The BBB acts as an interface between the periphery and the CNS and tightly regulates the components of the immune response to prevent unnecessary inflammation/pathology in the healthy brain. It is known that the nature of the vasculature and associated functions differ greatly depending upon their location in different CNS compartments 
. In a number of CNS infections, pial vessels of the BBB are particularly prone to disruption with leakage of leukocytes and serum components leading to meningitis 
. This increased vulnerability is possibly due to lack of additional barrier components and potential exposure to antigens compared with parenchymal vessels 
. Previously, gene expression analysis of endothelial cells has been performed either in an in vitro
setting or with whole brain endothelial cells 
, but not with endothelial cells present in specific anatomical compartments. Moreover, the effect of parasitic infection on endothelial cell biology has not been studied. The focus of this study was to characterize the infection-induced molecular signature of LCM isolated pial endothelial cells by evaluating global gene expression by microarray analyses.
LCM allowed us to isolate cells present in a specific location which has an added advantage over other marker-based techniques such as FACS. However, one pitfall is that the potential contamination of the BBB endothelium with the leukocyte that may be extravasating or adhering to the endothelial cells. Our data analysis confirmed that differential gene expression data obtained through microarray hybridization experiment is mainly contributed by endothelial cells comprising the BBB as common lymphoid or myeloid cell markers were not detectable in the data set. In addition, the expression of the following BBB specific transporter markers were induced during infection: TFRC (related to iron metabolism), ABCG1 (cholesterol homeostasis), SLC15A3 (proton oligopeptide co-transporters), SLC7A5 (cationic amino acid transporters and the glycoprotein-associated amino acid transporters), ABCC3 (multidrug resistance associated protein 3) and ABCC5 (multidrug resistance associated protein 5). Other BBB specific markers were downregulated including SLC9A3R2 (sodium/hydrogen exchanger), SLC6A9 (neurotransmitter transporter, glycine, sodium and chloride dependent neurotransmitter) 
Network analysis shows that apart from transporters several other sets of immune related genes including MRC1, complements (C3, C6, and C1R and complement factor properdin), TNF super family members and interferon inducible genes including STAT1 are induced in NCC infection which can potentially lead to endothelial cell activation 
. Interferon inducible genes have been shown to be induced in an in vitro
study with endothelial cells in HIV and Cryptococcus neoformans
infection model 
. STAT1 has been shown to promote inflammatory mediators and leukocyte transmigration at the BBB 
. Interferon signaling mediated through the Jak Stat pathway is critical to induce several of these genes in endothelial cells including chemokines and MHC class I antigen presentation related genes 
Among immune related genes chemokines play a critical role in leukocyte trafficking, differentiation and angiogenesis or angiostasis 
. Leukocyte trafficking is a multistep process in which chemokines induce the migration of leukocytes toward a chemokine gradient. Interaction between chemokines expressed by endothelial cells with their receptors on leukocytes triggers a signaling process that increases the avidity of integrin to their receptors on endothelial cells causing firm adhesion of leukocytes and facilitated transmigration towards chemokine gradient 
. Chemokines are divided into C, CC, CXC, and CX3C subgroups based on conserved cysteine residues 
. The present study advances the understanding about chemokine expression profile in endothelial cells comprising the BBB which are the first CNS cells to encounter peripheral leukocytes in vivo
. Many of the chemokines upregulated (Table S1
) in response to infection are summarized in along with their putative receptor and influence on specific leukocyte subsets.
Parasite infection induced chemokines in pial endothelial cells and their known role in leukocyte trafficking.
Our in vivo
and in vitro
data shows that CCL9 is expressed abundantly by endothelial cells and appears to coat the strands in a gradient fashion. Such strands have been observed in the areas of inflammation in other disease conditions such as EAE and toxoplasmic encephalitis 
. The origin and composition of these strands are still not clear. They have been described to extend from blood vessels to parenchyma and are thought to provide structural support for leukocytes migration 
. In the case of NCC, these strands coated with CCL9 might also provide a physical scaffold structure with a chemotactic signal for migration of leukocytes into the CNS. The functional correlation for CCL9 in terms of leukocyte subset recruitment remains to be defined in the CNS. However, in the periphery CCL9 has been implicated in recruitment of myeloid cells to peyers' patches and osteoclasts through the CCR1 receptor. Furthermore, it is also critical to recruit immature myeloid cells through CCR1receptor during liver metastasis 
. In addition, CCL17 and CCL22 are also noteworthy as they have been implicated in trafficking of CCR4 positive regulatory and Th2 T cells subsets 
. Chemokine can selectively influence the trafficking of leukocyte subsets. Therefore, the expression profile of chemokines in the BBB provides insight into the trafficking of different leukocyte subsets such as M1 and M2 macrophages, granulocytes, γδ T cells, αβ T cells and B cells known to infiltrate during NCC 
In summary, our data delineate infection-induced changes in the expression of genes associated with both immunity and disease, and collectively provide insight into the dysfunction of the BBB and mechanisms associated with leukocyte infiltration during murine NCC.