Gaucher iPSC-derived macrophages produce elevated levels of inflammatory mediators and serve as a new platform for therapeutic development

Abstract

Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the acid β-glucocerebrosidase (GCase; GBA) gene. The hallmark of GD is the presence of lipid-laden Gaucher macrophages, which infiltrate bone marrow and other organs. These pathological macrophages are believed to be the sources of elevated levels of inflammatory mediators present in the serum of GD patients. The alteration in the immune environment caused by GD is believed to play a role in the increased risk of developing multiple myeloma and other malignancies in GD patients. To determine directly whether Gaucher macrophages are abnormally activated and whether their functional defects can be reversed by pharmacological intervention, we generated GD macrophages by directed differentiation of human induced pluripotent stem cells (hiPSC) derived from patients with types 1, 2, and 3 GD. GD hiPSC-derived macrophages expressed higher levels of tumor necrosis factor α, IL-6, and IL-1β than control cells, and this phenotype was exacerbated by treatment with lipopolysaccharide. In addition, GD hiPSC macrophages exhibited a striking delay in clearance of phagocytosed red blood cells, recapitulating the presence of red blood cell remnants in Gaucher macrophages from bone marrow aspirates. Incubation of GD hiPSC macrophages with recombinant GCase, or with the chaperones isofagomine and ambroxol, corrected the abnormal phenotypes of GD macrophages to an extent that reflected their known clinical efficacies. We conclude that Gaucher macrophages are the likely source of the elevated levels of inflammatory mediators in the serum of GD patients and that GD hiPSC are valuable new tools for studying disease mechanisms and drug discovery.

Keywords: Cell biology; Experimental models; Induced pluripotent stem cells; Monocyte; Reprogramming.

Figure 1. Generation and characterization of GD hiPSC, and directed differentiation to monocytes and macrophages

A) Staining of W184R/D409H (type 2-b) GD hiPSC with antibodies to the stem cell and pluripotency markers OCT4, SSEA4, NANOG, TRA-1-60, TRA-1-81, and SOX2. Magnification, 10X. B) GD hiPSC gave rise to benign cystic teratomas in NOG-SCID mice. (a–d) H&E staining of teratoma cells from the three germ layers. (a) Ectodermal structures (pigmented neural epithelium and neuronal rosettes); (b) Endodermal structures (glandular or intestinal epithelium); c and d) Mesodermal structures (connective tissues, cartilage or skeletal muscle). Magnification, 20X. C) Normal karyotype of type 2-b GD hiPSC. D and E) FACS analysis of GD hiPSC monocytes and macrophages stained with the indicated antibodies. In the histograms, blue and red show the percentage of cells stained with specific markers and isotype controls, respectively. (D) CD14 expression in type 2 GD hiPSC monocytes. (E) Expression of CD68 and CD163 in type 2 GD hiPSC macrophages. F) Low levels of GCase enzymatic activity and GCase protein in types 1, 2 and 3 GD hiPSC macrophages vs. control hiPSC macrophages. G) May-Grünwald-Giemsa stain and phagocytic activity of GD hiPSC macrophages. (a) May-Grünwald-Giemsa stain of type 2-b GD hiPSC macrophages. (b) Live cell image of type 2-b GD hiPSC macrophages after phagocytosis of opsonized RBC. Magnification, 40X.

Figure 2. GD hiPSC macrophages secrete elevated levels of inflammatory cytokines

A–D) ELISA analysis showing the secretion of TNF alpha, IL-1beta, IL-6, and IL-10 by untreated and LPS-treated type 2 GD hiPSC vs. control hiPSC macrophages. Macrophages were treated with 100 ng/ml LPS for the indicated times, after which the culture supernatants were analyzed to measure the secretion of TNF alpha, IL-1beta, IL-6, and IL-10. Numbers in the ordinates represent pg/ml of secreted cytokines in culture supernatants. P values (A–C) for type 2-a and 2-b hiPSC vs. control hiPSC macrophages at the 3 time points are: TNF alpha (<0.001); IL1-beta (<0.001); IL-6 (<0.001, <0.01); and for D) IL-10 (<0.001). Results shown in panels A–D are representative of three independent experiments.

