Unexpected macrophage-independent dyserythropoiesis in Gaucher disease

Abstract

Gaucher disease is a rare inherited disease caused by a deficiency in glucocerebrosidase leading to lipid accumulation in cells of mononuclear-macrophage lineage known as Gaucher cells. Visceral enlargement, bone involvement, mild anemia and thrombocytopenia are the major manifestations of Gaucher disease. We have previously demonstrated that the red blood cells from patients exhibit abnormal properties, which indicates a new role in Gaucher disease pathophysiology. To investigate whether erythroid progenitors are affected, we examined the in vitro erythropoiesis from the peripheral CD34⁺ cells of patients and controls. CD34- cells were differentiated into macrophages and co-cultivated with erythroblasts. We showed an accelerated differentiation of erythroid progenitors without maturation arrest from patients compared to controls. This abnormal differentiation persisted in the patients when the same experiments were performed without macrophages, which strongly suggested that dyserythropoiesis in Gaucher disease is secondary to an inherent defect in the erythroid progenitors. The accelerated differentiation was associated with reduced cell proliferation. As a result, less mature erythroid cells were generated in vitro in the Gaucher disease cultures compared to the control. We then compared the biological characteristics of untreated patients according to their anemic status. Compared to the non-anemic group, the anemic patients exhibit higher plasma levels of growth differentiation factor-15, a marker of ineffective erythropoiesis, but they had no indicators of hemolysis and similar reticulocyte counts. Taken together, these results demonstrated an unsuspected dyserythropoiesis that was independent of the macrophages and could participate, at least in part, to the basis of anemia in Gaucher disease.

Figure 1.

Clonogenic potential of CD34+ cells. A. Number of total colony counts including erythroid (BFU-E: burst forming unit), non-erythroid (granulocyte/macrophage colony forming units: CFU-GM, CFU-G and CFU-M) and multilineage progenitors (CFU-GEMM) on day 14 of culture. B. Number of BFU-E burst-forming units (BFU-E). C. Number of early and late erythroid BFU-E. The colonies were scored by direct microscopic visualization on day 14 of culture for the CTL (n=13) and GD (n=13) subjects, which are shown as gray and black boxes, respectively. CTL: control; GD: Gaucher disease.

Figure 2.

In vitro erythroid differentiation of Gaucher disease (GD) erythroblasts cultivated with macrophages. Erythroblasts (EBs) derived from the CD34⁺ peripheral blood cells of GD patients (GD) or healthy controls (CTL) were cultivated with macrophages (MPs) derived from CD34− cells from the GD or CTL subjects. The GD EBs cultivated with GD MPs were compared with the CTL EBs cultivated with CTL MPs. Expression of erythroid surface markers was measured by flow cytometry on day 12 and 15 of erythroid differentiation. A. Representative flow cytometry plots of glycophorin A (GPA) and c-Kit (CD117) surface expression on days 12 and 15 in the GD and CTL cultures. The GPA⁺ CD117⁻ cell population represents the differentiating EBs. B. Percentage of GPA⁺ CD117⁻ cells derived from CTL or GD patients on days 12 and 15 of erythroid differentiation. C. Percentage of band 3^hi cells on day 15 of erythroid differentiation. These cells represent the mature EBs. D. The boxes represent the percentage of GD or CTL progenitors on day 15 of erythroid differentiation. Morphological analysis after May–Grünwald–Giemsa (MGG) staining was used. ProEB: proerythroblasts; Baso: basophilic cells; Polych: polychromatophilic cells; Acido+Retic: acidophilic cells and reticulocytes E. The boxes represent the terminal maturation index (on day 15 of differentiation) as defined in the Online Supplementary Methods section. F. CTL or GD MPs were co-cultivated with CTL EBs. The percentages of GPA⁺ CD117⁻ cells on days 12 and 15 of erythroid differentiation were compared. The results are presented as box-and-whisker plots. Gray boxes, culture of CTL EBs with CTL MPs; black boxes, culture of GD EBs with GD MPs; tiled boxes, culture of CTL EBs with GD MPs. For B, C, D, E and F, n=8 for each condition. The medians are represented as horizontal bars (−); the upper and lower quartiles are represented as the top and the bottom of the box, respectively; and the maximum and minimum data values are shown by dashes (−) at the top and the bottom, respectively, of the whiskers. The P values were determined using the Wilcoxon signed-rank test to compare the parameters of erythroid differentiation between the CTL and GD cultures on days 12 and 15 (*P<0.05). ns= non significant.

