AKT signaling modulates latent viral reservoir viability in HIV-1-infected blood-brain barrier pericytes.

Despite antiretroviral therapy (ART), chronic forms of HIV-associated neurocognitive disorders (HAND) affect an estimated 50% of individuals living with HIV, greatly impacting their quality of life. The prevailing theory of HAND progression posits that chronic inflammation arising from the activation of latent viral reservoirs leads to progressive damage in the central nervous system (CNS). Recent evidence indicates that blood-brain barrier (BBB) pericytes are capable of active HIV-1 infection; however, their latent infection has not been defined. Given their location and function, BBB pericytes are poised to be a key viral reservoir in the development of HAND. We present the first transcriptional analysis of uninfected, active, and latent human BBB pericytes, revealing distinct transcriptional phenotypes. In addition, we demonstrate that latent infection of BBB pericytes relies on AKT signaling for reservoir survival. These findings provide insight into the state of reservoir maintenance in the CNS during HIV-1 infection and provide novel targets for reservoir clearance.

Rift Valley fever virus is able to cross the human blood-brain barrier in vitro by direct infection with no deleterious effects.

Rift Valley fever (RVF) is a zoonotic arboviral disease that causes recurrent epidemics in Africa that may trigger fatal neurological disorders. However, the mechanisms of neuroinvasion by which the RVF virus (RVFV) reaches the human central nervous system (CNS) remain poorly characterized. In particular, it is not clear how RVFV is able to cross the human blood-brain barrier (hBBB), which is a neurovascular endothelium that protects the brain by regulating brain and blood exchanges. To explore these mechanisms, we used an in vitro hBBB model to mimic in vivo hBBB selectiveness and apicobasal polarity. Our results highlight the ability of RVFV to cross the hBBB by direct infection in a non-structural protein S (NSs)-independent but strain-dependent manner, leading to astrocyte and pericyte infections. Interestingly, RVFV infection did not induce hBBB disruption and was associated with progressive elimination of infected cells with no impairment of the tight junction protein scaffold and barrier function. Our work also shows that NSs, a well described RVFV virulence factor, limited the establishment of the hBBB-induced innate immune response and subsequent lymphocyte recruitment. These results provide in vitro confirmation of the ability of RVFV to reach human CNS by direct infection of the hBBB without altering its barrier function, and provide new directions to explore human RVFV neurovirulence and neuroinvasion mechanisms.IMPORTANCEThe RVF virus (RVFV) is capable of infecting humans and inducing severe and fatal neurological disorders. Neuropathogenesis and human central nervous system (CNS) invasion mechanisms of RVFV are still unknown, with only historical studies of autopsy data from fatal human cases in the 1980s and exploration studies in rodent models. One of the gaps in understanding RVFV human pathogenesis is how RVFV is able to cross the blood-brain barrier (BBB) in order to reach the human CNS. For the first time, we show that RVFV is able to directly infect cells of the human BBB in vitro to release viral particles into the human CNS, a well-characterized neuroinvasion mechanism of pathogens. Furthermore, we demonstrate strain-dependent variability of this neuroinvasion mechanism, identifying possible viral properties that could be explored to prevent neurological disorders during RVFV outbreaks.

Procoagulant phenotype of virus-infected pericytes is associated with portal thrombosis and intrapulmonary vascular dilations in fatal COVID-19.

