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Effects of AGP on PD-L1 expression and STAT signaling cascade in macrophages. HMDMs were incubated with the indicated concentrations of AGP for 24 h (A) or with AGP (1 mg/mL) for the indicated times (B), followed by the determination of PD-L1 and β-actin expression by western blot analysis. HMDMs were incubated with the indicated concentrations of AGP for 24 h (C) or with AGP (1 mg/mL) for the indicated times (D), followed by the determination of PD-L1, p-STAT1, STAT1, p-STAT3, STAT3, and β-actin expression by western blot analysis. HMDMs were incubated with AGP (1 mg/mL) in the presence of nifuroxazide (200 μM) for 24 h, followed by the determination of PD-L1, pSTAT1, STAT1, and β-actin expression by western blot analysis (E). The experiments were repeated three times with nearly identical results.

Blood levels of acute-phase protein α1-acid glycoprotein (AGP, orosmucoid) increase in patients with cancer. Although AGP is produced from hepatocytes following stimulation by immune cell–derived cytokines under conditions of inflammation and tumorigenesis, the functions of AGP in tumorigenesis and tumor progression remain unknown. In the present study, we revealed that AGP contributes directly to tumor development by induction of programmed death ligand 1 (PD-L1) expression and IL6 production in macrophages. Stimulation of AGP induced PD-L1 expression in both human monocyte–derived macrophages through STAT1 activation, whereas AGP had no direct effect on PD-L1 expression in tumor cells. AGP also induced IL6 production from macrophages, which stimulated proliferation in tumor cells by IL6R-mediated activation of STAT3. Furthermore, administration of AGP to AGP KO mice phenocopied effects of tumor-associated macrophages (TAM) on tumor progression. AGP decreased IFNγ secretion from T cells and enhanced STAT3 activation in subcutaneous tumor tissues. In addition, AGP regulated PD-L1 expression and IL6 production in macrophages by binding with CD14, a coreceptor for Toll-like receptor 4 (TLR4), and inducing TLR4 signaling. These results provide the first evidence that AGP is directly involved in tumorigenesis by interacting with TAMs and that AGP might be a target molecule for anticancer therapy.Significance:AGP-mediated suppression of antitumor immunity contributes to tumor progression by inducing PD-L1 expression and IL6 production in TAMs.

Effect of macrophage depletion on tumor development in AGP-treated tumor-bearing mouse model. AGP (2 mg/mouse) was administered to LM8 bearing C3H HeN mice treated with clodronate liposome to deplete macrophages, followed by the determination of subcutaneous tumor weights (A) (n=10 mice per group). Iba-1-positive cells in LM8 subcutaneous tumor tissues were evaluated by immunostaining (B). Statistical differences between groups were calculated by non-repeated-measures ANOVA.

Effects of GROα and CXCL8 on STAT3 activation in tumor cells, and the effect of AGP on tumor proliferation in tumor cells. Several tumor cells were incubated with GROα (1 ng/mL) or CXCL8 (1 ng/mL) for 24 h, followed by the determination of p-STAT3, STAT3, and β-actin expression by western blot analysis (A). Several human tumor cells were incubated with AGP (1 mg/mL) (NCI-H358, n=4 per group; SK-MEL-28, n=4 per group; 786-O, n=4 per group; ES2, n=3 per group; SBC3, n=3 per group; and HepG2, n=4 per group) for 24 h, followed by the determination of tumor proliferation by Cell Counting Kit-8 (B). The experiments were repeated three times with nearly identical results.

Involvement of IL-6R in AGP-MCM-induced STAT3 activation in tumor cells. IL-6R expression in macrophages and several tumor cells were determined by western blot analysis (A). HMDMs were incubated with IL-6 (10 ng/mL) and soluble IL-6R (20 ng/ml) for 24 h, followed by the determination of p-STAT3, STAT3, and β-actin expression by western blot analysis (B).

