Abstract
In many human cancers, including hepatocellular carcinoma (HCC), high density of infiltrating tumor-associated macrophages (TAM) is associated with poor prognosis. Most TAMs express a M2 phenotype subsequently supporting tumor growth. How tumor cells polarize these TAMs to a pro-tumor M2 phenotype is still poorly understood. Our previous studies have revealed that a Toll-like receptor 2 (TLR2)-dependent autophagy triggered by hepatoma-derived factors down-regulates NF-κB p65 and drives M2 macrophage differentiation. However, the underlying mechanisms and potential hepatoma-derived TLR2 ligands are not clear. Here, we provide evidence to reveal that NADPH oxidase 2 (NOX2)-dependent reactive oxygen species (ROS) generation is crucial for HCC-induced autophagy, NF-κB p65 down-regulation and M2 phenotype polarization in primary macrophages. This NOX2-generated ROS production in abolished in TLR2-deficient macrophages. HCC-derived or recombinant high-mobility group box 1 (HMGB1) is able to trigger this TLR2-mediated M2 macrophage polarization. Blockage of HMGB1 and ROS by inhibitors, ethyl pyruvate and N-acetylcysteine amide, respectively, significantly reduces both M2 macrophage accumulation and liver nodule formation in HCC-bearing mice. Our findings uncover a HMGB1/TLR2/NOX2/autophagy axis to trigger M2 macrophage polarization in HCC that can be considered as a novel therapeutic target for treating HCC.
Highlights
Leukocyte subpopulations have been found to accumulate within tumors
Cell lysates were collected to determine the expression of NF-κB p65, LC3-I/II, and p62 by Western blotting. (d) Wild type (WT) or g p91−/− bone marrow-derived macrophages (BMDMs) were treated with ML-14a conditioned medium (MCM) for the indicated time and the reactive oxygen species (ROS) production was determined by flow cytometry. (e) WT or gp91−/− BMDMs were treated with MCM for 24 h and supernatants were collected to analyze the production of IL-10 by ELISA. (f) NADPH oxidase 2 (NOX2)-depedent autophagy is induced by MCM
Inhibition of ROS by N-acetylcysteine amide (NAC) suppressed MCMtriggered LC3 II accumulation and degradation of NF-κB p65, a cargo of autophagosomes in MCM-polarized macrophages[5], or p62 (Fig. 1c). These results revealed that generation of ROS is involved in hepatoma-triggered autophagy and M2 macrophage polarization
Summary
Leukocyte subpopulations have been found to accumulate within tumors. One prominent population is tumorassociated macrophages (TAM), and immunologists think that the presence of TAM is evidence of a host immune response against growing tumors, many studies have demonstrated that these TAMs promote tumor progression[1]. NOX2-generated ROS is responsible for hepatoma-induced autophagy and M2 macrophage polarization. (d) Wild type (WT) or g p91−/− BMDMs were treated with MCM for the indicated time and the ROS production was determined by flow cytometry. (g) WT or gp91−/− BMDMs were treated with MCM for the indicated time Their cell lysates were collected to determine the expression of NF-κB p65, LC3-I/II, and p62 by Western blotting. We demonstrated that hepatoma cell-derived factors are able to trigger M2 macrophage polarization via toll-like receptor (TLR) 2 signaling. This TLR2 signaling pathway was found to down-regulate NF-κB p65, a M1-promoting transcription factor, via p62-mediated selective autophagy[5]. The contribution of NOX-derived ROS in hepatoma-induced M2 macrophage polarization remains unclear
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