BACKGROUND & AIMS: The underlying mechanisms and clinical impact of portal microthrombosis in severe COVID-19 are unknown. Intrapulmonary vascular dilation (IPVD)-related hypoxia has been described in severe liver diseases. We hypothesised that portal microthrombosis is associated with IPVD and fatal respiratory failure in COVID-19. METHODS: Ninety-three patients who died from COVID-19 were analysed for portal microvascular damage (histology), IPVD (histology and chest-computed tomography, CT), and hypoxemia (arterial blood gas). Seventeen patients who died from COVID-19-unrelated pneumonia served as controls. Vascular lesions and microthrombi were phenotyped for endothelial (vWF) and pericyte (αSMA/PDGFR-ß) markers, tissue factor (TF), viral spike protein and nucleoprotein (SP, NP), fibrinogen, and platelets (CD41a). Viral particles in vascular cells were assessed by transmission electron microscopy. Cultured pericytes were infected with SARS-CoV-2 to measure TF expression and tubulisation of human pulmonary microvascular endothelial cells was assessed upon vWF treatment. RESULTS: IPVD was present in 16/66 patients with COVID-19, with available liver and lung histology, and was associated with younger age (62 vs. 78 years-old), longer illness (25 vs. 14 days), worsening hypoxemia (PaO2/FiO2 from 209 to 89), and an increased requirement for ventilatory support (63% vs. 22%) compared to COVID-19/Non-IPVD. IPVD, absent in controls, was confirmed by chest CT. COVID-19/IPVD liver histology showed portal microthrombosis in >82.5% of portal areas, with a thicker wall of αSMA/PDGFR-ß+/SP+/NP+ pericytes compared with COVID-19/Non-IPVD. Thrombosed portal venules correlated with αSMA+ area, whereas infected SP+/NP+ pericytes expressed TF. SARS-CoV-2 viral particles were observed in portal pericytes. In vitro SARS-CoV-2 infection of pericytes upregulated TF and induced endothelial cells to overexpress vWF, which expanded human pulmonary microvascular endothelial cell tubules. CONCLUSIONS: SARS-CoV-2 infection of liver pericytes elicits a local procoagulant response associated with extensive portal microthrombosis, IPVD and worsening respiratory failure in fatal COVID-19. IMPACT AND IMPLICATIONS: Vascular involvement of the liver represents a serious complication of COVID-19 infection that must be considered in the work-up of patients with long-lasting and progressively worsening respiratory failure, as it may associate with the development of intrapulmonary vascular dilations. This clinical picture is associated with a procoagulant phenotype of portal venule pericytes, which is induced by SARS-CoV-2 infection of pericytes. Both observations provide a model that may apply, at least in part, to other vascular disorders of the liver, featuring obliterative portal venopathy, similarly characterised at the clinical level by development of hypoxemia and at the histological level by phlebosclerosis and reduced calibre of the portal vein branches in the absence of cirrhosis. Moreover, our findings shed light on an overlooked player in the pathophysiology of thrombosis, i.e. pericytes, which may present a novel therapeutic target.

SARS-CoV-2 infects neurons, astrocytes, choroid plexus epithelial cells and pericytes of the human central nervous system in vitro.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with neurological sequelae including haemorrhage, thrombosis and ischaemic necrosis and encephalitis. However, the mechanism by which this occurs is unclear. Neurological disease associated with COVID-19 has been proposed to occur following direct infection of the central nervous system and/or indirectly by local or systemic immune activation. We evaluated the expression of angiotensin-converting enzyme-2 and transmembrane protease, serine 2 (TMPRSS2) in brain tissue from five healthy human donors and observed low-level expression of these proteins in cells morphologically consistent with astrocytes, neurons and choroidal ependymal cells within the frontal cortex and medulla oblongata. Primary human astrocytes, neurons, choroid plexus epithelial cells and pericytes supported productive SARS-CoV-2 infection with ancestral, Alpha, Delta and Omicron variants. Infected cells supported the full viral life cycle, releasing infectious virus particles. In contrast, primary brain microvascular endothelial cells and microglia were refractory to SARS-CoV-2 infection. These data support a model whereby SARS-CoV-2 can infect human brain cells, and the mechanism of viral entry warrants further investigation.

SARS-CoV-2 induces blood-brain barrier and choroid plexus barrier impairments and vascular inflammation in mice.

