Critical roles of lysosomal acid lipase in myelopoiesis

Abstract

Lysosomal acid lipase (LAL) is a key enzyme that cleaves cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in lysosomes. Genetic ablation of the lal gene (lal(-/-)) in mice has resulted in a systemic increase of macrophages and neutrophils, causing severe inflammation and pathogenesis in multiple organs. We hypothesized that aberrant growth and differentiation of myeloid cells in lal(-/-) mice arises from dysregulated production of progenitor cells in the bone marrow. Indeed, lal(-/-) mice displayed increased numbers of primitive lin(-)Sca-1(+)c-Kit(+) (LSK) cells and granulocyte-macrophage precursors (GMP). Increased high proliferative potential colony-forming cells (HPP-CFC) were enumerated from cultured lal(-/-) bone marrow cells, as were significantly more CFU-GM, CFU-G, and CFU-M colonies. As a consequence, lal(-/-) mice developed significant myeloid infiltration, particularly with CD11b+/Gr-1+ myeloid-derived suppressive cells in multiple organs. Both decreased apoptosis and increased proliferation contribute to the systemic increase of myeloid cells in lal(-/-) myeloid cells. These lal(-/-) CD11b(+)/Gr-1(+) cells displayed suppressive activity on T cell proliferation and function in vitro. Bone marrow chimeras confirmed that the myeloproliferative disorder in lal(-/-) mice was primarily attributable to autonomous defects in myeloid progenitor cells, although the hematopoietic microenvironment in the lal(-/-) mice did not support hematopoiesis normally. These results provide evidence that LAL is an important regulator of myelopoiesis during hematopoietic development, differentiation, and homeostasis.

Figure 1

LAL deficiency affects hematopoietic progenitor populations. A: Representative FACS profiles of progenitor populations, including LSKs, LKs, CMPs, MEPs, GMPs, and CLPs from the bone marrow of lal^+/+ or lal^−/− mice at 3 months old. The gating strategy is described in Materials and Methods. B and C: The percentages and total numbers of LSK, LK, CMP, MEP, GMP, and CLP populations in the bone marrow of lal^+/+ and lal^−/− mice are calculated according to A by flow cytometry. Values were derived from four mice of each groups (n = 4). *P < 0.05; **P < 0.01. D: Proliferation and apoptosis of bone marrow progenitor cells were measured by BrdU and Annexin V labeling in gated areas of progenitor cells. Values were derived from four mice of each groups (n = 4). *P < 0.05. A representative of BrdU FACS analysis is presented. LK indicates IL7Rα⁻Lin⁻c-Kit⁺Sca-1⁻ progenitors; LSK, Lin⁻Sca-1⁺c-kit⁺ progenitors; CMP, common myeloid progenitors; GMP, granulocyte-monocyte progenitors; MEP, megakaryocyte–erythroid progenitor; CLP, common lymphoid progenitors.

Figure 2

Increased differentiation of progenitor cells in lal^−/− mice. A: Recipient wild-type mice (CD45.1) were transplanted with bone marrow cells from lal^+/+ and lal^−/− mice (CD45.2). CFU-S colony formation on the spleen surface of recipient mice was counted 14 days after transplantation. The frequency of committed progenitor colonies (CFU-S₁₄) in lal^+/+ and lal^−/− bone marrow is shown as the mean ± SD and represents the results of three experiments (n = 5). *P < 0.05. B: The frequency of HPP-CFC in lal^+/+ and lal^−/− bone marrow is shown as the mean ± SD and represents the results of four experiments (n = 4). *P < 0.05. C: The frequency of macrophage and granulocyte progenitor cells (CFU-GM) in lal^+/+ and lal^−/− bone marrow is shown as the mean ± SD and represents the results of three experiments (n = 3). *P < 0.05. D: Macrophage colony formation in 5 × 10⁴ bone marrow cells from lal^+/+ and lal^−/− mice in methylcellulose containing 100 ng/ml M-CSF. Colonies were sorted at day 10. Results are mean ± SD, **P < 0.01. E: Granulocyte colony formation in 5 × 10⁴ bone marrow cells from lal^+/+ and lal^−/− mice in methylcellulose containing 10 ng/ml G-CSF. Colonies were sorted at day 10. Results are mean ± SD, **P < 0.01.

