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Pathways regulating response to Trypanosoma congolense
infectionHarry Noyes
University of Liverpool
Participants• ILRI, Nairobi• Morris Agaba• John Gibson• Fuad Iraqi• Steve Kemp• Hassan Musa• Joel Mwakya• Daniel Mwanga• Jan Naessens• Joseph Nganga• Moises Ogugu• John Wambugu
• University of Manchester
• Andy Brass• Helen Hulme• Leo Zeef• Leanne Wardlesworth
• University of Liverpool
• Anthea Broadhead• Derek Daly• Harry Noyes• Katie Rennie
• Roslin Institute• Alan Archibald• Susan Anderson• Laurence Hall
Trypanosomosis
Is a fatal disease of livestock.The livestock equivalent
of sleeping sickness in humans
T brucei rhodesiense T gambiense
T. congolense, T. vivax
Bovins
Bovins et GlossinesGlossines
CattleTsetseCattle and tsetse
Origins of N’Dama and Boran cattle
N’DamaBoran
Mouse models of trypanotolerance
.
Survival of F6 and parental strains
0
10
20
30
40
50
60
70
80
90
100
1 21 41 61 81 101 121
Days Post Challenge
% S
urv
ival
F6
AJ
C57BL
Creating mapping populations
X =
C57BL6 resistant
Balb/c susceptible
Mapping single locus traits Resistant
Resistant
Susceptible
Resistant
Resistance locus
SusceptibleSusceptible
F1
F2
F2
Susceptible
F0
F2 linkage map
• Trypanotolerance was associated with three loci
• These loci accounted for almost all the genetic variation in susceptibility.
• Chromosome 17 and chromosome 5 loci were 20cM
• Chromosome 1 locus was 30cM
Tir2 locus in the F2
10cM
BalbxC57
A/JxC57
5
4
3
2
1
LO
D s
core
Limitations of F2 crosses
• 10cM intervals correspond to about 10Mb
• 10cM likely to contain 50-250 genes
• Too large an interval to easily be cloned
Advanced Intercross Lines
• Crossing the most susceptible and resistant F2 mice for four more generations to create F6
• Predicted to improve resolution by 3x
D17
MIT
408
D17
MIT
029
DI1
7MIT
234 D
17M
IT17
7
D17
MIT
091
D17
MIT
072
5CM
40
35
30
25
20
15
10
5
0
LO
D S
CO
RE
D17MIT16
Tir1 locus in the F6
Trypanosoma infection response (Tir) loci
C57/BL6 x AJ and C57/BL6 x BALB/C
Iraqi et al Mammalian Genome 2000 11:645-648 Kemp et al. Nature Genetics 1997 16:194-196
Contribution of 10 genes from Boranand N’Dama
cattle to reduction in degree of trypanosomosisBoran (relatively susceptible)
The N’Dama and Boran each contribute trypanotolerance alleles at 5 of the 10 most significant QTL, indicating that a synthetic breed could
have even higher tolerance than the N’Dama.
N’Dama (tolerant)
-15-10-505
1015
-15-10-50
51015
Development of Congenic miceC57BL/6 DNA
AJ DNA
QTL
Survival of congenic mice
Response of Congenic mice to T. congolense infection
0
20
40
60
80
100
120
21-30 31-40 41-50 51-60 61-70 71-80 81-90 91-100
Days post intection
% s
urv
ivin
g
Tir1AA (n = 20)Tir1CC (n = 25)Tir2AA (n = 60)Tir2CC (n = 120)Tir3AA (n = 20)Tir3CC (n = 90)TirnAA (n = 100)
Finding the causative difference
• Difference might be in primary structure of a gene– Sequence all genes in QTL
• Difference might be in regulatory regions– Test expression of all genes
Sequence comparisonsChromosome 5, 73-83Mb
Microarray design at each time point
Resistant C57BL/6Susceptible AJ
Harvesting Tissues
Cholesterol metabolism
Endogenous cholesterol production increases after infection
Tir loci C57/BL6 x AJHDL after 6 weeks high fat diet AIL C57BL6 x NZB/BIN
Iraqi et al Mammalian Genome 2000 11:645-648 Wang et al. Genome Research 2003 13:1654-1664Kemp et al. Nature Genetics 1997 16:194-196
Trypanotolerance QTL are the right of each pair of chromosomes HDL QTL are the left hand of each pair
Total Cholesterol levelsCHOLESTEROL
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
0 8 21 4
DAYS POST INFECTION
AJ _HIGH_FAT
AJ _LOW_FAT
BALB_HIGH_FAT
BALB_LOW_FAT
c57_HIGH_FAT
C57_LOW_FAT
ApoA1 is major lipoprotein of HDL
Scavenger Receptor B1 expression
• SRBI is involved in cholesterol uptake by macrophages
Saa acute phase response marker
AJ express less HMGCoAR
Cholesterol goes to foam cells?
• Macrophages are scavengers of cholesterol
• Foam cells: Activated macrophages loaded with lipids
• Erythrocytes and parasites may be source of cholesterol.
Leukocytes involved in response to Trypanosomiasis
• T cell knock out or T cell depleted mice are no more susceptible to infection than mice with intact T-cells
• Control of parasitaemia appears to be mediated by anti-body
• Opsonised parasites are phagacytosed by Liver macrophages
CD4 and CD8 cells do not respond to infection
Increase in FcGR1 suggests expansion of
macrophage populations in spleen and liver
Inflammatory counter inflammatory switch
C57
AJ/Balb
0 7 93 17
Classically activated macrophages
Alternatively activated macrophages
Th2 signal (IL4, IL10)
AJ and Balb/c produce alternatively activated macrophages early in infection
Intersection of Cholesterol and Inflammatory pathways
Dunn et al Journal of Experimental MedicineVol. 203, No. 2, February 20, 2006 401–412
Th2 bias
Suppression of cholesterol synthesis
NATURE MEDICINE • VOLUME 9 • NUMBER 2 • FEBRUARY 2003
LXR agonsists lower cholesterol and inhibit NFkB mediated inflammatory signals
Macrophage response to LXR agonist and LPS
Repressedinterleukin-6MCP-1high affinity IgG receptor (Fc-γ RI)interleukin-1-βanaphylatoxin C3a receptorCOX-2thrombospondin (THBS1)suppressor of cytokine signaling -3dendritic cell immunoreceptorsuppressor of cytokine signaling -1cytokine receptor-like molecule (EBl3)interferon inducible protein (IP-10)G-CSF
InducedABCA1Apo CIIfatty acid synthaseCTP:phosphocholine cytidylytransferase3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR)plasma phospholipid transfer proteinperoxisome membrane protein (PEX2)stearoyl-CoA desaturaselong-chain acyl-CoA dehydrogenasestearoyl-CoA
Conclusions
• Tissue arrays show bulk changes in organs
• Liver is responding by changes in lipid profile and inflammation
• SIRS/CARS
• Identify regulators of innate immune response
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