Targeting glutamine metabolism rescues mice from late-stage cerebral malaria Emile B. Gordona,1,2, Geoffrey T. Harta,1, Tuan M. Trana,1,3, Michael Waisberga,4, Munir Akkayaa, Ann S. Kima, Sara E. Hamiltonb, Mirna Penaa, Takele Yazewa, Chen-Feng Qia, Chen-Fang Leec,d, Ying-Chun Loc, Louis H. Millere,5, Jonathan D. Powellc,5, and Susan K. Piercea,5 aLaboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852; bDepartment of Laboratory Medicine and Pathology, Center for Immunology, University of Minnesota Medical School, Minneapolis, MN 55454; cSidney Kimmel Comprehensive Cancer Research Center, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21287; dChang-Gung Transplantation Institute, Department of Liver and Transplantation Surgery, Chang-Gung Memorial Hospital, Taoyuan 333, Taiwan; and eLaboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852 Contributed by Louis H. Miller, August 26, 2015 (sent for review June 19, 2015; reviewed by James R. Mitchell) + The most deadly complication of Plasmodium falciparum infection intravasculature leukocytes, including CD8 T cells, sequestered in the + + is cerebral malaria (CM) with a case fatality rate of 15–25% in brain vessels (13, 14). In ECM monocytes and both CD4 and CD8 T African children despite effective antimalarial chemotherapy. cells have been shown to accumulate in the brain by both flow There are no adjunctive treatments for CM, so there is an urgent cytometry and by intravital imaging (15). Current evidence indicates + need to identify new targets for therapy. Here we show that the that CD8 T cells are the major mediators of death in ECM (16) and + glutamine analog 6-diazo-5-oxo-L-norleucine (DON) rescues mice that antigen-specific CD8 T cells engage parasite antigens cross-pre- from CM when administered late in the infection a time at which sented on MHC class I molecules on brain endothelium, resulting in mice already are suffering blood–brain barrier dysfunction, brain endothelial cell dysfunction by a perforin-dependent mechanism (17). swelling, and hemorrhaging accompanied by accumulation of par- A critical role for metabolic reprogramming in regulating immune + asite-specific CD8 effector T cells and infected red blood cells in responses is becoming increasingly appreciated. Upon activation, T cells the brain. Remarkably, within hours of DON treatment mice showed undergo metabolic reprogramming to meet the increased energetic and blood–brain barrier integrity, reduced brain swelling, decreased biosynthetic demands of growth and effector T-cell functions (18–20). function of activated effector CD8+ T cells in the brain, and levels Reprogramming involves a shift to aerobic glycolysis and increased of brain metabolites that resembled those in uninfected mice. glutaminolysis. Activated T cells import large quantities of Gln and These results suggest DON as a strong candidate for an effective increase their expression of glutaminase (21–23). Because the pathology adjunctive therapy for CM in African children. leading to death in CM is believed to be in part immune mediated, we hypothesized that blocking T-cell metabolism might effectively mitigate + cerebral malaria | adjunctive therapy | CD8 T cells | glutamine the pathology leading to death in HCM. To this end, in the present metabolism | DON Significance MICROBIOLOGY he World Health Organization estimates that there are nearly T200 million clinical cases of Plasmodium falciparum malaria Cerebral malaria (CM) is a deadly complication of Plasmodium annually (1). For most individuals living in endemic areas, malaria falciparum infection in African children despite effective anti- is uncomplicated and resolves with time. However, in about 1% of malarial treatment. Once signs of neurologic disease have cases, almost exclusively among young children, malaria becomes commenced, there is no adjunctive treatment for CM, and severe and life threatening, resulting in 525,000 deaths each year overall mortality remains high. Thus, a treatment that arrests in Africa alone. One of the most deadly complications of P. fal- disease and promotes healing in the late stages is urgently ciparum infection in humans is cerebral malaria (HCM) charac- needed. Here we report, in an animal model of CM, that the terized by the onset of severe neurological signs such as altered glutamine analog 6-diazo-5-oxo-L-norleucine (DON) is an ef- consciousness, seizures, and coma (2). Autopsy and MRI analyses fective therapy even when treatment is initiated after infected of brains of children with HCM indicate sequestration of infected animals show neurological signs of disease. Within hours of – red blood cells (iRBCs), microhemorrhaging, breakdown