Inflammation has been functionally defined as the host response to injury, meant to eliminate the cause of damage and promote tissue healing. However, several studies have incriminated inflammation in the pathogenesis of numerous chronic diseases such as cancer, diabetes, or neurodegenerative conditions. The limited regenerative capacity of neurons makes the CNS particularly susceptible to damage mediated by inflammation. For a long time it was assumed that the CNS is protected from extensive immunological responses due to restricted leukocyte trafficking from the periphery to the CNS, a process erroneously referred to as “immune-privilege”. Initially, CNS immune-privilege was attributed to CNS isolation from the immune system by the BBB, the lack of draining lymphatics, and the apparent immune-incompetence of microglia, the resident CNS macrophages. However, this view has been revisited based on data showing that peripheral immune cells can cross the intact BBB.
The CNS is populated by three different glial cell populations: (1) astrocytes, (2) microglia, and (3) oligodendrocytes. Glial cells are believed to form the immune system in the brain. Astrocytes are star-shaped and constitute the most abundant cell type found in the CNS; it has been suggested that there are approximately 10-times more astrocytes than neurons [58
], although a ratio of only 1.4 [60
] has also been indicated. Astrocytes have a number of important functions in the brain homeostasis, including maintenance of the BBB, regulation of neuronal blood flow, regulation of the extracellular potassium balance, providing metabolic support to neurons, and stimulation of myelin formation by oligodendrocytes. Although astrocytes can produce acute-phase proteins and some pro-inflammatory cytokines, in general they have limited innate immune properties. Astrocytes contribute to the maintenance of the BBB by creating a network of foot processes called the glia limitans. In this regard, astrocytes lie at the gateway to the CNS parenchyma and play a crucial role in controlling leukocyte influx.
Microglia are the resident macrophages of the brain and comprise 10–20% of all glial cells. Their main function is to monitor and maintain neuronal health. They are very sensitive and become readily activated by most neuropathologic conditions, including inflammatory disease and neuronal injury. When activated, microglia display distinct functional plasticity, such as changes in cell morphology, cell number, cell surface receptor expression, and production of growth factors and cytokines. It has been shown that microglia are immunocompetent, but differ from macrophages and dendritic cells in their ability to orchestrate neuroprotective lymphocyte responses. In addition, CNS neurons and glial cells have been shown to be capable of actively regulating macrophage and lymphocyte responses [61
Oligodendrocytes are a type of neuroglia found in the CNS of vertebrates and invertebrates. Their function is to produce myelin, which acts as an insulating sheath on the axons of nerve fibers. Oligodendrocytes can be distinguished from astrocytes by their greater density of both the cytoplasm and the nucleus, the absence of fibrils and glycogen in the cytoplasm, and the large numbers of microtubules in the processes. A single oligodendrocyte can extend its processes to approximately 50 axons, wrapping around approximately 1 μm of myelin sheath of each axon.
In mice, WNV crosses the BBB and infects the CNS after the peak viremia (around day 3) [62
]. Several studies showed that immune responses in the brain are necessary to clear the infection. How exactly recruitment of leukocytes from blood into the brain parenchyma is regulated is however not completely understood. Recent data suggest that leukocytes have to pass two barriers in order to arrive in the brain parenchyma: the endothelial cell vascular wall and the glial limitans, collectively referred to as BBB (). After transmigration of the vascular wall, the majority of infiltrating leukocytes are retained in the perivascular space, but the factors that regulate this process have not yet been elucidated.
Figure 2. Regulation of Blood-brain barrier permeability, entry of WNV in the brain and factors involved in the pathogenesis of WNV-induced neuroinvasisve disease. Following inoculation of WNV in the dermis, virus infects and replicates in cells of the mononuclear (more ...)
The chemokine system is a critical aspect of the immune response that controls the migration of leukocytes into the brain. During infection chemokines are expressed and specific subsets of leukocytes migrate from blood into the brain. The importance of chemokines in controlling WNV infection is exemplified by the fact that chemokine CXC motif receptor 3 (CXCR3) and C-C chemokine receptor type 5 (CCR5) knock-out mice cannot clear infection and result in increased mortality. There is a clear need to define and understand the relative importance of the molecules that control leukocyte entry into the brain. For instance, it has been shown that TNF-α increases the vascular permeability and allows penetration of leukocytes in the perivascular space.
Once in the perivascular space, immune cells need to pass the second barrier, the glia limitans. Few studies have focused on understanding the molecular and cellular mechanisms regulating leukocyte penetration through the glia limitans. Expression of the C-X-C motif chemokine 12 (CXCL12) at the perivascular space is probably responsible for retention of cells in the perivascular spaces of the CNS [64
]. In this regard, neutralization of CXCL12 was shown to promote leukocyte entry into the brain parenchyma [65
]. On the other hand, matrix metalloproteinases (MMP), produced both by monocytes and glia cells, have been shown to be involved in migration of leukocytes to the perivascular space as well as migration through the glia limitans [66
A study performed by Verma et al.
using HBMVE and HBCA, showed that several MMPs were significantly induced in WNV-infected HBCA cells [45
]. Incubation of naïve HBMVE cells with the supernatant from WNV-infected HBCA cells resulted in loss of tight junctions. These data provided evidence that astrocytes represent a source of MMP in the brain, which may lead to disruption of the BBB. Degradation of components of the glia limitans is another mechanism facilitating migration of leukocytes into the brain parenchyma. Collagen (a component of glia limitans) could be degraded by extracellular proteases such as the cysteine protease cathepsins K, S, and L [67
], whereas conversion of plasminogen into plasmin may lead to degradation of laminin or fibronectin, other important components of the glia limitans [67
]. When the integrity of the BBB is compromised, immune cells may enter the brain, thereby contributing to WNV viral clearance and immune mediated damage.