Macrophages: Key regulators of steady-state and demand-adapted hematopoiesis  Amanda McCabe, Katherine C. MacNamara  Experimental Hematology  Volume 44, Issue 4, Pages 213-222 (April 2016) DOI: 10.1016/j.exphem.2016.01.003 Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 1 Granulocyte colony-stimulating factor and IFN-γ differentially modulate BM Mϕ numbers, HSPC pool size, and lineage fate. Mϕs reside within the HSPC niche, where they efferocytose senescent neutrophils, suppress granulopoiesis, and mobilize HSPCs via LXR signaling and circadian rhythms. Circulating HSPC levels are also influenced by their retention in the spleen via Mϕ-dependent VCAM-1 binding to VLA-4. GG-CSF (left column) reduces BM Mϕ number, which correlates with HSPC expansion in the BM, blood, and spleen. Reduced Mϕ number also correlates with a lift on the suppression of granulopoiesis. IFN-γ (right column) increases BM Mϕs, and signaling in Mϕs restricts the pool of HSPCs, limits circulating HSPCs, and increases monopoiesis. Splenic Mϕs and IFN-γ also enhance EMH in the spleen, which may also sequester circulating HSPCs. Experimental Hematology 2016 44, 213-222DOI: (10.1016/j.exphem.2016.01.003) Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 2 Macrophage depletion reduces splenic cellularity and abrogates infection-induced splenomegaly. Mice were infected with 5e4 copies of Ehrlichia muris and administered liposome-encapsulated phosphate-buffered saline (PBS) or clodronate (Clod) on days 4 and 6 postinfection (250 μL iv). On day 11 postinfection, spleens were homogenized with frosted slides, and cells were enumerated. (A) Graph representing the numbers of spleen cells in control uninfected (white bar) and E. muris (Em)-infected (black bar) mice. (B) Photograph of spleens from E. muris-infected mice treated with liposome-encapsulated PBS or clodronate. Means ± SEM are illustrated. A two-tailed Student t test was used for comparisons between groups. *p < 0.05, **p < 0.01, ***p <0.001. Experimental Hematology 2016 44, 213-222DOI: (10.1016/j.exphem.2016.01.003) Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions

Figure 3 Interferon-γ signaling in Mϕ-lineage cells restricts immature granulocytes in the BM and mature neutrophils in circulation. Mice were infected with 5e4 copies of Ehrlichia muris via intraperitoneal injection, and tissues were collected on day 11 post-E. muris infection. Blood was collected in 0.5 mol/L EDTA, and BM was flushed from one femur and tibia. Red blood cells were lysed with ammonium chloride Tris buffer, and single-cell suspensions were surface stained with fluorescence-conjugated antibodies, and analyzed by flow cytometry on an LSR II and with FlowJo software. (A) Graph representing the absolute frequency of immature granulocytes (CD11b+Ly6Cint.Ly6Glow) in the BM of control uninfected (white bar) and E. muris-infected (black bar) MIIG and LC mice (left panel) or 7 days post-administration of liposome-encapsulated PBS or clodronate (right panel, 250 μL iv). Means ± SEM are illustrated. A two-tailed Student t test was used for comparisons between groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) Graph representing complete blood cell count (CBC) for white blood cells in E. muris-infected MIIG or LC mice. CBCs were performed on an automated hematology analyzer (Advia 120, Bayer, Norwood, MA). Experimental Hematology 2016 44, 213-222DOI: (10.1016/j.exphem.2016.01.003) Copyright © 2016 ISEH - International Society for Experimental Hematology Terms and Conditions