**4. Astrocytes**

90 Non-Flavivirus Encephalitis

determine the reasons underlying increased monocyte migration into brain following lentiviral infection. HIV-infected leukocytes are primed for adhesion (Hallett, 1995), having already shed L-selectin, and increased expression of CD11b/CD18 compared with monocytes from healthy controls (Elbim et al., 1999). Therefore, it is possible that even barely increased levels of chemokines expressed within the parenchyma would lead to increased migration of monocytes. Recent studies have shown that glial cells are stimulated to produce chemokines in response to inflammatory cytokines (Renner et al., 2011, Thompson and Van Eldik, 2009) known to be secreted by SIV-infected macrophages (Orandle et al., 2002). Therefore, the role played by glial cells and tight junctions requires

Fig. 1. Schematic of key players in the development of SIV encephalitis. At left, a cutaway section of a cerebral microvessel showing circulating blood cells. At center, normal blood vessel with tight junctions (solid lines) evident between endothelial cells. Astrocyte foot processes are highly evident with occasional microglia. Low levels of cytokines and chemokines are expressed. At right a microvessel in an encephalitic lesion, showing disrupted junctions (dashed lines), leakage of serum proteins into parenchyma, displaced astrocyte foot processes and increased cytokines/ chemokines concomitant with increased

The primary defining feature of the blood brain barrier (BBB) is the presence of tight junction proteins between brain microvascular endothelial cells (BMEC). Immediately

further discussion.

numbers of perivascular macrophages.

**3.1 Tight junction proteins** 

### **4.1 Summary of astrocytes in encephalitis**

As illustrated in Figure 2, astrocyte foot processes are closely apposed to BMEC and ensheath more than 60% of the vessel exterior (Mathiisen et al., 2010). Through these contacts astrocytes are able to affect changes in BBB integrity during health and disease, and recruit or repel inflammatory cells through cytokines (Figure 2).

### **4.2 Role of astrocytes in BBB physiology**

The BBB is formed during early infancy in primates (Bayer et al., 1993). The exact mechanisms underlying BBB formation are not clear, but it is known that astrocytes are critical in both maturation and maintenance of the barrier integrity (Willis et al., 2004, Al Ahmad et al., 2010). Astrocytes also act to repel circulating immune cells through secretion

Blood-Brain Barrier Disruption and Encephalitis in Animal Models of AIDS 93

(McKimmie and Graham, 2010). Below we discuss key cytokines and chemokines that are

Productively-infected macrophages in the encephalitic brain express Tumor Necrosis Factor alpha (TNF- (Orandle et al., 2002). TNF- receptors are present in the non-encephalitic brain (Shaw and Greig, 1999), such that normal brains are primed to respond quickly to low levels of TNF-. TNF- induces increased chemokine production and secretion by astrocytes, and these chemokines induce monocyte migration preferentially over

Vascular Endothelial Growth Factor (VEGF) promotes proliferation of BMEC, resulting in reorganization of the cytoskeleton and TJ proteins. This induces a decrease in BBB integrity, creating a permissive environment for monocyte migration, and also bidirectional leakage of proteins across the BBB. A possible mechanism for the VEGF pathway could be as follows: tat binds to the VEGF receptor (Nyagol et al., 2008). The VEGF receptor binds to focal adhesion kinase (Garces et al., 2006), increases of which have been implicated in BBB

Other pro-inflammatory cytokines, including interferon- and IL-6 are upregulated in the encephalitic brain, with far-reaching effects in neuroinflammatory events (Roberts et al., 2004a). The complement pathway is also known to be induced through interferon- and IL-6

An early study of chemokine expression in brains of macaques infected with SIV showed increased CCLs 3-5 & 7, and CXCL10 (Sasseville et al., 1996), although no increase in CCLs 2 or 8 nor CXCL8 was observed in this definitive study, other later studies have "muddied the waters" somewhat: Penton-Rol used dexamethasone to stimulate cells to have increased CCL2 receptors before infecting with HIV 89.6 (Penton-Rol et al., 1999). The Clements group at Johns Hopkins has shown increased CCL2 mRNA in brain extracts using a highly accelerated encephalitis model (Witwer et al., 2009), although mRNA does not always equate with secreted protein. Additionally, the Berman group at Einstein College of Medicine has shown numerous effects of CCL2 on HIV-infected macrophages (Eugenin et al., 2003, Eugenin et al., 2006). CCL2 was among several chemokines in CSF that was not upregulated in one study using humans infected with HIV (Kolb et al., 1999), although IP-10 was upgregulated. In contrast, CCL2 was increased in pigtail macaques that develop