Figure 3.

In vitro erythroid differentiation of Gaucher disease (GD) erythroblasts cultivated without macrophages. CD34⁺ cells derived from the peripheral blood cells from GD patients (GD) or healthy controls (CTL) were cultivated without macrophages (MPs) and differentiated into the erythroid lineage until day 15 as described in methods. The surface expression of the erythroid differentiation markers was measured using flow cytometry during erythroblast (EB) differentiation. A. Representative flow cytometry plots of the cell surface expression of glycophorin A (GPA) and c-Kit (CD117) on days 12 and 15 of the erythroid differentiation culture. The GPA⁺ CD117⁻ cells represent the differentiating EBs. B. Percentage of GPA⁺ CD117⁻ cells derived from CTL or GD patients on days 12 and 15 of the erythroid differentiation performed without MPs (CTL n=18; GD n=24). C. Percentage of band 3^hi cells on day 15 of erythroid differentiation (CTL n=13; GD n=13). These cells represent the mature EBs. D. The boxes represent the percentage of GD or CTL erythroid cells on day 15 of the erythroid differentiation performed without MPs (CTL n=12; GD n=17). Morphological analysis after MGG (May-Grünwald-Giemsa) staining was used. ProEB, proerythroblasts; Baso: basophilic cells; Polych: polychromatophilic cells; Acido+Retic: acidophilic cells and reticulocytes. E. The boxes represent the terminal maturation index as defined in the Online Supplementary Methods section (CTL n=12; GD n=17). F. Representative morphological analysis of erythroid differentiation as indicated by MGG staining on day 15 of cell culture (magnification 60×). The results are presented as box-and-whisker plots. Gray boxes (CTL EBs); black boxes (GD EBs). The medians are represented as horizontal bars (−); the upper and lower quartiles are represented as the top and the bottom of the box, respectively; and the maximum and minimum data values are shown as dashes (−) at the top and the bottom, respectively, of the whiskers. The P values were determined using the Wilcoxon signed-rank test to compare the parameters of erythroid differentiation between the CTL and GD cultures on days 12 and 15 (*P<0.05; *** P<0.001).

Figure 4.

Cell proliferation during in vitro erythropoiesis. A. Absolute numbers of cells derived from CD34⁺ cells from the GD and CTL subjects on day 0 and day 15 of erythroid differentiation without MPs (macrophages) (n=24 for each GD and control group). B. Absolute numbers of mature cells derived from the CD34⁺ cells from the GD and CTL subjects on day 15 of erythroid differentiation without MPs (n=17 for each GD and control group). This value has been calculated from the percentage of band 3^hi cells multiplied by the total number of cells at day 15. Gray boxes and curve show the results for the CTL erythoblasts (EBs), and black boxes and curve show the results for the GD EBs. The P values were determined using the Wilcoxon signed-rank test to compare the expansion of cells between CTL and GD cultures. For figure A, the ratios of the cell numbers on day 15 to those on day 0 were compared by a Wilcoxon signed-rank test (**** P<0.0001). For figure B, the P value was determined using the Mann-Whitney test to compare the number of Band 3^hi erythroblasts (***P<0.001). For figure B, the P value was determined using the Mann-Whitney test to compare the number of Band 3^hi erythroblasts (***P<0.001). CTL: control; GD: Gaucher disease.

Figure 5.

Plasma growth differentiation factor-15 (GDF-15) levels in GD patients. A. Increased level of GDF-15 in the plasma from untreated GD patients (UT GD, n=15) compared to CTL (CTL, n=8). B. GDF-15 level measured in GD patients according to their anemic status (n=7 anemic patients vs. n=8 non-anemic patients). The P values were determined using the Mann-Whitney test to compare the GDF-15 levels between control and GD subjects. (**P<0.01, ****P<0.0001). The bars represent the medians. CTL: control; GD: Gaucher disease.