Effect of AGP on PD-L1 expression and STAT3 activation in both mouse macrophages and mouse tumor cells. Mouse peritoneal macrophages were incubated with AGP (1 mg/mL) for 24 h, followed by the determination of PD-L1 expression by western blot analysis and flow cytometry (A). Mouse tumor cells were incubated with AGP (1 mg/mL) for 24 h, followed by the determination of PD-L1 and β-actin expression by western blot analysis (B). Mouse peritoneal macrophages from AGP KO mice (C) and LLC cells (D) were incubated with serum derived from MC38-bearing AGP-KO mice treated with or without AGP for 24 h, followed by the determination of PD-L1 and β-actin expression by western blot analysis. LLC cells were incubated with AGP (1 mg/mL) (n=5 per group) for 24 h, followed by the determination of tumor proliferation by Cell Counting Kit-8 (E). MC38 cells were incubated with macrophages culture supernatant prepared from peritoneal macrophages of AGP-KO mice stimulated with serum derived from MC38-bearing AGP-KO mice treated with or without AGP for 24 h (F). IL-6 production in macrophages culture supernatant were evaluated by ELISA (F and G). pSTAT3, STAT3, and β-actin expression in the lysate of MC38 cells were evaluated by western blot analysis (F and H).

RNA-seq transcriptome analysis of macrophages vs macrophages with AGP samples. The heat map represents the normalized gene level RNA-Seq expression data in TPM (Transcripts per Million) for 1204 significant genes (q-value < 0.05) between macrophages vs macrophages with AGP samples (A). The Scatter blot represents the normalized gene level RNA-Seq expression data for all genes. Grey circle represented non-significant genes. Magenta circle represented significantly up-regulated genes. Cian circle represented down-regulated genes. In total 1204 genes were up- or down regulated, of which 503 genes were up-regulated genes (macrophages > macrophages with AGP, P

The list of antibodies for western blot analysis and primers for RT-qPCR

Effects of AGP-treated macrophage-conditioned medium on STAT3 activation in tumor cells. Several tumor cells were incubated with AGP-MCM for the indicated times, followed by the determination of p-STAT3, STAT3, and β-actin expression by western blot analysis.

Blood levels of acute-phase protein α1-acid glycoprotein (AGP, orosmucoid) increase in patients with cancer. Although AGP is produced from hepatocytes following stimulation by immune cell–derived cytokines under conditions of inflammation and tumorigenesis, the functions of AGP in tumorigenesis and tumor progression remain unknown. In the present study, we revealed that AGP contributes directly to tumor development by induction of programmed death ligand 1 (PD-L1) expression and IL6 production in macrophages. Stimulation of AGP induced PD-L1 expression in both human monocyte–derived macrophages through STAT1 activation, whereas AGP had no direct effect on PD-L1 expression in tumor cells. AGP also induced IL6 production from macrophages, which stimulated proliferation in tumor cells by IL6R-mediated activation of STAT3. Furthermore, administration of AGP to AGP KO mice phenocopied effects of tumor-associated macrophages (TAM) on tumor progression. AGP decreased IFNγ secretion from T cells and enhanced STAT3 activation in subcutaneous tumor tissues. In addition, AGP regulated PD-L1 expression and IL6 production in macrophages by binding with CD14, a coreceptor for Toll-like receptor 4 (TLR4), and inducing TLR4 signaling. These results provide the first evidence that AGP is directly involved in tumorigenesis by interacting with TAMs and that AGP might be a target molecule for anticancer therapy. Significance: AGP-mediated suppression of antitumor immunity contributes to tumor progression by inducing PD-L1 expression and IL6 production in TAMs.