Figure 3. Induction of cytokines and ChT1 in GD hiPSC macrophages

A–D) Control and type 2 GD hiPSC macrophages were either left untreated or were treated with 100 ng/ml LPS for the indicated times, after which RT-PCR analysis was carried out to measure induction of TNF alpha (A), IL-6 (B), IL1-beta (C), and IL-10 (D) mRNA. Numbers in the ordinates represent fold-activation of the corresponding cytokines compared with the non-treated condition. A-C) P values for type 2-a and 2-b hiPSC vs. control hiPSC macrophages at the 3 time points are: TNF alpha (<0.001 and <0.01); IL-6 (<0.001 and <0.01); IL-1beta (<0.001). E) ChT1 activity in control and types 1, 2, and 3 GD hiPSC macrophages was assayed as described in the Materials and Methods. Numbers in the ordinate represent the fold-activation of ChT1 activity compared to the control. P values for GD compared to control hiPSC are: type 1-a (<0.0038), type 1-b (<0.0002), type 2-a (<0.0042), type 2-b (<0.0319) and type 3-b (<0.01).

Figure 4. Recombinant GCase and chaperones increase lysosomal GCase levels in GD hiPSC macrophages

(A) Control and type 2 GD hiPSC macrophages that were either left untreated (NT) or were treated with isofagomine (Isof, 60 μM), ambroxol (Amb, 100 μM), or recombinant GCase (0.24 U/ml) for 5d, were immunostained for GCase and LAMP1 and counterstained with DAPI. Red color (left panels), LAMP1; green color (middle panels), GCase; blue color (right panels), DAPI. Overlay (right panels), GCase, LAMP1 and DAPI stainings. Magnification, 40X. (B) Quantification of mean fluorescence intensity (MFI) of colocalized LAMP1 and GCase.

Figure 5. Reversal of cytokine induction in GD hiPSC macrophages by recombinant GCase, ambroxol and isofagomine

A–C) Control and type 2-a GD hiPSC macrophages were incubated with recombinant GCase (0.24 U/ml), ambroxol (100 μM), isofagomine (60 μM) or vehicle (NT) for 5d. Control and GD hiPSC macrophages were then either left untreated or were treated with 100 ng/ml LPS for 4h, after which RT-PCR analysis was carried out to measure induction of TNF alpha (A), IL-1beta (B), and IL-6 (C) mRNA. Numbers in the ordinates represent fold-activation of the corresponding cytokines compared with the non-treated condition. P values for all treatments in the presence or absence of LPS are between <0.01 to < 0.05.

Figure 6. Recombinant GCase, isofagomine and ambroxol induce IL-10 and reverse the elevation of ChT1 activity in GD hiPSC macrophages

A) Control, type 2-a and type 2-b GD hiPSC macrophages were incubated with recombinant GCase (0.24 U/ml), ambroxol (100 μM), isofagomine (60 μM) or vehicle (NT) for 5d, after which RT-PCR analysis was carried out to measure induction of IL-10. Numbers in the ordinate represent the IL-10 mRNA fold-activation compared with the non-treated condition. P values for GCase, ambroxol and isofagomine in treated vs. untreated cells are: <0.05. B) Control, type 2-a and type 2-b GD hiPSC macrophages that were either not treated or treated for 5d with recombinant GCase (0.24 U/ml), ambroxol (100 μM), or isofagomine (60 μM), were assayed for ChT1 enzyme activity in intact cells as described in the Materials and Methods. For all macrophages including control, P values for NT vs. treated are between <0.01and <0.05.

Figure 7. Reversal of the delayed clearance of RBC in GD hiPSC macrophages by recombinant GCase and chaperones

A–C) Type 2 (A), type 3 (B) and type 1 (C) GD hiPSC macrophages were either left untreated (NT) or were incubated with either recombinant GCase (0.24 U/ml) or ambroxol (100 μM) for 5d. The cells were then incubated with opsonized RBC for 2h, washed, and the time course of RBC clearance was followed by microscopic observation for the indicated times as described in the M&M. Numbers in the ordinates represent % of hiPSC macrophages containing visible RBC. On day 2, for all GD types, P values of GCase treated vs. NT are between <0.0001 and <0.0003; for type 2 and 3 hiPSC treated with ambroxol vs. NT, P values are <0.0001 and <0.0005, respectively; for type 1, P <0.0012. D and E) Type 2 (D) and type 3 (E) GD hiPSC macrophages were either left untreated (NT) or were incubated with recombinant GCase (0.24 U/ml), ambroxol (50 and 100 μM) or isofagomine (60 and 100 μM) for 5d, after which they were assayed for RBC clearance. On day 2, (type 2 cells) P values for GCase, ambroxol (50 and 100 μM), and isofagomine (100 μM) treated vs. NT are between <0.0001 and <0.0006; P value for isofagomine (60 μM) is <0.001. For type 3 cells, P values for GCase, ambroxol (100 μM) and isofagomine (60, 100 μM) treated vs. NT are between <0.0001 and <0.0006.