of the DON treatment blood brain barrier integrity was restored, and blood–brain barrier (BBB) (3), and a fatal increase in intracranial brain swelling was reduced. These results suggest DON as a pressure resulting from edema (4, 5). At present, despite effective strong candidate for an effective adjunctive therapy for CM in antimalarial drug treatment, mortality for children presenting with African children. HCM remains high, at 15–25%. HCM takes a second toll on Author contributions: E.B.G., G.T.H., T.M.T., M.W., L.H.M., J.D.P., and S.K.P. designed re- African children, leaving survivors at risk for debilitating neuro- search; E.B.G., G.T.H., T.M.T., M.W., M.A., A.S.K., S.E.H., M.P., T.Y., C.-F.Q., C.-F.L., and Y.-C.L. logical defects (6). Thus, there is an urgent need for the devel- performed research; E.B.G., G.T.H., T.M.T., M.W., M.A., A.S.K., C.-F.Q., C.-F.L., Y.-C.L., and J.D.P. opment of effective adjunctive therapies that can be used in analyzed data; and E.B.G., G.T.H., T.M.T., L.H.M., J.D.P., and S.K.P. wrote the paper. conjunction with antimalarials to treat children with HCM. Reviewers included: J.R.M., Harvard School of Public Health. Experimental cerebral malaria (ECM) in mice is a widely used The authors declare no conflict of interest. model of HCM and provides a valuable tool for elucidating the 1E.B.G., G.T.H., and T.M.T. contributed equally to this work. mechanisms involved in CM pathogenesis and identifying cellular 2Present address: Perelman School of Medicine, University of Pennsylvania, Philadelphia, and molecular targets for adjunctive therapy (7). In ECM, 6–7dafter PA 19104. infection with Plasmodium berghei ANKA (PbA), mice of susceptible 3Present address: Division of Infectious Diseases, Indiana University School of Medicine, strains, such as C57BL/6, develop ataxia, paralysis, seizures, and coma Indianapolis, IN 46202. and ultimately die (8). ECM displays key features of HCM, including 4Present address: Department of Pathology, University of Virginia School of Medicine, BBB breakdown, focal hemorrhaging, and brain swelling (9–11). Charlottesville, VA 22903. ’ ECM s pathology also requires sequestration of iRBCs in the brain 5To whom correspondence may be addressed. Email: [email protected], Jpowell@jhmi. vasculature (12), a hallmark of HCM (3). Histological analysis of the edu, or [email protected]. brains of children who died of HCM showed leukocytes, primarily This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. monocytes with phagocytized hemozoin and platelets but also 1073/pnas.1516544112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1516544112 PNAS | October 20, 2015 | vol. 112 | no. 42 | 13075–13080 Downloaded by guest on October 1, 2021 A C signs) to 10 (moribund) using previously described criteria (25). 15 No Rx Nearly all the untreated mice that were infected with PbA developed 100 No Rx DON Rx d5a DON Rx d5a neurological signs by day 5p p.i. that in most cases were severe 10 DON Rx d5p DON Rx d5p DON Rx d6a > DON Rx d6a (clinical score 6) by day 6a p.i. (Fig. 1B). Treatment with DON 50 5 beginning on day 5a p.i. prevented the development of neurological % Survival % symptoms in all PbA-infected mice (Fig. 1B). Treatment of mice with % Parasitemia 0 0 456789 DON beginning on day 5p p.i., a point at which most mice had 0510 Day p.i. Day/Time p.i. clinical scores of 2, not only prevented the worsening of clinical signs B D but promoted the rapid resolution of symptoms (Fig. 1B). Re- 10 4 No Rx n.s. markably, treatment of mice on day 6a p.i., when many mice already DON Rx d5a F.C. 3 DON Rx d5p had developed clinical scores of 5, blocked the progression of the 5 DON Rx d6a 2 disease and rapidly resolved the symptoms in half of the mice (Fig. A 18s 1B). Thus, DON was able to arrest disease and promote healing even (d5p to d6a) to (d5p Pb 1 Clinical Score Clinical when the mice already were displaying signs of neurologic damage. 0 0 5678910 PbA infected: + + A scatter plot showing the clinical scores of individual mice on the DON Rx day p.i.: - d5p Day/Time p.i. RNA day p.i.: d6a d6a day of treatment and the resulting outcome of treatment is given in Fig. S2. For mice treated with DON on d5p p.i., the clinical scores on Fig. 1. DON treatment reduced the mortality associated with ECM. C57BL/6 the day of DON treatment (which ranged from 0 to 6) did not dis- mice were infected with PbA on day 0 and were injected i.p. with saline tinguish the mice that survived from those that died, even though the = (NoRx) (n 49) or with DON (1.3 mg/kg) beginning on day 5 p.i. at 7:00 AM average clinical score was significantly higher for mice that died (DON Rx d5a) (n = 28), on day 5 p.i.