supporting the existence of another mechanism by which autophagy promotes resistance against T. gondii. Indeed, the studies with ATG7flox/flox-Lyz-Cre mice provide an in vivo correlate to our in vitro studies on autophagic killing of T. gondii in microglia/ macrophages and indicate that autophagy in those cells is likely required for resistance against toxoplasmosis in neural tissue. Beclin 1 plays a central role in autophagy by promoting the formation of autophagosomes [23]. Our studies identified CD40 as a regulator of Beclin 1 since engagement of CD40 resulted in a rapid increase in Beclin 1 expression and enhanced autophagy. Changes in Beclin 1 levels were functionally relevant because a moderate increase in Beclin 1 expression markedly facilitated CD40-induced killing of T. gondii while a moderate decrease in Beclin 1 expression profoundly inhibited this response. Moreover, T. gondii-infected CD40 deficient mice had lower Beclin 1 levels in their brain microglia/macrophages and Beclin 1-deficient mice were susceptible to cerebral and ocular toxoplasmosis. Studies in cancer and neurodegeneration also support the relevance of Beclin 1 levels. Various cancer cells exhibit decreased Beclin 1 levels and the anti-cancer agent tamoxifen increases expression of this protein [25,38]. Brain injury appears to upregulate Beclin 1 [39]. In contrast, Beclin 1 is deficient in Alzheimer’s dementia and this deficiency impairs neuronal autophagy promoting neurode- generation [40]. In this model, lentiviral-induced overexpression of Beclin 1 reduces amyloid b pathology [40]. Thus, upregulation of Beclin 1 may drive not only host protection in the CNS but may also activate autophagy as a homeostatic mechanism to reduce cellular damage. Beclin 1 is not only targeted by CD40-dependent adaptive immunity to stimulate autophagy but can also be targeted by a pathogen to impair autophagy. For example, neurovirulent HSV- 1 and HIV-1 encode an ICP34.5 protein and Nef respectively that bind Beclin 1 and block autophagy [11,41]. The influenza M2 protein blocks autophagosome maturation, an effect that may be mediated through interference with Beclin 1 [42]. Together, these studies and our work indicate that Beclin 1 is an important regulator of autophagy during host-pathogen interaction. Our results indicate that p21 acts as a link between CD40 and Beclin 1 upregulation and stimulation of autophagy. p21 is not only an inhibitor of cyclin-dependent kinases but it also diminishes expression of Beclin 1 and Atg5 [27]. C2-ceramide inhibits autophagy in mouse embryonal fibroblasts because it triggers p21- dependent protein degradation of Beclin 1 and Atg5 [27]. Prior studies addressed the effects of CD40 on p21 expression in B cells. CD40 down-regulates p21 in WEHI 231 immature B lymphoma cells treated with anti-IgM antibody [43]. The discordant results on the effects of CD40 on p21 expression reported in other studies may be explained by differences in the stages of B cell development [44]. We report that CD40 stimulation rapidly reduced p21 protein expression in microglia. Moreover, knock- down of p21 in microglia promoted upregulation of Beclin 1 and autophagic killing of T. gondii in response to CD40 stimulation while overexpression of p21 impaired the ability of CD40 to upregulate Beclin 1 and kill T. gondii. Mouse models of T. gondii infection are used to study immunity against the parasite. However, these models do not fully mimic the immune response in humans. While IFN-c is indispensable to control the parasite in mice, mechanisms of resistance against T. gondii that do not rely on IFN-c are likely more effective in humans. Children with an autosomal dominant defect in IFN-cR1 that causes a deletion in the STAT1 binding site do not develop disease when infected with T. gondii [45]. In contrast, STAT12/2 mice die within 1 week after infection with the parasite [46,47]. The differences between the immune response in humans and Adaptive Immunity, Autophagy mice also appear to apply to the downstream effectors of IFN-c, NOS2 and IRG. NOS2 is more tightly regulated in humans than in rodents and the production of nitric oxide appears to be weaker in humans. Although IRGM has been reported to mediate anti- bacterial activity in human macrophages [48], the role of IRG is likely more restricted in humans than in mice. Mice have 23 IRG genes among which Irgm1, Irgm3 and Irga6 are important mediators of anti-T. gondii activity in mouse cells [30,49], whereas IRG in humans have been reduced to IRGM and IRGC [50]. CD40 induces killing of T. gondii independently of IFN-c, STAT1, NOS2, Irgm1, Irgm3 and Irga6 [6,8,33,51]. CD40-induced killing of T. gondii may be an important contributor to control of T. gondii in humans. Defects in the CD40 pathway are relevant to at least 3 groups of patients that develop ocular and/or cerebral toxoplas- mosis: patients with X-linked Hyper IgM syndrome who lack functional CD154 [52], newborns since neonatal CD4+ T cells exhibit impaired expression of CD154 [53,54] and CD40 levels are reduced on neonatal dendritic cells [54], and HIV-1+ patients because they exhibit a defect in CD154 induction in their CD4+ T cells [55,56]. In conclusion, our studies revealed that immunity can stimulate autophagy by upregulating autophagy proteins in a p21-depen- dent manner, and the autophagic machinery was required for resistance against cerebral and ocular toxoplasmosis despite the induction of T. gondii-reactive T cells, IFN-c, TNF-a and NOS2. These results shed new light on the spectrum of mechanisms of host protection. Future studies should explore the possibility of modulation of Beclin 1, autophagy and/or CD40 signaling as an approach to improve pathogen eradication. Materials and Methods Ethics statement The animal experimentation guidelines of the US Department of Health and Human Services were followed. The study was approved by the Institutional Animal Care and Use Committee of Case Western Reserve University School of Medicine. Animals and parasites C57BL/6, CD402/2 and mice expressing the Cre recombinase from within the lysozyme M locus (Lyz-Cre mice) were purchased from Jackson Laboratories and bred at the Animal Resource Center (Case Western Reserve University). Becn1+/2 and ATG7flox/flox mice have been described [24,29]. ATG7flox/flox mice were bred with Lyz-Cre mice. All mice were female, on a B6 background and were 8–12 week old when used for the studies. Each experimental group consisted of 4–8 mice. Mice were infected i.p. with 10 cysts of ME49 strain of T. gondii (gift from Dr. George Yap, Brown University). Tachyzoites of the RH, P (gift from Dr. Louis Weiss, Albert Einstein College of Medicine) and PTG strains of T. gondii as well as parasites expressing cytoplasmic DsRed (RFP) were maintained as described [6]. Histopathology and Immunohistochemistry Unless otherwise stated, mice were anesthetized and perfused with PBS 4 weeks after infection. Four 5 mm sections at different areas of the brains and eyes were stained with periodic acid Schiff hematoxylin (PASH) stain and used to score histopathologic changes using previously described criteria [16,17]. Tissue cysts were counted in brain homogenates or in tissue sections as previously described [19]. Sections were incubated with anti- NOS2 or anti-TNF-a Ab (Millipore and Abcam respectively) followed by incubation with secondary antibody conjugated to PLoS ONE | www.plosone.org 11 December 2010 | Volume 5 | Issue 12 | e14472