Parasitol Res (2014) 113:1019–1027 DOI 10.1007/s00436-013-3736-1
ORIGINAL PAPER
Alpha-tocopherol transfer protein gene inhibition enhances the acquired immune response during malaria infection in mice Maria Shirley Herbas & Magloire Hamtandi Natama & Hiroshi Suzuki
Received: 20 November 2013 / Accepted: 4 December 2013 / Published online: 21 December 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Immune response to malaria infection is complex and seems to be regulated by innate and adaptive immune response as well as environmental factors such as host genetics and nutritional status. Previously, we have reported that αtocopherol transfer protein knockout (α-ttpΔ) mice, showing low concentrations of α-tocopherol in circulation, infected with Plasmodium berghei NK65 survived significantly longer as compared with the wild-type mice. In addition, Plasmodium yoelii XL-17, a lethal strain, showed non-lethal virulence in α-ttpΔ mice. Thus, we hypothesized that the ability of the α-ttpΔ mice to control P. yoelli XL-17 proliferation may allow them to build an efficient immune response against murine malaria infection. On 15 days after infection with P. yoelli XL-17, α-ttpΔ mice were challenged to infection with P. berghei NK65. Results indicated that α-ttpΔ mice infected with P. yoelli XL-17 built a protective immunity against P. berghei NK65 associated to extremely low levels of parasitemia, a controlled inflammatory response, and a robust antibody response. Moreover, the importance of α−tocopherol for parasite proliferation was remarkable. The results suggest that inhibition of α-tocopherol transfer protein activity is effective for the enhancement of acquired immunity in murine malaria infection. M. S. Herbas : M. H. Natama : H. Suzuki (*) Research Unit for Functional Genomics, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13, Inada, Obihiro 080-8555, Japan e-mail: [email protected] M. H. Natama Unité de Recherche Clinique de Nanoro, Centre National de la Recherche Scientifique et Technologique, Institut de Recherche en Sciences de la Santé, 11 BP 218, Ouagadougou CMS 11, Burkina Faso
Introduction Immune response against malaria is complex and seems to be regulated by innate and adaptive responses (Beeson et al. 2008). Evidently, several factors such as host genetic diversity (Mendonca et al. 2012) and the nutritional status play important roles (Shankar 2000). At present, there is a great interest to assess the influence that vitamins and minerals has on the outcome of malaria (Shankar 2000). It is widely accepted that vitamin E (α-tocopherol) deficiency is thought to protect against malaria infection because the absence of this antioxidant lead to an increase of oxygen radicals and makes Plasmodium parasites more susceptible to oxidative stress resulting from the immune response to malaria infection (Levander et al. 1995; Shankar 2000; Nussemblatt and Semba 2002; Friedman et al. 2005). Vitamin E is the generic term for a group of molecules that include four tocopherols (α, β, γ, and δ) and four tocotrienols (α, β, γ, and δ) (Herrera and Barbas 2001). However, the human body has a specific preference for α-tocopherol. Alpha-tocopherol plays important roles as a free radical scavenger within cell membranes (Niki and Traber 2012), gene expression, and so on (Zingg and Azzi 2004). Alpha-tocopherol is specifically selected among all the tocopherols through the α-tocopherol transfer protein (α-ttp) in liver. This protein facilitates α-tocopherol secretion from the hepatocytes, thus regulating its concentration in circulation (Manor and Morley 2007; Brigelious-flohe 2009). It is well recognized that concomitant species of different species of parasites are common in the field (Chen et al. 2013). However, the real influences that a primary infection has on a secondary infection in the host remain unclear. Murine malaria models have been extensively used to understand the nature of immune responses and their role in development of protective immunity to malaria. It has been reported
1020
that non-lethal Plasmodium strains or genetically modified parasites are capable to prevent lethal infections in mice (Mueller et al. 2005; Voza et al. 2005; Niikura et al. 2008; Ting et al. 2008). Also, recent studies had demonstrated that mixed infections of P. yoelli with Listeria monocytogenes enhanced the immunity against malaria (Qi et al. 2013). In previous studies, we have demonstrated that α-ttp gene disruption confers protection against murine malaria (Herbas et al. 2010a; Herbas et al. 2010b). In these studies, we have used different strains of murine Plasmodium, namely Plasmodium berghei NK65 and P. yoelli XL-17 (Herbas et al. 2010a) and P. berghei ANKA (Herbas et al. 2010b). These parasites are lethal in C57BL/6 J mice, although they show different outcomes such as malarial anemia and cerebral malaria. In contrast, P .berghei NK65 and P. berghei ANKA showed different parasitemia kinetics when they infected to αtocopherol transfer protein knockout mice (α-ttpΔ). Survival rates in the α-ttpΔ mice were significantly prolonged as compared with C57BL/6 J mice; nonetheless, both strains remained lethal for the knockout mice (Herbas et al. 2010a; Herbas et al. 2010b). Interestingly, P. yoelli XL-17 was nonlethal, and parasitemia was almost undetectable in α-ttpΔ mice (Herbas et al. 2010a). These results clearly showed that α-ttpΔ mice have the ability to control P. yoelli XL-17 infection better than P. berghei NK65 or P. berghei ANKA infection. Moreover, we have demonstrated that oxidative stress is clearly involved in this protective effect (Herbas et al. 2010a). Due to the ability of α-ttpΔ mice to control P. yoelli XL-17 infection, we hypothesized that the slower proliferation of the parasite may allow them to mount an efficient immune response in mice.
