The cannabinoid receptor 2 is involved in acute rejection of cardiac allografts

Life Sciences 138 (2015) 29–34

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The cannabinoid receptor 2 is involved in acute rejection of cardiac allografts Andrea M. Kemter a,1, Stefanie Scheu b,1, Norbert Hüser c,1, Christina Ruland d, Beatrix Schumak e, Matthias Findeiß a, Zhangjun Cheng f, Volker Assfalg c, Volker Arolt d, Andreas Zimmer a, Judith Alferink a,d,g,⁎ a

Institute of Molecular Psychiatry, University of Bonn, Bonn, Germany Institute of Medical Microbiology and Hospital Hygiene, University of Düsseldorf, Düsseldorf, Germany Department of Surgery, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany d Department of Psychiatry and Psychotherapy, University of Münster, Münster, Germany e Institute of Medical Microbiology, Immunology and Parasitology, University of Bonn, Bonn, Germany f Department of General Surgery, The Affiliated Zhongda Hospital, Southeast University, Nanjing 210009, China g Cells-in-Motion Cluster of Excellence EXC 1003, University of Münster, Münster , Germany b c

a r t i c l e

i n f o

Article history: Received 30 September 2014 Accepted 12 February 2015 Available online 2 March 2015 Keywords: Endocannabinoids Cannabinoid receptor 2 Solid organ transplantation Cardiac allografts Dendritic cells TH1 and TH17 cells Graft rejection

a b s t r a c t Aims: Acute rejection of cardiac allografts is a major risk factor limiting survival of heart transplant recipients. Rejection is triggered by dendritic cell (DC) mediated activation of host T cells, amongst others CD4+ T helper (TH)1- and TH17 cells. The cannabinoid receptor 2 (CB2) is an important modulator of cellular immune responses. However, its role in cardiac allograft rejection has not been studied so far. Main methods: Here, we examined the effect of CB2 on cytokine release by mature DCs and its impact on CD4+ T cell differentiation by utilizing in vitro generated bone marrow-derived DCs (BM-DCs) and CD4+ T cells from CB2 knockout (Cnr2−/−) mice. We further assessed the functional role of CB2 in acute allograft rejection using Cnr2−/− mice in a fully major histocompatibility complex-mismatched mouse cardiac transplantation model. Key findings: Cardiac allograft rejection was accelerated in Cnr2−/− mice compared to wild type recipients. In vitro stimulation of BM-DCs showed enhanced secretion of the pro-inflammatory cytokines interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF) and the immunomodulatory cytokine TGF-β. Furthermore, secretion of the TH 1/TH 17 promoting cytokines IL-12 and IL-23 was increased in Cnr2 −/− BM-DCs. In addition, Cnr2 −/− CD4+ T cells showed an enhanced capacity to differentiate into interferon (IFN)-γ- or IL-17-producing effector cells. Significance: These results demonstrate that CB2 modulates in vitro cytokine responses via DCs and directly via its influence on TH1/TH17 differentiation. These findings and the fact that allograft rejection is enhanced in Cnr2−/− mice suggest that CB2 may be a promising therapeutic target in organ transplantation. © 2015 Elsevier Inc. All rights reserved.

1. Introduction The transplantation of cardiac allografts is a therapeutic strategy in patients with end-stage heart failure. Acute rejection of allografts is a life-threatening complication and the primary cause of failure of transplantation [1]. Heterotopic cardiac transplantation in mice is a wellaccepted model to study immunological mechanisms of transplant rejection [2]. Pathogenesis of acute allograft rejection is characterized by a sequence of events that is triggered by emigration of donor-derived DCs out of the transplant leading to the induction of T cell-mediated

⁎ Corresponding author at: Department of Psychiatry and Psychotherapy, University of Münster, Albert Schweitzer Campus 1, 48159 Münster, Germany. Tel.: +49 251 8356630; fax: +49 251 8356612. E-mail address: [email protected] (J. Alferink). 1 Equal contribution.

http://dx.doi.org/10.1016/j.lfs.2015.02.012 0024-3205/© 2015 Elsevier Inc. All rights reserved.

