Neuroinflammatory disease disrupts the blood-CNS barrier via crosstalk between proinflammatory and endothelial-to-mesenchymal-transition signaling.

Breakdown of the blood-central nervous system barrier (BCNSB) is a hallmark of many neuroinflammatory disorders, such as multiple sclerosis (MS). Using a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), we show that endothelial-to-mesenchymal transition (EndoMT) occurs in the CNS before the onset of clinical symptoms and plays a major role in the breakdown of BCNSB function. EndoMT can be induced by an IL-1ß-stimulated signaling pathway in which activation of the small GTPase ADP ribosylation factor 6 (ARF6) leads to crosstalk with the activin receptor-like kinase (ALK)-SMAD1/5 pathway. Inhibiting the activation of ARF6 both prevents and reverses EndoMT, stabilizes BCNSB function, reduces demyelination, and attenuates symptoms even after the establishment of severe EAE, without immunocompromising the host. Pan-inhibition of ALKs also reduces disease severity in the EAE model. Therefore, multiple components of the IL-1ß-ARF6-ALK-SMAD1/5 pathway could be targeted for the treatment of a variety of neuroinflammatory disorders.

The CSF1R-Microglia Axis Has Protective Host-Specific Roles During Neurotropic Picornavirus Infection.

Viral encephalitis is a major cause of morbidity and mortality, but the manifestation of disease varies greatly between individuals even in response to the same virus. Microglia are professional antigen presenting cells that reside in the central nervous system (CNS) parenchyma that are poised to respond to viral insults. However, the role of microglia in initiating and coordinating the antiviral response is not completely understood. Utilizing Theiler's murine encephalomyelitis virus (TMEV), a neurotropic picornavirus, and PLX5622, a small molecule inhibitor of colony-stimulating factor 1 receptor (CSF1R) signaling that can deplete microglia in the CNS; we investigated the role of the CSF1R-microglia axis in neurotropic picornavirus infection of C57BL/6J and SJL/J mice. These mouse strains differ in their ability to clear TMEV and exhibit different neurological disease in response to TMEV infection. CSF1R antagonism in C57BL/6J mice, which normally clear TMEV in the CNS, led to acute fatal encephalitis. In contrast, CSF1R antagonism in SJL/J mice, which normally develop a chronic CNS TMEV infection, did not result in acute encephalitis, but exacerbated TMEV-induced demyelination. Immunologically, inhibition of CSF1R in C57BL/6J mice reduced major histocompatibility complex II expression in microglia, decreased the proportion of regulatory T cells in the CNS, and upregulated proinflammatory pathways in CNS T cells. Acute CSF1R inhibition in SJL/J mice had no effect on microglial MHC-II expression and upregulated anti-inflammatory pathways in CNS T cells, however chronic CSF1R inhibition resulted in broad immunosuppression. Our results demonstrate strain-specific effects of the CSF1R-microglia axis in the context of neurotropic viral infection as well as inherent differences in microglial antigen presentation and subsequent T cell crosstalk that contribute to susceptibility to neurotropic picornavirus infection.

Molecular patterns from a human gut-derived Lactobacillus strain suppress pathogenic infiltration of leukocytes into the central nervous system.

BACKGROUND: Multiple sclerosis (MS) is an inflammatory demyelinating disease that affects 2.5 million people worldwide. Growing evidence suggests that perturbation of the gut microbiota, the dense collection of microorganisms that colonize the gastrointestinal tract, plays a functional role in MS. Indeed, specific gut-resident bacteria are altered in patients with MS compared to healthy individuals, and colonization of gnotobiotic mice with MS-associated microbiota exacerbates preclinical models of MS. However, defining the molecular mechanisms by which gut commensals can remotely affect the neuroinflammatory process remains a critical gap in the field. METHODS: We utilized monophasic experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice and relapse-remitting EAE in SJL/J mice to test the effects of the products from a human gut-derived commensal strain of Lactobacillus paracasei (Lb). RESULTS: We report that Lb can ameliorate preclinical murine models of MS with both prophylactic and therapeutic administrations. Lb ameliorates disease through a Toll-like receptor 2-dependent mechanism via its microbe-associated molecular patterns that can be detected in the systemic circulation, are sufficient to downregulate chemokine production, and can reduce immune cell infiltration into the central nervous system (CNS). In addition, alterations in the gut microbiota mediated by Lb-associated molecular patterns are sufficient to provide partial protection against neuroinflammatory diseases. CONCLUSIONS: Local Lb modulation of the gut microbiota and the shedding of Lb-associated molecular patterns into the circulation may be important physiological signals to prevent aberrant peripheral immune cell infiltration into the CNS and have relevance to the development of new therapeutic strategies for MS.

