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Loss of IKKβ but Not NF-κB p65 Skews Differentiation towards Myeloid over Erythroid Commitment and Increases Myeloid Progenitor Self-Renewal and Functional Long-Term Hematopoietic Stem Cells.

Zhang J, Li L, Baldwin AS, Friedman AD, Paz-Priel I - PLoS ONE (2015)

Bottom Line: Accordingly, we found a significantly reduced frequency of proerythroblasts and basophilic and polychromatic erythroblasts, and IKKβ-deficient bone marrow cells yielded a significantly decreased number of BFU-E compared to wild type.In contrast, no effect on erythropoiesis or expression of lineage-related transcription factors was found in marrow lacking NF-κB p65.In summary, loss of IKKβ resulted in significant effects on hematopoiesis not seen upon NF-κB p65 deletion.

View Article: PubMed Central - PubMed

Affiliation: Division of Pediatric Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
NF-κB is an important regulator of both differentiation and function of lineage-committed hematopoietic cells. Targeted deletion of IκB kinase (IKK) β results in altered cytokine signaling and marked neutrophilia. To investigate the role 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 we now also noted decreased monocytes and modest anemia. Granulocyte-macrophage progenitors (GMPs) accumulated markedly in bone marrow of IKKβ deleted mice whereas the proportion and number of megakaryocyte-erythrocyte progenitors (MEP) decreased. Accordingly, we found a significantly reduced frequency of proerythroblasts and basophilic and polychromatic erythroblasts, and IKKβ-deficient bone marrow cells yielded a significantly decreased number of BFU-E compared to wild type. These changes are associated with elevated expression of C/EBPα, Gfi1, and PU.1 and diminished Gata1, Klf1, and SCL/Tal1 in IKKβ deficient Lineage-Sca1+c-Kit+ (LSK) cells. In contrast, no effect on erythropoiesis or expression of lineage-related transcription factors was found in marrow lacking NF-κB p65. Bone marrow from IKKβ knockout mice has elevated numbers of phenotypic long and short term hematopoietic stem cells (HSC). A similar increase was observed when IKKβ was deleted after marrow transplantation into a wild type host, indicating cell autonomous expansion. Myeloid progenitors from IKKβ- but not p65-deleted mice demonstrate increased serial replating in colony-forming assays, indicating increased cell autonomous self-renewal capacity. In addition, in a competitive repopulation assay deletion of IKKβ resulted in a stable advantage of bone marrow derived from IKKβ knockout mice. In summary, loss of IKKβ resulted in significant effects on hematopoiesis not seen upon NF-κB p65 deletion. These include increased myeloid and reduced erythroid transcription factors, skewing differentiation towards myeloid over erythroid differentiation, increased progenitor self-renewal, and increased number of functional long term HSCs. These data inform ongoing efforts to develop IKK inhibitors for clinical use.

No MeSH data available.


Related in: MedlinePlus

Deletion of IKKβ is associated with increased number of myeloid progenitors and hematopoietic stem cells.A) Bone marrow cells were harvested from the tibias of wild type or IKKβΔ/Δ mice, and the average number of cells is presented (n = 4). B) Bone marrow cells from wild-type (WT), IKKβΔ/Δ or p65Δ/Δ mice were stained for lineage markers, Sca-1, c-Kit, CD34, CD16/32 and CD135. The indicated progenitor populations were identified and representative plots and the percent ± SEM of each population relative to total Lin- marrow cells are shown (n = 4). C) The actual number of cells for the indicated progenitor populations per hind leg was calculated and averages from at least 4 experiments are shown. D) The average absolute number of multipotential progenitors (MPP) and long and short term hematopoietic stem cells (LT- and ST-HSC) per hind leg are shown (n = 4). E) CD45.1 mice were lethally irradiated and then intravenously injected with 1E6 CD45.2 marrow cells from wild type, IKKβ(f/f);Mx1-Cre, or p65(f/f);Mx-1-Cre mice. Eight wks after transplant mice were intraperitoneally injected with poly(I:C) for 7 doses starting four weeks after transplantation. Marrow harvested 6 week later and the average absolute number of the indicated progenitor and stem cell populations per hind leg are shown (n = 3).
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pone.0130441.g002: Deletion of IKKβ is associated with increased number of myeloid progenitors and hematopoietic stem cells.A) Bone marrow cells were harvested from the tibias of wild type or IKKβΔ/Δ mice, and the average number of cells is presented (n = 4). B) Bone marrow cells from wild-type (WT), IKKβΔ/Δ or p65Δ/Δ mice were stained for lineage markers, Sca-1, c-Kit, CD34, CD16/32 and CD135. The indicated progenitor populations were identified and representative plots and the percent ± SEM of each population relative to total Lin- marrow cells are shown (n = 4). C) The actual number of cells for the indicated progenitor populations per hind leg was calculated and averages from at least 4 experiments are shown. D) The average absolute number of multipotential progenitors (MPP) and long and short term hematopoietic stem cells (LT- and ST-HSC) per hind leg are shown (n = 4). E) CD45.1 mice were lethally irradiated and then intravenously injected with 1E6 CD45.2 marrow cells from wild type, IKKβ(f/f);Mx1-Cre, or p65(f/f);Mx-1-Cre mice. Eight wks after transplant mice were intraperitoneally injected with poly(I:C) for 7 doses starting four weeks after transplantation. Marrow harvested 6 week later and the average absolute number of the indicated progenitor and stem cell populations per hind leg are shown (n = 3).

