Fig 2: Tumor infiltrating macrophages express EP4 in a murine C3L5 breast cancer model. (a) Day 12 vehicle-treated tumor sections showing F4/80-positive cells (green), expressing EP4 (red) in single channels and yellow in merged channel. The inset is shown enlarged in the next panel. Every nucleated cell (nuclei stained blue with DAPI) inclusive of tumor cells was EP4-positive. (b) Cytoplasmic staining of EP4 in C3L5 cells in culture. (c, d) Treatments with celecoxib and RQ-15986 (EP4A) reduce the incidence of macrophages and vascular endothelial growth factor (VEGF)-C/D-producing macrophages. Merged immunofluorescence images of tumor sections in vehicle-treated control or drug (celecoxib or EP4A)-treated mice showing staining for F4/80 (green), (c) VEGF-C (red, left panel), or (d) VEGF-D (red, right panel) and nuclei (blue for DAPI). Double-stained cells appear yellow. As the vehicle controls for both drugs were similar, a single control is shown. Bottom charts showing incidence of macrophages expressing VEGF-C (bottom left) or VEGF-D (bottom right). Scale bar = 50 μm. (n = 16, using the mean of three hot spots from each of the 16 tumors per group) ± SE. *P < 0.005; **P < 0.0005. (e, f) Treatments with celecoxib and EP4A reduce the VEGF-A/C/D production of macrophages. RAW 264.7 cells treated for 24 h with celecoxib and EP4A significantly reduced VEGF-A/C/D production tested with (e) quantitative PCR and (f) Western blots. Data represents mean fold change (n = 3) ± SE. *P < 0.05; **P < 0.005; #P < 0.0005.

Fig 3: The effect of metformin on ameliorating diet-induced steatohepatitis is diminished in Sirt 1 +/- mice. After metformin treatment, (A) Serum alanine transaminase (ALT), (B) Aspartate transaminase (AST) and (E) the relative mRNA levels of M1 markers were significantly decreased (p < 0.05) while (F) the relative mRNA levels of M2 markers were unaffected in DIO mice (p > 0.05). SIRT1 deficiency impaired the effect of metformin on decreasing (C) serum ALT levels and downregulating (G) the relative mRNA expressions of M1 markers (p > 0.05). (H) The relative mRNA levels of M2 markers were also unaffected by metformin in Sirt11 +/− mice (p > 0.05). (I–L) The F4/80, M1 markers iNOS, M2 markers CD206 immunohistochemical staining, and the quantification of those protein expressions of the liver in Sirt11 +/− mice also confirmed that the metformin-induced downregulation of pro-inflammatory factors was impaired in Sirt1 +/− mice (p > 0.05). Data are shown as mean ± SEM. In C57BL/6 mice, *p < 0.05, HFD group vs Chow diet group; # p < 0.05, HFD group vs HFD + Metformin group. In Sirt11 +/− mice, *p < 0.05, WT + HFD + Metformin or Sirt1 +/− + HFD vs. WT + HFD group, # p < 0.05, Sirt1 +/− + HFD + Metformin vs. Sirt1 +/− + HFD group.

Fig 4: The cell-specific expression pattern of NLRP3 in the fibrotic liver of BDL-treated mice. (A) Mice were anesthetized and sacrificed at 2 weeks after sham or BDL operation. NLRP3 positive cells of the mouse liver non-parenchymal cells were analyzed for T cells, endothelial cells, dendritic cells, and macrophages-specific markers (CD3e, CD31, CD11c, and F4/80, respectively). Mice were lethally irradiated and received whole bone marrow cell transplants from EGFP transgenic mice, followed by sham or BDL operation. At 2 weeks after sham or BDL operation, the proportion of NLRP3+ cells in BMMs (F4/80+ EGFP+) was analyzed by FACS. (B) Representative images of immunofluorescence analysis by confocal microscopy to track the expression of NLRP3 (red) in macrophages (F4/80+, pink) of bone marrow origin (EGFP+, green) in mouse liver tissue. Data are presented as the mean ± SEM. n = 6 per group. Student's t-test was used in panel (A). *p < 0.05 vs. sham group.

Fig 5: The immunofluorescence staining of a7nAChR and F4/80 in each group. This figure demonstrates that LL-TS can significantly increase the a7nAChR expression on cardiac macrophages, indicating that LL-TS can improve sepsis-induced myocardial dysfunction by activating cardiac cholinergic anti-inflammatory pathway and regulating macrophages.