(Figure 5C). The effects of p21 were of functional relevance because knockdown of p21 promoted anti-T. gondii activity in microglia stimulated with a suboptimal concentration of anti- CD40 mAb (Figure 5B) while overexpression of p21 inhibited anti- T. gondii activity induced by CD40 (Figure 5C) (p,0.004). These results indicate that p21 regulates Beclin 1 expression and autophagy protein-dependent killing of T. gondii in CD40- stimulated microglia (Figure 5D). Autophagy-deficient mice are susceptible to ocular toxoplasmosis and toxoplasmic encephalitis Our in vitro studies indicate that the levels of Beclin 1 modulate killing of T. gondii. Based on these findings, we hypothesized that mice with a partial defect in Beclin 1 would exhibit impaired killing of T. gondii and susceptibility to ocular and cerebral toxoplasmosis. We used mice with heterozygous deletion of Beclin 1 (Becn1+/2 mice) to begin to test the in vivo relevance of the autophagic pathway in protection against cerebral and ocular toxoplasmosis. Becn1+/2 mice exhibit diminished Beclin 1 protein levels and reduced autophagy in vivo [24]. Becn1+/2 mice infected with T. gondii exhibited decreased survival compared to littermate controls (Becn1+/+) (Figure 6A) (p,0.001). Moreover, infected Becn1+/2 mice exhibited higher parasite loads in the brain and eye (Figure 6B) (p,0.01). Similar to CD402/2 animals, Becn1+/2 mice exhibited more pronounced retinal histopathology (p,0.01) that included distortion of the retinal architecture, hypertrophy of retinal pigmented epithelial cells, retinal invasion by these cells and inflammatory infiltrates (Figure 6C–E). In the brain, Becn1+/2 mice had more extensive parenchymal inflammatory foci, perivascular cuffing as well as more numerous tissue cysts than control mice (Figure 6F–G) (p,0.01). This increased susceptibility to toxoplasmosis was not explained by diminished expression of IFN-c, NOS2 and TNF-a in the brain and eye (Figure 6H). Mice with Atg52/2 hematopoietic cells exhibit defects in T cell homeostasis [28]. However, Becn1+/2 mice have a partial defect in Beclin 1 expression. Indeed, the phenotypic composition of lymphoid tissue in these mice as well as in CD402/2 mice was similar to that of control mice (Figure S5A). Moreover, these groups of mice exhibited similar percentages of T. gondii-reactive IFN-c- or IL-4-producing CD4+ and CD8+ T cells as well as equivalent in vitro secretion of IFN-c after stimulation with T. gondii lysate antigen (TLA) (Figure S5B and S5C). Next, we studied microglial anti-microbial activity. Microglia from Becn1+/2 mice had diminished Beclin 1 protein levels compared to control mice (Figure 6I). In marked contrast to the cytokine studies, microglia from Becn1+/2 mice had a defect in killing of T. gondii induced by CD40 but not in anti-T. gondii activity triggered by IFN-c/TNF-a (Figure 6I). This defect occurred despite normal expression of CD40 on microglia from Becn1+/2 mice (cMFI: Control = 103610; Becn1+/2 = 112615). To further confirm that Becn1+/2 mice exhibit defective autophagic killing of T. gondii, macrophages from controls and Becn1+/2 mice were incubated with rapamycin, an agent that induces anti-T. gondii activity via autophagy [6]. Macrophages from Becn1+/2 mice exhibited impaired killing induced by rapamycin (Figure 6I). Thus, in spite of upregulation of IFN-c, TNF-a and NOS2, and the development of T cell- mediated immunity against T. gondii, mice with defective autophagy and with impaired autophagy protein-dependent killing of the parasite are susceptible to toxoplasmosis. To further evaluate the role of autophagy for in vivo control of T. gondii in the brain and eye, we conducted experiments using mice where another autophagy gene, ATG7, was inactivated in macrophages/microglia. ATG7 was inactivated in myelomonocyt- ic cells by breeding of ATG7flox/flox mice [29] with mice that express Adaptive Immunity, Autophagy the Cre recombinase