Cannabinoid Type 2 Receptor Activation Downregulates Stroke-Induced Classic and Alternative Brain Macrophage/Microglial Activation Concomitant to Neuroprotection
Background and Purpose—Ischemic stroke continues to be one of the main causes of death worldwide. Inflammation accounts for a large part of damage in this pathology. The cannabinoid type 2 receptor (CB2R) has been proposed to have neuroprotective properties in neurological diseases. Therefore, our aim was to determine the effects of the activation of CB2R on infarct outcome and on ischemia-induced brain expression of classic and alternative markers of macrophage/microglial activation.
Methods—Swiss wild-type and CB2R knockout male mice were subjected to a permanent middle cerebral artery occlusion. Mice were treated with either a CB2R agonist (JWH-133), with or without a CB2R antagonist (SR144528) or vehicle. Infarct outcome was determined by measuring infarct volume and neurological outcome. An additional group of animals was used to assess mRNA and protein expression of CB2R, interleukin (IL)-1β, IL-6, tumor necrosis factor α (TNF-α), monocyte chemoattractant protein–1 (MCP-1), macrophage inflammatory peptide (MIP) –1α, RANTES, inducible nitric oxide synthase (iNOS), cyclooxygenase-2, IL-4, IL-10, transforming growth factor β (TGF-β), arginase I, and Ym1.
Results—Administration of JWH-133 significantly improved infarct outcome, as shown by a reduction in brain infarction and neurological impairment. This effect was reversed by the CB2R antagonist and was absent in CB2R knockout mice. Concomitantly, administration of JWH-133 led to a lower intensity of Iba1+ microglia/macrophages and a decrease in middle cerebral artery occlusion–induced gene expression of both classic (IL-6, TNF-α, MCP-1, MIP-1α, RANTES, and iNOS) and alternative mediators/markers (IL-10, TGF-β, and Ym1) of microglial/macrophage activation after permanent middle cerebral artery occlusion.
Conclusions—The inhibitory effect of CB2R on the activation of different subpopulations of microglia/macrophages may account for the protective effect of the selective CB2R agonist JWH-133 after stroke.
The endocannabinoid system, integrated by endogenous ligands, cannabinoid receptors, and degrading enzymes, has been proposed as an important pharmacological target in several neurological diseases.1,2 The most-studied cannabinoid receptors are cannabinoid receptor type 1 (CB1R)3,4 and cannabinoid receptor type 2 (CB2R).5 Whereas CB1R is predominantly expressed by neurons (reviewed in6), CB2R is mainly expressed by immune cells, regulating migration, cytokine production, and antigen presentation.7,8
Indeed, increasing evidence demonstrates the role of CB2R in cannabinoid-mediated regulation of the immune system (reviewed in1,2,7,8). Stimulation of CB2R has been shown to inhibit cytokine release,9,10 to regulate B and T cell differentiation balance,11 and to modulate macrophage/microglial migration and proliferation.12–14
Considering the important role of inflammation in ischemic stroke pathophysiology,15,16 modulation of immune cells by CB2R activation has arisen as an interesting pharmacological approach toward neuroprotection after brain ischemia. In this context, the protective effect of selective CB2R agonists has been tested in different animal models of focal brain ischemia.17–20 In these studies, CB2R activation resulted in neuroprotection concomitant to a decreased peripheral response as shown by inhibition of leukocyte rolling and adhesion in venules and arterioles18,20 and inhibition of neutrophil recruitment17; attenuation of blood-brain barrier disruption has been also observed.20 However, local brain effects of CB2R activation have not been explored.
In the present study, we decided to investigate effects of the administration of a single dose of the selective CB2R agonist JWH-133 on immune cell activation, infarct size, and functional outcome after permanent middle cerebral artery occlusion (pMCAO).
Materials and Methods
The selective CB2R agonist (6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (JWH133) was purchased from Tocris Bioscience. JWH-133 has a very high affinity for the CB2R (Ki=3.4 nmol/L), but low affinity for the CB1R (Ki=677 nmol/L).21 N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528), a selective CB2R antagonist, was a kind gift from Dr. Francis Barth, Sanofi-Synthélabo (Montpellier, France). JWH-133 and SR144528 were dissolved in DMSO:Tween:PBS (1:1:18) and the total volume injected intraperitoneally into each animal was 200 μL. The vehicle was DMSO:Tween:PBS (1:1:18) and was also injected in a total volume of 200 μL.