The precise cell types producing these chemokines were not identified in these studies. CCL2 mRNA was upregulated in cultured astrocytes, but remained at low levels compared

Even under noninflamed conditions CCL7 is expressed in the brain (Renner et al., 2011, Sasseville et al., 1996), which could contribute to basal levels of monocyte migration into the brain for "routine surveillance" (Williams and Hickey, 1995). That CCL7 is upregulated by astrocytes in response to cytokines present in encephalitic brains gives a potential role for controlling monocyte migration during encephalitis as well (Sasseville et al., 1996, Renner et

to CCL7, suggesting a role for CCL7 in HIV-related encephalitis (Renner et al., 2011).

signaling, propagating inflammation in the area surrounding a lesion/lesions.

**4.3.3 Expression and secretion of selected chemokines** 

thought to play a role in SIVE/HIVE.

lymphocytes (Renner et al., 2011).

disruption (Ivey et al., 2009b).

encephalitis (Mankowski et al., 2004).

al., 2011).

**4.3.2 Expression and secretion of selected cytokines** 

of eotaxin (Cardona et al., 2003), reinforcing the brain's immune-privileged status in conjunction with the selective physical properties of the BBB.

Fig. 2. Schematic of role of astrocytes in pathogenesis. Normal astrocytes (at left) have foot processes ensheathing over 60% of the endothelium and express low levels of GFAP and cytokines / chemokines. In encephalitis, there can a loss of connection to the endothelial cells, increased cytokine / chemokine secretion and altered expression of intermediate filaments, including GFAP and peripherin (right).

### **4.3 Astrocytes and signaling in encephalitis**

Astrocytes are the primary cell type found in glia scar formation (Voskuhl et al., 2009, Kielian, 2004), and secrete cytokines and chemokines to elicit increased trafficking of leukocytes into the brain (Renner et al., 2011, Cota et al., 2000, Eugenin et al., 2006). Astrocytes also may provide a role for the resolution of inflammation by reducing the secretion of pro-inflammatory cytokines and increasing anti-inflammatory processes (Kielian, 2004, Hauwel et al., 2005, Park et al., 2003).

Decreased BBB integrity early in SIV/HIV infection allows latently-infected monocytes to enter the brain (Fischer-Smith and Rappaport, 2005). Circulating virus could induce BMEC to express CD106 diffusely (Sasseville et al., 1995, Sasseville et al., 1992) leading to increased monocyte migration into brain, where they become productively infected. Astrocytes respond to these macrophages resulting in a wide-range of cellular changes referred to as astrogliosis.

#### **4.3.1 Astrogliosis**

On activation, astrocytes undergo a morphological change: most notably an increase in ramification concomitant with upregulation of GFAP, and thickened processes. We have also observed some astrocytes in the proximity of SIV lesions to express peripherin, an alternative type III intermediate filament not normally expressed in brain (Mathew et al., 2001). Immunologically, astrocytes respond to HIV/ SIV infection through increased production of inflammatory cytokines. As outlined above, the predominate inflammatory cell type in HIVE/SIVE is the monocyte-derived macrophage. The chemokines upregulated by astrocytes in HIVE/SIVE are largely specific to monocyte/macrophages (Renner et al., 2011, Sasseville et al., 1996). This suggests the possibility of a positive feedback system being initiated: a productively-infected macrophage induces nearby astrocytes to upregulate secretion of macrophage-specific chemokines, leading to lesion formation.

The cytokine response of astrocytes includes a cornucopia of molecules including a variety of cytokines and chemokines. It is intriguing that astrocytes will secrete a different "barcode" of cytokines and chemokines in response to different classes of stimuli (McKimmie and Graham, 2010). Below we discuss key cytokines and chemokines that are thought to play a role in SIVE/HIVE.

