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The role of cholesterol in the pathology of African
animal trypanosomiasis
Hassan, M.1 & 2, Kemp, S.J. 1 & 3, Agaba, M.1, Noyes, H.A.3, Naessens, J.1, Iraqi, F.1, Gibson, J.5, Rennie, C.4, Hulme, H.4, Hinsley, T.4, & Brass, A.4,
1 InternationalLivestock Research Institute, P. O. Box 30709, Nairobi 00100, Kenya, 2 Department of Biochemistry, University of Nairobi, P.O Box 30197, Nairobi 00100, Kenya, 3 School of
Biological Sciences, Biosciences Building, Crown Street, Liverpool L69 7ZB, UK, 4 Dept. of Computer Science, School of Biological Sciences, University of Manchester, UK, 5 The Institute for
Genetics and Bioinformatics Hawkins Homestead University of New England Armidale, NSW 2351 Australia.
Introduction
African animal trypanosomiasis is often associated with a significant decrease in the host plasma cholesterol levels which often coincide with a rise in parasitaemia
(Katunguka-Rwakishaya et al., 1992; Traore-Leroux et al., 1987) . Decreases in plasma cholesterol levels and serum phospholipids have been reported in sheep infected
with T. congolense (Katunguka-Rwakishaya et al., 1991) and man infected with T. brucei (Huet et al., 1990). On the other hand, blood stream forms of African trypanosomes
lack the ability to synthesise lipids de novo. They acquire lipids from the host through the uptake of mainly high density lipoprotein (HDL) and low density lipoprotein
(LDL) particles (Heather et al., 2003). Genetic studies have mapped the Quantitative Trait Loci (QTLs) for Trypanotolerance (Tir2) (Kemp et al., 1997) and plasma HDL-
cholesterol levels (Wang et al., 2003) to the same region on chromosome 5. Therefore, the circulating levels of cholesterol seem to play a critical role in the pathology of
African Animal trypanosomiasis. The aim of this study was to correlate the expression levels of genes involved in cholesterol metabolism, and the plasma cholesterol
levels in laboratory mice with their susceptibility to T. congolense infections. In the first experiment we investigated the expression levels of genes associated with
cholesterol metabolism in mice fed normal laboratory diet and infected with T. congolense. In a second experiment we used characterised high and low fat diets to
manipulate the plasma cholesterol levels in mice and followed their response to T. congolense infection.
Materials and Methods
Mice : In the first experiment, male and female C57BL/6J, BALB/c, and A/J mice were used while in the second experiment only male mice were used. All the mice were
obtained from Harlan UK Ltd (Bicester, UK) at six weeks of age. They were fed on a normal laboratory mouse diet until time of use.
Diets: The high and low fat diets used to manipulate plasma cholesterol levels in the second experiment were supplied by Purina Mills (USA) and were matched for
calories and all other nutrients except the fat content.
Tissues and plasma collection: Liver used for RNA extraction was collected at 0, 3, 7,9 and 17 days post infection from 30 male and female mice infected with 104 T. congolense,
and fed a normal diet. In the second experiment, plasma for lipid profiling was collected at infection, 8, and 21 days post infection.
Infection parameters: Mice in the second experiment were followed for parasitaemia, body weight changes and anaemia. Parasitaemia was scored on a scale of 0 to 5 using
wet smears on dark background microscopy.
Microarray assay: cDNA synthesis was carried out using an Invitrogen SuperScript double-stranded cDNA synthesis kit using 100 pmol T7-(dT)24 primer (MWG). For each
target, a hybridization cocktail was made using the standard array recipe. The cocktail was hybridized to mouse genome Mouse430_2 chips. Gene expression analysis
were performed using RMAExpress.
Results
Gene Expression: There was no difference in the expression levels of the genes involved in cholesterol biosynthesis (Hmgcr) between the resistant and susceptible strains but
there was significant difference in the genes involved in cholesterol degradation (Cyp7A1) and transport (Scarb-1, and ATP10D) (P<0.001).
