Mechanisms Controlling Anaemia in Trypanosoma congolense Infected Mice

Abstract

Trypanosoma congolense are extracellular protozoan parasites of the blood stream of artiodactyls and are one of the main constraints on cattle production in Africa. In cattle, anaemia is the key feature of disease and persists after parasitaemia has declined to low or undetectable levels, but treatment to clear the parasites usually resolves the anaemia.
The progress of anaemia after Trypanosoma congolense infection was followed in three mouse strains. Anaemia developed rapidly in all three strains until the peak of the first wave of parasitaemia. This was followed by a second phase, characterized by slower progress to severe anaemia in C57BL/6, by slow recovery in surviving A/J and a rapid recovery in BALB/c. There was no association between parasitaemia and severity of anaemia. Furthermore, functional T lymphocytes are not required for the induction of anaemia, since suppression of T cell activity with Cyclosporin A had neither an effect on the course of infection nor on anaemia. Expression of genes involved in erythropoiesis and iron metabolism was followed in spleen, liver and kidney tissues in the three strains of mice using microarrays. There was no evidence for a response to erythropoietin, consistent with anaemia of chronic disease, which is erythropoietin insensitive. However, the expression of transcription factors and genes involved in erythropoiesis and haemolysis did correlate with the expression of the inflammatory cytokines Il6 and Ifng.
The innate immune response appears to be the major contributor to the inflammation associated with anaemia since suppression of T cells with CsA had no observable effect. Several transcription factors regulating haematopoiesis, Tal1, Gata1, Zfpm1 and Klf1 were expressed at consistently lower levels in C57BL/6 mice suggesting that these mice have a lower haematopoietic capacity and therefore less ability to recover from haemolysis induced anaemia after infection.

Full text

Mechanisms Controlling Anaemia in
Trypanosoma
congolense
Infected Mice
Harry A. Noyes
1.
, Mohammad H. Alimohammadian
4.
, Morris Agaba
5
, Andy Brass
2,3
, Helmut Fuchs
6
,
Valerie Gailus-Durner
6
, Helen Hulme
3
, Fuad Iraqi
5¤
, Stephen Kemp
1,5
, Birgit Rathkolb
6,7
, Eckard Wolf
7
,
Martin Hrabe
´de Angelis
7,8
, Delnaz Roshandel
3
, Jan Naessens
5
*
1School of Biological Sciences, University of Liverpool, Liverpool, United Kingdom, 2Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom,
3School of Computer Science, University of Manchester, Manchester, United Kingdom, 4Immunology Department, Pasteur Institute of Iran, Tehran, Iran, 5International
Livestock Research Institute, Nairobi, Kenya, 6GMC at the Helmholtz Zentrum Mu
¨nchen, Munich/Neuherberg, Germany, 7Chair for Molecular Animal Breeding and
Biotechnology, Gene Center, LMU Munich, Munich, Germany, 8Chair for Experimental Genetics, Center of Life and Food Science Weihenstephan, Technische Universita
¨t
Mu
¨nchen, Freising, Germany
Abstract
Background:
Trypanosoma congolense are extracellular protozoan parasites of the blood stream of artiodactyls and are one
of the main constraints on cattle production in Africa. In cattle, anaemia is the key feature of disease and persists after
parasitaemia has declined to low or undetectable levels, but treatment to clear the parasites usually resolves the anaemia.
Methodology/Principal Findings:
The progress of anaemia after Trypanosoma congolense infection was followed in three
mouse strains. Anaemia developed rapidly in all three strains until the peak of the first wave of parasitaemia. This was
followed by a second phase, characterized by slower progress to severe anaemia in C57BL/6, by slow recovery in surviving
A/J and a rapid recovery in BALB/c. There was no association between parasitaemia and severity of anaemia. Furthermore,
functional T lymphocytes are not required for the induction of anaemia, since suppression of T cell activity with Cyclosporin
A had neither an effect on the course of infection nor on anaemia. Expression of genes involved in erythropoiesis and iron
metabolism was followed in spleen, liver and kidney tissues in the three strains of mice using microarrays. There was no
evidence for a response to erythropoietin, consistent with anaemia of chronic disease, which is erythropoietin insensitive.
However, the expression of transcription factors and genes involved in erythropoiesis and haemolysis did correlate with the
expression of the inflammatory cytokines Il6 and Ifng.
Conclusions/Significance:
The innate immune response appears to be the major contributor to the inflammation
associated with anaemia since suppression of T cells with CsA had no observable effect. Several transcription factors
regulating haematopoiesis, Tal1,Gata1,Zfpm1 and Klf1 were expressed at consistently lower levels in C57BL/6 mice
suggesting that these mice have a lower haematopoietic capacity and therefore less ability to recover from haemolysis
induced anaemia after infection.
Citation: Noyes HA, Alimohammadian MH, Agaba M, Brass A, Fuchs H, et al. (2009) Mechanisms Controlling Anaemia in Trypanosoma congolense Infected
Mice. PLoS ONE 4(4): e5170. doi:10.1371/journal.pone.0005170
Editor: Gordon Langsley, INSERM U567, Institut Cochin, France
Received December 5, 2008; Accepted March 5, 2009; Published April 13, 2009
Copyright: ß2009 Noyes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding to HAN, MA, SJK, HH, AB, WellcomeTrust (GR066764MA to SJK.). The German Mouse Clinic Core (VG-D, HF, MHdA) and clinical chemistry
laboratory (BR, EW) received funding from the EU (EUMODIC grant #LSHG-2006-037188) and the Federal Ministry of Education and Research (National Genome
Research Net, NGFNplus grants 01GS0850 and 01GS0851). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: j.naessens@cgiar.org
¤ Current address: Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel
.These authors contributed equally to this work.
Introduction
African trypanosomes are protozoan parasites that cause severe
diseases in humans and livestock with fatal consequences unless
treated. Whilst trypanosomiasis due to Trypanosoma brucei spp causes
significant morbidity and mortality in humans, trypanosomiasis
caused by T. congolense and T. vivax is one of the most significant
constraints on cattle production in Africa and the cause of major
economic losses with serious effects on human health and welfare [1].