Parasitol Res (2014) 113:1019–1027
Parasites and experimental infections Stocks of Plasmodium berghei NK65 and P. yoelii XL-17 were kept at −80 °C in 50 % of glycerol. For the infectious experiments, infected red blood cells (iRBCs) from P. berghei NK65 or P. yoelli XL-17 were recovered twice in B6 mice, and then 0.2 ml of 1×105 iRBCs/ml was intraperitoneally inoculated to mice in each experimental group. In a primary infection, α-ttpΔ mice (n =8) were inoculated with P. yoelli XL-17, and then were challenged with 0.2 ml of 1× 105 iRBCs/ml of P. berghei NK65 on 15 days after infection considered as the secondary infection. As controls, B6 (n =6) and α-ttpΔ (n =8) mice were single-infected with P. yoelli XL-17 or P. berghei NK65 at primary infection or secondary infection time point. Parasitemia was determined by microscopic examination of Giemsa-stained blood smears collected from the mice tail vein every 2 days. Survival rates were monitored daily. The experimental design is described in Fig. 1. Additionally, to assess potential for parasite clearance in the α-ttpΔ mice immunized with P. yoelli XL17 and then re-infected with P. berghei NK65, SCID mice (n =5) were inoculated with total blood recovered from those double-infected α-ttpΔ mice at 90 days postsecondary infection. Briefly, total blood was recovered from double-infected α-ttpΔ mice showing 0.2 % of parasitemia and from wild-type mice infected only with P. berghei NK65 showing 2 % of parasitemia. Then each sample was diluted with phosphate-buffered saline (PBS) until each preparation had a concentration of 30 % of hematocrit. Thereafter, 0.2 ml containing 0.2×102 or 2×102 iRBCs were intraperitoneally inoculated into SCID mice. Survival rate was monitored daily and parasitemia every 2 days.
Materials and methods Mice Alpha-ttp knockout mice with a C57BL/6 J genetic background (α-ttpΔ) (Jishage et al. 2001), C57BL/6 J (B6) wildtype mice, and severe combined immunodeficiency (SCID) mice were bred in the specific pathogen-free facility of the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan. The animal room was light-controlled (lights on from 07:00 to 19:00) and air-conditioned. The temperature was set at 24±1 °C, and humidity was set at 50 ±10 %. Mice had free access to a standard laboratory nonpurified diet (CE-2; CLEA Japan, Tokyo, Japan) and tap water. All mice were used at 8 to 10 weeks of age in this study. All the experimental infections in this study were performed in accordance with the Guidance and Principles for the Care and Use of Research Animals of Obihiro University of Agriculture and Veterinary Medicine, Japan.
