Endoplasmic reticulum stress induced LOX-1+ CD15+ polymorphonuclear myeloid-derived suppressor cells in hepatocellular carcinoma

Abstract

A recent study indicated that Lectin-type oxidized LDL receptor-1 (LOX-1) was a distinct surface marker for human polymorphisms myeloid-derived suppressor cells (PMN-MDSC). The present study was aimed to investigate the existence LOX-1 PMN-MDSC in hepatocellular carcinoma (HCC) patients. One hundred and twenty-seven HCC patients, 10 patients with mild active chronic hepatitis B, 10 liver cirrhosis due to hepatitis B, 10 liver dysplastic node with hepatitis B and 50 health control were included. LOX-1⁺ CD15⁺ PMN-MDSC were significantly elevated in HCC patients compared with healthy control and patients with benign diseases. LOX-1⁺ CD15⁺ PMN-MDSC in circulation were positively associated with those in HCC tissues. LOX-1⁺ CD15⁺ PMN-MDSCs significantly reduced proliferation and IFN-γ production of T cells with a dosage dependent manner with LOX-1^- CD15⁺ PMNs reached negative results. The suppression on T cell proliferation and IFN-γ production was reversed by ROS inhibitor and Arginase inhibitor. ROS level and activity of arginase of LOX-1 ⁺ CD15⁺ PMN were higher in LOX-1⁺ CD15⁺ PMN-MDSCs than LOX-1^- CD15⁺ PMNs, as well as the expression of the NADPH oxidase NOX2 and arginase I. RNA sequence revealed that LOX-1⁺ CD15⁺ PMN-MDSCs displayed significantly higher expression of spliced X-box -binding protein 1 (sXBP1), an endoplasmic reticulum (ER) stress marker. ER stress inducer induced LOX-1 expression and suppressive function for CD15⁺ PMN from health donor. For HCC patients, LOX-1⁺ CD15⁺ PMN-MDSCs were positively related to overall survival. Above all, LOX-1⁺ CD15⁺ PMN-MDSC were elevated in HCC patients and suppressed T cell proliferation through ROS/Arg I pathway induced by ER stress. They presented positive association with the prognosis of HCC patients.

Keywords: endoplasmic reticulum stress; hepatocelluar carcinoma patients; lectin-type oxidized LDL receptor-1; polymorphonuclear myeloid-derived suppressor cell; prognosis.

Figure 1

Expansion of lectin‐type oxidized LDL receptor‐1 (LOX‐1)⁺  CD15⁺ polymorphonuclear myeloid‐derived suppressor cells (PMN‐MDSC) in peripheral blood mononuclear cells (PBMC) and whole blood (WB) in hepatocellular carcinoma (HCC) patients. (a) Gating strategy and statistical analysis of LOX‐1⁺  CD15⁺ PMN‐MDSCs by flow cytometry analysis in WB of HCC patients and health control. (b) Expression of LOX‐1 of human leucocyte antigen D‐related (HLA‐DR) ^−/low CD11b⁺  CD15⁺  CD14⁻ PMN‐MDSCs in PBMC from HCC patients and healthy controls. ***P < 0·001. (c) Typical morphology of sorted LOX‐1⁺ and LOX‐1⁻ PMN from a patient with HCC. (d) the frequency of LOX‐1⁺  CD15⁺ PMN‐MDSC in the PBMC and WB from the same patient. (e) Immunohistochemistry analysis of LOX‐1 (red) and CD15 (brown) expression. Brown arrow indicates LOX‐1⁻  CD15⁺ PMN‐MDSC. Red arrow indicates LOX‐1⁺  CD15⁺ PMN‐MDSC.

Figure 2

Lectin‐type oxidized LDL receptor‐1 (LOX‐1)⁺  CD15⁺ polymorphonuclear myeloid‐derived suppressor cells (PMN‐MDSC) from whole blood (WB) and peripheral blood mononuclear cells (PBMC) of hepatocellular carcinoma (HCC) patients suppressed T‐cell proliferation and activation. CD3⁺ T cells from PBMC were stimulated with anti‐CD3/anti‐CD28, cocultured with LOX‐1⁺  CD15⁺ PMN‐MDSCs (a) or LOX‐1⁻  CD15⁺ PMN (b) from the same donors at different ratios for 3 days, evaluated for CD4⁺ and CD8⁺ T cell proliferation by 5,6‐carboxyfluorescein diacetate, succinimidyl ester (CFSE) labelling and interferon (IFN)‐γ production in supernatants by ELISA. Down cumulative data (n = 4) and concentration of IFN‐γ in the media (n = 4). **P < 0·01; ***P < 0·001.

