Genes and Immunity (2010) 11, 310–318 & 2010 Macmillan Publishers Limited All rights reserved 1466-4879/10 $32.00 www.nature.com/gene ORIGINAL ARTICLE Identification of a novel cerebral malaria susceptibility locus (Berr5) on mouse chromosome 19 J Berghout1, G Min-Oo1,MTam2, S Gauthier1, MM Stevenson2 and P Gros1 1Department of Biochemistry, McGill University, Montreal, Quebec, Canada and 2Research Institute of the Montreal General Hospital, McGill University Health Center, Montreal, Quebec, Canada Cerebral malaria (CM) is an acute, generally lethal condition characterized by high fever, seizures and coma. The genetic component to CM can be investigated in mouse models that vary in degree of susceptibility to infection with Plasmodium berghei ANKA. Using survival time to measure susceptibility in an informative F2 cross (n ¼ 257), we identified linkage to chromosome 19 (Berr5 (Berghei resistance locus 5), LOD ¼ 4.69) controlling, in part, the differential response between resistant BALB/c and susceptible C57BL/6 progenitors. BALB/c alleles convey increased survival through the cerebral phase of infection but have no quantitative effect on parasitemia during the later, anemic phase. The Berr5 locus colocalizes with three other immune loci, including Trl-4 (tuberculosis resistance), Tsiq2 (T-cell secretion of IL-4) and Eae19 (experimental allergic encephalitis 19), suggesting the possibility of a common genetic effect underlying these phenotypes. Potential positional candidates include the family of Ifit1–3 (interferon-inducible protein with tetratricopeptide repeats 1–3) and Fas. Genes and Immunity (2010) 11, 310–318; doi:10.1038/gene.2009.79; published online 29 October 2009 Keywords: genetics; malaria; QTL; inflammation; Plasmodium berghei; Ifit Introduction strains such as C57BL/6 (B6) or CBA developing an acute cerebral syndrome within 6–7 days characterized Malaria is a major global health problem with approxi- by ataxia, paraplegia, seizures and coma, leading to mately 40% of the world’s population living in at risk uniform lethality by day 8–10 after infection. On the areas. In Africa, malaria is responsible for 20% of other hand, resistant mice including the BALB/c and childhood deaths with a cumulative mortality of 1–3 WLA strains do not develop neurological symptoms but million people each year.1 The most severe complication die 2–4 weeks after infection due to severe anemia of Plasmodium falciparum infection is cerebral malaria caused by high levels of blood parasitemia. Current (CM). CM is caused by infected erythrocytes becoming evidence suggests that CM in mice infected with PbA is trapped in brain microvasculature, leading to an caused in part by a robust but detrimental localized excessive, localized host-driven inflammatory response inflammation in response to parasitized erythrocytes and resulting in hypoxia, loss of blood–brain barrier trapped in the brain microvasculature.2 Transcript (BBB) integrity and cell death.2–4 Patients develop profiling experiments have documented a substantial seizures, paralysis, ataxia and unrousable coma that pro-inflammatory response that is more pronounced in often ends in death. The cellular mechanisms of the susceptible mice.7–9 In addition, loss-of-function muta- pathological response are poorly understood and must tions in genes coding for pro-inflammatory molecules be better characterized to develop strategies for preven- such as interferon-g (Ifng), lymphotoxin (Lta), hemolytic tion and treatment of this condition. complement (C5a) and interferon regulatory factor 1 The host genetic background has a major effect (Irf1) have been shown to protect against CM.10–13 These on response to malaria in humans (reviewed by observations support the hypothesis of for a central Kwiatkowski5 and Mazier et al.6). The genetic component function for immune-mediated pathology in CM, with T of susceptibility to CM is multigenic and further cells, platelets and resident macrophages implicated in modified by parasite virulence and other environmental inflammatory response and tissue injury.14–16 determinants. Inbred strains of mice exhibit different Quantitative trait locus (QTL) mapping studies by responses to P. berghei ANKA (PbA), with susceptible whole-genome scanning in mice have detected five chromosomal loci affecting the host response to PbA infection. Using survival as a phenotypic trait—an Correspondence: Dr P Gros, Department of Biochemistry and unnamed locus on chromosome 18 in F2 mice derived Complex Traits Program, McGill University, Bellini Life Sciences from susceptible B6 and resistant DBA/2 was first Building, 3649 Promenade Sir William Osler, Room 366, Montreal, identified.17 Subsequently, QTLs designated Berr1 (chro- Quebec, Canada H3G-0B1. mosome 1), Berr2 (chromosome 11), Berr3 (chromosome 9) E-mail: [email protected] Received 26 May 2009; revised 17 September 2009; accepted 22 and Berr4 (suggestive linkage to chromosome 4) affecting September 2009; published online 29 October 2009 the presence and