Involvement of hepcidin in iron metabolism dysregulation in Gaucher disease

Abstract

Gaucher disease (GD) is an inherited deficiency of glucocerebrosidase leading to accumulation of glucosylceramide in tissues such as the spleen, liver, and bone marrow. The resulting lipid-laden macrophages lead to the appearance of "Gaucher cells". Anemia associated with an unexplained hyperferritinemia is a frequent finding in GD, but whether this pathogenesis is related to an iron metabolism disorder has remained unclear. To investigate this issue, we explored the iron status of a large cohort of 90 type I GD patients, including 66 patients treated with enzyme replacement therapy. Ten of the patients treated with enzyme replacement were followed up before and during treatment. Serum levels of hepcidin, the iron regulatory peptide, remained within the physiological range, while the transferrin saturation was slightly decreased in children. Inflammation-independent hyperferritinemia was found in 65% of the patients, and Perl's staining of the spleen and marrow smear revealed iron accumulation in Gaucher cells. Treated patients exhibited reduced hyperferritinemia, increased transferrin saturation and transiently increased systemic hepcidin. In addition, the hepcidin and ferritin correlation was markedly improved, and, in most patients, the hemoglobin level was normalized. To further explore eventual iron sequestration in macrophages, we produce a Gaucher cells model by treating the J774 macrophage cell line with a glucocerebrosidase inhibitor and showed induced local hepcidin and membrane retrieval of the iron exporter, ferroportin. These data reveal the involvement of Gaucher cells in abnormal iron sequestration, which may explain the mechanism of hyperferritinemia in GD patients. Local hepcidin-ferroportin interaction was involved in this pathogenesis.

Figure 1.

Hemoglobin levels, serum ferritin and serum hepcidin values in untreated Gaucher disease (GD) patients. Correlation of the hemoglobin concentration (Hb) with serum ferritin (A–C) and serum hepcidin (E–F) in men (A and D), women (B and E) and children (C and F), respectively. According to the Spearman test, no significant correlation was found (r<0.3; P>0.05) between hemoglobin and ferritin but correlations were positive between hemoglobin and hepcidin in women and children. The dotted lines represent the high normal value of ferritin on the x-axis and low normal level of hemoglobin on the y-axis.

Figure 2.

Serum hepcidin level and transferrin saturation (TS) are independent of the ferritin status. Serum hepcidin (A) and TS (B) levels were measured in the normal ferritin (NF) and hyperferritin (HF) untreated patient groups. The means are represented by the horizontal line. According to a two-tailed Mann-Whitney test, no significant differences were observed.

Figure 3.

Iron sequestration in Gaucher cells. Tissue iron content was determined by Perl’s staining of medullar smear (A) and spleen sections (B). The large cells with a laminated aspect were Gaucher cells. The representative images show iron deposition mostly in Gaucher cells. The classical description of Gaucher cells (black arrows) is limited to cells 20–100 μm in diameter with eccentrically placed nuclei and cytoplasm with characteristic crinkles and striations. Images were taken at 20X magnification and a higher magnification (40X).

Figure 4.

Impact of glucocerebrosidase activity inhibition on ferroportin (FPN) and hepcidin expression in the J774 cell line. J774 cells were incubated with 1 mM CBE (+CBE) or with vehicle (-CBE) for 96 hours (h). (A) Staining of FPN and actin were performed as described in the Methods section. FPN is labeled in green and actin in red. In the absence of CBE, FPN is stained mostly at the plasma membrane with some intracellular extent. In the presence of CBE, FPN membrane staining was reduced, and its localization was mostly intracellular. The cross-section images demonstrated a significant overlap of FPN and actin staining in the -CBE condition but not in the +CBE condition. Images were taken by confocal microscopy (60X). (B and C) Quantification of the mRNA expression levels of FPN (B) and hepcidin (C) from treated and untreated J774 cells. Data were normalized by the housekeeping transcript Hprt1 and expressed as the percentage of the -CBE group mean±Standard Error of Mean. Mann-Whitney test was used to compare RNA levels. (D) Quantification of ferritin in cellular extracts from treated and untreated J774 cells. Data were expressed as percentage of the control mean. Medians are represented by the horizontal line. According to a two-tailed Mann-Whitney test, the level of cellular ferritin was significantly higher in the CBE treated cells; P=0.021.

Figure 5.

Impact of CBE treatment on J774 inflammatory profile. IL-1β, IL-10, MCP-1, CCL-5 and TNF-α were quantified in supernatant of J774 cells after incubation with (+CBE) and without (-CBE) CBE for 96 hours (h). Mann-Whitney test was used to compare cytokine levels.

Figure 6.

Impact of ERT on iron metabolism. Paired comparison of the levels of ferritin (A), TS (B) and hemoglobin (C) in patients before (pre-ERT) and after initiation of enzyme replacement treatment (ERT) (from 6 months at least). (D) The hepcidin level was measured in untreated (NT) and treated patients during different periods of time. Each group was compared with the NT group. The hepcidin level was transiently increased during the first five years of ERT (<5). (E) Comparison of the levels of serum soluble transferrin receptor in untreated (n=24) versus treated (ERT; n=22) patients. (A–C) A paired Wilcoxon test was used; (D and E) an unpaired Mann-Whitney test was used.

Figure 7.

Recovery of hepcidin control on iron metabolism in treated Gaucher disease (GD) patients. (A and B) Time course of iron-related parameters in 2 treated patients from enzyme replacement therapy (ERT) initiation until 18–25 months after ERT initiation. Patient 1: 18-year-old woman; Patient 2: 52-year-old woman. (C) According to the Spearman test, hepcidin/ferritin correlation in the untreated and treated patients r and P were shown for each condition and slope of linear regression.