The coronavirus disease of 2019 (COVID-19) pandemic has led to more than 700 million confirmed cases and nearly 7 million deaths. Although severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus mainly infects the respiratory system, neurological complications are widely reported in both acute infection and long-COVID cases. Despite the success of vaccines and antiviral treatments, neuroinvasiveness of SARS-CoV-2 remains an important question, which is also centered on the mystery of whether the virus is capable of breaching the barriers into the central nervous system. By studying the K18-hACE2 infection model, we observed clear evidence of microvascular damage and breakdown of the blood-brain barrier (BBB). Mechanistically, SARS-CoV-2 infection caused pericyte damage, tight junction loss, endothelial activation and vascular inflammation, which together drive microvascular injury and BBB impairment. In addition, the blood-cerebrospinal fluid barrier at the choroid plexus was also impaired after infection. Therefore, cerebrovascular and choroid plexus dysfunctions are important aspects of COVID-19 and may contribute to neurological complications both acutely and in long COVID.

Robust CXCL10/IP-10 and CCL5/RANTES Production Induced by Tick-Borne Encephalitis Virus in Human Brain Pericytes Despite Weak Infection.

Tick-borne encephalitis virus (TBEV) targets the central nervous system (CNS), leading to potentially severe neurological complications. The neurovascular unit plays a fundamental role in the CNS and in the neuroinvasion of TBEV. However, the role of human brain pericytes, a key component of the neurovascular unit, during TBEV infection has not yet been elucidated. In this study, TBEV infection of the primary human brain perivascular pericytes was investigated with highly virulent Hypr strain and mildly virulent Neudoerfl strain. We used Luminex assay to measure cytokines/chemokines and growth factors. Both viral strains showed comparable replication kinetics, peaking at 3 days post infection (dpi). Intracellular viral RNA copies peaked at 6 dpi for Hypr and 3 dpi for Neudoerfl cultures. According to immunofluorescence staining, only small proportion of pericytes were infected (3% for Hypr and 2% for Neudoerfl), and no cytopathic effect was observed in the infected cells. In cell culture supernatants, IL-6 production was detected at 3 dpi, together with slight increases in IL-15 and IL-4, but IP-10, RANTES and MCP-1 were the main chemokines released after TBEV infection. These chemokines play key roles in both immune defense and immunopathology during TBE. This study suggests that pericytes are an important source of these signaling molecules during TBEV infection in the brain.

Powassan Viruses Spread Cell to Cell during Direct Isolation from Ixodes Ticks and Persistently Infect Human Brain Endothelial Cells and Pericytes.