Differential transcriptional profiles identify microglial- and macrophage-specific gene markers expressed during virus-induced neuroinflammation.

BACKGROUND: In the healthy central nervous system (CNS), microglia are found in a homeostatic state and peripheral macrophages are absent from the brain. Microglia play key roles in maintaining CNS homeostasis and acting as first responders to infection and inflammation, and peripheral macrophages infiltrate the CNS during neuroinflammation. Due to their distinct origins and functions, discrimination between these cell populations is essential to the comprehension of neuroinflammatory disorders. Studies comparing the gene profiles of microglia and peripheral macrophages, or macrophages in vitro-derived from bone marrow, under non-infectious conditions of the CNS, have revealed valuable microglial-specific genes. However, studies comparing gene profiles between CNS-infiltrating macrophages and microglia, when both are isolated from the CNS during viral-induced neuroinflammation, are lacking. METHODS: We isolated, via flow cytometry, microglia and infiltrating macrophages from the brains of Theiler's murine encephalomyelitis virus-infected C57BL/6 J mice and used RNA-Seq, followed by validation with qPCR, to examine the differential transcriptional profiles of these cells. We utilized primary literature defining subcellular localization to determine whether or not particular proteins extracted from the transcriptional profiles were expressed at the cell surface. The surface expression and cellular specificity of triggering receptor expressed on myeloid cells 1 (TREM-1) protein were examined via flow cytometry. We also examined the immune response gene profile within the transcriptional profiles of these isolated microglia and infiltrating macrophages. RESULTS: We have identified and validated new microglial- and macrophage-specific genes, encoding cell surface proteins, expressed at the peak of neuroinflammation. TREM-1 protein was confirmed to be expressed by infiltrating macrophages, not microglia, at the peak of neuroinflammation. We also identified both unique and redundant immune functions, through examination of the immune response gene profiles, of microglia and infiltrating macrophages during neurotropic viral infection. CONCLUSIONS: The differential expression of cell surface-specific genes during neuroinflammation can potentially be used to discriminate between microglia and macrophages as well as provide a resource that can be further utilized to target and manipulate specific cell responses during neuroinflammation.

Microglial cell depletion is fatal with low level picornavirus infection of the central nervous system.

Microglia are the only resident myeloid cell in the central nervous system (CNS) parenchyma, but the role of microglia in the context of neurotropic viral infection is poorly understood. Using different amounts of Theiler's murine encephalomyelitis virus (TMEV) in a preclinical model of epilepsy and PLX5622, a colony stimulating factor-1 receptor inhibitor that selectively depletes microglia in the CNS, we report that microglia-depleted, TMEV-infected mice develop seizures, manifest paralysis, and uniformly succumb to fatal encephalitis regardless of viral amount. CNS demyelination correlates with viral amount; however, viral amount does not correlate with axon damage and TMEV antigen in the CNS.

Positive modulation of mGluR5 attenuates seizures and reduces TNF-α+ macrophages and microglia in the brain in a murine model of virus-induced temporal lobe epilepsy.

Viral encephalitis markedly increases the risk for the development of epilepsy. The Theiler's murine encephalomyelitis virus (TMEV)-induced model of seizures/epilepsy is a murine model of both viral-induced seizures/epilepsy and human Temporal Lobe Epilepsy. The inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α have been shown to play a role in seizure development in the TMEV-induced model of seizures/epilepsy, and infiltrating macrophages along with microglia have been shown to be major producers of these cytokines. The metabotropic glutamate receptor 5 (mGluR5) is a G-protein coupled receptor that has been shown to reduce IL-6 and TNF-α and to provide neuroprotection in other disease models. Therefore, we hypothesized that stimulation of mGluR5 would not only reduce seizures but attenuate IL-6 and TNF-α production in microglia and macrophages in the TMEV model. We found that pharmacological stimulation of mGluR5 with the selective positive allosteric modulator VU0360172 not only reduced acute seizure outcomes, but also reduced the percent of microglia and macrophages producing TNF-α 3 days post infection. Furthermore, treatment with VU0360172 did not alter the level of viral antigen, compared to controls, showing that this treatment does not compromise viral clearance. These results establish that mGluR5 may represent a therapeutic target in the TMEV-induced model of seizures/epilepsy.

