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Human Immunodeficiency Virus Infection of Human Astrocytes Disrupts Blood-Brain Barrier Integrity by a Gap Junction-Dependent Mechanism
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The Journal of Neuroscience, June 29, 2011
Eliseo A. Eugenin,1 Janice E. Clements,3 M. Christine Zink,3 and Joan W. Berman1,2 Departments of 1Pathology and 2Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, and 3Department of Molecular and Comparative Pathobiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Abstract
HIV infection of the CNS is an early event after primary infection, resulting in neurological complications in a significant number of individuals despite antiretroviral therapy (ART). The main cells infected with HIV within the CNS are macrophages/microglia and a small fraction of astrocytes. The role of these few infected astrocytes in the pathogenesis of neuroAIDS has not been examined extensively. Here, we demonstrate that few HIV-infected astrocytes (4.7 ± 2.8% in vitro and 8.2 ± 3.9% in vivo) compromise blood-brain barrier (BBB) integrity. This BBB disruption is due to endothelial apoptosis, misguided astrocyte end feet, and dysregulation of lipoxygenase/cyclooxygenase, BKCa channels, and ATP receptor activation within astrocytes. All of these alterations in BBB integrity induced by a few HIV-infected astrocytes were gap junction dependent, as blocking these channels protected the BBB from HIV-infected astrocyte-mediated compromise. We also demonstrated apoptosis in vivo of BBB cells in contact with infected astrocytes using brain tissue sections from simian immunodeficiency virus-infected macaques as a model of neuroAIDS, suggesting an important role for these few infected astrocytes in the CNS damage seen with HIV infection. Our findings describe a novel mechanism of bystander BBB toxicity mediated by low numbers of HIV-infected astrocytes and amplified by gap junctions. This mechanism of toxicity contributes to understanding how CNS damage is spread even in the current ART era and how minimal or controlled HIV infection still results in cognitive impairment in a large population of infected individuals.
Introduction
Human immunodeficiency virus type 1 (HIV) invades the CNS early after primary infection and causes HIV-associated neurological disorders in 40-60% of infected individuals even in the current antiretroviral therapy era (Anthony et al., 2005). In vivo, microglial cells and perivascular macrophages are the predominant cell types productively infected by HIV (Wiley et al., 1986; Cosenza et al., 2002). However, a small population of HIV-infected astrocytes has been detected using diverse techniques both in vivo and in vitro (Wiley et al., 1986; Conant et al., 1994; Nuovo et al., 1994; Tornatore et al., 1994b; Bagasra et al., 1996; Takahashi et al., 1996; Ohagen et al., 1999; Eugenin and Berman, 2007; Churchill et al., 2009). The importance of these few infected astrocytes in the pathogenesis of neuroAIDS has not been examined extensively. Our group demonstrated that, although only few astrocytes become infected in vitro (4.7 ± 2.8% of the total astrocytes in the culture) and HIV production is extremely low or undetectable, gap junction (GJ) channels amplify and spread toxic signals to uninfected cells in culture (Eugenin and Berman, 2007). We now demonstrate that these few HIV-infected astrocytes, with no detectable viral production, cause dramatic alterations in blood-brain barrier (BBB) physiology in vitro. We detected similar toxic effects on the BBB in vivo, using brain tissue sections obtained from simian immunodeficiency virus (SIV)-infected macaques.
The BBB is composed of highly specialized microvascular endothelial cells on top of an extracellular matrix, the basal lamina. Astrocyte end feet contact the basal lamina and are close to the endothelial cells as well as accessory cells including pericytes, perivascular macrophages, and microglia (Abbott, 2002; Ballabh et al., 2004). Astrocytes and their end feet processes surround the brain capillaries, providing factors important in the development and maintenance of the BBB (Goldstein, 1988; Risau and Wolburg, 1990; Risau, 1991; Hayashi et al., 1997). Astrocytes and their end feet regulate BBB physiology by soluble factors (Rubin and Staddon, 1999; Hawkins and Davis, 2005; Iadecola and Nedergaard, 2007), and by direct intercellular communication through GJ channels, and perhaps by connexin/pannexin hemichannels on the surface of the cells that connect the cytoplasm and the extracellular environment. GJs are conglomerates of channels that enable the exchange of small molecules up to 1.2 kDa between the cytoplasm of adjacent cells (Saez et al., 2003). Each channel is formed by the docking of two hemichannels, located in apposing cell membranes, and each hemichannel is an assembly of six connexins (Cxs). In pathological conditions including inflammation and viral infections, GJ communication is reduced (Danave et al., 1994; Faccini et al., 1996; Rouach et al., 2002; Kielian and Esen, 2004; Knabb et al., 2007). However, HIV infection of astrocytes maintains expression of Cx43 and functional GJ communication allowing the spread of toxic signals generated from the few infected astrocytes to neighboring uninfected astrocytes (Eugenin and Berman, 2007). We now demonstrate that these few infected astrocytes have profound effects, through a GJ-dependent mechanism, on BBB integrity by dysregulating endothelial survival, the stability of astrocyte end feet, and their signaling.
