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Hepcidin and Trypanosoma brucei infection
cellular products, such as hemolysins,10,11 direct mechanical erythrocyte injury,12 lipid peroxidation,13-15 and extracellu- lar vesicles that can fuse with erythrocytes resulting in rapid clearance and anemia.16 Furthermore, although there are clear indications that iron metabolism has a significant role in the establishment of anemia during trypanosomal infections,17,18 the overall molecular mechanisms that lead to it are still poorly understood, and this includes, in par- ticular, the involvement of hepcidin.
Hepcidin is a small antimicrobial peptide and a key reg- ulator of iron metabolism.19-21 During infectious/inflamma- tory processes, hepcidin leads to a systemic decrease in iron mobilization by blocking iron release from hepato- cytes, enterocytes and macrophages. This impacts the proliferation of the pathogens but also affects the host by impairing erythropoiesis. The scarcity of iron and the sub- sequent impairment of erythropoiesis are thought to lead to a condition known as anemia of inflammation. This mechanism of response has been established for several bacterial infections22-26 and some intracellular parasites.27,28 However, studies of hepcidin involvement in the develop- ment of anemia in infections with extracellular parasites are extremely limited.
The present study was undertaken to determine the possible role of hepcidin in the regulation of iron metabo- lism during trypanosomal infections and its contribution to the onset, development and recovery from anemia.
Methods
Mice, parasites and infections
Five-week-old C57BL/6 and BALB/c female mice were pur- chased from Charles River Laboratories (Saint-Germain-Nuelles, France). Female hepcidin knockout (Hamp-/-) mice29 were bred at the institute facilities. The Trypanosoma brucei brucei, GVR35 strain, was used to infect the mice. All experiments were carried out in accordance with the IBMC.INEB Animal Ethics Committees and the Portuguese National Authorities for Animal Health guidelines according to the statements on the directive 2010/63/EU of the European Parliament and Council.
Hematologic and serum parameters, tissue iron content
Hematologic and serum parameters were blindly determined by a certified laboratory (CoreLab, Centro Hospitalar do Porto, Portugal). Liver and spleen iron content was evaluated by the bathophenanthroline method30 and Perls staining.
Cytokine profile analysis
Cytokine levels were measured in the serum using the BD CBA Mouse Inflammation Kit (BD Biosciences, San Jose, CA, USA).
Flow cytometry
Bone marrow (BM) cells were stained with anti-CD3e (17A2), anti-CD19 (6D5), anti-TER119, anti-CD71 (RI7217), anti-CD11c (N418), and anti-CD11b (M1/70) antibodies, and run in a BD FACSCanto II Flow Cytometer (BD Biosciences). Data were ana- lyzed with FlowJo software (FlowJo LCC, Ashland, OR, USA).
RNA isolation and cDNA synthesis
Total RNA was isolated from tissues and cells with the PureLink RNA Mini Kit (Thermo Fisher Scientific) and converted to cDNA using the NZY First-Strand cDNA Synthesis Kit (NZYTech, Lisbon, Portugal).
Analysis of gene expression by quantitative-polymerase chain reaction
Relative levels of several genes mRNAs were quantified in rele- vant organs of control and infected animals, by quantitative-poly- merase chain reaction (qPCR). The comparative CT method (2-ΔΔCT method) was used to analyze gene expression levels.
Analysis of ferroportin levels by Western blot
Levels of FPN1 protein were evaluated in the liver, spleen and duodenum of C57BL/6 and Hamp-/- mice by Western blot, with GAPDH being used as housekeeping protein. Primary antibodies used were rabbit anti-Ferroportin/SLC40A1 (Novus, Littletown, CO, USA; catalog #NPB1-21502), (1:1000) 1 hour (h) RT, rabbit anti-GAPDH (Abcam, Cambridge, UK, catalog #EPR16891), (1:1000) 1 h RT.
Statistical analysis
Statistical analysis was carried out using GraphPad Prism 8 (GraphPad Software Inc., La Jolla, CA, USA). Multiple compar- isons were performed with one-way ANOVA and post hoc Student Newman-Keuls test. P<0.05 was considered statistically signifi- cant.
Further details of the study methods are available in the Online Supplementary Appendix.
Results
T.b. brucei infection in mice leads to macrocytic anemia, decreased erythropoietic activity and iron redistribution
The course of the infection with T.b. brucei GVR35 strain expressing luciferase was followed in BALB/c mice by bio- luminescence imaging and counting the parasites in the blood (Online Supplementary Figure S1). A significant reduc- tion in the number of red blood cells (RBC), reticulocytes, hematocrit and hemoglobin levels was observed up to day 7, with a gradual return to normal levels (Figure 1A-D), indicating an early onset of acute anemia, followed by a later recovery. A significant increase in the mean corpus- cular volume (MCV) was also observed, indicative of macrocytic anemia (Figure 1E). Alterations in the develop- ment of erythroid lineage in the BM were evaluated by flow cytometry. Overall, a decreased total number of mature and developing erythrocytes was observed (Figure 1F), as well as decreased numbers of pro-erythroblasts (Figure 1G), basophilic erythroblasts (Figure 1H), and polychromatic erythroblasts (Figure 1, panel I).
Trypanosomal infection also caused a significant decrease in circulating serum iron levels (Figure 1J), trans- ferrin saturation (Figure 1K), and increased total iron bind- ing capacity (TIBC) (Figure 1L) and serum ferritin (Figure 1M), highlighting the inflammatory status of the animals and thus, indirectly, a condition of anemia of inflamma- tion. The lower systemic iron concentration was accom- panied by the accumulation of iron in the liver (Figure 1N) and spleen (Figure 1, panel O).
Circulating cytokine levels indicate the development of an acute infection
We evaluated the impact of T.b. brucei infection in the expression of several inflammatory cytokines. IL-6 levels were elevated as early as 1 day post infection and remained high up to day 7, with a gradual recovery to con- trol levels (Figure 2A). IL-6 not only acts as a pro-inflam-
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