The CD40-Autophagy Pathway Is Needed for Host Protection Despite IFN-C-Dependent Immunity and CD40 Induces Autophagy via Control of P21 Levels Jose-Andres C. Portillo1,2, Genevieve Okenka1,2, Erin Reed 3 , Angela Subauste4, Jennifer Van Grol1,2,5, Katrin Gentil1, Masaaki Komatsu6, Keiji Tanaka6, Gary Landreth3, Beth Levine7, Carlos S. Subauste1,2,5* 1 Department of Ophthalmology and Visual Sciences, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America, 2 Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America, 3 Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America, 4 Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, Michigan, United States of America, 5 Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio, United States of America, 6 Laboratory for Frontier Science, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan, 7 Department of Internal Medicine, Department of Microbiology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America Abstract Autophagy degrades pathogens in vitro. The autophagy gene Atg5 has been reported to be required for IFN-c-dependent host protection in vivo. However, these protective effects occur independently of autophagosome formation. Thus, the in vivo role of classic autophagy in protection conferred by adaptive immunity and how adaptive immunity triggers autophagy are incompletely understood. Employing biochemical, genetic and morphological studies, we found that CD40 upregulates the autophagy molecule Beclin 1 in microglia and triggers killing of Toxoplasma gondii dependent on the autophagy machinery. Infected CD402/2 mice failed to upregulate Beclin 1 in microglia/macrophages in vivo. Autophagy- deficient Beclin 1+/2 mice, mice with deficiency of the autophagy protein Atg7 targeted to microglia/macrophages as well as CD402/2 mice exhibited impaired killing of T. gondii and were susceptible to cerebral and ocular toxoplasmosis. Susceptibility to toxoplasmosis occurred despite upregulation of IFN-c, TNF-a and NOS2, preservation of IFN-c-induced microglia/macrophage anti-T. gondii activity and the generation of anti-T. gondii T cell immunity. CD40 upregulated Beclin 1 and triggered killing of T. gondii by decreasing protein levels of p21, a molecule that degrades Beclin 1. These studies identified CD40-p21-Beclin 1 as a pathway by which adaptive immunity stimulates autophagy. In addition, they support that autophagy is a mechanism through which CD40-dependent immunity mediates in vivo protection and that the CD40- autophagic machinery is needed for host resistance despite IFN-c. Citation: Portillo J-AC, Okenka G, Reed E, Subauste A, Van Grol J, et al. (2010) The CD40-Autophagy Pathway Is Needed for Host Protection Despite IFN-C- Dependent Immunity and CD40 Induces Autophagy via Control of P21 Levels. PLoS ONE 5(12): e14472. doi:10.1371/journal.pone.0014472 Editor: Gordon Langsley, INSERM U1016, Institut Cochin, France Received July 6, 2010; Accepted December 3, 2010; Published December 31, 2010 Copyright: 2010 Portillo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health (NIH) grants EY018341 (C.S.S) and AI48406 (C.S.S), the American Heart Association Ohio Valley Affiliate (C.S.S), NIH grant P30 EY11373, and Case/University Hospitals of Cleveland (UHC) Center for AIDS Research NIH grant AI36219. J-A. P. is a recipient of a post-doctoral fellowship from the Ohio Lions Eye Research Foundation. J.V.G. is a recipient of a pre-doctoral fellowship from Prevent Blindness Ohio. J.V.G. was supported by T32 EY007157-07. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: carlos.subauste@case.edu Introduction The lysosomal system is an effector of microbial degradation. Unfortunately, many pathogens including the intracellular protozoan Toxoplasma gondii have developed various strategies to avoid lysosomal degradation. Autophagy is a mechanism that can re-route pathogens to lysosomes. Autophagy is a process where an isolation membrane encircles portions of cytosol and organelles leading to the formation of autophagosomes [1,2]. Fusion between autophagosomes and endosomes-lysosomes culminates in the formation of autolysosomes and degradation of their contents. Autophagy can mediate in vitro anti-microbial activity against various pathogens [3,4,5,6,7]. In the case of T. gondii infection, the CD40 – CD154 pathway triggers killing of the parasite within macrophages that is dependent on the autophagy pathway [6,8]. CD40 is a member of the TNF receptor superfamily expressed on antigen presenting cells as well as on some non-hematopoietic cells, while CD154 (CD40 ligand) is expressed primarily on activated CD4+ T cells. The interaction between T. gondii-reactive T cells and infected macrophages results in CD40-dependent killing of the parasite through the autophagy pathway [6]. Innate immunity can activate autophagy to mediate host protection in vivo. Autophagy protects Drosophila against Listeria monocytogenes and Vesicular stomatitis virus [9,10]. In the case of Herpes simplex virus 1 (HSV-1), the virus prevents autophagy by producing the neurovirulence factor ICP34.5 that binds and blocks the effect of the autophagy protein Beclin 1 [11]. A strain of HSV-1 deficient in ICP34.5 does not cause encephalitis in wild type mice but causes disease in mice deficient in PKR, a signaling molecule linked to autophagy [11]. Similarly, in a model of Salmonella infection in Caenorhabditis elegans and Dictyostelium discoideum, autophagy gene inactivation results in increased bacterial replication and decreased animal lifespan [12]. PLoS ONE | www.plosone.org 1 December 2010 | Volume 5 | Issue 12 | e14472