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. 2012 Apr;30(4):709-18.
doi: 10.1002/stem.1050.

Noncanonical NF-κB signaling regulates hematopoietic stem cell self-renewal and microenvironment interactions

Chen Zhao  1 , Yan Xiu, John Ashton, Lianping Xing, Yoshikazu Morita, Craig T Jordan, Brendan F Boyce
Affiliations

Noncanonical NF-κB signaling regulates hematopoietic stem cell self-renewal and microenvironment interactions

Chen Zhao et al. Stem Cells. 2012 Apr.

Abstract

RelB and nuclear factor κB (NF-κB2) are the main effectors of NF-κB noncanonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-κB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Furthermore, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-κB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment.

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Figures

Figure 1
Figure 1. Characterization of hematopoiesis in RelB/NF-κB2 dKO mice
(A, B) Frequency of KLS cells in control and dKO mice (n=8, *p<0.01), assessed by flow cytometry. (C, D) Frequency of long-term stem cells (CD150+CD48−KLS) in control and dKO mice (n=8, *p=0.04). (E, F) Cell cycle analysis using Hoechst 33342 and Pyronin Y staining. G0 phase defined as PyroninlowHoechstlow and G1 phase defined as PyroninhighHoechstlow. (n=3, *p<0.01 **p=0.012). (G, H) Analysis of proliferation of KLS cells using BrdU labeling. (n=4, *p<0.01). (I, J) Apoptosis analysis of the stem cell population (CD150+KLS cells). (n=3, *p<0.01), assessed by annexin V positivity. (K) Real time PCR analysis of the expression of Ki67, cyclin D1, and the cell cycle inhibitor p27. (n=3, *p<0.01 for Ki67 and cyclin D1 and p=0.025 for p27). Dot plots in (A, C, E, G and I) are shown for one representative control (left) and dKO (right). Values are means +/− SD.
Figure 2
Figure 2. RelB/NF-κB2 intrinsically regulate HSPC self-renewal
(A) Limiting-dilution competitive repopulation analysis of control (Con) and dKO mice. Five to eight mice were transplanted at each dose (10, 50, 100 and 500 KLS cells/recipient for both Con and dKO groups). Peripheral blood cells of the recipients were analyzed 16 weeks after transplantation and recipient mice containing less than 1% donor cells in any one lineage were scored as negative. (B) Engraftment of control and dKO (3 × 107 whole bone marrow cells/recipient). (n=7, *p<0.01). (C) Secondary bone marrow transplantation using whole bone marrow cells isolated from primary recipients transplanted with control or dKO cells as in (B). (n=6, *p<0.01). (D) dKO and control bone marrow cells have equivalent homing ability. 2×106 whole bone marrow cells from control or dKO mice were transplanted into lethally irradiated recipients. Data are chimerism after 20 hours (n=3 for control and 4 for dKO). (E) Kinetic lineage contribution of dKO cells (gated on CD45.2+CD45.1−) after transplantation (n=3 at 4 weeks and n=2 at 8 and 16 weeks). Values are means +/− sem.
Figure 3
Figure 3. dKO bone marrow microenvironment controls donor cell fate
(A) Scheme for analysis of bone marrow microenvironment. (B) Average donor and host chimerism (left) and the ratio of donor to host cells (right) after transplantation. (n=6, *p < 0.01). (C) Donor cell differentiation in the peripheral blood (PB). Bar graphs shown are donor-derived myeloid and lymphoid cells at 3 weeks after transplantation into control or dKO mice. (D) Normal peripheral blood lineage differentiation from control and dKO mice. (E) Donor chimerism in the bone marrow (BM) after transplantation. (F) Frequency of donor-derived myeloid cells in the bone marrow after transplantation. (G) Frequency of donor-derived lymphoid cells in the bone marrow after transplantation. (H, I) Analysis of the frequency of host-(H) and donor-(I) derived KLS cells after transplantation. (J) BrdU labeling of donor-derived KLS cells. (n=6 for D, F, H and G. n=3 for E, I, J, and K). Except for D and E, data were obtained at 16 weeks after transplantation.
Figure 4
Figure 4. The instructive effect of dKO microenvironment is reversible
Chimerism and lineage commitment after “educated” donor cells were transplanted back to a wild-type microenvironment. 1×106 FACS-sorted “educated” donor cells from control or dKO mice were transplanted back to lethally irradiated wild-type recipients (n=5). Donor chimerism and lineage differentiation at 4-weeks (A) and 16-weeks (B) are shown. Each dot represents an individual mouse. Values are means +/− SD.
Figure 5
Figure 5. RelB/NF-κB2 positively regulate bone marrow stromal and bone-lining cell functions
(A, B) Analysis of the frequency of bone marrow stromal cells (CD45−Ter119−CD31−). (n=6, *p < 0.01). (C) Assessment of proliferation of bone marrow stromal cells using BrdU labeling. (D) Analysis of the frequency of bone-lining cells from collagenase-digested control or dKO bone fragments (left) and the expression of N-Cadherin (right). (E) Alkaline phosphate (ALP) staining of bone-lining cells after 5 days culture with osteogenic medium (1×105/96-well). (F) Co-culture of collagenase-digested bone-lining cells from control or dKO mice with sorted lineage− wild-type bone marrow cells. 40 hours later, bone marrow cells were collected for colony forming assay. (G) Real time PCR analysis of the expression of Tpho, Cxcl12, osteopontin (OPN), SCF and Osteocalcin (Bglap) mRNA in control and dKO bone-lining cells. (n=3, *p< 0.01).
Figure 6
Figure 6. Increased expression of inflammatory cytokines in dKO marrow cells induces myeloid differentiation and HSPC expansion
(A, B and C) Real time PCR analysis of the expression of G-CSF, GM-CSF and IL-6 mRNA levels in control and dKO whole bone marrow cells. (n=3, *p< 0.01). (D) Myeloid differentiation after WT whole bone marrow cells were cultured with 20% control or dKO mouse serum. (E) Analysis of the frequency of lineage (left panel) and KLS cells (right panel) after WT whole bone marrow cells cultured with 20% of control or dKO mouse serum.
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