Powassan viruses (POWVs) are neurovirulent tick-borne flaviviruses emerging in the northeastern United States, with a 2% prevalence in Long Island (LI) deer ticks (Ixodes scapularis). POWVs are transmitted within as little as 15 min of a tick bite and enter the central nervous system (CNS) to cause encephalitis (10% of cases are fatal) and long-term neuronal damage. POWV-LI9 and POWV-LI41 present in LI Ixodes ticks were isolated by directly inoculating VeroE6 cells with tick homogenates and detecting POWV-infected cells by immunoperoxidase staining. Inoculated POWV-LI9 and LI41 were exclusively present in infected cell foci, indicative of cell to cell spread, despite growth in liquid culture without an overlay. Cloning and sequencing establish POWV-LI9 as a phylogenetically distinct lineage II POWV strain circulating in LI deer ticks. Primary human brain microvascular endothelial cells (hBMECs) and pericytes form a neurovascular complex that restricts entry into the CNS. We found that POWV-LI9 and -LI41 and lineage I POWV-LB productively infect hBMECs and pericytes and that POWVs were basolaterally transmitted from hBMECs to lower-chamber pericytes without permeabilizing polarized hBMECs. Synchronous POWV-LI9 infection of hBMECs and pericytes induced proinflammatory chemokines, interferon-ß (IFN-ß) and proteins of the IFN-stimulated gene family (ISGs), with delayed IFN-ß secretion by infected pericytes. IFN inhibited POWV infection, but despite IFN secretion, a subset of POWV-infected hBMECs and pericytes remained persistently infected. These findings suggest a potential mechanism for POWVs (LI9/LI41 and LB) to infect hBMECs, spread basolaterally to pericytes, and enter the CNS. hBMEC and pericyte responses to POWV infection suggest a role for immunopathology in POWV neurovirulence and potential therapeutic targets for preventing POWV spread to neuronal compartments. IMPORTANCE We isolated POWVs from LI deer ticks (I. scapularis) directly in VeroE6 cells, and sequencing revealed POWV-LI9 as a distinct lineage II POWV strain. Remarkably, inoculation of VeroE6 cells with POWV-containing tick homogenates resulted in infected cell foci in liquid culture, consistent with cell-to-cell spread. POWV-LI9 and -LI41 and lineage I POWV-LB strains infected hBMECs and pericytes that comprise neurovascular complexes. POWVs were nonlytically transmitted basolaterally from infected hBMECs to lower-chamber pericytes, suggesting a mechanism for POWV transmission across the blood-brain barrier (BBB). POWV-LI9 elicited inflammatory responses from infected hBMEC and pericytes that may contribute to immune cell recruitment and neuropathogenesis. This study reveals a potential mechanism for POWVs to enter the CNS by infecting hBMECs and spreading basolaterally to abluminal pericytes. Our findings reveal that POWV-LI9 persists in cells that form a neurovascular complex spanning the BBB and suggest potential therapeutic targets for preventing POWV spread to neuronal compartments.

Zika virus infects pericytes in the choroid plexus and enters the central nervous system through the blood-cerebrospinal fluid barrier.

Zika virus (ZIKV) can infect and cause microcephaly and Zika-associated neurological complications in the developing fetal and adult brains. In terms of pathogenesis, a critical question is how ZIKV overcomes the barriers separating the brain from the circulation and gains access to the central nervous system (CNS). Despite the importance of ZIKV pathogenesis, the route ZIKV utilizes to cross CNS barriers remains unclear. Here we show that in mouse models, ZIKV-infected cells initially appeared in the periventricular regions of the brain, including the choroid plexus and the meninges, prior to infection of the cortex. The appearance of ZIKV in cerebrospinal fluid (CSF) preceded infection of the brain parenchyma. Further the brain infection was significantly attenuated by neutralization of the virus in the CSF, indicating that ZIKV in the CSF at the early stage of infection might be responsible for establishing a lethal infection of the brain. We show that cells infected by ZIKV in the choroid plexus were pericytes. Using in vitro systems, we highlight the possibility that ZIKV crosses the blood-CSF barrier by disrupting the choroid plexus epithelial layer. Taken together, our results suggest that ZIKV might exploit the blood-CSF barrier rather than the blood-brain barrier to invade the CNS.

The emerging role of perivascular cells (pericytes) in viral pathogenesis.

Viruses may exploit the cardiovascular system to facilitate transmission or within-host dissemination, and the symptoms of many viral diseases stem at least in part from a loss of vascular integrity. The microvascular architecture is comprised of an endothelial cell barrier ensheathed by perivascular cells (pericytes). Pericytes are antigen-presenting cells (APCs) and play crucial roles in angiogenesis and the maintenance of microvascular integrity through complex reciprocal contact-mediated and paracrine crosstalk with endothelial cells. We here review the emerging ways that viruses interact with pericytes and pay consideration to how these interactions influence microvascular function and viral pathogenesis. Major outcomes of virus-pericyte interactions include vascular leakage or haemorrhage, organ tropism facilitated by barrier disruption, including viral penetration of the blood-brain barrier and placenta, as well as inflammatory, neurological, cognitive and developmental sequelae. The underlying pathogenic mechanisms may include direct infection of pericytes, pericyte modulation by secreted viral gene products and/or the dysregulation of paracrine signalling from or to pericytes. Viruses we cover include the herpesvirus human cytomegalovirus (HCMV, Human betaherpesvirus 5), the retrovirus human immunodeficiency virus (HIV; causative agent of acquired immunodeficiency syndrome, AIDS, and HIV-associated neurocognitive disorder, HAND), the flaviviruses dengue virus (DENV), Japanese encephalitis virus (JEV) and Zika virus (ZIKV), and the coronavirus severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2; causative agent of coronavirus disease 2019, COVID-19). We touch on promising pericyte-focussed therapies for treating the diseases caused by these important human pathogens, many of which are emerging viruses or are causing new or long-standing global pandemics.

