SummaryThe transfer of antigens from oligodendrocytes to immune cells has been implicated in the pathogenesis of autoimmune diseases. Here, we show that oligodendrocytes secrete small membrane vesicles called exosomes, which are specifically and efficiently taken up by microglia both in vitro and in vivo. Internalisation of exosomes occurs by a macropinocytotic mechanism without inducing a concomitant inflammatory response. After stimulation of microglia with interferon-, we observe an upregulation of MHC class II in a subpopulation of microglia. However, exosomes are preferentially internalised in microglia that do not seem to have antigenpresenting capacity. We propose that the constitutive macropinocytotic clearance of exosomes by a subset of microglia represents an important mechanism through which microglia participate in the degradation of oligodendroglial membrane in an immunologically 'silent' manner. By designating the capacity for macropinocytosis and antigen presentation to distinct cells, degradation and immune function might be assigned to different subtypes of microglia. Journal of Cell Sciencethrough which microglia participate in the degradation of oligodendroglial membrane. Results Internalisation of oligodendroglia-derived exosomes by microgliaTo obtain exosomes in large quantities, we isolated exosomes from the culture medium of the mouse oligodendroglial precursor cell line Oli-neu. Sequential centrifugation steps with increasing centrifugal forces up to 100,000 g yielded a pellet, which we have previously shown to contain small membrane vesicles with a diameter of about 50-100 nm (Trajkovic et al., 2008). Further analysis of the 100,000 g pellet with continuous sucrose density gradients revealed that PLP and myelin oligodendrocyte glycoprotein (MOG) are recovered from fractions with the characteristic density of exosomes that contain the exosomal marker proteins Alix (ALG-2-interacting protein 1), TSG101 and flotillin-2 (Fig. 1A). Hence, we refer to the 100,000 g membrane fractions as exosomes.To follow the fate of exosomes, suspensions of purified exosomes were labelled with the dye PKH67 and added to primary cultures of mouse oligodendrocytes, cortical neurons, astrocytes and microglia. After incubating the exosomes for 2 hours at 37°C, cells were fixed and analysed by confocal microscopy. We observed efficient internalisation of exosomes by microglia, whereas uptake by astrocytes, neurons or oligodendrocytes was almost absent (Fig. 1B). By confocal microscopy, we confirmed that exosomes were inside microglia rather than attached to the cell surface. In addition, we observed that exosomes colocalised with Lamp1, a marker for late endosomes or lysosomes, verifying that exosomes had been endocytosed and transported to endosomal organelles ( Fig. 2A). When exosomes were added to mixed brain cultures that contained all major glial cell types, we again observed that exosomes were almost exclusively internalised by Iba-1-positive microglia (supplementary material Fig. S1). Similar results wer...

No abstract

Although there is increasing evidence that blood-derived macrophages support tumor progression, it is still unclear whether specialized resident macrophages, such as brain microglia, also play a prominent role in metastasis formation. Here, we show that microglia enhance invasion and colonization of brain tissue by breast cancer cells, serving both as active transporters and guiding rails. This is antagonized by inactivation of microglia as well as by the Wnt inhibitor Dickkopf-2. Proinvasive microglia demonstrate altered morphology, but neither upregulation of M2-like cytokines nor differential gene expression. Bacterial lipopolysacharide shifts tumor-educated microglia into a classical M1 phenotype, reduces their proinvasive function, and unmasks inflammatory and Wnt signaling as the most strongly regulated pathways. Histological findings in human brain metastases underline the significance of these results. In conclusion, microglia are critical for the successful colonization of the brain by epithelial cancer cells, suggesting inhibition of proinvasive microglia as a promising antimetastatic strategy. V V C