Dexmedetomidine, an Alpha 2a Adrenergic Receptor Agonist

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Dexmedetomidine, an alpha 2a adrenergic receptor agonist, mitigates experimental autoimmune encephalomyelitis by desensitization of CXCR7 in microglia Yi Huang, Shite Hu, Yongliang Li, Dan Xue, and Xiuguo Wu Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00430 • Publication Date (Web): 13 Jun 2018 Downloaded from http://pubs.acs.org on June 14, 2018

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Biochemistry

Dexmedetomidine, an alpha 2a adrenergic receptor agonist, mitigates experimental autoimmune encephalomyelitis by desensitization of CXCR7 in microglia

Yi Huang a, Shite Hu a,*, Yongliang Li a, Dan Xue b, Xiuguo Wu a

a

Department of Anesthesiology, The Third Affiliated Hospital of Wenzhou Medical University (Ruian People’s Hospital), Ruian, Zhejiang 325200, China

b

Department of Plastic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310000, China

*

Corresponding author: Shite Hu, TEL: 86-577-58815881. FAX: 86-577-65866586. E-mail: [email protected].

Key Words CXCR7; Desensitization; Microglia; Experimentally-induced autoimmune encephalomyelitis (EAE); Dexmedetomidine, Protein kinase C (PKC)

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Abstract In the autoimmune disease multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), characterized by an ascending paralysis that is characterized by extensive infiltration of the central nervous system by inflammatory cells. Although several studies to some extent uncover the cellular mechanisms of microglia that govern EAE pathogenesis, the molecular mechanisms that orchestrate microglia movement remain unknown and potential novel therapeutic strategies are still required. In the current study, we report that dexmedetomidine, an alpha 2a adrenergic receptor agonist, attenuates clinical severity of EAE with less infiltration of microglia. During EAE, dexmedetomidine inhibits SDF-1 and I-TAC-induced chemotaxis of microglia mediated by CXCR7 but not CXCR4 or CXCR3. Most importantly, Alpha 2a adrenergic receptor is essential in dexmedetomidine-induced CXCR7 desensitization in microglia. Further experiments confirmed that CXCR7 desensitization required atypical PKCζ activation, while conventional and novel PKC isoforms were not involved. Altogether our data elucidate the mechanism of dexmedetomidine-induced CXCR7 desensitization in microglia and amelioration in EAE, that may lead to a better understanding of the therapeutic effects of dexmedetomidine as well as the its implication of CXCR7 desensitization in autoimmune disease.


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1. Introduction Multiple sclerosis is an inflammatory disorder of the central nervous system (CNS) characterized by demyelination and axonal damage. It is estimated to affect up to two million people worldwide 1. Experimental autoimmune encephalomyelitis (EAE) is a most commonly used animal model for multiple sclerosis, classically manifests as an ascending paralysis that is characterized by extensive inflammatory cells infiltration in CNS 2. Among kinds of inflammatory cells, microglia are the brain-resident immune cells and represent about 10% of the total brain cell population 3. Accumulative evidence indicates that activated microglia predominate in demyelinated areas and their numbers correlate to tissue lesions of MS brain 4-6

. Several studies reported that the activation and movement of microglia are crucial during

EAE 7, however, the chemoattractant molecules that orchestrate microglia movement remain ill defined. Knowledge of these molecules and mechanism is of considerable interest for the development of therapies that would limit the development of inflammatory infiltrates in patients with MS. CXCR7 (also known as RDC-1) belongs to the superfamily of G-protein-coupled receptor (GPCR). Loss of function of one GPCR is often via desensitization, which can be considered as the loss of response subsequent to prolonged or repeated exposure of an agonist [6]. There are two types of GPCR desensitization. Firstly, homologous desensitization is always including adaptive changes at the level of the GPCR itself, phosphorylated by G protein– coupled receptor kinases (GRK) and subsequent binding of β-arrestins. Secondly, heterologous desensitization is due to phosphorylation of the receptor by second messenger– dependent protein kinases, such as PKA and PKC 8. Interestingly, conflicting data raise the


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debate whether CXCR7 acts as a signaling or non-signaling “decoy” receptor 9. On one hand, CXCR7 was reported to be a decoy receptor and functions as a scavenger to remove extracellular SDF-1, indirectly regulating other chemokine receptors signaling

9-11

. On the

other, CXCR7 was shown to mediate SDF-1-mediated neuron or microglia migration 6, 9, 12, 13. Until now, most studies focus on the decoy role of CXCR7. To the best of our knowledge, only one paper has demonstrated that CXCR7 modulates microglial chemotaxis during EAE 6. However, CXCR7-mediated chemotaxis and its precise regulation mechanism in microglia in the pathogenesis of EAE remain unclear. Dexmedetomidine is a selective and potent alpha 2a adrenergic receptor agonist, which was approved for sedation of intensive care units by the US Food and Drug Administration in 1999. Since then, a growing body of research papers have emerged reporting other possible applications, including use as a protective agent for ischemia/reperfusion injury in various organs 14-16. Recently, dexmedetomidine was shown to have additional neuroprotective effects and exert anti-inflammation role

