Chemokine receptor CXCR3 and its ligands CXCL9 and CXCL10 are required for the development of murine cerebral malaria

Transcription

1 Chemokine receptor CXCR3 and its ligands CXCL9 and CXCL10 are required for the development of murine cerebral malaria Gabriele S. V. Campanella, Andrew M. Tager, Joseph K. El Khoury, Seddon Y. Thomas*, Tabitha A. Abrazinski, Lindsay A. Manice, Richard A. Colvin, and Andrew D. Luster Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA Communicated by Jeffrey Ravetch, The Rockfeller University, New York, NY, February 15, 2008 (received for review December 18, 2007) Cerebral malaria is a significant cause of global mortality, causing an estimated two million deaths per year, mainly in children. The pathogenesis of this disease remains incompletely understood. Chemokines have been implicated in the development of cerebral malaria, and the IFN-inducible CXCR3 chemokine ligand IP-10 (CXCL10) was recently found to be the only serum biomarker that predicted cerebral malaria mortality in Ghanaian children. We show that the CXCR3 chemokine ligands IP-10 and Mig (CXCL9) were highly induced in the brains of mice with murine cerebral malaria caused by Plasmodium berghei ANKA. Mice deficient in CXCR3 were markedly protected against cerebral malaria and had far fewer T cells in the brain compared with wild-type mice. In competitive transfer experiments, CXCR3-deficient CD8 T cells were 7-fold less efficient at migrating into the infected brains than wild-type CD8 T cells. Adoptive transfer of wild-type CD8 effector T cells restored susceptibility of CXCR3-deficient mice to cerebral malaria and also restored brain proinflammatory cytokine and chemokine production and recruitment of T cells, independent of CXCR3. Mice deficient in IP-10 or Mig were both partially protected against cerebral malaria mortality when infected with P. berghei ANKA. Brain immunohistochemistry revealed Mig staining of endothelial cells, whereas IP-10 staining was mainly found in neurons. These data demonstrate that CXCR3 on CD8 T cells is required for T cell recruitment into the brain and the development of murine cerebral malaria and suggest that the CXCR3 ligands Mig and IP-10 play distinct, nonredundant roles in the pathogenesis of this disease. T cells Plasmodium IP-10 brain trafficking Malaria remains one of the most common infectious diseases in the world with an estimated 500 million clinical cases per year. One of the most lethal complications of infection with Plasmodium falciparum is cerebral malaria (CM), which causes more than one million deaths per year, mainly in African children under the age of 5. Although CM has been studied extensively, the pathogenesis of this disease is not fully understood. Sequestration of parasitized red blood cells in the microvasculature of the brain has long been thought to be the main factor leading to CM. However, sequestration does not seem to be the sole cause of CM. Instead, loss of blood brain barrier integrity and activation of the inflammatory response have been well documented in CM and likely contribute to the complex pathogenesis of this disease (1). Mouse models of CM have been widely used to gain insight into the pathogenesis of CM. Although no animal model fully recapitulates the human disease, infection of certain mouse strains with Plasmodium berghei ANKA induces murine CM with many characteristics of the human disease. Infected mice develop cerebral complications, including paralysis, ataxia, convulsion, coma, and death. CD8 T cell sequestration in the brain has been shown to play a critical role in the development of murine CM (2 4). However, the mechanisms directing these cells to the brain have not been fully elucidated, with two studies showing that the chemokine receptor CCR5 partially guides CD8 T cells into the brain (3, 4). Chemokines are a superfamily of chemotactic cytokines that play important roles in the generation and delivery of immune and inflammatory responses. They are also involved in most disease processes, including infectious, autoimmune, inflammatory, and malignant diseases (5). Chemokines orchestrate the movement of leukocytes and other cells by activating specific seven transmembrane-spanning G protein-coupled receptors expressed on responsive cells. IP-10 (IFN-induced protein of 10 kda), or