Page 75 - 2021_02-Haematologica-web

Low-dose erythropoietin induces erythroferrone
(<20%), did not change (Figure 4C). Consistent with the low Tfsat, hepcidin concentration at sea level was below the detection limit in 15 subjects and was unchanged after exposure to high altitude in the subjects with detectable baseline values (Figure 4D). Lack of correlation between ERFE and hepcidin was previously found in patients with chronic kidney disease, in whom increased ERFE levels were not accompanied by lower hepcidin.9 In line with a previous report showing unaltered iron availability and no signs of inflammation at high altitude,15 the inflammatory marker interleukin-6 was not affected by the subjects’ exposure to high altitude (1.28 ± 1.04 pg/mL vs. 1.25 ± 0.9 pg/mL at sea level), whereas the concentration of ferritin increased slightly (from 125 ± 8 to 132 ± 7 ng/mL), although remaining within the normal range (Figure 4E).
Discussion
Recently, mouse studies have shown that acute rhEpo treatment downregulates hepcidin in an ERFE-indepen- dent manner by decreasing serum iron and Tfsat.16,17 Moreover, the demonstration that rhEpo administration also downregulates hepcidin in mice lacking ERFE18 sug- gests that prolonged erythropoietic stimulation inhibits hepcidin expression in mice by depleting iron stores, whereas ERFE represents an acute regulator of stress ery- thropoiesis.18 Conversely, the present results show that in healthy humans ERFE responds even to low Epo levels which are not associated with an expansion of Hbmass, a functional marker of erythropoietic response.19 This con- clusion is also supported by our findings in a physiological condition such as high-altitude hypoxia (Figure 4). Furthermore, our data showing no alterations in Tfsat and a progressive decrease in ferritin with repeated rhEpo injections are consistent with the view that ERFE may inhibit hepcidin transcription directly in the absence of changes in serum and liver iron.7 In fact, the ERFE-hep- cidin axis was affected early, i.e. 24 h after the first rhEpo injection, while other serum iron parameters were unchanged at that time, as t test analysis showed no dif- ference in ferritin between groups at 24 and 48 h after the first injection (see Online Supplementary Material, Statistical analysis section). However, it is well conceivable that under conditions of strong erythropoietic stimulation, such as in mice treated with high doses of rhEpo (8000 IU/kg),16-18 increased iron consumption for erythropoiesis leads to iron depletion and repression of hepcidin.
The introduction of the Athlete’s Blood Passport20 has improved the detection of blood doping, although the Passport does have several limitations,21 in particular in detecting micro-dose rhEpo doping.22 A study in which
ERFE was measured in six subjects receiving relatively high doses of rhEpo or analogs, intravenously or subcuta- neously, using an assay different from the one used in this study did not suggest that ERFE would be a reliable mark- er for rhEpo doping,23 although (while our manuscript was under revision) the same group reported that a different enzyme-linked immunosorbent assay was able to detect increased ERFE levels in the same samples.24 Conversely, increased erythropoiesis induced by training did not affect ERFE and hepcidin levels in runners.25 Our results demon- strate that ERFE is sensitive enough to flag even micro- dose rhEpo, correlates with Epo levels (Figure 3A, B) and has a detection window longer than that of Epo, thereby indicating that ERFE holds promise as a novel biomarker of doping for implementation in the ABP, although addi- tional studies are required. In view of our results, ferritin or hepcidin could also be considered as potential biomark- ers of the use of micro-dose rhEpo, although both factors may be confounded by iron supplementation, a legal prac- tice commonly used by athletes.
In summary, the present results demonstrate that in healthy humans ERFE is promptly enhanced in response to moderately increased Epo levels and represses hepcidin in an iron-independent way. Assaying ERFE levels may provide additional analytical support for the fight against doping.
Disclosures
No conflicts of interests to disclose.
Contributions
PR designed and coordinated the project, planned and per- formed experiments and co-wrote the manuscript. EG collected and interpreted data and co-wrote the manuscript. SR, DG and AC collected and analyzed data. MR analyzed data and per- formed the statistical analysis, A-K L, PBa, PBo, SV, GS and MU collected data, CL analyzed data and revised the manu- script, CC provided clinical support, PS designed and coordinat- ed the project, discussed the results and co-wrote the manuscript. GC conceived and coordinated the study, interpreted data and co- wrote the manuscript. All authors discussed the results and com- mented on the manuscript.
Acknowledgments
The authors thank Dr. Catherine Mercier (Hospices Civils de Lyon) for expert statistical advice, as well as Dr. Jean-Pierre Herry, Dr. Alice Gavet and Laura Oberholzer for their assistance during the protocol. The work involving rhEpo administration was supported by a grant from Partnership for Clean Competition to GC. The altitude study was supported by the Fédération Française des Clubs Alpins et de Montagne and Fondation Petzl through grants to PBo.
References
1.Ganz T. Erythropoietic regulators of iron metabolism. Free Radic Biol Med. 2019;133:69-74.
2.Camaschella C, Pagani A. Advances in understanding iron metabolism and its crosstalk with erythropoiesis. Br J Haematol. 2018;182(4):481-494.
3.Robach P, Recalcati S, Girelli D, et al. Alterations of systemic and muscle iron
metabolism in human subjects treated with low-dose recombinant erythropoietin. Blood. 2009;113(26):6707-6715.
4.Ashby DR, Gale DP, Busbridge M, et al. Erythropoietin administration in humans causes a marked and prolonged reduction in circulating hepcidin. Haematologica. 2010;95(3):505-508.
5. Robach P, Recalcati S, Girelli D, et al. Serum hepcidin levels and muscle iron proteins in humans injected with low- or high-dose erythropoietin. Eur J Haematol. 2013;91
(1):74-84.
6.Kautz L, Jung G, Valore EV, Rivella S,
Nemeth E, Ganz T. Identification of ery- throferrone as an erythroid regulator of iron metabolism. Nat Genet. 2014;46(7): 678-684.
7.Arezes J, Foy N, McHugh K, et al. Erythroferrone inhibits the induction of hepcidin by BMP6. Blood. 2018;132(14): 1473-1477.
8.Ganz T, Jung G, Naeim A, et al. Immunoassay for human serum erythrofer-
haematologica | 2021; 106(2)
389