alloreactive responses through presentation of alloantigens by donor and recipient DCs [3]. A number of cytokines have been shown to play a role in the acute rejection of solid organ grafts. For example, it has been suggested that cytokines of the TNF superfamily initiate and orchestrate the rejection response. Enhanced expression levels of TNF and Lymphotoxin α (LTα) in human cardiac allografts correlate with severity of rejection and deficiency or neutralization of these cytokines [4-6] or knock-out of their receptors attenuates rejection [7]. Another cytokine that affects graft rejection is IL-6, a key modulator of TH17 and regulatory T cell differentiation [8,9]. Neutralization of IL-6 decreases the infiltration of immune cells into the cardiac graft and thus delays the onset of acute rejection [10]. Similarly, neutralization of IL-12/23p40 attenuates acute cardiac allograft rejection [11]. IL-12 and IL-23 are primarily produced by DCs and regulate differentiation of CD4+ T cells into T H1 and maintenance of TH 17 cells, respectively [12]. Both T cell subsets have been

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implicated in acute allograft rejection together with their signature cytokines IFN-γ and IL-17 [13-15]. The endocannabinoid-system (eCBS) plays a key immunomodulatory role by signaling via CB2 [16]. The eCBS is comprised of lipid-signaling molecules (endocannabinoids) synthesized from membrane phospholipids that bind to and activate the G-protein coupled CB1 and CB2 [17]. In contrast to CB1, which is mainly expressed in the central nervous system (CNS), CB2 is predominantly expressed on immune cells and does not mediate the psychotropic effects of 9-tetrahydrocannabinol (9THC) binding to both CB1 and CB2 [18-20]. It was demonstrated that CB2 activation negatively regulates pro-inflammatory cytokine production but enhances IL-10 release in lipopolysaccharide (LPS)-activated macrophages [21,22]. It was also shown that it affects T helper cell differentiation, as treatment with a CB2 agonist ameliorates clinical severity in experimental autoimmune encephalomyelitis by inhibiting IFN-γ and IL17 production by CD4+ T cells [23]. Also, CB2 agonists were shown to downregulate in vitro differentiation of CD4 T cells into TH17 lymphocytes [24]. Thus, CB2 is an important modulator of DC and T cell-dependent responses involved in allograft rejection and therefore an attractive therapeutic target. However, the functional role of CB2 in acute allograft rejection has not been studied so far. 2. Material and methods 2.1. Mice CB2-deficient (Cnr2−/−, N10 backcross to C57BL/6) and wild type mice were bred locally [25]. DBA/2 (H-2d) female mice were purchased from Janvier (France) and/or bred locally. All animals were bred and housed in the Specific Pathogen Free (SPF) animal facility of the House for Experimental Therapy (University of Bonn, Germany) according to German guidelines for animal care. Ethical approval for the use of all mice in this study was obtained from the German government. 2.2. Heart transplantation Vascularized allogeneic BALB/c (H-2d) were transplanted heterotopically into C57BL/6 or C57BL/6 Cnr2−/− recipients (age 10–12 weeks) according to microsurgical techniques as previously described [2]. Briefly, the harvested donor hearts were stored in 4°C cardioplegic solution. Under isoflurane-induced anesthesia, the abdominal aorta and inferior vena cava were separated from surrounding fatty tissue. An end-to-side anastomosis of donor aorta and pulmonary artery to the host's infrarenal aorta and vena cava was performed using running 10–0 nylon suture. The abdominal wall was closed using 5–0 nylon suture in a running fashion. Graft function was monitored daily by abdominal palpation. Rejection was defined as complete cessation of cardiac muscle contraction, visually confirmed after laparotomy. 2.3. Generation of BM-DCs Bone marrow cells were cultured as described before [26]. In brief, bone marrow cells were cultured at 5 × 105 cells/ml in DMEM supplemented with 10% FCS (PromoCell), 1% MEM/NEAA, 1% Penicillin/ Streptavidin, 0.1% β-Mercaptoethanol (Invitrogen if not otherwise stated) and 10% conditioned medium of B16 cells. Medium was exchanged on day three and cells stimulated on day five with 100 ng/ml Escherichia coli Lipopolysaccharide (LPS) Serotype 0127:B8 (Sigma-Aldrich) or 1 nmol/ml CpG (TIB MOLBIOL). Supernatants were collected after 16 h of stimulation. 2.4. ELISA TNF, IL-6, IL-1β, IL-12 and IL-23 concentrations in supernatants of BM-DC stimulated for 16 h with LPS or CpG were analyzed using the

