Hepatocyte-Specific Expression of Human Lysosome Acid Lipase Corrects Liver Inflammation and Tumor Metastasis in lal(-/-) Mice

Abstract

The liver is a major organ for lipid synthesis and metabolism. Deficiency of lysosomal acid lipase (LAL; official name Lipa, encoded by Lipa) in mice (lal(-/-)) results in enlarged liver size due to neutral lipid storage in hepatocytes and Kupffer cells. To test the functional role of LAL in hepatocyte, hepatocyte-specific expression of human LAL (hLAL) in lal(-/-) mice was established by cross-breeding of liver-activated promoter (LAP)-driven tTA transgene and (tetO)7-CMV-hLAL transgene with lal(-/-) knockout (KO) (LAP-Tg/KO) triple mice. Hepatocyte-specific expression of hLAL in LAP-Tg/KO triple mice reduced the liver size to the normal level by decreasing lipid storage in both hepatocytes and Kupffer cells. hLAL expression reduced tumor-promoting myeloid-derived suppressive cells in the liver of lal(-/-) mice. As a result, B16 melanoma metastasis to the liver was almost completely blocked. Expression and secretion of multiple tumor-promoting cytokines or chemokines in the liver were also significantly reduced. Because hLAL is a secretory protein, lal(-/-) phenotypes in other compartments (eg, blood, spleen, and lung) also ameliorated, including systemic reduction of myeloid-derived suppressive cells, an increase in CD4(+) and CD8(+) T and B lymphocytes, and reduced B16 melanoma metastasis in the lung. These results support a concept that LAL in hepatocytes is a critical metabolic enzyme in controlling neutral lipid metabolism, liver homeostasis, immune response, and tumor metastasis.

Figure 1

Human LAL (hLAL) expression in wild-type (WT), lal^−/− (KO), and liver-activated promoter (LAP)–driven tTA transgene and (tetO)₇-CMV-hLAL transgene with lal^−/− (LAP-Tg/KO) triple mice. A: RT-PCR for hLAL mRNA expression in the liver, lung, spleen, and bone marrow (BM) of WT, KO, and LAP-Tg/KO triple mice treated with or without doxycycline (DOX). The housekeeping gene β-actin was used as an internal control. B: RT-PCR for hLAL mRNA expression in isolated primary hepatocytes and Ly6G⁺ cells from WT and LAP-Tg/KO triple mouse liver without DOX. The housekeeping gene β-actin was used as an internal control. C: Western blot analysis of LAL protein in the liver, lung, spleen, and bone marrow of WT, lal^−/−(KO), and LAP-Tg/KO triple mice, treated or untreated with DOX. D: Immunohistochemical staining of hLAL and F4/80 in the livers of DOX-treated (+DOX) or DOX-untreated (–DOX) LAP-Tg/KO triple mice. White arrows indicate representative hepatocytes that express hLAL. Black arrows indicate the F4/80⁺ Kupffer cells that are also positive for hLAL. Without hLAL expression, there is accumulation of enlarged F4/80-positive storage cells in DOX-treated LAP-Tg/KO triple mice. Original magnification: ×200. Hepa, hepatocyte, Ly6G⁺, Ly6G⁺ cells from the liver (B).

Figure 2

Hepatic expression of hLAL in liver-activated promoter (LAP)–driven tTA transgene and (tetO)₇-CMV-hLAL transgene with lal^−/− (LAP-Tg/KO) mice corrects abnormality in the liver, spleen, and small intestine. A: Gross view of the liver and spleen of lal^+/+ [wild-type (WT)], lal^−/− (KO), and doxycycline-untreated (DOX-Off) and doxycycline-treated (DOX-On) LAP-Tg/KO mice. B–M: Hematoxylin and eosin staining of the liver, spleen, and small intestine paraffin sections from WT (B, F, and J), lal^−/− (KO) (C, G, and K), DOX-ON (D, H, and L), and DOX-OFF (E, I, and M) LAP-Tg/KO mice. Original magnification: ×200 (B–M).

Figure 3

Hepatic expression of human lysosomal acid lipase (hLAL) in liver-activated promoter (LAP)–driven tTA transgene and (tetO)₇-CMV-hLAL transgene with lal^−/− (LAP-Tg/KO) mice corrects neutral lipid storage in the liver, spleen, and small intestine. Oil Red-O staining of liver, spleen, and small intestine frozen sections from wild-type (WT) (A, E, and I), lal^−/− (KO) (B, F, and J), doxycycline-treated (DOX-On) (C, G, and K), and doxycycline-untreated (DOX-Off) (D, H, and L) LAP-Tg/KO mice. Original magnification: ×200 (A–L).

Figure 4

Quantitative analyses of cholesterol and triglycerides in the liver, spleen, and small intestine of human lysosomal acid lipase (hLAL) in liver-activated promoter (LAP)–driven tTA transgene and (tetO)₇-CMV-hLAL transgene with lal^−/− (LAP-Tg/KO) mice. Concentrations of cholesterol and triglycerides in the liver, spleen, and small intestine of hLAL in LAP-Tg/KO mice were determined as described in Materials and Methods. Data are expressed as means ± SEM from five mice in each group. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. WT, wild type.

