Dipeptidylpeptidase 4 negatively regulates colony-stimulating factor activity and stress hematopoiesis

Abstract

Enhancement of hematopoietic recovery after radiation, chemotherapy, or hematopoietic stem cell (HSC) transplantation is clinically relevant. Dipeptidylpeptidase (DPP4) cleaves a wide variety of substrates, including the chemokine stromal cell-derived factor-1 (SDF-1). In the course of experiments showing that inhibition of DPP4 enhances SDF-1-mediated progenitor cell survival, ex vivo cytokine expansion and replating frequency, we unexpectedly found that DPP4 has a more general role in regulating colony-stimulating factor (CSF) activity. DPP4 cleaved within the N-termini of the CSFs granulocyte-macrophage (GM)-CSF, G-CSF, interleukin-3 (IL-3) and erythropoietin and decreased their activity. Dpp4 knockout or DPP4 inhibition enhanced CSF activities both in vitro and in vivo. The reduced activity of DPP4-truncated versus full-length human GM-CSF was mechanistically linked to effects on receptor-binding affinity, induction of GM-CSF receptor oligomerization and signaling capacity. Hematopoiesis in mice after radiation or chemotherapy was enhanced in Dpp4(-/-) mice or mice receiving an orally active DPP4 inhibitor. DPP4 inhibition enhanced engraftment in mice without compromising HSC function, suggesting the potential clinical utility of this approach.

Figure 1

Deletion or inhibition of Dpp4 enhances SDF-1’s effects on survival and ex vivo expansion of HPCs. (a) Colony formation assays in WT and Dpp4^−/− bone marrow after 1 d delayed addition of growth factors. SDF-1 was added to plates at day 0, and growth factors were added either on day 0 or on day 1 (one of two reproducible experiments with 3 plates per point scored for each bar; each experiment used bone marrow pooled from three mice). *P < 0.05 compared to day 0 WT; **P < 0.05 compared to WT on day 1; ***P < 0.05 compared to WT + SDF-1 on day 1. (b) Effect of diprotin A on survival of human cord blood CFU-GEMM after 1 d delayed addition of growth factors (GF) as in a for mouse cells (one of three reproducible experiments scoring three plates per point; each experiment used bone marrow pooled from three mice). Human cord blood was pretreated with 5 mM diprotin A, and the cells were either washed or not prior to plating in semisolid culture medium in the absence or presence of SDF-1 at day 0 with GFs added on day 0 or day 1. Only the day 1 results are shown, as there was no effect of SDF-1 or of diprotin treatment on cells plated with growth factors at day 0. Control refers to PBS (no SDF-1) in culture plates at day 0. *P < 0.05 compared to day 0 without SDF-1; **P < 0.05 compared to day 1 control. (c) Ex vivo expansion of HPCs (average results of eight experiments, each using a different cord blood collection, with three plates scored per point for each experiment). CD34⁺ cells isolated from fresh cord blood were pretreated with 5 mM diprotin A or PBS, and the cells were then plated in suspension culture with SCF, FL and TPO and either PBS (control medium) or SDF-1. After 7 d, the cells were washed and plated in semisolid culture medium and scored for colonies. Results are expressed as fold change from the diprotin A and PBS group cultured with SCF, FL and TPO. *P < 0.05 compared to control; **P < 0.05 compared to SDF-1 without diprotin A pretreatment. FL, full-length; TPO, thrombopoietin. (d) Same experiment as in c except that CD34⁺ cells were purified from an unseparated cord blood that had been thawed after 21 years in a frozen cryopreserved state and the actual numbers of progenitors for input colony-forming cells (prior to suspension culture) were compared to colony-forming cells generated after 7-d culture with PBS, SDF-1 or SDF-1 with diprotin A. The numbers in parentheses above the bars refer to the fold increase compared to input colony-forming cells (n = 1 experiment with three plates scored per point). *P < 0.05 compared to input; **P < 0.05 compared to PBS control; ***P < 0.05 compared to SDF-1 without diprotin A. All results are mean ± s.e.m.

