Slava Rom

Cannabinoid Receptor 2: Potential Role in Immunomodulation and Neuroinflammation

An accumulating body of evidence suggests that endocannabinoids and cannabinoid receptors type 1 and 2 (CB1, CB2) play a significant role in physiologic and pathologic processes, including cognitive and immune functions. While the addictive properties of marijuana, an extract from the Cannabis plant, are well recognized, there is growing appreciation of the therapeutic potential of cannabinoids in multiple pathologic conditions involving chronic inflammation (inflammatory bowel disease, arthritis, autoimmune disorders, multiple sclerosis, HIV-1 infection, stroke, Alzheimer’s disease to name a few), mainly mediated by CB2 activation. Development of CB2 agonists as therapeutic agents has been hampered by the complexity of their intracellular signaling, relative paucity of highly selective compounds and insufficient data regarding end effects in the target cells and organs. This review attempts to summarize recent advances in studies of CB2 activation in the setting of neuroinflammation, immunomodulation and HIV-1 infection.

CCL8/MCP-2 is a target for mir-146a in HIV-1-infected human microglial cells

MicroRNA‐mediated regulation of gene expression appears to be involved in a variety of cellular processes, including development, differentiation, proliferation, and apoptosis. Mir‐146a is thought to be involved in the regulation of the innate immune response, and its expression is increased in tissues associated with chronic inflammation. Among the predicted gene targets for mir‐146a, the chemokine CCL8/ MCP‐2 is a ligand for the CCR5 chemokine receptor and a potent inhibitor of CD4/CCR5‐mediated HIV‐1 entry and replication. In the present study, we have analyzed changes in the expression of mir‐146a in primary human fetal microglial cells upon infection with HIV‐1 and found increased expression of mir‐146a. We further show that CCL8/MCP‐2 is a target for mir‐146a in HIV‐1 infected microglia, as overexpression of mir‐146a prevented HIV‐induced secretion of MCP‐2 chemokine. The clinical relevance of our findings was evaluated in HIV‐encephalitis (HIVE) brain samples in which decreased levels of MCP‐2 and increased levels of mir‐146a were observed, suggesting a role for mir‐146a in the maintenance of HIV‐mediated chronic inflammation of the brain.—Rom, S., Rom, I., Passiatore, G., Pacifici, M., Radhakrishnan, S., Del Valle, L., Piña‐Oviedo, S., Khalili, K., Eletto, D., Peruzzi, F. CCL8/MCP‐2 is a target for mir‐146a in HIV‐1 infected human microglial cells. FASEB J. 24, 2292–2300 (2010). www.fasebj.org

Emerging roles of pericytes in the regulation of the neurovascular unit in health and disease.

Pericytes of the central nervous system (CNS) are uniquely positioned within a multicellular structure termed the neurovascular unit (NVU) to provide crucial support to blood brain barrier (BBB) formation, maintenance, and stability. Numerous CNS diseases are associated with some aspect of BBB dysfunction. A dysfunction can manifest as one or multiple disruptions to any of the following barriers: physical, metabolic, immunological and transport barrier. A breach in the BBB can notably result in BBB hyper-permeability, endothelial activation and enhanced immune-endothelial interaction. How the BBB is regulated within this integrated unit remains largely unknown, especially as it relates to pericyte-endothelial interaction. We summarize the latest findings on pericyte origin, possible marker expression, and availability within different organ systems. We highlight pericyte-endothelial cell interactions, concentrating on extra- and intra- cellular signaling mechanisms linked to platelet derived growth factor-B, transforming growth factor -β, angiopoietins, Notch, and gap junctions. We discuss the role of pericytes in the NVU under inflammatory insult, focusing on how pericytes may indirectly affect leukocyte CNS infiltration, the direct role of pericyte-mediated basement membrane modifications, and immune responses. We review new findings of pericyte actions in CNS pathologies including Alzheimer’s disease, stroke, multiple sclerosis, diabetic retinopathy, and HIV-1 infection. The uncovering of the regulatory role of pericytes on the BBB will provide key insight into how barrier integrity can be re-established during neuroinflammation.

Selective Activation of Cannabinoid Receptor 2 in Leukocytes Suppresses Their Engagement of the Brain Endothelium and Protects the Blood-Brain Barrier

Cannabinoid receptor 2 (CB2) is highly expressed in immune cells and stimulation decreases inflammatory responses. We tested the idea that selective CB2 activation in human monocytes suppresses their ability to engage the brain endothelium and migrate across the blood-brain barrier (BBB), preventing consequent injury. Intravital videomicroscopy was used to quantify adhesion of leukocytes to cortical vessels in lipopolysaccharide-induced neuroinflammation, after injection of ex vivo CB2-activated leukocytes into mice; CB2 agonists markedly decreased adhesion of ex vivo labeled cells in vivo. In an in vitro BBB model, CB2 activation in monocytes largely attenuated adhesion to and migration across monolayers of primary human brain microvascular endothelial cells and diminished BBB damage. CB2 stimulation in monocytes down-regulated active forms of integrins, lymphocyte function-associated antigen 1 (LFA-1), and very late antigen 4 (VLA-4). Cells treated with CB2 agonists exhibited increased phosphorylation levels of inhibitory sites of the actin-binding proteins cofilin and VASP, which are upstream regulators of conformational integrin changes. Up-regulated by relevant stimuli, Rac1 and RhoA were suppressed by CB2 agonists in monocytes. CB2 stimulation decreased formation of lamellipodia, which play a key role in monocyte migration. These results indicate that selective CB2 activation in leukocytes decreases key steps in monocyte-BBB engagement, thus suppressing inflammatory leukocyte responses and preventing neuroinflammation.

