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