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Identification of gene networks associated with
anaemia caused by Trypanosoma congolense
Alimohammadian M4; Anderson, S5, Brass A3; Gibson, JP1; Hulme, H3, Iraqi, FA1,
2
1
1
2
Kemp, SJ ; Mburu D ; Naessens, J ; Noyes, HA ;
1International
Livestock Research Institute, P. O. Box 30709, Nairobi, Kenya
2School of Biological Sciences, University of Liverpool, L69 7ZB, UK
3Department of Computer Science, University of Manchester, UK
4Dept. of Immunology, Pasteur Institute of Iran
5Roslin Institute, Roslin Biocentre, Midlothian
0
Fall in PCV
20
40
60
80
-10
-15
N'Dama
(Trypanotolerant)
-20
Zebu (Suceptible)
-25
-30
-35
Days Post infection
Bovine trypanosomiasis caused by Trypanosoma congolense is one of the most important limitations on cattle production
in Africa. The disease is similar to sleeping sickness in humans which is caused by Trypanosoma brucei. The most
important feature of the disease is anaemia which is used as an indicator of when to treat infected animals. Some ancient
African breeds of cattle such as N’Dama can survive and remain productive after infection and are described as
trypanotolerant whilst more recently introduced breeds are highly susceptible. Both breeds develop anaemia after infection
but trypanotolerant animals do not develop such severe anaemia and spontaneously recover whilst susceptible animals die
if not treated.
Relative hemoglobin (OD 540 nm)
% fall in PCV after T. congolense infection .
-5 0
Mice infected with T. congolense also develop
anaemia but C57BL/6 mice which are the most
resistant to trypanosomiasis develop the most
severe anaemia, whilst AJ and BALB/c
develop relatively mild anaemia. Anaemia may
be part of the host protective response as it
effectively denies essential iron to the parasite.
We have measured the expression of genes
associated with erythropoesis and iron
recycling on Affymetrix microarrays to
identify the processes regulating anaemia in
these mice.
0.70
0.60
0.50
0.40
0.30
0.20
0
10
20
30
40
days post-infection
Materials and Methods
Groups of 210 AJ, BALB/c and C57BL/6 mice were challenged with 104 T. congolense IL1180 parasites and groups of 30 of each strain were sacrificed at each of seven time
points and spleen, liver and kidney were harvested in liquid nitrogen. Haematocrit was determined at the time of sacrifice. RNA was prepared from individual samples and
mixed in pools of five samples. Five independent pools for each strain were hybridised to Affymetrix 420_2 arrays which have approximately 45000 probe sets. Data
presented is the mean ± SD of the five replicate measurements.
Relative hemoglobin titres in BL/6 (circles,
full line), A/J (triangles, broken line) and
BALB (squares, dotted line) after infection
with T. congolense, and uninfected BL/6
mice (open circles, broken line). Each point
is an average of ten mice.
Iron recycling through macrophages
Hemoglobin
Haptoglobin
Fe2+
Transferrin
C57
NRAMP
Tfr2
CD163
Tfr1
CD163
IL6
Iron storage and recycling.
Anaemia is a common correlate of inflammatory conditions and has been associated with
increases in iron stored in macrophages as ferritin or the insoluble haemosiderin. Storage is
believed to be mainly regulated by hepcidin which negatively regulates the export of iron
from macrophages by ferroportin by binding ferroportin RNA and targeting it for destruction.
Hepcidin
Tal1
Tal1, Gata1, Lmo2 and Fog1 form a
multimeric DNA binding complex which
regulates primitive erythropoeisis. All three
genes have similar transcription patterns,
declining in production in the spleen post
infection and by most in C57BL/6.
EKLF is involved in erythroid cell
proliferation and is also lower in C57BL/6.
All these expression patterns are suggestive
of suppressed erythropoeisis in C57BL/6.
Gata1
Fog1
EKLF
Hepcidin
IL6
TRANSFERRIN
Transferin receptor
Ferroportin
Tissue Size
Gene expression was studied on constant RNA sample sizes.
However Liver and Spleen tissues increased significantly in size over
the course of infection. The spleen of BALB/c was larger than that of
AJ and C57BL/6 suggestive of greater erythropoetic potential in
BALB/c.
Conclusion
In four out of five systems tested gene expression was consistent with
C57BL/6 mice having the most severe anaemia. The exception was
enzymes for catabolism of erythrocyte proteins which were most
upregulated in AJ mice.
The overall expression patterns would also have predicted that AJ mice
would have the mildest anaemia after infection with T. congolense when
it appeared that BALB/c mice had the milder anaemia than BALB/c but
possibly not significantly different. This apparent anomaly may be
attributable to the 50% larger size of the spleen in BALB/c mice.
The range of systems in which C57BL/6 gene expression was consistent
with more severe anaemia suggests that there is coordinated regulation of
the anaemia associated with inflammation. The identification of those
regulatory factors will be the focus of future work.
Receptors for haematopoietic growth factors
Blvra
Hmox1
EpoR
Erythrocyte Degradation
Biliveridin reductase (Blvra) and
Heme oxygenase (Hmox1) are both
involved in degradation of
erythrocytes and both increased
substantially in the liver. By the
most in AJ and least in C57BL/6.
This is the only system that is not
consistent with most severe anaemia
in C57BL/6.
FERROPORTIN
Nramp
Igf1r
There was some evidence for decline
in transcription of three receptors for
haematopoetic growth factors. Epo
receptor transcription declined in AJ
and C57BL/6 and was lowest in
C57BL/6. Kit declined in all strains
but by most in C57BL/6.
KIT
12
8
8
SPLEEN
LIVER
weight as % of body weight
Erythroid differentiation
factors
Haemosiderin
Ferritin
Hepcidin in turn is regulated by IL6. Splenic IL6 production increases most in C57BL/6 post
infection, concomitant with this, hepcidin expression increases approximately twofold post
infection and by more in C57BL/6 than AJ or BALB/c. However ferroportin transcription
also increases but by more in AJ than C57BL/6 leading to a two fold higher ratio of hepcidin
to ferroportin in C57BL/6 than in AJ with the likely outcome that C57BL/6 will export
significantly less iron that AJ macrophages. However the hepcidin ferroportin ratio falls over
the course of infection which might have the effect of permitting greater iron export in all
strains by day 17.
Iron uptake also changes post infection. Transferrin which imports iron carried on ferritin
changes little, but Nramp which transports molecular iron ions increases 8-16 fold post
infection and by most in AJ mice. Most surprisingly transcription of CD163 is almost
completely abolished post infection in all strains. CD163 scavenges haptoglobin from
plasma. Haptoglobin scavenges the products of haemolysis, which is extensive in the early
stages of infection, so it is remarkable that its receptor is reduced in expression.
Fe2+
KIDNEY
10
6
6
8
4
4
6
2
2
4
0
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15
20
0
0
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10
15
20
0
0
5
10
15
20
days post-infection
Mean weights of internal organs during T. congolense infection in
A/J mice (triangles), BALB/c (squares) and C57BL/6J (circles)
mice.
Erythrocyte structural proteins
Spectrin alpha and beta (Spna1 and Spnb1), Glycophorin (Gypa) and erythrocyte
protein band 7 Epb7.2 all declined in production in the spleen but stayed
constant in the liver. In each case C57BL/6 had the lowest levels of transcription
consistent with relatively low levels of haematopoesis.
Spna1
Gypa
Spnb1
Epb7.2