Ready-SET-Go! enzyme-linked immunosorbent assay (eBioscience) according to the manufacturer's recommendations. Free active TGF-β1 levels in supernatants of BM-DCs stimulated with LPS for 16 h were determined using the LEGEND MAX™ Free Active TGF-β1 ELISA Kit (Biolegend) as prespecified by the manufacturer. Plates were read at 450 nm using the Dynex Technologies MRX TC II reader. 2.5. CD4 TH differentiation CD4 TH differentiation was performed as described before [27]. Briefly, CD4+ T cells were isolated by immunomagnetic separation using CD4-MACS beads (Miltenyi Biotec) and stimulated with plate-bound 4 μg/ml CD3 antibody (145-2C11) and 4 μg/ml CD28 antibody (3751) together with 5 ng/ml TGF-β and 20 ng/ml IL-6 (PeproTech) for Th17 differentiation or with IL-12 (10 ng/ml) for Th1. 2.6. Flow cytometry Fluorescence staining was performed using the following FITC, PE or APC labeled or biotinylated antibodies purchased from BD Biosciences: anti-CD4, anti-IFN-γ and anti-IL-17 with SA-PerCP-Cy5.5 as appropriate. Intracellular cytokine production was measured according to the manufacturer's instructions (BD Biosciences). Fluorescence was analyzed using a FACSCalibur™ flow cytometer and CELLQuest™ software (both Becton Dickinson). 2.7. Statistical analyses Cardiac survival of donor hearts in the animal groups was compared using a two-sided logrank test at a 0.05 level of significance. Due to the skewed distributions of organ rejection, the median event-free interval was reported with 95% confidence interval. Kaplan–Meier curves are provided to depict differences in event times between the groups. In all other experiments, statistical significance was determined using Prism 4 (GraphPad Software, La Jolla, CA, USA) by unpaired Student's t-test. A P value of b0.05 was considered significant. 3. Results 3.1. Enhanced release of TNF, IL-6, IL-1β and TGF-β by TLR-ligand stimulated Cnr2−/− BM-DCs DCs are key cellular mediators of allograft rejection by producing cytokines such as IL-1β, IL-6 and TNF that play decisive roles in allograft rejection [28,9]. We therefore studied whether CB2 modulates cytokine release by DCs. Bone marrow derived (BM)-DCs were generated from Cnr2−/− and WT mice using granulocyte–macrophage colonystimulating factor (GM-CSF) followed by stimulation with Toll-like receptor (TLR) ligands such as the Gram-negative bacterial compound LPS and the immunostimulatory CpG oligodeoxynucleotide 1668 (CpG-ODN). Increased levels of TNF were detected in the supernatants of Cnr2−/− DCs upon stimulation with LPS and CpG when compared to WT DCs (Fig. 1A). In addition, IL-6 levels were enhanced in the supernatants of LPS and CpG stimulated Cnr2−/− DC cultures. Furthermore, mildly but significantly higher IL-1β levels were detected in CpG stimulated DC cultures in the absence of CB2. In conjunction with IL-6 the cytokine transforming growth factor (TGF)-β may drive generation and differentiation of TH17 cells, a CD4+ TH cell subset involved in transplant rejection [3]. We therefore measured active TGF-β1 levels in supernatants of BM-DCs from WT and Cnr2−/− mice after stimulation with LPS. We found enhanced levels of active TGF-β in the Cnr2−/− DC cultures when compared to WT controls (Fig. 1B). Thus, secretion of proinflammatory cytokines and TGF-β by DCs in response to TLR ligands is enhanced in the absence of CB2.