#### **4.3.2 Expression and secretion of selected cytokines**

92 Non-Flavivirus Encephalitis

of eotaxin (Cardona et al., 2003), reinforcing the brain's immune-privileged status in

Fig. 2. Schematic of role of astrocytes in pathogenesis. Normal astrocytes (at left) have foot processes ensheathing over 60% of the endothelium and express low levels of GFAP and cytokines / chemokines. In encephalitis, there can a loss of connection to the endothelial cells, increased cytokine / chemokine secretion and altered expression of intermediate

Astrocytes are the primary cell type found in glia scar formation (Voskuhl et al., 2009, Kielian, 2004), and secrete cytokines and chemokines to elicit increased trafficking of leukocytes into the brain (Renner et al., 2011, Cota et al., 2000, Eugenin et al., 2006). Astrocytes also may provide a role for the resolution of inflammation by reducing the secretion of pro-inflammatory cytokines and increasing anti-inflammatory processes

Decreased BBB integrity early in SIV/HIV infection allows latently-infected monocytes to enter the brain (Fischer-Smith and Rappaport, 2005). Circulating virus could induce BMEC to express CD106 diffusely (Sasseville et al., 1995, Sasseville et al., 1992) leading to increased monocyte migration into brain, where they become productively infected. Astrocytes respond to these macrophages resulting in a wide-range of cellular changes referred to as

On activation, astrocytes undergo a morphological change: most notably an increase in ramification concomitant with upregulation of GFAP, and thickened processes. We have also observed some astrocytes in the proximity of SIV lesions to express peripherin, an alternative type III intermediate filament not normally expressed in brain (Mathew et al., 2001). Immunologically, astrocytes respond to HIV/ SIV infection through increased production of inflammatory cytokines. As outlined above, the predominate inflammatory cell type in HIVE/SIVE is the monocyte-derived macrophage. The chemokines upregulated by astrocytes in HIVE/SIVE are largely specific to monocyte/macrophages (Renner et al., 2011, Sasseville et al., 1996). This suggests the possibility of a positive feedback system being initiated: a productively-infected macrophage induces nearby astrocytes to upregulate

The cytokine response of astrocytes includes a cornucopia of molecules including a variety of cytokines and chemokines. It is intriguing that astrocytes will secrete a different "barcode" of cytokines and chemokines in response to different classes of stimuli

secretion of macrophage-specific chemokines, leading to lesion formation.

conjunction with the selective physical properties of the BBB.

filaments, including GFAP and peripherin (right).

(Kielian, 2004, Hauwel et al., 2005, Park et al., 2003).

astrogliosis.

**4.3.1 Astrogliosis** 

**4.3 Astrocytes and signaling in encephalitis** 

Productively-infected macrophages in the encephalitic brain express Tumor Necrosis Factor alpha (TNF- (Orandle et al., 2002). TNF- receptors are present in the non-encephalitic brain (Shaw and Greig, 1999), such that normal brains are primed to respond quickly to low levels of TNF-. TNF- induces increased chemokine production and secretion by astrocytes, and these chemokines induce monocyte migration preferentially over lymphocytes (Renner et al., 2011).

Vascular Endothelial Growth Factor (VEGF) promotes proliferation of BMEC, resulting in reorganization of the cytoskeleton and TJ proteins. This induces a decrease in BBB integrity, creating a permissive environment for monocyte migration, and also bidirectional leakage of proteins across the BBB. A possible mechanism for the VEGF pathway could be as follows: tat binds to the VEGF receptor (Nyagol et al., 2008). The VEGF receptor binds to focal adhesion kinase (Garces et al., 2006), increases of which have been implicated in BBB disruption (Ivey et al., 2009b).

Other pro-inflammatory cytokines, including interferon- and IL-6 are upregulated in the encephalitic brain, with far-reaching effects in neuroinflammatory events (Roberts et al., 2004a). The complement pathway is also known to be induced through interferon- and IL-6 signaling, propagating inflammation in the area surrounding a lesion/lesions.

