Fig 2: ZQT reduced the population of MDSCs but increased the population of CD8+ T cells in tumor tissue. Cells from the tumor tissue of tumor-bearing mice treated with normal saline or ZQT were analyzed by flow cytometry after staining with CD11b, Gr-1, Ly6G, Ly6C, CD3, CD4, and CD8 antibodies. (a–d) Percentages of MDSCs and T cells in tumor. (e, f) Percentages of MDSCs in tumor on days 7, 14, 21, and 28 after inoculation. n = 7. Data are expressed as the mean ± SD. n/s: nonstatistical significance. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 3: As compared to wild-type mice, leukocyte infiltration is not altered in Mer null regenerating TA muscles following cardiotoxin injury, but the pro-inflammatory to healing phenotypic conversion of macrophages is delayed.Alterations a in the number of CD45+ leukocytes per injured muscle, b ratio of anti-F4/80 antibody-stained MFs and anti-Ly6G/Ly6C- (GR-1) stained neutrophils within the CD45+ leukocyte population in Mer+/+ and Mer-/- TA muscles during the first 4 days of regeneration following CTX-induced injury (n = 3). c MCP-1 mRNA expression levels of muscle-derived wild type or Mer null CD45+ leukocytes determined by qRT-PCR following CTX-induced injury (n = 3). d Changes in Mer mRNA expressions of CD45+ infiltrating leukocytes isolated from Mer+/+ and Mer-/- regenerating TA muscles following CTX injury determined by qRT-PCR at the indicated time points (n = 6). e Representative scatter plots of CD206- and Ly6C-stained muscle-derived F4/80+ cells determined at the indicated days following CTX-induced injury (n = 3). f Mer, IL-1ß, and TNF-a mRNA expressions in Ly6Chigh and Ly6Clow F4/80+ cells isolated from TA muscles determined by qRT-PCR at days 2 and 5 post injury (n = 3). g Necrotic C2C12 myoblast cell phagocytic capacity of muscle-derived Mer+/+ and Mer-/- MFs (MMF) isolated at day 4 post-CTX-induced injury determined by flow cytometric analysis (n = 3). h Changes in the mRNA expressions of the indicated cytokines in CD45+ infiltrating leukocytes isolated from Mer+/+ and Mer-/- regenerating TA muscles following CTX-induced injury determined by qRT-PCR (n = 6). i Representative scatter plots of MHCII-stained muscle-derived F4/80+ cells determined at the indicated days following CTX-induced injury (n = 3). j mRNA expressions of the indicated M2 phenotypic markers in Mer+/+ and in Mer-/- muscle-derived MFs isolated at day 4 post CTX-induced injury determined by qRT-PCR (n = 6). All data are expressed as mean ± SEM. Asterisks indicate statistical significance (*P < 0.05, **P < 0.01, Student’s t-test and ANOVA test).

Fig 4: EPO increases recruitment of Ly6Chi monocytes and neutrophils during AILI. (A) Liver non-parenchymal cells were analyzed by multi-parameter flow cytometry. The gating strategy for the definition of CD11b+ (myeloid cells) or CD11b- (lymphocytes) in the APAP-challenged livers was defined by using CD45, F4/80 and CD11b. Bar graphs indicate CD11b+/ CD11b- cell ratio from diluent (control) or EPO-injected mice, 24 h or 48 h post injection with APAP, N = 5, **p < 0.01, *p < 0.05. (B) Serum CCL2 levels detected by ELISA in diluent (control) or EPO-injected mice, 24 h or 48 h after treatment with APAP, N = 4–10, *p = 0.05. (C) Liver non-parenchymal cells were analyzed by flow cytometry. The gating strategy for the definition of Ly6Chi monocytes or neutrophils in the APAP-challenged liver was defined by using CD45, F4/80, CD11b, Ly6C, CX3CR1 and MHCII. Bar graphs indicate Ly6Chi monocytes or neutrophils as a percent of CD45+ liver cells from diluent (control) or EPO-injected mice, 24 h or 48 h post injection with APAP, N = 4–10, *p < 0.05. All graphs represent mean ± SEM.

Fig 5: PGE2 partially rescues Cox2-dependent immune-mediated senescence surveillance(A) Experimental outline; Ptgs2fl/fl mice underwent HDTV injection of transposons containing oncogenic NRASG12V with or without Cre recombinase, leading to Ptgs2 knockout in the same cells. Mice received daily intra-peritoneal injections of either vehicle or dimethyl-PGE2 (dmPGE2; 200 µg/kg) between day 5 and day 8 post-injection. Mice were harvested at day 9 after the HDTV injection.(B) Illustrative photomicrographs of NRAS IHC in indicated conditions.(C) Quantification of NRAS-positive area by IHC. Dots indicate individual mice, and bars represent means. Data were analyzed by one-way ANOVA with Sidak’s multiple comparisons test; *p = 0.05, ***p = 0.001.(D) Representative immunofluorescence images of NRAS and Cxcl1 in mouse livers at day 9 post-HDTV injection of the indicated constructs. Scale bar, 100 µm. White arrowheads demonstrate same Cxcl1+ cells between images. Quantification of the number of NRAS+ hepatocytes that were also positive for Cxcl1 staining in the indicated conditions is shown. Dots indicate individual mice, and bars represent means. Data were analyzed by one-way ANOVA with Sidak’s multiple comparisons test; **p = 0.01.(E) Mass cytometry data showing live, lineage-negative, non-granulocytic/non-DC immune cells gated as in Figure S6C, before analysis of different monocyte populations based on CD68 and F4/80 expression, identifying iMCs as CD68INT and F4/80LO. Dots indicate individual mice, and bars represent means. Data were analyzed by one-way ANOVA with Sidak’s multiple comparisons test; *p = 0.05.(F and G) Expression of Ly6C in the liver by IHC in same mice used for mass cytometry; (F) example photomicrographs of Ly6C IHC from indicated mice demonstrating clusters of Ly6C+ cells surrounding NRAS-positive hepatocytes in NRASG12V + Cre are mostly abrogated in mice treated with dmPGE2. Arrowheads show residual Ly6C+ cells. Scale bar, 100 µm. (G) quantification of Ly6C+ cells. Dots indicate individual mice, and bars represent means. Data were analyzed by one-way ANOVA with Sidak’s multiple comparisons test; ***p = 0.001.See also Figure S5.