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A

B

Fig. 1. Enhanced release of TNF, IL-6, IL-1β and TGF-β by Cnr2−/− BM-DCs stimulated with TLR-ligands. (A) BM-DCs were incubated for 16 h with LPS or CpG. Supernatants of BM-DC cultures were harvested and TNF, IL-6 and IL-1β quantified by ELISA. (B) BM-DCs were incubated for 16 h with LPS. Supernatants of BM-DC cultures were harvested and TGF-β quantified by ELISA. Results show mean ± S.E.M. Representative data of two experiments (n = 5 mice per group). *: p b 0.05, **: p b 0.01.

3.2. Enhanced secretion of IL-12 and IL-23 by TLR-ligand stimulated Cnr2−/ − BM-DCs In addition to secretion of pro-inflammatory cytokines, DCs are the major sources of IL-12 and IL-23 [29]. Neutralization of their shared subunit IL-12/23p40 has been shown to inhibit acute cardiac allograft rejection [11]. To test whether secretion of IL-12 or IL-23 production by DCs is modulated by CB2, supernatants from TLR ligand stimulated BM-DCs from WT and Cnr2−/− mice were analyzed by ELISA. Stimulation with LPS or CpG induces significantly increased secretion of IL-12 by Cnr2−/− BM-DCs compared to WT cells. Upon stimulation with CpG, IL-23 levels were slightly but significantly elevated in supernatants of Cnr2−/− BM-DCs compared to WT cells (Fig. 2). Thus, CB2 deficiency in DCs results in an enhanced secretion of IL-12 and IL-23 by DCs. 3.3. TH1 and TH17 differentiation is enhanced in Cnr2−/− T cells Both IL-12 and IL-23 are known to modulate the differentiation of CD4+ T cells into two of the main subsets implicated in acute allograft rejection. While IL-12 has been shown to drive differentiation into TH1 cells [30], IL-23 is required for the expansion and survival of TH17 cells [31]. To investigate whether CB2 deficiency has an impact on the differentiation of CD4+ T cells into TH1 or TH17 cells, naïve T cells were isolated from WT and Cnr2−/− mice and subjected to TH1 (IL-12) or TH17 (TGF-β and IL-6) differentiation conditions. Differentiation of CD4 T cells was assessed by intracellular stainings for IFN-γ or IL-17. The

percentage of IFN-γ+ CD4+ TH1 cells was significantly increased in Cnr2−/− compared to WT cultures. Similarly, upon differentiation with TGF-β and IL-6, a higher percentage of Cnr2−/− than WT CD4+ T cells secreted IL-17. In conclusion, CB2 deficiency enhances differentiation efficiency of naïve CD4+ T cells into TH1 or TH17 cells (Fig. 3). These data indicate that CB2 modulates cytokine release by DCs and T cell differentiation, both critical immune functions involved in acute graft rejection [32−34].

3.4. Allogeneic cardiac graft rejection is enhanced in Cnr2−/− mice We next studied the impact of CB2 in acute cardiac graft rejection in vivo in a model of heterotopic heart transplantation. For this, fully mismatched BALB/c allogeneic cardiac grafts (H2d) were heterotopically transplanted into Cnr2−/− mice and WT controls (both H2b). Graft survival was monitored daily by abdominal palpation of donor hearts and considered rejected when contractions ceased as confirmed by laparotomy. Indefinite survival of cardiac grafts was seen in a syngeneic donor/ recipient strain combination serving as a control. Allogeneic grafts transplanted into WT recipients were acutely rejected at day 8 (95% CI: 7.63 to 8.65 days) after transplantation (Fig. 4) as previously shown [26]. In contrast, allograft survival in Cnr2−/− recipient mice was significantly reduced with cardiac grafts being rejected at day 6 (95% CI: 5.48 to 6.81 days; P-logrank test = 0.001). These data demonstrate that CB2 plays a role in allograft rejection and therefore may

Fig. 2. Enhanced secretion of IL-12 and IL-23 by TLR-ligand stimulated Cnr2−/− BM-DCs. BM-DCs from Cnr2−/− or WT mice were stimulated with LPS or CpG for 16 h and cytokine production was determined by ELISA. Diagrams show mean ± S.E.M. Representative data of two experiments (n = 5 mice per group). *: p b 0.05, **: p b 0.01.