Objectives Cardiovascular disease is one of the major causes of death in patients with end-stage kidney disease who have undergone kidney transplantation. Since the complication of cardiovascular disease in patients with chronic kidney disease is strongly linked to oxidative stress, understanding the oxidative stress condition after kidney transplantation would be of great importance for the prevention of cardiovascular disease. This study examined whether improvement of renal function after kidney transplantation has an impact on the redox state of the Cys34 residue of albumin that reflects the level of oxidative stress in blood. Design & methods We enrolled 23 patients with end-stage renal failure who received kidney transplantation. All patients were followed for 180 days after transplantation. The fractions of albumin isoforms were determined by the electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) method. Results Serum creatinine decreased significantly immediately after kidney transplantation, suggesting successful transplantations. The ESI-TOFMS method identified three albumin isoforms cysteinylated at the Cys34 residue (Cys-Cys34-albumin) and the three corresponding albumin isoforms without Cys34 cysteinylation. The fraction of total Cys-Cys34-albumin decreased transiently after kidney transplantation, and was followed by an elevation at day 7 and gradual decrease thereafter until day 180. Meanwhile, reduced albumin concentration did not change until day 14 after kidney transplantation, then showed a significant increase compared to pre-transplant level at day 30 and remained stably elevated until day 180. Conclusions Actual reduced albumin levels were found to exceed pre-transplant levels on or after day 30 following kidney transplantation unlike immediate restoration of renal function. Renal function was recovered immediately following kidney transplantation, but reduced albumen concentration increased above the pre-transplant levels only from day 30 after transplantation.

Kupffer cells are a major producer of reactive oxygen species and have been implicated in the development of liver fibrosis during chronic hepatitis in non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH). We recently reported on the development of a polythiolated and mannosylated human serum albumin (SH-Man-HSA) that functions as a Kupffer cell-targeting nanoantioxidant. In this material, the albumin is mannosylated, which permits it to be taken up by mannose receptor C type 1 expressed on Kupffer cells, and is also polythiolated to have antioxidant activity. To clarify the anti-fibrotic property of this nanoantioxidant, we repeatedly administered SH-Man-HSA to a liver fibrosis mouse model that was induced by the repeated treatment of the concanavalin-A, which mimics the liver fibrosis observed in NASH and ASH. SH-Man-HSA dramatically improved the survival rate and suppressed liver fibrosis in the experimental model. In addition, SH-Man-HSA suppressed hepatic oxidative stress levels, thereby decreasing the numbers of apoptotic cells. In contrast, N-acetylcysteine, which contains the same thiol content as the SH-Man-HSA, failed to show a substantial therapeutic effect in these mice. The expression levels of inflammatory genes including epidermal growth factor module-containing mucin-like receptor (Emr-1/F4/80), Toll-like receptor-4 (TLR-4), high mobility group box-1 (HMGB-1), CC chemokine ligand-5 (CCL-5), tumor necrosis factor-α (TNF-α), CCL-2, interleukin-6 (IL-6), and IL-1β, as well as fibrotic (α-smooth muscle actin (α-SMA), transforming growth factor-β (TGF-β), and Snail) and extracellular matrix genes (collagen, type Iα2 (Col1α2), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase 1 (TIMP-1)), showed some decreasing trends by the SH-Man-HSA administration. These findings suggest that the repeated administration of the Kupffer cell-targeting nanoantioxidant, SH-Man-HSA, ameliorates liver fibrosis in mice by suppressing the level of oxidative stress and a portion of the inflammation, and has a potential therapeutic effect against NASH and ASH.

Nanosize plasma proteins could be used as a biomimetic drug delivery system (DDS) for cancer treatment when loaded with anticancer drugs based on the fact that plasma proteins can serve as a source of nutrients for cancer cells. This prompted us to investigate the potential of α1-acid glycoprotein (AGP) for this role because it is a nanosize plasma protein and binds a variety of anticancer agents. Pharmacokinetic analyses indicated that AGP is distributed more extensively in tumor tissue than human serum albumin, which was already established as a cancer DDS carrier. AGP is possibly being incorporated into tumor cells via endocytosis pathways. Moreover, a synthetic AGP-derived peptide which possesses a high ability to form an α-helix, as deduced from the primary structure of AGP, was also taken up by the tumor cells. AGP loaded with anticancer agents, such as paclitaxel or nitric oxide, efficiently induced tumor cell death. These results suggest that AGP has the potential to be a novel DDS carrier for anticancer agents.

Blood levels of acute-phase protein α

Nanosize plasma proteins could be used as a biomimetic drug delivery system (DDS) for cancer treatment when loaded with anticancer drugs based on the fact that plasma proteins can serve as a source of nutrients for cancer cells. This prompted us to investigate the potential of α