African trypanosomes are extracellular parasites that survive in
the blood stream. In cattle, anaemia is the key feature of disease
and persists after the first wave of parasitaemia when parasite
numbers have declined to low or undetectable levels. Anaemia
rather than parasitaemia is best correlated with productivity and is
used as the primary indicator of when to treat the infection [2].
Treatment to clear the parasites usually resolves the anaemia.
Trypanotolerance, or the capacity of some ancient West African
cattle breeds such as the N’dama to remain productive despite
being infected, is correlated with a genetic capacity to limit
anaemia [3,4,5]. In breeds that have been introduced to the
continent more recently, such as the Boran, erythrocyte counts
continue to decrease after parasitaemia has been controlled and,
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unless treated, the animals die with very low Packed Cell Volume
(PCV).
Because of the importance of the anaemia in trypanosomiasis
many studies have been carried out to describe its nature and
discover its causes. The major mode of red blood cell elimination
appears to be extravascular destruction due to a massive
erythrophagocytosis in spleen and liver [6]. The observation of
hyperactivated macrophages and erythrophagocytosis in tissues of
infected cattle [7] suggests that they may be a major cause of
anaemia and haemophagocytic syndrome [4]. However, evidence
has been provided for the contribution of other mechanisms in
different host-parasite combinations, such as haemolysins (reviewed
in [6]), differences in type and amounts of sialic acids [8,9], binding
of autologous or polyreactive antibodies or complement C3 to
erythrocyte surfaces [10–12] or the passive absorption of trypano-
some molecules in the erythrocyte membrane [13]. Yet, immuno-
logical competence is not essential for the development of anaemia.
Irradiated rats still became anaemic after T. brucei infection [5,6]
and in vivo T-cell depletion did not affect anaemia in cattle [14,15].
Anaemia is also a feature in some murine trypanosomiasis models
[16–20]. A comparison of anaemia and parasitaemia between A/J
and more resistant C57BL/6 mice revealed that anaemia
development was more severe in the C57BL/6 strain, despite the
fact that this strain acquired lower parasitaemias and survived
longer after infection than A/J mice [17]. A comparison of different
host-parasite combinations revealed no correlation between pathol-
ogy and survival [19]. Such data suggest that anaemia is a
consequence of host responses to the infection, and not directly
induced by the parasite products. Studies with T. brucei infected
C3H/He mice suggested an involvement of nitric oxide (NO) [20].
In some murine models, but not others, anaemia was mediated by
TNF [18], which seems to achieve its function via binding with
TNFR2 [19]. And an extensive evaluation of anaemia related genes
responding to infection of C57BL/6 mice with T. brucei was
interpreted as evidence for increased iron storage and reduced
erythropoiesis as a consequence of restricted iron availability [16].
In order to assess the parameters that influence anaemia in
murine T. congolense infections we compared survival, parasitaemia
and development of anaemia and pathology in three mouse strains
A/J, BALB/c and C57BL/6 that differ in their susceptibility to
trypanosomiasis with gene expression data from Affymetrix
microarrays.
Materials and Methods
Animals
C57BL/6JOlaHSD, BALB/cOlaHsd, A/JOlaHsd mice (here-
after C57BL/6, BALB/c and A/J) were purchased from Harlan
UK. Mice were kept in the small animal unit of the ILRI institute
and treated in accordance with the Institute’s Animal Care and
Use Committee (IACUC) policies.
12 weeks old A/J, BALB/c and C57BL/6 mice were infected
with 10
4
T. congolense IL1180 parasites [18]. Parasites per ml of tail
blood were enumerated using a haemocytometer. Mice were killed
by cervical dislocation or CO
2
euthanasia at appropriate time
points post infection and spleen, liver and kidney were collected
into liquid nitrogen.
The role of T cells in the response to infection was determined
by treating six C57BL/6 mice with Cyclosporin A (CsA) and
following the course of infection with T. congolense clone IL1180.
CsA was a gift from Sandoz Ltd, Basel, Switzerland, and was
solubilised in pure ethyl alcohol at 10 mg/ml and diluted in sterile
saline (0.9% NaCl). A volume of 200 ml containing 400 mg CsA/
mouse (about 20 mg/kg) was injected ip every other day for 10
days. Four control mice were injected with the same diluent
without CsA.
Blood parameters
Blood samples were tested for erythrocyte counts and relative
haemoglobin concentration. Erythrocyte numbers were enumer-
ated by haemocytometer under phase-contrast microscopy. The
haemoglobin concentrations were measured spectrophotometri-
cally at 540 nm [21]. Samples of 2 ml of blood were collected from
the tail and diluted in 150 ml of distilled water in a plate with 96
round bottom wells (Costar 3799, Corning Incorporated, Corning
NY, USA). After 30 minutes at room temperature, the plate was
centrifuged at 6006g for 10 minutes, after which 100 mlof
supernatant was transferred to a new plate and the optical density
measured at 540 nm in an ELISA plate reader (Multiscan MCC/
340, Titertek Instruments, Huntsville, AL, USA). Measurements
were carried out in triplicate.
Additionally, blood was collected post mortem by opening the
thoracic cavity, removing the sternum, cutting the vena cava
caudalis and the aorta cranial to the diaphragm and collecting the
leaking blood from the thoracic cavity using a pipette. Blood was left
for two hours at room temperature to clot, then stirred and
centrifuged at 46006g for 10 minutes. The serum was collected,
frozen and sent to the German Mouse Clinic (GMC). Iron
metabolism related serum parameters (ferritin, transferrin) were
determined from serum samples by the clinical chemical laboratory
of the GMC using an AU 400 autoanalyzer (Olympus, Germany)
and Olympus kits developed for the analysis of human samples that
had been adapted for analysis of small volume mouse samples.
Analysis of variance (ANOVA) was performed on anaemia
parameters (RBC counts and relative haemoglobin titres) and
tissue weights using Genstat 11 software, with fixed effects for
mouse genotype, days post infection (dpi) and genotype x dpi. For
organ weight analysis, data were log transformed before analysis
for dpi, strain, sex and interactions between them, sex x dpi, strain
x dpi and strain x sex.
