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N.U. Stoffel et al.
non-anemic), serum IL-6 increased within 3 h after injec- tion, and urinary hepcidin peaked at 6 h, followed by sig- nificant hypoferremia.10 In a study in Gambian newborns, routine immunizations at birth did not affect serum IL-6 or SHep at 72-96 h post vaccination, but the inflammation- hepcidin axis was already activated, likely due to the birth process.18 However, these studies did not compare respons- es between anemic and non-anemic subjects or measure effects on iron absorption.
In our study, vaccination induced a rapid and sustained inflammatory response reflected in an approximately 2- to 3-fold increase in IL-6 apparent at 8 h after vaccination and persisting at 36 h in both the IDA and non-anemic groups (Figure 2A). Despite this, in the women with IDA, SHep did not significantly increase (Figure 2A and B). Several factors likely contributed to this effect. High circulating diferric transferrin and high liver iron stores increase hepatic hep- cidin synthesis via stimulation of the bone morphogenic protein [sons of mothers against decapentaplegic (BMP- SMAD) pathway].1 EPO, the main driver of erythropoiesis, stimulates a hepcidin-suppressing factor synthesized in the bone marrow; this factor may be erythroferrone. In a recent study in mice, erythroferrone suppressed hepcidin by inhibiting hepatic BMP/SMAD signaling through BMP5, BMP6, and BMP7.19 However, the role of erythroferrone during IDA remains uncertain.20 In the IDA group, BMP- SMAD signaling was likely suppressed by low TSAT, depleted liver iron stores, and high erythropoietic drive, as indicated by high EPO and sTfR concentrations (Table 1). Our data suggest that moderate IL-6 stimulation of the Janus kinase/ signal transducer and activator of transcrip- tion (JAK/STAT) and BMP-SMAD pathway was unable to overcome this suppression, and SHep remained low. In contrast, in the non-anemic group, BMP-SMAD signaling was not suppressed, and, as a result, IL-6 activation resulted in a rapid increase in SHep. These data suggest that, in mild IDA, low iron status and erythropoietic drive can keep SHep low even in the face of an acute inflammatory stimu- lus. In addition to the suppression of erythropoiesis by iron restriction through hepcidin, cytokines may directly affect erythropoiesis.21 In both the IDA and the non-anemic groups, erythropoiesis appeared to be mildly suppressed 24 h after vaccination, as indicated by a vaccination effect to decrease sTfR and increase EPO (Table 3).
In animal studies, inflammation is a strong inducer of hepcidin, but its effects can be blunted by iron deficiency and/or enhanced erythropoiesis.3-5 In mice, erythropoietic drive down-regulated hepcidin even during inflammation induced by LPS injection.3 Ferroportin transcription in macrophages may be attenuated by inflammation inde- pendent of hepcidin,22,23 but activation of Nrf2 reverses this attenuation,24 suggesting that iron may dominate over inflammatory stimuli. Conversely, other studies suggest erythroid and inflammatory regulators dominate over iron stores: iron-deficient mice injected with LPS up-regulated hepcidin expression,2 while iron-loaded mice with experi- mentally induced anemia down-regulated hepcidin expres- sion.25
Studies in humans with the anemia of chronic inflamma- tion (ACD) and/or IDA suggested erythroid demand for iron is a stronger regulator of hepcidin expression than mild inflammation: SHep was similar between ACD/IDA and IDA, but was higher in ACD; duodenal ferroportin expres- sion was inversely related to SHep and SF, but not to IL-6; and IL-6 levels were similar between ACD (with high
SHep) and ACD/IDA subjects (with low Shep).4 In African children, Hb and SF were positively associated with hep- cidin while IL-6 levels were not.26 In another study in African children, erythropoietic drive (sTfR) was a much stronger negative predictor of SHep than inflammation.27 The differing results of these studies suggest that either inflammation or IDA can be the dominant factor regulating hepcidin, depending on the varying strengths of the oppos- ing stimuli.
In our study, there was a significant and sustained increase in SHep after vaccination in the non-anemic group, with median SHep (nM) more than twice baseline values at 8, 24 and 36 h after vaccination. We anticipated this would decrease erythrocyte iron incorporation because in our pre- vious studies, acute SHep increases of similar magnitude and duration (approx. 1-2 nM) in non-anemic women after the administration of high oral iron doses reduced iron absorption by approximately 40%.28,29 Contrary to our hypothesis, in the present study, there was no significant change in erythrocyte iron incorporation from a labeled test meal given at 24 h after vaccination, at the peak of the SHep increase, although the women developed mild hypofer- remia. Several mechanisms may explain this effect. Hepcidin promotes rapid degradation of ferroportin in liver cells and macrophages reducing iron recycling and serum iron,30 but enterocyte ferroportin may be less sensitive to acute changes in hepcidin.31-34 Another potential explanation why high SHep did not effect iron absorption in our study is that inflammation also induced hypoferremia, and the reduction in circulating diferric transferrin was sensed by the enterocyte through TfR1, leading to changes within the enterocyte35,36 that induced divalent metal transporter 1 (DMT-1) and ferroportin expression. This may have offset ferroportin degradation by SHep, and allowed continued iron export from the enterocyte. This is supported by our findings that, at 24 h after vaccination, serum iron was a sig- nificant predictor of erythrocyte iron incorporation in the non-anemic group, but not in the IDA group.
The strengths of this study are: 1) we prospectively stud- ied the effects of a standardized (and safe) inflammatory stimulus in both non-anemic women and women with IDA; 2) our subjects were young women who were other- wise healthy and free of potential confounding comorbidi- ties; 3) we precisely quantified erythrocyte iron incorpora- tion (absorption and utilization) using iron stable isotopic labels. Limitation of the study are: 1) we included women in the IDA group who were only mildly anemic, and we induced only a moderate acute inflammatory state. More severe, chronic inflammation and/or anemia may have resulted in differing effects; 2) using the stable iron isotope method we measured erythrocyte iron incorporation, which reflects both iron absorption by enterocytes and iron utilization for production of erythrocytes in the bone marrow, and we could not differentiate between these. Finally, we were unable to distinguish whether the lack of SHep increase in response to the inflammatory stimulus in the women with IDA was due to the effect of erythropoi- etic drive, iron deficiency or both.
To our knowledge, this is the first human experimental study showing that erythrocyte iron incorporation (absorption and utilization of dietary iron) is not reduced in non-anemic subjects by an acute increase in SHep that induces hypoferremia. This finding suggests the enterocyte may be less sensitive to the effect of acute changes in SHep than macrophage iron recycling. This pattern of regulation
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haematologica | 2019; 104(6)