Chronic Liver Disease in Humans Causes Expansion and Differentiation of Liver Lymphatic Endothelial Cells

Abstract

Liver lymphatic vessels support liver function by draining interstitial fluid, cholesterol, fat, and immune cells for surveillance in the liver draining lymph node. Chronic liver disease is associated with increased inflammation and immune cell infiltrate. However, it is currently unknown if or how lymphatic vessels respond to increased inflammation and immune cell infiltrate in the liver during chronic disease. Here we demonstrate that lymphatic vessel abundance increases in patients with chronic liver disease and is associated with areas of fibrosis and immune cell infiltration. Using single-cell mRNA sequencing and multi-spectral immunofluorescence analysis we identified liver lymphatic endothelial cells and found that chronic liver disease results in lymphatic endothelial cells (LECs) that are in active cell cycle with increased expression of CCL21. Additionally, we found that LECs from patients with NASH adopt a transcriptional program associated with increased IL13 signaling. Moreover, we found that oxidized low density lipoprotein, associated with NASH pathogenesis, induced the transcription and protein production of IL13 in LECs both in vitro and in a mouse model. Finally, we show that oxidized low density lipoprotein reduced the transcription of PROX1 and decreased lymphatic stability. Together these data indicate that LECs are active participants in the liver, expanding in an attempt to maintain tissue homeostasis. However, when inflammatory signals, such as oxidized low density lipoprotein are increased, as in NASH, lymphatic function declines and liver homeostasis is impeded.

Keywords: alcoholic liver disease; cirrhosis; fibrosis; hepatitis C virus; interleukin-13; lymphatic endothelial cells; non-alcoholic steatohepatitis; oxidized low density lipoprotein.

Figure 1

Lymphatic vessels increase in fibrotic regions of cirrhotic livers independent of disease etiology. Liver explants were obtained from cirrhotic patients who received liver transplantation. Non-Alcoholic Steatohepatitis (NASH) (n = 8), Alcoholic liver disease (ALD) (n = 12), chronic Hepatitis C viral infection (HCV) (n = 5), Autoimmune hepatitis (AIH) (n = 1), Wilson's disease (n = 1), Primary sclerosing cholangitis (PSC) (n = 1) and four non-diseased livers. Representative images from non-diseased (A) or ALD (B) explants are shown. Five micrometer sections were stained with anti-podoplanin (lymphatic vessels D2/40-red), anti-CD3 (T cells-white), anti-CD68 (macrophages-green), and dapi (nuclei-blue) and imaged using the Perkin Elmer Vectra 3.0 imaging system and linear unmixed with inFORM software. (C) Lymphatic vessels density was determined using inFORM software and normalized to area in each disease listed and designated by color of dot. Statistical analysis was performed using a student's t-test. **P < 0.01.

Figure 2

Chronic liver disease induces the expansion of CCL21⁺ lymphatic vessels and immune cell recruitment to the liver. (A) Lymphatic vessel (PDPN-Red) expression of CCL21 (Yellow), along with Macrophages (CD68-Magenta), T cells (CD3-Green) and B cells (CD19-White) in non-diseased, NASH, and HCV explanted livers. (B) Zoomed in representative examples from (A) shown with and without PDPN. White dotted line denotes where lymphatic vessel appears in the image. (C) Quantification of CCL21⁺ lymphatic vessels in non-diseased (n = 3), HCV (n = 5), and NASH (n = 3) livers. (D) Quantification of B cells, T cells, and Macrophages in liver tissue from Non-diseased (black, n = 3), NASH (red, n = 3–5) or HCV (blue, n = 4). *P < 0.05, **P < 0.01.

Figure 3

Single cell analysis of liver lymphatic endothelial cells. (A) Sorting strategy used to identify LECs. (B) TSNE analysis from single cell mRNA sequencing of cells obtained from the sorting strategy in (A). (C) Expression of LEC specific genes expressed by isolated cell subsets from the liver (yellow = high, purple = low). (D) Normalized CCL21 mRNA expression by LECs from non-diseased (blue), NASH (green), or HCV (red) livers.

Figure 4

Chronic liver disease results in increased LEC proliferation. (A) Frequency of LECs (left) and PECs (right) in each stage of cell cycle based on gene expression from single cell sequencing data. (B) Representative flow cytometric profiles of LECs from non-diseased (left and red) or diseased (right and blue). (C) Quantification of (B). (D) Hierarchical clustering of differentially expressed genes from LECs sorted from non-diseased (ND), NASH, or HCV explanted livers.

Figure 5

Cholesterol regulates IL13 signaling in LECs. (A) Genes involved in IL13 signaling in LECs from Non-diseased (ND, blue), NASH (green), and HCV (red) (B). Representative images of lymphatic branching from hLECs treated for 24 h with Vehicle (DMSO), Ox-LDL (100 μg/ml) or PA (0.25 mM). (C) Quantification of (B). (D) Quantitative RT-PCR of hLEC treated with the indicated stimulus for 24 h. (E) Representative flow cytometric profiles of IL13 protein production by hLECs after 24 h with the indicated stimulus. (F) Quantification of (E). *P < 0.05. ***P < 0.001.

Figure 6

Cholesterol results in IL13 production by liver LECs in vivo. (A) Representative flow plots of cholesterol uptake and IL13 production by liver and lymph node LECs from Brefeldin A treated mice. LECs were gated as in 3A. (B) Quantification of A. *P < 0.05, **P < 0.01, ***P < 0.001.