Fig. 3.

Persistent heme effects in vivo. (A) Cytokine release of bone marrow–derived macrophages (BMDM), bone marrow (BM) isolated at day 7 after control (Ctrl; n = 8) or heme (n = 8) injection and 24 h after LPS (10 ng/mL) stimulation. Pool of two independent experiments. (B) Phagocytic capacity of BMDM, BM isolated at day 7 after Ctrl (n = 4) or heme (n = 4) injection and 2 h after pHrodo Red E. coli stimulation. Pool of two independent experiments. (C) Gating of myeloid cells. Neutrophils are CD11b⁺Ly6G⁺ and monocytes are CD11b⁺Ly6G^‒ and further distinguished as Ly6C^high (inflammatory monocytes) and Ly6C^low (patrolling monocytes). Peritoneal Mφ were gated as CD11b^highLy6G^‒F4/80⁺. (D) Cell count and flow cytometry analysis of peritoneal lavage fluid of vehicle- or heme-pretreated mice at day 7 and 6 h after endotoxemia. (E) Experimental setup. (F) Survival of polymicrobial sepsis induced 7 d after vehicle (n = 10) or heme (n = 10) treatment in mice. Pool of two independent experiments. Survival: Fisher’s exact test. (G) Pathogen load and (H) serology of vehicle- (n = 8) or heme- (n = 7) pretreated mice 24 h after sepsis induction. Pool of two independent experiments. Mann–Whitney U test; mean, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (I) Experimental setup. (J) Flow cytometry analysis of indicated HSPC cellular subsets of vehicle- or heme-pretreated mice at day 7. n = 5 to 6 animals per group derived from two independent experiments. Student’s t test; mean ± SD, *P ≤ 0.05; **P ≤ 0.01. Ae, aerob; ALT, alanin aminotransferase; An, anaerob; h, hours; hi, high; IFN, interferon; IL, interleukin; LDH, lactate dehydrogenase; LSK, Lin⁻Sca1⁺cKit⁺; Neutrop, neurophils; n.s., non-significant; PCI, peritoneal contamination and infection; p.i., post injection; PLF, peritoneal lavage fluid.