P. yoelii XL17 Primary infection
P. berghei NK65 Secondary infection
Fig. 1 Experimental design for the infectious experiments. In the first experiment, α-ttpΔ and B6 mice were infected with P. yoelli XL-17 (0.2 ml of 1×105 IRBCs/ml). Fifteen days after primary infection, these mice were challenged with P. berghei NK65 (0.2 ml of 1×105 IRBCs/ ml). Control groups of α-ttpΔ and C57BL/J mice were single infected with either P. yoelli XL-17 or P. berghei NK65. The experimental groups are designated below: Group I: α-ttpΔ mice infected with P. yoelli XL-17 and then challenged with P. berghei NK65. Group II: α-ttpΔ mice singleinfected with P. yoelli X-L17. Group III: C57BL/6 J mice single-infected with P. yoelli XL-17. Group IV: α-ttpΔ mice single-infected with P. berghei NK65. Group V: C57BL/6 J mice single-infected with P. berghei NK65. Liver and spleen were collected on days 7, 15, 22, and 30 after infection for further analysis
Parasitol Res (2014) 113:1019–1027
1021
Blood and organs collection
Table 1 Primers and probes used for real-time quantitative PCR
Blood, liver, and spleen were collected from α-ttpΔ mice (n =3) infected with P. yoelli XL-17 and then re-infected with P. berghei NK65 on days 7, 15, 22, and 30 post-infection. In addition, samples were also collected from α-ttpΔ (n =3) and B6 mice (n =3) uninfected or single-infected with P. yoelli XL-17 or P. berghei NK65. Blood and organs were aseptically removed and immediately placed in liquid nitrogen and then kept at −80 °C. Collected blood was centrifuged at 5,000 rpm for 5 min, and the plasma was stored at −80 °C for antibody detection.
Gene
Primer/probe
IFN-α TNF-α IL-10 β-Actin
Mm00801778_m1 (Applied Biosystems Inc, USA) Mm00443258_m1 (Applied Biosystems Inc, USA) Mm00439616_m1 (Applied Biosystems Inc, USA) 5′-GCTCTGGCTCCTAGCACCAT-3′F 5′-GCCACCGATCCACACAGAGT-3′R 5′-FAM-ATCAAGATCATTGCTCCTC-MGB-3′ 5′-CGGCTACCACATCCAAGGAA-3′F 5′-GCTGGAATTACCGCGGCT-3′R 5′-FAM-TGCTGGCACCAGACTTGCCCTC-MGB-3′ 5′-CGTCGCGAAGGATACTCT-3′F 5′-GGCAGCAGATTTCACTGTGAAG-3′R 5′-FAM-TCGTCAACGGCCACCG-MGB-3′
Gene expression analysis by real-time quantitative polymerase chain reaction (RT-PCR) Total RNA from liver and spleen was extracted by using Tri reagent (Sigma, St Louis, MO). RNA concentration was obtained by using a spectrophotometer (Ultrospec 2100 pro), and the RNA quality was assessed by using Experion automatized chromatography (Experion TM RNA StdSens Analysis Kit; Bio-Rad, Hercules, CA). The mRNA expression of interleukin (IL)-10, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α was monitored. The RT-PCR was performed with specific double-labeled probes in an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA). Briefly, the reaction was carried out in a final volume of 20 μL containing 10 μL of 2× master mix without UNG (uracil-N -glycosylase), 0.5 μL of 40× multiscribe and RNase inhibitor mix, 1 μL of TaqMan gene expression assay probe, 4 μL of 50 ng/μL RNA template, and 1.1 μL of RNase-free doubled-distilled water. The reaction condition was as follows: 48 °C for 30 min, 95 °C for 10 min, 45 cycles with 95 °C for 15 s, and 60 °C for 1 min. The data were analyzed by using SDS 2.1 software (Applied Biosystems). Mouse β-actin, GAPDH, and 18SrRNA were used as internal control genes. The most stable i n t e r n a l c on t r o l g e ne w a s d et e r m i n e d b y u s i n g Genorm_win_3.5 software (Vandesompele et al. 2002). Primers and probes used for target genes and for internal control genes are described in Table 1. Crude parasite antigen preparation and detection of total IgG, and IgG2c B6 mice infected with P. yoelli XL-17 or P. berghei showing 20 to 25 % of parasitemia were killed (n =3). Then blood was obtained by cardiac puncture and treated with Histopaque 1119 and 1077 (Sigma, St Louis, USA) following the manufacturer’s instructions in order to isolate the mononuclear and polymorphonuclear cells. Packed red blood cells (RBCs) were washed with cold PBS twice. Packed RBCs were lysed with diethyl pyrocarbonate-treated water (Ambion, TX, USA) and
