A Jak3 Mutation Protects against Cerebral Malaria Figure 2. Resistance to cerebral malaria in pedigree 48 maps to the central portion of chromosome 8. Genome-wide linkage analysis of the CM-resistance trait (survival) was conducted in 44 G3 (15 resistant, 29 susceptible) mice from pedigree 48, using polymorphic markers informative for the B6 and B10 progenitors. (A) LOD score traces identifying significant linkage to chromosome 8 (p = 0.05, genome-wide significance shown as dotted line); the position of informative markers is shown, including rs33080067 and rs32729089 (LOD score ,5.8). (B) Haplotype analysis of the central portion of chromosome 8 in CM-resistant and CM-susceptible G3 animals from pedigree 48 (A, B6 homozygotes; H, B6/B10 heterozygotes; B, B10 homozygotes) showing exclusion of homozygote B6 haplotypes from the CM-susceptible group. The positions of the markers (in Mb) from the centromere (shown as a grey dot) are shown. (C) The G1 male from pedigree 48 was out-crossed to 129S1/SvlmJ to generate an F2 population (n = 211) that was phenotyped for response to P. berghei ANKA infection. Survival of F2 mice as well as parental 129S1/SvlmJ and C57BL/10J controls is shown. doi:10.1371/journal.pone.0031012.g002 have atrophied thymuses, as well as low numbers of splenic CD8+ T cells, NK cells and a defect in B cell maturation [28]. Compound (Jak3W81R/2) heterozygotes were generated by cross- ing Jak3W81R homozygotes to Jak32/2 mice, and these together with Jak3W81R/+ and Jak3+/2 heterozygotes were phenotyped for susceptibility to P. berghei-induced CM. Compound Jak3W81R/2 heterozygotes were found to be as CM-resistant as Jak3W81R homozygotes and as Jak32/2 animals, confirming that the Jak3W81R mutation is indeed responsible for protection against CM in P48. On the other hand, Jak3+/2 heterozygotes were as susceptible to P. berghei induced CM as the B10 WT controls. Finally, we noted that 50% of Jak3W81R/+ heterozygotes were resistant to CM, in agreement with haplotype analyses of the original G3 animals. This observation together with the uniform susceptibility of Jak3+/2 heterozygotes, confirmed the co-domi- nant mode of inheritance of the Jak3W81R mutation, and suggested that it may have a dominant negative effect (Figure 5). Splenocytes from infected B10 WT mice restore CM susceptibility to Jak3W81R homozygous mutant mice Sequestration of parasitized red blood cells at the brain microvasculature together with local inflammatory response in situ, have been shown to be necessary for development and progression of P. berghei-induced CM [15,29]. We aimed to establish in cell transfer experiments, which of the immune cell population(s) missing from the Jak3W81R mutant mice may be involved in CM pathogenesis, and which absence results in a CM- protective effect. First, total splenocytes from normal B10, from Jak3W81R/+ heterozygote or from Jak3W81R homozygote mutants, harvested 5 days following infection with P. berghei ANKA, were transferred into Jak3W81R homozygotes. Two hours later, recipient and control un-treated mice were infected with P. berghei and the effect of cell transfer on appearance of CM-associated phenotypes and lethality were monitored. The transfer of total spleen cells from wild type B10 controls into Jak3W81R mutants eliminated CM-resistance in these animals and they succumbed quickly in the cerebral phase of the disease, just like un-treated wild type controls (Figure 6A and 6B). On the other hand, the transfer of total spleen cells from Jak3W81R homozygote and, interestingly, from Jak3W81R/+ heterozygotes into Jak3W81R homozygote mutants had no consequence on the CM-resistance trait of these mice (Figure 6A and 6B). To determine which of the absent spleen cell populations is associated with CM-resistance in Jak3W81R mutants, we carried out further cell fractionation and transfer experiments (Figure 6C). While the transfer of NK cells from infected B10 mice into Jak3W81R mutants had no effect on CM-resistance, the transfer of either total T cells or of CD8+ T cells caused a similar and significant decrease in survival time following P. berghei infection of the transferred animals, with 50% of the transferred mice succumbing by day 9. Together, these results indicate that CD8+ T cells are critically important for the pathogenesis of P. berghei-induced CM, and that their absence in the Jak3W81R homozygous mutants is in part responsible for the CM-resistance phenotype of these mice. Jak3W81R mutant mice are susceptible to infection with Mycobacterium and with Citrobacter Pro-inflammatory Th1 cytokines (IFN-c, TNF-a and LT-a) play an important role in CM pathogenesis, and inactivating mutations in these molecules have a protective effect against P. berghei-induced CM. Jak3W81R mutant mice lack CD8+ splenic T cells, and total spleen cells from Jak3W81R mutants do not produce IFN-c in response to activation along the Th1 pathway (data not shown). On the other hand, CD4+ and CD8+ T cells, as well as Th1 cytokines produced by these and other cells are absolutely required for protection against intracellular pathogenic mycobac- teria [30]. Therefore, we evaluated the response of Jak3W81R mice to infection with mycobacteria (Figure 7). Mice were infected with low dose M. bovis BCG and bacterial replication was measured 6 weeks following infection (Figure 7A). Jak3W81R mutants showed splenomegaly and increased spleen bacterial counts (by a factor of 10 fold) compared to control B6 mice. Likewise, a large proportion (.75%) of Jak3W81R mutants succumbed within 45 days following aerosol infection with virulent M. tuberculosis H37Rv, while all of the control B6 mice survived over the same period (Figure 7B). Together, these results indicate that inactivation of Jak3 kinase causes susceptibility to mycobacterial infection. Independently, it is known that effective protection against enteropathogenic bacteria such as Citrobacter rodentium requires intact CD4+ T and B lymphocyte compartments [31], and is associated with a robust production of Th1 cytokines such as IFN-c and TNF-a [32,33]. Therefore, we assessed the response of Jak3W81R homozygote and Jak3W81R/+ heterozygote mutants to infection with C. rodentium (Figure 7C). Inactivation of Jak3 caused a dramatic increase in susceptibility to infection, leading to progressive mortality within 15 days of infection, compared to B6 and Jak3W81R/+ heterozy- gotes which developed transient disease symptoms but survived the infection. These results indicate that full activation of Jak3 is required for effective Th1-driven host response to infections with extracellular and intracellular pathogens. Discussion Cerebral malaria has a devastating impact on global health. The sudden appearance, rapid progression, and irreversible nature of the CM pathology together with the paucity of treatment options (limited to high dose parenteral administration of quinine and artemisinin derivatives [34]), underlie the high rate of mortality and morbidity associated with CM. Therefore, there is a desperate need for novel therapeutic interventions. CM has a complex pathology that involves multiple host tissues and physiological pathways, including the eyrthroid replicative niche of the parasite, the structure and secretion products of endothelial cells of the microvasculature, and different cell populations, cytokines and associated pathways of the innate and acquired immune system, the activity of which is triggered by variable parasite virulence determinants and further modulated by intrinsic genetic factors of the host [3,4,5]. A better characterization of the molecular pathways involved is required to identify novel targets PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e31012