The choroid plexus in progressive multiple sclerosis

The choroid plexus is a tissue located in the brain ventricles with secretory, immunological and barrier functions in the central nervous system (CNS). The choroid plexus is highly vascularized and produces cerebrospinal fluid (CSF) that nourishes and protects the CNS. The choroidal stroma is surrounded by a layer of epithelial cells forming an interface with the periphery known as the blood-CSF barrier (BCSFB). Immune cells densely populate the choroid plexus and can traffic in and out of the CNS. The strategic position of the choroid plexus facilitates the monitoring of signals from the CSF and the response to neurological damage. Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the CNS that affects 2.8 million people worldwide. In MS, inflammatory processes destroy the myelin sheaths around neuronal axons. Damaged axons and inflammation eventually lead to neuronal death, particularly in progressive phases of the disease. MS lesions frequently occur in periventricular regions of the brain bathed by the CSF. The choroid plexus, as the primary producer of CSF and a barrier for immune cell trafficking, may have a role in MS. However, an in-depth characterization of the choroid plexus and its potential alterations in progressive MS was lacking. Sequencing technologies are a powerful tool to screen the transcriptome. In chapter 2, we review the knowledge and guidelines for using RNA-sequencing in brain barrier research. In chapter 3, we performed RNA-sequencing at the choroid plexus in progressive MS. We discovered alterations related to hypoxia, secretion and neuroprotection, but only subtle immunological and no barrier changes. A subset of the genes upregulated in progressive MS code for secreted proteins. These proteins have been assigned neuroprotective properties and may reach the brain via the flow of CSF. Several of the genes upregulated in progressive MS belong to the hypoxia-inducible factor (HIF) pathway (chapter 3). The exposure of choroid plexus explants to hypoxia resulted in a similar transcriptional profile to that seen in progressive MS. Thus, in progressive MS, a state of (virtual) hypoxia at the choroid plexus may activate the HIF pathway, leading to the secretion of neuroprotective factors. Moreover, our sequencing study detected a higher expression of HIF1A-AS3, a long non-coding RNA antisense of the HIF1A gene, in the choroid plexus from progressive MS donors relative to that of controls. Our initial results from chapter 4 suggest that HIF1A-AS3 is a target gene of the transcription factor HIF-1ɑ that exerts negative feedback control over the HIF pathway. Inflammation has also been reported at the choroid plexus in MS. First, we characterized the main immune cell populations with immunohistochemistry. Both in progressive MS donors and non-neurological controls, we found macrophages and dendritic cells abundantly populating the stroma (chapter 5). These antigen-presenting cells were often close to T cells, suggesting constitutive immunosurveillance. In progressive MS, CD8+ T cells, granulocytes, and CD56bright natural killer (NK) cells accumulated in the choroid plexus (chapters 5 and 6). Choroid plexus immune cells can influence periventricular regions of the brain locally –after migration across the BCSFB– or remotely –by the secretion of cytokines from the choroidal stroma. Using mass cytometry, we investigated the immune populations in the periventricular brain, choroid plexus and blood from MS donors and controls with or without neurological disease (chapter 6). We detected a CD56bright NK cell signature in the brain of progressive MS donors. Circulating CD56bright NK cells presented an activated and migratory phenotype suggesting direct and indirect infiltration through the blood-brain and blood-CSF barriers, respectively. In sum, this thesis provides a comprehensive overview of the choroid plexus immune populations and uncovers secretory and immunological alterations in progressive MS that can influence periventricular pathology.