Figure 3

Lectin‐type oxidized LDL receptor‐1 (LOX‐1)⁺  CD15⁺ polymorphonuclear myeloid‐derived suppressor cells (PMN‐MDSC) suppress functional T cells in a reactive oxygen species/arginase 1 (ROS/Arg1)‐dependent manner. (a) Effect of arginase inhibitor N omega‐hydroxy‐L‐arginine (NOHA), L‐arginine supplementation or ROS inhibitor N‐acetyl‐L‐cysteine (NAC) on LOX‐1⁺  CD15⁺ PMN‐MDSC function. T cells from end stage renal disease (ESRD) patients were stimulated with anti‐CD3/anti‐CD28, cocultured with LOX‐1⁺  CD15⁺ PMN‐MDSCs from whole blood at a 2 : 1 ratio with treatments as indicated, evaluated for T cell proliferation by CFSE labelling and IFN‐γ production in supernatants by enzyme‐linked immunosorbent assay (ELISA). Representative flow cytometry data, cumulative data (n = 3) and concentration of IFN‐γ in the media (n = 3). (b) ROS level illustrated by 2′,7′‐dichlorofluorescein diacetate (DCFDA) and NOX2 expression in LOX‐1⁺  CD15⁺ PMN‐MDSCs and LOX‐1⁻  CD15⁺ PMN. Right: representative flow cytometry data. Middle: cumulative data (n = 6). Left: NOX2 expression. (c) Expression of Arg1 and arginase activity in LOX‐1⁺  CD15⁺ PMN‐MDSCs and LOX‐1⁻  CD15⁺ PMN. (n = 6). *P < 0·05; **P < 0·01; ***P < 0·001. [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 4

Endoplasmic reticulum stress (ER) stress and clinical significance of lectin‐type oxidized LDL receptor‐1 (LOX‐1)⁺  CD15⁺ polymorphonuclear myeloid‐derived suppressor cells (PMN‐MDSC) in hepatocellular carcinoma (HCC) patients. (a) Hierarchical clustering of samples based on the expression levels of genes differentially expressed between LOX‐1⁺ and LOX‐1⁻ PMN in HCC patients. (b) Cell components analysis of different genes expressed. (c) Molecular function of different genes expressed. (d) Expression of X‐box–binding protein 1 (XBP1), CCAAT/enhancer binding protein (CHOP) and activating transcription factor 3 (ATF3) in LOX‐1⁺  CD15⁺ PMN‐MDSCs and LOX‐1⁻  CD15⁺ PMN by quantitative reverse transcription–polymerase chain reaction (qRT‐PCR). (n = 6). (e) Expression of XBP1, CHOP and ATF3 in LOX‐1⁺  CD15⁺ PMN‐MDSCs and LOX‐1⁻  CD15⁺ PMN by Western blot.

Figure 5

Endoplasmic reticulum stress (ER) stress induced lectin‐type oxidized LDL receptor‐1 (LOX‐1) expression and suppressive function of LOX‐1‐CD15⁺ polymorphonuclear cells (PMN). (a) Percentage and suppression to T cell proliferation of LOX‐1⁺  CD15⁺ PMN‐myeloid‐derived suppressor cells (MDSC) derived from CD15⁺ PMN isolated from six healthy donors and treated with 1 mM thapsigargin (THG) with or without 20 mM B‐I09. (b) X‐box–binding protein 1 (XBP1) binding sites in the origin of light‐strand replication (OLR) promoter. (c) Chromatin immunoprecipitation (ChIP) assays were performed with LOX‐1⁺  CD15⁺ PMN‐MDSC with antibodies against XBP1 or with an immunoglobulin (Ig)G control. Recruitment of XBP1 to the LOX‐1 promoter was determined by qPCR in eluted DNA. Data were normalized to the input DNA and are presented as fold increases over the values obtained with IgG. (d) Percentage of LOX‐1⁺ CD15⁺ PMN‐MDSC derived from CD15⁺ PMN isolated from six healthy donors treated with serum from hepatocellular carcinoma (HCC) patients with increased LOX‐1⁺  CD15⁺ PMN‐MDSC and conditioned medium from HCC tumour cells (TCM) was used at 20% (v/v) concentration. [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 6

Prognostic value of lectin‐type oxidized LDL receptor‐1 (LOX‐1)⁺  CD15⁺ polymorphonuclear myeloid‐derived suppressor cells (PMN‐MDSC) in hepatocellular carcinoma (HCC) patients. (a) Association between LOX‐1⁺  CD15⁺ PMN‐MDSC and clinical parameters by linear regression. (b) Kaplan–Meier survival curves are shown for overall survival in low and high LOX‐1⁺  CD15⁺ PMN‐MDSC level HCC patients. Hazard ratios (HRs) were calculated using the unadjusted Cox proportional hazards model. P‐values were calculated using the unadjusted log‐rank test; 95% CI indicates 95% confidence interval. [Colour figure can be viewed at http://wileyonlinelibrary.com]