Fig 2: Changes in the proportion of spleen Ter119+ cell subpopulations during repeated restraint stress and macrophage depletion. The total Ter119+ splenocytes were analyzed for their size (forward-scatter parameter) and CD71 expression, and accordingly divided into three increasingly mature cell subpopulations (E1, E2 and E3). (a) Representative dot plots of Ter119+ cell subpopulations in the bone marrow of control mice, (b) animals subjected to daily restraint stress for 7 consecutive days, (c) animals treated with clodronate-liposomes, (d) animals treated with clodronate-liposomes and subjected to daily restraint, (e) animals received control liposomes and subjected to daily restraint, (f) percentage participation of E1 subpopulation, (g) E2 subpopulation, and (h) E3 subpopulation of Ter119+ splenocytes. Data are expressed as mean ± SEM (n = 6–8/group). * p < 0.05, ** p < 0.01 (one-way ANOVA following Games–Howell as appropriate).

Fig 3: Minihepcidin-Induced Serum Iron Deficiency Inhibits Immune Responses(A) Experimental design for investigation of the effect of combined minihepcidin injection and low iron diet on the primary immune response to immunization. Serum iron levels and liver hepcidin mRNA were measured 24 h after the last minihepcidin injection. Means ± SDs. Student’s 2-tailed t test, unpaired.(B) Left: representative flow cytometry plots showing frequency of OVA (SIINFEKL) tetramer-positive CD8 effector T cells (Tetramer+ CD44+ CD8 T cells) as a percentage of CD8s T cells. Right: number of splenic OVA-specific CD8 effector cells 7 days after immunization with AdHu5-OVA. Means ± SDs. Student’s 2-tailed t test, unpaired.(C) Number of splenic OVA-specific CD8 effector cells 7 days after OVA protein in adjuvant immunization. Means ± SDs. Student’s 2-tailed t test, unpaired.(D) Number of T follicular helper cells (PD-1+ CXCR5+ CD44+ CD4 T cells), germinal center (GC) B cells (IgD- CD95+ B220+ B cells), plasma cells (CD138+ IgD- cells), and anti-OVA IgG titer (a.u., arbitrary unit) 7 days after immunization with OVA in adjuvant. Means ± SDs. Student’s 2-tailed t test, unpaired.(E) Experimental design to determine whether parenteral iron supplementation can reverse the effect of minihepcidin injection; FAC = ferric ammonium citrate. Left: the frequency of antigen-specific splenic OT-I CD8 T cells; center: normalized relative mean fluorescence intensity (MFI) of surface CD71 expression by OT-I CD8 T cells 5 days after OVA and adjuvant immunization were detected with flow cytometry; right: liver hepcidin mRNA. Means ± SDs. One-way ANOVA with correction for multiple comparisons.

Fig 4: Effects of repeated restraint stress and macrophage depletion on erythroid precursors in the spleen: (a) representative dot plots of erythroid precursors at different maturational stages in the bone marrow of control mice, (b) animals subjected to daily restraint stress for 7 consecutive days, (c) animals treated with clodronate-liposomes, (d) animals treated with clodronate-liposomes and subjected to daily restraint, and (e) animals received control liposomes and subjected to daily restraint. The main erythroid cell populations, (f) CD71+/Ter119+ and (g) CD71-/Ter119+, are presented as a percentage of all splenocytes. Data are expressed as mean ± SEM (n = 6–8/group). * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA following Games–Howell and Bonferroni-corrected t test as appropriate).

Fig 5: Changes in the proportion of bone marrow Ter119+ cell subpopulations during repeated restraint stress and macrophage depletion. The total Ter119+ bone marrow cells were analyzed for their size (forward-scatter parameter) and CD71 expression, and accordingly divided into three increasingly mature cell subpopulations (E1, E2 and E3). (a) Representative dot plots of Ter119+ cell subpopulations in the bone marrow of control mice; (b) animals subjected to daily restraint stress for 7 consecutive days; (c) animals treated with clodronate-liposomes; (d) animals treated with clodronate-liposomes and subjected to daily restraint; and (e) animals received control liposomes and subjected to daily restraint. Percentage participation of (f) E1 subpopulation, (g) E2 subpopulation, and (h) E3 subpopulation of bone marrow Ter119+ cell subpopulations. Data are expressed as mean ± SEM (n = 6–8/group). * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA following Bonferroni-corrected t test as appropriate).