Dietary lipids and Trypanosomiasis: The mice on the high fat diet had low levels of parasitaemia but only A/J mice showed a significant difference. Following infection, the
lipid levels decreased, but only HDL-cholesterol showed statistically significant decrease (P<0.001). There was no significant difference in the severity of anaemia between
the high fat and the low fat fed mice.
8.0
1.5
Plasma Total-C (mmol/l)
7.0 5.0
6.0 5.0
Plasma HDL-C (mmol/l)
Plasma LDL-C (mmol\l)
4.0
5.0
Plasma TG (mmol\l)
1.0 4.0
4.0 3.0
3.0
3.0 2.0
2.0 0.5 2.0
1.0
1.0 1.0
0.0 0.0 0.0 0.0
0 8 21 46 Uninfected 0 8 21 46 Uninfected 0 8 21 46 Uninfected 0 8 21 46 Uninfected
Days post infection Days Post infection
Days post infection Days Post Infection
C57-HF C57-LF Balbc-HF Balbc-LF AJ-HF AJ-LF C57-HF C57-LF Balbc-HF Balbc-LF AJ-HF AJ-LF C57-HF C57-LF Balbc-HF Balbc-LF AJ-HF AJ-LF
-AJ-HF -AJ-LF C57-HF C57-LF BALB/c-HF BALB/c-LF
Fig. 5a-d: Plasma lipid levels in three mouse strains before (point 0) and after infection. The plotted lipid levels represent means of plasma lipid levels obtained from 5 individual mice.
1.0
1.9
6 0.9
1.7 0.8
Weights indexes
5
0.7
Means of parasite scores
1.5
Hb-Readings
4 0.6
1.3
0.5
1.1 3
0.4
0.9 2 0.3
0.7 0.2
1
0.1
0.5 0 0.0
-69 -62 -55 -48 -41 -31 -24 -17 -10 -3 4 11 18 25 32 39 46 4 7 10 14 17 21 24 28 31 35 38 42 46 4 7 13 17 21 28 31 35 37 41 46
Days post infection.
Days post infection
Days post infection
BALB/c_HF BALB/c_LF AJ_HF AJ_LF C57BL/6J_HF C57BL/6J_LF HF-Diet LF-Diet
AJ-HF AJ-LF BALB\c-HF BALB\c-LF C57-HF C57-LF
Fig. 6: Weight index of laboratory mouse strains fed on high or low fat Fig. 7: The mean parasitaemia scores in A/J mice infected with T. congolense.
diet before infection with T. congolense. The weight indices were The mice fed on a high fat diet had low parasite scores than the low fat fed mice, Fig. 8: The mean haemoglobin readings from mice infected with T. congolense. There was
calculated by dividing the weights through by the weight at infection. but only the A/J strain showed significant difference (p<0.001) significant differences in the Hb concentrations between the low and high fat fed mice.
There was a significant difference the weights between the low and high
fat fed mice.
Conclusion. References.
1. Heather et al., 2003: Evidence for Trypanosoma brucei lipoprotein scavenger receptor.
This results indicate that the difference in cholesterol metabolism between the susceptible and tolerant
J. Biol. Chem. 278 (1) 422-27.
mouse strains lie not in the biosynthesis but in cholesterol degradation and transport. Following infection, 2. Katunguka-Rwakishaya et al., 1992: The pathophysiology of ovine trypanosomiasis:
haematological and blood biochemical changes. Vet. Parasitol. 45: 17-32.
resistant (C57BL/6) mice down regulate Cyp7A1 and Scarb-1 hence able to retain cholesterol better than
3. Huet, G., J. L. Lemsre, G. Grard, F. Boutignon, M. C. Dieu, J. Jannin, & P. Degand.
the A/J and BALB/c. Whether this is an effect of or caused by trypanasome infection is not yet clear. The (1990). "Serum lipid and lipoprotein abnormalities in human African
mice with elevated cholesterol had lower levels of parasitaemia compared to those with low cholesterol trypanosomiasis." Trans. Roy. Soc. Trop. Med. Hyg. 84: 792 - 794.
levels. This again indicates the ability of cholesterol to determine the pathology of the trypanosomiasis Acknowledgments: This work was funded by grants from
and perhaps explains why resistant strains of mice down regulate cholesterol clearance from their plasma.