The SARS-CoV-2 Spike protein disrupts human cardiac pericytes function through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a broad range of clinical responses including prominent microvascular damage. The capacity of SARS-CoV-2 to infect vascular cells is still debated. Additionally, the SARS-CoV-2 Spike (S) protein may act as a ligand to induce non-infective cellular stress. We tested this hypothesis in pericytes (PCs), which are reportedly reduced in the heart of patients with severe coronavirus disease-2019 (COVID-19). Here we newly show that the in vitro exposure of primary human cardiac PCs to the SARS-CoV-2 wildtype strain or the α and δ variants caused rare infection events. Exposure to the recombinant S protein alone elicited signalling and functional alterations, including: (1) increased migration, (2) reduced ability to support endothelial cell (EC) network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm, and (4) production of pro-apoptotic factors causing EC death. Next, adopting a blocking strategy against the S protein receptors angiotensin-converting enzyme 2 (ACE2) and CD147, we discovered that the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in PCs. The neutralisation of CD147, either using a blocking antibody or mRNA silencing, reduced ERK1/2 activation, and rescued PC function in the presence of the S protein. Immunoreactive S protein was detected in the peripheral blood of infected patients. In conclusion, our findings suggest that the S protein may prompt PC dysfunction, potentially contributing to microvascular injury. This mechanism may have clinical and therapeutic implications.

Infection of Brain Pericytes Underlying Neuropathology of COVID-19 Patients.

A wide range of neurological manifestations have been associated with the development of COVID-19 following SARS-CoV-2 infection. However, the etiology of the neurological symptomatology is still largely unexplored. Here, we used state-of-the-art multiplexed immunostaining of human brains (n = 6 COVID-19, median age = 69.5 years; n = 7 control, median age = 68 years) and demonstrated that expression of the SARS-CoV-2 receptor ACE2 is restricted to a subset of neurovascular pericytes. Strikingly, neurological symptoms were exclusive to, and ubiquitous in, patients that exhibited moderate to high ACE2 expression in perivascular cells. Viral dsRNA was identified in the vascular wall and paralleled by perivascular inflammation, as signified by T cell and macrophage infiltration. Furthermore, fibrinogen leakage indicated compromised integrity of the blood-brain barrier. Notably, cerebrospinal fluid from additional 16 individuals (n = 8 COVID-19, median age = 67 years; n = 8 control, median age = 69.5 years) exhibited significantly lower levels of the pericyte marker PDGFRß in SARS-CoV-2-infected cases, indicative of disrupted pericyte homeostasis. We conclude that pericyte infection by SARS-CoV-2 underlies virus entry into the privileged central nervous system space, as well as neurological symptomatology due to perivascular inflammation and a locally compromised blood-brain barrier.

Role of angiotensin-converting enzyme 2 and pericytes in cardiac complications of COVID-19 infection.

The prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) quickly reached pandemic proportions, and knowledge about this virus and coronavirus disease 2019 (COVID-19) has expanded rapidly. This review focuses primarily on mechanisms that contribute to acute cardiac injury and dysfunction, which are common in patients with severe disease. The etiology of cardiac injury is multifactorial, and the extent is likely enhanced by preexisting cardiovascular disease. Disruption of homeostatic mechanisms secondary to pulmonary pathology ranks high on the list, and there is growing evidence that direct infection of cardiac cells can occur. Angiotensin-converting enzyme 2 (ACE2) plays a central role in COVID-19 and is a necessary receptor for viral entry into human cells. ACE2 normally not only eliminates angiotensin II (Ang II) by converting it to Ang-(1-7) but also elicits a beneficial response profile counteracting that of Ang II. Molecular analyses of single nuclei from human hearts have shown that ACE2 is most highly expressed by pericytes. Given the important roles that pericytes have in the microvasculature, infection of these cells could compromise myocardial supply to meet metabolic demand. Furthermore, ACE2 activity is crucial for opposing adverse effects of locally generated Ang II, so virus-mediated internalization of ACE2 could exacerbate pathology by this mechanism. While the role of cardiac pericytes in acute heart injury by SARS-CoV-2 requires investigation, expression of ACE2 by these cells has broader implications for cardiac pathophysiology.

Blood-brain barrier pericytes as a target for HIV-1 infection.

Pericytes are multifunctional cells wrapped around endothelial cells via cytoplasmic processes that extend along the abluminal surface of the endothelium. The interactions between endothelial cells and pericytes of the blood-brain barrier are necessary for proper formation, development, stabilization, and maintenance of the blood-brain barrier. Blood-brain barrier pericytes regulate paracellular flow between cells, transendothelial fluid transport, maintain optimal chemical composition of the surrounding microenvironment, and protect endothelial cells from potential harmful substances. Thus, dysfunction or loss of blood-brain barrier pericytes is an important factor in the pathogenesis of several diseases that are associated with microvascular instability. Importantly, recent research indicates that blood-brain barrier pericytes can be a target of HIV-1 infection able to support productive HIV-1 replication. In addition, blood-brain barrier pericytes are prone to establish a latent infection, which can be reactivated by a mixture of histone deacetylase inhibitors in combination with TNF. HIV-1 infection of blood-brain barrier pericytes has been confirmed in a mouse model of HIV-1 infection and in human post-mortem samples of HIV-1-infected brains. Overall, recent evidence indicates that blood-brain barrier pericytes can be a previously unrecognized HIV-1 target and reservoir in the brain.

HIV-1 infection renders brain vascular pericytes susceptible to the extracellular glutamate.

Reduced pericytes' coverage of endothelium in the brain is one of the structural changes leading to breach of the blood-brain barrier during HIV infection. We previously showed in central memory T (TCM) cells that HIV latency increases cellular susceptibility to DNA damage. In this study, we investigated susceptibility of primary brain pericytes infected with HIV-1 to DNA damage in response to glutamate and TNF-α, both known to induce neuronal death during chronic inflammatory conditions. To infect pericytes, we used a single-cycle HIV-1 pseudotyped with VSV-G envelope glycoprotein and maintained the cultures until latency was established. Our data indicate that pericytes silence HIV-1 expression at similar rate compared to primary TCM cells. TNF-α and IL-1ß caused partial reactivation of the virus suggesting that progression of disease and neuroinflammation might facilitate virus reactivation from latency. Significant increases in the level of γH2AX, which reflect DNA damage, were observed in infected cultures exposed to TNF-α and glutamate at day 2 post-infection. Glutamate, an excitatory neurologic stimuli, also caused increases in the γH2AX level in latently infected pericytes, whereas PARP and DNA-PK inhibitors caused reductions in cell population suggesting that HIV-1 latency affects repairs of single- and double-strand DNA breaks. For comparison, we also analyzed latently infected astrocytes and determined that DNA damage response in astrocytes is less affected by HIV-1. In conclusion, our results indicate that productive infection and HIV-1 latency in pericytes interfere with DNA damage response, rendering them vulnerable to the agents that are characteristic of chronic neuroinflammatory disease conditions.

