Anandamide inhibits Theiler's virus induced VCAM-1 in brain endothelial cells and reduces leukocyte transmigration in a model of blood brain barrier by activation of CB1 receptors

<p>Abstract</p> <p>Background</p> <p>VCAM-1 represents one of the most important adhesion molecule involved in the transmigration of blood leukocytes across the blood-brain barrier (BBB) that is an essential step in the pathogenesis of MS. Several evidences have suggested the potential therapeutic value of cannabinoids (CBs) in the treatment of MS and their experimental models. However, the effects of endocannabinoids on VCAM-1 regulation are poorly understood. In the present study we investigated the effects of anandamide (AEA) in the regulation of VCAM-1 expression induced by Theiler's virus (TMEV) infection of brain endothelial cells using <it>in vitro </it>and <it>in vivo </it>approaches.</p> <p>Methods</p> <p>i) <it>in vitro</it>: VCAM-1 was measured by ELISA in supernatants of brain endothelial cells infected with TMEV and subjected to AEA and/or cannabinoid receptors antagonist treatment. To evaluate the functional effect of VCAM-1 modulation we developed a blood brain barrier model based on a system of astrocytes and brain endothelial cells co-culture. ii) <it>in vivo</it>: CB₁receptor deficient mice (Cnr1^-/-) infected with TMEV were treated with the AEA uptake inhibitor UCM-707 for three days. VCAM-1 expression and microglial reactivity were evaluated by immunohistochemistry.</p> <p>Results</p> <p>Anandamide-induced inhibition of VCAM-1 expression in brain endothelial cell cultures was mediated by activation of CB₁receptors. The study of leukocyte transmigration confirmed the functional relevance of VCAM-1 inhibition by AEA. <it>In vivo </it>approaches also showed that the inhibition of AEA uptake reduced the expression of brain VCAM-1 in response to TMEV infection. Although a decreased expression of VCAM-1 by UCM-707 was observed in both, wild type and CB₁receptor deficient mice (Cnr1^-/-), the magnitude of VCAM-1 inhibition was significantly higher in the wild type mice. Interestingly, Cnr1^-/-mice showed enhanced microglial reactivity and VCAM-1 expression following TMEV infection, indicating that the lack of CB₁receptor exacerbated neuroinflammation.</p> <p>Conclusions</p> <p>Our results suggest that CB₁receptor dependent VCAM-1 inhibition is a novel mechanism for AEA-reduced leukocyte transmigration and contribute to a better understanding of the mechanisms underlying the beneficial role of endocannabinoid system in the Theiler's virus model of MS.</p

Respuesta inmune innata en el SNC CD200-CD200R en un modelo viral de esclerosis múltiple

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, Departamento de Fisiología (Fisiología Animal II), leída el 25/10/2010.Depto. de Genética, Fisiología y MicrobiologíaFac. de Ciencias BiológicasTRUEProQuestpu

The endocannabinoid anandamide downregulates IL-23 and IL-12 subunits in a viral model of multiple sclerosis: Evidence for a cross-talk between IL-12p70/IL-23 axis and IL-10 in microglial cells

Theiler's virus (TMEV) infection of the central nervous system (CNS) induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis (MS). The endocannabinoid system represents a novel therapeutic target for autoimmune and chronic inflammatory diseases due to its anti-inflammatory properties by regulating cytokine network. IL-12p70 and IL-23 are functionally related heterodimeric cytokines that play a crucial role in the pathogenesis of MS. In the present study we showed that the endocannabinoid anandamide (AEA) downregulated the gene expression of IL-12p70 and IL-23 forming subunits mRNAs in the spinal cord of TMEV-infected mice and ameliorated motor disturbances. This was accompanied by significant decreases on the serological levels of IL-12p70/IL-23 and more interestingly, of IL-17A. In contrast, serum levels of IL-10 resulted elevated. In addition, we studied the signalling pathways involved in the regulation of IL-12p70/IL-23 and IL-10 expression in TMEV-infected microglia and addressed the possible interactions of AEA with these pathways. AEA acted through the ERK1/2 and JNK pathways to downregulate IL-12p70 and IL-23 while upregulating IL-10. These effects were partially mediated by CB2 receptor activation. We also described an autocrine circuit of cross-talk between IL-12p70/IL-23 and IL-10, since endogenously produced IL-10 negatively regulates IL-12p70 and IL-23 cytokines in TMEV-infected microglia. This suggests that by altering the cytokine network, AEA could indirectly modify the type of immune responses within the CNS. Accordingly, pharmacological modulation of endocannabinoids might be a useful tool for treating neuroinflammatory diseases. © 2011 Elsevier Inc.This work was supported by grants from the Spanish Ministerio de Educación y Ciencia [SAF 2007/60038]; the Comunidad Autónoma de Madrid (CAM) [S/SAL0261/2006] and Red Española de Esclerosis Multiple (RD07/0060; REEM; RETICS; ISCIII). F Correa is a CAM FPI Fellow.Peer Reviewe

CD200R1 agonist attenuates glial activation, inflammatory reactions, and hypersensitivity immediately after its intrathecal application in a rat neuropathic pain model

