Discovering the genes controlling 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• Kate Goodheart• Harry Noyes• Katie Rennie

Roslin InstituteAlan ArchibaldSusan AndersonLaurence Hall

FundingWellcome Trust

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

Distribution of Trypanosomiasis

N’DamaBoran

Trypanosoma brucei

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Mouse models of trypanotolerance.

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Days Post Challenge

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F6

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C57BL

Survival of F6 and parental mice

X =

C57BL6 resistant

AJ and Balb/c susceptible

Creating mapping populations

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

D17

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Tir1 locus in the F6

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

-15-10-505

1015

From QTL to gene

• Sequence variants– Resequencing

• Structural variants– Agilent tiling arrays

• Expression variation– Affymetrix microarrays

• Congenic mice• Gene networks

Sequence comparisonsChromosome 5, 73-83Mb

From QTL to gene

• Sequence variants– Resequencing

• Structural variants– Agilent tiling arrays

• Expression variation– Affymetrix microarrays

• Congenic mice• Gene networks

Microarray design at each time point

Resistant C57BL/6Susceptible AJ

Harvesting Tissues

From QTL to gene

• Sequence variants– Resequencing

• Structural variants– Agilent tiling arrays

• Expression variation– Affymetrix microarrays

• Congenic mice• Gene networks

Congenic Mice

• Three congenic lines

• One line for each trypanotolerance QTL

• F1 Cross between AJ and C57BL6

• Backcrossed to AJ for six generations selecting for C57 allele at each generation

Development of Congenic mice

C57BL/6 DNA

AJ DNA

QTL

Position of Mmu17 QTL (Tir1)

Position of Mmu5 QTL (Tir2)

Survival of congenic mice

Response of Congenic mice to T. congolense infection

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Days post intection

% survivingTir1AA (n = 20)Tir1CC (n = 25)Tir2AA (n = 60)Tir2CC (n = 120)Tir3AA (n = 20)Tir3CC (n = 90)TirnAA (n = 100)

From QTL to gene

• Sequence variants– Resequencing

• Structural variants– Agilent tiling arrays

• Expression variation– Affymetrix microarrays

• Congenic mice• Gene networks

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

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AJ_HIGH_FAT

AJ_LOW_FAT

BALB_HIGH_FAT

BALB_LOW_FAT

c57_HIGH_FAT

C57_LOW_FAT

Tir2 QTL controls weight

Collaboration with GSF

• Clinical chemistry and cytokines

• InbredC57BL/6, Balb/c, A/J mice

• Congenic Tir1, Tir2, Tir3 mice plus controls

• Serum samples collected pre-infection and at days 3, 9, 17 and 35 post-infection

Inflammatory counter inflammatory switch

C57

AJ/Balb

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Classically activated macrophages

Alternatively activated macrophagesTh2 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

Innate immune response controls survival after infection

with T. congolense • Deletion of T cells does not effect survival

time• C57BL/6 (resistant) mice have strong

inflammatory response and high cholesterol• Identification of regulators of both

mechanisms may lead us to the Quantative trait gene (QTG)