Fig 2: EPO-treatment increases KC number in vivo. Livers were harvested from C57BL/6 J mice injected 3 times on alternating days with 180 U EPO or diluent (control). (A) Schematic outline of EPO/diluent injections. (B) Left: EPO-R transcript levels in total liver (Liver) and in non-parenchymal cells (NP) from diluent and EPO-injected mice. Right: EPO-R transcript levels in sorted KCs (defined as CD45+/CD11bint/F4/80hi/CD64+ cells39) from diluent (KCs) or EPO-injected mice (KCs + EPO), were evaluated by RT-PCR. Graph represents EPO-R transcript levels ± SEM. BMDM served as a positive control27. Ly6Chi = Ly6Chi monocytes, Ly6Clo = Ly6Clo monocytes. (C) Frozen sections of the livers were stained with a purified antibody directed against F4/80 surface marker followed by Cy3 labeled Goat anti-Rat antibody, and viewed on confocal microscopy. Left: Representative analysis of livers from diluent (control) and EPO-injected mice. Right: Bars represent mean ± SEM, N = 5–6, **p < 0.01. Bar = 25 µm. (D) Liver non-parenchymal cells were analyzed by flow cytometry for CD45, F4/80 and CD11b. Left: Representative analysis of livers from diluent (control) and EPO-injected mice. Right: KCs or CD45+/CD11b- (lymphocytes) cell number per gram liver tissue. Graphs represent mean ± SEM. For KCs: N = 12-13, *p < 0.05. For CD45+/CD11b- cells: N = 7–8. (E) Livers of EPO-overexpressing mice (Tg6) or their WT littermates were harvested. Liver non-parenchymal cells were analyzed by flow cytometry for CD45, F4/80 and CD11b. Left: Representative analysis of livers from Tg6 versus WT. Right: Graphs represent mean ± SEM, N = 6, **p < 0.01 (F) Left: Flow cytometry analysis of Ki67 labelling of gated KCs (representative plot). Right: quantification of Ki67 positive KCs, N = 4, *p < 0.05. (G) Graph represents liver weight as mean ± SEM, N = 7–9, **p < 0.01.

Fig 3: EPO effect on KC levels is abrogated in LysM cre -Epor fl/fl. Epor fl/fl or LysM cre -Epor fl/fl mice were injected 6 times on alternating days over 2 weeks, with 180 U EPO or diluent (control). (A) EPO-R transcript levels in total liver cells and in sorted KCs (CD45+/F4/80hi/CD11blo) from Epor fl/fl mice or LysM cre -Epor fl/fl mice were evaluated by RT-PCR. Graph represents EPO-R transcript levels ± SEM, N = 4–6, **p < 0.01. (B) Left: Representative analysis of livers from control and EPO-injected Epor fl/fl or LysM cre -Epor fl/fl mice. Right: KC number per liver weight (gr). Graphs represent mean ± SEM, N = 4–9, *p < 0.05.

Fig 4: (A) IHC staining of CD8a in tumor of the MC38 tumor model treated with anti-PD-1 or anti-PCSK9 antibody and quantitative analysis of positive particles and (B) for the CT26 tumor model. (C) Flow cytometry analysis of CD45+, CD3+, and CD8+ T-cell infiltration in MC38 tumors. "*" means p-value < 0.05 and "**" means p-value < 0.01.

Fig 5: NELF potentiates inflammatory responses secondary to constraining the production of IL-10.a ELISA for IL-6 (n = 5, P = 0.0051), TNF (n = 5, P = 0.0402) and IL-12p40 (n = 4, P = 0.0061) protein levels in WT and Nelfb KO BMDM stimulated with LPS for 6 h. *P < 0.05, **P < 0.01 by two-sided paired Student’s t test. b WT and Nelfb KO BMDM pretreated with 2 µg/ml of anti-IL10R for 0.5 h, where indicated, were activated with LPS for 6 h. IL-6 levels in culture supernatants were measured by ELISA and shown as relative values normalized to levels in WT cells that were set as 1 in each independent experiment (n = 3). c FACS analysis of the macrophage population (CD45+CD11b+F4/80+) in peritoneal exudates of WT and Nelfb KO mice 9 h after intraperitoneal injection of LPS (100 ng/mouse). Representative FACS distribution (left) and cumulative (n = 4) percentage (middle, P = 0.0002) as well as absolute numbers (right, P = 0.0014) are shown. d Cumulative data showing total peritoneal cell numbers from WT and Nelfb KO mice 9 h after LPS injection (n = 7, P = 0.8232). e RT-qPCR analysis of Il6 in peritoneal cells of WT and Nelfb KO mice 9 h after intraperitoneal injection of LPS (n = 6, P = 0.0074). **P < 0.01 by two-sided paired Student’s t test. f FACS analysis of macrophage population (CD45+CD11b+F4/80+) in peritoneal exudates of WT and Nelfb KO mice 9 h after intraperitoneal injection of LPS or LPS + IL-6 (40 ng/mouse). Representative FACS distribution (left) and percentage of macrophages from independent experiments (n = 4–6) (right, WT + LPS/Nelfb KO + LPS P = 0.0008; Nelfb KO + LPS/Nelfb KO + LPS + IL-6 P = 0.0105) are shown. For c, d, and f, *P < 0.05, **P < 0.01, ***P < 0.001, N.S. P > 0.05 by two-sided unpaired Student’s t test. n represents biologically independent animals. Error bars indicate SEM. Source data are provided as a Source Data file.