The triterpenoid CDDO-Me promotes hematopoietic progenitor expansion and myelopoiesis in mice

Abstract

The synthetic triterpenoid CDDO-Me has been shown to directly inhibit the growth of myeloid leukemias and lends itself to a wide array of therapeutic indications, including inflammatory conditions, because of its inhibition of NF-κB. We have previously demonstrated protection from acute graft-versus-host disease after CDDO-Me administration in an allogeneic bone marrow transplantation model. In the current study, we observed that CDDO-Me promoted myelopoiesis in both naive and transplanted mice. This effect was dose dependent, as high doses of CDDO-Me inhibited myeloid growth in vitro. All lineages (granulocyte macrophage colony-forming unit, BFU-E) were promoted by CDDO-Me. We then compared the effects with granulocyte colony-stimulating factor, a known inducer of myeloid expansion and mobilization from the bone marrow. Whereas both drugs induced terminal myeloid expansion in the spleen, peripheral blood, and bone marrow, granulocyte colony-stimulating factor only induced granulocyte macrophage colony-forming unit precursors in the spleen, while CDDO-Me increased these precursors in the spleen and bone marrow. After sublethal total-body irradiation, mice pretreated with CDDO-Me further displayed an accelerated recovery of myeloid progenitors and total nucleated cells in the spleen. A similar expansion of myeloid and myeloid progenitors was noted with CDDO-Me treatment after syngeneic bone marrow transplantation. Combined, these data suggest that CDDO-Me may be of use posttransplantation to accelerate myeloid recovery in addition to the prevention of graft-versus-host disease.

Figure 1. CDDO-Me stimulates CFU-GM formation at low concentrations, but is toxic at higher concentrations

Naïve mouse splenocytes were plated in a CFU-GM with a suboptimal amount of colony-stimulating cytokines (0.3ng/mL GM-CSF). CDDO-Me was added the Petric dishes at the indicated concentrations while keeping the amount of vehicle in each sample normalized. Plates were incubated for 7 days at which time colonies of greater that 50 cells were counted. Colony counts from individual experiments were normalized to the vehicle control in each experiment and pooled. *p<0.05 by Dunnett’s multiple comparison test.

Figure 2. CDDO-Me administration causes a myeloid expansion in the spleen and bone marrow

C57BL/6 mice were treated for 7 days with vehicle control (VC) or CDDO-Me, or 5 days with G-CSF after which mice were sacrificed and spleens and bone marrow were harvested. Single cell suspensions were stained with anti-CD11b and anti-Gr-1. A, Representative flow cytometry panels showing percentages of cells in either organ. B and C, total CD11b⁺Gr-1⁺ cell numbers in the spleen and bone marrow were enumerated. P values were determined by Dunnett’s multiple comparison test.

Figure 3. CDDO-Me administration results in a significant increase of CFU-GM and BFU-e in the BM and periphery

Mice were treated with vehicle control (VC), CDDO-me or G-CSF as before. CFU-GM and BFU-e colony assays were performed on bone marrow, spleens, and peripheral blood of treated animals. The data is representative of three independent experiments with three mice per group per experiment. Statistical differences were determined by one-way ANOVA with Tukey’s post-test and a p-value<0.05 was considered significant.

Figure 4. CDDO-Me administration results in increased CFU-HPP in the spleen and BM

Mice were treated with vehicle control or CDDO-me (120 μg/dose BID) a day intraperitoneally (i.p) for 7 days. Bone marrow and spleen were collected at day 7, processed into single cell suspensions and used for colony assays. Recombinant hG-CSF (2.5 μg/dose BID) was used as a control. The number of CFU-HPP colonies were enumerated in both the spleen (A) and femurs (B). The data is representative of three independent experiments with three mice per group per experiment. Statistical differences were determined by One-way ANOVA (Tukey) and p-value<0.05 was considered significant. TNTC = too numerous to count

Figure 5. CDDO-me administration increases the number of hematopoietic progenitor cells in the bone marrow and spleen

Mouse bone marrow and spleens were collected after the previously described treatment schedule and stained for lineage markers, c-kit, Sca-1 and 7-AAD to exclude dead cells. 7-AAD emits in the same fluorescent channel as the lineage markers used in this study (PE-Cy-5); thus dead cells were eliminated from gating during lineage exclusion. A, Representative flow plots of spleens and bone marrow HPCs are displayed. B and C, total bone marrow and spleen hematopoietic progenitor cells were determined. Statistical differences were determined by one-way ANOVA with Tukey’s post-test and a p-value<0.05 was considered significant.

Figure 6. CDDO-Me is radioprotective when administered before sublethal TBI

Mice were treated with CDDO-Me or vehicle control on days -7 through -1 before sublethal total body irradiation. Mice were harvested on day 8 after irradiaiton. A, Total nucleated cell counts in the spleen were quantified. B, leukocyte cell types in the spleen were quantified by flow cytometry for vehicle control (white bars) and CDDO-Me (black bars) treted mice. C, The total number of CFU-GM cells per spleen were quantified. Statistical differences were determined by one-way ANOVA with Tukey’s post-test and a p-value<0.05 was considered significant.

Figure 7. CDDO-Me administration after HSCT accelerates myeloid recovery

C57BL/6 (CD45.2) mice underwent a congenic BMT with 10⁷ BMC from C57BL/6 Ly5.2 (CD45.1) donors. Mice received 60μg CDDO-Me daily on days 1 through 13 after BMT. Mouse spleens and femurs were collected on day 14 and were assessed for CFU-GM and BFU-e activity. Donor myeloid cells (CD45.1⁺/CD11b⁺/Gr-1⁺) were also assessed by flow cytometry. Statistical differences were determined by Student’s t-test and a p-value<0.05 was considered significant.