The autophagy gene ATG5 mediates autophagosome-indepen- dent host protection. Studies in mice deficient in ATG5 in phagocytes revealed that this autophagy gene was required for IFN-c-mediated in vivo host protection likely because ATG5 was required for the induction of IFN-c-dependent anti-microbial activity in macrophages [13]. ATG5 was required for recruitment of the Immunity- Related GTPase (IRG) Irga6 to the para- sitophorous vacuole, the damage to this structure and clearance of the parasite [13]. This process occurred independently of classical autophagosome formation. In addition, mice with inactivation of ATG5 in dendritic cells revealed that this gene promoted the induction of a Th1 response and protection against HSV-1 [14]. The role of ATG5 in dendritic cells was to enhance MHC II processing of phagocytosed antigens that contain TLR agonists [14]. Processing of antigens for MHC II did not appear to be dependent on canonical autophagy [14]. These studies indicate that ATG5 regulates IFN-c-dependent host protection and it does so through a manner that is does not rely on classical autophagy. Thus, the in vivo role of autophagy in mediating protection conferred by adaptive immunity is still not completely understood. In addition, it is unclear how adaptive immunity activates autophagy. T. gondii can cause disease that manifests as cerebral and/or ocular toxoplasmosis. IFN-c, TNF-a and their downstream effector molecule NOS2 are considered the mediators of resistance against ocular and cerebral toxoplasmosis [15,16,17,18,19,20]. We report herein that despite the induction of pathogen-reactive T cells, IFN-c, TNF-a, NOS2, and the preservation of IFN-c- induced antimicrobial activity in microglia/macrophages, adap- tive immunity still required the autophagic pathway to confer in vivo protection against cerebral and ocular toxoplasmosis. CD40 upregulates Beclin 1, triggers autophagy and killing of T. gondii in microglia independently of IFN-c, nitric oxide and Irga6. This pathway is required for host protection since mice deficient in Beclin 1, mice with defective expression of Atg7 in microglia/ macrophages and CD402/2 mice exhibit impaired autophagy protein-dependent killing of T. gondii and are susceptible to cerebral and ocular toxoplasmosis despite induction of T. gondii- specific T cell response, upregulation of IFN-c, TNF-a and NOS2 and unimpaired IFN-c-induced anti-microbial activity in microg- lia/macrophages. We also identified a pathway whereby adaptive immunity enhances autophagy. CD40 down-regulates p21 leading to increased levels of Beclin 1 and enhanced autophagy. Results CD402/2 mice are susceptible to ocular toxoplasmosis and toxoplasmic encephalitis despite upregulation of IFN-c, TNF-a and NOS2 B6 and CD402/2 mice were infected with T. gondii. As shown in Figure 1A, CD402/2 died during the chronic phase of infection between weeks 4 to 6 while B6 mice survived at least 11 weeks (p,0.001). We conducted histopathologic studies of the eye and brain. The histological features of the choroid and retina from uninfected B6 and CD402/2 mice were similar. At 4 weeks post- infection, infected B6 mice revealed mild histopathologic changes in the retina and choroid (Figure 1B). In contrast, CD402/2 mice revealed more pronounced histopathology (p,0.01) that included distortion of the retinal architecture, hypertrophy of retinal pigmented epithelial cells, invasion of the retina by these cells, inflammatory infiltrates as well as occasional tissue cysts (Figure 1C–E). Next, we examined the parasite load to determine if worse histopathology in CD402/2 mice was linked to inability to control the parasite. Compared to B6 mice, CD402/2 mice Adaptive Immunity, Autophagy exhibited a significantly higher parasite load in the eye as assessed by quantification of parasite DNA (Figure 1F) (p,0.02). Histopathologic studies of the brain at 4 weeks post-infection showed that CD402/2 mice had more extensive parenchymal inflammatory foci, perivascular cuffing and more numerous tissue cysts than infected B6 mice (Figure 1G, H). Histopathologic changes were more pronounced in CD402/2 mice than in B6 mice (p,0.01). A significantly higher parasite load in the brain as assessed by the number of tissue cysts accompanied worsened histopathology in CD402/2 mice (Figure 1I) (p,0.001). CD402/2 mice also exhibited increased parasite load and more pronounced histopathology at 2 weeks post-infection (not shown). Thus, CD40 is critical for control of ocular and cerebral toxoplasmosis. These results are similar to those obtained with CD1542/2 mice [21] and indicate that in contrast to studies with Mycobacterium tuberculosis [22], both components of the CD40 – CD154 pathway are critical for resistance against the chronic phase of T. gondii infection. Next, we examined whether susceptibility to toxoplasmosis in CD402/2 mice could be explained by impaired expression of IFN-c, TNF-a and NOS2. Compared to uninfected mice, T. gondii infected animals exhibited at least a 2-log increase in mRNA levels of these genes (not shown) and eyes and brains from both infected CD402/2 and infected B6 mice exhibited upregulation of mRNA of these genes at 2 and 4 weeks post-infection (Figure 2A and not shown). Immunohistochemical studies of the brain and immuno- blot of brain lysates at 4 weeks post-infection revealed protein expression of NOS2 and TNF-a in both CD402/2 and control mice (Figure 2B and 2C). Thus, CD402/2 mice are susceptible to ocular and cerebral toxoplasmosis despite upregulation of key mediators of host protection: IFN-c, TNF-a and NOS2. CD40 upregulates autophagy proteins and stimulates autophagy Microglia are considered central mediators of resistance against T. gondii in neural tissue [16]. Microglia were incubated with a stimulatory anti-CD40 or control mAb. Primary brain or retinal microglia treated with a stimulatory anti-CD40 mAb exhibited a significant lower parasite load than control microglia (p,0.02) (Figure 3A). Similar results were obtained with 2 mouse microglia cell lines (BV-2 and N9) or by treating cells with mouse CD154 (not shown). Anti-T. gondii activity induced by CD40 was not impaired by a neutralizing anti-IFN-c mAb, or NMA, an inhibitor of NOS2 (p.0.5) (Figure S1A and S1B). In contrast, anti-IFN-c mAb or NMA blocked the anti-T. gondii activity induced by IFN- c/TNF-a (p,0.001). The autophagy pathway was required for CD40-induced anti-T. gondii activity in microglia because CD40 stimulation caused recruitment of the autophagy marker LC3 around the parasite (Figure 3B) and silencing of Beclin 1 using siRNA ablated CD40-induced anti-T. gondii activity (p,0.01) (Figure 3C). In contrast, silencing of Beclin 1 had no effect on IFN- c/TNF-a induced anti-T. gondii activity. The role of the autophagy pathway was also studied in primary microglia and macrophages. Treatment of microglia with the autophagy inhibitor 3-methyl adenine (3-MA) inhibited toxoplasmacidal activity induced by CD40 stimulation (p,0.01) (Figure S2A). Silencing of Atg5 prevented CD40 stimulation from decreasing the parasite load in primary macrophages and microglia (p,0.01) (Figures S2B and S2C). Similarly, silencing of Atg5 in BV-2 cells ablated toxoplasmacidal activity induced by CD40 (p,0.01) (Figure S2D). Atg5 mediates anti-T. gondii activity in IFN-c-activated macrophages independent of autophagosome formation [13]. IFN-c induces killing of type II strains of the parasite through Atg5-dependent recruitment of Irga6 to the membrane of the PLoS ONE | www.plosone.org 2 December 2010 | Volume 5 | Issue 12 | e14472