Gliomas attract brain-resident (microglia) and peripheral macrophages and reprogram these cells into immunosuppressive, pro-invasive cells. M-CSF (macrophage colony-stimulating factor, encoded by the CSF1 gene) has been implicated in the control of recruitment and polarization of macrophages in several cancers. We found that murine GL261 glioma cells overexpress GM-CSF (granulocyte-macrophage colony-stimulating factor encoded by the CSF2 gene) but not M-CSF when compared to normal astrocytes. Knockdown of GM-CSF in GL261 glioma cells strongly reduced microglia-dependent invasion in organotypical brain slices and growth of intracranial gliomas and extended animal survival. The number of infiltrating microglia/macrophages (Iba1(+) cells) and intratumoural angiogenesis were reduced in murine gliomas depleted of GM-CSF. M1/M2 gene profiling in sorted microglia/macrophages suggests impairment of their pro-invasive activation in GM-CSF-depleted gliomas. Deficiency of M-CSF (op/op mice) did not affect glioma growth in vivo and the accumulation of Iba1(+) cells, but impaired accumulation of Iba1(+) cells in response to demyelination. These results suggest that distinct cytokines of the CSF family contribute to macrophage infiltration of tumours and in response to injury. The expression of CSF2 (but not CSF1) was highly up-regulated in glioblastoma patients and we found an inverse correlation between CSF2 expression and patient survival. Therefore we propose that GM-CSF triggers and drives the alternative activation of tumour-infiltrating microglia/macrophages in which these cells support tumour growth and angiogenesis and shape the immune microenvironment of gliomas.

CNS endothelial cells (CNS-ECs), one of the main non-neural CNS cell populations, play a vital role in physiology, pathology, and regeneration of the nervous system. Therefore, there is an urgent need to enhance our knowledge on their biology to elucidate mechanisms responsible for the blood brain barrier function in normal and pathological conditions, interaction between brain endothelium and neural stem cells in the neurogenic niche, the paracrine processes in the brain and spinal cord, etc. Here, we described a novel, simple, and efficient protocol for isolation of endothelial, vesselforming cells from the murine CNS, which is based on Sca-1 expression. Using this newly described protocol we were able to detect and sort viable, highly pure CNS-ECs with minimal contamination by cells of non-endothelial origin. This method will increase the availability of CNS-ECs for in vitro research. Moreover, we compared phenotype of CNS-ECs isolated from neonatal mice and adult intact and injured brain looking for the cells of endothelial precursor characteristic, such as those found in the bone marrow and circulating in the bloodstream after organ injuries. We have found that neonatal brain capillaries contain proportion of endothelial precursor cells (Sca-1 1 , CD452 , c-Kit. Such precursors were also found in adult brain cortex, both in intact and injured brain. Finally, we discuss several crucial technical issues concerning CNS tissue preparation for flow cytometry analysis and cell sorting as well as nonspecific antibody binding caused by inflammatory microglia/macrophages which should be avoided in order to reliable isolation of pure CNS cells for downstream procedures including cell transplantation-based translational studies. V C 2015 International Society for Advancement of CytometryKey terms

Remyelination, a highly efficient central nervous system (CNS) regenerative process, is performed by oligodendrocyte progenitor cells (OPCs), which are recruited to the demyelination sites and differentiate into mature oligodendrocytes to form a new myelin sheath. Microglia, the specialized CNS-resident phagocytes, were shown to support remyelination through secretion of factors stimulating OPC recruitment and differentiation, and their pharmacological depletion impaired remyelination. Macrophage colony-stimulating factor (Csf1) has been implicated in the control of recruitment and polarization of microglia/macrophages in injury-induced CNS inflammation. However, it remains unclear how Csf1 regulates a glial inflammatory response to demyelination as well as axonal survival and new myelin formation. Here, we have investigated the effects of the inherent Csf1 deficiency in a murine model of remyelination. We showed that remyelination was severely impaired in Csf1-/- mutant mice despite the fact that reduction in monocyte/microglia accumulation affects neither the number of OPCs recruited to the demyelinating lesion nor their differentiation. We identified a specific inflammatory gene expression signature and found aberrant astrocyte activation in Csf1-/- mice. We conclude that Csf1-dependent microglia activity is essential for supporting the equilibrium between microglia and astrocyte pro-inflammatory vs. regenerative activation, demyelinated axons integration and, ultimately, reconstruction of damaged white matter.