17, 18

. More importantly, dexmedetomidine at the

concentration ranging from 10 to 100 ng/mL was demonstrated to be a potent suppressor of inflammatory stimuli in microglia of which activation and movement are crucial during EAE 19

. Therefore, it is tempting to hypothesize that the anti-inflammatory dexmedetomidine might

have a therapeutic role in EAE, with microglia involved in the pathogenesis of EAE. These intriguing results promoted us to explore that whether dexmedetomidine have a therapeutic role in EAE and its regulatory mechanism. In this study, we provide evidences revealing that dexmedetomidine mitigates EAE by modulating microglial chemotaxis through


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CXCR7 desensitization. Further, we uncovered the main signaling molecules and pathway in the process during CNS autoimmunity.


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2. Materials and methods 2.1. Animals, induction, treatment and clinical assessment of EAE C57BL/6 mice were purchased from Wenzhou Medical University Animal Facility. All mice were housed in a specific pathogen-free facility at the campus. All experimental procedures were conducted in conformity with institutional guidelines for the care and use of laboratory animals and obtained ethics approval from The Third Affiliated Hospital of Wenzhou Medical University. EAE was induced by MOG35-55 in female mice used between 8 and 10 weeks of age. Briefly, each mouse was immunized subcutaneously with 300 µg of MOG35-55 emulsified with an equal volume of complete Freund’s adjuvant (CFA, total 300 µg of Mycobacterium tuberculosis, strain H37RA, Difco) and then injected with 400 ng of pertussis toxin (Sigma) intraperitoneally at the time of immunization and 2 days later. Mice were weighed and examined for clinical scoring daily by the same investigator after immunization. For dexmedetomidine treatment, mice were divided randomly into five groups (n = 8 in each group) (a) sham; EAE induction treated with (b) saline control; (c) dexmedetomidine (Jiangsu

Hengrui Medicine) (5 µg/kg); (d) dexmedetomidine (10 µg/kg); (e) dexmedetomidine (20 µg/kg). Treatments began on the day of immunization, and were given every day until day 25. Saline control and dexmedetomidine were given by intraperitoneal (i.p.) injection. At the end of

the study, mice were sacrificed and isolated microglia in the equal amount of spinal cords were counted by chemiluminescence using the luciferase-containing reagent Cell-Titer Glo (Promega).


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Neurological assessments were reported using a five-point standardized rating scale to evaluate motor deficit: 0, no deficit; 1, tail paralysis; 2, incomplete hind limb paralysis; 3, complete hind limb paralysis; 4, complete hind limb paralysis and partial forelimb paralysis; 5, moribund state or death.

2.2. Cell preparation, culture and stimulation The spinal cords of normal or EAE mice were isolated, homogenized, filtrated, centrifuged, and suspended in 70% Percoll (GE Healthcare) and overlaid with 37 and 30% gradient. After centrifugation, the majority of mononuclear cells, found in the interface of 37 and 70% Percoll, were collected, and then were washed twice and resuspended in PBS containing 0.5% bovine serum albumin (BSA) (Sigma). Untouched primary cells were purified by positive selection using Microglia Isolation Kit (Miltenyi Biotec). Subsequently, the purity of isolated monocytes was above 95%, as confirmed by fluorescence-activated cell sorter analysis. Isolated microglia were cultured in microglial culture medium (Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum), penicillin (50 U/mL), streptomycin (50 µg/mL), sodium pyruvate (1 mM) and L-glutamine (2 mM). Cultures were maintained in an incubator at 37 ºC in an atmosphere of 5% CO2 and 95% air.

2.3. Real-time RT-PCR Total RNA was isolated using Trizol reagent according to the manufacturer's protocol, and 1 µg of total RNA was converted to cDNA by SuperScriptTM III First-Strand Synthesis System for RT-PCR (Invitrogen, Life Technologies). Brilliant SYBR-Green was combined with


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cDNA and corresponding primers (Table. S1) specific for indicated genes and real-time PCR amplification was performed using the ABI Prism 7000 (Applied Biosystems). Template cDNA was denatured at 95 ºC for 10 min followed by 40 cycles (denaturation for 1 min at 95 ºC, annealing for 1 min at 60 ºC, and extension for 2 min at 72 º C). The expression levels of target genes were measured using the comparative Ct method (ddCt) normalized against GAPDH.