Figure 2

Inhibition of DPP4 enhances the in vitro activity of selected CSFs with DPP4 truncation sites. (a) Mass spectrometry analysis of recombinant human GM-CSF and recombinant human IL-3 before and after exposure of the CSFs to soluble DPP4. The shift in molecular weight after DPP4 action correlates with the presence of an N-terminal alanine or proline DPP4 truncation site. (b) Mass spectrometry analysis of recombinant human GM-CSF. Same as in a, with additional time points after DPP4 addition shown. (c) Influence of diprotin A (DA) inhibition of DPP4 on human cord blood cell CFU-GM colony formation with and without washing the cells prior to addition of different concentrations (ng ml⁻¹ in parentheses) of recombinant human (rhu) human growth factors to semisolid culture medium (one of three reproducible experiments). Control refers to no DA treatment of cells. (d) Influence of diprotin A inhibition of DPP4 on unseparated mouse bone marrow cells with and without washing the cells prior to addition of different concentrations (ng ml⁻¹ in parentheses) of recombinant mouse (rm) growth factors to semisolid culture medium (one of three reproducible experiments). For c and d, *P < 0.05 compared to control without diprotin A for that growth factor. (e) Lack of effect of diprotin A pretreatment of target cells on activity of potent co-stimulating cytokines SCF or Flt3-L, when used alone or in combination with a CSF. Mouse bone marrow cells were pretreated with PBS (control) or diprotin A and then plated with the cytokines shown. *P < 0.05 compared to control of that group. Exp., experiment; ND, not done. (f) N-terminal sequences for G-CSF. (g) Influence of diprotin A treatment on mouse bone marrow BFU-E colony formation with washing of cells prior to plating of cells with recombinant mouse or recombinant human erythropoietin (EPO), and on human cord blood cells with washing of cells prior to plating of cells with recombinant human erythropoietin. *P < 0.05 compared to without diprotin A treatment of cells. (h) Influence of diprotin A or PBS (control) pretreatment of cells on colony formation by bone marrow cells from Dpp4^−/− and WT mice. Three plates per point were scored in one experiment, for which cells were pooled from three different mice. Shown is one of two reproducible experiments. *P < 0.05 compared to control without diprotin A for that growth factor. All results are mean ± s.e.m.

Figure 3

Influence of soluble DPP4 on activities of recombinant mouse CSFs in vitro, and effects of full-length and truncated CSFs alone and in combination on hematopoiesis in vivo in mice. (a) Influence of soluble DPP4 on activity of CSFs. Top, mouse bone marrow cells were treated with recombinant mouse GM-CSF, IL-3 or EPO which had been pretreated with PBS, soluble DPP4 or DPP4 first heat-treated at 56 °C for 1 h (DPP4-Δ*) to inactivate DPP4. Bottom, bone marrow was pretreated with diprotin A. Then, as indicated in the sequences shown on the y axis, cells were added to culture dishes with cytokines that had been first treated with PBS, DPP4 or DPP4-Δ* (as indicated in the first column in the sequence) and then with either PBS or diprotin A (second column); these treated cytokines were added with either PBS or the corresponding full-length cytokine (third column). *P < 0.05 compared to control medium (PBS, PBS, PBS) in that group. (b) Influence of full-length (FL) and truncated (T) recombinant mouse GM-CSF in vivo in WT and Dpp4^−/− mice on absolute numbers and cycling status of bone marrow HPCs. n = 4 mice per group. (c) Influence of full-length and truncated recombinant human erythropoietin in vivo on reticulocyte release to the blood of WT and Dpp4^−/− mice 18 h after a single (experiment 1, blood assessed 24 h later; n = 4 mice per group) or multiple injections of erythropoietin (experiments 2 and 3; n = 4 mice per group for each experiment). Experiment 3 used blood obtained from tail bleeds. The control treatment was PBS. (d) Influence of erythropoietin on absolute numbers of bone marrow HPCs in WT and Dpp4^−/− mice (n = 4 mice per group). The control treatment was PBS. For experiments 2 and 3 in c and for d, erythropoietin was given s.c. twice per day for 3 d at 10 U per injection, and mice were assessed 24 h after the last injection. For b–d, × = fold change from the indicated comparisons; *P < 0.05. All results are mean ± s.e.m.

