Bone Marrow Niche Attenuation by Non-Steroidal Anti-Inflammatory Drugs Mobilizes Hematopoietic Stem and Progenitor Cells by Differing Mechanisms
Authors: Hoggatt Jonathan , Mohammad Khalid S , Chitteti Brahmananda Reddy , Singh Pratibha , Speth Jennifer M , Hu Peirong , Poteat Bradley , Srour Edward , Guise Theresa , Pelus Louis M. 
Published: October 2019

Abstract
Abstract 724
Hematopoeitic stem (HSC) and progenitor cells (HPC) are localized in niches contained within the bone marrow (BM) microenvironment. To facilitate acquisition of HSC/HPC for hematopoietic transplantation, donors can be treated with granulocyte-colony stimulating factor (G-CSF) to “mobilize” HSC/HPC from the BM niche to the peripheral system. Our laboratory has recently discovered that short term administration of non-steroidal anti-inflammatory drugs (NSAIDs) also mobilizes HSC/HPC and works in synergy with G-CSF. To understand the mechanisms underlying this novel therapeutic utility of NSAIDs we analyzed the BM niche post-NSAID and G-CSF treatment.
Femurs of mice were analyzed after a 4 day regimen of the NSAID Meloxicam (3mg/kg, bid) and compared to control or G-CSF treated mice. Gross histological analysis showed a remarkable osteoblast (OB) “flattening” similar to that seen with G-CSF. Goldners trichrome staining revealed significantly reduced osteoid bone surfaces, with ∼3-fold increase in quiescent surfaces. Similarly, dynamic bone formation analysis using calcein/tetracycline labeling demonstrated reductions in mineral apposition rate and bone formation rate. There were no significant alterations in trabecular bone as determined by MicroCT. TRAP+ osteoclasts (OCs) were slightly elevated in both Meloxicam and G-CSF treated groups. To further assess the role of OCs, mice were mobilized with or without zoledronic acid (ZA) treatment, which inhibits OC activity. Similar to a recent report (Winkler, Blood, 2010), ZA resulted in an increase in HSC/HPC mobilization by both Meloxicam and G-CSF, suggesting that increased OC activity is not a mitigating mechanism for NSAID-mediated mobilization. Immunohistochemical (IHC) staining showed marked reductions in osteopontin (OPN), stromal-derived factor-1 (SDF-1) and N-cadherin expression by Meloxicam and G-CSF treated mice. In a separate set of experiments, OBs and mesenchymal stem cells (MSCs) were sorted by flow cytometry and gene expression assessed by Taq-Man assay. Similar to IHC analysis, Meloxicam treatment resulted in reduced SDF-1, OPN, Jagged-1, Runx2 and VCAM-1 gene expression.
While the interaction of SDF-1 with its hematopoietically expressed receptor CXCR4 is a well known mediator of niche retention, both HSC and HPC were significantly mobilized by Meloxicam in CXCR4 knockout (KO) mice, suggesting that while reduced SDF-1 expression may concurrently play a role in NSAID mediated mobilization, it is not the definitive mechanism. In contrast, when OPN KO mice were mobilized with Meloxicam or G-CSF, Meloxicam unexpectedly increased mobilization of HPC only, and not HSC, while both HPC and HSC were mobilized by G-CSF. This surprising result indicates that NSAID-mediated OPN reduction is specifically responsible for the observed HSC mobilization, while HPC mobilization appears to be mediated by another mechanism(s).
Recently, it has been reported that G-CSF reduces resident F4/80+ monocytes/macrophages (MOs), which normally support niche OBs and MSCs, and this reduction is at least partially responsible for niche attenuation and hematopoietic mobilization. However, in contrast to G-CSF, IHC analysis showed no reduction in F4/80+ cells after Meloxicam treatment, nor was there a reduction in CD169+ BM macrophages as assessed by flow cytometry. Therefore, while NSAIDs and G-CSF attenuate the niche microenvironment similarly, these agents function through independent mechanisms, perhaps explaining the synergistic mobilization seen by the two.
In conclusion, NSAID treatment results in significant attenuation of the BM niche, including the niche components SDF-1, OPN, and N-cadherin. NSAID-mediated mobilization is independent of reduced SDF-1/CXCR4 signaling, as CXCR4 KO mice can be mobilized by NSAID. OPN KO specifically blocks the NSAID mobilization of HSC, but not HPC, suggesting differing mechanisms, or possibly multiple niche locations for these two populations; an intriguing finding suggesting that other mobilization strategies may be able to specifically target HSC or HPC. While G-CSF treatment results in reduced MOs, NSAIDs do not alter BM levels of these supportive cells. These results define not only a novel strategy for mobilization of HSC/HPC, but also suggest a possible strategy for targeted niche attenuation for other therapeutic applications, such as reduced conditioning regimens.

Disclosures:
Hoggatt: Fate Therapeutics: Consultancy. Pelus:Fate Therapeutics: Consultancy.