CXCL10, one of the very first chemokines identified, directs the trafficking of activated effector CD4 and CD8 T lymphocytes and other effector lymphocytes, such as natural killer (NK) and natural killer T (NKT) cells. It does so by binding to its high-affinity receptor CXCR3, which it shares with two other ligands, monokine-induced by IFN- (Mig/CXCL9) and IFN-inducible T cell- chemoattractant (I-TAC/CXCL11). CXCR3 and its ligands have been implicated in neurological diseases caused by other pathogens, including Toxoplasma gondii (6), West Nile virus (7), and HIV (8). To date, little has been done to investigate the role of CXCR3 and its ligands in CM. A recent study measuring 36 biomarkers found that IP-10 was the only serum marker independently associated with CM mortality in Ghanaian children (9). In addition, IP-10 was one of eight biomarkers in the cerebral spinal fluid (CSF) significantly upregulated in the CM group. Furthermore, in a mouse model of CM, NK cells were shown to induce the recruitment of CXCR3 T cells to the brain (10). However, the role of IP-10 and CXCR3 in CM pathogenesis has not been fully established. We therefore investigated chemokine expression in the brain and spleen of mice infected with P. berghei ANKA and studied the effects of IP-10, Mig, and CXCR3 in murine CM, by using mice genetically deficient for CXCR3, IP-10, and Mig. Results CXCR3-Binding Chemokines Are Highly Up-Regulated in the Brains of Mice with CM. We examined the expression of 21 chemokines in the brain and spleen during murine CM induced by P. berghei ANKA in C57BL/6 mice. Eight days after infection, when the mice displayed signs of CM, the two CXCR3 ligands, IP-10 and Mig, were the most highly expressed of the tested chemokines in the brain in terms of total mrna levels (Fig. 1A). In addition, Author contributions: G.S.V.C., A.M.T., and A.D.L. designed research; G.S.V.C., T.A.A., and L.A.M. performed research; G.S.V.C., J.K.E.K., S.Y.T., and R.A.C. analyzed data; and G.S.V.C., A.M.T., and A.D.L. wrote the paper. The authors declare no conflict of interest. *Present address: Howard Hughes Medical Institute, Committee on Immunology, Department of Pathology, University of Chicago, Chicago, IL To whom correspondence should be addressed at: Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA aluster@mgh.harvard.edu by The National Academy of Sciences of the USA PNAS March 25, 2008 vol. 105 no cgi doi pnas

2 Fig. 1. Chemokine mrna and protein levels in brain and spleen during murine CM. C57BL/6 mice were infected with P. berghei ANKA ( parasitized red blood cells), and their brains (A C) and spleens (D F) were harvested at the indicated time points. mrna levels were analyzed by qpcr and are shown normalized to GAPDH (A and D) and as fold induction compared with uninfected mice (B, C, E, and F). Brain and spleen IP-10 and Mig protein levels were determined by ELISA (C and F, Inset). Results are representative of two independent experiments with two or three mice per group. they were also the most highly induced chemokines. Mig was induced 500-fold and IP fold after infection (Fig. 1B). Although a small increase in I-TAC mrna expression was observed, we did not study further the effect of I-TAC on the development of murine CM. C57BL/6 mice do not produce functional I-TAC protein because of a mutation that introduces a stop codon in the I-TAC leader sequence (GenBank accession numbers NM and AA174767). Sequencing of PCR products obtained from our IP-10- and Mig-deficient mice in the C57BL/6 background revealed the same mutations, demonstrating that the IP-10- and Mig-deficient mice do not express functional I-TAC protein (R.A.C., G.S.V.C., and A.D.L., unpublished data). In addition to the CXCR3 ligands, the CC chemokines MCP-1, MCP-3, RANTES, MIP-1, MIP-1, and the CXC chemokine MIP-2 were also induced but to lower levels than the CXCR3 ligands. Chemokine levels in the spleen in general did not increase as much as in the brain during murine CM (Fig. 1 D and E). In the spleen, Mig increased 1.8-fold and IP fold, whereas the mrna levels of a number of other chemokines, including RANTES, SDF-1, and ELC decreased after P. berghei ANKA infection. IP-10 and Mig were the initial chemokines induced in the brain 6 days after infection (50-fold and 165-fold, respectively) and increased