© 2016 Hernangómez et al.[Background]: Interaction of CD200 with its receptor CD200R has an immunoregulatory role and attenuates various types of neuroinflammatory diseases.[Methods]: Immunofluorescence staining, western blot analysis, and RT-PCR were used to investigate the modulatory effects of CD200 fusion protein (CD200Fc) on activation of microglia and astrocytes as well as synthesis of pro- (TNF, IL-1β, IL-6) and anti-inflammatory (IL-4, IL-10) cytokines in the L4-L5 spinal cord segments in relation to behavioral signs of neuropathic pain after unilateral sterile chronic constriction injury (sCCI) of the sciatic nerve. Withdrawal thresholds for mechanical hypersensitivity and latencies for thermal hypersensitivity were measured in hind paws 1 day before operation; 1, 3, and 7 days after sCCI operation; and then 5 and 24 h after intrathecal application of artificial cerebrospinal fluid or CD200Fc.[Results]: Seven days from sCCI operation and 5 h from intrathecal application, CD200Fc reduced mechanical and thermal hypersensitivity when compared with control animals. Simultaneously, CD200Fc attenuated activation of glial cells and decreased proinflammatory and increased anti-inflammatory cytokine messenger RNA (mRNA) levels. Administration of CD200Fc also diminished elevation of CD200 and CD200R proteins as a concomitant reaction of the modulatory system to increased neuroinflammatory reactions after nerve injury. The anti-inflammatory effect of CD200Fc dropped at 24 h after intrathecal application.[Conclusions]: Intrathecal administration of the CD200R1 agonist CD200Fc induces very rapid suppression of neuroinflammatory reactions associated with glial activation and neuropathic pain development. This may constitute a promising and novel therapeutic approach for the treatment of neuropathic pain.This work was supported by the projects CEITEC from European Regional Development Fund (CZ.1.05/1.1.00/02.0068) and Employment of Newly Graduated Doctors of Science for Scientific Excellence (CZ.1.07/2.3.00/30.0009)

Anandamide enhances IL-10 production in activated microglia by targeting CB2 receptors: Roles of ERK1/2, JNK, and NF-κB

The endocannabinoid system exhibits anti-inflammatory properties by regulating cytokine production. Anandamide (AEA) down-regulates proinflammatory cytokines in a viral model of multiple sclerosis (MS). However, little is known about the mechanisms by which AEA exerts these effects. Microglial cells are the main source of cytokines within the brain and the first barrier of defense against pathogens by acting as antigen presenting cells. IL-10 is a key physiological negative regulator of microglial activation. In this study we show that AEA enhances LPS/IFNg-induced IL-10 production in microglia by targeting CB2 receptors through the activation of ERK1/2 and JNK MAPKs. AEA also inhibits NF-jB activation by interfering with the phosphorylation of IjBa, which may result in an increase of IL-10 production. Moreover, endogenously produced IL-10 negatively regulates IL-12 and IL-23 cytokines, which in its turn modify the pattern of expression of transcription factors involved in Th commitment of splenocytes. This suggests that by altering the cytokine network, AEA could indirectly modify the type of immune responses within the central nervous system (CNS). Accordingly, pharmacological modulation of AEA uptake and degradation might be a useful tool for treating neuroinflammatory diseases. © 2009 Wiley-Liss, Inc.The authors thank Dr. E. Muñoz (University of Córdoba, Spain) for κB factor (KBF)‐Luc. F Correa is a CAM FPI fellow.Peer Reviewe

Brain-borne IL-1 adjusts glucoregulation and provides fuel support to astrocytes and neurons in an autocrine/paracrine manner

It is still controversial which mediators regulate energy provision to activated neural cells, as insulin does in peripheral tissues. Interleukin-1β (IL-1β) may mediate this effect as it can affect glucoregulation, it is overexpressed in the ‘healthy’ brain during increased neuronal activity, and it supports high-energy demanding processes such as long-term potentiation, memory and learning. Furthermore, the absence of sustained neuroendocrine and behavioral counterregulation suggests that brain glucose-sensing neurons do not perceive IL-1β-induced hypoglycemia. Here, we show that IL-1β adjusts glucoregulation by inducing its own production in the brain, and that IL-1β-induced hypoglycemia is myeloid differentiation primary response 88 protein (MyD88)-dependent and only partially counteracted by Kir6.2-mediated sensing signaling. Furthermore, we found that, opposite to insulin, IL-1β stimulates brain metabolism. This effect is absent in MyD88-deficient mice, which have neurobehavioral alterations associated to disorders in glucose homeostasis, as during several psychiatric diseases. IL-1β effects on brain metabolism are most likely maintained by IL-1β auto-induction and may reflect a compensatory increase in fuel supply to neural cells. We explore this possibility by directly blocking IL-1 receptors in neural cells. The results showed that, in an activity-dependent and paracrine/autocrine manner, endogenous IL-1 produced by neurons and astrocytes facilitates glucose uptake by these cells. This effect is exacerbated following glutamatergic stimulation and can be passively transferred between cell types. We conclude that the capacity of IL-1β to provide fuel to neural cells underlies its physiological effects on glucoregulation, synaptic plasticity, learning and memory. However, deregulation of IL-1β production could contribute to the alterations in brain glucose metabolism that are detected in several neurologic and psychiatric diseases.This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG RE 1451/3-1) to AdR