Fig 1: IL-6 promotes PD-L1 expression in macrophage both directly and IFN-γ dependently by deregulating protein tyrosine phosphatase, receptor type O (PTPRO). (A) Macrophages isolated from wild-type (WT) and PTPRO KO mice were treated with IFN-γ (50 ng/mL), IL-6 (50 ng/mL), and IFN-γ+IL-6 (50 ng/mL) for 72 hours and the transcription and protein expression of Pd-L1 were determined by using real-time PCR and western blotting, respectively. (B) U937-, THP-1–, U937 PTPRO shRNA–, and THP-1 PTPRO shRNA–derived macrophages were treated with IFN-γ (50 ng/mL), IL-6 (50 ng/mL), or IFN-γ+IL-6 (50 ng/mL each), and the transcription and protein expression of PD-L1 were determined by real-time PCR and western blotting, respectively. (C) Macrophages isolated from WT and PTPRO KO mice were treated with IL-6, and the expression of PD-L1 and PTPRO was detected by real-time PCR and western blotting at different times after IL-6 treatment. (D) U937-, THP-1–, U937 PTPRO shRNA–, and THP-1 PTPRO shRNA–derived macrophages were treated with IL-6, and the expression of PD-L1 and PTPRO was detected by real-time PCR and western blotting at different times after IL-6 treatment. Each experiment was performed in triplicate. Data are presented as the mean±SEM and were analyzed by Student’s t-test (**p<0.01).
Fig 2: Schematic diagram of protein tyrosine phosphatase, receptor type O (PTPRO)–related PD-L1 upregulation in macrophages. The increased serum IL-6 in patients with hepatocellular carcinoma activated STAT3/c-MYC signaling in peripheral monocytes, enhanced the transcription of miR-25–3 p, suppressed PTPRO expression, promoted PD-L1 through both JAK2/STAT3/c-MYC and JAK2/STAT1 signaling in tumor-infiltrated macrophages, and promoted tumor growth by enhancing T-cell exhaustion.
Fig 3: IL-6 downregulates protein tyrosine phosphatase, receptor type O (PTPRO) in hepatocellular carcinoma (HCC) monocytes by upregulating miR-25–3 p via STAT3/c-MYC. (A, B) Heatmap and volcano plot indicating significantly expressed miRNA within monocytes in patients with HCC compared with the healthy controls. (C) Anti-CD14 magnetic beads were used to isolate human monocytes from 165 patients with HCC and 155 healthy controls. Transcription of miR-25–3 p was detected using real-time PCR and compared between the two groups. **p<0.01, Student’s t-test. (D, E) The linear correlations between expression of miR-25–3 p and PTPRO or PD-L1 were analyzed in HCC monocytes. (F) The potential binding site and mutation in the 3′ untranslated region (UTR) are indicated in the schematic figure. A luciferase reporter gene assay was carried out to test the promoter activity of PTPRO regulated by miR-25–3 p on the 3′UTR of PTPRO. (G, H) Linear correlations between serum IL-6 and monocyte PTPRO, and serum IL-6 and monocyte miR-25–3 p were analyzed in patients with HCC. (I) THP-1–derived and U937-derived macrophages were treated as indicated, and expression of miR-25–3 p was determined by real-time PCR. **p<0.01, Student’s t-test, compared with control group. (J) Cell signaling was analyzed by western blotting. Each experiment was performed in triplicate. Data are presented as the mean±SEM and were analyzed with Student’s t-test (**p<0.01).
Fig 4: The JAK2/STAT3 Pathway Contributes to STING Dysfunction in Tumor Cells. (A) A549, HeLa, HCT116 and DU145 cells were treated with PBS (−), 2 μg/ml cGAMP and/or 7 μM WP1066 for 4 h. Treated cells were analyzed for the expression of IFNA (black columns) and IFNB (white column) transcripts by real-time PCR. Expression values were normalized to PBS-treated cells. Data are representative of 3 independent experiments. Scale bars denote 10 μm. (B) A549, HeLa, HCT116 and DU145 cells were cultured in presence of 1 μg/ml IL-6 neutralizing or isotype control antibodies for 24 h followed by stimulation with 2 μg/ml cGAMP or PBS for 4 h. Treated cells were analyzed for the expression of IFNA (black columns) and IFNB (white column) transcripts by real-time PCR. Expression values were normalized to PBS-treated cells. (C) Representative confocal microscopy images of DU145, A549, HeLa and HCT116 cells stained for phosphorylated STAT3 (red) in the presence of DAPI (blue). Cells were cultured in the presence of 1 μg/ml IL-6 neutralizing or isotype control antibodies. Bar graph below shows quantification of the number of nuclear phosphorylated STAT3 foci in control and treated cancer cells.
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