Fig 2: Characterization of erythroblast island forming macrophages (EBIMØs) in sham and burn mice. (A) EBIMØs in total bone marrow (TBM) cells isolated from sham and post burn day 7 (PBD7) mice were characterized by the presence of cell surface markers-F4/80, ERHR3, Siglec1, Vcam1, and Ly6G. F4/80+ERHR3+Siglec1+Vcam1+Ly6G± macrophages constituted the EBI forming population as analyzed by flow cytometry. (B) Overlay of EBIMØs contour plots (Sham = blue, PBD7 = red) gated on Siglec1 and Ly6G shows equal distribution between Ly6Gpos and Ly6Gneg cells. (C) Histogram of Siglec1 expression as measured by MFI (X-axis) in EBIMØs of burn (red) compared to Sham (blue) from both the Ly6G pos and Ly6G neg subsets (D) The EBIMØs were analyzed for the presence of erythroblast (EB) markers. CD71+Ter119− cells represent early EBs (EEBs) and CD71±Ter119+ cells represent late EBs (LEBs). (E) Data from 6 animals were analyzed using t-SNE plots to find the association of Ly6G with EBs. F4/80+ MØs were gated on Ly6G marker (color scale blue to red indicates Ly6G low to high subsets) was checked for CD71 (X-axis) and Ter119 (Y-axis) expressions in sham (top panel) and PBD7 (bottom panel) mice. Ly6G low subset was more associated with EB markers when compared to Ly6G high subset in both the treatment groups. Among the F4/80+Ly6Glo MØs in sham, there were 3 times more LEB and 2.5 times less EEB association compared to the burn group. Data represent mean values containing 6 mice/group.

Fig 3: Burn injury perturbs myelopoiesis in bone marrow and spleen. (A,B) Line graphs represent CD71+Ter119± cells as percentage of total spleen (A) and total bone marrow cells (B) in sham (blue) and burn injury (red) after 3, 7, 14, and 21 days. (C) Maturation index (MI) calculated as the ratio of reticulocytes to nucleated BM erythroblasts (represented as y-axis of the line graph) at post burn days 7, 14, and 21 (x-axis), showing a significant decrease in MI throughout the course of burn injury. (D) Mean fluorescent intensities of adhesion molecules ERHR3, Siglec1, and Vcam1 in F4/80+ MØs after 7, 14, and 21 days of burn injury are depicted as bar graphs (sham = blue/gray, burn = red). Reduced expression of the three adhesion molecules is seen at post burn day 7. Error bars indicate standard error of mean of >3 independent experiments. *P < 0.05, **P < 0.001; ***P < 0.0001 vs. burn by one-way ANOVA.