#### **4.3.3 Expression and secretion of selected chemokines**

An early study of chemokine expression in brains of macaques infected with SIV showed increased CCLs 3-5 & 7, and CXCL10 (Sasseville et al., 1996), although no increase in CCLs 2 or 8 nor CXCL8 was observed in this definitive study, other later studies have "muddied the waters" somewhat: Penton-Rol used dexamethasone to stimulate cells to have increased CCL2 receptors before infecting with HIV 89.6 (Penton-Rol et al., 1999). The Clements group at Johns Hopkins has shown increased CCL2 mRNA in brain extracts using a highly accelerated encephalitis model (Witwer et al., 2009), although mRNA does not always equate with secreted protein. Additionally, the Berman group at Einstein College of Medicine has shown numerous effects of CCL2 on HIV-infected macrophages (Eugenin et al., 2003, Eugenin et al., 2006). CCL2 was among several chemokines in CSF that was not upregulated in one study using humans infected with HIV (Kolb et al., 1999), although IP-10 was upgregulated. In contrast, CCL2 was increased in pigtail macaques that develop encephalitis (Mankowski et al., 2004).

The precise cell types producing these chemokines were not identified in these studies. CCL2 mRNA was upregulated in cultured astrocytes, but remained at low levels compared to CCL7, suggesting a role for CCL7 in HIV-related encephalitis (Renner et al., 2011).

Even under noninflamed conditions CCL7 is expressed in the brain (Renner et al., 2011, Sasseville et al., 1996), which could contribute to basal levels of monocyte migration into the brain for "routine surveillance" (Williams and Hickey, 1995). That CCL7 is upregulated by astrocytes in response to cytokines present in encephalitic brains gives a potential role for controlling monocyte migration during encephalitis as well (Sasseville et al., 1996, Renner et al., 2011).

Blood-Brain Barrier Disruption and Encephalitis in Animal Models of AIDS 95

endothelial cells to form tubes with astrocytes extending processes to induce tight junction proteins (Al Ahmad et al., 2010). Collagen gels were used to create 3D cultures with BMEC and astrocytes. Al Ahmad et al. showed that BMEC alone were unable to localize tight junction proteins to the cell border. Coculture with astrocytes corrected this, with Claudin5 and ZO1 localized to functionally relevant positions. This clearly demonstrates the necessity for including astrocytes in BBB culture models. As of yet, these models have not been

A further model that has been utilized is slice cultures (Renner et al., 2011, Noraberg, 2004). These *ex vivo* models are essentially a complex co-culture that preserves cell:cell ratios, and functional spatial relationships. This model allows one to determine precise cell types secreting chemokines in response to viral-infected cells. It will also prove useful for

Under normal conditions the brain allows only limited access by immune cells. Early in HIV infection the virus enters the brain through normal trafficking. This leads to a transient increase in BBB permeability, and a localized immune response. As the disease progresses to encephalitis the immune response is dramatically increased, marked by a loss of tight junction integrity, gliosis, and formation of multinucleated giant cells in the parenchyma. The parallel between the neuropathogenesis of HIV in humans, and SIV in the rhesus macaque has led to the establishment of rhesus macaque as the predominant *in vivo* model for HIVE. The use of *in vitro* models allows for precise control for investigating pathways of

Supported by: This work was supported in part by PHS grants RR00164, MH077544 (AGM),

al Ahmad, A., Taboada, C. B., Gassmann, M. & Ogunshola, O. O. 2010. Astrocytes and

Andras, I. E., Pu, H., Deli, M. A., Nath, A., Hennig, B. & Toborek, M. 2003. HIV-1 Tat protein

Annunziata, P. 2003. Blood-brain barrier changes during invasion of the central nervous system by HIV-1. Old and new insights into the mechanism. *J Neurol,* 250, 901-6. Bayer, S. A., Altman, J., Russo, R. J. & Zhang, X. 1993. Timetables of neurogenesis in the

Biegel, D. & Pachter, J. S. 1994. Growth of brain microvessel endothelial cells on collagen

pericytes differentially modulate blood-brain barrier characteristics during

alters tight junction protein expression and distribution in cultured brain

human brain based on experimentally determined patterns in the rat.

gels: applications to the study of blood-brain barrier physiology and CNS

Louisiana Board of Regents Fellowship LEQSF(2007-2012)-GF15 (NAR).

development and hypoxic insult. *J Cereb Blood Flow Metab*.

inflammation. *In Vitro Cell Dev Biol Anim,* 30A, 581-8.

endothelial cells. *J Neurosci Res,* 74, 255-65.

*Neurotoxicology,* 14, 83-144.

applied to encephalitis studies.

lentiviral neuropathogenesis.

**8. Acknowledgements** 

**9. References** 

mechanistic studies of neuropathogenesis.

**7. Summary of SIV model of encephalitis** 