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Fig. 3. TH1 and TH17 differentiation is enhanced in Cnr2−/− T cells. Purified Cnr2−/− or WT CD4+ T cells were subjected to TH1 or TH17 differentiation conditions and cytokine-producing CD4+ T cells were determined by flow cytometry. Diagrams show mean percentages ± S.E.M of cytokine producing cells. One representative of two experiments is shown (n = 5 mice per group). *: p b 0.05; **: p b 0.01.

represent a modulator of immune reactions after solid organ transplantation. 4. Discussion Cardiac allograft transplantation triggers immune responses that may lead to acute graft rejection even under sustained immunosuppressive therapy [1]. Cannabinoids have been discussed as potentially promising therapeutics in solid organ transplantation based on multiple studies documenting their immunosuppressive effects through signaling via CB2 [35]. In vitro studies utilizing the mixed lymphocyte reaction [36] pointed to a potential role of the eCBS in graft rejection and led to the implication of cannabinoids as potential therapeutics in transplantation medicine. However, in vivo data confirming these findings have been missing up to now. Here we show for the

WT (n=7)

% Survival of Heart Allografts

100

Cnr2 -/- (n=7) 75 50 25 0 0

2

4

6

8

10

Days after HTx Fig. 4. Decreased survival of heart allografts in the absence of CB2. Cnr2−/− or WT mice were transplanted with a second allogeneic heart and survival of transplants was monitored daily. Survival curves of heart transplants are shown (n = 7 mice per group; p = 0.0015).

first time in an in vivo model of cardiac allograft rejection using Cnr2 −/− mice that CB2 is an important regulator of immune responses in solid organ transplantation. Transplant rejection involves a complex cascade of immune responses of the recipient and the donor organ leading to the destruction of donor cells and the rejection of the graft. Donor- as well as hostderived DCs are essential inducers of immune responses to solid organ allografts [33]. Cytokines secreted by DCs play a central role in T cell activation and subsequent differentiation [37]. Here we found that upon TLR stimulation BM-DCs generated from Cnr2−/− mice produced higher levels of the pro-inflammatory cytokines TNF, IL6 and IL-1β when compared to WT BM-DCs. Several studies suggest an involvement of these cytokines in allograft rejection. Thus, TNF and IL-1β protein levels are elevated in acutely rejected heart transplants while survival of the graft is increased upon neutralization of these cytokines or in mice deficient in TNF receptors [4,7,28,38]. It was further demonstrated that elevated levels of IL-6 protein in graft biopsies as well as in the serum of patients correlated with severity of rejection [9,39]. Using IL-6 deficient mice, it was demonstrated that IL-6 production by the host, rather than grafts, is necessary for allograft rejection [10]. We further demonstrated that Cnr2−/− BM-DCs secreted enhanced levels of active TGF-β upon LPS challenge. Multiple studies suggest an anti-inflammatory role of TGF-β and beneficial effects of this cytokine in transplantation tolerance by induction of regulatory CD4+ T cells [40]. However, TGF-β together with IL-6 further plays an essential role in the differentiation of CD4+ T cells into pathogenic TH17 cells, a phenotype that is stabilized by IL-23 [3,29]. IL-12, which shares a subunit with IL-23, induces TH1 differentiation [12]. Both TH1 and TH17 cells have been implicated in graft rejection and neutralization of the shared IL12/23p40 subunit resulted in prolonged graft survival in an allograft model [11]. Based on our finding that IL-6 and TGF-β secretion are elevated in Cnr2−/− BM-DCs, we investigated if CB2 deficiency leads to a more generalized increase in cytokine production by DCs creating a milieu that favors differentiation of

