Fig 2: Interferon-stimulated gene 15 (ISG15) induced the M2-like phenotype and promoted NPC cell migration and tumorigenicity. (A, B) Human peripheral blood mononuclear cell (PBMC)-derived macrophages treated with 59 nM and 295 nM rISG15 for 24 h, and then were detached for fluorescence activated cell sorting (FACS) analysis, or were extracted mRNA for qPCR analysis. (A) Representative data of the switched surface markers profile of M2 (CD163)/M1 (HLA-DR) in ISG15-treated macrophages measured by FACS. (B) rISG15 induced macrophage mRNA expression of M2 cytokines, such as IL-10 and TGF-β, but not of M1 cytokines, such as iNOS, by qPCR analysis. IFN-γ/LPS and IL-4 served as positive control for M1 and M2 polarization. Heat-inactivated recombinant ISG15 (HI ISG15) protein was boiled within 100°C for 15 min. (C) Cytokine panel indicated the increased secretion of M2-polarizing cytokine IL-4 in culture supernatants of ISG15-treated macrophages, but not of M1-polarizing cytokine IL-6 and TNF-α. The culture supernatants were harvested from the macrophages upon rISG15 treatment at indicated dose for 24 h and replaced with serum-free culture for 24 h. (D) Representative photography of transwell filters in HK1 NPC cells cultured with the culture medium (CM) of macrophages. Quantification analysis revealed the increased number of transwell cell in the presence of ISG15 treatment. Data are expressed as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p< 0.0001. (E) Photographic illustration of the grow tumors established by a tumor model within BALB-c mice through subcutaneous inoculation with a mixture of human HK1 NPC cells and PBMC-derived macrophages (at ratio of 3:1) with or without rISG15 pre-treatment. All (10 in 10) mice with ISG15 treatment had tumor grow, but only 8 of 10 mice in the control group had tumor grow. The growth curve showed a significant larger tumor volume in the ISG15 treatment group compared with that of the control. ns, not significant.

Fig 3: ISG15+ CD163+ TAMs suppressed cytotoxic leukocyte (CTL) response in nasopharyngeal carcinoma (NPC). (A) Single cell suspensions from NPC tissues (n=9) were stained with antibodies for ISG15-PE and CD163-APC. Total frequency of ISG15+ CD163+ cells were determined by FACS analysis and were indicated as percentage in upper right quadrant. Left penal showed single cell suspensions of NPC tissue were gated. Right panel showed representative FACS photograph of low or high CD163+ ISG15+ cells. (B) Representative FACS analysis and quantification of the percentage of IFN-γ+, perforin+, and Granzyme B+ cells within CD8+ T cells between high and low CD163+ ISG15+ cells groups in NPC tissue suspensions. (C) Flow cytometric quantification of the percentage of IFN-γ+, perforin+, and Granzyme B+ cells within CD8+ T cells in vitro cocultured with or without the conditional media from ISG15-treated macrophages over the indicated time. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Fig 4: Interferon-stimulated gene 15 (ISG15)-treated macrophages promoted nasopharyngeal carcinoma (NPC) cell migration through CCL18 secretion. (A) Cytokine array of the CM of macrophages induced with rISG15 at the indicated doses for 24 h. The lower table summarizing the relative signal intensity of the indicated cytokines is presented. The red marker box indicates the site of CCL18. (B) Increased level of CCL18 in the supernatant of ISG15-treated macrophages was confirmed by ELISA. Macrophages were treated with the indicated dose of rISG15 for 24 h. The CCL18-inducing effect of ISG15 was blocked by pretreatment with a CCL18 neutralizing antibody, and was not observed in heat-inactivated rISG15. (C) The increased number of infiltrated NPC cells, as measured by transwell assay, in the presence of CM from ISG15-treated macrophages. The enhancement was abolished by anti-CCL18 antibody. Data are expressed as mean ± SD of three independent experiments. **p < 0.01 and ***p < 0.001.

Fig 5: Interferon-stimulated gene 15 (ISG15)-induced CCL18 secretion by macrophages was dependent on the ISG15 receptor, leukocyte function-associated antigen-1 (LFA-1), and SRC family kinase (SFK) signaling. (A) Immunofluorescence demonstrated that the ISG15 receptor CD11a/CD18, also called LFA-1, was expressed on the membranes of human macrophages. Macrophages were treated with 295nM rISG15 for 24 h. Scale bars, 10 μm. (B) ELISA results showed small molecular inhibitors of LFA-1, A286982, inhibited CCL18 secretion by macrophages. (C) Small molecular inhibitors of LFA-1 and SRC, A286982 and PP2, hindered the ISG15-induced activation of SFK signaling. (D) PP2 reduced the CCL18 secretion by ISG15-treated macrophages in a dose-dependent manner, as determined by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.