Analysis based on CD86 expression identifies the activation of erythropoiesis early after infection. (A) Population sizes of MEPs (CD150⁺FcγR⁻ LKs) and GMPs (CD150⁻FcγR^high LKs) before and 18 hours after LPS treatment (5 mg/kg). Representative FCM plots of the Lin⁻c-kit⁺CD86⁻ fraction are shown. (B) Kinetic analysis of CD150⁺ cells (MEPs) in the Lin⁻c-kit⁺Sca-1⁻ (left panel) and Lin⁻c-kit⁺CD86⁻ fractions (middle panel) during the 72 hours after LPS treatment. Data during the first 18 hours (shown in left and middle panels) were merged in the right panel (numbers of MEPs in the Lin⁻c-kit⁺Sca-1⁻ and Lin⁻c-kit⁺CD86⁻ fractions are shown as black and red lines, respectively); n = 3 per time point. (C) Kinetic analysis of the number of proerythroblasts (CD71⁺TER119^int) during the 72 hours after LPS treatment; n = 3 per time point. (D) Representative image of hemolysates of the BM before and after LPS treatment. (E) Kinetic analysis of TER119⁺CD45⁻ erythrocytes in the BM (left panel), blood (middle panel), and spleen (right panel) after LPS treatment; n = 3 per time-point. (F) Gata1 mRNA expression in CMPs obtained before and 5 hours after LPS treatment. Gene expression was examined by real-time PCR; n = 3 per group. (G) CMPs, isolated as in panel F, were cultured in methylcellulose medium for 10 days, and the number of TER119⁺ cells generated was examined by FCM; n = 3 per group. (H) Gata1 mRNA expression in CMPs stimulated with LPS. CMPs were isolated from naive CD45.1⁺ mice and cocultured with CD45.2⁺ total BM cells in the absence or presence of LPS (100 ng/mL) for 12 hours. CD45.1⁺ CMPs were sorted again, and gene expression was examined by real-time PCR; n = 3 per group. (I) Kinetics for the frequency of mRBCs (CD45⁻TER119⁺CD71⁻FSC^low) in TER119⁺ cells after LPS treatment; n = 3 per time-point. (J) Relative amount of hemoglobin in mRBCs obtained before and 18 hours after LPS treatment. The amount of hemoglobin was evaluated by measuring the optical density (OD)₅₆₀ of the hemolysates; n = 5 per group. (K-L) mRBCs were isolated from the BM of WT mice before and 18 hours after LPS treatment and were cultured in 20% FBS-containing RPMI medium for 48 hours. The cells were harvested, and the ratio of Annexin V⁺ apoptotic cells was examined by FCM. Representative FCM plots are shown in panel L; n = 3 per group. (M-N) TER119⁺CD45⁻ erythrocytes were isolated from the BM before and 18 hours after LPS treatment and incubated with the indicated concentration of PBS for 30 minutes. Hemolysis was evaluated by measuring the OD₅₆₀ of the supernatants. Data are statistically analyzed in panel N. DW, distilled water. (O-Q) Activation of erythropoiesis in Stat1^−/− mice after LPS treatment. Representative image of hemolysates (O) and the numbers of erythrocytes (P) and MEPs (Q) in WT and/or Stat1^−/− mice before and 18 hours after LPS treatment. The numbers on FCM plots indicate the frequencies of the gated populations. *P < .05, Student t test (F-H, J, L, N, P, and Q) or 1-way ANOVA (D and E). Data are representative of 2 (F-H and K-Q) or 3 (A-E and I) independent experiments or from 2 independent experiments (J). Error bars in panels B, C, E-H, and J-N represent SEM.