Fig 2: EPZ6438 reversed M2 macrophage re-polarization toward the M1 type. (A) Schematic illustration of EZH2 inhibitor treatment schedule. (B) RAW264.7 cells were pre-treated for 24 h with IL-4 (20 ng/ml) to induce M2 macrophages. Then, fresh medium was added with EPZ6438 (10, 20 μM) or GSK126 (5, 10 μM) and treated for another 48 h. Control cells were treated with DMSO. The scatter diagram of flow cytometry on CD86+ F4/80+ cells ratio. (C) The statistical graph of flow cytometry on CD86+ macrophages ratio in CD11b+ F4/80+ cells. (D) The histogram of mean fluorescence intensity of CD86+ cells. Gray color represents the control sample; red and green colors represent 5 μM and 10 μM GSK126, respectively; blue and yellow colors represent 10 μM and 20 μM EPZ6438, respectively. (E) mRNA level of Arg1 was analyzed by RT-PCR according to the EZH2 inhibitor treatment schedule described in (A). Data are shown as mean ± SEM and are representative of one out of three independent experiments with similar results. *p < 0.05, **p < 0.01, ****p < 0.0001, ns, no significance by one-way ANOVA. (F) The protein level of Arg1 was detected by Western blot. The protein level of Arg1 was significantly downregulated after different doses of GSK126 and EZP6438.

Fig 3: Macrophages protect PTPN2fl/flxCD11cCre mice from elevated colitis. PTPN2fl/fl (WT) and PTPN2fl/flxCD11cCre (KO) mice received 2.5% DSS for 7 days and were treated with vehicle liposomes or clodronate liposomes on day-1, day 2, and day 4 to deplete macrophages: (A) weight development post injection; (B) representative pictures from colonoscopy and assessment of colitis severity according to the murine endoscopic index of colitis severity (MEICS); (C,D) representative pictures from H&E staining of the terminal colon and histological scoring; (E) colon length, MPO activity, and spleen weight; (F) IHC staining for F4/80+ macrophages in the colon; (G) frequencies of IFNγ+ and IL17+ CD4+ T cells in the lamina propria. * p < 0.05, ** p < 0.01, *** p < 0.001; unpaired Mann–Whitney test. Data are representative for one out of two independent experiments with 3–4 mice per experimental group.

Fig 4: Early lesions developed in AKT/TAZ mice display an inflammatory infiltrate. (A) Two weeks after hydrodynamic injection of AKT and TAZ, numerous transfected cells (indicated by black arrows) were encircled by an inflammatory infiltrate. The transfected cells and the inflammatory cells could be better appreciated at higher magnification (B), with the inflammatory cells characterized by small spherical nuclei and forming a fence around the transfected cells. The transfected cells displayed elevated TAZ (C) and CK19 (D) immunoreactivity. The inflammatory cells consisted of lymphocytes, characterized by immunoreactivity for CD4 and CD45 markers (E, F), and macrophages (immunoreactivity for the F4/80 marker) (G). Some small cells encircled by the inflammatory reaction showed signs of apoptosis, as revealed by positive immunoreactivity of apoptotic bodies (indicated by black arrows) for cleaved Caspase 3 (H) and cleaved PARP (I), implying their destruction by the inflammatory response. Original magnification: 100x in (A), 400x in (B-I); scale bar: 200 μm in (A), 50 μm in (B-I).

Fig 5: Comparative analysis of lung pathology and immune cell infiltration. (A) Lung tissue harvested at 0,0.25,1,2,3, and 7 dpi. from mice that had been infected H7N9 or H9N2 avian influenza virus was stained with H&E. (B) Quantified pathological scores derived from the calculation of lung histology data. (C) Immuno-histochemical detection of total macrophages in the lung sections by staining with anti-F4/80 antibody, with nuclei counterstained by hematoxylin. Brown signals indicate detected macrophages. (D) Summation data of the measurements of lung macrophages. (E) Immunofluorescence detection of CD4+ plus CD8+ T cells in the lung sections by sequential staining with anti-CD4 and anti-CD8 antibodies, with nuclei counterstained by DAPI. Red and green signals indicate detected CD8+ and CD4+ T cells, respectively. (F) Summation data of the measurements of CD4+ and CD8+ T cells in the lung. n=3 mice for each group per time point. **P < 0.01 by unpaired t test.