18S rRNA
GAPDH
then washed with PBS twice. The pellet was sonicated three times for 30 s each time. Crude antigen concentration was adjusted to the same value for all the samples using the BCATM Protein assay kit (Thermo scientific, Rockford, USA). The resulting crude antigen was used in an enzymelinked immunosorbent assay. Briefly, 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated overnight at 4 °C with 50 μl of P. berghei NK65 or P. yoelli XL17 crude antigen at a concentration of 20 μg/ml in a coating buffer (50 μM carbonate-bicarbonate buffer, pH 9.6). The plates were washed with 0.05 % Tween 20-PBS (PBST) and then incubated with 100 μl of a blocking solution (3 % skim milk in PBS) for 1 h at 37 °C. Subsequently, the plates were washed with PBST and then incubated with 50 μl of mouse sera diluted in 1:100 with the blocking solution for 1 h at 37 °C. The plates were washed six times with PBST and then were incubated with 50 μl of the horseradish peroxidaseconjugated goat anti-mouse immune-globulins IgG and IgG2c (Bethyl Laboratories, TX, USA) diluted to 1:4,000 with the blocking solution for 1 h at 37 °C as a secondary antibody. The plates were washed for six times with PBST. Then 100 μl of the substrate solution [0.1 M citric acid, 0.2 M sodium phosphate, 0.003 % H202, and 0.3 mg/ml 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid); Sigma, St Louis, USA] per well was added. After incubation for 1 h at room temperature, absorbance was measured using an MTP microplate reader (Corona Electric, Tokyo, Japan) at 415 nm. Preparation of dead P. yoelli XL-17 for mice immunization Fixed parasite was prepared as described previously (Li et al. 2012). Briefly, P. yoelli XL17 infected blood having 60 % of parasitemia was collected from heart using sodium citrate (WAKO, Tokyo, Japan) as anticoagulant, and then infected and un-infected RBCs were isolated using
1022 Table 2 Composition of standard diet 100g (CLEA, Tokyo, Japan)
Parasitol Res (2014) 113:1019–1027
Nutrients and calories
Minerals
Vitamins
Moisture (%) Crude protein (%) Crude fat (%) Crude fiber (%) Crude ash (%)
8.9 24.9 4.6 4.1 6.6
Ca (g) P (g) Mg (g) K (g) Mn (mg)
1.06 0.99 0.34 1.02 9.96
Retinol (mg) Vitamin B1 (mg) Vitamin B2 (mg) Vitamin B6 (mg) Vitamin B12 (μg)
0.6 2.0 1.3 1.4 5.6
NFE (%) Energy (kcal) Hardness (kg/cm2)
51.0 344.9 25.8
Fe (mg) Cu (mg) Zn (mg) Na (g)
30.64 0.71 5.45 0.31
Vitamin C (mg) Vitamin E (mg) Pantothenic acid (mg) Niacin (mg)
25.0 8.5 3.0 18.9
α-Tocopherol supplementation, 600 mg/kg
Histopaque 1119 and 1077 (Sigma, St Louis, USA) following the manufacturer’s instructions. Packed RBCs including infected and un-infected RBCs were washed with cold PBS for three times, and then parasites were fixed with 0.25 % of glutaraldehyde for 15 min at room temperature; thereafter, they were washed with cold PBS for three times; 0.2 ml of this preparation at a concentration of 1×105 IRBCs/ml was inoculated into B6 (n =6) and αttpΔ mice (n =6). Also, un-infected blood was used as a control. At 15 days after fixed parasite inoculation, mice were challenged with 0.2 ml of 1 × 10 5 IRBCs/ml of P. berghei NK65. Then survival rate was monitored daily and parasitemia every 2 days.
2 weeks and then was challenged with P. berghei NK65. Survival rate was monitored daily and parasitemia every 2 days. Diet composition is described in Table 2. Statistical analysis Statistical analysis was performed using Kruskal-Wallis all pairwise comparisons–Conover-Inman tests (Stats-Direct, version 2.7.5, software for Windows). For survival rate analysis, Kaplan–Meier method was performed. A p value less than 0.05 was considered as statistically significant.
