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Considerable clinical evidence supports that increased blood–brain barrier (BBB) permeability is linked to immune extravasation of CNS parenchyma during neuroinflammation. Although BBB permeability and immune extravasation are known to be provoked by vascular endothelial growth factor-A (i.e., VEGF-A) and C-X-C motif chemokine ligand 12 (CXCL12), respectively, the mechanisms that link both processes are still elusive. The interleukin-20 (i.e., IL-20) cytokine signaling pathway was previously implicated in VEGF-mediated angiogenesis and is known to induce cellular response by way of signaling through IL-20 receptor subunit β (i.e., IL-20RB). Dysregulated IL-20 signaling is implicated in many inflammatory pathologies, but it’s contribution to neuroinflammation has yet to be reported. We hypothesize that the IL-20 cytokine, and the IL cytokine subfamily more broadly, play a key role in CNS neuroinflammation by signaling through IL-20RB, induce VEGF activity, and enhance both BBB-permeability and CXCL12-mediated immune extravasation. To address this hypothesis, we actively immunizedIL-20RB–/–mice and wild-type mice to induce experimental autoimmune encephalomyelitis (EAE) and found thatIL-20RB–/–mice showed amelioration of disease progression compared to wild-type mice. Similarly, we passively immunizedIL-20RB–/–mice and wild-type mice with myelin-reactive Th1 cells from eitherIL-20RB–/–and wild-type genotype. HostIL-20RB–/–mice showed lesser disease progression than wild-type mice, regardless of the myelin-reactive Th1 cells genotype. Using multianalyte bead-based immunoassay and ELISA, we found distinctive changes in levels of pro-inflammatory cytokines betweenIL-20RB–/–mice and wild-type mice at peak of EAE. We also found detectable levels of all cytokines of the IL-20 subfamily within CNS tissues and specific alteration to IL-20 subfamily cytokines IL-19, IL-20, and IL-24, expression levels. Immunolabeling of CNS region-specific microvessels confirmed IL-20RB protein at the spinal cord microvasculature and upregulation during EAE. Microvessels isolated from macaques CNS tissues also expressed IL-20RB. Moreover, we identified the expression of all IL-20 receptor subunits: IL-22 receptor subunit α-1 (IL-22RA1), IL-20RB, and IL-20 receptor subunit α (IL-20RA) in human CNS microvessels. Notably, human cerebral microvasculature endothelial cells (HCMEC/D3) treated with IL-1β showed augmented expression of the IL-20 receptor. Lastly, IL-20-treated HCMEC/D3 showed alterations on CXCL12 apicobasal polarity consistent with a neuroinflammatory status. This evidence suggests that IL-20 subfamily cytokines may signal at the BBBviaIL-20RB, triggering neuroinflammation.

Background Current methods for murine brain microvasculature isolation requires the pooling of brain cortices while disregarding the rest of the CNS, making the analysis of single individuals non feasible. New method Efficient isolation of brain microvessels requires the elimination of meninges, vessels of high caliber vessels and choroid plexus, commonly done by rolling the over filter paper, but can’t be done on other CNS regions. We overcome this hurdle by using a double-pronged pick, as well as elution and filtration through cell strainers after centrifugation. Results We were able to develop a region-specific murine CNS microvessels isolation, that allows for the comparison of the neurovascular unit from these regions both within the same individual and between multiple individuals and/or treatment groups without pooling. Additionally, we were able to adapt this method to macaque CNS tissue. Comparison with existing method(s) Although similar to a previously published method that requires no enzymatic dissociation and no ultracentrifugation, it does differ in its ability to isolate from a single experimental animal and from non-cortical tissues. However, it relies heavily on the researcher dissecting skills and careful elution and filtration of re-suspended samples. Conclusions CNS region-specific microvessels comparison can inform of molecular and/or cellular differences that would otherwise be obscured by excluding non-cortical tissue. Additionally, it allows for the unmasking of variations between individuals that remained hidden when pooling of multiple samples is the norm. Lastly, isolation of region-specific microvessels for non-human primate CNS allows for more translationally relevant studies of the BBB.

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often rodents. This approach limits the interrogation of blood-brain barrier (BBB) properties to the cortex and does not allow for individual comparison. This project focuses on the development of an isolation method that allows for the comparison of the neurovascular unit (NVU) from multiple CNS regions: cortex, cerebellum, optic lobe, hypothalamus, pituitary, brainstem, and spinal cord. Moreover, this protocol, originally developed for murine samples, was successfully adapted for use on CNS tissues from small and large vertebrate species from which we are also able to isolate microvessels from brain hemisphere white matter. This method, when paired with immunolabeling, allows for quantitation of protein expression and statistical comparison between individuals, tissue type, or treatment. We proved this applicability by evaluating changes in protein expression during experimental autoimmune encephalomyelitis (EAE), a murine model of a neuroinflammatory disease, multiple sclerosis. Additionally, microvessels isolated by this method could be used for downstream applications like qPCR, RNA-seq, and Western blot, among others. Even though this is not the first attempt to isolate CNS microvessels without the use of ultracentrifugation or enzymatic dissociation, it is unique in its adeptness for the comparison of single individuals and multiple CNS regions. Therefore, it allows for investigation of a range of differences that may otherwise remain obscure: CNS portions (cortex, cerebellum, optic lobe, brainstem, hypothalamus, pituitary, and spinal cord), CNS tissue type (gray or white matter), individuals, experimental treatment groups, and species.