further by day 8 (Fig. 1C). RANTES, MIP-1, MCP-1, and IFN- levels were still low on day 6 and increased by day 8. In contrast, in the spleen, the highest chemokine levels were seen 6 days after infection, with MCP-1 and IFN- showing the highest induction (Fig. 1F). By day 8 after infection, spleen RNA levels had decreased for all chemokines analyzed. IP-10 and Mig protein levels in the brain and spleen were analyzed by ELISA. Consistent with RNA data, IP-10 and Mig protein levels increased by day 6 after infection (Fig. 1C Inset) and increased further on day 8. Spleen IP-10 and Mig protein levels were increased day 6 after infection and declined by day 8 (Fig. 1F Inset). CXCR3 KO Are Markedly Protected from CM. To determine the role of the CXCR3 ligands in murine CM, we infected CXCR3- deficient (CXCR3 KO) mice with P. berghei ANKA. Wild-type mice developed murine CM between days 6 and 10 after infection, and their mortality was % (Fig. 2A). In contrast, CXCR3 KO mice were markedly protected from CM, with mortality of only 10 30% (Fig. 2A). Levels of parasitemia for wild-type and CXCR3 KO mice were found to be similar through day 10 after infection, by which time most wild-type mice had died (Fig. 2B). CXCR3 KO mice that did not develop CM had increasing levels of parasitemia, developed anemia, and were killed at day 21 after infection. Typical signs of CM were evident in H&E-stained brain sections from wild-type mice, with occluded and disrupted blood vessels and leukocyte accumulation, whereas these findings were absent in CXCR3 KO mice (Fig. 2C). T Cell Recruitment to the Brain Is Reduced in CXCR3 KO Mice. Brain-infiltrating CD8 T cells have been shown to be crucial mediators of CM induced by P. berghei ANKA. We therefore compared the recruitment of CD3 T cells and CD3 CD8 T Fig. 2. CXCR3 KO mice are protected from murine CM. Wild-type and CXCR3 KO C57BL/6 mice were infected with P. berghei ANKA. (A) Mortality was checked twice daily. The experiment is representative of four separate infections, each with at least eight mice per group. (B) Parasitemia was monitored every other day by Giemsa-stained thin blood smears. The experiment is representative of two independent infections. (C) Brain histology was assessed by H&E staining. Brains were removed after heart perfusion with 10% formalin of uninfected mice or mice 8 days after infection, as indicated. (Scale bars: 20 m.) IMMUNOLOGY Campanella et al. PNAS March 25, 2008 vol. 105 no

3 Fig. 4. CXCR3 is required for efficient CD8 T cell trafficking to the brain. Wild-type (Thy1.1) and CXCR3 KO (Thy1.2) OT-I cells were activated in vitro for 5 days and were adoptively cotransferred ( each) into the same Thy1.1 Thy1.2 wild-type mouse by i.v. injection. Mice were infected 5 days before adoptive transfer with P. berghei ANKA. Eight days after infection, when mice developed signs of murine CM, spleens (A) and brains (B) were harvested, and Thy1.1 and Thy1.2 single positive cells were analyzed by flow cytometry as shown. Results are representative of three independent experiments. *, P 0.05 compared with wild-type OT-I cells. (Right) Representative primary flow cytometric dot plots are shown after lymphocyte gating. The percentage of lymphocyte gate is shown for the indicated cell populations. Fig. 3. CD3 CD8 T cell sequestration is reduced in the brains of CXCR3 KO mice infected with P. berghei ANKA. Flow cytometric analysis of brainsequestered T lymphocytes and NK cells is shown. Wild-type and CXCR3 KO mice were infected with P. berghei ANKA, and brains were harvested at different days after infection. (Left) Number of brain sequestered CD3 (Top), CD3 CD8 (Middle), and NK (Bottom) cells were averaged over four independent experiments and are shown SD. (Right) Representative primary flow cytometric dot plots are shown after lymphocyte gating for T cells (Top and Middle) and NK/NKT cells (Bottom). The percentage of lymphocyte gate is shown for the indicated cell populations. cells to the brains of infected wild-type and CXCR3 KO mice. T cell infiltration to the brain peaked at day 8, when mice developed signs of severe CM (Fig. 3). Infiltration of both CD3 and CD8 T cells into the brain was higher in wild-type mice than in CXCR3 KO mice. The number of brain-sequestered CD8 T cells at day 8 after infection when CM was