H WT and LMP7-deficient 
mice at 5 days after infection with PyL. The degree of activation was lower in LMP7-deficient mice in terms of expression of DC activation markers (Fig. 3A). These results suggested that DCs were less activated in response to lower levels of parasites, because the level of parasitemia was significantly low at this time point in LMP7-deficient mice. Thus, activation of DCs could not explain the ��-Sitosterol ��-D-glucoside cost enhanced protection in LMP7-deficient mice. Therefore, we tried to examine more primitive host defense mechanism against malaria parasites, the phagocytosis of pRBCs by macrophages. Macrophages are thought to be crucial effectors for eliminating pRBCs or free merozoites, by phagocytosis followed by their digestion in phagosomes. Schizont-rich pRBCs purified from WT mice using Percoll gradient were labeled with CFSE, cultured with macrophages, and their phagocytic ability assessed by CFSE incorporation. Phagocytosis of pRBCs by LMPdeficient macrophages was comparable to WT macrophages (Fig. 3B). Macrophages phagocytosed low numbers of RBCs fromMalaria Resistance in LMP7-Deficient MiceFigure 2. Comparable adaptive immune responses to malaria parasites in LMP7-deficient mice. Spleen cells isolated from WT and LMP7deficient mice 5 days after infection were analyzed. (A) Splenocytes stained with fluorochrome-conjugated anti-CD3, anti-CD4, and anti-CD69 were analyzed for activation of T cells. Gated CD3+ cells were separated by CD4 and CD69 expression. The CD42 cell population contained mostly CD8+ cells. Numbers represent the percentage of all cells in each quadrant. (B) mRNA encoding IFN-c in total RNA extracted from splenocytes of the indicated mice was quantified by real-time PCR. Values represent the relative quantities of mRNA encoding genes of interest to that of b-actin and mean 6 SD of 3 mice. (C) Production of IFN-c in splenic T cells of the indicated mice was analyzed. Gated CD3+ cells were separated by CD4 and IFN-c expression. Numbers represent the percentage of all cells in each quadrant. (D) Pentagastrin Absolute numbers of IFN-c-producing cells were also calculated (bar graph). Values indicate mean 6 SD of 3 mice. Results are representative of at least two independent experiments. doi:10.1371/journal.pone.0059633.guninfected mice (Fig. 4A), suggesting that they specifically recognize some alterations in RBCs associated with infection by malaria parasites.RBCs of LMP7-deficient Mice are More Susceptible to Phagocytosis by MacrophagesSurprisingly, phagocytosis was remarkably enhanced when pRBCs from LMP7-deficient mice were used compared with those from WT mice (Fig. 4A), indicating that LMP7-deficient RBCs could be phagocytosed 18204824 more easily by macrophages. This enhancement of phagocytosis was observed only when mice wereinfected with malaria parasites, as RBCs from uninfected LMP7deficient mice were phagocytosed comparably to that from uninfected WT mice (Fig. 4A). These results suggested a difference in structure between WT and LMP7-deficient RBCs after infection. To explore the reasons why LMP7-deficient pRBCs were more sensitive to phagocytosis, we evaluated the morphology of RBCs infected with PyL using SEM. Before infection, there was no visible difference in RBCs from WT or LMP7-deficient mice, which showed a typical discoid form (Fig. 4B). Percoll gradient purified schizont-rich pRBCs composed of late trophozoites and schizonts, contained many spherical RBCs, indicating that in-Malaria Resistance in LMP7-Deficient MiceFig.H WT and LMP7-deficient mice at 5 days after infection with PyL. The degree of activation was lower in LMP7-deficient mice in terms of expression of DC activation markers (Fig. 3A). These results suggested that DCs were less activated in response to lower levels of parasites, because the level of parasitemia was significantly low at this time point in LMP7-deficient mice. Thus, activation of DCs could not explain the enhanced protection in 
LMP7-deficient mice. Therefore, we tried to examine more primitive host defense mechanism against malaria parasites, the phagocytosis of pRBCs by macrophages. Macrophages are thought to be crucial effectors for eliminating pRBCs or free merozoites, by phagocytosis followed by their digestion in phagosomes. Schizont-rich pRBCs purified from WT mice using Percoll gradient were labeled with CFSE, cultured with macrophages, and their phagocytic ability assessed by CFSE incorporation. Phagocytosis of pRBCs by LMPdeficient macrophages was comparable to WT macrophages (Fig. 3B). Macrophages phagocytosed low numbers of RBCs fromMalaria Resistance in LMP7-Deficient MiceFigure 2. Comparable adaptive immune responses to malaria parasites in LMP7-deficient mice. Spleen cells isolated from WT and LMP7deficient mice 5 days after infection were analyzed. (A) Splenocytes stained with fluorochrome-conjugated anti-CD3, anti-CD4, and anti-CD69 were analyzed for activation of T cells. Gated CD3+ cells were separated by CD4 and CD69 expression. The CD42 cell population contained mostly CD8+ cells. Numbers represent the percentage of all cells in each quadrant. (B) mRNA encoding IFN-c in total RNA extracted from splenocytes of the indicated mice was quantified by real-time PCR. Values represent the relative quantities of mRNA encoding genes of interest to that of b-actin and mean 6 SD of 3 mice. (C) Production of IFN-c in splenic T cells of the indicated mice was analyzed. Gated CD3+ cells were separated by CD4 and IFN-c expression. Numbers represent the percentage of all cells in each quadrant. (D) Absolute numbers of IFN-c-producing cells were also calculated (bar graph). Values indicate mean 6 SD of 3 mice. Results are representative of at least two independent experiments. doi:10.1371/journal.pone.0059633.guninfected mice (Fig. 4A), suggesting that they specifically recognize some alterations in RBCs associated with infection by malaria parasites.RBCs of LMP7-deficient Mice are More Susceptible to Phagocytosis by MacrophagesSurprisingly, phagocytosis was remarkably enhanced when pRBCs from LMP7-deficient mice were used compared with those from WT mice (Fig. 4A), indicating that LMP7-deficient RBCs could be phagocytosed 18204824 more easily by macrophages. This enhancement of phagocytosis was observed only when mice wereinfected with malaria parasites, as RBCs from uninfected LMP7deficient mice were phagocytosed comparably to that from uninfected WT mice (Fig. 4A). These results suggested a difference in structure between WT and LMP7-deficient RBCs after infection. To explore the reasons why LMP7-deficient pRBCs were more sensitive to phagocytosis, we evaluated the morphology of RBCs infected with PyL using SEM. Before infection, there was no visible difference in RBCs from WT or LMP7-deficient mice, which showed a typical discoid form (Fig. 4B). Percoll gradient purified schizont-rich pRBCs composed of late trophozoites and schizonts, contained many spherical RBCs, indicating that in-Malaria Resistance in LMP7-Deficient MiceFig.