Adult male Swiss mice (age 8–10 weeks) were used. All animals were kept in a room with controlled temperature, a 12-hour dark/light cycle and fed with standard food and water ad libitum. Male CB2R knockout mice (CB2R-KO) on a Swiss congenic background were used.22 All experimental protocols adhered to the guidelines of the Animal Welfare Committee of the Universidad Complutense (EU directives 86/609/CEE and 2003/65/CE).
Induction of Permanent Focal Ischemia
Surgery leading to focal cerebral ischemia was performed as described in Supplemental Materials and Methods (http://stroke.ahajournals.org).
All the groups were performed and quantified in a randomized fashion by investigators blinded to treatment groups for determination of infarct outcome; all treatments were administered 10 minutes after occlusion. Animals were allocated by randomization (coin toss) to 4 different groups. One group received an intraperitoneal injection of JWH-133 (1.5 mg/kg; n=12). A second and third group were treated with SR144528 (3–5 mg/kg) and 3 minutes later with JWH-133 (1.5 mg/kg) intraperitoneally (n=12 in each group); a fourth group of animals was injected with vehicle (DMSO:Tween:PBS) and was considered the control group (n=12). In addition, a group of CB2R-KO mice was subjected to pMCAO and treated either with vehicle (n=12) or with JWH-133 1.5 mg/kg (n=12). An additional group of animals was treated 3 hours after pMCAO with either JWH-133 (1.5 mg/kg; n=8) or vehicle.
Infarct Outcome Determination
Infarct volume determination and neurological score were used as measures of infarct outcome as described in Supplemental Materials and Methods.
Quantitative Real-Time Polymerase Chain Reaction
Total RNA was extracted from samples collected either 15 or 5 to 18 hours after pMCAO (n=5 for each group) for inflammatory mediators or cannabinoid receptors expression, respectively, using TRIzol reagent (Invitrogen). Sample preparation and quantitative real-time polymerase chain reaction were performed as described in Supplemental Materials and Methods.
Cytometric Bead Array and Western Blotting
Protein homogenates from brain peri-infarct tissue obtained 24 hours after pMCAO (n=4–8 in each group) were used to measure the protein levels of IL-1β, tumor necrosis factor α (TNF-α), IL-6, macrophage inflammatory peptide (MIP) –1α, MCP-1, RANTES, IL12/IL-23p40, and IL-10 by a cytometric bead array (Beckton Dickinson), and of iNOS and COX-2 by western blotting, as described in Supplemental Materials and Methods.
Immunofluorescence and Confocal Microscopy
Free-floating coronal brain slices (30 mm) were processed 24 hours after pMCAO (n=4 in each group) and immunofluorescence, confocal microscopy, and image analysis were performed as described in Supplemental Materials and Methods.
Student t test was used to compare 2 groups, and 1-way ANOVA with Newman-Keuls or Bonferroni post hoc tests were used to compare more than 2 groups. Data are expressed as mean±SD, and a P<0.05 was assumed as statistically significant difference.
Expression of CB1R and CB2R After pMCAO
pMCAO increased CB2R mRNA expression in the peri-infarct area 18 hours after injury and decreased CB1R mRNA levels at 5 hours (Supplemental Figure IA). The CB2R agonist JWH-133 decreased ischemia-induced mRNA expression of CB2R 15 hours after pMCAO, whereas CB1R mRNA expression was not affected by this treatment (Supplemental Figure IB).
Immunofluorescence characterization of brain sections 24 hours after MCAO and subsequent confocal analysis were performed to elucidate the nature of the CB2R-expressing cells. A large proportion of microglial cells (Iba-1+) in the peri-infarct area coexpressed CB2R (Figure 1 and Supplemental Figure IIB). In both peri-infarct (Figure 1) and ischemic core (Supplemental Figure IIB), all neurons (NeuN+ cells) were negative for CB2R. Some isolated astrocytes (GFAP+ cells) present in the corpus callosum showed CB2R expression (Figure 1 and Supplemental Figure IIB). In addition, the ischemic core appeared infiltrated with neutrophils (NIMP-R14+ cells), many of them expressing CB2R (Figure 1 and Supplemental Figure IIB). In contrast, we could not detect a significant CB2R expression in brain sections from sham-operated animals (Supplemental Figure IIA).
