An Engineered Adeno-Associated Virus Capsid Mediates Efficient Transduction of Pericytes and Smooth Muscle Cells of the Brain Vasculature.

Neurodegeneration and cerebrovascular disease share an underlying microvascular dysfunction that may be remedied by selective transgene delivery. To date, limited options exist in which cellular components of the brain vasculature can be effectively targeted by viral vector therapeutics. In this study, we characterize the first engineered adeno-associated virus (AAV) capsid mediating high transduction of cerebral vascular pericytes and smooth muscle cells (SMCs). We performed two rounds of in vivo selection with an AAV capsid scaffold displaying a heptamer peptide library to isolate capsids that traffic to the brain after intravenous delivery. One identified capsid, termed AAV-PR, demonstrated high transduction of the brain vasculature, in contrast to the parental capsid, AAV9, which transduces mainly neurons and astrocytes. Further analysis using tissue clearing, volumetric rendering, and colocalization revealed that AAV-PR enabled high transduction of cerebral pericytes located on small-caliber vessels and SMCs in the larger arterioles and penetrating pial arteries. Analysis of tissues in the periphery indicated that AAV-PR also transduced SMCs in large vessels associated with the systemic vasculature. AAV-PR was also able to transduce primary human brain pericytes with higher efficiency than AAV9. Compared with previously published AAV capsids tropisms, AAV-PR represents the first capsid to allow for effective transduction of brain pericytes and SMCs and offers the possibility of genetically modulating these cell types in the context of neurodegeneration and other neurological diseases.

The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in-vitro models of the human blood-brain barrier.

As researchers across the globe have focused their attention on understanding SARS-CoV-2, the picture that is emerging is that of a virus that has serious effects on the vasculature in multiple organ systems including the cerebral vasculature. Observed effects on the central nervous system include neurological symptoms (headache, nausea, dizziness), fatal microclot formation and in rare cases encephalitis. However, our understanding of how the virus causes these mild to severe neurological symptoms and how the cerebral vasculature is impacted remains unclear. Thus, the results presented in this report explored whether deleterious outcomes from the SARS-CoV-2 viral spike protein on primary human brain microvascular endothelial cells (hBMVECs) could be observed. The spike protein, which plays a key role in receptor recognition, is formed by the S1 subunit containing a receptor binding domain (RBD) and the S2 subunit. First, using postmortem brain tissue, we show that the angiotensin converting enzyme 2 or ACE2 (a known binding target for the SARS-CoV-2 spike protein), is ubiquitously expressed throughout various vessel calibers in the frontal cortex. Moreover, ACE2 expression was upregulated in cases of hypertension and dementia. ACE2 was also detectable in primary hBMVECs maintained under cell culture conditions. Analysis of cell viability revealed that neither the S1, S2 or a truncated form of the S1 containing only the RBD had minimal effects on hBMVEC viability within a 48 h exposure window. Introduction of spike proteins to invitro models of the blood-brain barrier (BBB) showed significant changes to barrier properties. Key to our findings is the demonstration that S1 promotes loss of barrier integrity in an advanced 3D microfluidic model of the human BBB, a platform that more closely resembles the physiological conditions at this CNS interface. Evidence provided suggests that the SARS-CoV-2 spike proteins trigger a pro-inflammatory response on brain endothelial cells that may contribute to an altered state of BBB function. Together, these results are the first to show the direct impact that the SARS-CoV-2 spike protein could have on brain endothelial cells; thereby offering a plausible explanation for the neurological consequences seen in COVID-19 patients.

HIV infects astrocytes in vivo and egresses from the brain to the periphery.