Results Effect of α-tocopherol supplementation on Plasmodium proliferation To examine the effect of α-tocopherol supplementation on the parasite proliferation, α-ttpΔ mice were infected with P. yoelli XL-17 and fed with standard diet containing 85 mg/kg of αtocopherol. Two weeks after the primary infection, the diet was shifted to a supplemented diet of α-tocopherol (600 mg/kg) for
Fig. 2 Alpha-tocopherol transfer protein knockout mice infected with P. yoelii XL-17 develop protective immunity against P. berghei NK65 infection. a : Parasitemia kinetics indicates that the double-infected knockout mice control parasite proliferation efficiently. Moreover, a lethal
A protective immunity against P. berghei NK65 infection was developed in the α-ttpΔ mice pre-infected with P. yoelli XL-17 Parasitemia in B6 mice infected with P. yoelli XL17 increased dramatically from day 5 post-infection, and 100 % of death was observed on day 15 post-infection. In contrast,
strain such as P. berghei NK65 behave as no lethal in this mouse model. b: Mortality in the double infected knockout mice was 0 %. *p
ORIGINAL PAPER
Alpha-tocopherol transfer protein gene inhibition enhances the acquired immune response during malaria infection in mice Maria Shirley Herbas & Magloire Hamtandi Natama & Hiroshi Suzuki
Received: 20 November 2013 / Accepted: 4 December 2013 / Published online: 21 December 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Immune response to malaria infection is complex and seems to be regulated by innate and adaptive immune response as well as environmental factors such as host genetics and nutritional status. Previously, we have reported that αtocopherol transfer protein knockout (α-ttpΔ) mice, showing low concentrations of α-tocopherol in circulation, infected with Plasmodium berghei NK65 survived significantly longer as compared with the wild-type mice. In addition, Plasmodium yoelii XL-17, a lethal strain, showed non-lethal virulence in α-ttpΔ mice. Thus, we hypothesized that the ability of the α-ttpΔ mice to control P. yoelli XL-17 proliferation may allow them to build an efficient immune response against murine malaria infection. On 15 days after infection with P. yoelli XL-17, α-ttpΔ mice were challenged to infection with P. berghei NK65. Results indicated that α-ttpΔ mice infected with P. yoelli XL-17 built a protective immunity against P. berghei NK65 associated to extremely low levels of parasitemia, a controlled inflammatory response, and a robust antibody response. Moreover, the importance of α−tocopherol for parasite proliferation was remarkable. The results suggest that inhibition of α-tocopherol transfer protein activity is effective for the enhancement of acquired immunity in murine malaria infection. M. S. Herbas : M. H. Natama : H. Suzuki (*) Research Unit for Functional Genomics, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13, Inada, Obihiro 080-8555, Japan e-mail: [email protected] M. H. Natama Unité de Recherche Clinique de Nanoro, Centre National de la Recherche Scientifique et Technologique, Institut de Recherche en Sciences de la Santé, 11 BP 218, Ouagadougou CMS 11, Burkina Faso
Introduction Immune response against malaria is complex and seems to be regulated by innate and adaptive responses (Beeson et al. 2008). Evidently, several factors such as host genetic diversity (Mendonca et al. 2012) and the nutritional status play important roles (Shankar 2000). At present, there is a great interest to assess the influence that vitamins and minerals has on the outcome of malaria (Shankar 2000). It is widely accepted that vitamin E (α-tocopherol) deficiency is thought to protect against malaria infection because the absence of this antioxidant lead to an increase of oxygen radicals and makes Plasmodium parasites more susceptible to oxidative stress resulting from the immune response to malaria infection (Levander et al. 1995; Shankar 2000; Nussemblatt and Semba 2002; Friedman et al. 2005). Vitamin E is the generic term for a group of molecules that include four tocopherols (α, β, γ, and δ) and four tocotrienols (α, β, γ, and δ) (Herrera and Barbas 2001). However, the human body has a specific preference for α-tocopherol. Alpha-tocopherol plays important roles as a free radical scavenger within cell membranes (Niki and Traber 2012), gene expression, and so on (Zingg and Azzi 2004). Alpha-tocopherol is specifically selected among all the tocopherols through the α-tocopherol transfer protein (α-ttp) in liver. This protein facilitates α-tocopherol secretion from the hepatocytes, thus regulating its concentration in circulation (Manor and Morley 2007; Brigelious-flohe 2009). It is well recognized that concomitant species of different species of parasites are common in the field (Chen et al. 2013). However, the real influences that a primary infection has on a secondary infection in the host remain unclear. Murine malaria models have been extensively used to understand the nature of immune responses and their role in development of protective immunity to malaria. It has been reported
1020
that non-lethal Plasmodium strains or genetically modified parasites are capable to prevent lethal infections in mice (Mueller et al. 2005; Voza et al. 2005; Niikura et al. 2008; Ting et al. 2008). Also, recent studies had demonstrated that mixed infections of P. yoelli with Listeria monocytogenes enhanced the immunity against malaria (Qi et al. 2013). In previous studies, we have demonstrated that α-ttp gene disruption confers protection against murine malaria (Herbas et al. 2010a; Herbas et al. 2010b). In these studies, we have used different strains of murine Plasmodium, namely Plasmodium berghei NK65 and P. yoelli XL-17 (Herbas et al. 2010a) and P. berghei ANKA (Herbas et al. 2010b). These parasites are lethal in C57BL/6 J mice, although they show different outcomes such as malarial anemia and cerebral malaria. In contrast, P .berghei NK65 and P. berghei ANKA showed different parasitemia kinetics when they infected to αtocopherol transfer protein knockout mice (α-ttpΔ). Survival rates in the α-ttpΔ mice were significantly prolonged as compared with C57BL/6 J mice; nonetheless, both strains remained lethal for the knockout mice (Herbas et al. 2010a; Herbas et al. 2010b). Interestingly, P. yoelli XL-17 was nonlethal, and parasitemia was almost undetectable in α-ttpΔ mice (Herbas et al. 2010a). These results clearly showed that α-ttpΔ mice have the ability to control P. yoelli XL-17 infection better than P. berghei NK65 or P. berghei ANKA infection. Moreover, we have demonstrated that oxidative stress is clearly involved in this protective effect (Herbas et al. 2010a). Due to the ability of α-ttpΔ mice to control P. yoelli XL-17 infection, we hypothesized that the slower proliferation of the parasite may allow them to mount an efficient immune response in mice.