Figure 3

Systemic expansion and accumulation of myeloid cells in lal^−/− mice. A: Representative FACS analysis of bone marrow (BM), peripheral blood (PBMC), and spleen from 3-month-old lal^+/+ and lal^−/− mice by CD11b and GR-1 antibody staining. B: The percentage number of CD11b⁺/GR-1⁺ double-positive cells in the bone marrow, blood, and spleen of lal^+/+ and lal^−/− mice at 1, 3, and 6 months old from five independent experiments (n = 5). C: The percentage number of CD11b⁺/GR-1⁻ cells in the bone marrow, blood, and spleen of lal^+/+ and lal^−/− mice at 1, 3, and 6 months old from five independent experiments (n = 5). Results in B and C were mean ± SD, *P < 0.05; **P < 0.01. D: Morphological analysis of peripheral blood neutrophils by transmission electron microscopy (original magnification, ×9300). E: CFSE labeled wild type CD4⁺ T cells were stimulated with anti-CD3 mAb plus anti-CD28 mAb for 4 days in the presence or absence of CD11b⁺/Gr-1⁺ cells isolated from the spleens of wild-type mice (wild-type MDSCs) or lal^−/− mice (lal^−/− MDSCs) at different ratios between MDSC: CD4⁺ T cells. Proliferation of labeled CD4⁺ T cells was analyzed by FACS. Peaks represent cell division cycles. PBS was negative control. The concentrations of secreted IL-2 in the cultured medium were measured by ELISA. n = 4.

Figure 4

Counts of red blood cells, neutrophils, lymphocytes, and platelet in the blood. Red blood cells, lymphocytes, neutrophils, and platelets were counted from 1-, 3-, 6-, and 9-month-old lal^+/+ and lal^−/− mice. Results were mean ± SD.

Figure 5

Myeloid cell infiltration in the lal^−/− lung. A: Representative FACS analysis of whole lung cells from 3-month-old lal^+/+ and lal^−/− mice by CD11b and GR-1 antibody staining. B: The percentage number of CD11b⁺/GR-1⁺ and CD11b⁺/GR-1⁻ positive cells in the lung of lal^+/+ and lal^−/− mice at 1, 3, and 6 months old from five independent experiments (n = 5). Results are mean ± SD, *P < 0.05; **P < 0.01. C: H&E staining of the lung in lal^+/+ and lal^−/− mice (original magnification, ×200). Alveolar lumen was filled with inflammatory cells. D: Kwik-Diff staining of the same amount of bronchioalveolar larvage fluid from lal^+/+ and lal^−/− mice (original magnification, ×400). Both macrophages and neutrophils were increased. Arrows point to neutrophils.

Figure 6

Apoptotic inhibition of myeloid cells in lal^−/− mice. A: Representative Annexin V staining in CD11b⁺/GR-1⁺ cells of the bone marrow from 3-month-old lal^+/+ and lal^−/− mice. B: The percentage numbers of Annexin V⁺ CD11b⁺/GR-1⁺ cells or CD11b⁺/GR-1⁻ cells in the bone marrow, blood, and spleen from four independent experiments (n = 4). Results were mean ± SD, *P < 0.05; **P < 0.01. C: The percentage numbers of BrdU⁺ CD11b⁺/GR-1⁺ cells or CD11b⁺/GR-1⁻ cells in the bone marrow, blood, and spleen from four independent experiments (n = 4). Results were mean ± SD, *P < 0.05; **P < 0.01.

Figure 7

Systemic expansion and accumulation of myeloid cells in lal^−/− bone marrow chimeric mice. A: Representative FACS analysis of bone marrow (BM), peripheral blood (PBMC), spleen, and lung cells from lal^+/+ and lal^−/− bone marrow–transplanted recipient mice by CD11b and GR-1 antibody staining. B: Statistic analyses of donor CD11b⁺/GR-1⁺ myeloid cell populations in lal^+/+ and lal^−/− bone marrow–transplanted recipient mice. Values were derived from four mice in each group (n = 4). Results are mean ± SD, *P < 0 0.05; **P < 0.01.