Erythroid differentiation Identification of gene networks associated with anaemia caused by Trypanosoma congolense 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 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. 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. 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. 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. FERROPORTIN TRANSFERRIN Tfr1 Tfr2 NRAMP CD163 Fe2+ Transferrin Hemoglobin Haptoglobin Ferritin Haemosiderin C57 Hepcidin IL6 Iron recycling through macrophages 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 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. Mean weights of internal organs during T. congolense infection in A/J mice (triangles), BALB/c (squares) and C57BL/6J (circles) mice. 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. Erythroid differentiation Factors 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. Tal1 GATA1 Lmo2 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. ZFPM1 KLF1 Blvra Hmox1 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. 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. SPNA1 GYPA EpoR KIT Epb7.2 SPNB1