Human Vascular Pericytes and Cytomegalovirus Pathobiology.

Pericytes are multipotent cells of the vascular system with cytoplasmic extensions proximal to endothelial cells that occur along the abluminal surface of the endothelium. The interactions between endothelial cells and pericytes are essential for proper microvascular formation, development, stabilization, and maintenance. Pericytes are essential for the regulation of paracellular flow between cells, transendothelial fluid transport, angiogenesis, and vascular immunosurveillance. They also influence the chemical composition of the surrounding microenvironment to protect endothelial cells from potential harm. Dysregulation or loss of pericyte function can result in microvascular instability and pathological consequences. Human pericytes have been shown to be targets for human cytomegalovirus (HCMV) infection and lytic replication that likely contribute to vascular inflammation. This review focuses on human vascular pericytes and their permissiveness for HCMV infection. It also discusses their implication in pathogenesis in the blood⁻brain barrier (BBB), the inner blood⁻retinal barrier (IBRB), the placenta⁻blood barrier, and the renal glomerulus as well as their potential role in subclinical vascular disease.

Inflammation and outer blood-retina barrier (BRB) compromise following choroidal murine cytomegalovirus (MCMV) infections.

Purpose: The purpose of this study was to determine whether the blood-retina barrier is compromised by choroidal murine cytomegalovirus (MCMV) infection, using electron microscopy. Methods: BALB/c mice were immunosuppressed with methylprednisolone and monoclonal antibodies to CD4 and CD8. At several time points post-MCMV intraperitoneal inoculation, the eyes were removed and analyzed with western blotting and immunoelectron microscopy for the presence of MCMV early antigen (EA) and the host protein RIP3. Posterior eyecups from RIP3-/- and RIP3+/+ mice were cultured and inoculated with MCMV. At days 4, 7, and 11 post-infection, cultures were collected and analyzed with plaque assay, immunohistochemical staining, and real-time PCR (RT-PCR). Results: MCMV EA was observed in the nuclei of vascular endothelial cells and pericytes in the choriocapillaris. Disruption of Bruch's membrane was observed, especially at sites adjacent to activated platelets, and a few RPE cells containing some enlarged vesicles were found directly beneath disrupted Bruch's membrane. Some virus particles were also observed in the enlarged vesicles of RPE cells. Levels of the RIP3 protein, which was observed mainly in the RPE cells and the basement membrane of the choriocapillaris, were greatly increased following MCMV infection, while depletion of RIP3 resulted in greatly decreased inflammasome formation, as well as expression of downstream inflammation factors. Conclusions: The results suggest that systemic MCMV spreads to the choroid and replicates in vascular endothelia and pericytes of the choriocapillaris during immunosuppression. Choroidal MCMV infection is associated with in situ inflammation and subsequent disruption of Bruch's membrane and the outer blood-retina barrier.

Occludin controls HIV transcription in brain pericytes via regulation of SIRT-1 activation.

HIV invades the brain early after infection; however, its interactions with the cells of the blood-brain barrier (BBB) remain poorly understood. Our goal was to evaluate the role of occludin, one of the tight junction proteins that regulate BBB functions in HIV infection of BBB pericytes. We provide evidence that occludin levels largely control the metabolic responses of human pericytes to HIV. Occludin in BBB pericytes decreased by 10% during the first 48 h after HIV infection, correlating with increased nuclear translocation of the gene repressor C-terminal-binding protein (CtBP)-1 and NFκB-p65 activation. These changes were associated with decreased expression and activation of the class III histone deacetylase sirtuin (SIRT)-1. Occludin levels recovered 96 h after infection, restoring SIRT-1 and reducing HIV transcription to 20% of its highest values. We characterized occludin biochemically as a novel NADH oxidase that controls the expression and activation of SIRT-1. The inverse correlation between occludin and HIV transcription was then replicated in human primary macrophages and differentiated monocytic U937 cells, in which occludin silencing resulted in 75 and 250% increased viral transcription, respectively. Our work shows that occludin has previously unsuspected metabolic properties and is a target of HIV infection, opening the possibility of designing novel pharmacological approaches to control HIV transcription.

