benjamim CF, Henriques M. Involvement of CC chemokines in gammadelta T lymphocyte trafficking during allergic inflammation: the role of CCL2/CCR2 pathway. Int Immunol. 2008;20:129–139. doi: 10.1093/intimm/dxm128. [DOI] [PubMed] [Google Scholar]
  • 32.Lanca T, Costa MF, Goncalves-Sousa N, Rei M, Grosso AR, Penido C, Silva-Santos B. Protective role of the inflammatory CCR2/CCL2 chemokine pathway through recruitment of type 1 cytotoxic gammadelta T lymphocytes to tumor beds. J Immunol. 2013;190:6673–6680. doi: 10.4049/jimmunol.1300434. [DOI] [PubMed] [Google Scholar]
  • 33.Biswas SK, Mantovani A. Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm. Nat Immunol. 2010;11:889–896. doi: 10.1038/ni.1937. [DOI] [PubMed] [Google Scholar]
  • 34.Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol. 2011;11:723–737. doi: 10.1038/nri3073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Wang X, Wang W, Xu J, Le Q. Effect of rapamycin and interleukin-2 on regulatory CD4+CD25+Foxp3+ T cells in mice after allogenic corneal transplantation. Transplant Proc. 2013;45:528–537. doi: 10.1016/j.transproceed.2012.06.064. [DOI] [PubMed] [Google Scholar]
  • 36.Wang Y, Camirand G, Lin Y, Froicu M, Deng S, Shlomchik WD, Lakkis FG, Rothstein DM. Regulatory T cells require mammalian target of rapamycin signaling to maintain both homeostasis and alloantigen-driven proliferation in lymphocyte-replete mice. J Immunol. 2011;186:2809–2818. doi: 10.4049/jimmunol.0903805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Shin HJ, Baker J, Leveson-Gower DB, Smith AT, Sega EI, Negrin RS. Rapamycin and IL-2 reduce lethal acute graft-versus-host disease associated with increased expansion of donor type CD4+CD25+Foxp3+ regulatory T cells. Blood. 2011;118:2342–2350. doi: 10.1182/blood-2010-10-313684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Liesz A, Suri-Payer E, Veltkamp C, Doerr H, Sommer C, Rivest S, Giese T, Veltkamp R. Regulatory T cells are key cerebroprotective immunomodulators in acute experimental stroke. Nat Med. 2009;15:192–199. doi: 10.1038/nm.1927. [DOI] [PubMed] [Google Scholar]
  • 39.Thornton AM, Korty PE, Tran DQ, Wohlfert EA, Murray PE, Belkaid Y, Shevach EM. Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells. J Immunol. 2010;184:3433–3441. doi: 10.4049/jimmunol.0904028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Yilmaz G, Granger DN. Leukocyte recruitment and ischemic brain injury. Neuromolecular Med. 2010;12:193–204. doi: 10.1007/s12017-009-8074-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Wang Q, Tang XN, Yenari MA. The inflammatory response in stroke. Journal of neuroimmunology. 2007;184:53–68. doi: 10.1016/j.jneuroim.2006.11.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Kleinschnitz C, Schwab N, Kraft P, Hagedorn I, Dreykluft A, Schwarz T, Austinat M, Nieswandt B, Wiendl H, Stoll G. Early detrimental T-cell effects in experimental cerebral ischemia are neither related to adaptive immunity nor thrombus formation. Blood. 2010;115:3835–3842. doi: 10.1182/blood-2009-10-249078. [DOI] [PubMed] [Google Scholar]
  • 43.Iadecola C, Anrather J. The immunology of stroke: from mechanisms to translation. Nat Med. 2011;17:796–808. doi: 10.1038/nm.2399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Kleinschnitz C, Kraft P, Dreykluft A, Hagedorn I, Gobel K, Schuhmann MK, Langhauser F, Helluy X, Schwarz T, Bittner S, Mayer CT, Brede M, Varallyay C, Pham M, Bendszus M, Jakob P, Magnus T, Meuth SG, Iwakura Y, Zernecke A, Sparwasser T, Nieswandt B, Stoll G, Wiendl H. Regulatory T cells are strong promoters of acute ischemic stroke in mice by inducing dysfunction of the cerebral microvasculature. Blood. 2013;121:679–691. doi: 10.1182/blood-2012-04-426734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Ren X, Akiyoshi K, Vandenbark AA, Hurn PD, Offner H. CD4+FoxP3+ regulatory T-cells in cerebral ischemic stroke. Metabolic brain disease. 