evident was 14-fold higher in wild-type mice than on day 0, with overall CD3 T cells being increased 7-fold (Fig. 3 Left). This response was markedly attenuated in CXCR3 KO mice, which only had a 4-fold increase in CD3 CD8 T cell and 2.7-fold increase in CD3 T cell recruitment compared with uninfected mice. CD8 T cell recruitment to the infected brain in CXCR3 KO mice was reduced 300% compared with wild-type mice. In addition to T lymphocytes, NK and NKT cells have been shown to play an important role in the development of murine CM. Recruitment of NK cells to the brain in CXCR3 KO mice was 2-fold lower than for wild-type mice, whereas NKT cells were not significantly different (Fig. 3 Bottom). CXCR3 Is Required for CD8 T Cell Trafficking to the Brain. To determine directly the role for CXCR3 in CD8 T cell homing to the brain in murine CM, we used competitive adoptive transfer experiments. In these studies, in vitro-generated wildtype and CXCR3 KO effector CD8 T cells were cotransferred into wild-type mice that were infected 5 days earlier with P. berghei ANKA. We used CD8 T cells from ovalbumin-specific T cell receptor-transgenic mice (OT-I) crossed with wild-type Thy1.1 or CXCR3 KO Thy1.2 mice. When transferred into wild-type Thy1.1 Thy1.2 mice, both wild-type (Thy1.1) and CXCR3 KO (Thy1.2) OT-1 CD8 T cells can be analyzed in the same recipient mouse. In addition, the use of OT-I cells allowed us to examine the role of CXCR3 in migration to the brain independent of antigen-induced proliferation. Both wild-type and CXCR3 KO OT-I effector CD8 T cells were confirmed to be similarly activated, with high CD25 expression and intermediate CD62L expression, as determined by FACS analysis (data not shown). When recipient mice developed CM, brain and spleen were harvested and analyzed for the presence of transferred cells by using the Thy1.1 and Thy1.2 markers. In the spleen, wild-type and CXCR3 KO cells were present in similar numbers (Fig. 4A). In contrast, in the brain there were 7-fold fewer CXCR3 KO OT-I cells than wild-type cells (Fig. 4B), which clearly demonstrates that CXCR3-deficient CD8 T cells could not traffic to the brain as efficiently as wild-type CD8 T cells. Wild-Type CD8 T Cells Restore CXCR3KO Mice CM Mortality. To define more specifically the involvement of CXCR3 in this disease, splenocytes from wild-type or CXCR3 KO mice infected 5 days earlier with P. berghei ANKA were adoptively transferred i.v. into CXCR3 KO recipients. Two hours later, recipient and control CXCR3 KO mice were infected with P. berghei ANKA. CXCR3 KO mice that had received wild-type splenocytes developed CM at nearly the same rate as wild-type mice, whereas only a very low proportion of CXCR3 KO mice that had received CXCR3 KO splenocytes developed CM, similar to CXCR3 KO mice that did not receive any transferred cells (Fig. 5A). This finding demonstrates that CXCR3 on leukocytes is required for the development of murine CM and that wild-type leukocytes can restore CM mortality in CXCR3 KO mice. Because CXCR3 is expressed on different leukocyte subsets among splenocytes, we next isolated CD3 CD8 T cells from wild-type splenocytes 5 days after infection by FACS sorting and then adoptively transferred them into CXCR3 KO mice before infection. Mice that had received CD8 T cells developed CM at rates similar to those of wild-type mice (Fig. 5A), indicating that CXCR3 on CD8 T cells is required to induce CM. Using the Thy1.1 allele, we found that adoptively transferred wild-type CD8 T cells (Thy1.1) were recruited to the brain of CXCR3 KO mice (Thy1.2) at the CM stage ( % of lymphocyte gate) (Fig. 5 B and C). CXCR3 KO mice that had received wild-type CD8 T cells also had a marked increase in the numbers of their own CXCR3-deficient CD8 T cells (Thy1.2) in the brain compared with CXCR3 KO mice that had not received wild-type CD8 T cells (Fig. 5C). The number of CXCR3KO CD8 T cells (Thy1.2) in the brain of CXCR3 KO mice that had received adoptively transferred wild-type CD8 T cells was 3-fold higher than in CXCR3 KO mice without transferred cells. To investigate the mechanism of this CXCR3- independent recruitment of CD8 T cells, we measured chemokine and IFN- expression in the brains of wild-type, CXCR3 KO, and CXCR3 KO mice that had received adoptively trans cgi doi pnas Campanella et al.