Interplay of inflammatory gene expression in pericytes following Japanese encephalitis virus infection.

Neuroinflammation is a pathological hallmark and has been implicated in the pathogenesis of Japanese encephalitis. Although brain pericytes show regulatory effects on neuroinflammation, their involvement in Japanese encephalitis-associated neuroinflammation is not understood. Here, we demonstrated that brain microvascular pericytes could be an alternative cellular source for the induction and/or amplification of neuroinflammation caused by Japanese encephalitis virus (JEV) infection. Infection of cultured pericytes with JEV caused profound production of IL-6, RANTES, and prostaglandin E2 (PGE2). Mechanistic studies revealed that JEV infection elicited an elevation of the toll-like receptor 7 (TLR7)/MyD88 signaling axis, leading to the activation of NF-κB through IKK signaling and p65 phosphorylation as well as cAMP response element-binding protein (CREB) via phosphorylation. We further demonstrated that extracellular signal-regulated kinase (ERK) could be an alternative regulator in transducing signals to NF-κB, CREB, and cytosolic phospholipase A2 (cPLA2) through the phosphorylation mechanism. Released IL-6 and RANTES played an active role in the disruption of endothelial barrier integrity and leukocyte chemotaxis, respectively. cPLA2/PGE2 had a role in activating NF-κB and CREB DNA-binding activities and inflammatory cytokine transcription via the EP2/cAMP/PKA mechanism in an autocrine loop. These inflammatory responses and biochemical events were also detected in the brain of JEV-infected mice. The current findings suggest that pericytes might have pathological relevance in Japanese encephalitis-associated neuroinflammation through a TLR7-related mechanism. The consequences of pericyte activation are their ability to initiate and/or amplify inflammatory cytokine expression by which cellular function of endothelial cells and leukocytes are regulated in favor of CNS infiltration by leukocytes.

Tat 101-mediated enhancement of brain pericyte migration involves platelet-derived growth factor subunit B homodimer: implications for human immunodeficiency virus-associated neurocognitive disorders.

In the era of antiretroviral therapy, although the human immunodeficiency virus (HIV) replication can be successfully controlled, complications of the CNS continue to affect infected individuals. Viral Tat protein is not only neurotoxic but has also been shown to disrupt the integrity of the blood-brain barrier (BBB). Although the role of brain microvascular endothelial cells and astrocytes in Tat-mediated impairment has been well documented, pericytes, which are important constituents of the BBB and play a key role in maintaining the integrity of the barrier, remain poorly studied in the context of HIV-associated neurocognitive disorders (HAND). In the present study, we demonstrated that exposure of human brain microvascular pericytes and C3H/10T1/2 cells to HIV-1 Tat101 resulted in increased expression of platelet-derived growth factor subunit B homodimer (PDGF-BB) and increased migration of the treated cells. Furthermore, we also demonstrated that this effect of Tat was mediated via activation of mitogen-activated protein kinases and nuclear factor-κB pathways. Secreted PDGF-BB resulted in autocrine activation of the PDGF-BB/PDGF ß receptor signaling pathway, culminating ultimately into increased pericyte migration. Ex vivo relevance of these findings was further corroborated in isolated microvessels of HIV Tg26 mice that demonstrated significantly increased expression of PDGF-BB in isolated brain microvessels with a concomitant loss of pericytes. Intriguingly, loss of pericyte coverage was also detected in sections of frontal cortex from humans with HIV-encephalitis compared with the uninfected controls. These findings thus implicate a novel role of PDGF-BB in the migration of pericytes, resulting in loss of pericyte coverage from the endothelium with a subsequent breach of the BBB.