Fig 2: DU325 treatment hampered the viability of splenocytes, both CD11b+/Ly6C+ and CD11b+/Ly6G+ splenic MDSCs. Cells were treated with 5 µM, 1 µM, 200 nM, 40 nM, and 20 nM DU325 for 72 h and assayed by the resazurin viability test (A). The percentage of AnnV+/PI+ late apoptotic cells was increased after treatment with 200 nM DU325 for 72 h (B). The immunofluorescent staining showed a reduction of both monocytic CD11b+/Ly6C+ and granulocytic CD11b+Ly6G+ MDSCs treated with 200 nM DU325 for 72 h ex vivo (C). Data are shown as arithmetic mean values ± standard deviation from triplicate experiments. Statistical significance was set to *** p < 0.001.

Fig 3: In situ clearance of EMT6-Luc cells in EMT6 tumor-primed mice. Primary tumor-bearing (EMT6 or 4T1) or the naive BALB/c mice were injected with EMT6-Luc cells (100K) via the tail vein 3-weeks post implantation. a, b Live animal and lung ex vivo bioluminescent images show entrapped EMT6-Luc cells after 2 h of tail vein injection in naive, EMT6, and 4T1 tumor-bearing mice, respectively. c, d Live imaging of mice and ex vivo imaging of lungs at 3 days post injection of indicated animals. The reduced signal intensity of EMT6-Luc cells in the lungs of EMT6 tumor-bearing mice compared with naive and 4T1 tumor-bearing mice is associated with less number of tumor cells. e, f Pan-keratin staining of tumor cells and TUNEL-positive apoptotic cells in the lungs of the indicated animals at day 3. g Flow-cytometry analyses of CD8+ T cells in the lungs of naive, EMT6, and 4T1 tumor-bearing mice. h, i Flow-cytometry analysis shows an elevated CD11b+Ly6Cint cell population (gMDSC) in the lungs of 4T1 tumor-bearing mice compared with naive and EMT6 tumor-bearing mice. j The CD11b−Ly6C+ population is significantly increased in only EMT6 tumor-bearing mice at day 3 post i.v. injection. Results are presented as mean ± SD (n = 3). Scale bar: 50 µm. *P < 0.05, **P < 0.005, ***P < 0.0005, one-way analysis of variance test

Fig 4: PLX5622 treatment, when post-injury, reduces microglia and infiltrating cells in both intraspinal cord and brain at 7 days after SCI. (A) PLX5622 diet or vehicle chow were fed to animals on day 1 post-injury and maintained up to 7 days. (B-C) Body weight and food intake were recorded before and 1, 3, 5, 7 days after injury. All injured mice displayed a significant decrease in both body weight and food consumption. PLX had no effects on animal weight and food intake after injury. n = 5 mice/group, ***p < 0.001, vs. Sham/Veh group. 2-way ANOVA with repeated measurements following Sidak's multiple comparisons test. (D) Representative dots plots show the relative immune cell composition in the impact site of the spinal cord at 7 days post-injury. (E) The number of living CD45intCD11b+Ly6C- microglia (left) and CD45hiCD11b+ infiltrating cells (right) in each group is shown, n = 5 mice/group. (F) Representative dot plots depict the composition of immune cells in the brain after SCI. (G) The number of brain-resident microglia (top) and infiltrating myeloid cells (bottom) is quantified, n = 4-5 mice/group. *p < 0.01, ***p < 0.001, ++++p < 0.0001, vs. Sham/Veh group. ####p < 0.0001, vs. SCI/Veh group. 2-way ANOVA following Tukey's multiple comparisons test.

Fig 5: Splenectomy reduces cardiac infiltration of monocytes and heart failure in 5/6Nx mice.a Induction of GPR68 expression in circulating monocytes in 5/6Nx mice. Flow cytometry analysis was performed to detect high-GPR68-expressing CD11b+/Ly6C+ cells in the spleen, blood, and bone marrow of Sham and 5/6Nx mice. b Decrease in the number of cardiac-infiltrated CD11b+/Ly6C+ cells in splenectomized (Splx)-5/6Nx mice. Left panel shows the flow cytometry histogram of CD11b+/Ly6C+ cells isolated from the cardiac ventricle of Sham, 5/6Nx, Splx, and Splx-5/6Nx mice. The number of CD11b+/Ly6C+ cells, indicated by red circles, was quantified and the data are shown in the right panel. c Serum concentrations of BNP in Sham, 5/6Nx, Splx, and Splx-5/6Nx mice. d The mRNA levels of inflammatory cytokines (Tnf-a and IL-6) and fibrosis-related factors (aSma and Col1a2) in the cardiac ventricle of Sham, 5/6Nx, Splx, and Splx-5/6Nx mice. The mean value of the sham-operated wild-type group was set as 1.0. e Double immunofluorescence labeling of GPR68 with F4/80 in the ventricle slices prepared from Sham, 5/6Nx, Splx, and Splx-5/6Nx mice. High-GPR68-expressing cells are double-labeled (yellow) with F4/80. The scale bar indicates 50 µm. f Decrease in the number of cardiac-infiltrated high-GPR68-expressing monocytes in Splx-5/6Nx mice. For all panels, graphs show the mean ± SD of individual mice in independent experiments. Statistical significance was determined using two-way ANOVA with Tukey–Kramer post hoc tests (b–d, f). Numbers and P-values are shown in each graph. Source data are provided as a Source Data file.