Abrogation of IL-1 signaling attenuates TLR1/2 agonist-induced multipotent HSPC expansion. (A) DCs were sorted from the bone marrow of Cx3cr1^gfp/+ mice treated with one dose of 100 μg PAM₃CSK₄, and RNA-sequencing was performed. Expression of selected inflammatory cytokines/chemokines is shown. LSK (B) or LSK-SLAM (C) cell number per femur. (D) Number of multipotent progenitor-2 (MPP2), MMP3, and MMP4 per femur. (E) Number of common myeloid progenitors (CMPs), GMPs, and megakaryocyte-erythroid progenitors (MEPs) per femur. (F) Bone marrow cells from wild-type (WT) or Il1r1^−/− (Ly5.2) mice treated with vehicle alone (Ctrl) or PAM₃CSK₄ were transplanted along with an equal number of WT competitor (Ly5.1) bone marrow. Shown is the percentage of Ly5.2 donor chimerism in peripheral blood. (G) LSK Ly5.2 donor chimerism 12 weeks after transplantation. (H) Peripheral blood Ly5.2 donor chimerism after secondary transplantation. (I) LSK Ly5.2 donor chimerism 12 weeks after secondary transplantation. (J) CM from bone marrow–derived DC cultures stimulated with vehicle alone (Ctrl), PAM₃CSK₄ (10 ng/mL) alone, or PAM₃CSK₄ with anakinra (1 μg/mL) was prepared. HSCs (LSK-SLAM cells) were sorted into the different DC CM and cultured for 7 days with supportive cytokines. (K) IL-1β protein level in DC CM. (L) Number of lineage-negative (CD11b^– Gr1^–) Kit⁺ cells on day 7 of culture. Data represent the mean ± standard error of the mean. Statistical significance determined by using unpaired t-test (panels A and K), two-way analysis of variance (panels B-E and L), and one-way analysis of variance (panels G and I). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.