Extraction of total RNA and Microarrays
Ex vivo tissue samples were ground under liquid nitrogen prior to
RNA extraction. RNA was extracted from tissue with Trizol
reagent (Invitrogen). RNA quantity and quality was determined
with an Agilent 2100 bioanalyzer. Pools of RNA from five mice
were prepared for hybridisation to Affymetrix 430_2 arrays. For
each condition five independent pools of five RNA samples were
hybridised to the arrays. The Affymetrix 430_2 arrays contain
45,000 probe sets for over 39,000 transcripts and variants from
over 34,000 mouse genes. Labeling and hybridisation was done
according to the manufacturers instructions.
Technical quality control was performed with dChip (V2005)
(www.dchip.org) [22] using the default settings. Background
correction, quantile normalization, and gene expression analysis
were performed using RMA in BioConductor [23]. Statistical tests
were conducted in SPSS (V16).
All microarray data has been deposited at ArrayExpress under
the accession numbers E-MEXP-1190. The expression data and
plots like those presented here are also available for all genes on
the microarrays from the authors’ website ‘‘Expression viewer’’ at
http://www.genomics.liv.ac.uk/tryps/resources.html.
Results
Survival
As described previously [17], C57BL/6 mice survived a mean of
25 days longer than A/J mice after T. congolense infection (p = 0.04
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Log Rank Survival Test; Fig. 1A). About 25% of A/J mice died
around day 10–11 post-infection. This corresponds to the time
when the first parasitaemic wave reached a peak. A second wave of
mice died after day 50, again when parasitaemia increased to very
high levels. BALB/c mice were not included in this experiment but
they have a survival time that is intermediate between A/J and
C57BL/6 mice [24–27]. T. congolense IL1180 is a relatively low
virulence strain with long survival times after infection. T. congolense
Tc13, which has been used in many other studies, kills BALB/c
mice in about 12 days [28], therefore the responses to the two
parasite strains may not be comparable.
Parasitaemia
The first wave of parasitaemia peaked around day 9 (Fig. 1B)
and parasitaemia was higher in A/J than in C57BL/6 mice.
Anaemia
The change in red blood cell counts (RBC) was expressed as a
percentage change of the pre-infection number, as uninfected A/J
mice had a higher erythrocyte density. The number of RBC in the
blood decreased after infection in both mouse strains (Fig. 1C).
However, A/J mice recovered quickly after the first parasitaemic
wave, while RBC numbers in C57BL/6 mice kept falling with
time. C57BL/6 mice started dying when RBC density dropped
below 60% of its normal value, suggesting that death in C57BL/6
mice was correlated with severe anaemia.
Relative haemoglobin concentration
Three additional T. congolense infection experiments were carried
out in order to compare anaemia development between
susceptible A/J, tolerant C57BL/6 and intermediately susceptible
BALB/c mice. Mean haemoglobin titres were measured in ten
mice of each strain up to day 30 post infection (pi). After infection,
a decrease in haemoglobin titres was almost immediately
noticeable in all three mouse strains (Fig. 2). This initial anaemia
reached a nadir by day 10 pi in A/J mice and haemoglobin titres
partially recovered thereafter. C57BL/6 mice never recovered and
developed severe anaemia. The haemoglobin titre in BALB/c
mice recovered even faster than the titre in A/J mice. Comparison
of the mean haemoglobin titres in the three mouse strains on day
17 pi in three different infection experiments (Table 1) confirmed
that C57BL/6 mice are the most susceptible to anaemia
development, while BALB/c mice are the most resistant. In a
previous evaluation of anaemia in A/J and C57BL/6 over a
shorter time period (18 days) we also found that A/J had higher
haemoglobin titres than C57BL/6 [29]. However, in that case A/J
had higher titres from the start and the differences remained
constant over the 18 days of the experiment. The difference in
timing and size of the relative haemoglobin levels between strains
between experiments may be due to the high variability in
haemoglobin levels (note the large error bars in figure 2) but in
both cases C57BL/6 developed a more severe anaemia than A/J.
Role of T lymphocytes in anaemia development
To find out whether T lymphocytes play a role in the
development of anaemia, haemoglobin levels were compared
between control and CsA-treated C57BL/6 mice. CsA is an
immunosuppressive agent that induces defective T cells. No
significant difference was observed in anaemia development
between the two groups of mice (Fig. 3).
Hepato-splenomegaly
Haematopoiesis normally occurs in the bone marrow. However,
in any situation in which the number of blood cells significantly
decreases and bone marrow cannot compensate for this loss alone,
haematopoiesis may occur in extramedullary (outside bone
marrow) sites including both liver and spleen [30,31].
To see whether there was a correlation between anaemia and
hepato-splenomegaly, the weights of liver, spleen and kidney were
monitored in all three mouse strains during the first 17 days of
infection. Weights of liver, spleen and kidney increased 1.9, 10.3
and 1.7 fold respectively over this period, and the change in
weight, both absolute and relative to body weight, was highly
significant in all cases (ANOVA p,0.001) (Figure 4a). The
increase in liver and spleen weights, but not kidney, was
significantly (p,0.001) higher in females than in males
Figure 1. Comparison of survival (A), parasitaemia (B) and % change in red blood cell numbers (C) between susceptible A/J mice
(red) and resistant C57BL/6 mice (green) after infection with a
T. congolense
IL1180, shown as mean6SD. The plots show that A/J mice
have a shorter survival time (mean 57 days) than C57BL/6 (mean 71 days), higher parasitaemia but less severe anaemia.
doi:10.1371/journal.pone.0005170.g001
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(Figure 4b). There was a significant difference between strains in
spleen weight (ANOVA p,= 0.005) pre-infection and at each
sampling day post infection except day 5. BALB/c had the highest
weight at all days; this may be associated with particularly high
haematopoietic potential in this organ in this strain. There were
also significant differences in liver, but not in kidney weight
between strains (p,0.05) at most time points. However, the
differences in weight were not large and may represent differences
in timing of responses as much as fundamental differences in
response. Total bodyweight increased slightly over the course of
the infection but by less than the total increase in organ weight.
This may reflect a loss of muscle mass and be a consequence of the
cachexia that is a well-known consequence of the disease.
Anaemia related metabolites
The measurement of serum iron was precluded by high levels of
haemolysis after infection. Ferritin levels did not differ significantly
between strains or over time, due to a very high variance.