Induced CNS expression of CXCL1 augments neurologic disease in a murine model of multiple sclerosis via enhanced neutrophil recruitment.

Increasing evidence points to an important role for neutrophils in participating in the pathogenesis of the human demyelinating disease MS and the animal model EAE. Therefore, a better understanding of the signals controlling migration of neutrophils as well as evaluating the role of these cells in demyelination is important to define cellular components that contribute to disease in MS patients. In this study, we examined the functional role of the chemokine CXCL1 in contributing to neuroinflammation and demyelination in EAE. Using transgenic mice in which expression of CXCL1 is under the control of a tetracycline-inducible promoter active within glial fibrillary acidic protein-positive cells, we have shown that sustained CXCL1 expression within the CNS increased the severity of clinical and histologic disease that was independent of an increase in the frequency of encephalitogenic Th1 and Th17 cells. Rather, disease was associated with enhanced recruitment of CD11b+ Ly6G+ neutrophils into the spinal cord. Targeting neutrophils resulted in a reduction in demyelination arguing for a role for these cells in myelin damage. Collectively, these findings emphasize that CXCL1-mediated attraction of neutrophils into the CNS augments demyelination suggesting that this signaling pathway may offer new targets for therapeutic intervention.

The immune response to picornavirus infection and the effect of immune manipulation on acute seizures.

Viral infection of the central nervous system can result in encephalitis. About 20% of individuals who develop viral encephalitis go on to develop epilepsy. We have established an experimental model where virus infection of mice with Theiler's murine encephalomyelitis virus (TMEV) leads to acute seizures, followed by a latent period (no seizures/epileptogenesis phase) and then spontaneous recurrent seizures-epilepsy. Infiltrating macrophages (CD11b+CD45hi) present in the brain at day 3 post-infection are an important source of interleukin-6, which contributes to the development of acute seizures in the TMEV-induced seizure model. Time course analysis of viral infection and inflammatory [CD11b+CD45hiLy-6Chi] and patrolling [CD11b+CD45hiLy-6Clow] monocyte and T cell infiltration into the brains of TMEV-infected C57BL/6J mice over the entire course of the acute viral infection was performed to elucidate the role of virus and the immune response to virus in seizures and viral clearance. The infiltrating inflammatory macrophages were present early following infection but declined over the course of acute viral infection, supporting a role in seizure development, while the lymphocyte infiltration increased rapidly and plateaued, advocating that they play a role in viral clearance. In addition, we showed for the first time that, while TMEV infection of RAG1-/- mice did not alter the number of mice experiencing acute seizures, TMEV infection of C57BL/6J mice depleted of macrophages resulted in a significant decrease in the number of mice experiencing seizures, again supporting a role for infiltrating macrophages in the development of acute seizures in the TMEV-induced seizure model.

The role of peripheral interleukin-6 in the development of acute seizures following virus encephalitis.

Seizure disorders are often associated with infectious etiologies. Infection, via the intracerebral (i.c.) route, of C57BL/6J mice with the Daniels (DA) strain of Theiler's murine encephalomyelitis virus (TMEV) results in approximately 50% of the mice developing acute behavioral seizures. TMEV-DA is the wild-type strain of the virus that replicates within the parenchyma of the brain. A variant of TMEV-DA, TMEV-H101, does not replicate within the parenchyma of the brain. However, infection with TMEV-H101 via the i.c. route still results in approximately 40% of the mice developing acute behavioral seizures. Infiltrating macrophages producing interleukin-6 (IL-6) have been implicated in the induction of acute seizures following TMEV-DA infection. We examined macrophage infiltration and microglial activation within the brain and cytokine levels in the periphery in mice infected with TMEV-DA or TMEV-H101 and assessed the effects of the addition of recombinant IL-6 to the periphery in wild-type and IL-6 knockout mice infected with TMEV-DA. We found that pathologic levels of IL-6 in the periphery may play a role in the development of seizures when viral replication within the brain is limited. Examination of the role played by the peripheral immune system in the development of seizures/epilepsy in the TMEV-induced seizure model, the first viral infection driven model for epilepsy, could lead to the elucidation of novel therapeutics.

Complement Components Are Expressed by Infiltrating Macrophages/Activated Microglia Early Following Viral Infection.