Parasitol Res (2014) 113:1019–1027
Parasites and experimental infections Stocks of Plasmodium berghei NK65 and P. yoelii XL-17 were kept at −80 °C in 50 % of glycerol. For the infectious experiments, infected red blood cells (iRBCs) from P. berghei NK65 or P. yoelli XL-17 were recovered twice in B6 mice, and then 0.2 ml of 1×105 iRBCs/ml was intraperitoneally inoculated to mice in each experimental group. In a primary infection, α-ttpΔ mice (n =8) were inoculated with P. yoelli XL-17, and then were challenged with 0.2 ml of 1× 105 iRBCs/ml of P. berghei NK65 on 15 days after infection considered as the secondary infection. As controls, B6 (n =6) and α-ttpΔ (n =8) mice were single-infected with P. yoelli XL-17 or P. berghei NK65 at primary infection or secondary infection time point. Parasitemia was determined by microscopic examination of Giemsa-stained blood smears collected from the mice tail vein every 2 days. Survival rates were monitored daily. The experimental design is described in Fig. 1. Additionally, to assess potential for parasite clearance in the α-ttpΔ mice immunized with P. yoelli XL17 and then re-infected with P. berghei NK65, SCID mice (n =5) were inoculated with total blood recovered from those double-infected α-ttpΔ mice at 90 days postsecondary infection. Briefly, total blood was recovered from double-infected α-ttpΔ mice showing 0.2 % of parasitemia and from wild-type mice infected only with P. berghei NK65 showing 2 % of parasitemia. Then each sample was diluted with phosphate-buffered saline (PBS) until each preparation had a concentration of 30 % of hematocrit. Thereafter, 0.2 ml containing 0.2×102 or 2×102 iRBCs were intraperitoneally inoculated into SCID mice. Survival rate was monitored daily and parasitemia every 2 days.
Materials and methods Mice Alpha-ttp knockout mice with a C57BL/6 J genetic background (α-ttpΔ) (Jishage et al. 2001), C57BL/6 J (B6) wildtype mice, and severe combined immunodeficiency (SCID) mice were bred in the specific pathogen-free facility of the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan. The animal room was light-controlled (lights on from 07:00 to 19:00) and air-conditioned. The temperature was set at 24±1 °C, and humidity was set at 50 ±10 %. Mice had free access to a standard laboratory nonpurified diet (CE-2; CLEA Japan, Tokyo, Japan) and tap water. All mice were used at 8 to 10 weeks of age in this study. All the experimental infections in this study were performed in accordance with the Guidance and Principles for the Care and Use of Research Animals of Obihiro University of Agriculture and Veterinary Medicine, Japan.
P. yoelii XL17 Primary infection
P. berghei NK65 Secondary infection
Fig. 1 Experimental design for the infectious experiments. In the first experiment, α-ttpΔ and B6 mice were infected with P. yoelli XL-17 (0.2 ml of 1×105 IRBCs/ml). Fifteen days after primary infection, these mice were challenged with P. berghei NK65 (0.2 ml of 1×105 IRBCs/ ml). Control groups of α-ttpΔ and C57BL/J mice were single infected with either P. yoelli XL-17 or P. berghei NK65. The experimental groups are designated below: Group I: α-ttpΔ mice infected with P. yoelli XL-17 and then challenged with P. berghei NK65. Group II: α-ttpΔ mice singleinfected with P. yoelli X-L17. Group III: C57BL/6 J mice single-infected with P. yoelli XL-17. Group IV: α-ttpΔ mice single-infected with P. berghei NK65. Group V: C57BL/6 J mice single-infected with P. berghei NK65. Liver and spleen were collected on days 7, 15, 22, and 30 after infection for further analysis
Parasitol Res (2014) 113:1019–1027
1021
Blood and organs collection
Table 1 Primers and probes used for real-time quantitative PCR
Blood, liver, and spleen were collected from α-ttpΔ mice (n =3) infected with P. yoelli XL-17 and then re-infected with P. berghei NK65 on days 7, 15, 22, and 30 post-infection. In addition, samples were also collected from α-ttpΔ (n =3) and B6 mice (n =3) uninfected or single-infected with P. yoelli XL-17 or P. berghei NK65. Blood and organs were aseptically removed and immediately placed in liquid nitrogen and then kept at −80 °C. Collected blood was centrifuged at 5,000 rpm for 5 min, and the plasma was stored at −80 °C for antibody detection.