However the mean values increased from day 0 to day 9 in all
three strains, as it can be expected in haemolytic anemia. By day
17 ferritin concentration was declining in A/J and BALB/c mice
but it increased further in C57BL/6 mice; all three strains showed
normal values at day 35. Transferrin levels increased in all strains
after infection (Fig 5a) and stayed relatively constant from day 3
(BALB/c) or day 9 (A/J and C57BL/6). The largest increase was
seen in A/J mice.
Genes regulating haematopoiesis
A large microarray gene expression data set was reviewed in
order to identify the role of haematopoietic genes in the
development of the differences in anaemia between the strains.
The primary regulator of normal erythropoiesis is erythropoi-
etin (EPO) expressed in the kidney. The erythropoietin gene was
not included in the Affymetrix array. However genes that respond
to erythropoietin were included; Ptp4a1 (Prl1) and Tnfrs11a (Rank)
have both been shown to respond to EPO and may further
propagate EPO signals [32]. Neither of these genes appeared to
respond to infection or to be differentially expressed, although
Ptp4a1 was highly expressed throughout the infection (not shown).
Other Epo responsive genes did appear to respond to infection:
Eif1a (Eukaryotic translation initiation factor) and Kif3a (kinesin
family member 3A) were up regulated in all three mouse strains at
day 7 and 9 respectively (Fig 6). However, since these genes
participate in multiple signal transduction pathways, their up-
regulation may be related to inflammation rather than erythro-
poiesis [32].
Erythropoietin primarily acts through the erythropoietin
receptor (Epor) that is exclusively expressed on cells from the
erythroid lineage. So the level of expression of Epor may be related
to the number of erythroid cells in the tissues. In uninfected mice,
Epor transcription was highest in A/J mice. After infection
C57BL/6 mice tended to have lower levels of expression than
either A/J or BALB/c mice although this was only significant if
the expression levels were compared over the whole time course
(p = 0.00003 with respect to BALB/c mice and p = 0.043 with
respect to A/J mice). This was consistent with lower erythropoiesis
and haemoglobin titre in C57BL/6 mice. However, given the
small difference in expression and the lack of evidence for changes
in Epo response genes this may not be an important mechanism
driving anaemia after T. congolense infection.
Interferon gamma (Ifng) down regulates Kit ligand (Kitl) and Epor
and may be an important contributor to anaemia of infection [33].
Kit (CD117) is a receptor for Kitl (SCF or Stem Cell Factor), which
acts synergistically with Epo in the promotion of erythropoeisis
[33]. Ifng expression increased approximately 8-fold after infection
in all mouse strains and then declined fastest in BALB/c and
remained highest in C57BL/6 consistent with the observed
haemoglobin titres (Fig 7). Consistent with the inhibitory activity
of IFN-c, the expression of Kit and Epor all declined somewhat at
Figure 2. Haemoglobin titres in C57BL/6 (green), A/J (red) and
BALB/c mice (blue) after infection with
T. congolense
, and
uninfected C57BL/6 mice (grey, broken line), shown as
mean6SD. Each point is an average of ten mice. Haemoglobin
declines rapidly in all mouse strains until the first peak of parasitaemia
after which it recovers to almost baseline levels in BALB/c mice, partially
recovers in A/J mice but continues to decline in C57BL/6 mice.
doi:10.1371/journal.pone.0005170.g002
Table 1. Mean relative haemoglobin titres (OD at 540 nm)
and standard deviation in blood from susceptible A/J,
intermediately susceptible BALB/c and more resistant C57BL/6
mice 17 days post-infection in three T. congolense infection
experiments (n = 10 per experiment).
Mouse strain A/J BALB/c C57BL/6
Infection 1 0.2360.13 0.3160.04 0.2160.06
Infection 2 0.2560.01 0.3360.06 0.2260.05
Infection 3 0.3960.13 0.4560.08 0.3260.03
Uninfected 0.5360.06 0.5360.05 0.5560.06
In each experiment, haemoglobin titres remained highest in BALB/c and fell to
the lowest level in C57BL/6.
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Figure 4. (A) Mean weights of internal organs relative to body weight during T. congolense infection in A/J mice (red), BALB/c mice (blue) and C57BL/
6 (green) mice, shown as mean6SD. The mean relative weights of liver, spleen and kidney increased 1.9, 10.3 and 1.7 fold over the course of the
infection (p,0.001). There were statistically significant (ANOVA p,0.05) differences in weight between strains at most time points but the largest and
perhaps biologically most significant difference was in the spleen where the relative weight in BALB/c mice increased 12 fold and in A/J and C57BL/6
mice it increased about 9.4 fold. (B) The increase in mean spleen and liver weights (6StErr) is higher in female (red, circles) than male (blue, squares)
mice (p,0.001).
doi:10.1371/journal.pone.0005170.g004
Figure 3. Mean haemoglobin titres in four C57BL/6 mice (green) and six CsA-treated C57BL/6 mice (magenta) after infection with
T.
congolense
, and four uninfected C57BL/6 mice (black, broken line), shown as mean6SD. CsA induces defective T cells. Since there was no
difference in anaemia after CsA treatment it is unlikely that T cells play a major role in the development of anaemia in C57BL/6 mice.
doi:10.1371/journal.pone.0005170.g003
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day 7 pi and C57BL/6 had the lowest levels of expression after day
three (Fig 7). Higher levels of Kitl may be indicative of higher levels
of erythropoiesis. In the spleen Kitl expression was highest in A/J
and lowest in C57BL/6 from day 3 to 9 (Fig 7).
Insulin like growth factor (Igf1) appears to be more important
than Epo for regulation of erythropoiesis in some anaemic patients
[34,35,36]. Expression of Igf1 declined in C57BL/6 mice till day 7
pi, while it increased in A/J mice and was more than twice as high
as in C57BL/6 on day 7 (Fig 7). However, differences at other
days were small and no correlations with anaemia could be made.
Igf2 is the predominant regulator of erythropoietin-independent
erythroid colony formation by neonatal progenitor cells and has
antiapoptotic effects. Its expression was approximately 2–4 fold
higher in A/J than C57BL/6 or BALB/c in the kidney, consistent
with better recovery of A/J but not BALB/c from the initial drop
in haematocrit.
