IL-1 receptor antagonist or antibiotic treatment of WT SPF donor mice increases lymphoid differentiation of older HSC populations upon transplantation. (A) Experimental approach to test the effects of blocking IL-1 signaling and reducing bacterial load. First, 2mo and 2y WT SPF mice were given water containing antibiotics (ABx) (ampicillin 1 mg/mL, vancomycin 1 mg/mL, neomycin sulfate 0.5 mg/mL, metronidazole 1 mg/mL) for 8 weeks. Alternatively, mice were injected once per day with PBS or anakinra (hIL-1ra 37 μg per injection per mouse) for 3 weeks. CD45.2⁺ LT-HSCs (LKS CD34^–Flt3^–CD48^–CD150⁺) were isolated from treated animals and 100 or 200 fluorescence-activated cell sorted cells were transplanted into lethally irradiated recipients. BM LT-HSC donor chimerism, PB CD45⁺ donor chimerism, and PB lineage contribution of donor LT-HSCs were assessed 4 months after transplantation. (B) BM chimerism frequency analysis of donor (CD45.2⁺) LT-HSCs from anakinra- or PBS-treated 2mo and 2y WT SPF donor mice 4 months after transplantation. PBS 2mo, n = 5; hIL-1ra 2mo, n = 5; PBS 2y, n = 5; hIL-1ra 2y, n = 6. In (B-D), “n” refers to recipient animals. For donors, 3 young PBS-, 3 young anakinra-, 3 older PBS-, and 3 older anakinra-treated animals were used. (C) PB chimerism frequency analysis of donor (CD45.2⁺) leukocytes from anakinra- or PBS-treated 2mo and 2y WT SPF donor mice 4 months after transplantation. PBS 2mo, n = 5; hIL-1ra 2mo, n = 5; PBS 2y, n = 5; hIL-1ra 2y, n = 6. Engraftment levels of >0.1% were considered. (D) Lineage contribution of LT-HSCs from 2mo and 2y WT SPF donor mice treated with anakinra or PBS 4 months after transplantation. Donor cell contribution in myeloid cells (CD45.2⁺ CD11b⁺CD3e^–CD19^–) is shown in red, T cells (CD45.2⁺ CD3e⁺CD19^–) yellow, B cells (CD45.2⁺ CD3e^–CD19⁺) green. PBS 2mo, n = 5; hIL-1ra 2mo, n = 5; PBS 2y, n = 5; hIL-1ra 2y, n = 6. (E) BM chimerism frequency analysis of donor (CD45.2⁺) LT-HSCs from 2mo and 2y WT SPF donor mice treated with antibiotics or normal drinking water (ie, untreated [Untr]) 4 months after transplantation. Untr 2mo, n = 7; ABx 2mo, n = 7; Untr 2y, n = 7; ABx 2y, n = 7. In (E-G), “n” refers to recipient animals. For donors, 3 young PBS-, 3 older PBS-, 5 young ABx-, and 5 older ABx-treated animals were used. (F) PB chimerism frequency analysis of donor (CD45.2⁺) leukocytes from 2mo and 2y WT SPF donor mice treated with antibiotics or normal drinking water 4 months after transplantation. Untr 2mo, n = 7; ABx 2mo, n = 7; Untr 2y, n = 7; ABx 2y, n = 7. (G) Lineage contribution of HSCs from young and old WT SPF donor mice treated with antibiotics or normal drinking water (untreated) 4 months after transplantation. Donor cell contributions in myeloid cells (CD45.2⁺ CD11b⁺CD3e^–CD19^–) are shown in red, in T cells (CD45.2⁺ CD3e⁺CD19^–) yellow, and in B cells (CD45.2⁺ CD3e^–CD19⁺) light green. Untr 2mo, n = 7; ABx 2mo, n = 7; Untr 2y, n = 7; ABx 2y, n = 7. Error bars represent SD in (B-C,E-F) and SEM in (D,G). P values were calculated using Student t test. *P < .05; **P < .01; ***P < .001; ****P < .0001.