from endogenous lysozyme M locus (Lyz- Cre). Indeed, microglia and macrophages from ATG7flox/flox-Lyz-Cre mice had impaired expression of Atg7 (Figure 7A and not shown). Moreover, microglia as well as macrophages from these mice exhibited defective killing of T. gondii induced by CD40 but unimpaired parasite killing in response to IFN-c/TNF-a (Figure 7A). Compared to control mice (ATG7flox/flox), ATG7flox/flox- Lyz-Cre mice infected with T. gondii were unable to control the parasite in the brain and eye (Figure 7B) (p,0.01) and exhibited more prominent histopathology in these organs (Figure 7C–F) (p,0.01). Similar to the studies with Becn1+/2 mice, ATG7flox/flox- Lyz-Cre mice were susceptible to toxoplasmosis despite upregulation of IFN-c, TNF-a and NOS2 mRNA (Figure 7G) and the generation of T. gondii-specific T cell response (Figure 7H). Thus, mice with defective expression of Atg7 targeted to microglia/macrophages and impaired killing of T. gondii exhibit defective in vivo control of T. gondii in neural tissue. Discussion The cascades by which adaptive immunity activates autophagic killing of an intracellular pathogen and the in vivo role of autophagy in protection conferred by adaptive immunity are incompletely understood. We report that CD40 rapidly diminishes the levels of p21, a protein that degrades Beclin 1 [27]. As a result, CD40 causes upregulation of this autophagy protein and stimulates killing of T. gondii in microglia dependent on the autophagy pathway. Moreover, mice with diminished expression of Beclin 1 and mice with inactivation of Atg7 in microglia/macrophages are susceptible to cerebral and ocular toxoplasmosis despite upregulation of IFN-c, TNF-a and NOS2 as well as the induction of T cell-mediated immunity. Susceptibility to the chronic phase of T. gondii infection in these mice occurred in the setting of defective autophagy pathway- dependent killing of the parasite while IFN-c-induced killing of the parasite was unimpaired in these mice. These results contrast the report of autophagosome-independent in vivo protection conferred by Atg5, where Atg5 expression in phagocytic cells was required for IFN-c-mediated killing of the parasite [13]. The findings reported herein strongly support the notion that adaptive immunity utilizes autophagy to confer resistance against an intracellular pathogen because: i) CD40 induced entrapment of the parasite by an LC3+ structure; ii) Beclin 1, Atg5 and Atg7 were required for killing of T. gondii within CD40-activated microglia and macrophages; iii) Deficiency of two autophagy proteins, Beclin 1 and Atg7, led to susceptibility to toxoplasmosis while leaving unaffected IFN-c- induced anti-parasite activity and the induction of an IFN-c- mediated immune response. One study reported that autophagosomes surround T. gondii in primed macrophages [7,13]. This event followed the disruption of the parasitophorous vacuole membrane raising the possibility that formation of autophagosomes was a response to the presence of disrupted structures within host cells [7,13]. However, recent work in macrophages did not find evidence of autophagosome formation around the parasite in IFN-c-activated cells [13]. In addition, studies in astrocytes including work using live-cell imaging revealed that IFN-c induced killing of T. gondii without localization of LC3 to the parasitophorous vacuole [30,31]. Interestingly, while Atg5 is required in killing of the parasite in IFN-c-activated cells, this effect occurs in association with recruitment of IRG to the parasitophor- ous vacuole membrane rather than autophagic entrapment of the parasite [13,32]. IRG disrupt the parasitophorous vacuole mem- brane indicating that the anti-T. gondii effect of Atg5 in IFN-c- activated cells is likely dependent on IRG loading onto the parasitophorous vacuole rather that classical autophagy [13]. Our PLoS ONE | www.plosone.org 8 December 2010 | Volume 5 | Issue 12 | e14472