Gene
Primer/probe
IFN-α TNF-α IL-10 β-Actin
Mm00801778_m1 (Applied Biosystems Inc, USA) Mm00443258_m1 (Applied Biosystems Inc, USA) Mm00439616_m1 (Applied Biosystems Inc, USA) 5′-GCTCTGGCTCCTAGCACCAT-3′F 5′-GCCACCGATCCACACAGAGT-3′R 5′-FAM-ATCAAGATCATTGCTCCTC-MGB-3′ 5′-CGGCTACCACATCCAAGGAA-3′F 5′-GCTGGAATTACCGCGGCT-3′R 5′-FAM-TGCTGGCACCAGACTTGCCCTC-MGB-3′ 5′-CGTCGCGAAGGATACTCT-3′F 5′-GGCAGCAGATTTCACTGTGAAG-3′R 5′-FAM-TCGTCAACGGCCACCG-MGB-3′
Gene expression analysis by real-time quantitative polymerase chain reaction (RT-PCR) Total RNA from liver and spleen was extracted by using Tri reagent (Sigma, St Louis, MO). RNA concentration was obtained by using a spectrophotometer (Ultrospec 2100 pro), and the RNA quality was assessed by using Experion automatized chromatography (Experion TM RNA StdSens Analysis Kit; Bio-Rad, Hercules, CA). The mRNA expression of interleukin (IL)-10, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α was monitored. The RT-PCR was performed with specific double-labeled probes in an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA). Briefly, the reaction was carried out in a final volume of 20 μL containing 10 μL of 2× master mix without UNG (uracil-N -glycosylase), 0.5 μL of 40× multiscribe and RNase inhibitor mix, 1 μL of TaqMan gene expression assay probe, 4 μL of 50 ng/μL RNA template, and 1.1 μL of RNase-free doubled-distilled water. The reaction condition was as follows: 48 °C for 30 min, 95 °C for 10 min, 45 cycles with 95 °C for 15 s, and 60 °C for 1 min. The data were analyzed by using SDS 2.1 software (Applied Biosystems). Mouse β-actin, GAPDH, and 18SrRNA were used as internal control genes. The most stable i n t e r n a l c on t r o l g e ne w a s d et e r m i n e d b y u s i n g Genorm_win_3.5 software (Vandesompele et al. 2002). Primers and probes used for target genes and for internal control genes are described in Table 1. Crude parasite antigen preparation and detection of total IgG, and IgG2c B6 mice infected with P. yoelli XL-17 or P. berghei showing 20 to 25 % of parasitemia were killed (n =3). Then blood was obtained by cardiac puncture and treated with Histopaque 1119 and 1077 (Sigma, St Louis, USA) following the manufacturer’s instructions in order to isolate the mononuclear and polymorphonuclear cells. Packed red blood cells (RBCs) were washed with cold PBS twice. Packed RBCs were lysed with diethyl pyrocarbonate-treated water (Ambion, TX, USA) and
18S rRNA
GAPDH
then washed with PBS twice. The pellet was sonicated three times for 30 s each time. Crude antigen concentration was adjusted to the same value for all the samples using the BCATM Protein assay kit (Thermo scientific, Rockford, USA). The resulting crude antigen was used in an enzymelinked immunosorbent assay. Briefly, 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated overnight at 4 °C with 50 μl of P. berghei NK65 or P. yoelli XL17 crude antigen at a concentration of 20 μg/ml in a coating buffer (50 μM carbonate-bicarbonate buffer, pH 9.6). The plates were washed with 0.05 % Tween 20-PBS (PBST) and then incubated with 100 μl of a blocking solution (3 % skim milk in PBS) for 1 h at 37 °C. Subsequently, the plates were washed with PBST and then incubated with 50 μl of mouse sera diluted in 1:100 with the blocking solution for 1 h at 37 °C. The plates were washed six times with PBST and then were incubated with 50 μl of the horseradish peroxidaseconjugated goat anti-mouse immune-globulins IgG and IgG2c (Bethyl Laboratories, TX, USA) diluted to 1:4,000 with the blocking solution for 1 h at 37 °C as a secondary antibody. The plates