Adaptive Immunity, Autophagy Figure 3. Autophagy mediates anti-T. gondii activity induced by CD40 in microglia. A, Primary retinal and brain microglia obtained from B6 mice were incubated with a stimulatory anti-CD40 or control mAb, or with IFN-c/TNF-a followed by infection with tachyzoites of RH T. gondii. The numbers of tachyzoites per 100 microglia were determined at 18 h post-infection. B, BV-2 cells were transfected with LC3-EGFP followed by incubation with anti-CD40 or control mAb. Cells were infected with RFP-T. gondii. Cells were examined at 5 h to determine the percentages of cells where LC3 accumulated around the parasites (arrow head). C, BV-2 cells were transfected with control siRNA or siRNA against Beclin 1. Protein expression of Beclin 1 and actin were examined by immunoblot. BV-2 cells transfected with control siRNA or siRNA against Beclin 1 were incubated with a stimulatory anti-CD40 mAb, control mAb or IFN-c/TNF-a followed by RH T. gondii infection. Results are representative of 3 independent experiments. *#0.05; **p#0.01. doi:10.1371/journal.pone.0014472.g003 independently of this cytokine but also independently of Irga6 and nitric oxide. Moreover, CD40 but not IFN-c requires Beclin 1, an autophagy protein that controls autophagosome formation [23]. The level of Beclin 1 expression has been linked to autophagic activity [24,25]. We examined whether CD40 modulates Beclin 1 expression. As shown in Figure 4A, CD40 stimulation caused a rapid increase in Beclin 1 protein expression in primary microglia (2.8-fold increase on average; n = 4). CD40 stimulation also caused an apparently less pronounced upregulation of the Atg5 expression (average 1.7-fold) but did not appear to affect expression of Atg7 (Figure 4A). We examined whether CD40 affects the in vivo levels of Beclin 1 during T. gondii infection. While we did not detect differences in Beclin 1 protein expression between brain microglia from uninfected B6 and CD402/2 mice, Beclin 1 expression in microglia/macrophages from T. gondii-infected B6 mice was higher than in microglia/macrophages from infected CD402/2 mice (Figure 4B). We determined if CD40 not only increased expression of autophagy protein but also enhanced basal levels of autophagy. BV-2 cells were transfected with LC3-EGFP and were incubated with or without anti-CD40 mAb. CD40 stimulation increased the percentage of microglia with LC3+ punctae (Figure 4C, D). An increase in these structures could be due to increased autophago- some formation or decreased degradation. Microglia were treated with bafilomycin A1, an inhibitor of vacuolar ATPase, which prevents autophagosome degradation [26]. CD40 stimulation still upregulated LC3+ punctae when autophagosome degradation was prevented. CD40 stimulated autophagy through Beclin 1 because knock-down of this autophagy protein inhibited upregulation of LC3+ punctae (Figure 4E) (p,0.001). Together, CD40 increases expression of Beclin 1 and Atg5 in microglia and enhances autophagy. Next, we tested whether changes in the level of autophagy protein expression affect toxoplasmacidal activity induced by CD40. We centered on Beclin 1 given that levels of expression of this protein can affect autophagy. BV-2 cells were transfected with a Beclin 1-encoding plasmid or with empty plasmid (Figure 4F). A suboptimal concentration of anti-CD40 mAb (1 mg/ml) did not affect Beclin 1 expression and did not induce anti-T. gondii activity in BV-2 cells transfected with the empty plasmid. In contrast, Beclin 1-transfected BV-2 cells exhibited on average a 2.4-fold increase in Beclin 1 protein expression that was accompanied by anti-T. gondii activity even if stimulated with 1 mg/ml of anti-CD40 mAb (Figure 4F). Conversely, BV-2 cells transfected with a suboptimal concentration of Beclin 1 siRNA exhibited on average a 53% reduction in Beclin 1 protein levels and ablated CD40- induced anti-T. gondii activity despite treatment with a fully activating dose of anti-CD40 mAb (10 mg/ml) (Figure 4G). Thus, PLoS ONE | www.plosone.org 5 December 2010 | Volume 5 | Issue 12 | e14472