G-CSF regulates hematopoietic stem cell activity, in part, through activation of Toll-like receptor signaling

Abstract

Recent studies demonstrate that inflammatory signals regulate hematopoietic stem cells (HSCs). Granulocyte colony-stimulating factor (G-CSF) is often induced with infection and has a key role in the stress granulopoiesis response. However, its effects on HSCs are less clear. Herein, we show that treatment with G-CSF induces expansion and increased quiescence of phenotypic HSCs, but causes a marked, cell-autonomous HSC repopulating defect associated with induction of Toll-like receptor (TLR) expression and signaling. The G-CSF-mediated expansion of HSCs is reduced in mice lacking TLR2, TLR4 or the TLR signaling adaptor MyD88. Induction of HSC quiescence is abrogated in mice lacking MyD88 or in mice treated with antibiotics to suppress intestinal flora. Finally, loss of TLR4 or germ-free conditions mitigates the G-CSF-mediated HSC repopulating defect. These data suggest that low-level TLR agonist production by commensal flora contributes to the regulation of HSC function and that G-CSF negatively regulates HSCs, in part, by enhancing TLR signaling.

Figure 1. G-CSF treatment increases the number of phenotypic HSCs in the bone marrow, but results in a loss of HSC repopulating and self-renewal activity

Bone marrow cells were harvested from mice treated with G-CSF or saline alone (PBS) for 7 days and analyzed by flow cytometry. (A) Representative dot plots showing the gating strategy used to identify phenotypic HSCs in the bone marrow. The frequency (B) and absolute number (C) of KSL CD34- CD41- Flk2-and the frequency (D) and absolute number (E) of KSL CD150+ CD48- CD41- cells in the bone marrow are shown. Data represent >5 independent experiments and 24–26 mice per condition. Competitive repopulation assays were performed using bone marrow cells from saline- or G-CSF-treated mice (Ly5.2), which was transplanted in a 1:1 ratio with untreated wild-type marrow (Ly5.1) into lethally irradiated recipients (Ly5.1/Ly5.2). (F) Shown is the percentage of donor (Ly5.2⁺) leukocytes in the blood. Data represent 6–12 mice per cohort from three pooled experiments. (G) The frequency of donor (Ly5.2⁺) B cells (B220⁺), T-cells (CD3e⁺), and neutrophils (Gr-1^high) in the peripheral blood 22 weeks after transplantation for a representative experiment is shown. (H) CD150⁺ CD48⁻ CD41⁻ lineage- cells were sorted by flow cytometry from the bone marrow. Sixty of these cells (Ly5.1⁺) were transplanted along with 500,000 competitor (Ly5.1/5.2) bone marrow cells into irradiated recipients (Ly5.2⁺). Shown is the percentage of donor (Ly5.1⁺) cells in the blood 20 weeks after transplantation (n=5 mice per condition). (I) Bone marrow cells harvested from primary recipients at 22 weeks as shown in (F) were transplanted into secondary recipients and the percentage of donor (Ly5.2⁺) leukocytes in the blood at 20 weeks was determined (n=3–4 mice per condition). Data represent the mean ± SEM. **p<0.01; ***p<0.001.

Figure 2. G-CSF treatment does not impair homing to the bone marrow

(A) GFP⁺ KSL cells (1 × 10⁵) were injected retroorbitally into lethally irradiated recipient mice, and viable GFP⁺ cells in the bone marrow quantified by flow cytometry 16 hours later. Shown is the number of GFP⁺ KSL cells per 1×10⁶ whole bone marrow cells analyzed (n=4 mice per condition). (B) Bone marrow cells from untreated or G-CSF treated mice (Ly5.2) were mixed with an equal number of competitor bone marrow cells (Ly5.1) and injected intrafemorally into irradiated recipients (Ly5.1/5.2). Shown is the percentage of donor (Ly5.2) cells 16 weeks after transplantation relative to the input percentage of donor cells injected intrafemorally (n = 9 from 3 independent experiments). Data represent the mean ± SEM. **p<0.01

Figure 3. G-CSF treatment induces HSC quiescence

Mice were treated with G-CSF or saline alone (PBS) for 12 hours to 7 days and the cell cycle status of HSCs assessed. (A) Shown are representative dot plots gated on KSL SLAM cells showing Ki-67 and DAPI staining. The percentage of KSL SLAM cells in G₀ (B) and S/G₂/M (C) are shown; data from mice 7 and 14 days after completing a 7 day course of G-CSF also is shown (n= 5–20 mice per time point). The percentage of CD34⁻ Flk2⁻ CD41- KSL cells in G₀ (D) and S/G₂/M (E) are shown (n=5–7 mice per time point). (F) Mice were treated with BrdU over the last 48 hours of a 7 day course of G-CSF. Shown is the percentage of BrdU⁺ CD150⁺ CD48⁻ CD41⁻ lineage⁻ cells (n=15–16 mice per group). Data represent the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.