severity of neurological symptoms Berr5 in cerebral malaria resistance J Berghout et al 311 following infection were mapped in F2 mice derived from BALB/c brains showed very little uptake of the blue dye, C57BL/6 (susceptible) and the resistant wild-derived with levels similar to those seen in uninfected controls WLA strain.18,19 Finally, the Cmsc locus mapping to the H- (data not shown). These results confirm increased PbA- 2 region of chromosome 17 was found to affect suscept- induced BBB permeability as a distinguishing feature ibility in progeny of CBA mice crossed with the resistant between B6 and BALB/c animals. DBA/2 strain.20 In all cases, the detected QTLs span large To study the genetic complexity of the B6 vs BALB/c chromosomal segments, and the causative genes under- differential susceptibility to CM, we infected 257 lying their effect on survival time in P. berghei-infected (C57BL/6 Â BALB/c)F2 mice (CB6F2) with PbA. CB6F2 animals have not been identified. mice segregated into two groups, with a majority of In this study, we have investigated the genetic basis of animals (B75%) dying in the early cerebral phase (before differential susceptibility of the commonly compared B6 day 13), and the remainder surviving past day 17. The strain and the BALB/c strain to infection with P. berghei experiment was terminated at day 22 (Figures 2a and b). ANKA. All animals dying from early CM (susceptible, CM-S) had low parasitemia (o20%), whereas those dying late (resistant, CM-R; including those killed at day 22) developed high parasitemia (40–90%). In addition, the Results predictive value of blood parasitemia at day 6 (Figure 2c) We compared the response of the inbred C57BL/6 and and serum IFNg levels at day 5 (Figure 2d) on overall BALB/c strains of mice to infection with PbA. B6 were susceptibility was determined. We observed that CM found to be susceptible with the majority of animals susceptibility as measured by survival was significantly dying during the early cerebral phase of infection correlated (P ¼ 0.0001) with both higher early parasite- (Figures 1a and b). Neurological symptoms and death mia values and higher IFNg levels. in these mice occurred while levels of blood A genome-wide scan was carried out on all 257 CB6F2 parasitemia were o20% (data not shown). Conversely, mice, using 252 informative markers with survival as a BALB/c mice were resistant to PbA-induced CM, with marker of CM susceptibility. Both a binary (CM-S vs 475–80% of the animals surviving the cerebral phase. CM-R) and a parametric model of survival time (two- Surviving mice did not develop any subsequent cerebral part model) were used in this analysis that yielded symptoms. Instead, they developed high levels of blood similar results. The two-part model takes into account parasitemia (460%; data not shown) and died of severe both the binary trait (probability of survival to the malarial anemia. experimental end point) and the approximately normally CM in susceptible PbA-infected mice has been distributed survival within the cerebral phase (survival previously associated with disruption of the integrity of time contingent on not reaching the end point).22 Both the BBB,21 which we monitored using a dye extravasa- analyses identified a single genetic effect on chromosome tion assay (Figure 1c). Examination of brains from Evan’s 19, with maximum linkage to the CEL-19_32349880 Blue-injected mice revealed that in B6 mice the entire marker at chromosomal position 32.3 Mb (Figures 3a tissue was dyed blue with several darker foci whereas and c). Using the two-part model, we found that the CM-S 100 100 CM-R 80 80 60 60 40 40 Proportion (%) Percent survival 20 BALB/c 20 C57BL/6 0 0 0 5 10 15 20 C57BL/6 BALB/c Time (days) n=22 n=25 C57BL/6 BALB/c Figure 1 Differential susceptibility of inbred mouse strains to Plasmodium berghei-induced cerebral malaria. (a) Mice were infected by the intraperitoneal route with 5 Â 104 P. berghei ANKA parasitized erythrocytes and survival was monitored. Neurological symptoms began to appear at day 6 in susceptible (CM-S) C57BL/6 mice. The majority of BALB/c mice were categorized as resistant (CM-R), surviving well past day 12. (b) Proportion of CM-S (filled bars; dead before day 10) and CM-R (empty bars; survive past day 12) mice detected in each strain. (c) Integrity of the blood–brain barrier in PbA-infected B6 and BALB/c mice was evaluated 8 days after infection using an Evan’s Blue dye permeability assay in vivo (see Materials and methods section). Genes and Immunity Berr5 in cerebral malaria resistance J Berghout et al 312 100 100 20 7000 *p=0.0001 6000 *p=0.0001 80 15 75 5000 4000 60 10 (pg/ml) 50 γ 3000 IFN 2000 40 (%) Parasitemia 5 Proportion (%) 25 1000 Peak Parasitemia (%) Parasitemia Peak 20 0 0 0 /c B CB6F2 L BL/6 0 BALB/c BA CM-S C57BL/6 C57 2 : CM-R 0 3 6 9 12 15 18 21 6F2: CM-S CM-R CB CB6F2: CM-R Survival day CB6F2 CB6F: CM-S Figure 2 Segregation analysis of susceptibility to Plasmodium berghei-induced cerebral malaria in (C57BL/6 Â BALB/c)F2 mice.