Involvement of galectin-3 in inflammation-associated immunopathology elicited during African trypanosomiasis

African trypanosomiasis is a disease which affects both humans as well as animals in sub-Saharan Africa and which causes severe economical losses in this continent. It is caused by the extracellular, hemoflagellated parasite, called trypanosome, which is transmitted to a mammalian host through the bite of an infected tsetse fly. Trypanosome infections are typically characterized by a pro-inflammatory immune response during the early stage of infection, needed to control the parasitemia. Yet, persistence of this type of immune response during the later stages of infection leads to severe immunopathology, whereby anemia is considered to be the most prominent parameter. Using experimental trypanosome infection (T. brucei brucei) in mice it was found that this can be used as a model system to study inflammation-associated pathology. In addition, we have shown that the anemia associated with these infections resembles anemia of inflammation, also known as anemia of chronic disease. We have observed that under the inflammatory conditions caused by the infection, iron is mainly withheld inside the cells, leading to a decreased bio-availability and thus leading to anemia development. Interestingly, it was shown that by reducing this inflammatory environment, the iron modulation was restored and anemia alleviated. These results indicated that the study of key regulators of inflammation could be important to reduce inflammation-associated anemia. Hereby, galectin-3 could be a potential candidate. From literature it was known that galectin-3 is involved in the regulation of inflammation. Furthermore, previous experiments in the lab have shown that there is a strong upregulation of galectin-3 during trypanosome infections. Using galectin-3 deficient mice (Gal3-/-, C57BL/6 background) to study the role of galectin-3 in our model it could be concluded that these mice had a reduced inflammatory immune response (lower serum TNF-? and higher IL10 levels), less severe anemia and survived twice as long as compared to the Gal3+/+ mice. In addition, these mice exhibited unaffected serum iron levels during infection, less liver iron-accumulation and less liver damage. Given that the liver-associated macrophages, i.e. Kupffer cells, play a key role in iron regulation, we focused in a second part of the work on these cells. Therefore, an isolation protocol was optimized based on the surface expression of CD11b and F4/80 on these cells. The identity of the isolated cells was confirmed by using known markers for Kupffer cells from literature, such as CRIg and CD68. Furthermore, we compared the gen-expression levels of genes involved in the iron metabolism as well as several chemokines between Gal3-/- and Gal3+/+ mice. Hereby, we found that the differences in iron metabolism-related genes observed in the total liver samples, was not due to the Kupffer cells. On the other hand, it was observed that Gal3+/+ Kupffer cells had a higher expression of chemokines than the Gal3-/- mice. This might be related to the less pronounced pro-inflammatory environment in the livers of the Gal3-/- mice. Furthermore, we could also discriminate subpopulations in the isolated Kupffer cells, which are different between infected Gal3+/+ and Gal3-/- mice. This suggests that Kupffer cells might be differentially regulated. In a last part of the work, whereby we aimed at neutralizing extracellular galectin-3, we generated anti-galectin-3 VHHs. From experiments with a polyclonal anti-galectin-3 antibody, we knew that this affects mainly the survival, but not the anemia. The VHHs had a low affinity and in an attempt to increase this (through avidity effects), we chose two binders a