Acute viral infection of the central nervous system can induce a variety of disease states. A disease state of particular interest to our group stems from the ability of viruses to induce meningitis (or inflammation of the lining of the brain). It is estimated that viral meningitis is induced with a peak monthly incidence of 1 in 100,000 persons, particularly in temperate climates. The disease is associated with symptoms that include fever, headache, stiffness of the neck, and seizures. Enteroviruses are the most common cause of viral meningitis, accounting for approximately 75-90% of the cases. Other common meningitis-inducing viruses in humans include herpesviruses, human immunodeficiency virus-1, arbovirus, mumps virus, and lymphocytic choroimeningitis virus (LCMV). While complications associated with enterovirus-induced meningitis (the most common viral meningitis) in adults are rare, and are often seen in the immunocompromised, studies have shown that infection of children less that one year of age can result in mild to moderate neurological disability by the age of 5. On the other end of the spectrum, herpesviruses induce an array of CNS disorders that include encephalitis, myelitis, and meningitis, and these disorders have a very high mortality rate if left untreated. Because so many viruses have the capacity to induce meningitis and injure the CNS, it is important to uncover potential routes to pathogenesis in murine model systems. To gain novel mechanistic insights into CNS viral pathogenesis, our laboratory studies the LCMV model system. LCMV is an ambisense negative stranded RNA virus that is both a murine and human pathogen. Intracerebral inoculation of immunocompetent mice with LCMV induces a fatal choriomeningitis and is the disease for which LCMV is named. Importantly, LCMV does not kill the cells it infects in vivo, meaning that all cellular injury observed following infection is caused by the host immune system. The immune system is absolutely required for the convulsive seizures and fatalities observed during LCMV-induced meningitis. In fact, this disease is considered a prototypic cytotoxic lymphocyte (CTL)-dependent disorder because depletion of CTL converts LCMV infected mice to asymptomatic carriers. However, at the outset of our studies, the mechanism by which CTL caused fatal meningitis in the LCMV system was unknown. Over the past year, our laboratory set out to gain real time mechanistic insights into this disease process with the hope of better understanding the pathogenesis of viral meningitis in general. To accomplish this aim we utilized intravital two photon microscopy to watch viral meningitis unfold in real time. By injecting a fluorescent dye in the blood supply and labeling LCMV-specific CTL with green fluorescent protein (GFP), we were able to visualize through a surgically thinned skull the activities of the cells thought to be responsible for the disease. Our real time studies revealed a surprising and unexpected twist in the pathogenesis of this classic CTL-dependent disorder. Imaging of meningeal vasculature revealed obvious evidence of immunopathology. Many vessels were observed to be leaking blood-derived material into the subarachnoid space, which can cause fatal seizures. However, the GFP-labeled CTL were not associated in any way with the vascular injury. Moreover, genetic knockout of all the pathways known to be involved in CTL effector function (i.e., perforin, TNF-alpha, interferon-gamma, granzymes, Fas, degranulation) did not have any effect on the disease. These data suggested that CTL were not directly responsible for disease pathogenesis and might be operating through an indirect mechanism. To test this supposition, we examined the composition of the CNS infiltrate in LCMV-infected mice and observed an influx of monocytes and neutrophils (referred to as myelomonocytic cells) that coincided with the arrival of CTL. To define the activities of these cells at the site of disease, we examined GFP-tagged myelomonocytic cells at the peak of meningitis using two photon microscopy. Our real time studies revealed that the activities of myelomonocytic cells caused significant injury to meningeal blood vessels resulting in the leakage of blood-derived material into the subarachnoid space. Interestingly, neutrophils and monocytes appeared to accomplish this through non-overlapping mechanisms. Neutrophils caused sustained leakage of meningeal blood vessels by adhering to vascular endothelium and synchronously extravasating. Monocytes, on the other hand, caused transient breaches in blood vessel integrity by localizing perivascularly. We proved that both of these mechanisms of vascular injury were required for fatal convulsive seizures through depletion studies. Depletion of neutrophils or monocytes alone had no impact on disease;however, depletion of both populations simultaneously prevented fatal convulsive seizures and preserved vascular integrity. These data indicated that myelomonocytic cells rather than CTL were directly responsible for rapid onset disease observed during LCMV-induced meningitis. To examine the mechanistic link between CTL (adaptive immunity) and myelomonocytic cells (innate immunity), we used gene arrays to quantify chemoattractants that might draw myelomonocytic cells into the CNS following infection. Our results revealed that six chemokines (CCL2, CCL3, CCL4, CCL5, CCL7, and CXCL2) were upregulated at the peak of disease, and flow cytometry studies revealed that three of these chemokines (CCL3, 4, and 5) were directly produced by CNS-infiltrating virus-specific CTL. These data suggested that CTL caused disease by recruiting a massive number of myelomonocytic cells into the meningeal space, which then induced fatal vascular injury. It is presently unclear why CTL recruit myelomonocytic cells to a site of viral infection, but we predict that disconnecting (or at least reducing) the pathogenic link between the innate and adaptive immune system might represent a viable treatment option for viral meningitis as well as other infections of the CNS. We also predict that this novel mode of pathogenesis revealed in the LCMV model system exists in other disease states involving CTL. We are presently examining whether it is possible to decouple these two arms of the immune system to limit pathology and are trying to determine precisely how myelomonocytic cells cause such severe injury to meningeal vasculature. We are also examining interactions between CTL and the meningeal stromal network. During our studies we made the exciting observation that LCMV primarily infects stromal cells in the meninges. From an immunological perspective, the meningeal stromal network remains completely undefined, and these cells likely play a major role in shaping immune responses within the subarachnoid space. The overall goal of this project is to use a combination virological, molecular, immunological, and intravital imaging approaches to provide a comprehensive mechanistic understanding of how the innate and adaptive immune systems operate in the meninges following acute viral infection. Our long term hope is that these studies will translate into therapies to better manage acute CNS viral infections in humans.

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Russo, Matthew V; Latour, Lawrence L; McGavern, Dorian B (2018) Distinct myeloid cell subsets promote meningeal remodeling and vascular repair after mild traumatic brain injury. Nat Immunol 19:442-452
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