2011;26:87–90. doi: 10.1007/s11011-010-9226-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Wohler JE, Smith SS, Zinn KR, Bullard DC, Barnum SR. Gammadelta T cells in EAE: early trafficking events and cytokine requirements. Eur J Immunol. 2009;39:1516–1526. doi: 10.1002/eji.200839176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Lakhan SE, Kirchgessner A, Hofer M. Inflammatory mechanisms in ischemic stroke: therapeutic approaches. Journal of translational medicine. 2009;7:97. doi: 10.1186/1479-5876-7-97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Frenkel D, Huang Z, Maron R, Koldzic DN, Moskowitz MA, Weiner HL. Neuroprotection by IL-10-producing MOG CD4+ T cells following ischemic stroke. Journal of the neurological sciences. 2005;233:125–132. doi: 10.1016/j.jns.2005.03.022. [DOI] [PubMed] [Google Scholar]
  • 49.Strle K, Zhou JH, Shen WH, Broussard SR, Johnson RW, Freund GG, Dantzer R, Kelley KW. Interleukin-10 in the brain. Critical reviews in immunology. 2001;21:427–449. [PubMed] [Google Scholar]
  • 50.Lee JH, Lydon JP, Kim CH. Progesterone suppresses the mTOR pathway and promotes generation of induced regulatory T cells with increased stability. Eur J Immunol. 2012;42:2683–2696. doi: 10.1002/eji.201142317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Yurchenko E, Shio MT, Huang TC, Da Silva Martins M, Szyf M, Levings MK, Olivier M, Piccirillo CA. Inflammation-driven reprogramming of CD4+ Foxp3+ regulatory T cells into pathogenic Th1/Th17 T effectors is abrogated by mTOR inhibition in vivo. PLoS One. 2012;7:e35572. doi: 10.1371/journal.pone.0035572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Battaglia M, Stabilini A, Tresoldi E. Expanding human T regulatory cells with the mTOR-inhibitor rapamycin. Methods Mol Biol. 2012;821:279–293. doi: 10.1007/978-1-61779-430-8_17. [DOI] [PubMed] [Google Scholar]
  • 53.Delgoffe GM, Kole TP, Zheng Y, Zarek PE, Matthews KL, Xiao B, Worley PF, Kozma SC, Powell JD. The mTOR kinase differentially regulates effector and regulatory T cell lineage commitment. Immunity. 2009;30:832–844. doi: 10.1016/j.immuni.2009.04.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Zhang P, Tey SK, Koyama M, Kuns RD, Olver SD, Lineburg KE, Lor M, Teal BE, Raffelt NC, Raju J, Leveque L, Markey KA, Varelias A, Clouston AD, Lane SW, MacDonald KP, Hill GR. Induced regulatory T cells promote tolerance when stabilized by rapamycin and IL-2 in vivo. J Immunol. 2013;191:5291–5303. doi: 10.4049/jimmunol.1301181. [DOI] [PubMed] [Google Scholar]
  • 55.Zhang W, Zhang D, Shen M, Liu Y, Tian Y, Thomson AW, Lee WP, Zheng XX. Combined administration of a mutant TGF-beta1/Fc and rapamycin promotes induction of regulatory T cells and islet allograft tolerance. J Immunol. 2010;185:4750–4759. doi: 10.4049/jimmunol.1000769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Park Y, Jin HS, Lopez J, Elly C, Kim G, Murai M, Kronenberg M, Liu YC. TSC1 regulates the balance between effector and regulatory T cells. J Clin Invest. 2013;123:5165–5178. doi: 10.1172/JCI69751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Haxhinasto S, Mathis D, Benoist C. The AKT-mTOR axis regulates de novo differentiation of CD4+Foxp3+ cells. J Exp Med. 2008;205:565–574. doi: 10.1084/jem.20071477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Collison LW, Workman CJ, Kuo TT, Boyd K, Wang Y, Vignali KM, Cross R, Sehy D, Blumberg RS, Vignali DA. The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature. 2007;450:566–569. doi: 10.1038/nature06306. [DOI] [PubMed] [Google Scholar]
  • 59.Tsuji-Takayama K, Suzuki M, Yamamoto M, Harashima A, Okochi A, Otani T, Inoue T, Sugimoto A, Toraya T, Takeuchi M, Yamasaki F, Nakamura S, Kibata M. The production of IL-10 by human regulatory T cells is enhanced by IL-2 through a STAT5-responsive intronic enhancer in the IL-10 locus. J Immunol. 2008;181:3897–3905. doi: 10.4049/jimmunol.181.6.3897. [DOI] [PubMed] [Google Scholar]
  • 60.Tsuji-Takayama K, Suzuki M, Yamamoto M, Harashima A, Okochi A, Otani T, Inoue T, Sugimoto A, Motoda R, Yamasaki F, Nakamura S, Kibata M. IL-2 activation of STAT5 enhances production of IL-10 from human cytotoxic regulatory T cells, HOZOT. Exp Hematol. 2008;36:181–192. doi: 10.1016/j.exphem.2007.09.010. [DOI] [PubMed] [Google Scholar]