HIV invades the brain during acute infection. Yet, it is unknown whether long-lived infected brain cells release productive virus that can egress from the brain to re-seed peripheral organs. This understanding has significant implication for the brain as a reservoir for HIV and most importantly HIV interplay between the brain and peripheral organs. Given the sheer number of astrocytes in the human brain and their controversial role in HIV infection, we evaluated their infection in vivo and whether HIV infected astrocytes can support HIV egress to peripheral organs. We developed two novel models of chimeric human astrocyte/human peripheral blood mononuclear cells: NOD/scid-IL-2Rgc null (NSG) mice (huAstro/HuPBMCs) whereby we transplanted HIV (non-pseudotyped or VSVg-pseudotyped) infected or uninfected primary human fetal astrocytes (NHAs) or an astrocytoma cell line (U138MG) into the brain of neonate or adult NSG mice and reconstituted the animals with human peripheral blood mononuclear cells (PBMCs). We also transplanted uninfected astrocytes into the brain of NSG mice and reconstituted with infected PBMCs to mimic a biological infection course. As expected, the xenotransplanted astrocytes did not escape/migrate out of the brain and the blood brain barrier (BBB) was intact in this model. We demonstrate that astrocytes support HIV infection in vivo and egress to peripheral organs, at least in part, through trafficking of infected CD4+ T cells out of the brain. Astrocyte-derived HIV egress persists, albeit at low levels, under combination antiretroviral therapy (cART). Egressed HIV evolved with a pattern and rate typical of acute peripheral infection. Lastly, analysis of human cortical or hippocampal brain regions of donors under cART revealed that astrocytes harbor between 0.4-5.2% integrated HIV gag DNA and 2-7% are HIV gag mRNA positive. These studies establish a paradigm shift in the dynamic interaction between the brain and peripheral organs which can inform eradication of HIV reservoirs.

Reward and immune responses in adolescent females following experimental traumatic brain injury.

The pathology of traumatic brain injury (TBI) adversely affects many brain regions, often resulting in the development of comorbid psychiatric disorders including substance use disorders (SUD). Although traditionally thought to be an epidemic that predominantly affects males, recent clinical studies report females have higher rates of concussions and longer recovery times than males. Yet, how neurotrauma, particularly deep within the brain, between the sexes is differentially manifested remains largely unknown. The risk of TBI peaks during adolescence when neuronal networks that regulate reward behaviors are not fully developed. Previously, using the conditioned place preference (CPP) assay, we found that adolescent TBI increased susceptibility to the rewarding effects of cocaine in male mice. Further, we observed augmented inflammatory profiles, increased microglial phagocytosis of neuronal proteins, and decreased neuronal spine density in the NAc. Notably, the extent of sex differences in SUD susceptibility following TBI has not be investigated. Thus, here we ask the central question of whether the adolescent TBI-induced neuroinflammatory profile at reward centers is divergent in a sex-dependent manner. Using the CPP assay, we found that female mice with high levels of female sex hormones at the time of adolescent TBI demonstrated neuroprotection against increased sensitivity to the rewarding effects of cocaine. These studies also provide evidence of significantly reduced microglial activation and phagocytosis of neuronal proteins within the NAc of females. Overall, our results offer crucial insight into how adolescent TBI impacts the reward pathway in a sex depending manner that could explain a vulnerability to addiction-like behavior.

Selection of an Efficient AAV Vector for Robust CNS Transgene Expression.

Adeno-associated virus (AAV) capsid libraries have generated improved transgene delivery vectors. We designed an AAV library construct, iTransduce, that combines a peptide library on the AAV9 capsid with a Cre cassette to enable sensitive detection of transgene expression. After only two selection rounds of the library delivered intravenously in transgenic mice carrying a Cre-inducible fluorescent protein, we flow sorted fluorescent cells from brain, and DNA sequencing revealed two dominant capsids. One of the capsids, termed AAV-F, mediated transgene expression in the brain cortex more than 65-fold (astrocytes) and 171-fold (neurons) higher than the parental AAV9. High transduction efficiency was sex-independent and sustained in two mouse strains (C57BL/6 and BALB/c), making it a highly useful capsid for CNS transduction of mice. Future work in large animal models will test the translation potential of AAV-F.

Characterization of cancer-associated IDH2 mutations that differ in tumorigenicity, chemosensitivity and 2-hydroxyglutarate production.

The family of isocitrate dehydrogenase (IDH) enzymes is vital for cellular metabolism, as IDH1 and IDH2 are required for the decarboxylation of isocitrate to α-ketoglutarate. Heterozygous somatic mutations in IDH1 or IDH2 genes have been detected in many cancers. They share the neomorphic production of the oncometabolite (R)-2-hydroxyglutarate [(R)-2-HG]. With respect to IDH2, it is unclear whether all IDH2 mutations display the same or differ in tumorigenic properties and degrees of chemosensitivity. Here, we evaluated the three most frequent IDH2 mutations occurring in cancer. The predicted changes to the enzyme structure introduced by these individual mutations are supported by the observed production of (R)-2-HG. However, their tumorigenic properties, response to chemotherapeutic agents, and baseline activation of STAT3 differed. Paradoxically, the varying levels of endogenous (R)-2-HG produced by each IDH2 mutant inversely correlated with their respective growth rates. Interestingly, while we found that (R)-2-HG stimulated the growth of non-transformed cells, (R)-2-HG also displayed antitumor activity by suppressing the growth of tumors harboring wild type IDH2. The mitogenic effect of (R)-2-HG in immortalized cells could be switched to antiproliferative by transformation with oncogenic RAS. Thus, our findings show that despite their shared (R)-2-HG production, IDH2 mutations are not alike and differ in shaping tumor cell behavior and response to chemotherapeutic agents. Our study also reveals that under certain conditions, (R)-2-HG has antitumor properties.

