Loss of IKKβ Increases the Number and Self-Renewal Capacity of Hematopoietic Stem Cells

NF-kB is an important regulator of both differentiation and function of lineage-committed hematopoietic cells. Targeted deletion of IkB kinase (IKK) β results in altered cytokine signaling and marked neutrophilia. To investigate the roles of IKKβ in regulation of hematopoiesis, we employed Mx1-Cre mediated IKKβ conditional knockout mice. As previously reported, deletion of IKKβ in hematopoietic cells results in neutrophilia, and decreased monocytes and. We now show that bone marrow cells from IKKβ deleted mice display accumulation of granulocyte-macrophage progenitors (GMPs, Lin^-Sca-1^-c-kit⁺CD34⁺CD16/32⁺), with 1.8-fold increase compared to wild-type control. Accordingly, methylcellulose-based colony-forming assay demonstrated that IKKβ-deficient marrow produced increased proportion of CFU-G and decreased CFU-M compared with wild type control. Importantly, pharmacologic inhibition of IKKβ activity in wild type murine bone marrow cells results in significant increase in the total number of colonies and the number and proportion of CFU-G colonies. In addition, loss of IKKβ is associated with a modest decrease of baseline hematocrit levels (52.5 ± 0.64 vs. 43.9 ± 4.3, p<0.0001) and blunted recovery after challenge with 5-FU. Accordingly, IKKβ deleted mice have 3-fold decreased megakaryocyte-erythrocyte progenitors population (MEPs, Lin^-Sca-1^-c-kit⁺CD34^-CD16/32^-). Using FACS analysis erythroid progenitor subsets in the marrow were characterized based on Ter119, CD71, and forward scatter. We found a significantly reduced frequency of proerythroblasts, basophilic and polychromatic erythroblasts. When cultured in methylcellulose in the presence of hEPO IKKβ-deficient bone marrow cells yielded a significantly decreased number of BFU-E compared to wild type. Accordingly, pharmacologic inhibition of IKKβ in wild type marrow cells resulted in diminished BFU-E colonies formation. We next studied the role of IKKβ in early hematopoietic progenitors. Bone marrow from IKKβ knockout mice displays the accumulation of phenotypic hematopoietic stem cells (HSCs), including LT-HSCs (Lin^-Sca-1⁺c-kit⁺CD135^-CD34^-) and ST-HSCs (Lin^-Sca-1⁺c-kit⁺CD135^-CD34⁺), with 1.5-fold increase as compared to the wild-type control. Functionally, Lin^- bone marrow cells from IKKβ deleted mice show increased serial replating in colony-forming assays, indicating increased cell autonomous long-term self-renewal capacity. Accordingly, competitive transplantation studies demonstrated that deletion of IKKβ greatly increases the repopulation ability of HSCs resulting in a stable advantage of bone marrow derived from IKKβ knockout mice. Quantitative real-time PCR assay demonstrated that compared to Lin^- bone marrow from wild-type control mice, Lin^- bone marrow cells from IKKβ deficient mice have up-regulation of genes related to HSC self-renewal in early stage of hematopoiesis and granulocytic lineage commitment such as GFI1, HOXA9, PU.1, C/EBPα, CEBPε; but down regulation of genes involved in megakaryocytic-erythroid lineage determination such as GATA1, GATA2, Tal-1, and Klf1. In summary, our data indicate that loss of IKKβ results in a cell autonomous alteration of expression of key regulators of hematopoiesis, leads to increased self-renewal of HSC, and drives hematopoietic development towards GMP lineage, favoring granulopoiesis over monopoiesis.