1. Discovering the genes controlling response to Trypanosoma congolense infection Harry Noyes University of Liverpool

2. 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

3. Trypanosomosis Is a fatal disease of livestock. The livestock equivalent of sleeping sickness in humans T brucei rhodesiense T gambiense T. congolense, T. vivax

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

5. Trypanosoma brucei

7. Mouse models of trypanotolerance. 0 10 20 30 40 50 60 70 80 90 100 121416181101121 Days Post Challenge % Survival F6 AJ C57BL Survival of F6 and parental mice

8. X=X= C57BL6 resistant AJ and Balb/c susceptible Creating mapping populations

9. 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

10. D17MIT408D17MIT029DI17MIT234D17MIT177D17MIT091D17MIT072 5CM 40 35 30 25 20 15 10 5 0 LOD SCORE D17MIT16 Tir1 locus in the F6

11. 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)

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

16. Sequence comparisons Chromosome 5, 73-83Mb

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

18. Microarray design at each time point Resistant C57BL/6Susceptible AJ

19. Harvesting Tissues

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

23. 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

24. Development of Congenic mice C57BL/6 DNA AJ DNA QTL

25. Position of Mmu17 QTL (Tir1)

26. Position of Mmu5 QTL (Tir2)

27. Survival of congenic mice

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

29. Cholesterol metabolism

30. Endogenous cholesterol production increases after infection

31. Tir loci C57/BL6 x AJ HDL 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 13:1654-1664 Kemp 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

32. Total Cholesterol levels

33. Tir2 QTL controls weight

34. 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

39. Inflammatory counter inflammatory switch C57 AJ/Balb 079317 Classically activated macrophages Alternatively activated macrophages Th2 signal (IL4, IL10)

40. AJ and Balb/c produce alternatively activated macrophages early in infection

41. Intersection of Cholesterol and Inflammatory pathways Dunn et al Journal of Experimental Medicine Vol. 203, No. 2, February 20, 2006 401–412 Th2 bias Suppression of cholesterol synthesis

42. NATURE MEDICINE VOLUME 9 NUMBER 2 FEBRUARY 2003 LXR agonsists lower cholesterol and inhibit NFkB mediated inflammatory signals

43. 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)