  • 61.Brandenburg S, Takahashi T, de la Rosa M, Janke M, Karsten G, Muzzulini T, Orinska Z, Bulfone-Paus S, Scheffold A. IL-2 induces in vivo suppression by CD4(+)CD25(+)Foxp3(+) regulatory T cells. Eur J Immunol. 2008;38:1643–1653. doi: 10.1002/eji.200737791. [DOI] [PubMed] [Google Scholar]
  • 62.Thomson AW, Turnquist HR, Raimondi G. Immunoregulatory functions of mTOR inhibition. Nat Rev Immunol. 2009;9:324–337. doi: 10.1038/nri2546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Dodge IL, Demirci G, Strom TB, Li XC. Rapamycin induces transforming growth factor-beta production by lymphocytes. Transplantation. 2000;70:1104–1106. doi: 10.1097/00007890-200010150-00020. [DOI] [PubMed] [Google Scholar]
  • 64.Wang X, Wang W, Xu J, Hong J, Le Q. Pretreatment of rapamycin before allogenic corneal transplant promotes graft survival through increasing CD4(+)CD25(+)Foxp3(+) regulatory T cells. Exp Clin Transplant. 2013;11:56–62. doi: 10.6002/ect.2012.0025. [DOI] [PubMed] [Google Scholar]
  • 65.Wang C, Yi T, Qin L, Maldonado RA, von Andrian UH, Kulkarni S, Tellides G, Pober JS. Rapamycin-treated human endothelial cells preferentially activate allogeneic regulatory T cells. J Clin Invest. 2013;123:1677–1693. doi: 10.1172/JCI66204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Turnquist HR, Raimondi G, Zahorchak AF, Fischer RT, Wang Z, Thomson AW. Rapamycin-conditioned dendritic cells are poor stimulators of allogeneic CD4+ T cells, but enrich for antigen-specific Foxp3+ T regulatory cells and promote organ transplant tolerance. J Immunol. 2007;178:7018–7031. doi: 10.4049/jimmunol.178.11.7018. [DOI] [PubMed] [Google Scholar]
  • 67.Hu X, Li P, Guo Y, Wang H, Leak RK, Chen S, Gao Y, Chen J. Microglia/macrophage polarization dynamics reveal novel mechanism of injury expansion after focal cerebral ischemia. Stroke. 2012;43:3063–3070. doi: 10.1161/STROKEAHA.112.659656. [DOI] [PubMed] [Google Scholar]
  • 68.Perego C, Fumagalli S, De Simoni MG. Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice. J Neuroinflammation. 2011;8:174. doi: 10.1186/1742-2094-8-174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Lambertsen KL, Biber K, Finsen B. Inflammatory cytokines in experimental and human stroke. J Cereb Blood Flow Metab. 2012;32:1677–1698. doi: 10.1038/jcbfm.2012.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70.Shih AY, Fernandes HB, Choi FY, Kozoriz MG, Liu Y, Li P, Cowan CM, Klegeris A. Policing the police: astrocytes modulate microglial activation. J Neurosci. 2006;26:3887–3888. doi: 10.1523/JNEUROSCI.0936-06.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71.Viscomi MT, D'Amelio M, Cavallucci V, Latini L, Bisicchia E, Nazio F, Fanelli F, Maccarrone M, Moreno S, Cecconi F, Molinari M. Stimulation of autophagy by rapamycin protects neurons from remote degeneration after acute focal brain damage. Autophagy. 2012;8:222–235. doi: 10.4161/auto.8.2.18599. [DOI] [PubMed] [Google Scholar]
  • 72.Lisi L, Navarra P, Feinstein DL, Dello Russo C. The mTOR kinase inhibitor rapamycin decreases iNOS mRNA stability in astrocytes. J Neuroinflammation. 2011;8:1. doi: 10.1186/1742-2094-8-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73.Codeluppi S, Svensson CI, Hefferan MP, Valencia F, Silldorff MD, Oshiro M, Marsala M, Pasquale EB. The Rheb-mTOR pathway is upregulated in reactive astrocytes of the injured spinal cord. J Neurosci. 2009;29:1093–1104. doi: 10.1523/JNEUROSCI.4103-08.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 74.Banerjee S, Gianino SM, Gao F, Christians U, Gutmann DH. Interpreting mammalian target of rapamycin and cell growth inhibition in a genetically engineered mouse model of Nf1-deficient astrocytes. Mol Cancer Ther. 2011;10:279–291. doi: 10.1158/1535-7163.MCT-10-0654. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS587595-supplement-1.pdf (746.6KB, pdf)

RESOURCES