Effects of the CB2R Agonist JWH-133 on Infarct Outcome
Administration of the CB2R agonist JWH-133 (1.5 mg/kg) 10 minutes after pMCAO caused a decrease in the infarct size determined 48 hours after the ischemic injury when compared with the vehicle group (n=12; P<0.05). Lower and higher doses of the agonist (0.5 mg/kg and 5 mg/kg) did not significantly affect infarct volume (P>0.05;Figure 2A). Similarly, animals treated with the CB2R agonist (1.5 mg/kg) had a lower score in the modified Neurological Severity Score (mNSS) 48 hours after the pMCAO (Figure 2D). The effect of JWH-133 on infarct volume was reversed in a dose-dependent manner by the administration of the CB2R selective antagonist SR144528 (3–5 mg/kg;Figure 2B,D). Likewise, the administration of JWH-133 (1.5 mg/kg), 3 hours after pMCAO, also decreased the infarct volume compared with the vehicle-treated group (Figure 2C).
To test further the selectivity of JWH-133 on CB2R, a group of CB2R-KO mice (n=12) were subjected to pMCAO and treated either with vehicle or the CB2R agonist. Our results show that there are no differences in infarct outcome when wild-type and CB2R-KO mice are compared. Furthermore, the CB2R agonist did not affect either infarct size or neurological impairment in the CB2R-KO mice (Figure 2B, D).
Effects of JWH-133 on Gene and Protein Expression of Inflammatory Molecules
Exposure to pMCAO caused an increase in mRNA expression of the proinflammatory cytokines and chemokines IL-1β, IL-6, MCP-1, MIP-1α, RANTES, and IL-12/IL-23p40 as well as expression of the inflammatory enzymes myeloperoxidase (MPO), iNOS, and COX-2 in peri-infarct tissue 15 hours after the insult (Figure 3). Treatment with the CB2R agonist significantly decreased MCAO-induced mRNA expression of IL-6, IL-12/IL-23p40, MCP-1, MIP-1α, RANTES, and iNOS. No significant differences were observed in TNF-α, IL-1β, and MPO mRNA expression between the groups at the time studied (Figure 3).
Ischemia also increased brain protein expression of MCP-1, IL-6, TNF-α, MIP-1α, RANTES, IL-12/IL-23p40, and iNOS, but not of COX-2, in the cortical peri-infarct brain tissue of vehicle-treated mice 24 hours after pMCAO (Figure 4). Treatment with the CB2R agonist JWH-133 significantly reduced iNOS, IL-6, TNF-α, MIP-1α, and RANTES, but not IL-12 or MCP-1 protein expression 24 hours after the ischemic insult (Figure 4). Protein expression of IL-1β was not detectable with the assay used.
Effects of JWH-133 on the Gene Expression of Alternative Microglia/Macrophage Phenotype Markers
Ischemia induced mRNA expression of the alternative microglia/macrophage phenotype markers TGF-β, Ym1, and IL-10 (but not of arginase I and IL-4) in samples obtained from the peri-infarct area 15 hours after pMCAO. This effect was decreased by the CB2R agonist JWH-133 (Figure 5).
Effects of JWH-133 on Microglial Activation in the Ischemic Boundary
Ischemia induced the activation of microglia/macrophages in the injured cortex, as demonstrated by a slight hypertrophy of the processes of Iba1-positive cells and an increased intensity of Iba1 expression in the peri-infarct microglia. Moreover, the group treated with the CB2R agonist had a lower expression of Iba1 in the ischemic boundary when compared with the vehicle-treated group (Figure 6A–B).
Effects of JWH-133 on Peripheral Blood Cells and on Neutrophil Infiltration
Ischemia did not change the percentage of neutrophils, monocytes, and lymphocytes in peripheral blood 4 hours after pMCAO compared with the sham control group. Treatment with JWH-133 (1.5 mg/kg) did not alter the number of blood immune cells at the time observed (Supplemental Figure IV).
Similarly, treatment with JWH-133 (1.5 mg/kg) did not affect the number of neutrophils (NIMP-R14+ cells) infiltrated into the ischemic core 24 hours after MCAO (Supplemental Figure V).
In this study, we have explored the effects of CB2R activation in experimental stroke by using a permanent model of focal ischemia in mice. Our data show that a single administration of a CB2R receptor agonist after the onset of ischemic injury improves stroke outcome concomitant to reduced classic and alternative microglial activation in the injured cortex.