4 Fig. 5. Wild-type CD8 T cells restore CXCR3 KO murine CM mortality. (A) Wild-type or CXCR3 KO splenocytes (Left) orcd8 T cells (Right) were prepared from P. berghei ANKA-infected mice 5 days after infection and i.v. transferred into CXCR3 KO mice 2 h before infection with P. berghei ANKA. (B) Brains of wild-type or CXCR3 mice with or without CD8 T cell transfer were harvested on day 8 after infection, and brain-sequestered recipient CD8 T (Thy1.2) cells were analyzed by flow cytometry. (C) Representative primary flow cytometric dot plots are shown after lymphocyte gating. The percentage of lymphocyte gate is shown for the indicated cell populations. (D) Chemokine and IFN- expression in the brain was analyzed for mice from experiments shown in C. ferred wild-type CD8 T cells (Fig. 5D). The marked induction of IP-10, Mig, RANTES, MCP-1, and IFN- seen in wild-type mice was reduced in CXCR3 KO mice. Adoptive transfer of wild-type CD8 T cells restored the expression of these chemokines and IFN- in CXCR3 KO mice to levels observed in wild-type mice. This finding suggests that CXCR3 guides CD8 T cells into the brain, which results in the production of additional chemokines that then amplifies CD8 T cell recruitment to the brain independently of CXCR3. IP-10 KO and Mig KO Are Partially Protected from CM. Because we found that both IP-10 and Mig were highly induced in the brain by murine CM, we sought to evaluate the individual contributions of these two ligands to the development of this syndrome. To do so, IP-10- and Mig-deficient mice were infected with P. berghei ANKA and observed for mortality from CM. Both IP-10- and Mig-deficient mice were partially protected from CM, with only 30 40% mortality (Fig. 6 A and B) compared with % for wild-type mice, suggesting that both ligands are independently involved in the disease. To explore further the respective roles of IP-10 and Mig in CM, we performed immunhistochemistry on brains of mice on days 0 and 8 after infection. Uninfected mice showed very little staining for either ligand. Mig- and IP-10-deficient mice that had developed signs of CM were used as controls to ensure specific antibody staining. After infection and the development of CM, brains of wild-type mice showed marked Mig staining on endothelial vessels (Fig. 6C). In contrast, IP-10 staining was found widely disseminated in neurons throughout the brain parenchyma and only occasionally on endothelial cells (Fig. 6D). Immunofluorescence double staining with antibodies specific for neurons (TuJ-1) and astrocytes (GFAP) confirmed that IP-10 was expressed by neurons but not astrocytes (Fig. 6E). These data suggest that IP-10 and Mig have distinct functions in the pathogenesis of CM. Discussion Mortality rates in CM remain very high (10 30%) because of the lack of timely and effective treatments, which relates, in part, to our lack of understanding of the pathogenesis of this syndrome. To advance the understanding of this complex disease, both human and animal studies are required, which complement and direct each other. In recent years, human studies have found associations between levels of certain chemokines and CM mortality, which we are now studying further in a mouse model of this disease. In particular, a recent study found that among 36 biomarkers, serum IP-10 levels were the only variable independently associ- IMMUNOLOGY Fig. 6. IP-10 and Mig KO are protected from murine CM and are expressed on different cells in the brain. (A and B) Wild-type and Mig KO (A) or IP-10 KO (B) in the C57BL/6 background were infected with P. berghei ANKA, and mortality from murine CM was monitored twice daily. One representative experiment from three independent infections is shown with at least eight mice per group. (C E) Representative immunohistochemistry (C and D) and immunofluorescence (E) of mouse brains harvested day 0 or day 8 after infection, staining for Mig (C) or IP-10 (D and E). Arrows show endothelial cells staining, arrowheads show neuronal staining. (Scale bars: 20 m.) Campanella et al. PNAS March 25, 2008 vol. 105 no