were washed for six times with PBST. Then 100 μl of the substrate solution [0.1 M citric acid, 0.2 M sodium phosphate, 0.003 % H202, and 0.3 mg/ml 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid); Sigma, St Louis, USA] per well was added. After incubation for 1 h at room temperature, absorbance was measured using an MTP microplate reader (Corona Electric, Tokyo, Japan) at 415 nm. Preparation of dead P. yoelli XL-17 for mice immunization Fixed parasite was prepared as described previously (Li et al. 2012). Briefly, P. yoelli XL17 infected blood having 60 % of parasitemia was collected from heart using sodium citrate (WAKO, Tokyo, Japan) as anticoagulant, and then infected and un-infected RBCs were isolated using
1022 Table 2 Composition of standard diet 100g (CLEA, Tokyo, Japan)
Parasitol Res (2014) 113:1019–1027
Nutrients and calories
Minerals
Vitamins
Moisture (%) Crude protein (%) Crude fat (%) Crude fiber (%) Crude ash (%)
8.9 24.9 4.6 4.1 6.6
Ca (g) P (g) Mg (g) K (g) Mn (mg)
1.06 0.99 0.34 1.02 9.96
Retinol (mg) Vitamin B1 (mg) Vitamin B2 (mg) Vitamin B6 (mg) Vitamin B12 (μg)
0.6 2.0 1.3 1.4 5.6
NFE (%) Energy (kcal) Hardness (kg/cm2)
51.0 344.9 25.8
Fe (mg) Cu (mg) Zn (mg) Na (g)
30.64 0.71 5.45 0.31
Vitamin C (mg) Vitamin E (mg) Pantothenic acid (mg) Niacin (mg)
25.0 8.5 3.0 18.9
α-Tocopherol supplementation, 600 mg/kg
Histopaque 1119 and 1077 (Sigma, St Louis, USA) following the manufacturer’s instructions. Packed RBCs including infected and un-infected RBCs were washed with cold PBS for three times, and then parasites were fixed with 0.25 % of glutaraldehyde for 15 min at room temperature; thereafter, they were washed with cold PBS for three times; 0.2 ml of this preparation at a concentration of 1×105 IRBCs/ml was inoculated into B6 (n =6) and αttpΔ mice (n =6). Also, un-infected blood was used as a control. At 15 days after fixed parasite inoculation, mice were challenged with 0.2 ml of 1 × 10 5 IRBCs/ml of P. berghei NK65. Then survival rate was monitored daily and parasitemia every 2 days.
2 weeks and then was challenged with P. berghei NK65. Survival rate was monitored daily and parasitemia every 2 days. Diet composition is described in Table 2. Statistical analysis Statistical analysis was performed using Kruskal-Wallis all pairwise comparisons–Conover-Inman tests (Stats-Direct, version 2.7.5, software for Windows). For survival rate analysis, Kaplan–Meier method was performed. A p value less than 0.05 was considered as statistically significant.
Results Effect of α-tocopherol supplementation on Plasmodium proliferation To examine the effect of α-tocopherol supplementation on the parasite proliferation, α-ttpΔ mice were infected with P. yoelli XL-17 and fed with standard diet containing 85 mg/kg of αtocopherol. Two weeks after the primary infection, the diet was shifted to a supplemented diet of α-tocopherol (600 mg/kg) for
Fig. 2 Alpha-tocopherol transfer protein knockout mice infected with P. yoelii XL-17 develop protective immunity against P. berghei NK65 infection. a : Parasitemia kinetics indicates that the double-infected knockout mice control parasite proliferation efficiently. Moreover, a lethal
A protective immunity against P. berghei NK65 infection was developed in the α-ttpΔ mice pre-infected with P. yoelli XL-17 Parasitemia in B6 mice infected with P. yoelli XL17 increased dramatically from day 5 post-infection, and 100 % of death was observed on day 15 post-infection. In contrast,
strain such as P. berghei NK65 behave as no lethal in this mouse model. b: Mortality in the double infected knockout mice was 0 %. *p