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CD4+ T cells into pathogenic subsets. After stimulation with LPS and CpG, Cnr2−/− BM-DCs showed significantly increased production of IL-12 and IL-23. These findings show similarities with earlier studies showing a CB2 dependent inhibition of IL-12 production in Legionella pneumophila infected DCs by 9-THC [41-43]. Furthermore, treatment of human DCs with the endocannabinoid anandamide or a synthetic CB2 agonist inhibited the secretion of IL-6, IL-12 and TNF after stimulation with the TLR7 ligand R848. This could be reversed by treatment with a CB2 antagonist [44]. It is well established that markers for the pathogenic CD4+ TH1 and TH17 subsets are enriched in rejected grafts [13,15,45,46]. It was therefore of interest to investigate whether CB2 deficiency directly influences the differentiation of CD4+ T cells. Here, we found that stimulation of naïve CD4+ T cells from WT and Cnr2−/− mice in the presence of IL-12 resulted in a higher percentage of Cnr2−/− T cells producing IFNγ, indicating a higher potential to differentiate to TH1 cells. Similarly, if stimulated in the presence of IL-6 and TGF-β, Cnr2−/− T cells demonstrated a higher potential to differentiate into TH17 cells, indicated by an increased percentage of IL-17+ cells. This led to the conclusion that in addition to Cnr2−/− DCs inducing a shift towards TH1 and TH17 responses by secreting increased amounts of the appropriate cytokines, T cells from Cnr2−/− mice also have an inherently higher potential to differentiate into these subsets. In line with our findings on the direct impact of CB2 on the differentiation of pathogenic CD4+ TH subsets, earlier studies indicated that pharmacological CB2 activation reduces TH17 differentiation. In models for delayed type hypersensitivity and multiple sclerosis, treatment with cannabinoids reduce the secretion of IFN-γ and IL-17 by CD4+ T cells and ameliorate disease [23,47,48]. Furthermore, Guillot et al. have shown that Cnr2−/− mice express elevated levels of IL-17 and TH17 markers after bile duct ligation, conversely treatment of WT mice with a CB2 agonist inhibited differentiation of CD4+ T cells into TH17 cells [24]. Taken together we show here that cells crucial for both the induction phase as well as the effector phase of the immune response against the graft display functional differences in Cnr2−/− mice compared to WT mice. We therefore hypothesize that signaling through CB2 in recipient DCs dampens their production of pro-inflammatory as well as TH1 and TH17 inducing cytokines and thereby limits the activation of the adaptive immune response against the transplant. In addition, signaling via CB2 on naïve CD4+ T cells directly limits their potential to differentiate towards TH1 and TH17 cells. There have been numerous suggestions on how these two subsets contribute to transplant rejection, including activation of alloreactive B cells and recruitment of further graft damaging cells into the transplanted organ [3]. Elucidating the exact mechanism of CB2 mediated allograft protection requires further studies including the investigation of the role of CB2 in chronic rejection models. This identifies CB2 as a potential target for new treatment regimes to decrease the risk of graft rejection and the improvement of survival of transplant recipients.

5. Conclusions In this study, we show that CB2 is functionally involved in allograft rejection. Our findings demonstrate a reduced survival of fully allogeneic cardiac grafts transplanted into Cnr2−/− mice. We further show that CB2 modulates immune responses that play a critical role in allograft rejection such as the secretion of proinflammatory and TH1/TH17promoting cytokines by DCs and CD4+ T cell differentiation. Targeting CB2 may therefore be an effective immunomodulatory therapy in solid organ transplantation and a promising therapeutic approach for prevention of allograft rejection.

Conflict of interest statement The authors declare no conflict of interest.

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Acknowledgments We thank K. Poppensieker and Özlem Mutluer for excellent technical assistance. Funding: This study was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft [DFG], FOR926) to J.A. and A.Z.; DFG EXC 1003, Grant FF-2014-01 Cells in Motion– Cluster of Excellence, Münster, Germany to J.A.; A.Z. and B.S. are members of the DFG Cluster of Excellence ImmunoSensation (EXC 1023); Grants 2012-1-22, 2013-1-29 (BONFOR intramural funding scheme of the Medical Faculty at Bonn University) to B.S.; DFG SCHE692/3-1, SCHE692/4-1 to S.SCH.; Strategic Research Fund of the Heinrich Heine University Düsseldorf (SFF-F2012/79-5-Scheu) to S.SCH.; Grant IDL121204 (The Intramural Münster IMF funding) to J.A.

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