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. Electronic supplementary material The online version of this article (10.1186/s12974-019-1545-x) contains supplementary material, which is available to authorized users.
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.
Purpose:Mutational activation of GNAQ or GNA11 (GNAQ/11), detected in >90% of uveal melanomas, leads to constitutive activation of oncogenic pathways, including MAPK and YAP. To date, chemo- or pathway-targeted therapies, either alone or in combination, have proven ineffective in the treatment of patients with metastatic uveal melanoma.Experimental Design:We tested the efficacy of chloroquine or hydroxychloroquine, in combination with MAPK pathway inhibition in GNAQ/11-mutated cells in vitro and in vivo and identified mechanisms of MEK1/2 inhibitor plus chloroquine-induced cytotoxicity.Results:Inhibition of GNAQ/11-mediated activation of MAPK signaling resulted in the induction of autophagy. Combined inhibition of Gα and autophagy or lysosome function resulted in enhanced cell death. Moreover, the combination of MEK1/2 inhibition, using trametinib, with the lysosome inhibitor, chloroquine, also increased cytotoxicity. Treatment of mice bearing GNAQ/11-driven melanomas with trametinib plus hydroxychloroquine resulted in inhibition of tumor growth and significantly prolonged survival. Interestingly, lysosomal- and autophagy-specific inhibition with bafilomycin A1 was not sufficient to promote cytotoxicity in combination with trametinib. However, the addition of YAP inhibition with trametinib plus bafilomycin A1 resulted in cell death at comparable levels to trametinib plus chloroquine (T/CQ) treatment. Furthermore, T/CQ-treated cells displayed decreased YAP nuclear localization and decreased YAP transcriptional activity. Expression of a constitutively active YAP5SA mutant conferred resistance to T/CQ-induced cell death.Conclusions:These results suggest that YAP, MEK1/2, and lysosome function are necessary and critical targets for the therapy of GNAQ/11-driven melanoma, and identify trametinib plus hydroxychloroquine as a potential treatment strategy for metastatic uveal melanoma.
Multiple sclerosis (MS) is a metabolically demanding disease involving immune-mediated destruction of myelin in the central nervous system. We previously demonstrated a significant alteration in disease course in the experimental autoimmune encephalomyelitis (EAE) preclinical model of MS due to diet. Based on the established crosstalk between metabolism and gut microbiota, we took an unbiased sampling of microbiota, in the stool, and metabolites, in the serum and stool, from mice (Mus musculus) on the two different diets, the Teklad global soy protein-free extruded rodent diet (irradiated diet) and the Teklad sterilisable rodent diet (autoclaved diet). Within the microbiota, the genus Lactobacillus was found to be inversely correlated with EAE severity. Therapeutic treatment with Lactobacillus paracasei resulted in a significant reduction in the incidence of disease, clinical scores and the amount of weight loss in EAE mice. Within the metabolites, we identified shifts in glycolysis and the tricarboxylic acid cycle that may explain the differences in disease severity between the different diets in EAE. This work begins to elucidate the relationship between diet, microbiota and metabolism in the EAE preclinical model of MS and identifies targets for further study with the goal to more specifically probe the complex metabolic interaction at play in EAE that may have translational relevance to MS patients.
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