Mechanisms controlling anaemia in Trypanosoma congolense infected mice

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.

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 mean±SD.

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.

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 mean±SD.

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.

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 mean±SD.

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.

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 mean±SD. 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 (±StErr) is higher in female (red, circles) than male (blue, squares) mice (p<0.001).

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 mean±SD. (C) Expression of serum amyloid P (Apcs), the major murine acute phase protein, in the liver post infection.

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.

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.

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.

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.

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.

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.

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.