CCR2 of Tumor Microenvironmental Cells Is a Relevant Modulator of Glioma Biology.

Glioblastoma multiforme (GBM) shows a high influx of tumor-associated macrophages (TAMs). The CCR2/CCL2 pathway is considered a relevant signal for the recruitment of TAMs and has been suggested as a therapeutic target in malignant gliomas. We found that TAMs of human GBM specimens and of a syngeneic glioma model express CCR2 to varying extents. Using a Ccr2-deficient strain for glioma inoculation revealed a 30% reduction of TAMs intratumorally. This diminished immune cell infiltration occurred with augmented tumor volumes likely based on increased cell proliferation. Remaining TAMs in Ccr2-/- mice showed comparable surface marker expression patterns in comparison to wildtype mice, but expression levels of inflammatory transcription factors (Stat3, Irf7, Cox2) and cytokines (Ifnß, Il1ß, Il12α) were considerably affected. Furthermore, we demonstrated an impact on blood vessel integrity, while vascularization of tumors appeared similar between mouse strains. The higher stability and attenuated leakiness of the tumor vasculature imply improved sustenance of glioma tissue in Ccr2-/- mice. Additionally, despite TAMs residing in the perivascular niche in Ccr2-/- mice, their pro-angiogenic activity was reduced by the downregulation of Vegf. In conclusion, lacking CCR2 solely on tumor microenvironmental cells leads to enhanced tumor progression, whereby high numbers of TAMs infiltrate gliomas independently of the CCR2/CCL2 signal.

Lymphocytes modulate innate immune responses and neuronal damage in experimental meningitis.

In bacterial meningitis, excessive immune responses carry significant potential for damage to brain tissue even after successful antibiotic therapy. Bacterial meningitis is regarded primarily as the domain of innate immunity, and the role of lymphocytes remains unclear. We studied the contribution of lymphocytes to acute inflammation and neurodegeneration in experimental Toll-like receptor 2-driven meningitis, comparing wild-type mice with RAG-1-deficient mice that have no mature T and B lymphocytes. At 24 h after intrathecal challenge with the synthetic bacterial lipopeptide Pam(3)CysSK(4), RAG-1-deficient mice displayed more pronounced clinical impairment and an increased concentration of neutrophils, reduced expression of interleukin-10 (IL-10) mRNA, and increased expression of CXCL1 mRNA in the cerebrospinal fluid. Conversely, neuronal loss in the dentate gyrus was reduced in RAG-1-deficient mice, and expression of IL-10, transforming growth factor ß and CCL2 mRNA by microglia was increased compared to wild-type mice. Adoptive transfer of wild-type lymphocytes reversed the enhanced meningeal inflammation and functional impairment observed in RAG-1-deficient mice. Our findings suggest compartment-specific effects of lymphocytes during acute bacterial meningitis, including attenuation of meningeal inflammation and shifting of microglial activation toward a more neurotoxic phenotype.

Thrombopoietin contributes to neuronal damage in experimental bacterial meningitis.

Thrombopoietin (Tpo), which primarily regulates megakaryopoiesis, and its receptor (c-Mpl) are expressed in the brain, where Tpo exhibits proapototic effects on neurons. In the present study, we investigated the implication of Tpo in experimental pneumococcal meningitis. Following intrathecal infection with the encapsulated Streptococcus pneumoniae strain D39, we observed upregulation of Tpo mRNA expression at 12 h and 24 h in brain homogenates of wild-type C57BL/6 mice. c-Mpl mRNA expression was upregulated at 12 h and returned to baseline at 24 h. Compared to wild-type mice, mutants with homozygous Tpo receptor ablation (c-Mpl(-/-)) displayed reduced microglial activation and neuronal apoptosis in the dentate gyrus. Concentrations of bacteria in blood or cerebrospinal fluid (CSF), as well as CSF pleocytosis, were not significantly different between wild-type and c-Mpl(-/-) mice. In human postmortem brain, Tpo protein was colocalized to macrophages during encephalitis. In murine primary microglia and RAW264.7 macrophages, upregulation of Tpo mRNA was induced by D39-conditioned medium but not by bacterial lipopeptide or by medium conditioned by pneumococcal mutants defective in hydrogen peroxide formation (ΔspxB) or pneumolysin (Δpln). We conclude that Tpo acts as a mediator of neuronal damage in bacterial meningitis.

Inflammation stimulates thrombopoietin (Tpo) expression in rat brain-derived microvascular endothelial cells, but suppresses Tpo in astrocytes and microglia.

Thrombopoietin (Tpo) and its receptor (c-Mpl; TpoR), which primary regulate megakaryopoiesis and platelet production, are also expressed in the central nervous system (CNS). Increased Tpo concentrations are present in the cerebrospinal fluid (CSF) of some patients with bacterial or viral meningitis. Since previous data implicated a proapoptotic role of Tpo on newly generated neuronal cells, we herein elucidated the regulation of Tpo in primary rat neurons (e17), astrocytes, and microglia (p0-p3), as well as in brain-derived vascular endothelial cells of 3-week-old rats after exposure to bacterial lipopolysaccharide (LPS). LPS inhibited Tpo gene expression in astrocytes and microglia, but not in neurons, most likely due to absence of Toll-like receptor 4 in neurons. While Tpo mRNA expression recovered in astrocytes after 24 h, it remained suppressed in microglia. Furthermore, we detected Tpo mRNA expression in primary brain-derived vascular endothelial cells, which also express the TpoR. In these cells, LPS significantly up-regulated Tpo mRNA expression. TpoR mRNA and protein expression remained constitutive in all cell types. Thus, our data provide evidence for a cell-type-specific modulation of Tpo mRNA expression by inflammation in brain-derived cells. Transient down-regulation of Tpo expression in astrocytes and microglia may limit Tpo-induced neuronal cell death in inflammatory brain disorders.