Figure 4. G-CSF acts in a cell intrinsic fashion to inhibit HSC function

(A) Mixed Csf3r^−/− (G-CSFR deficient) bone marrow chimeras were generated by transplanting a 3:1 ratio of Csf3r^−/− to wild type (WT) bone marrow cells into irradiated recipients. This resulted in mixed chimeras with approximately 50% Csf3r^−/− cells (data not shown). After stable hematopoietic reconstitution (6 weeks), mice were treated with G-CSF or PBS for 7 days, and bone marrow was harvested after the last dose and transplanted into secondary recipients. Peripheral blood chimerism for B220⁺ cells was assessed starting at 9 weeks (B). After 12 weeks, pooled marrow from secondary recipients was transplanted into tertiary recipients, and peripheral blood chimerism for B220⁺ cells was assessed starting at 6 weeks (C). Data are representative of 2 independent experiments, with 5 mice per condition. **p<0.01

Figure 5. G-CSF treatment upregulates TLR expression and signaling

KSL SLAM cells were sorted from mice that were treated with PBS or G-CSF for 7 days and RNA expression profiling performed. (A) Shown are all genes significantly upregulated after 7 days of G-CSF. Arrows indicate genes whose expression has previously been reported to be regulated by TLR signaling. (B) Gene Set Enrichment Analysis showed an enrichment of TLR signaling (FDR q-value: 0.15). (C). Shown is the Affymetrix expression values for TLR family members in KLS SLAM cells after 7 days of G-CSF. ***p<0.001. Data represent the mean ± SEM of three independent arrays for each condition. (D) Representative histograms showing increased cell surface TLR2 expression on CD34⁻ Flk2⁻ KSL cells. Data are representative of 5–8 mice from two independent experiments.

Figure 6. TLR2 regulates HSC numbers in response to G-CSF

Bone marrow cells were harvested from Tlr2^−/− mice treated with G-CSF or saline alone for 7 days. Data for wild-type mice treated similarly are shown again for comparison. The number of CD34⁻ Flk2⁻ CD41⁻ KSL cells (A) in the bone marrow is shown. Data represent the mean ± SEM of 7–26 mice. The cycling status of CD34⁻ Flk2⁻ CD41⁻ KSL cells was determined using Ki-67 and DAPI; shown are the percentages of cells in G₀ (B) (n=4–5 mice per group). Competitive repopulation assays were performed to assess repopulating activity in the bone marrow of saline (PBS)- or G-CSF-treated Tlr2^−/− mice. (C) The percentage of donor (Ly5.2⁺) leukocytes in the blood over time is shown; data for wild type bone marrow is shown again to facilitate comparison. (D) The frequency of donor (Ly5.2⁺) B cells (B220⁺), T-cells (CD3e⁺), and neutrophils (Gr-1^high) in the peripheral blood 12 weeks after transplantation is shown. The data represent the mean ± SEM of 5 mice per group. *p<.0.05, **p<0.01, ***p<0.001, and reflect the comparison to the PBS group.

Figure 7. TLR signaling affects HSC repopulating activity at baseline and in response to G-CSF

Bone marrow cells were harvested from MyD88^−/− and Tlr4^−/− mice treated with G-CSF or saline alone for 7 days. Data for wild-type mice treated similarly are shown again for comparison. The number of CD34⁻ Flk2⁻ CD41⁻ KSL cells (A) in the bone marrow is shown. Data represent the mean ± SEM of 6–26 mice. The cycling status of CD34⁻ Flk2⁻ CD41⁻ KSL cells was determined using Ki-67 and DAPI; shown are the percentages of cells in G₀ (B) (n=4–10 mice per group). Competitive repopulation assays were performed to assess repopulating activity in the bone marrow of saline (PBS)- or G-CSF-treated Myd88^−/− or Tlr4^−/− mice. (C) The percentage of donor (Ly5.2⁺) leukocytes in the blood over time is shown; data for wild type bone marrow is shown again to facilitate comparison. The data represent the mean ± SEM of 4–10 mice per group from 2–3 independent transplants. (D) The frequency of donor (Ly5.2⁺) B cells (B220⁺), T-cells (CD3e⁺), and neutrophils (Gr-1^high) in the peripheral blood 22 weeks after transplantation is shown for a representative experiment from each group. In (E), the average repopulating units from 1×10⁶ test donor cells were determined for each group using the equation RU=([%chimerism of test donor-derived cells] × [# of competitor cells] × 10⁻⁵)/ %chimerism of competitor-derived cells. To determine RU/ bone marrow, this value was then multiplied by the total marrow WBCs/10⁶. *p<.0.05, **p<0.01, ***p<0.001, and reflect the comparison to the PBS group.

Figure 8. Commensal flora regulates HSCs at baseline and in response to G-CSF

(A) Bone marrow cells were harvested from wild type mice treated with ciprofloxacin and polymyxin B and then treated with saline alone or G-CSF for 7 days as shown. The number of KSL CD34⁻ Flk2⁻ cells (B) in the bone marrow is shown (n = 5–26 mice); SPF: wild-type mice housed under standard pathogen-free conditions. The cycling status of CD34⁻ Flk2⁻ CD41⁻ KSL cells was determined using Ki-67 and DAPI; shown are the percentages of cells in G₀ (C) (n= 4–10 mice per group). (D) Competitive repopulation assays were performed using bone marrow from saline- or G-CSF treated mice. The percentage of donor (Ly5.2⁺) leukocytes in the blood over time is shown (n = 5–7 mice per group); data for wild type bone marrow is shown again to facilitate comparison. (E) The frequency of donor (Ly5.2⁺) B cells (B220⁺), T-cells (CD3e⁺), and neutrophils (Gr-1^high) in the peripheral blood 12 weeks after transplantation is shown for germ-free mice. (F) The absolute number of KSL SLAM cells in the spleen of mice following treatment with saline alone or G-CSF for 7 days is shown (n= 4–10 mice per group). *p<.0.05, **p<0.01, ***p<0.001.