Inhibition of Glycogen Synthase Kinase 3β Promotes Tight Junction Stability in Brain Endothelial Cells by Half-Life Extension of Occludin and Claudin-5

Neuroinflammatory conditions often involve dysfunction of the Blood-Brain Barrier (BBB). Therefore, identifying molecular targets that can maintain barrier fidelity is of clinical importance. We have previously reported on the anti-inflammatory effects that glycogen synthase kinase 3β (GSK3β) inhibition has on primary human brain endothelial cells. Here we show that GSK3β inhibitors also promote barrier tightness by affecting tight junction (TJ) protein stability. Transendothelial electrical resistance (TEER) was used to evaluate barrier integrity with both pharmacological inhibitors and mutants of GSK3β. Inhibition of GSK3β produced a gradual and sustained increase in TEER (as much as 22% over baseline). Analysis of subcellular membrane fractions revealed an increase in the amount of essential tight junction proteins, occludin and claudin-5, but not claudin-3. This phenomenon was attributed to a decrease in TJ protein turnover and not transcriptional regulation. Using a novel cell-based assay, inactivation of GSK3β significantly increased the half-life of occludin and claudin-5 by 32% and 43%, respectively. A correlation was also established between the enhanced association of β-catenin with ZO-1 as a function of GSK3β inhibition. Collectively, our findings suggest the possibility of using GSK3β inhibitors as a means to extend the half-life of key tight junction proteins to promote re-sealing of the BBB during neuroinflammation.

miR-98 and let-7g* Protect the Blood-Brain Barrier Under Neuroinflammatory Conditions:

Pathologic conditions in the central nervous system, regardless of the underlying injury mechanism, show a certain level of blood-brain barrier (BBB) impairment. Endothelial dysfunction is the earliest event in the initiation of vascular damage caused by inflammation due to stroke, atherosclerosis, trauma, or brain infections. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators. The relationship between neuroinflammation and miRNA expression in brain endothelium remains unexplored. Previously, we showed the BBB-protective and anti-inflammatory effects of glycogen synthase kinase (GSK) 3β inhibition in brain endothelium in in vitro and in vivo models of neuroinflammation. Using microarray screening, we identified miRNAs induced in primary human brain microvascular endothelial cells after exposure to the pro-inflammatory cytokine, tumor necrosis factor-α, with/out GSK3β inhibition. Among the highly modified miRNAs, let-7 and miR-98 were predicted to target the inflammatory molecules, CCL2 and CCL5. Overexpression of let-7 and miR-98 in vitro and in vivo resulted in reduced leukocyte adhesion to and migration across endothelium, diminished expression of pro-inflammatory cytokines, and increased BBB tightness, attenuating barrier ‘leakiness’ in neuroinflammation conditions. For the first time, we showed that miRNAs could be used as a therapeutic tool to prevent the BBB dysfunction in neuroinflammation.

Poly(ADP-ribose) Polymerase-1 Inhibition in Brain Endothelium Protects the Blood—Brain Barrier under Physiologic and Neuroinflammatory Conditions

Blood—brain barrier (BBB) dysfunction seen in neuroinflammation contributes to mortality and morbidity in multiple sclerosis, encephalitis, traumatic brain injury, and stroke. Identification of molecular targets maintaining barrier function is of clinical relevance. We used a novel in vivo model of localized aseptic meningitis where tumor necrosis factor alpha (TNFα) was introduced intracerebrally and surveyed cerebral vascular changes and leukocyte—endothelium interactions by intravital videomicroscopy. Poly(ADP-ribose) polymerase-1 (PARP) inhibition significantly reduced leukocyte adhesion to and migration across brain endothelium in cortical microvessels. PARP inactivation diminished BBB permeability in an in vivo model of systemic inflammation. PARP suppression in primary human brain microvascular endothelial cells (BMVEC), an in vitro model of BBB, enhanced barrier integrity and augmented expression of tight junction proteins. PARP inhibition in BMVEC diminished human monocyte adhesion to TNFα-activated BMVEC (up to 65%) and migration (80–100%) across BBB models. PARP suppression decreased expression of adhesion molecules and decreased activity of GTPases (controlling BBB integrity and monocyte migration across the BBB). PARP inhibitors down-regulated expression of inflammatory genes and dampened secretion of pro-inflammatory factors increased by TNFα in BMVEC. These results point to PARP suppression as a novel approach to BBB protection in the setting of endothelial dysfunction caused by inflammation.