5 ated with CM mortality and that CSF IP-10 concentrations were also up-regulated in Ghanaian children who died of CM (9). Although the mouse model of CM caused by P. berghei ANKA does not reproduce all of the features of human CM, these human findings are very similar to what we report here for IP-10 and the other CXCR3 ligand Mig, which were the most highly expressed chemokines in the brains of mice with CM. In addition, we also found that splenic levels of both chemokines were up-regulated 2-fold, similar to what was found for serum levels in Ghanaian children. Interestingly, the human study also found that CM induced a greater fold increase in IP-10 levels in the CSF than in the serum, which is also similar to what we found in our murine study. Different human studies have reported that decreased RANTES serum levels were independently associated with CM mortality in Uganda (11), whereas another study reported increased RANTES expression in the brain in children who died of CM in Ghana (12). We similarly found decreased RANTES expression in the spleen and increased expression in the brain during murine CM. Taken together, these data suggest that chemokine regulation is similar in the P. berghei ANKA mouse model to what has been described for children dying of CM. Although the human studies have shown that chemokine levels are associated with mortality from CM, they cannot provide information about the functional roles of different chemokines in the pathogenesis of this disease. Employing the mouse model has allowed us to interrogate the potential contribution of specific chemokines and chemokine receptors. We found that mice deficient in CXCR3 were highly protected against murine CM. CD3 and CD3 CD8 T cell recruitment to the brain was markedly reduced in CXCR3 KO mice, whereas NK cell recruitment was only modestly reduced. To establish whether CXCR3 is directly involved in directing CD8 T cells to the brain during murine CM, we used a competitive transfer model in which both wild-type and CXCR3 KO CD8 T cells were i.p. injected into the same mouse. Similarly to what we found in the CXCR3 KO mice, a small number of CXCR3 KO OT-I cells were found sequestered in the brain, but wild-type cells were 7-fold more efficient in migrating to the brain. Adoptive transfers of in vivo activated wild-type CD8 T cells restored murine CM susceptibility to CXCR3 KO mice and induced CXCR3-independent recruitment CD8 T cells to the brain. Early IP-10 and Mig expression in the brain might be induced by the innate immune system, possibly in response to Toll-like receptor signaling, as one study reported that brain IP-10 and Mig expression depended on MyD88 signaling after P. berghei ANKA infection (13). IP-10 and Mig were the earliest chemokines up-regulated in the brain, 6 days after infection, before CD8 T cell infiltration to the brain. Our findings therefore suggest that CD8 T cells recruited to the brain by IP-10 and Mig amplify the induction of cytokines and additional chemokines that leads to the recruitment of CD8 T cells independent of CXCR3. One intriguing question in the chemokine field is the apparent redundancy of the chemokine system, with most receptors being activated by more than one chemokine and many chemokines binding to more than one receptor. To determine the respective roles of the CXCR3 ligands in CM, we analyzed IP-10 and Mig KO mice in the P. berghei ANKA model. We found that IP-10 and Mig KO mice were partially protected from CM, demonstrating that both of these CXCR3 ligands were needed for the development of CM. Immunohistochemistry revealed that the two chemokines may play distinct roles. Mig staining was found mainly on brain endothelial cells, whereas IP-10 was expressed mainly on neurons. While our article was in review, Miu et al. (14) reported similar findings regarding the role of CXCR3 in murine CM induced by P. berghei ANKA. However, by using in situ RNA hybridization, Miu et al. detected Mig expression in microglia and endothelial cells and IP-10 expression in astrocytes and endothelial cells, but not neurons. Using immunohistochemistry, we also detected Mig and IP-10 expression in endothelial cells. However, we detected IP-10 expression in neurons but not astrocytes, which we confirmed by using immunofluorescence double staining. In West Nile virus infection, IP-10 is expressed in neurons and directs the migration of CD8 T cells into the brain (7). In HIV infection, IP-10 is expressed by neurons and has a direct neurotoxic effect, and its CSF levels were found to correlate with HIV-associated dementia (15, 16). It is possible that IP-10 may contribute to the neurological impairment seen in human CM in an analogous manner through direct neurotoxicity in addition to its role in CD8 T cell recruitment. In summary, we have shown that CXCR3 and its two ligands IP-10 and Mig are required for the development of murine CM. CXCR3 is critically required for early CD8 T cell sequestration in the brain of infected mice. CXCR3 T cells induce the production of a second wave of chemokines in the brain, which in turn induces further recruitment of CD8 T cells independently of CXCR3. Our findings suggest that CXCR3 and its ligands link innate immune cell activation and the delivery of an immunopathogenic adaptive immune response to the brain. Methods