Mentions: We next utilized FACS to further compare the frequency of various hematopoietic progenitor populations in marrow cells from wild type, IKKβΔ/Δ, and p65Δ/Δ mice, including the LSK, common myeloid progenitor (CMP), granulocyte/monocyte progenitor (GMP), megakaryocyte/erythroid progenitor (MEP), and common lymphoid progenitor (CLP). Deletion of IKKβ using Mx1-Cre leads to a 2-fold increase in the total number of cells retrieved from the marrow (Fig 2A), in contrast to p65 deleted mice. Therefore, both the proportion of each subpopulation and the absolute number of cells in each fraction per hind leg is presented. As expected from their neutrophilia and as previously reported [25], IKKβΔ/Δ mice had significantly increased proportion and total number of GMPs. However, the absolute number of CMPs was similar and their proportion reduced (Fig 2B and 2C). As previously noted, loss of p65 is associated with a significantly reduced number of CMPs (Fig 2C) [26]. In addition, comparing wild type to mice lacking IKKβ or p65 we noted a significant decrease in the number and percentage of MEPs but no effect on the number of CLPs (1940 ± 490, 2540 ± 1700, or 3750 ± 1690 per hind leg, respectively) (Fig 2C). Notably, total numbers of multipotent progenitors (MPP) and the ST-HSC subpopulations of the LSK fraction are significantly increased in IKKβΔ/Δ mice (Fig 2B and 2D). The trend towards increased number of LT-HSC did not reach statistical significance (p = 0.1). To confirm a cell autonomous expansion of myeloid progenitors we harvested marrow from CD45.2 wild type, IKKβ(f/f);Mx1-Cre, or p65(f/f);Mx1-Cre mice and injected these into CD45.1, lethally irradiated congenic mice. Mice were exposed to poly(I:C) only after engraftment. Analysis of myeloid progenitors 6 weeks later revealed a significant 2-fold expansion of the LSK and ST_HSC fractions in the absence of IKKβ but not p65, and in addition revealed an increased numbers of MPPs or LT-HSC approached statistical significance in the absence of IKKβ (p = 0.1) (Fig 2E). Of note, mice transplanted with p65Δ/Δ marrow do not reconstitute hematopoiesis and die.


Loss of IKKβ but Not NF-κB p65 Skews Differentiation towards Myeloid over Erythroid Commitment and Increases Myeloid Progenitor Self-Renewal and Functional Long-Term Hematopoietic Stem Cells.

Zhang J, Li L, Baldwin AS, Friedman AD, Paz-Priel I - PLoS ONE (2015)

Deletion of IKKβ is associated with increased number of myeloid progenitors and hematopoietic stem cells.A) Bone marrow cells were harvested from the tibias of wild type or IKKβΔ/Δ mice, and the average number of cells is presented (n = 4). B) Bone marrow cells from wild-type (WT), IKKβΔ/Δ or p65Δ/Δ mice were stained for lineage markers, Sca-1, c-Kit, CD34, CD16/32 and CD135. The indicated progenitor populations were identified and representative plots and the percent ± SEM of each population relative to total Lin- marrow cells are shown (n = 4). C) The actual number of cells for the indicated progenitor populations per hind leg was calculated and averages from at least 4 experiments are shown. D) The average absolute number of multipotential progenitors (MPP) and long and short term hematopoietic stem cells (LT- and ST-HSC) per hind leg are shown (n = 4). E) CD45.1 mice were lethally irradiated and then intravenously injected with 1E6 CD45.2 marrow cells from wild type, IKKβ(f/f);Mx1-Cre, or p65(f/f);Mx-1-Cre mice. Eight wks after transplant mice were intraperitoneally injected with poly(I:C) for 7 doses starting four weeks after transplantation. Marrow harvested 6 week later and the average absolute number of the indicated progenitor and stem cell populations per hind leg are shown (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4477978&req=5

