Pattern recognition receptor (PRR) detection of pathogen-associated molecular patterns (PAMPs), such as viral RNA, drives innate immune responses against West Nile virus (WNV), an emerging neurotropic pathogen. Here we demonstrate that WNV PAMPs orchestrate endothelial responses to WNV via competing innate immune cytokine signals at the blood-brain barrier (BBB), a multicellular interface with highly specialized brain endothelial cells that normally prevents pathogen entry. While Th1 cytokines increase the permeability of endothelial barriers, type I interferon (IFN) promoted and stabilized BBB function. Induction of innate cytokines by pattern recognition pathways directly regulated BBB permeability and tight junction formation via balanced activation of the small GTPases Rac1 and RhoA, which in turn regulated the transendothelial trafficking of WNV. In vivo, mice with attenuated type I IFN signaling or IFN induction (Ifnar−/− Irf7−/−) exhibited enhanced BBB permeability and tight junction dysregulation after WNV infection. Together, these data provide new insight into host-pathogen interactions at the BBB during neurotropic viral infection.

During CNS autoimmunity, brain endothelial cell CXCR7 internalizes CXCL12 from the perivascular space, thereby permitting leukocyte migration into the CNS parenchyma.

Multiple sclerosis (MS) is an inflammatory disease of the CNS that is characterized by BBB dysfunction and has a much higher incidence in females. Compared with other strains of mice, EAE in the SJL mouse strain models multiple features of MS, including an enhanced sensitivity of female mice to disease; however, the molecular mechanisms that underlie the sex- and strain-dependent differences in disease susceptibility have not been described. We identified sphingosine-1-phosphate receptor 2 (S1PR2) as a sex- and strain-specific, disease-modifying molecule that regulates BBB permeability by destabilizing adherens junctions. S1PR2 expression was increased in disease-susceptible regions of the CNS of both female SJL EAE mice and female patients with MS compared with their male counterparts. Pharmacological blockade or lack of S1PR2 signaling decreased EAE disease severity as the result of enhanced endothelial barrier function. Enhanced S1PR2 signaling in an in vitro BBB model altered adherens junction formation via activation of Rho/ROCK, CDC42, and caveolin endocytosis-dependent pathways, resulting in loss of apicobasal polarity and relocation of abluminal CXCL12 to vessel lumina. Furthermore, S1PR2-dependent BBB disruption and CXCL12 relocation were observed in vivo. These results identify a link between S1PR2 signaling and BBB polarity and implicate S1PR2 in sex-specific patterns of disease during CNS autoimmunity.

Background: A number of prostaglandins (PGs) sensitize dorsal root ganglion (DRG) neurons and contribute to inflammatory hyperalgesia by signaling through specific G protein-coupled receptors (GPCRs). One mechanism whereby PGs sensitize these neurons is through modulation of "thermoTRPs," a subset of ion channels activated by temperature belonging to the Transient Receptor Potential ion channel superfamily. Acrid, electrophilic chemicals including cinnamaldehyde (CA) and allyl isothiocyanate (AITC), derivatives of cinnamon and mustard oil respectively, activate thermoTRP member TRPA1 via direct modification of channel cysteine residues.

The immortalized human cerebral microvascular endothelial cell line HCMEC/D3 presents a less expensive and more logistically feasible alternative to primary human brain microvascular endothelial cells (HBMEC’s) for use in constructing in vitro models of the blood brain barrier (BBB). However, the fidelity of the HCMEC/D3 cell line to primary HBMEC’s in studies of immune transmigration has yet to be established. Flow cytometric analysis of primary human leukocyte migration across in vitro BBB’s generated with either HCMEC/D3 or primary HBMEC’s revealed that HCMEC/D3 maintains the immune barrier properties of primary HBMEC’s. Leukocyte migration responses and inflammatory cytokine production were statistically indistinguishable between both endothelial cell types, and both cell types responded similarly to astrocyte coculture, stimulation of leukocytes with phorbol myristate acetate (PMA) and ionomycin, and inflammatory cytokine treatment. This report is the first to validate the HCMEC/D3 cell line in a neuroimmunological experimental system via direct comparison to primary HBMEC’s, demonstrating remarkable fidelity in terms of barrier resistance, immune migration profiles, and responsiveness to inflammatory cytokines. Moreover, we report novel findings demonstrating that interaction effects between immune cells and resident CNS cells are preserved in HCMEC/D3, suggesting that important characteristics of neuroimmune interactions during CNS inflammation are preserved in systems utilizing this cell line. Together, these findings demonstrate that HCMEC/D3 is a valid and powerful tool for less expensive and higher throughput in vitro investigations of immune migration at the BBB.