Discussion
In this report, we demonstrate that HIV infection of astrocyte cultures, despite low numbers of infected cells (4.7 ± 2.8% in vitro and 8.2 ± 3.9% in vivo using an SIV model) and undetectable viral replication, when used to establish a culture model of the human BBB, results in BBB disruption by a mechanism that involves EC apoptosis and alterations in astrocyte end feet formation and signaling. All of these effects are mediated by functional GJ channels, because blocking these channels reduced the BBB disruption triggered by few infected astrocytes.
In vivo, microglial cells and perivascular macrophages are the predominant cell types productively infected by HIV-1 (Wiley et al., 1986). However, HIV infection of astrocytes has also been reported in vivo and in vitro, characterized by low viral replication and few infected cells (Tontsch and Bauer, 1991; Conant et al., 1994; Saito et al., 1994; Tornatore et al., 1994a,b; Ranki et al., 1995; Ohagen et al., 1999; Eugenin and Berman, 2007). HIV-1 mRNA and DNA have been detected in astrocytes within the adult and pediatric brains of individuals with AIDS (Wiley et al., 1986, 1991; Tornatore et al., 1994b), representing 1-3% of the total astrocytes (Nuovo et al., 1994; Bagasra et al., 1996; Takahashi et al., 1996). Recently, with improved techniques, HIV DNA has been detected in up to 19% of astrocytes in human brain sections (Churchill et al., 2009), suggesting that HIV-infected cells are active participants in the dysregulation of the CNS observed in HIV-infected individuals and serve as an important viral reservoir within the CNS. Our data using primary cultures of human astrocytes demonstrated that 4.7 ± 2.8% of the cells are infected after virus exposure and using an animal model of accelerated neuroAIDS, SIV-infected macaques, we demonstrated that at least 8.2 ± 3.9% of the astrocyte population are SIV infected during the time course of the disease (21-56 d after infection in the mild and severe encephalitic animals).
Our previous data indicated that HIV infection of human astrocytes resulted in bystander killing of uninfected neighboring astrocytes (Eugenin and Berman, 2007) and neurons (E. A. Eugenin and J. W. Berman, unpublished data) by a mechanism that involves functional gap junction channels to spread toxic signals (Eugenin and Berman, 2007). In vivo, at the BBB, astrocytes almost completely cover the endothelial cells with their end foot processes separated by a basement membrane (Kacem et al., 1998). Our understanding of the communication systems between astrocytes and BMVECs is increasing, especially the contribution of astrocyte end feet to BBB physiology (Abbott, 2002; Simard et al., 2003; Iadecola and Nedergaard, 2007; Girouard et al., 2010). The potential effects of other cell types at the BBB, such as pericytes, smooth muscle cells, and perivascular macrophages that also participate in regulating BBB function in normal and pathological conditions, should be considered in future experiments (Rubin and Staddon, 1999). In this study, we addressed some of the basic mechanisms by which HIV infection of even small numbers of astrocytes alters BBB integrity and function.