2.4. Radioactive binding assay SDF-1 was labeled with [125I]. For competition binding assays, equivalent quantities of cell membrane extracts and [125I]-SDF-1 were incubated with varying quantities of unlabeled SDF-1 or dexmedetomidine. All reactions were incubated at 4˚C for 24 h. After the binding step, 25% polyethylene glycol (6000–8000) and 0.5% human-globulin were added, and the mixture was incubated at room temperature for 15 min. Binding complexes were obtained by centrifuging the mixture at 3500 rpm for 20 min at 4˚C, and radioactivity was evaluated. Results were corrected for “blank” back-ground radiation, defined as the radioactivity precipitated by polyethylene glycol-globulin from the same amount of radioactive ligand in binding buffer in the absence of cells.

2.5. Chemotaxis assay Chemotaxis assays were performed using 6.5-mm Transwell tissue culture inserts with a 5 µm pore size (Corning). Microglia were suspended at 5×106 cells/ml in RPMI 1640 with 0.1% BSA, and 100 µl of cell suspension was added to an insert in a well. The lower compartment


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was placed with 600 µL of medium containing indicated concentrations of SDF-1 or I-TAC, the plates were subsequently incubated for 150 min. Cells were fixed, and stained with the Three Step Stain Set (Richard-Allan Scientific). Microglia that had remained in the upper chamber were removed by wiping the filters with cotton swaps. Microglia found on the bottom of filters were counted as cells that had carried out chemotaxis. The migrant cells were counted in five randomly selected high-power fields (400×) per well. The chemotaxis index (CI) was calculated as the number of cells that migrated to the sample medium divided by the number of cells that migrated to the control medium. For the chemotaxis stimulation or inhibition assay, cells were pretreated with Clonidine (CR Double-Crane), LY294002, GF109203X, PKCζ pseudosubstrate inhibitors (Calbiochem), pertussis toxin, staurosporine (Cell Signaling), DMSO (dimethylsulfoxide), a neutralizing anti-CXCR7 antibody (R&D Systems, AF4227) or sheep IgG isotype control, and then loaded into the upper chamber.

2.6. Colorimetric PKCζ Kinase Assay PKCζ kinase activity was measured using HTScan PKCζ Kinase Assay Kit according to the manufacturer’s instructions (Cell Signaling). Briefly, cells under indicated pretreatments with

dexmedetomidine were prepared with the Kinase Lysis Buffer and were used as the source of enzymes for the analysis of PKCζ kinase activity. The reaction mixture was then added with the substrate peptide (1.5 µM) and ATP (200 µM) and incubated at room temperature for 30 min. The reaction was stopped with 50 mM EDTA (pH 8.0). Phosphorylation of the substrate peptide was quantified by colorimetric ELISA with Phospho-PKA Substrate antibody.


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2.7. Flow Cytometry Microglia were collected by gentle scrapping, washed with ice-cold phosphate buffered saline containing 1% BSA. The cells were then Fc-blocked by treatment with 1 µg of human IgG/105 cells for 30 min at 4 ºC. Next, the cells were incubated at 4 ºC for 30 min with antibodies against CXCR7 (Allophycocyanin (APC), clone 11G8, R&D systems), CXCR4 (APC, clone 247506, R&D systems), CXCR3 (APC, clone 220803, R&D systems), CD45 (APC, clone 30-F11, R&D systems) or alpha 2a adrenergic receptor (APC, ABIN2196814, antibodies-online) following the manufacturers’ instructions. Finally, the cells were washed and analyzed on a FACSCalibur flow cytometer (Becton Dickinson). Expression levels are referred to mean fluorescence intensity.

2.8. siRNAs transfection Murine CXCR7, CXCR4, CXCR3 and non-targeting (scrambled) siRNAs were obtained from Qiagen. Transfection of macrophage was performed using Transmessenger Transfection Reagent (Qiagen) as described by the manufacturer.

2.9. Western blot Whole-cell lysates were prepared and subjected to western blot analysis. Equal amounts of the cell lysates were resuspended in 5×Tris-glycine SDS sample buffer, electrophoresed on 12% SDS–PAGE, and transferred to nitrocellulose membranes (Bio-Rad). The detection of proteins was performed with anti-PKCζ antibody (1:1000), and anti-GAPDH antibody (1:3000) (Cell Signaling), followed by corresponding IDRy second antibody. All antibodies were from Cell Signaling Technology. The blots were scanned using an Odyssey Imaging


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Biochemistry

System (LI-COR Bioscience, USA).

2.10. Statistical analysis Data are provided as mean ± standard deviation values. Statistical differences between groups were analyzed by Student t-test and Mann Whitney-test and were considered significant when P

Dexmedetomidine, an Alpha 2a Adrenergic Receptor Agonist

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