Blockade of MCU-Mediated Ca2+ Uptake Perturbs Lipid Metabolism via PP4-Dependent AMPK Dephosphorylation.

Mitochondrial Ca2+ uniporter (MCU)-mediated Ca2+ uptake promotes the buildup of reducing equivalents that fuel oxidative phosphorylation for cellular metabolism. Although MCU modulates mitochondrial bioenergetics, its function in energy homeostasis in vivo remains elusive. Here we demonstrate that deletion of the Mcu gene in mouse liver (MCUΔhep) and in Danio rerio by CRISPR/Cas9 inhibits mitochondrial Ca2+ (mCa2+) uptake, delays cytosolic Ca2+ (cCa2+) clearance, reduces oxidative phosphorylation, and leads to increased lipid accumulation. Elevated hepatic lipids in MCUΔhep were a direct result of extramitochondrial Ca2+-dependent protein phosphatase-4 (PP4) activity, which dephosphorylates AMPK. Loss of AMPK recapitulates hepatic lipid accumulation without changes in MCU-mediated Ca2+ uptake. Furthermore, reconstitution of active AMPK, or PP4 knockdown, enhances lipid clearance in MCUΔhep hepatocytes. Conversely, gain-of-function MCU promotes rapid mCa2+ uptake, decreases PP4 levels, and reduces hepatic lipid accumulation. Thus, our work uncovers an MCU/PP4/AMPK molecular cascade that links Ca2+ dynamics to hepatic lipid metabolism.

Antibody blockade of CLEC12A delays EAE onset and attenuates disease severity by impairing myeloid cell CNS infiltration and restoring positive immunity.

The mechanism of dendritic cells (DCs) recruitment across the blood brain barrier (BBB) during neuroinflammation has been the least explored amongst all leukocytes. For cells of myeloid origin, while integrins function at the level of adhesion, the importance of lectins remains unknown. Here, we identified functions of one C-type lectin receptor, CLEC12A, in facilitating DC binding and transmigration across the BBB in response to CCL2 chemotaxis. To test function of CLEC12A in an animal model of multiple sclerosis (MS), we administered blocking antibody to CLEC12A that significantly ameliorated disease scores in MOG35-55-induced progressive, as well as PLP138-151-induced relapsing-remitting experimental autoimmune encephalomyelitis (EAE) mice. The decline in both progression and relapse of EAE occurred as a result of reduced demyelination and myeloid cell infiltration into the CNS tissue. DC numbers were restored in the spleen of C57BL/6 and peripheral blood of SJL/J mice along with a decreased TH17 phenotype within CD4+ T-cells. The effects of CLEC12A blocking were further validated using CLEC12A knockout (KO) animals wherein EAE disease induction was delayed and reduced disease severity was observed. These studies reveal the utility of a DC-specific mechanism in designing new therapeutics for MS.

Trafficking of adeno-associated virus vectors across a model of the blood-brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells.

Developing therapies for central nervous system (CNS) diseases is exceedingly difficult because of the blood-brain barrier (BBB). Notably, emerging technologies may provide promising new options for the treatment of CNS disorders. Adeno-associated virus serotype 9 (AAV9) has been shown to transduce cells in the CNS following intravascular administration in rodents, cats, pigs, and non-human primates. These results suggest that AAV9 is capable of crossing the BBB. However, mechanisms that govern AAV9 transendothelial trafficking at the BBB remain unknown. Furthermore, possibilities that AAV9 may transduce brain endothelial cells or affect BBB integrity still require investigation. Using primary human brain microvascular endothelial cells as a model of the human BBB, we performed transduction and transendothelial trafficking assays comparing AAV9 to AAV2, a serotype that does not cross the BBB or transduce endothelial cells effectively in vivo. Results of our in vitro studies indicate that AAV9 penetrates brain microvascular endothelial cells barriers more effectively than AAV2, but has reduced transduction efficiency. In addition, our data suggest that (i) AAV9 penetrates endothelial barriers through an active, cell-mediated process, and (ii) AAV9 fails to disrupt indicators of BBB integrity such as transendothelial electrical resistance, tight junction protein expression/localization, and inflammatory activation status. Overall, this report shows how human brain endothelial cells configured in BBB models can be utilized for evaluating transendothelial movement and transduction kinetics of various AAV capsids. Importantly, the use of a human in vitro BBB model can provide import insight into the possible effects that candidate AVV gene therapy vectors may have on the status of BBB integrity. Read the Editorial Highlight for this article on page 192.