Endothelial apoptosis in neuroAIDS has been detected (Shi et al., 1996; Ullrich et al., 2000; Huang et al., 2001; Acheampong et al., 2005; Yano et al., 2007); however, the mechanisms that contribute to this toxicity are not well understood. We demonstrated that endothelial compromise induced by few infected astrocytes is dependent on functional astrocytic gap junctions because blocking these channels resulted in BBB protection. Using uninfected or HIV-infected astrocyte cultures to establish the human BBB model, we demonstrated that only a few HIV-infected astrocytes are sufficient to cause a profound effect on BBB integrity and endothelial apoptosis. The BBB disruption induced by few HIV-infected astrocytes in these cultures altered the guidance and functions of astrocyte end feet close to BMVECs and was also gap junction dependent. Astrocytic end feet have specialized features that contribute to the regulation of water homeostasis, calcium signaling, and regulation of blood flow, suggesting that these structures play a key role in communicating or amplifying astrocytic signals into BMVECs, despite the presence of the basement membrane (Kim et al., 2006). We identified particular signaling pathways, mainly concentrated in astrocyte end feet that contribute to BBB disruption. Under normal conditions, these pathways participate in control of vascular tone by an equilibrated production of both dilatory and constrictor agents (Paemeleire and Leybaert, 2000; Abbott, 2002; Simard et al., 2003; Zonta et al., 2003; Bogatcheva et al., 2005; Yakubu and Leffler, 2005; Kim et al., 2006; Iadecola and Nedergaard, 2007; Girouard et al., 2008). Using our BBB model, we showed that activation or blocking the activation of pathways required for control of blood flow, in the astrocyte side of the model, resulted in no significant changes in BBB permeability when inhibition of lipoxygenase, COX, high-conductance Ca2+-activated K+ (BKCa) channels, and purinergic ATP receptors, in cocultures with uninfected astrocytes did not alter BBB permeability. Addition of AA or ATP to the astrocyte layer resulted in BBB disruption. Blocking these individual signaling pathways in cocultures with HIV-infected astrocytes resulted in protection against BBB disruption, suggesting that multiple signaling pathways contribute to the disruption induced by few infected astrocytes. In agreement with our previous data, GJ blockers, AGA or Car, completely abolished BBB disruption induced by HIV-infected cultures of astrocytes as well as by direct application of COX activation subproduct, AA, or direct activation of ATP receptors, ATP, to the BBB using uninfected astrocytes, suggesting that gap junctional signaling is required for amplification of BBB dysfunction in the context of HIV infection and inflammation in the CNS. During the pathogenesis of many CNS diseases, dysregulation of these signaling pathways has been described, but they had not been examined in the context of BBB disruption and/or HIV infection. Increased arachidonic acid release, adenosine/ATP, prostaglandins, increased K+, and activation of BKCa channels have been demonstrated to participate in control of blood flow within the CNS (Benarroch, 2005; Bogatcheva et al., 2005; Yakubu and Leffler, 2005; Kim et al., 2006; Girouard et al., 2010). All these changes in signaling are triggered by excessive neuronal signaling, such as excitotoxicity, energy failure, ischemia, and neurodegeneration, all features characteristic of neuroAIDS. Thus, we propose that toxic signals generated in few HIV-infected astrocytes are spread by GJ to neighboring cells, and additionally to the astrocyte end feet, resulting in aberrant glial-vascular signaling resulting in BBB dysregulation and disruption. In addition to the bystander killing mediated by GJ, we cannot rule out the participation of Cx hemichannels, especially at the end feet of the astrocytes, because GJ blockers also block these hemichannels (Spray et al., 2006; Giaume and Theis, 2010; Hamilton and Attwell, 2010). Thus, as techniques to block specifically GJ or hemichannels become available, these questions can be addressed.
To demonstrate the presence of infected astrocytes in vivo and the potential contribution of these infected cells to the amplification of toxicity to neighboring cells, we used an animal model of accelerated neuroAIDS, SIV-infected macaques. Using this model, we detected significant numbers of SIV-infected astrocytes, 8.2 ± 3.9%, in close interaction with the endothelium as well as in the CNS parenchyma. These numbers are in agreement with those described for HIV-infected astrocytes in vivo and in vitro (Wiley et al., 1986, 1991; Tontsch and Bauer, 1991; Conant et al., 1994; Nuovo et al., 1994; Saito et al., 1994; Tornatore et al., 1994a,b; Ranki et al., 1995; Bagasra et al., 1996; Takahashi et al., 1996; Ohagen et al., 1999; Eugenin and Berman, 2007; Churchill et al., 2009). In addition, as in our in vitro experiments (Eugenin and Berman, 2007), we detected that SIV-infected astrocytes are not susceptible to apoptosis but that uninfected cells surrounding these clusters of infected cells apoptose extensively. Thus, our data in vivo indicate that, despite minimal SIV infection of astrocytes, significant changes in BBB integrity and apoptosis in parenchymal cells are observed. Our compelling data in vivo are strongly supported by our in vitro data that cell-to-cell killing is GJ dependent between HIV-infected and uninfected astrocytes.
We demonstrated that a few HIV-infected astrocytes trigger bystander cellular dysfunction and endothelial apoptosis, resulting in BBB compromise. Thus, despite the low number of HIV-infected astrocytes, considerable damage can be spread to uninfected cells by a gap junction-dependent mechanism. Our data suggest that astrocytes participate actively in the CNS dysfunction observed in HIV-infected individuals and that gap junction channels amplify toxicity and dysfunctional signaling to other areas of the brain. These data represent a novel mechanism in neuroAIDS and indicate potential new targets for therapeutic interventions to reduce the ongoing CNS effects of HIV and to eradicate CNS viral reservoirs.
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