Figure 5

Hepatic expression of human lysosomal acid lipase (hLAL) in liver-activated promoter (LAP)–driven tTA transgene and (tetO)₇-CMV-hLAL transgene with lal^−/− (LAP-Tg/KO) mice reduces B16 melanoma metastasis. A: B16 melanoma cells (5 × 10⁵) were intravenously injected into doxycycline-treated (+Dox) or doxycycline-untreated (–DOX) LAP-Tg/KO triple mice for 2 weeks. Metastasized B16 melanoma colonies in the liver and lung are shown. B: Quantitative analysis of B16 melanoma colonies in the livers of doxycycline-treated or doxycycline-untreated LAP-Tg/KO triple mice. C: Representative hematoxylin and eosin staining of liver and lung sections. D: Representative immunohistochemical staining of metastasized livers and lungs using anti-Ki67 antibody. n = 10 to 16 (A and B). ^∗∗P < 0.01. Original magnification: ×200 (C and D).

Figure 6

Hepatic expression of human lysosomal acid lipase (hLAL) in liver-activated promoter (LAP)–driven tTA transgene and (tetO)₇-CMV-hLAL transgene with lal^−/− (LAP-Tg/KO) mice reduces CD11b⁺Ly6G⁺ cell expansion. The percentages (A) and total cell numbers (B) of CD11b⁺ Ly6G⁺ cells in the wild-type (WT), lal^−/− (KO), doxycycline-treated (+DOX), or doxycycline-untreated (–DOX) LAP-Tg/KO liver, bone marrow (BM), blood [peripheral blood mononuclear cells (PBMCs)], lung, and spleen (3 × 10⁴). A representative dot plot of CD11b⁺Ly6G⁺ cells in the blood is shown. Data are expressed as means ± SD from four mice in each group. n = 4. ^∗P < 0.05, ^∗∗P < 0.01.

Figure 7

Hepatic expression of human lysosomal acid lipase (hLAL) in liver-activated promoter (LAP)–driven tTA transgene and (tetO)₇-CMV-hLAL transgene with lal^−/− (LAP-Tg/KO) mice increases CD4⁺, CD8⁺, and B220⁺ cells. The percentages and total cell numbers of CD4⁺ T cells (A), CD8⁺ T cells (B), and B220⁺ B cells (C) in the wild-type (WT), lal^−/− (KO), doxycycline-treated (+DOX), or doxycycline-untreated (–DOX) LAP-Tg/KO bone marrow (BM), blood [peripheral blood mononuclear cells (PBMCs)], lung, and spleen. Representative dot plots or histograms of CD4⁺, CD8⁺, and B220⁺ cells in the blood (PBMCs) are shown, respectively. Data are expressed as means ± SD from four mice in each group. n = 4. ^∗P < 0.05, ^∗∗P < 0.01.

Figure 8

Hepatic expression of human lysosomal acid lipase (hLAL) in liver-activated promoter (LAP)–driven tTA transgene and (tetO)₇-CMV-hLAL transgene with lal^−/− (LAP-Tg/KO) mice reduces synthesis and secretion of cytokines and chemokines. A: The concentrations of IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein-1 (MCP-1), and chemokine ligand (CCL)-5 in the plasma of doxycycline-treated (+DOX) or doxycycline-untreated (–DOX) lal^+/+ [wild type (WT)], lal^−/− (KO), and LAP-Tg/KO mice were determined by enzyme-linked immunosorbent assay. B: Quantitative real-time PCR analyses of mRNA expression levels of cytokines and chemokines in the liver of lal^+/+ (WT), lal^−/− (KO), and +DOX or –DOX LAP-Tg/KO mice. The relative gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA, and analysis was performed by the 2^−ΔΔC_T method. Data are expressed as means ± SD. n = 5 to 6 (A); n = 4 (B). ^∗P < 0.05, ^∗∗P < 0.01. IFNγ, interferon-γ; TNFα, tumor necrosis factor-α.

Figure 9

In vitro doxycycline treatment of primary hepatocytes from untreated liver-activated promoter (LAP)–driven tTA transgene and (tetO)₇-CMV-hLAL transgene with lal^−/− (LAP-Tg/KO) triple mice induces synthesis and secretion of inflammatory cytokines and chemokines. Hepatocytes isolated from lal^+/+ [wild type (WT)] and doxycycline-untreated LAP-Tg/KO triple mice were treated with doxycycline in vitro for 5 days. A: The concentrations of IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein-1 (MCP-1), and chemokine ligand (CCL)-5 in the culture medium were determined by enzyme-linked immunosorbent assay. B: Quantitative real-time PCR analyses of mRNA expression levels of cytokines and chemokines in the isolated hepatocytes of lal^+/+ (WT) and LAP-Tg/KO mice treated with or without doxycycline. The relative gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA, and analyses were performed by the 2^−ΔΔC_T method. Data are expressed as means ± SD. n = 4. ^∗P < 0.05, ^∗∗P < 0.01. IFNγ, interferon-γ; TNFα, tumor necrosis factor-α.