Mechanical Injury Induces Brain Endothelial-Derived Microvesicle Release: Implications for Cerebral Vascular Injury during Traumatic Brain Injury.

It is well established that the endothelium responds to mechanical forces induced by changes in shear stress and strain. However, our understanding of vascular remodeling following traumatic brain injury (TBI) remains incomplete. Recently published studies have revealed that lung and umbilical endothelial cells produce extracellular microvesicles (eMVs), such as microparticles, in response to changes in mechanical forces (blood flow and mechanical injury). Yet, to date, no studies have shown whether brain endothelial cells produce eMVs following TBI. The brain endothelium is highly specialized and forms the blood-brain barrier (BBB), which regulates diffusion and transport of solutes into the brain. This specialization is largely due to the presence of tight junction proteins (TJPs) between neighboring endothelial cells. Following TBI, a breakdown in tight junction complexes at the BBB leads to increased permeability, which greatly contributes to the secondary phase of injury. We have therefore tested the hypothesis that brain endothelium responds to mechanical injury, by producing eMVs that contain brain endothelial proteins, specifically TJPs. In our study, primary human adult brain microvascular endothelial cells (BMVEC) were subjected to rapid mechanical injury to simulate the abrupt endothelial disruption that can occur in the primary injury phase of TBI. eMVs were isolated from the media following injury at 2, 6, 24, and 48 h. Western blot analysis of eMVs demonstrated a time-dependent increase in TJP occludin, PECAM-1 and ICAM-1 following mechanical injury. In addition, activation of ARF6, a small GTPase linked to extracellular vesicle production, was increased after injury. To confirm these results in vivo, mice were subjected to sham surgery or TBI and blood plasma was collected 24 h post-injury. Isolation and analysis of eMVs from blood plasma using cryo-EM and flow cytometry revealed elevated levels of vesicles containing occludin following brain trauma. These results indicate that following TBI, the cerebral endothelium undergoes vascular remodeling through shedding of eMVs containing TJPs and endothelial markers. The detection of this shedding potentially allows for a novel methodology for real-time monitoring of cerebral vascular health (remodeling), BBB status and neuroinflammation following a TBI event.

Methamphetamine induces trace amine-associated receptor 1 (TAAR1) expression in human T lymphocytes: role in immunomodulation.

The novel transmembrane G protein-coupled receptor, trace amine-associated receptor 1 (TAAR1), represents a potential, direct target for drugs of abuse and monoaminergic compounds, including amphetamines. For the first time, our studies have illustrated that there is an induction of TAAR1 mRNA expression in resting T lymphocytes in response to methamphetamine. Methamphetamine treatment for 6 h significantly increased TAAR1 mRNA expression (P < 0.001) and protein expression (P < 0.01) at 24 h. With the use of TAAR1 gene silencing, we demonstrate that methamphetamine-induced cAMP, a classic response to methamphetamine stimulation, is regulated via TAAR1. We also show by TAAR1 knockdown that the down-regulation of IL-2 in T cells by methamphetamine, which we reported earlier, is indeed regulated by TAAR1. Our results also show the presence of TAAR1 in human lymph nodes from HIV-1-infected patients, with or without a history of methamphetamine abuse. TAAR1 expression on lymphocytes was largely in the paracortical lymphoid area of the lymph nodes with enhanced expression in lymph nodes of HIV-1-infected methamphetamine abusers rather than infected-only subjects. In vitro analysis of HIV-1 infection of human PBMCs revealed increased TAAR1 expression in the presence of methamphetamine. In summary, the ability of methamphetamine to activate trace TAAR1 in vitro and to regulate important T cell functions, such as cAMP activation and IL-2 production; the expression of TAAR1 in T lymphocytes in peripheral lymphoid organs, such as lymph nodes; and our in vitro HIV-1 infection model in PBMCs suggests that TAAR1 may play an important role in methamphetamine -mediated immune-modulatory responses.