Latexin has been found to be a negative regulator of the size of
the haematopoietic stem cell population in mice [37]. Latexin
expression increased in the liver of all strains by 40–100% at day
7–9 and decreased but remained above baseline levels at day 17
(not shown). The expression levels in the spleen were higher than
in the liver but did not vary.
The CD34 membrane antigen is specifically expressed by
activated haematopoietic stem cells [38]. Therefore, higher levels
of CD34 signify the presence of higher numbers of haematopoietic
stem cells, and could be an activity index for haematopoiesis.
CD34 was approximately 30% more highly expressed on day 9
post-infection in both liver and spleen of A/J in comparison with
C57BL/6 mice (not shown), suggesting higher levels of haemato-
poiesis in A/J.
The function of synuclein-alpha (Snca) in erythropoiesis is not
known, however an analysis of its expression in 71 tissues and cell
types showed that it is expressed at maximum levels in early
erythroid CD71 cells (reticulocytes) and in a separate analysis of
human reticulocytes Snca was found in the top twenty most highly
expressed genes [39,40]. Snca was one of the genes with the
Figure 5. Acute phase proteins and ferritin. Titres of ferritin (A) and transferrin (B) in plasma from T. congolense-infected A/J, BALB/c and C57BL/
6 mice, shown as mean6SD. (C) Expression of serum amyloid P (Apcs), the major murine acute phase protein, in the liver post infection.
doi:10.1371/journal.pone.0005170.g005
Figure 6. EPO responsive genes. Kif3a and Eif1a are EPO responsive genes but respond to inflammatory signals as well. Consequently their
positive response to infection may not be related to induction of erythropoiesis.
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greatest difference in mRNA abundance between C57BL/6 mice
and the other two mouse strains, A/J and BALB/c, which had 60–
250 fold higher expression of Snca than C57BL/6 mice in the
spleen at all time points (Fig 8b). In the liver Snca expression levels
were similar in all three strains until day 9 when the expression
was about two fold higher in A/J and BALB/c mice than in
C57BL/6 mice (Fig 8a). The transient increase in the liver could
have been caused by circulating reticulocytes in anaemic animals,
however the gross differences in expression in the spleen, even
prior to infection, are suggestive of substantial differences in
extramedullary haematopoiesis in the spleen.
Transcription factors regulating erythropoiesis
Tal1,Gata1,Lmo2,Ldb1,TcfE2a and Zfpm1 (Fog1) form a
multimeric DNA binding complex that regulates primitive
haematopoiesis [41]. All six genes were highly expressed, declined
in production in the spleen post infection and returned to near
baseline levels by day 17, with the exception of Ldb1,Zfpm1 and
Tcfe2a in C57BL/6 (Fig 9). The transcription factor EKLF (Klf1)is
involved in erythroid cell proliferation and has a similar expression
profile suggesting that it might be co-regulated with the other six
genes. C57BL/6 had lower levels of Tal1,Gata1,Zfpm1 and Kif1,
which are suggestive of lower levels of haematopoiesis in C57BL/6
particularly at later time-points. The similarity of their expression
profiles is suggestive of co-ordinate regulation, which is consistent
with the requirement for stoichiometric binding of the multimeric
complex. The expression of Gata2, which acts earlier in
erythropoiesis [41], did not change during infection and did not
differ between strains (not shown).
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 post infection but recovered by day 17 (Fig 10). In
Figure 7. Genes that mediate haematopoiesis. Igf1 appears to be an important regulator of erythropoiesis in some anaemic patients. Epor,Kit
and Kitl are regulators of erythropoiesis. Ifng down-regulates Epor and Kitl and the expression of the three genes was consistent with this relationship.
doi:10.1371/journal.pone.0005170.g007
Figure 8. Expression of
Snca
in (A) liver and (B) spleen. Snca is strongly associated with reticulocytes and was the gene with largest expression
difference that correlated with anaemia response. The strong expression of Snca in the spleen of A/J and BALB/c is suggestive of extra medullary
haematopoiesis in this organ in these strains.
doi:10.1371/journal.pone.0005170.g008
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each case C57BL/6 had the lowest level of transcription consistent
with relatively low levels of haematopoiesis. The expression of
these genes tightly followed that of the regulatory genes described
above (Fig 9) in both expression levels and in the decline from day
zero to day seven followed by the recovery of expression to day 17.
Haemoglobin-alpha (Hba-a1) was the most highly expressed gene
in the spleen from day 0 and its expression changed little over the
course of the infection but was 2–4 fold higher in BALB/c than A/
J or C57BL/6 in both liver and spleen respectively (Fig 10).
Haemoglobin beta expression was invariant and similar amongst
all strains (not shown).
Erythrocyte degradation
Biliveridin reductase a and b (BLVRA and BLVRB) and Heme
oxygenase (HMOX1) are involved in degradation of erythrocytes
and both Blvra and Blvrb expression increased 2–4 fold in the liver
by day 9 post infection (Fig 11). HMOX1 cleaves the heme ring to
form biliveridin which is reduced to bilirubin by BLVRA and
BLVRB and which is then excreted in the bile. Hmox1 expression
increased 16 fold between days 3 and 9 in the liver from all strains,
expression of all three genes declined by day 17. The expression of
erythrocyte degradation genes correlated with expression of the
pan-leukocyte antigens Cd45 (Ptprc) and Cd14, which is primarily
expressed on macrophages. Macrophages are the principal cells
that destroy erythrocytes and trypanosomes are also cleared from
the circulation by macrophages in the liver [42]. Cd45 and Cd14
expression in the liver was similar in all strains. Therefore although
these data are consistent with the substantial increase in
haemolysis after infection there is no evidence that different rates
of haemolysis or haemophagocytosis are the cause of the more
severe anaemia seen in C57BL/6. Furthermore expression of these
genes declined to near baseline levels by day 17 in all strains
suggesting that the more chronic anaemia of C57BL/6 is not a
consequence of continuing haemolysis.