pone.0130441.g002: Deletion of IKKβ is associated with increased number of myeloid progenitors and hematopoietic stem cells.A) Bone marrow cells were harvested from the tibias of wild type or IKKβΔ/Δ mice, and the average number of cells is presented (n = 4). B) Bone marrow cells from wild-type (WT), IKKβΔ/Δ or p65Δ/Δ mice were stained for lineage markers, Sca-1, c-Kit, CD34, CD16/32 and CD135. The indicated progenitor populations were identified and representative plots and the percent ± SEM of each population relative to total Lin- marrow cells are shown (n = 4). C) The actual number of cells for the indicated progenitor populations per hind leg was calculated and averages from at least 4 experiments are shown. D) The average absolute number of multipotential progenitors (MPP) and long and short term hematopoietic stem cells (LT- and ST-HSC) per hind leg are shown (n = 4). E) CD45.1 mice were lethally irradiated and then intravenously injected with 1E6 CD45.2 marrow cells from wild type, IKKβ(f/f);Mx1-Cre, or p65(f/f);Mx-1-Cre mice. Eight wks after transplant mice were intraperitoneally injected with poly(I:C) for 7 doses starting four weeks after transplantation. Marrow harvested 6 week later and the average absolute number of the indicated progenitor and stem cell populations per hind leg are shown (n = 3).
Mentions: We next utilized FACS to further compare the frequency of various hematopoietic progenitor populations in marrow cells from wild type, IKKβΔ/Δ, and p65Δ/Δ mice, including the LSK, common myeloid progenitor (CMP), granulocyte/monocyte progenitor (GMP), megakaryocyte/erythroid progenitor (MEP), and common lymphoid progenitor (CLP). Deletion of IKKβ using Mx1-Cre leads to a 2-fold increase in the total number of cells retrieved from the marrow (Fig 2A), in contrast to p65 deleted mice. Therefore, both the proportion of each subpopulation and the absolute number of cells in each fraction per hind leg is presented. As expected from their neutrophilia and as previously reported [25], IKKβΔ/Δ mice had significantly increased proportion and total number of GMPs. However, the absolute number of CMPs was similar and their proportion reduced (Fig 2B and 2C). As previously noted, loss of p65 is associated with a significantly reduced number of CMPs (Fig 2C) [26]. In addition, comparing wild type to mice lacking IKKβ or p65 we noted a significant decrease in the number and percentage of MEPs but no effect on the number of CLPs (1940 ± 490, 2540 ± 1700, or 3750 ± 1690 per hind leg, respectively) (Fig 2C). Notably, total numbers of multipotent progenitors (MPP) and the ST-HSC subpopulations of the LSK fraction are significantly increased in IKKβΔ/Δ mice (Fig 2B and 2D). The trend towards increased number of LT-HSC did not reach statistical significance (p = 0.1). To confirm a cell autonomous expansion of myeloid progenitors we harvested marrow from CD45.2 wild type, IKKβ(f/f);Mx1-Cre, or p65(f/f);Mx1-Cre mice and injected these into CD45.1, lethally irradiated congenic mice. Mice were exposed to poly(I:C) only after engraftment. Analysis of myeloid progenitors 6 weeks later revealed a significant 2-fold expansion of the LSK and ST_HSC fractions in the absence of IKKβ but not p65, and in addition revealed an increased numbers of MPPs or LT-HSC approached statistical significance in the absence of IKKβ (p = 0.1) (Fig 2E). Of note, mice transplanted with p65Δ/Δ marrow do not reconstitute hematopoiesis and die.

Bottom Line: Accordingly, we found a significantly reduced frequency of proerythroblasts and basophilic and polychromatic erythroblasts, and IKKβ-deficient bone marrow cells yielded a significantly decreased number of BFU-E compared to wild type.In contrast, no effect on erythropoiesis or expression of lineage-related transcription factors was found in marrow lacking NF-κB p65.In summary, loss of IKKβ resulted in significant effects on hematopoiesis not seen upon NF-κB p65 deletion.

View Article: PubMed Central - PubMed

Affiliation: Division of Pediatric Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
NF-κB is an important regulator of both differentiation and function of lineage-committed hematopoietic cells. Targeted deletion of IκB kinase (IKK) β results in altered cytokine signaling and marked neutrophilia. To investigate the role 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 we now also noted decreased monocytes and modest anemia. Granulocyte-macrophage progenitors (GMPs) accumulated markedly in bone marrow of IKKβ deleted mice whereas the proportion and number of megakaryocyte-erythrocyte progenitors (MEP) decreased. Accordingly, we found a significantly reduced frequency of proerythroblasts and basophilic and polychromatic erythroblasts, and IKKβ-deficient bone marrow cells yielded a significantly decreased number of BFU-E compared to wild type. These changes are associated with elevated expression of C/EBPα, Gfi1, and PU.1 and diminished Gata1, Klf1, and SCL/Tal1 in IKKβ deficient Lineage-Sca1+c-Kit+ (LSK) cells. In contrast, no effect on erythropoiesis or expression of lineage-related transcription factors was found in marrow lacking NF-κB p65. Bone marrow from IKKβ knockout mice has elevated numbers of phenotypic long and short term hematopoietic stem cells (HSC). A similar increase was observed when IKKβ was deleted after marrow transplantation into a wild type host, indicating cell autonomous expansion. Myeloid progenitors from IKKβ- but not p65-deleted mice demonstrate increased serial replating in colony-forming assays, indicating increased cell autonomous self-renewal capacity. In addition, in a competitive repopulation assay deletion of IKKβ resulted in a stable advantage of bone marrow derived from IKKβ knockout mice. In summary, loss of IKKβ resulted in significant effects on hematopoiesis not seen upon NF-κB p65 deletion. These include increased myeloid and reduced erythroid transcription factors, skewing differentiation towards myeloid over erythroid differentiation, increased progenitor self-renewal, and increased number of functional long term HSCs. These data inform ongoing efforts to develop IKK inhibitors for clinical use.

No MeSH data available.


Related in: MedlinePlus