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 ß, CX-43) and found their downregulation after treatment with tumor necrosis factor-α (TNFα) or interleukin-1 ß (IL-1ß). Pericyte exposure to virus or cytokines resulted in decreased secretion of factors promoting BBB formation (angiopoietin-1, transforming growth factor-ß1) and mRNA for basement membrane components. TNFα and IL-1ß 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.

Activation of Cannabinoid Type Two Receptors (CB2) Diminish Inflammatory Responses in Macrophages and Brain Endothelium.

Chronic neuroinflammatory disorders (such as HIV associated neurodegeneration) require treatment that decreases production of inflammatory factors by activated microglia and macrophages and protection of blood brain barrier (BBB) injury secondary to activation of brain endothelium. Cannabioid type 2 receptor (CB2) is highly expressed on macrophages and brain microvasular enndothelial cells (BMVEC) and is upregulated in inflammation and HIV infection. It has been shown that CB2 activation dampened inflammatory responses in macrophages and BMVEC. In this study, we assessed by PCR array the expression of a wide range of genes increased in macrophages and BMVEC in inflammation. TNFα treatment upregulated 33 genes in primary human BMVEC, and two highly selective CB2 agonists diminished expression of 31 and 32 genes. These results were confirmed by functional assays (BBB protection after inflammatory insult and decreased migration of monocytes across BMVEC monolayers after CB2 stimulation). Similarly, CB2 stimulation in primary human macrophages led to the suppression of 35 genes out of the 50 genes upregulated by LPS. Such changes in gene expression paralleled diminished secretion of proinflammatory factors. These results indicate the potential utility of CB2 agonists for the treatment of 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.

Gene therapy for the nervous system: challenges and new strategies.

Current clinical treatments for central nervous system (CNS) diseases, such as Parkinson's disease and glioblastoma do not halt disease progression and have significant treatment morbidities. Gene therapy has the potential to "permanently" correct disease by bringing in a normal gene to correct a mutant gene deficiency, knocking down mRNA of mutant alleles, and inducing cell-death in cancer cells using transgenes encoding apoptosis-inducing proteins. Promising results in clinical trials of eye disease (Leber's congenital aumorosis) and Parkinson's disease have shown that gene-based neurotherapeutics have great potential. The recent development of genome editing technology, such as zinc finger nucleases, TALENS, and CRISPR, has made the ultimate goal of gene correction a step closer. This review summarizes the challenges faced by gene-based neurotherapeutics and the current and recent strategies designed to overcome these barriers. We have chosen the following challenges to focus on in this review: (1) delivery vehicles (both virus and nonviral), (2) use of promoters for vector-mediated gene expression in CNS, and (3) delivery across the blood-brain barrier. The final section (4) focuses on promising pre-clinical/clinical studies of neurotherapeutics.

Characterization of platelet-monocyte complexes in HIV-1-infected individuals: possible role in HIV-associated neuroinflammation.

HIV-1-associated neuroinflammation persists even with effective combined antiretroviral therapy, and it is associated with the presence of activated monocytes/macrophages within the CNS. To infiltrate the CNS, monocytes transmigrate across the selectively permeable blood-brain barrier, which is compromised during HIV-1 infection. Interestingly, platelet-derived excess soluble CD40 ligand found in the plasma and cerebrospinal fluid of HIV-1-infected individuals with cognitive impairment has previously been implicated in increased blood-brain barrier permeability. In this study we show that soluble CD40 ligand also promotes the formation of complexes between inflammatory monocytes and activated platelets (PMCs), which are detected by flow cytometry as monocytes that express excess of CD61, a platelet marker, and that these complexes are increased in individuals with HIV-1 infection. PMCs exhibit an enhanced ability to adhere to human brain microvascular endothelial cells as compared with monocytes alone, and they migrate across the transendothelial barrier. These complexes can be found marginalized in the lumen of postcapillary venules in postmortem brain tissue derived from cases of HIV-1-associated encephalitis. The extravasation of monocytes across the brain endothelium may exacerbate neuroinflammation, indicating that enhancing this event via platelet interaction may be a contributing factor in the development of cognitive impairment. Thus, dampening platelet activation, and in turn PMC formation, with antiplatelet agents may prove beneficial in developing adjunctive therapies for use in combination with combined antiretroviral therapy in an effort to reduce HIV-1-associated neurologic deficit.