Iron recycling by macrophages
Almost all iron for erythropoiesis is obtained by recycling
existing stores. Given the evidence for the large increase in
Figure 9. Transcription factors regulating erythropoiesis. Tal1,Gata1,Lmo2,Ldb1,TcfE2a and Zfpm1 (Fog1) form a multimeric DNA binding
complex, which regulates primitive haematopoiesis. All six genes were highly expressed and had similar patterns of expression consistent with co-
ordinate regulation. Klf1 is involved in erythroid cell proliferation and had similar levels and patterns of expression suggesting that it may be
regulated by the same mechanisms. In all cases C57BL/6 mice tended to have the lowest levels of expression after day 3.
doi:10.1371/journal.pone.0005170.g009
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haemolysis and haem breakdown in the liver there should be
evidence for a corresponding increase in iron recycling by liver
macrophages. Hepcidin is known as the master regulator of iron
storage in macrophages and its baseline levels are maintained by
BMP/SMAD [43]. However during inflammation hepcidin
appears to be primarily regulated by IL6 [44]. Hepcidin (HAMP)
inhibits the release of recycled iron from macrophages by binding
SLC40A1 (ferroportin) and targeting it for internalization and
Figure 10. Erythrocyte structural proteins. Expression of erythrocyte structural protein genes followed the expression of their transcription
factors (Fig 8) and C57BL/6 had lower expression levels than A/J or BALB/c.
doi:10.1371/journal.pone.0005170.g010
Figure 11. Erythrocyte degradation and leukocyte abundance. Blvra and Hmox1, which are involved in erythrocyte degradation, increased
dramatically after infection but then declined to near baseline by day 17. Cd14 and Cd45 (Ptprc) are markers of macrophage and leukocyte abundance
respectively. Macrophages are the main cells involved in haemolysis and it appears that expression of Blvra and Hmox1 was correlated with
macrophage abundance. C57BL/6 did not have consistently higher expression of any of these genes, suggesting that higher or more chronic
haemolysis is not the cause of the more chronic anaemia of this strain.
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degradation [45,46]. Expression of Slc40a1 is also known to be
repressed via a TLR4 mediated pathway after stimulation with
LPS and IFNG, suggesting that Slc40a1 expression is mediated by
both hepcidin-dependent and independent pathways and that the
latter may be more important in infections in which the TLR4
pathway is activated.
Il6 expression in the liver did not change (not shown). Hamp
expression increased slightly post infection in all strains before
declining to below baseline levels at day 17 (Fig. 12). Slc40a1
expression in the liver followed that of Hamp (Fig. 12) and declined
steadily in the spleen (Not shown). After export by SLC40A1, iron
is loaded onto transferrin by Hephaestin, the expression of which
remained constant until day 9 (Not shown). These data provide no
persuasive evidence for substantial change in iron recycling after
infection despite the evidence for a .10 fold increase in
macrophage numbers. The relatively steady state expression of
iron recycling genes compared with the large increase in
expression of macrophage associated genes suggests that iron
recycling by individual cells may have declined substantially.
Iron Uptake by macrophages
CD163 expression is a scavenger receptor for haptoglobin and
haem. Cd163 expression was high at day zero and then declined
.10 fold in both spleen and liver by day 3 to below the threshold
of detection (Fig 12). In contrast expression of Slc11a1 (Nramp1),
which is a transporter of divalent cations including Fe
++
, increased
about 20 fold between days three and seven (Fig. 12) following the
expression of the macrophage and leukocyte markers CD14 and
CD45 (Fig. 11). SLC11A1 is a macrophage protein and a metal
ion transporter. It has a role in macrophage defense against
microbial invasion and the increase in expression may be
correlated with macrophage activation to kill parasites by
oxidative stress as well as scavenging surplus Fe
++
to reduce its
abundance in the plasma.
Discussion
Development of anaemia and other infection parameters were
monitored during T. congolense infections in three inbred mouse
strains and associations were made between anaemia development
and gene expression profiles.
The characteristics of the anaemia in the mouse model used
here were very similar to the anaemia in trypanotolerant and
susceptible cattle and suggest that the causes of the anaemia are
similar in both species. First, the kinetics of the anaemia
development is similar in both species. The graph of haemoglobin
in the three mouse strains studied, indicates two phases of anaemia
development. The initial phase is characterized by a rapid decline
in haemoglobin titres in all three mouse strains up to around day
10 post-infection, the time of the first peak of parasitaemia. In the
second phase, haemoglobin levels continued to decrease in
C57BL/6 mice although at a slower rate, while they recovered
in A/J mice and even faster in BALB/c mice, accompanied by a
simultaneous increase of transferrin levels. These two phases were
also described in trypanosome infections in cattle, with the second
phase starting after control of the first wave of parasitaemia.
Stabilization and recovery of anaemia occurred in trypanotoler-
ant, but not in trypanosusceptible breeds [6]. Second, in both
species there is no evidence for a role of T cells in anaemia
development. The preliminary experiment with CsA, which
suppresses T lymphocyte functions, suggested that murine
anaemia is not T cell-mediated. A similar lack of response to T
cell suppression has been observed in cattle where depletion of
CD4 or CD8 T cells in both susceptible and trypanotolerant
breeds did not influence the severity of anaemia after T. congolense
infection [14,15]. Third, in cattle there is a degree of correlation
between severity of anaemia and death. This also seems to be the
case in C57BL/6 mice. Mortality started when RBC levels
dropped below 60% of normal values, suggesting that severe
anemia might be a contributory cause of death in this strain.
However there was no correlation between anaemia and survival
in the other two mouse strains. This has been observed previously
with different combinations of parasites and mice [19], indicating
that death in these strains is due to other causes, possibly related to
the high parasitaemia levels. Fourth, the capacities to control
parasitaemia and to limit anaemia in trypanotolerant cattle are the
result of two unrelated mechanisms [15] and data in this paper
and previous ones [17,19] suggest that this is also the case in the
Figure 12. Expression of genes involved in iron recycling in the liver. Hamp is a negative regulator of Slc40a1, which exports iron from
macrophages. Despite a decline in Hamp expression at day 17 there was no corresponding increase in Slc40a1.Cd163 and Slc11a1 are involved in
uptake by macrophages of haem and molecular iron from the plasma. Both responded strongly to infection but the increase in Slc11a1 may be for
acquisition of iron for generation of oxidative stress for parasite killing rather than iron recycling.
doi:10.1371/journal.pone.0005170.g012
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mouse model. C57BL/6 mice had the lowest parasitaemia, yet
developed the most severe anaemia, while A/J had high
parasitaemia, but had better anaemia control.
