a-Syn and Innate Immunity Figure 3. Pro-inflammatory interleukin profile of a-Syn-stimulated primary mixed glial and isolated microglial cultures. IL-6 (A) and IL-1b (B) release was measured by ELISA in culture supernantants of mixed glia (left) and microglia (right) after a 20-hour treatment with monomeric Wt or mutant a-Syn variants or lipopolysaccharide (LPS). Values are mean 6 S.E.M. (n = 4). * P,0.05, ** P,0.01, *** P,0.001. The results shown are representative of two or three independent experiments with microglia and mixed glial cultures, respectively. doi:10.1371/journal.pone.0013481.g003 showed a significant stimulatory effect. Interestingly, when tested on isolated microglial cells, while A30P did not cause a significant rise in IL-1b levels relative to the control, Wt a-Syn 2in agreement with a previous report of mRNA levels in a comparable experiment design [37]2 and especially E46K, showed a very significant increase in cytokine release (Figure 3B, right panel). Taken together, these results suggest that A30P and E46K, as opposed to either Wt a-Syn or the A53T variant, induce a pro- inflammatory response in glia. Remarkably, only two of the three PD-linked variants of a-synuclein were IL-1b inducers in primary glia, and contrary to expectations from previous studies performed using cell line cultures and other studies involving modified a- synuclein species, our results show that the A53T a-Syn variant has instead a very modest activity in regulating the innate immune response by primary glia and microglia. These findings indicate the need for further detailed studies on the role of the glia in early PD onset before detailed conclusions as to the role of the latter in PD can be drawn. Previously, the available data had shown that the A30P and A53T a-Syn variants, but not the Wt and the E46K forms, efficiently induced the release of IL-1b when added to THP-1 macrophage cell line cultures [52] and that all the a-Syn variants were able to increase IL-1b secretion in THP-1 cells only when co- treatments with INF-c were included, suggesting a pro-inflamma- tory response in already immune-primed THP-1 cells [52]. Our data, obtained with primary microglial cultures, indicate a very different behaviour. Thus, Wt and E46K a-Syn in our study appear to be pro-inflammatory in primary microglia, while the A30P and A53T variants seem to be unable to produce a significant response. These differential responses of these two studies point at the importance of the differentiation/maturation status of the primary microglia, which suggests that there could be multiple subtle but important differences in immune responses during the establishment of PD. It is interesting that these differences were only revealed in primary settings that were not over-primed, as was the case of IFN-c treated THP-1 cells. PLoS ONE | www.plosone.org 5 October 2010 | Volume 5 | Issue 10 | e13481