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Immune Privilege in the Brain, part II: The CNS Lymphatic System
There are more connections in the human body than there are stars in the galaxy. We possess a gigantic network of information to which we have almost no access.
– Professor Norman. Lucy, Universal Pictures.
In a previous blog, we discussed the blood-brain barrier (BBB) and how the endothelial cells surrounding cerebral capillaries create tight junctions that are very selective in what passes through. This creates a privileged immune state in the brain where peripheral immune cells are not able to communicate directly with the central nervous system (CNS). Instead, the CNS depends largely on glial cells to clear pathogens in addition to homeostatic functions. Though this system works well for the most part, a breach of the BBB can lead to infiltration of immune cells into the CNS, starting or propagating an immune response that can cause neuronal damage. The belief that the CNS and peripheral immune cells are completely separated, and that the brain doesn't have a method of access or communication with circulating immune cells, has been widely held for nearly a century. However, as evident in most of biology, this system is much more complex than previously thought.

Lord of the Rings: The Fellowship of the Ring, New Line Cinema.
Though the CNS has been widely thought to not have access to the lymphatic circulation, it has been known that the CNS was able to access and communicate with antigen-presenting cells (APCs) in order to provide CNS antigens. The CNS is able to do this because the interstitial fluid, the fluid that surrounds cells, of the CNS can contain soluble antigens that drain into the cerebrospinal fluid (CSF). The CSF can ultimately deliver these antigens to deep cervical lymph nodes in the nasal mucosa, where B cells produce CNS antigen-specific antibodies. Though the mechanism behind this was not known, it was thought that these antigens are presented by meningeal macrophages that travel with the CSF1.

Cross-section of meninges. Grey’s Anatomy, 2nd Edition.
In a groundbreaking paper recently published in Nature, Antoine Louveau and Jonathan Kipnis, and their associates at the University of Virginia, set out to further understand how immune surveillance occurs within the CNS and how it is able to share antigens with peripheral immune cells2. Seeking to further characterize the immune cell interaction that occurs in meningeal spaces, they performed whole-mounts on mouse meninges, a procedure developed by Louveau, a post-doc in the Kipnis Lab, which allowed examination of the meninges on a single slide3. To do this, Louveau fixed the meninges in place on the skullcap prior to dissection, allowing it to keep its structure. Through in-depth immunohistological analysis, they determined that there were restricted areas of the meninges in which T cells and APCs (as indicated by CD3 and MHCII expression, respectively) were clustered around the dural sinuses, which drain blood from veins in the brain to the jugular. Interestingly, staining for endothelial cells with CD31 showed that these T cells and APCs were linearly aligned with some sort of vessel.

Confocal image of CD3+ T cells trafficking along meningeal lymphatics. Lyve-1 (green), CD3e (red), and DAPI (blue). Source: Tjk4a
Upon seeing that few of these immune cells were in the actual blood vessels, the group next stained for lymphatic vessel endothelial hyaluronan receptor 1 (Lyve-1), a marker of lymphatic endothelial cells (LECs). Indeed, there existed lymphatic vessels in the dural sinuses that had characteristics of classical lymphatic vessels, including PROX1 (a LEC transcription factor), podoplanin, and VEGFR3 expression. This was incredibly exciting, as it completely rejected the well-established dogma of no lymphatic system existing within the brain.
Louveau did a number of additional experiments that confirmed the Lyve-1+ lymphatic vessels they found were separate from the cardiovascular system of their mice through intracranial injections of one dye in comparison to intravenous infusion of a different dye. Then, the group found that staining for CD3+ T cells, MHCII+ APCs, and B220+ B cells co-localized with Lyve-1 expression, suggesting that these cells could traffic along the lymphatic vessels of the brain, eventually draining to the deep cervical lymph nodes. For the first time, the field is able to explain how cerebrospinal fluid is able to initiate an immune response in other tissues, even though the CNS was considered separate from peripheral tissues and circulatory systems.

One might wonder that after over a century of research, why did it take so long to discover this missing link in CNS immunosurveillance, when all of the techniques of analysis (such as immunohistochemistry) have existed for decades. Hasn’t the entire body been mapped out by now? In this situation, the lymphatic vessels were so well hidden that they were easily mistaken for blood vessels, and the technique of whole-mounting meninges finally allowed for them to be examined separately.

The Matrix. Village Roadshow Pictures.
The implications of the discovery are limitless, and will require reanalysis of the current literature for a number of neurological disorders, such as multiple sclerosis and Alzheimer's. This could allow for the targeting of immune cells in the brain by honing therapies to the CNS lymphatic system, and potentially provide an additional method of therapeutic delivery to the brain by subverting the blood-brain barrier. It also opens up additional areas of research for any neurological condition that involves neuroinflammation, and allows us to re-examine questions regarding how the immune component of these diseases is communicating and affecting the neurons and other cells of the CNS.

Any comments on this groundbreaking discovery? Let us know at
  1. Anatomical and cellular basis of immune surveillance in CNS, Nature Reviews Immunology
  2. Structural and functional features of CNS lymphatic vessels, Nature
  3. Missing Link Between Brain and Immune System, Neuroscience News

Time to go to the next level of research...
Contributed by Ed Chen, PhD.
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