Discovering the genes controlling response to Trypanosoma congolense infection Harry 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 Institute Alan Archibald Susan Anderson Laurence Hall Funding Wellcome 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

Cattle Tsetse Cattle and tsetse Distribution of Trypanosomiasis N’Dama Boran

Trypanosoma brucei

Mouse models of trypanotolerance Days Post Challenge % Survival F6 AJ C57BL Survival of F6 and parental mice

X=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 : Kemp et al. Nature Genetics :

D17MIT408D17MIT029DI17MIT234D17MIT177D17MIT091D17MIT072 5CM LOD SCORE D17MIT16 Tir1 locus in the F6

Contribution of 10 genes from Boranand N’Dama cattle to reduction in degree of trypanosomosis Boran (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)

From QTL to gene Sequence variants –Resequencing Structural variants –Agilent tiling arrays Expression variation –Affymetrix microarrays Congenic mice Gene networks

Sequence comparisons Chromosome 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

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 AJ HDL after 6 weeks high fat diet AIL C57BL6 x NZB/BIN Iraqi et al Mammalian Genome : Wang et al. Genome Research : Kemp et al. Nature Genetics : Trypanotolerance QTL are the right of each pair of chromosomes HDL QTL are the left hand of each pair

Total Cholesterol levels

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 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 Medicine Vol. 203, No. 2, February 20, –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)