Figure legends
Fig. 1. IL-28B therapy efficiently suppressed subcutaneous MC38 tumors. C57BL/6 mice (6-8 weeks) were inoculated subcutaneously with 1×106 MC38 tumor cells. From day 0, tumor volume was measured every 48 h, and IL-28B (2.5 µg/mouse) was injected into the tissue surrounding the tumor once a day. All mice were assayed on day 18. (A) The schedule of IL-28B in the treatment of mouse colon tumors. (B) Representative images of each group of tumors after IL-28B treatment. (C) Based on tumor volume, the IL-28B effectively inhibited the tumor growth compared with the control group. (D) The weight of each group of tumors is shown. (E) H&E staining of paraffin-embedded tumor sections. IL-28B protein increased tumor tissue necrosis, ×200 original magnification; n = 5; **P < 0.01; *** P < 0.001.
Fig. 2. IL-28B reduces M2 macrophages in tumor tissues. (A, B) Representative
FACS plots and ratio of macrophages (CD11b+F4/80+CD206+) in tumors (CD45 events). (C-I) Changes in M1 macrophages (CD11b+F4/80+CD86+), MDSC (CD11b+Gr-1+), CD4 cells (CD3+CD4+) and CD8 cells (CD3+CD8+) in tumors. (J) Immunofluorescence staining of infiltrated F4/80+CD206+ M2 macrophages in tumors. Red, anti-F4/80 Ab; green, anti-CD206 Ab; yellow, F4/80 and CD206 merged; blue, DAPI. Original magnification: ×200. **P < 0.01; n.s. denotes, P > 0.05.
Fig. 3. Effect of IL-28B on M1 and M2 macrophage polarization in vitro . (A) IL-28B treatment of M2 macrophages and experimental workflow. Briefly, bone marrow cells were isolated from the femur and tibia of mice in PBS. Then, cells were cultured in complete DMEM containing GM-CSF (10 ng/mL), and IL-28B protein (0, 100, 200, and 400 ng/mL) in treatment groups. After four days, media was supplemented. On the seventh day, BMDMs were harvested, IL-4 (10 ng/mL) and IL-13 (10 ng/mL) were used to polarize BMDMs to M2 macrophages for subsequent experiments. M1 and M2 macrophages were similarly treated with IL-28. (B - E) The proportions of M1 and M2 in BMDMs were determined using flow cytometry. Data are expressed as mean ± SEM.*P < 0.05, **P < 0.01 and ***P < 0.001.
Fig. 4. IL-28B treatment inhibits the expression of marker cytokines in M2 macrophages. (A) Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of cytokines, TGF-β (supernatant), and Arg-1 (intracellular). The cell culture supernatants and total cellular proteins were collected after 24 h of polarization of M2 macrophages. (B) Real-time PCR was used to detect cytokine mRNA expression in M2 macrophages. The total cellular RNA was collected after 4 h of polarization of M2 macrophages. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01 and ***P < 0.001; n.s. denotes, P > 0.05.
Fig. 5. IL-28B protein downregulates the STAT3 and JNK signaling pathways in M2 macrophages. The total cell protein was collected after 1 h of polarization of M2 macrophages. The levels of STAT3, p-STAT3, p-JNK, and JNK were analyzed by western blot analysis.