a-Syn and Innate Immunity Figure 2. Evaluation of the purity of isolated microglial cell fractions. Immunofluorescence characterization of purified microglial cell cultures for the specific macroglial lineage marker GFAP (A), the pan haematopoietic lineage marker CD45 (B), and the mature macrophages markers CD11b (C), CD68 (D), and F4/80 (E). Nuclei are counterstained in blue with Hoechst 33342. Scale bar: 100 mm. mRNA expression (qRT-PCR) from isolated microglial preparations after no stimulation (control) or after stimulation with 1 mg/ml LPS for 20 hours (F). Specific primers for amplifying the GFAP and CD11b genes were used. doi:10.1371/journal.pone.0013481.g002 detached by trypsinization of the long-term cultures from neonatal mouse brains –expected to be macroglia– were found to be strongly immunostained for GFAP (not shown). As a second measure of the purity of microglial cell cultures, the majority of cells were found to express the pan haematopoietic lineage marker CD45 (Figure 2B), the monocyte-macrophage marker CD11b (Figure 2C), and the mature macrophage markers CD68 and F4/ 80 (Figures 2D and 2E, respectively). In addition, quantitative RT-PCR was employed to amplify specifically the GFAP and CD11b genes (Figure 2F), and the results indicate no detectable mRNA expression of the GFAP macroglial marker, and a clear up-regulation of the microglia-specific CD11b gene expression after stimulation by lipopolyssacharide (LPS). Taken together, the cell marker profiles observed confirms the high purity of the microglial cultures and hence to the reliability of the results obtained using our purified microglial cell culture model. Pro-inflammatory response of a-Syn-stimulated glia and microglia In the normal CNS, brain tissue provides an immunosuppres- sive environment, which seems to be important for the proper function of the CNS. Under circumstances that cause disruption of this environment, including chronic inflammatory conditions in neurodegenerative diseases, a variety of immune regulatory and inflammatory mediators can be activated [25]. Although various types of cells have been identified as sources of cytokines in the CNS, microglia appear to be a principal source of pro- inflammatory and immune regulatory cytokines [25,61]. In order to explore the immunological properties of extracellular, non- aggregated a-Syn, as well as to evaluate the importance of the cellular context in this process, we measured by ELISA the release of a series of key cytokines after incubation for 20 hours of mouse primary cultures of mixed glia and isolated microglia, with exogenously added Wt a-Syn or the early onset PD-linked a-Syn variants A30P, E46K, and A53T (at 0.2, 1 or 5 mg/ml). First, we assayed the release of IL-6, TNF-a, IFN-c and IL-1b, four key pro-inflammatory cytokines (Figure 3 and S2). Wild-type a-Syn moderately stimulated the release of IL-6 in mixed glial cultures (Figure 3A, left panel), but contrary to findings for aggregated Wt a-Syn [62], this effect was hardly detectable on isolated microglia (Figure 3A, right panel). And in contrast to the comparable IL-6 response observed for the four a-Syn variants reported in a study using the human U-373 MG astrocytoma cell line [53], we found remarkable differences in their behaviour, notably a very strong IL-6-mediated pro-inflammatory response induced by the A30P and E46K variants after stimulation of both mixed glial and microglial cultures. These differences could be explained by the nature of the cell line used by Klegeris and coworkers. Alternatively, given that astrocytes are the most abundant glial cell population of the CNS participating in local innate immune responses; this result could be consistent with our finding of less marked differences (Figure 3A) between the a-Syn variants in mixed glial cultures. The A53T variant, however, caused a weak but significant increase in IL-6 levels on total glia but not on isolated microglia, similar to Wt a-Syn but even less prominent. In the case of TNF-a and IFN-c levels measured in microglial cultures, only stimulation with A30P produced a significant increase (Figure S2), partially coinciding with the IL- 6 observed profile. Therefore, A30P and E46K appear to drive IL- 6, TNF-a, and IFN-c cytokine secretion in the context of PD- affected glia. When we assayed the levels of IL-1b from mixed glial cultures (Figure 3B, left panel), only the A30P and E46K a-Syn variants PLoS ONE | www.plosone.org 4 October 2010 | Volume 5 | Issue 10 | e13481