The observed effects on IL-1b secretion by primary glia and microglia are of particular interest considering the emerging role of an adaptive immune response in PD, in particular by CD4+ T cells [63]. Given IL-1’s known effects to promote T cells responses, our findings on IL-1b regulation by a-Syn variants in innate immunocompetent cells require further attention in view of the potential effects of the latter in mediating immune tolerance and T effector responses. IL-10 regulation by a-Syn-stimulated glia and microglia Although most studies in the past have focused on microglial production of pro-inflammatory cytokines, a large body of evidence has supported the notion that microglia also produce cytokines with anti-inflammatory or regulatory activities [25]. Indeed, a strong induction of IL-10 2recognized as an anti- inflammatory cytokine2 had been observed for microglial cells stimulated with nitrated, aggregated Wt a-Syn [62]. We therefore investigated the effects of non-aggregated and unmodified a-Syn on glial secretion of IL-10 2a Th2 immunoregulator2 which reduces cytokine production by Th1 cells (Figure 4). Our results show that only the A30P variant produced a significant increase in IL-10 levels in mixed glial cells (top panel) whilst, on the contrary, the A53T variant caused a significant reduction of IL-10 basal levels, also observed in microglia, likely suggesting a lack of microglial response, a differential uptake by microglia, or an effect of the uptaken a-Syn on the endogenous IL-10 when A53T is present. In this sense, it has been reported a link between a-Syn and the microglial activation features [64], including phagocytic ability. In microglial cells, on the other hand, only the E46K variant produced an increase in IL-10 levels as compared to the control (bottom panel). These results might suggest that microglial cells, in order to produce a-Syn-driven endogenous IL-10, require both IL-6 and IL-1b secretion. In this sense, while Wt a-Syn increased only IL-1b production in microglial cells, A30P increased only the IL-6 production. These facts could reflect the requirement of a doubly activated state of the microglia for IL-10 production. Although the general mechanism that generate IL-10 production within the CNS during neuroinflammation is still not well enough understood, our results support a role for a-Syn in the modulation of the microglial phenotype as suggested by Austin and collaborators [64]. Chemokine release profiles by a-Syn-stimulated glia and microglia Chemokines are involved in a wide variety of disorders in the CNS and their actions contribute to reactive glial changes and neuronal injury in neuroinflammatory conditions [65–67]. We therefore sought to determine the chemokine release profiles of glial and microglial cells induced by non-aggregated Wt and PD- linked a-Syn variants. In particular, we assayed the release of IP- 10/CXCL10, RANTES/CCL5, MCP-1/CCL2 and MIP-1a/ CCL3 chemokines (Figure 5). The regulation by a-Syn of IP-10/CXCL10 in CNS cells has not been previously reported. In addition, the only data available regarding RANTES/CCL5 levels in PD are from a transgenic rat model for A53T a-Syn [38], or from sera and thus relate to peripheral dysregulation in the cytokine network associated with PD patients [68–70], and although no information is given on the genetic background of these PD patients, these studies reported increased levels of circulating RANTES/CCL5. Our results show large and comparable effects for A30P and E46K variants on IP- 10/CXCL10 and RANTES/CCL5 release, both by mixed glial cultures and by microglia (Figures 5A and 5B). Remarkably, a-Syn and Innate Immunity Figure 4. Immunoregulatory effect of a-Syn-stimulation in primary mixed glial and isolated microglial cultures. IL-10 release was measured by ELISA in supernatants of a-Syn-stimulated mixed-glial cultures (top) and microglia (bottom) after a 20-hour treatment with monomeric Wt or mutant a-Syn variants, or lipopoly- saccharide (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.g004 neither Wt nor A53T in both glial cultures and microglia produced a relevant increase in IP-10/CXCL10 or RANTES/ CCL5 secretion, the latter result in agreement with the one reported for the striatum and SN in the (A53T) rat model [38]. These data suggest a specific role for A30P and E46K a-Syn variants associated with enhancement of Th1-cell recruitment, activation, and effector potential. Besides their role as chemoattractants, IP-10/CXCL10 and RANTES/CCL5 are known to induce T-cell proliferation and cytokine production [71], suggesting a role for A30P and E46K a- PLoS ONE | www.plosone.org 6 October 2010 | Volume 5 | Issue 10 | e13481