Differential stem- and progenitor-cell trafficking by prostaglandin E2

Abstract

To maintain lifelong production of blood cells, haematopoietic stem cells (HSCs) are tightly regulated by inherent programs and extrinsic regulatory signals received from their microenvironmental niche. Long-term repopulating HSCs reside in several, perhaps overlapping, niches that produce regulatory molecules and signals necessary for homeostasis and for increased output after stress or injury. Despite considerable advances in the specific cellular or molecular mechanisms governing HSC-niche interactions, little is known about the regulatory function in the intact mammalian haematopoietic niche. Recently, we and others described a positive regulatory role for prostaglandin E2 (PGE2) on HSC function ex vivo. Here we show that inhibition of endogenous PGE2 by non-steroidal anti-inflammatory drug (NSAID) treatment in mice results in modest HSC egress from the bone marrow. Surprisingly, this was independent of the SDF-1-CXCR4 axis implicated in stem-cell migration. Stem and progenitor cells were found to have differing mechanisms of egress, with HSC transit to the periphery dependent on niche attenuation and reduction in the retentive molecule osteopontin. Haematopoietic grafts mobilized with NSAIDs had superior repopulating ability and long-term engraftment. Treatment of non-human primates and healthy human volunteers confirmed NSAID-mediated egress in other species. PGE2 receptor knockout mice demonstrated that progenitor expansion and stem/progenitor egress resulted from reduced E-prostanoid 4 (EP4) receptor signalling. These results not only uncover unique regulatory roles for EP4 signalling in HSC retention in the niche, but also define a rapidly translatable strategy to enhance transplantation therapeutically.

Figures

Figure 1. NSAIDs mobilize hematopoietic stem and progenitor cells

Meloxicam enhances mobilization of HPC, a, and HSC b, into blood (n=4–5 mice/group/experiment; 3 experiments). c, Chimerism; d, competitive repopulating units (CRU); and e, LT-HSC frequency (Poisson distribution) 36 weeks after limiting dilution competitive transplants of peripheral blood mononuclear cells (PBMC) from mice treated with G-CSF and combination regimens (n=8 mice/group, assayed individually). Mice were treated with G-CSF or a staggered regimen of G-CSF + Meloxicam and PBMC transplanted into lethally irradiated mice. f, Neutrophil and g, platelet recovery were monitored for 90 days. *P<0.05, ** P<0.01, ***P<0.001; unpaired two-tailed t-test. All error bars represent mean ± s.e.m.

Figure 2. Non-human primates and healthy human volunteers mobilize HSC/HPC in response to NSAID treatment

a, Four baboons were treated with G-CSF +/- Meloxicam in a cross-over design and b, CD34+ cells and c, CFU-GM in peripheral blood (PB) determined. d, CD34+ cells and e, CFU-GM in PB determined pre- and post-5 days of meloxicam alone treatment. Seven healthy human volunteers were treated with 15 mg/day p.o. for 5 days, and were assessed for f, CD34+ cells; g, CFU-GM; h, BFU-E, and i, CFU-GEMM pre- and post-treatment. Statistics represent paired, two-tailed t-test.

Figure 3. Prostaglandin E2 EP4 receptor antagonism/knockout expands bone marrow HPC and enhances mobilization

a, HPC mobilization with G-CSF, G-CSF + meloxicam, G-CSF + EP receptor antagonists, or G-CSF + meloxicam + EP receptor agonists (n=5 mice/group, assayed individually). b, The EP4 antagonist L-161,982 enhanced HSC mobilization (n=4 mice/group, assayed individually), and c, long-term reconstitution 16 weeks post-transplant with d, multi-lineage reconstitution (n=5 mice/group, assayed individually). e, Meloxicam enhances mobilization of HPC, and f, SLAM SKL cells in WT littermates, but not in EP4 conditional knockouts (n=3,4 mice/group, assayed individually). *P<0.05, ** P<0.01, ***P<0.001; unpaired two-tailed t-test. †P<0.05 compared to G-CSF + meloxicam. All error bars represent mean ± s.e.m.

Figure 4. NSAIDs attenuate hematopoietic supportive molecules and differentially mobilize HSC and HPC in OPN knockout and EP4 conditional knockout mice

a, b, Assessment of Col2.3-GFP cells after vehicle or meloxicam demonstrates reduced c, percentages and d, number of osteolineage cells (n=4 mice/group, assayed individually). e, Immunohistochemical staining of hematopoietic supportive molecules after treatment with meloxicam (400X). f, Meloxicam enhances mobilization of HPC in OPN −/− mice, with g,h, no enhancement in long-term reconstitution 16 weeks post-transplant. i, Representation of chimera generation allowing conditional knockout of donor hematopoietic cells, or recipient stromal cells. EP4 was deleted with tamoxifen 8 weeks post-transplant and mice treated with G-CSF or G-CSF + meloxicam. j, Enhanced mobilization of HPC by meloxicam when EP4 is expressed on hematopoietic cells and k, enhanced mobilization of HSC when EP4 is expressed by stromal cells (n=4 mice/group, assayed individually). *P<0.05, **P<0.01, ***P<0.001; unpaired two-tailed t-test. Error bars represent mean ± s.e.m.