Attenuation of HIV-1 replication in macrophages by cannabinoid receptor 2 agonists

Infiltrating monocytes and macrophages play a crucial role in the progression of HIV‐1 infection in the CNS. Previous studies showed that activation of the CB2 can attenuate inflammatory responses and affect HIV‐1 infectivity in T cells and microglia. Here, we report that CB2 agonists can also act as immunomodulators on HIV‐1‐infected macrophages. First, our findings indicated the presence of elevated levels of CB2 expression on monocytes/macrophages in perivascular cuffs of postmortem HIV‐1 encephalitic cases. In vitro analysis by FACS of primary human monocytes revealed a step‐wise increase in CB2 surface expression in monocytes, MDMs, and HIV‐1‐infected MDMs. We next tested the notion that up‐regulation of CB2 may allow for the use of synthetic CB2 agonist to limit HIV‐1 infection. Two commercially available CB2 agonists, JWH133 and GP1a, and a resorcinol‐based CB2 agonist, O‐1966, were evaluated. Results from measurements of HIV‐1 RT activity in the culture media of 7 day‐infected cells showed a significant decrease in RT activity when the CB2 agonist was present. Furthermore, CB2 activation also partially inhibited the expression of HIV‐1 pol. CB2 agonists did not modulate surface expression of CXCR4 or CCR5 detected by FACS. We speculate that these findings indicate that prevention of viral entry is not a central mechanism for CB2‐mediated suppression in viral replication. However, CB2 may affect the HIV‐1 replication machinery. Results from a single‐round infection with the pseudotyped virus revealed a marked decrease in HIV‐1 LTR activation by the CB2 ligands. Together, these results indicate that CB2 may offer a means to limit HIV‐1 infection in macrophages.

Dysfunction of brain pericytes in chronic neuroinflammation.

Brain pericytes are uniquely positioned within the neurovascular unit to provide support to blood brain barrier (BBB) maintenance. Neurologic conditions, such as HIV-1-associated neurocognitive disorder, are associated with BBB compromise due to chronic inflammation. Little is known about pericyte dysfunction during HIV-1 infection. We found decreased expression of pericyte markers in human brains from HIV-1-infected patients (even those on antiretroviral therapy). Using primary human brain pericytes, we assessed expression of pericyte markers (α1-integrin, α-smooth muscle actin, platelet-derived growth factor-B receptor β, CX-43) and found their downregulation after treatment with tumor necrosis factor-α (TNFα) or interleukin-1 β (IL-1β). Pericyte exposure to virus or cytokines resulted in decreased secretion of factors promoting BBB formation (angiopoietin-1, transforming growth factor-β1) and mRNA for basement membrane components. TNFα and IL-1β enhanced expression of adhesion molecules in pericytes paralleling increased monocyte adhesion to pericytes. Monocyte migration across BBB models composed of human brain endothelial cells and pericytes demonstrated a diminished rate in baseline migration compared to constructs composed only of brain endothelial cells. However, exposure to the relevant chemokine, CCL2, enhanced the magnitude of monocyte migration when compared to BBB models composed of brain endothelial cells only. These data suggest an important role of pericytes in BBB regulation in neuroinflammation.

Glycogen Synthase Kinase 3β Inhibition Prevents Monocyte Migration across Brain Endothelial Cells via Rac1-GTPase Suppression and Down-Regulation of Active Integrin Conformation

Glycogen synthase kinase (GSK) 3β has been identified as a regulator of immune responses. We demonstrated previously that GSK3β inhibition in human brain microvascular endothelial cells (BMVECs) reduced monocyte adhesion/migration across BMVEC monolayers. Herein, we tested the idea that GSK3β inhibition in monocytes can diminish their ability to engage the brain endothelium and migrate across the blood-brain barrier. Pretreatment of primary monocytes with GSK3β inhibitors resulted in a decrease in adhesion (60%) and migration (85%), with similar results in U937 monocytic cells. Monocyte-BMVEC interactions resulted in diminished barrier integrity that was reversed by GSK3β suppression in monocytic cells. Because integrins mediate monocyte rolling/adhesion, we detected the active conformational form of very late antigen 4 after stimulation with a peptide mimicking monocyte engagement by vascular cell adhesion molecule-1. Peptide stimulation resulted in a 14- to 20-fold up-regulation of the active form of integrin in monocytes that was suppressed by GSK3β inhibitors (40% to 60%). Because small GTPases, such as Rac1, control leukocyte movement, we measured active Rac1 after monocyte activation with relevant stimuli. Stimulation enhanced the level of active Rac1 that was diminished by GSK3β inhibitors. Monocytes treated with GSK3β inhibitors showed increased levels of inhibitory sites of the actin-binding protein, cofilin, and vasodilator-stimulated phosphoprotein-regulating conformational changes of integrins. These results indicate that GSK3β inhibition in monocytes affects active integrin expression, cytoskeleton rearrangement, and adhesion via suppression of Rac1-diminishing inflammatory leukocyte responses.