The individual innate immune components, interleukin-6 and complement component C3, play a role in the development of acute seizures in the Theiler's murine encephalomyelitis virus-induced seizure model. We examined the mRNA expression of various other complement components, cytokines, chemokines, and major histocompatibility complex antigens both within brain and in isolated ramified microglial and infiltrating macrophage/activated microglial cell populations over a time course covering the first 3 days postinfection. We found that complement component C3 showed the greatest increase in expression in brain of all of the complement components assayed and its level of expression was higher in infiltrating macrophages/activated microglia than in ramified microglial cells.

The effects of diet on the severity of central nervous system disease: One part of lab-to-lab variability.

OBJECTIVE: Many things can impact the reproducibility of results from laboratory to laboratory. For example, food from various sources can vary markedly in composition. We examined the effects of two different food sources, the Teklad Global Soy Protein-Free Extruded Rodent Diet (irradiated diet) and the Teklad Sterilizable Rodent Diet (autoclaved diet), on central nervous system disease. METHODS: Three preclinical models for human disease: Two different experimental autoimmune encephalomyelitis models (multiple sclerosis) and the Theiler's murine encephalomyelitis virus-induced seizure model (epilepsy), were examined for the effects of two different food sources on disease. RESULTS: We found that mice fed the irradiated diet had more severe clinical disease and enhanced seizures compared with animals provided the autoclaved diet in both experimental autoimmune encephalomyelitis models examined and in the Theiler's murine encephalomyelitis virus-induced seizure model, respectively. CONCLUSIONS: Therefore, just altering the source of food (lab chow) can have marked effects on disease severity and outcome.

NBQX, a highly selective competitive antagonist of AMPA and KA ionotropic glutamate receptors, increases seizures and mortality following picornavirus infection.

Seizures occur due to an imbalance between excitation and inhibition, with the balance tipping towards excitation, and glutamate is the predominant excitatory neurotransmitter in the central nervous system of mammals. Since upregulation of expression and/or function of glutamate receptors can contribute to seizures we determined the effects of three antagonists, NBQX, GYKI-52466 and MK 801, of the various ionotropic glutamate receptors, AMPA, NMDA and KA, on acute seizure development in the Theiler's murine encephalomyelitis virus (TMEV)-induced seizure model. We found that only NBQX had an effect on acute seizure development, resulting in a significantly higher number of mice experiencing seizures, an increase in the number of seizures per mouse, a greater cumulative seizure score per mouse and a significantly higher mortality rate among the mice. Although NBQX has previously been shown to be a potent anticonvulsant in animal seizure models, seizures induced by electrical stimulation, drug administration or as a result of genetic predisposition may differ greatly in terms of mechanism of seizure development from our virus-induced seizure model, which could explain the opposite, proconvulsant effect of NBQX observed in the TMEV-induced seizure model.

DA virus mutant H101 has altered CNS pathogenesis and causes immunosuppression.

Viruses use various mechanisms to evade clearance by the host. Investigating how a few changes in the genome of a non-lethal virus can lead to altered disease, from survivable to immunosuppression/death, would provide valuable information into viral pathogenesis. The Daniels strain of Theiler's murine encephalomyelitis virus causes an asymptomatic infection or acute encephalitis followed by viral clearance. A mutant, H101, carries several alterations in the viral genome. H101 infection causes profound immunosuppression and death. Thus, a virus that is normally cleared by its natural host can become lethal due to just a few changes in the viral genome.

Infiltrating macrophages are key to the development of seizures following virus infection.

Viral infections of the central nervous system (CNS) can trigger an antiviral immune response, which initiates an inflammatory cascade to control viral replication and dissemination. The extent of the proinflammatory response in the CNS and the timing of the release of proinflammatory cytokines can lead to neuronal excitability. Tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), two proinflammatory cytokines, have been linked to the development of acute seizures in Theiler's murine encephalomyelitis virus-induced encephalitis. It is unclear the extent to which the infiltrating macrophages versus resident CNS cells, such as microglia, contribute to acute seizures, as both cell types produce TNF-α and IL-6. In this study, we show that following infection a significantly higher number of microglia produced TNF-α than did infiltrating macrophages. In contrast, infiltrating macrophages produced significantly more IL-6. Mice treated with minocycline or wogonin, both of which limit infiltration of immune cells into the CNS and their activation, had significantly fewer macrophages infiltrating the brain, and significantly fewer mice had seizures. Therefore, our studies implicate infiltrating macrophages as an important source of IL-6 that contributes to the development of acute seizures.