1. Pathways regulating 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 Harry Noyes Katie Rennie Roslin Institute Alan Archibald Susan Anderson Laurence Hall

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 Origins of N’Dama and Boran cattle N’Dama Boran

5. Mouse models of trypanotolerance.

6. Creating mapping populations X=X= C57BL6 resistant Balb/c susceptible

7. Mapping single locus traits Resistant Susceptible Resistant Resistance locus Susceptible F1 F2 Susceptible F0

8. 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

9. Tir2 locus in the F2 10cM BalbxC57 A/JxC57 5432154321 LOD score

10. 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

11. Advanced Intercross Lines Crossing the most susceptible and resistant F2 mice for four more generations to create F6 Predicted to improve resolution by 3x

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

13. 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

14. 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)

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

16. Survival of congenic mice

17. 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

18. Sequence comparisons Chromosome 5, 73-83Mb

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

20. Harvesting Tissues

21. Cholesterol metabolism

22. Endogenous cholesterol production increases after infection

23. 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

24. Total Cholesterol levels

25. ApoA1 is major lipoprotein of HDL

26. Scavenger Receptor B1 expression SRBI is involved in cholesterol uptake by macrophages

27. Saa acute phase response marker

28. AJ express less HMGCoAR

29. 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.

30. 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

31. CD4 and CD8 cells do not respond to infection

32. Increase in FcGR1 suggests expansion of macrophage populations in spleen and liver

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

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

39. 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

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

41. Macrophage response to LXR agonist and LPS Repressed interleukin-6 MCP-1 high affinity IgG receptor (Fc-γ RI) interleukin-1-β anaphylatoxin C3a receptor COX-2 thrombospondin (THBS1) suppressor of cytokine signaling -3 dendritic cell immunoreceptor suppressor of cytokine signaling -1 cytokine receptor-like molecule (EBl3) interferon inducible protein (IP-10) G-CSF Induced ABCA1 Apo CII fatty acid synthase CTP:phosphocholine cytidylytransferase 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) plasma phospholipid transfer protein peroxisome membrane protein (PEX2) stearoyl-CoA desaturase long-chain acyl-CoA dehydrogenase stearoyl-CoA

42. 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