An interesting observation was that the ability of A/J and
BALB/c mice to recover from anaemia during infection was
correlated with spleen size. BALB/c had the largest spleens and
the highest expression of Hba-a1 and the most rapid recovery from
anaemia, while A/J had intermediate sized spleen and interme-
diate anaemia. The size of the spleen may correlate with
haematopoietic capacity and account for the particularly rapid
recovery of BALB/c mice. Spleen and liver size were significantly
larger in female than male mice in the three strains, and differed in
absolute size by about 20%. A study of twelve different mouse lines
infected with T. brucei found females survived significantly longer
than males in the seven lines with longest survival and that the
difference was not X-linked [47]. However, in a study of A/J and
C57BL/6 mice infected with T. congolense IL1180 [17], differences
between the sexes in survival were observed but the effect differed
in direction between strains and was not statistically significant
(Nakamura personal communication), so any effect of sex on
survival is likely to be small.
Several gene expression patterns measured in the arrays suggest
higher erythropoietic activity in BALB/c compared to C57BL/6.
Expression of Epo receptor, Kit and Kit ligand were consistently
lower in C57BL/6. So was the expression of a number of erythroid
structural proteins (spectrin and glycophorin), suggesting a lower
density of erythroid precursors in tissues of C57BL/6. Further,
expression of the transcription factors Gata1,Lmo2,Fog1 (Zfpm1)
and Klf1 were lower in C57BL/6 than A/J and BALB/c,
particularly at later time points, and the expression of Ldb1,Zfpm1
and Tcfe2a had not returned to baseline levels by day 17, consistent
with suppressed haematopoiesis in C57BL/6 mice in later stages.
It is interesting to note that the more severe chronic anaemia of
C57BL/6 mice also correlates with the lower number of
Cobblestone Area Forming Cells that are in S phase in the femur
of 7 day old C57BL/6 mice. These cells are a marker of the
abundance of haematopoietic stem cells and A/J and BALB/c
mice have approximately twice the numbers of them as C57BL/6
[48]. Consequently the more chronic anaemia of C57BL/6 mice
may be a consequence of reduced numbers of haematopoietic
stem cells and a more limited capacity to replace erythrocytes
destroyed by haemolysis. In cattle, the capacity to recover from
anaemia during an infection in genetically tolerant cattle, depends
on the genetic background of the haematopoietic tissue, but not on
that of lymphoid tissue, suggesting a role of erythropoietic
responses in trypanotolerance [3].
The first phase of the anaemia development was characterized by
an immediate and rapid decline in erythrocyte numbers in all three
mouse strains (and in cattle breeds [3,4,5,6]). Studies in infected
cattle hinted that this may be related to phagocytosis of erythrocytes
associated with the rising parasitaemia [7]. The 2–3 fold increase in
Biliveridin expression and 16 fold increase in haem oxygenase
expression that was observed between days 7–17 post-infection in
the three mouse strains are consistent with substantially increased
recycling of erythrocyte components in this period. Furthermore,
A/J mice are known to be deficient in the C5 (Hc
0
) component of
the complement cascade (http://jaxmice.jax.org/strain/000646.
html). This is the component that forms the multimeric membrane
attack complex that can destroy erythrocytes directly or more
commonly by inducing phagocytosis of erythrocytes by macro-
phages. This would be expected to make A/J mice more resistant to
the development of anaemia but since all strains developed anaemia
at a similar rate in our experiment, the complement cascade may
not be an important participant in this process.
The second phase of anaemia development differed markedly
between the three mouse strains (and in previous studies between
cattle breeds [3,4,5,6], implying that this mechanism is dependent
on genetic and host background. As discussed above, erythropoi-
esis and innate responses, but not acquired immune responses,
may play an important role in this. Comparison of gene expression
between the mouse strains could therefore give a clue as to which
metabolic pathways might be responsible for the different
phenotypes.
The lack of evidence for a response by Ptp4a1 and Tnfrs11a to
infection is consistent with previous reports of a blunted response
to erythropoietin in the anaemia of chronic disease [49]. Kif3a and
Eif3a have been shown to respond to EPO by increased
expression, however Kif3a participates in multiple signaling
pathways including Wnt and Sonic Hedghog [50]. Eif3a is
required for maximal protein synthesis and is not a specific EPO
response gene. Therefore although these genes have been found to
respond to EPO [32,51], the increase in expression may be due to
the inflammatory stimulus rather than any EPO specific function.
The transcription factors Tal1,Gata1,Lmo2 and Fog1 (Zfpm1) all
had lower expression in C57BL/6 than BALB/c consistent with
more severe anaemia in C57BL/6. However, these transcription
factors appeared to recover to preinfection levels by day 17 and
correlated better with Ifng expression than with haemoglobin titre,
so they may be responding to the acute phase inflammatory
response rather than haemoglobin.
Il6 has been proposed as a key link between inflammation and
anaemia via its activation of hepcidin (Hamp), which acts as a
negative regulator of intestinal iron absorption and macrophage
iron release [51,52]. Both Il6 and hepcidin expression increased
transiently and correlated with the transient anaemia observed in
A/J and BALB/c mice, however by day 17 post infection hepcidin
expression had declined to below pre-infection levels in all strains,
indicating that hepcidin was unlikely to be responsible for the
persistent anaemia in C57BL/6. Ferritin heavy chain Fth1
expression declined 2–3 fold at day 17 post infection and ferritin
abundance in the plasma returned to baseline by day 35 suggesting
that there may not be a substantial increase in iron storage in
ferritin in the chronic phase, however it is possible that iron is
sequestered in haemosiderin deposits. In cattle Il6 expression in
blood mononuclear cells increased in all cattle breeds, but earlier
in the susceptible than the tolerant breeds, suggesting its
expression was correlated with disease severity [53].
