Alimohammadian M4 Anderson, S5, Brass A3 Gibson, JP1 Hulme, H3, Iraqi, FA1,

1
Alimohammadian M4 Anderson, S5, Brass A3
Gibson, JP1 Hulme, H3, Iraqi, FA1, Kemp, SJ2
Agaba M2 Naessens, J1 Noyes, HA2
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
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.

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 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.
IL6
CD163
Hepcidin
Nramp
Transferin receptor
Ferro-portin

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.
Tal1, GATA1, Lmo2 and ZFPM1 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. KLF1 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.
Erythroid differentiation Factors
Tal1
Lmo2
GATA1

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 bit stayed
constant in the liver. In each case C57BL/6 had
the lowest levels of transcription consistent
with relatively low levels of haematopoesis.
KLF1
ZFPM1

Receptors for haematopoietic growth factors
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.
GYPA
SPNA1
EpoR
KIT
Epb7.2
SPNB1