Page 46 - Haematologica-April 2018

590
T. Lefebvre et al.
ABC
DEF
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.
of the pro-inflammatory interleukin 6 (IL-6) in 22 patients (mix of treated and untreated patients) and found that this cytokine was not induced (data not shown), suggesting an absence of systemic inflammation. We conclude that hyperferritinemia is independent of the inflammatory state in our GD cohort. Furthermore, there was no corre- lation between serum ferritin and hemoglobin in untreat- ed patients (Figure 1). There was even a positive correla- tion between hemoglobin and serum hepcidin in women and children, who were more affected by anemia in our cohort, excluding the eventual presence of an anemia of inflammation.
Because some untreated GD patients have normal fer- ritinemia (NF), we compared their levels of serum hep- cidin and TS with those of untreated patients with hyper- ferritinemia (HF) (Figure 2). For both parameters, no signif- icant differences were found between NF and HF groups, and correlation between serum hepcidin and ferritin was not significant in any group of the cohort (Online Supplementary Figure S1). Thus, the ferritin level in GD patients is increased independently of systemic inflamma- tion, the iron pool and the serum hepcidin level.
Hyperferritinemia reflects local iron sequestration in Gaucher cells
Serum ferritin is a primary marker of tissue iron over- load; thus, we investigated whether hyperferritinemia might reflect local iron sequestration in Gaucher cells. Perl’s staining on the myelogram of one patient and on histological sections of the spleen from 2 patient biopsies showed high iron accumulation in Gaucher cells in both
organs (Figure 3A and B, respectively). We noticed a high variation in the amount of accumulated iron within these Gaucher cells, particularly in the spleen. To investigate the underlying mechanism of this iron sequestration, we decided to mimic the Gaucher phenotype in vitro using the J774 macrophagic cell line incubated with the GCerase inhibitor CBE. The inhibition of GCerase activity in the presence of CBE was confirmed by flow cytometry show- ing a decreased fluorescence of its PFB-FDGlu substrate (Online Supplementary Figure S2). We hypothesized that the inhibition of GCerase activity could affect the protein expression of the iron exporter FPN (Figure 4). In the absence of CBE, FPN protein staining was strongly observed both at the plasma membrane localized with actin and in the subapical compartment, reminiscent of the known intracellular diffuse distribution of the FPN protein.16 However, when added to the culture media at a final concentration of 1 mM for 96 h, CBE dramatically reduced the level of FPN membrane expression (Figure 4A). We also explored the expression pattern of CD11b, a macrophage cell surface protein, which was found not to be affected by CBE treatment (Online Supplementary Figure S3), confirming specificity in FPN regulation by CBE. The decrease in the FPN protein level was not associated with decreased FPN mRNA, the level of which was slightly but not significantly increased (Figure 4B), indicating a post- translational downregulation of FPN expression due to GCerase activity inhibition. Furthermore, FPN downregu- lation was associated with increased level of cellular fer- ritin in CBE-treated cells, reminiscent of an iron sequestra- tion in Gaucher cells (Figure 4D). Because the post-trans-
haematologica | 2018; 103(4)