Indeed, our experiments show that a single acute dose of the CB2R selective agonist JWH-133 administered after permanent focal ischemia (10 minutes or 3 hours) is neuroprotective by reducing infarct volume and improving neurological outcome. This effect was reversed by the CB2R selective antagonist SR144528 and was absent in CB2R-KO mice, thus demonstrating that JWH-133-induced neuroprotection is selectively dependent on CB2R receptor activation. In this vein, the existing literature shows a neuroprotective effect in different protocols of administration of CB2R agonists in animal models of transient and permanent brain ischemia.17–20 Here we have used a model of permanent occlusion of the middle cerebral artery (MCA), the clinical relevance of which is supported by the fact that the only approved treatment for stroke to date is thrombolysis with recombinant tissue-type plasminogen activator23; this is available only to 2% to 5% of the patients and only 32% of those patients undergo a complete recanalization.24 Thus, stroke models based on permanent vessel occlusions are the ones that more closely represent the human patient situation, at least in the first 24 hours after stroke onset.25,26 Furthermore, our data demonstrate a neuroprotective effect of a CB2R agonist administered postischemia (10 minutes or 3 hours), suggesting its utility in the clinical situation.
As shown in previous studies,17,20 the neuroprotective effect of the CB2R agonist was lost at the highest doses used. Although the exact mechanisms of the bell-shaped dose-response curves remain to be elucidated, it could be speculated that interactions with other cannabinoid receptors might be involved.17,20,27,28
As previously reported,17 lack of CB2R expression did not affect infarct outcome. It has been reported that no significant changes in N-arachidonoylethanolamine, one of the main endogenous cannabinoid agonists, was seen after 4, 12, or 24 hours of pMCAO in mice (reviewed in6). As to the other major endocannabinoid, 2-arachidonoylglycerol, its content did increase in the ipsilateral hemisphere after 4, but not after 12 or 24 hours of MCAO. Then, it is plausible that a poor endogenous CB2R activation caused by low endocannabinoid production might explain why CB2R deletion does not correlate with a clear effect in neuroprotection.
Our results illustrate the time-expression profile of cannabinoid receptors in mouse brains after pMCAO injury, showing an increased expression of CB2R that corroborates the presence of the drug target. A previous study reported that CB1R and CB2R are induced 6 and 24 hours, respectively, after an ischemia/reperfusion model in mice.19 In this context, we show that CB2R mRNA expression occurs earlier (18 hours after pMCAO) after a permanent occlusion in mice. Our data are consistent with several reports that demonstrate a brain upregulation of CB2R in animal models of different neurological diseases in which inflammation is involved.29 Conversely, CB1R is transiently downregulated, possibly because of neuronal damage, but levels are subsequently recovered, an effect that might be caused by increased CB1R expression in astrocytes because of gliosis and/or in surviving neurons in the ischemic boundary (additional details are discussed in Supplemental Discussion).
Regarding the mechanisms involved in the neuroprotective effect of CB2R, we have found that JWH-133 decreases activation of microglia after pMCAO, as shown by the reduced expression of Iba1 and the predominance of a resting morphology in microglial cells located within the ischemic boundary.30,31 This is in agreement with previous reports,29,32 pointing to a central inactivation of microglia/macrophage cells30 as the main mechanism by which CB2R mediates protection after stroke. Because we have shown that CB2R expression is induced in reactive microglia after pMCAO, downregulation of ischemia-induced CB2R expression after JWH-133 treatment further supports lower microglial activation. In addition, our results showed that disperse astrocytes at the corpus callosum displayed immunoreactivity for CB2R, the activation of which might also be involved in JWH-133-induced neuroprotection.
Focal ischemia induced mRNA and/or protein expression in brain parenchyma of several proinflammatory and inflammatory mediators, which include IL-1β, IL6, iNOS, COX-2, RANTES, MCP-1, MIP-1α, TNF-α, IL-12/IL-23p40, and MPO, consistent with neural injury, macrophage/microglial classic activation, and infiltration of immune cells (lymphocytes, neutrophils, macrophages); these are events that have detrimental results in the acute phase of stroke.15,16,33 Activation of CB2R decreased this effect, as demonstrated in other settings of central nervous system injury.34,35 This effect differed among the mediators studied, a fact likely caused by the specific time profiles of induction of each molecule following pMCAO. A recent study in mice subjected to permanent ischemia (MCA electrocoagulation) did not find any effect of JWH-133 on TNF-α, IL-6, and CXCL2 mRNA cortical expression, thus claiming a peripheral effect of the CB2R agonist because of reduced neutrophil infiltration.17 Our results are the first to demonstrate that a unique dose of JWH-133 (1.5 mg/kg), administered after the onset of ischemic injury, diminishes the expression of proinflammatory molecules in the cortical peri-infarct tissue. Moreover, we did not observe any effect of the treatment with JWH-133 either on the gene expression of the neutrophil marker MPO 15 hours after pMCAO, or in the number of neutrophils present in the ischemic core 24 hours after the ischemic occlusion. The apparent controversy with the aforementioned study could be explained by differences in the ischemia models, drug administration protocols, and doses used in the 2 studies. Whereas the acute inflammatory response is associated with molecules that contribute to the demise of neurons in the penumbra, inflammation may be also instrumental for lesion containment and repair (for review, see36,37). In this context, additional studies are needed to elucidate the role of CB2R-induced modulation of inflammation in the later, chronic phase of stroke.