Figure 4

Influence of DPP4 inhibition on colony formation, receptor binding and signaling in the TF-1 factor-dependent human cell line and in CD34⁺ cord blood cells. (a) Influence of the full length and truncated forms of GM-CSF, IL-3 and erythropoietin on colony formation by TF-1 cells pretreated with or without diprotin A (DA). *P < 0.05 compared to full-length GM-CSF, IL-3 or EPO in the absence of diprotin A; **P < 0.001, compared to full-length GM-CSF, IL-3 or EPO in the absence of diprotin A; ***P < 0.001, compared to GM-CSF, IL-3 or EPO in the presence of diprotin A. (b) GM-CSF receptor binding analysis by Scatchard plot, shown for TF-1 cells. Results of full-length ligand (FL) and truncated ligand (T) are shown from a representative experiment. High-affinity and low- affinity binding sites are readily observed. Inset, statistical analysis of K_ds from results of three experiments shown with error bars; *P < 0.05 for both affinity classes. (c) Scatchard analysis of CD34⁺ cord blood cells (one of two reproducible experiments). (d) Cold competition binding experiment. Concentration of cold (unlabeled) full-length or truncated ligand that was used to compete, or block, binding of the same amount (7 pM) of ‘hot’ ([¹²⁵I]-full-length GM-CSF) ligand per point is shown. IC₅₀, concentration of the cold ligand required to produce 50% inhibition of binding. Inset, full range of concentrations of cold competitor are shown, demonstrating that high concentrations of both FL and T ligands produce nearly 100% inhibition of binding of the labeled ligand. Diprotin A was added to all truncated samples to quench the DPP4 reaction before use. The arrows pointing to the left and right respectively refer to the IC₅₀ for truncated and full-length GM-CSF. (e,f) Phosphorylation of JAK2 (e) and STAT5 (f) in TF-1 cells. Influence of varying concentrations of truncated GM-CSF and/or full-length GM-CSF. The flow analysis at the left of e and f are one representative of three experiments; at right is shown quantitative data (mean ± s.e.m.) for all three experiments. The bottom graphs in e and f show the results of one experiment assessing effects of different ratios of T to FL cytokine. *P < 0.004; values for full-length GM-CSF are compared to no stimulation, and P values for T or T plus FL are compared to full-length GM-CSF. (g) Influence of full-length versus truncated recombinant human GM-CSF on phosphorylation of JAK2 and STAT5 in CD34⁺ cord blood cells (n = 6 experiments for pJAK2 and 3 experiments for pSTAT5). *P < 0.05 compared to truncated GM-CSF. (h) Influence of full-length and truncated recombinant human GM-CSF (shown as ng ml⁻¹) alone and in combination on colony formation by TF-1 cells in the presence and absence of diprotin A. *P < 0.002 for truncated CSF without diprotin A, or the combination of full-length plus truncated CSF without diprotin A, compared to the corresponding full-length CSF without diprotin A; **P < 0.001 for full-length CSF with diprotin A compared to the corresponding full-length CSF without diprotin A; ***P < 0.001 for truncated CSF or full-length plus truncated CSF with diprotin A compared to the corresponding full-length CSF with diprotin A. All results are mean ± s.e.m.

Figure 5

Modeling of the GM-CSF GM-CSFR interaction, and effects of DPP4 deficiency or inhibition on hematopoietic recovery. (a) Model for the GM-CSF–GM-CSFR interaction, indicating the inhibitory effect of the βc site 4 GM-CSFR–specific antibody on dodecamer complex formation. (b) Influence of βc site 4 GM-CSFR antibody (anti-R) on actions of full length versus truncated CSFs. Cord blood cells were pretreated in sequence in the order shown with PBS, diprotin A (DA) or antibody specific to the human GM-CSFR βc site 4 (anti-R) prior to plating cells with GM-CSF or G-CSF that was either not treated (no pretreatment) or treated with DPP4 and 1 h later with DA before adding the CSF to cells (one of two reproducible experiments in which three plates were scored per point). *P < 0.05 compared to PBS, no treatment. (c) Time course of the effects of radiation on DPP4 activity in plasma and lysates of unseparated bone marrow cells from WT mice. DPP4 activity assays were done in duplicate, and data are expressed as mean ± s.d. for plasma or cell lysates from three mice per group at each day. DPP4 enzyme activity is shown in relative light units. *P < 0.05 compared to time 0. (d,e) Effects of sitagliptin treatment of WT mice or Dpp4^−/− on in vivo recovery of nucleated cellularity and HPC numbers from treatment of mice with 400 cGy radiation (d) or 5-FU (e). For d and e, × = fold change compared to WT numbers of that day. *P < 0.05; a = one mouse only for these groups, all others n = 3. ND, not done. All results are mean ± s.e.m.

Figure 6

Influence of DPP4 inhibition in vivo on engraftment of HSCs and of CXCR4 deficiency on sitagliptin-enhanced hematopoietic recovery. (a,b) Effects of pretreating mice with diprotin A (DA) (a) or sitagliptin (b) on engraftment of C57BL/6 (CD45.2⁺) cells in a competitive assay with B6.BoyJ mice (CD45.1). Number of mice used is given in the Online Methods section. For a and b, *P < 0.05. (c) Inducible Cxcr4 knockout. Wild-type mice are represented by a single 430-bp PCR product and knockout mice are represented by a single 510-bp PCR product due to deletion of the exon 2 region of the Cxcr4 gene, as diagrammed. (d) Nucleated cellularity and progenitors per femur in WT control mice and mice with induced Cxcr4^−/− in the absence or presence of oral administration of sitagliptin before (top) and 7 d after receiving 5-FU treatment (bottom). Results are based on an analysis of four mice per group. *P < 0.05. NS, not significant (P > 0.05), and × = fold change from cells from the indicated mice. All results are mean ± s.e.m.