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.

Glycogen synthase kinase 3ß inhibition prevents monocyte migration across brain endothelial cells via Rac1-GTPase suppression and down-regulation of active integrin conformation.

Glycogen synthase kinase (GSK) 3ß has been identified as a regulator of immune responses. We demonstrated previously that GSK3ß inhibition in human brain microvascular endothelial cells (BMVECs) reduced monocyte adhesion/migration across BMVEC monolayers. Herein, we tested the idea that GSK3ß inhibition in monocytes can diminish their ability to engage the brain endothelium and migrate across the blood-brain barrier. Pretreatment of primary monocytes with GSK3ß 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 GSK3ß 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 GSK3ß 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 GSK3ß inhibitors. Monocytes treated with GSK3ß 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 GSK3ß inhibition in monocytes affects active integrin expression, cytoskeleton rearrangement, and adhesion via suppression of Rac1-diminishing inflammatory leukocyte responses.

Activation of cannabinoid receptor 2 attenuates leukocyte-endothelial cell interactions and blood-brain barrier dysfunction under inflammatory conditions.

Previous studies have shown that modulation of the receptor-mediated cannabinoid system during neuroinflammation can produce potent neuroprotective and anti-inflammatory effects. However, in this context, little is known about how selective activation of the cannabinoid type-2 receptor (CB2R) affects the activated state of the brain endothelium and blood-brain barrier (BBB) function. Using human brain tissues and primary human brain microvascular endothelial cells (BMVECs), we demonstrate that the CB2R is highly upregulated during inflammatory insult. We then examined whether the CB2R agonists could attenuate inflammatory responses at the BBB using a mouse model of LPS-induced encephalitis and highly selective CB2R agonists. Visualization by intravital microscopy revealed that administration of JWH133 [(6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran] or a novel resorcinol-based compound, O-1966 (1-[4-(1,1-dimethyl-heptyl)-2,6-dimethoxy-phenyl]-3-methyl-cyclohexanol), greatly attenuated leukocyte adhesion in surface pial vessels and in deep ascending cortical postcapillary venules. BBB permeability assessments with small and large fluorescent tracers showed that CB2R agonists were effective at preventing barrier leakiness after LPS administration. To determine whether the effects by CB2R agonists on barrier protection are not only due to the CB2R modulation of immune cell function, we tested the agonists in vitro with barrier-forming primary BMVECs. Remarkably, the addition of CB2R agonist increased transendothelial electrical resistance and increased the amount of tight junction protein present in membrane fractions. Furthermore, CB2R agonists decreased the induction of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 surface expression in BMVECs exposed to various proinflammatory mediators. Together, these results suggest that pharmacological CB2R ligands offer a new strategy for BBB protection during neuroinflammation.

Dyad of CD40/CD40 ligand fosters neuroinflammation at the blood-brain barrier and is regulated via JNK signaling: implications for HIV-1 encephalitis.

Human immunodeficiency virus 1 (HIV-1) infection may result in activation of peripheral monocytes followed by their infiltration into the CNS, where the release of proinflammatory mediators causes neurologic disease. Previously, we detected high levels of soluble CD40 ligand (CD40L) in CSF and plasma of HIV-infected patients with cognitive impairment. We now show that CD40, a receptor for CD40L, is highly expressed in brain endothelial cells of patients affected by HIV-1 encephalitis (HIVE), suggesting an important role for the CD40/CD40L dyad in regulating blood-brain barrier (BBB) functions. This concept was further supported by in vitro experiments. Exposure of primary human brain microvascular endothelial cells (BMVECs) to CD40L upregulated the expression of adhesion molecules intracellular adhesion molecule-1 and vascular cell adhesion molecule-1, which caused a fourfold increase in monocyte adhesion to BMVECs and stimulated migration across an in vitro BBB model. Investigations into the intracellular signaling pathways that govern these events revealed that cJUN-N-terminal kinase (JNK) is critical to CD40 activation in the BMVECs. CD40L induced activation of mixed-lineage-kinase-3 and JNK, leading to the subsequent activation of cJUN/AP-1 (activating-protein-1). JNK inhibition in the BMVECs prevented CD40L-mediated induction of adhesion molecules, monocyte adhesion, and transendothelial migration. These new findings support the concept that the CD40/CD40L dyad plays an important role in HIVE neuroinflammation.