As commented on above, these inflammatory mediators are characteristic features of the classic activation of macrophages/microglial cells (reviewed in38,39). This classic, or “M1,” phenotype arises as a rapid response to tissue injury and is associated with expression of molecules that will contribute to the demise of neurons in the penumbra; therefore, this inflammatory response is detrimental to neurological outcome. Consequently, its inhibition by CB2R activation is likely to contribute to the neuroprotective effect reported here. Interestingly, microglial cells and macrophages are able to acquire other states to arrest the killing phase and to restore tissue homeostasis. In this context, the switch to an alternative phenotype, characterized by anti-inflammatory and resolutive mediators, provides the scenario for repair and tissue reconstruction. Mirroring the Th1/Th2 nomenclature of T helper cells, the term “M2” was proposed, given that its polarization is caused by anti-inflammatory mediators such as Th2-derived IL-4 and IL-13 (toward M2a, alternative, or wound-healing macrophages/microglia), or Treg-derived IL-10 and TGF-β (toward M2c, regulatory, or deactivated macrophages/microglia). A third subgroup, M2b, includes regulatory macrophages activated by immune complexes+Toll-like receptors/IL-1R ligands (reviewed in38–41). Because the CB2R receptor regulates the balance of Th1-proinflammatory to Th2-anti-inflammatory cytokines,11 as well as enhances the expression of IL-10 in vitro,32,42 we decided to explore the effects of JWH-133 on anti-inflammatory and M2 markers. Interestingly, our data show that brain ischemia causes the expression of the M2-polarizing cytokines TGF-β and IL-10, which are in turn required for M2c activation, and also of Ym1, a secretory chitinase-like protein marker of the M2a state38,41,43; this is consistent with the coexistence of different subsets of macrophage/microglial cells in the ipsilesional hemisphere and suggests an endogenous protective response. Surprisingly, acute administration of the CB2R agonist decreased pMCAO-induced expression of these mediators at the time studied, 15 hours after the ischemic insult. To our knowledge, this is the first report of the induction of the M2a or alternative marker Ym1 in experimental stroke, and the inhibitory effect of CB2R activation on anti-inflammatory mediators in this setting. It is known that IL-10 and TGF-β are induced in focal brain ischemia, where they mediate neuroprotection.16,44 The reason for which CB2R causes the downregulation of these molecules deserves additional study, but it might be caused by an action on microglia toward an inactivated, quiescent state; this is in agreement with the immunofluorescence studies showing decreased microglial activation, with features close to the cells present in the contralesional healthy hemisphere. Our results open an interesting line for future studies aimed at the study of the role of CB2R activation on microglial cell phenotypes in the chronic phase of ischemia, and specifically on its involvement on neurorepair processes at this late stage.
In summary, the results of the present study demonstrate the important role of the CB2R receptor in neuroprotection after focal brain ischemia, which appears to be caused by central microglia/macrophage inactivation and subsequently an anti-inflammatory mechanism with the inhibition of TNF-α, IL-6, IL-12/IL-23p40, MCP-1, MIP-1α, and RANTES mediated by the CB2R receptor. Finally, the effects of CB2R activation on the expression of both mediators or markers of alternatively activated macrophages indicate a general inactivation of different subpopulations of microglia/macrophages.
Sources of Funding
This work was supported by grants from Spanish Ministry of Science and Innovation (MICINN) SAF2009-08145 (M.A.M.), SAF2011-23354 (I.L.), SAF 2008-01106 (J.M.), and from both MICINN and Fondo Europeo de Desarrollo Regional (FEDER) grants Consolider CSD2010-00045 (M.A.M.) and Red Neurovascular (RENEVAS) RD06/0026/0005 (I.L.), RD06/0026/0001 (M.T.) and RD06/0026/0006 (M.C.B.). J.G.Z. was supported by the Programme Alban, scholarship No. E07D400805CO. M.S.G.-G. is a predoctoral fellow of the Ministry of Science and Innovation. M.C.B. was supported by a postdoctoral grant from the Spanish Ministry of Health CD07/00236.
The online-only Data Supplement is available at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.111.631044/-/DC1.
- Received June 29, 2011.
- Accepted August 23, 2011.
- © 2012 American Heart Association, Inc.
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