The dramatic reduction in Cd163 expression occurred before
any other inflammatory markers, except the serum amyloid genes,
had responded to infection. Cd163 is exclusively expressed on
monocytes and macrophages and is the scavenger for haem bound
to haptoglobin. The decline in expression is particularly striking
given the evidence for the substantial increase in macrophage
numbers. Plasma haptoglobin levels have been found to rise
rapidly in mice infected with T. congolense and this increase was the
most sensitive marker of infection [54], the very early decline in
CD163 expression could cause a reduction in haptoglobin uptake
and the observed increase in plasma concentration. LPS and
inflammatory cytokines such as IFN-care known to repress Cd163
expression whereas anti-inflammatory cytokines such as IL4 and
IL10 induce expression [55]. In the current study Cd163
expression appeared to be an exquisitely sensitive marker of
infection since expression declined .10 fold within three days of
infection when parasites are hard to detect by microscopy and
before any increase in expression of inflammatory cytokines. The
GPI-anchor of the surface coat glycoprotein of Trypanosoma cruzi
[51] and Trypanosoma brucei [56,57] have been reported to have
potent LPS-like properties, and the surface coat of T. congolense
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may have similar properties, so Cd163 expression may be
responding directly to parasite antigens. The down regulation of
CD163 would be expected to lead to an increase of haem-
haptoglobin complexes in the plasma, which has been observed
[54], and a concomitant increase in oxidative stress which would
have inflammatory and anti-parasitic effects [58]. In humans the
haptoglobin-related protein HPR has been implicated in the sterile
immunity of humans to T. brucei brucei by acting as a carrier for
APOL1 [59].
The 16-fold increase in Slc11a1 expression would be expected to
be associated with a large increase in uptake of molecular iron.
However Slc11a1 expression after infection may be correlated with
macrophage numbers and the control of oxidative stress rather
than iron storage. The increase in abundance of transferrin in the
plasma (Fig 5) in all strains but particularly A/J suggests that iron
recycling is not impaired by the infection although the extensive
haemolysis caused by the infection makes quantative studies
difficult.
Overall there appears to be increased uptake of erythrocytes for
degradation, reduced uptake of haptoglobin and decreased export
of iron from the liver. The massive phagocytosis of erythrocytes is
likely to exceed the available capacity of ferritin for storage and in
such circumstances iron is removed from circulation by deposition
as insoluble haemosiderin. The evidence for a large increase in
erythrocyte degradation but no concomitant increase in iron
export or ferritin production suggests that there may be a
substantial increase in iron in haemosiderin, which is insoluble,
and might restrict the availability of iron for erythropoiesis. An
analysis of anaemia in C57BL/6 mice infected with T. brucei
concluded that increased iron storage might be a cause of the
chronic anaemia in that model [16]. However in the present study
the expression of Hamp declined in all strains by day 17, which
would be expected to permit an increase in iron recycling. Further
studies are required to determine whether iron stored as insoluble
haemosiderin is restricting availability for erythropoiesis as
previously proposed [16] or if massive haemolysis and phagocy-
tosis combined with reduced erythropoiesis is causing iron to be
stored until required.
The strong association between chronic anaemia and inflam-
mation would suggest that C57BL/6 mice might be maintaining a
more persistent inflammatory state. However the relative resis-
tance of C57BL/6 mice to T. congolense infection appears to be
associated with the ability to switch from an initial Type 1 cytokine
response (IFN-c, TNF) to Type-2 cytokine production (first IL10,
followed by IL4 and IL13). In contrast a continuing Type-1
cytokine response or an early mixed Type-1/Type-2/regulatory
cytokine production confers susceptibility to trypanosome infec-
tions [60]. Consequently although it is quite likely that
inflammation plays a role in the anaemia of C57BL/6 mice it
seems unlikely that differences in inflammatory state, as measured
by cytokine activity, can adequately account for the differences in
anaemia after T. congolense infection.
Conclusions
The data presented here showed that although A/J, BALB/c
and C57BL/6 all developed anaemia in response to infection with
Trypanosoma congolense, A/J and BALB/c mice were able to control
the anaemia whilst C57BL/6 were not. Multiple genes involved in
erythropoiesis responded to infection and correlated with the
expression of genes that are markers of inflammation. The
expression levels of almost all these genes returned to near baseline
levels by day 17 post infection. This was consistent with the
anaemia of A/J and BALB/c being regulated by the inflammatory
process. The innate immune response may have been the major
contributor to the inflammation associated with anaemia in these
mouse strains since suppression of T cells with CsA had no
observable effect. However the anaemia of C57BL/6 persisted
long after the initial inflammatory stimulus possibly as a
consequence of a more persistent inflammatory state in these
mice. The transcription factors Tal1,Gata1,Zfpm1 and Klf1 all
tended to be expressed at consistently lower levels in C57BL/6.
The lowered expression of these genes may provide a valuable
molecular marker for chronic anaemia and may be correlated with
the small increase in spleen size and hence haematopoietic
potential in C57BL/6 relative to A/J and BALB/c and may best
account for the differences in anaemia in these mice.
Acknowledgments
The authors would like to thank Percy Ritho Mukuthu and Joseph Nthale
of ILRI and Leanne Wardlesworth and Leo Zeef of the University of
Manchester Core Services unit as well as Elfi Holupirek of the German
Mouse Clinic for excellent technical assistance. We thank Sonal Nagda of
ILRI for assistance with statistics and Kate Goodheart of the University of
Liverpool for assistance with preparation of the figures.
Author Contributions
Conceived and designed the experiments: HN AB HF VGD SK JN.
Performed the experiments: MHA MA FI BR JN. Analyzed the data: HN
AB HF HH BR EW MHdA DR JN. Wrote the paper: HN BR JN. Study
design: HN HF VGD SK. Supervised challenges: HN. Drafted manuscript:
HN. In vitro assays: MHA. Reviewed paper: MHA. Mouse challenges: MA
FI. Laboratory supervision: HF. Revised manuscript: HF BR DR.
Logistics: VGD. Primary analysis of microarray data: HH. Clinical
chemistry data collection and interpretation: BR. Analysis and interpre-
tation of clinical chemical results: EW. Result interpretation: MHdA. Data
interpretation: DR.
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Anaemia of Trypanosomiasis
PLoS ONE | www.plosone.org 13 April 2009 | Volume 4 | Issue 4 | e5170