Materials and Mice. C57BL/6 wild-type mice were purchased from the National Cancer Institute, National Institutes of Health (NIH), Bethesda, MD. Breeder pairs of CXCR3-deficient (CXCR3 KO) mice were a kind gift from G. Gerard (Children s Hospital, Harvard Medical School, Boston, MA), and breeder pairs of Mig KO mice were a kind gift from J. M. Farber (National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD). IP-10 KO mice were generated in our laboratory and backcrossed into C57BL/6 for nine generations (17); CXCR3 KO and Mig KO mice were backcrossed into C57BL/6 mice 10 and nine generations, respectively. All three KO mice strains were rederived to be free of Helicobacter and Pasteurella pneumotropica. The OT-I TCR mice in the C57BL/6 background were obtained from Jackson Immunoresearch Laboratories and crossed with CXCR3 KO mice. All protocols were approved by the Massachusetts General Hospital Subcommittee on Research and Animal Care. Antibodies were from BD Biosciences, unless stated otherwise. Infections. Six- to 8-week-old female mice were injected i.p. with P. berghei ANKA-infected red blood cells. Mortality was checked twice daily, and mice showing signs of severe CM (ataxia, convulsion, coma) between days 6 and 10 after infection were killed. Parasitemia was assessed by tail blood smears stained with Giemsa every other day. Mice without CM developed anemia because of rising parasitemia and were killed on day 21. Quantitative PCR. Total RNA from spleens and brains of mice either uninfected or at different points after infection with P. berghei ANKA was isolated by using TRIzol and subsequently with an RNAeasy kit (Qiagen). RNA was converted to cdna and analyzed by qpcr as described before (18) by using the MX4000 multiplex quantitative PCR system (Stratagene). Brain and Spleen Chemokine ELISA. Brains and spleens were homogenized in HBSS and protease inhibitor mixtures (Roche Applied Science) and centrifuged at 11,000 g for 20 min. IP-10 and Mig levels in the supernatants were determined by using murine Quantikine immunoassay kits (R&D Systems). Brain-Sequestered Leukocyte Isolation and Spleen Cell Isolation. Brain and spleen cells were obtained from mice at different times after infection. Brains were removed after intracardial perfusion with 35 ml of PBS; they were minced and digested for 45 min with Blendzyme (Roche) and DNase (Sigma Aldrich) at 37 C with shaking. The cells were passed through a 70- m cell strainer and centrifuged on a 35% Percol for 40 min at 450 g. The pellets were washed, and the red blood cells were lysed (Sigma), counted, and stained for flow cytometry. Spleens were removed, passed through a 70- m strainer, and red blood cells lysed and counted. For flow cytometry, cells were incubated for 10 min with 2.4G2 anti-fc III/II receptor (BD PharMingen) and stained with the following antibodies: FITC-conjugated anti-murine CD3, PE-Cy5.5 or allophycocyanin (APC)-conjugated anti-murine CD8, APCconjugated anti-murine NK1.1, PE- or PercP-conjugated anti-murine Thy1.1, or APC-conjugated anti-murine Thy1.2 at 4 C for 20 min. Cells were fixed with 1% paraformaldehyde, and cytofluorometry was performed by using a FACS cgi doi pnas Campanella et al.

6 Caliber Cytometer (Becton Dickinson) and analyzed with CellQuest or Flowjo software. In Vivo OT-I Recruitment. OT-I cells from OT-I wild-type Thy1.1 and CXCR3 KO OT-I Thy1.2 were prepared and cultured with IL-2, IL-12, anti-cd28 and ovalbumin antigen as described in ref. 19, and harvested after 5 days of in vitro stimulation with Lympholyte (Cedarlane). FACS analysis confirmed equal activation of both cell types. Wild-type and CXCR3 KO cells were mixed ( each per mouse) and coinjected i.p. in 500 l of HBSS into Thy1.1 Thy1.2 mice infected 5 days earlier with P. berghei ANKA. When mice were at CM stage, the spleen and brain were harvested and processed as above. Transferred cells were detected with Thy1.1 PercP and Thy1.2 APC antibodies. Splenocyte and CD8 T Cell Transfer. Spleens of wild-type Thy1.1 mice or CXCR3 KO mice were removed 5 days after infection with P. berghei ANKA and put through a 70- m cell strainer. Red blood cells were lysed, and splenocytes were washed three times. For CD8 T cell transfer, splenocytes were stained with FITC-conjugated anti-murine CD3 and APC-conjugated anti-murine CD8 as described above. CD3 CD8 cells in the lymphocyte gate were sorted on a fluorescence-activated Aria cell-sorting instrument (BD Biosciences) and were 99% pure after sorting. Splenocytes (10 7 )orcd8 T cells (10 6 ) were injected by tail vein into CXCR3 KO mice. Mice were infected with P. berghei ANKA 2 h after cell transfer as described above. Immunohistochemistry (IHC). Brains were removed after intracardial perfusion with 35 ml of buffered formalin, sectioned (5 m), and stained for H&E. For IP-10 and Mig staining, paraffin-embedded 5- m sections were deparaffinized, and for IHC they were incubated in 3% H 2 O 2 in methanol and subsequently in 70% ethanol. Slides were pretreated with antigen decloaker in the decloaker (Biocare Medical) and blocked with horse serum/avidin (Vector Laboratories). The anti-mip-10 or anti-mmig antibodies (both from R&D Systems) were added at 1:200 dilution overnight. Slides were incubated first with biotin, then biotinylated horse anti-goat IgG (1:200, 35 min; Vector Laboratories). For IHC, slides were treated with Elite (Vector Laboratories) for 1 h, followed by Perm AEC solution (Biocare Medical), and counterstained with hematoxylin and lithium bicarbonate. For immunofluorescence, slides were incubated with TuJ-1 antibody (1:200, overnight; R&D Systems) for neuronal staining, employing the MoM kit (Vector Laboratories), then rabbit anti-mouse Alexa Fluor 488 (1:500, 20 min; Invitrogen). For astrocyte staining, slides were incubated with anti-gfap (1:200, 1 h; Dakocytomation) followed by donkey anti-rabbit Alexa Fluor 488 (1:500, 20 min). IP-10 staining was performed as above, employing rabbit serum, biotinylated rabbit anti-goat IgG (Vector Laboratories), and strep-alexa Fluor 555 (1:250, 20 min; Invitrogen). Slides were counterstained with DAPI. Statistical Analysis. Statistical analysis was performed by Student s two-tailed t test (unpaired) for means and by Cox Mantel log-rank test for differences in mortality. A P value of 0.05 was considered significant. ACKNOWLEDGMENTS. We thank Nicole Brousaides for technical assistance. This work was supported by National Institutes of Health Grant R01-CA69212 (to A.D.L.). 1. Coltel N, Combes V, Hunt NH, Grau GE (2004) Cerebral malaria: A neurovascular pathology with many riddles still to be solved. Curr Neurovasc Res 1: Hermsen C, van de Wiel T, Mommers E, Sauerwein R, Eling W (1997) Depletion of CD4 or CD8 T cells prevents Plasmodium berghei-induced cerebral malaria in end-stage disease. Parasitology 114: Nitcheu J, et al. (2003) Combadiere B Perforin-dependent brain-infiltrating cytotoxic CD8 T lymphocytes mediate experimental cerebral malaria pathogenesis. J Immunol 170: Belnoue E, et al. (2003) CCR5 deficiency decreases susceptibility to experimental cerebral malaria. Blood 101: Viola A, Luster AD (2007) Chemokines and their receptors: Drug targets in immunity and inflammation. Annu Rev Pharmacol Toxicol 48: Khan IA, et al. (2000) IP-10 is critical for effector T cell trafficking and host survival in Toxoplasma gondii infection. Immunity 12: Klein RS, et al. (2005) Neuronal CXCL10 directs CD8 T cell recruitment and control of West Nile virus encephalitis. J Virol 79: Sui Y, et al. (2004) Neuronal apoptosis is mediated by CXCL10 overexpression in simian human immunodeficiency virus encephalitis. Am J Pathol 164: Armah HB, et al. (2007) Cerebrospinal fluid and serum biomarkers of cerebral malaria mortality in Ghanaian children. Malar J 6:147 (abstr). 10. Hansen DS, Bernard NJ, Nie CQ, Schofield L (2007) NK cells stimulate recruitment of CXCR3 T cells to the brain during Plasmodium berghei-mediated cerebral malaria. J Immunol 178: John CC, Opika-Opoka R, Byarugaba J, Idro R, Boivin MJ (2006) Low levels of RANTES are associated with mortality in children with cerebral malaria. J Infect Dis 194: Sarfo BY, et al. (2004) The cerebral malaria-associated expression of RANTES, CCR3, and CCR5 in post-mortem tissue samples. Ann Trop Med Parasitol 98: Griffith JW, et al. (2007) Toll-like receptor modulation of murine cerebral malaria is dependent on the genetic background of the host. J Infect Dis 196: Miu J, et al. (2008) Chemokine gene expression during fatal murine cerebral malaria and protection due to CXCR3 deficiency. J Immunol 180: Kolb SA, et al. (1999) Identification of a T cell chemotactic factor in the cerebrospinal fluid of HIV-1-infected individuals as interferon- -inducible protein 10. J Neuroimmunol 93: Cinque P, et al. (2005) Cerebrospinal fluid interferon- -inducible protein 10 (IP-10, CXCL10) in HIV-1 infection. J Neuroimmunol 168: Dufour JH, et al. (2002) IFN- -inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking. J Immunol 168: Means TK, Hayashi F, Smith KD, Aderem A, Luster AD (2003) The Toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine production in human dendritic cells. J Immunol 170: Medoff BD, et al. (2005) BLT1-mediated T cell trafficking is critical for rejection and obliterative bronchiolitis after lung transplantation. J Exp Med 202: IMMUNOLOGY Campanella et al. PNAS March 25, 2008 vol. 105 no