Page 170 - 2021_03-Haematologica-web

J.V. Neves et al.
and by others.36 Additionally, there is an increase in iron storage both in the liver, the major organ for iron accumu- lation, and the spleen, where iron recycling from senes- cent erythrocytes occurs. However, this redistribution of iron with the goal of limiting its mobilization and avail- ability to pathogens is actually a double-edged sword; at the same time potentially limiting iron availability for ery- thropoiesis in the BM and leading to the condition known as anemia of inflammation,37,38 thus contributing to the overall trypanosome-related anemia.
On the inflammatory side, it has been well documented that the response to infectious/inflammatory stimuli involves the expression of numerous pro- and anti-inflam- matory cytokines that have various effects on different leukocyte populations, from lymphocytes to macrophages, with the latter also being involved in the modulation of iron homeostasis. We evaluated the levels of some relevant circulating inflammatory cytokines, where we observed a strong type I cytokine response in all models, with increases in the levels of IL-6, IFN-g and TNF-a. IL-6, which is mostly produced by macrophages but also by Th2 T cells in response to the extracellular par- asites, is a major inducer of hepcidin expression by the liver during inflammatory processes.37,40,41 In trypanosomi- asis, increased levels of IFN-g can inhibit BM proliferation and suppress erythropoiesis,42 whereas TNF-a is known to be a key mediator involved in parasitemia control but can also contribute to enhanced erythrophagocytosis.43,44 Furthermore, these cytokines favor the maturation of naïve T cells into Th1 T cells, which are involved in cell- mediated immunity. We also observed extremely high lev- els of MCP1, a chemokine that plays an important role in monocyte recruitment.45 Contrary to other protozoan infections, such as those from Leishmania major,46 Toxoplasma gondii47 or Plasmodium chabaudi,48 where this recruitment is essential for the effective control of the infection, in T. brucei infections expression of MCP1 and other chemokines seem to have deleterious effects, espe- cially during early infection, contributing to enhanced pathogenesis.49,50 However, such effects might be mitigat- ed by the production of the type II cytokine IL-10, which potentially limits MCP1 expression and reduces monocyte recruitment from the BM.51 IL-10 is also known to down- regulate IFN-g and TNF-a, and, depending on the balance between these cytokines, it may contribute to attenuate the severity of the anemia.52
During the development of the immune response to var- ious pathogens, hepcidin is known to be key in the regu- lation of iron metabolism, leading to reduced mobilization and redistribution of iron in order to limit its access by pathogens, and in turn, to the so-called anemia of inflam- mation.37,38 However, there are cases where iron redistrib- ution and anemia occur but by mechanisms that are hep- cidin-independent.22 As such, we investigated the possible role of hepcidin in the development of trypanosome-relat- ed anemia and further looked into the molecular mecha- nisms subjacent to the transition from a status of acute anemia to a status of recovery/chronic anemia.
Increases in hepcidin expression were observed in BALB/c and C57BL/6 mice, with no discernible expression in Hamp-/- mice. The liver is long known to be the major contributor for systemic hepcidin levels, and thus the mas- ter regulator of iron homeostasis. In response to an infec- tious/inflammatory stimulus, an increased expression of hepcidin is triggered in the liver, mostly mediated by IL-6.
Hepcidin then binds to ferroportin, leading to its internal- ization and degradation, effectively blocking iron release from hepatocytes, intestinal enterocytes and macrophages.19-21,37,53 In prolonged infections, this limits iron availability for the pathogens, but also for the host itself, thus leading to the aforementioned anemia of inflammation. However, since there is no hepcidin in Hamp-/- mice, there is no limitation in iron availability, so the milder anemia observed in these animals is likely mediated by hepcidin-independent mechanisms, which is not always required for the onset of early inflammatory hypoferremia.54,55
The increased hepcidin expression in the spleen, kidney and BM is expected to have a low impact on systemic iron homeostasis, but may have an important role in the con- trol of local iron fluxes. As with the hematologic parame- ters, there is a turning point at around day 7 when the infectious stimulus that leads to increased hepcidin expression seems to be replaced by an inhibitory signal that suppresses hepcidin. This could partially be explained by a decrease in IL-6 levels, but there are likely other sig- naling pathways contributing to this suppression. As such, we also evaluated the expression of genes that are influ- enced by hepcidin or, in turn, influence hepcidin expres- sion.
Ferroportin is the major target for hepcidin, being removed from the cell surface and also inhibited at the expression level.21,56 As the sole known iron exporter, this interaction will severely limit iron release and mobiliza- tion, especially by the intestinal enterocytes, recycling macrophages and hepatocytes, leading to hypoferremia. In both BALB/c and C57BL/6 mice, ferroportin expression correlates both negatively with hepcidin expression and positively with the development of anemia, being down- regulated at the earlier days of infection. This limits iron release for the production of new erythrocytes and leads to anemia. It is subsequently up-regulated over the follow- ing days when iron is again being released and enhanced erythropoiesis occurs, allowing a recovery from anemia. However, in Hamp-/- mice there is no such control of ferro- portin due to the lack of hepcidin, so iron is readily avail- able to allow for the faster recovery from anemia observed in these animals. A similar regulation of ferroportin was observed at the protein level. Levels in the liver of C57BL/6 mice closely matched variations in mRNA expression, and also mirrored hepcidin expression, with a decrease up to day 7. This is then followed by a recovery and an increase over subsequent days, indicating an early iron retention and a later release from the liver. In Hamp-/- mice, liver FPN1 levels also closely matched mRNA expression, and were kept elevated throughout the exper- iment, with the zenith at day 7. These results show that there was no limitation in iron release from the liver dur- ing infection, thus supporting the hypothesis of a faster erythrocyte recovery when compared with C57BL/6 mice. In the spleen, a similar response was observed for both C57BL/6 and Hamp-/-, with a very significant decrease in ferroportin levels, followed by a later increase in the second stage of infection, albeit faster and higher in the Hamp-/- mice. During anemia of inflammation, after erythrophagocytosis, iron is not properly released from macrophages due to ferroportin internalization mediated by hepcidin (hence the development of anemia). But at a later stage, during recovery, ferroportin levels are normal- ized, iron mobilized and erythropoiesis also normalizes,
816
haematologica | 2021; 106(3)