Gdf15^−/−defects in steady state and in splenic niche after 75% dosage of PHZ challenge. (A) Analysis of the concentration and percentage of monocytes in the peripheral blood at steady state. n = 9 for WT and n = 11 for Gdf15^−/−. (B) Analysis of F4/80⁻CD11b⁺Ly6C⁺ monocytes in the homeostatic bone marrow. (C) Flow cytometry analysis of steady-state spleen resident red pulp macrophages, F4/80⁺Vcam1⁺ (left) and percentage of F4/80⁺Vcam1⁺ cells (right). (D-N) WT and Gdf15^−/− mice were challenged with 75 mg/kg body weight PHZ through intraperitoneal injection. (D-F) Spleen cells were collected 24 hours after PHZ injection and stained for intracellular Ccl2 and cell surface CD11b and Ly6C. (D) Percentage of Ccl2⁺ splenocytes. (E) Flow cytometry analysis of CD11b and Ly6C expression on Ccl2⁺ cells (left) and the percentage of CCL2⁺CD11b⁺Ly6C⁺ monocytes in the spleen (right). (F) MFI of F4/80 expression on CCL2⁺CD11b⁺Ly6C⁺ monocyte population. (G-N) Blood and spleens were collected 48 hours after PHZ challenge. (G) Hematocrit. (H) Monocyte frequency (left) and percentage (right) in peripheral blood. (I) Flow cytometry analysis of spleen cells stained with anti-F4/80, anti-CD11b and anti-Ly6C antibodies (left) and the percentage of CD11b⁺Ly6C⁺ monocytes in the spleen (right). (J) The percentage of F4/80⁺CD11b⁻Ly6C⁻ macrophages in the spleen. (K) Flow cytometry analysis of macrophage surface markers, F4/80 and Vcam1 (left), and the percentage of F4/80⁺Vcam1⁺ red pulp macrophages. (L) Flow cytometry analysis CD169 expression in splenocytes (left) and the percentage of F4/80⁺CD169⁺ macrophages in the spleen. (M) Changes in macrophage and monocyte populations between 24-hour and 48-hour time points after PHZ treatment. Percentage of CD11b⁺Ly6C⁺ monocytes (far left), percentage of F4/80⁺CD11b⁻Ly6C⁻ macrophages (mid-left), percentage of F4/80⁺Vcam1⁺ RPMs (mid-right) and percentage of F4/80⁺CD169⁺ macrophages (far right) are shown as fold changes. (N) Change in relative expression levels of CD163, Hmox1, and Spic in the spleen between 24-hour and 48-hour time points are shown as fold changes. Data are shown as individual subject and the mean ± SEM. *P < .05, **P < .01, and ***P < .001. (O) Schematic of differences of monocyte mobilization in the peripheral blood, homing to the spleen, and monocytes differentiation into RPMs in WT and Gdf15^−/− mice. MFI, mean fluorescent intensity.