Adipogenic Chemokine Inhibition of Hematopoietic Cell Adherence and Transmigration, a Hurdle to Homing

Abstract

The hematopoietic cell niche serves to maintain hematopoiesis and recruit cells after hematopoietic cell transplant. The determinants of homing and engraftment after transplant are incompletely understood, but are often mediated by small chemokines secreted by the "niche" cells (stromal, endothelial, reticular, etc). We have shown that the hematopoietic cell homing to adipocyte-rich marrow is decreased by 50% compared to adipocyte-poor regions at 20 hours after adoptive transfer in irradiated recipients (n = 9, p < 0.05). To search for chemokines responsible for homing inhibition, we performed an unsupervised cluster analysis of the transcriptomes of adipocytes versus other niche cells in the marrow including CD45+, osteoblasts, endothelial cells, etc. We found that a small protein (26 kDa), dermatopontin (DPT), was highly expressed in adipocytes (10-fold versus endothelial cells and 7.5-fold versus osteocytes, ANOVA p = 0.02). Immunohistochemistry (IHC) and ELISA confirmed that DPT expression was predominately localized to adipocyte-rich regions of the marrow. We also found the intramarrow DPT concentrations were 57.1-fold elevated compared to circulating levels of DPT (n = 10 p = < 0.0001). After radiation, DPT was increased 20-fold in the bone marrow as shown by RT-PCR (p < 0.01). Homing assays in DPT pre-treated irradiated mice showed a 90% reduction (n = 12, p = 0.02) in whole marrow and a 40% reduction (n = 6, p < 0.05) lin-sca1+kit+ cells that migrated to the marrow 20 hours post-transplant, respectively. This reduction in homing translated to decreases in short- and long-term multilineage engraftment when assessed in primary and secondary transplant recipients. DPT contains a heparan-binding domain at its N-terminus, and co-treatment with DPT and heparin completely abrogated DPT's ability to inhibit homing. Colony forming unit assays and apoptosis assays of LSK cells revealed no toxic effects of DPT on hematopoietic cells suggesting a specific defect in cell migration. Adhesion assays showed a 55% decrease in the number of hematopoietic cells that adhered to an endothelial monolayer when DPT was present (n = 6, p = 0.01), and IHC revealed that DPT binds to the inner endothelial surface of the vascular after myeloablative conditioning. Furthermore, DPT was able to cause previously attached hematopoietic cells to be released from endothelial monolayers. We performed CFU assays of circulating cells 20 hours after adoptive transfer and found that DPT led to an increased number of circulating CFU (149 v 83 CFU/ml, p = 0.002 indicating that transplanted cells remained in circulation instead of efficiently homing to the marrow likely due to loss of adherence. These data suggest that DPT retards the ability of hematopoietic cells to adhere and transmigrate across the endothelial barrier during the homing process particularly in regions of increase adiposity. Thus DPT is a new and novel regulator of hematopoietic cell trafficking, albeit with negative consequences. While there have been several recent attempts to expand cell products or increase hematopoietic cell homing efficiency prior to transplant, overcoming or circumventing the "hurdles" of homing may also be an approach toward improving engraftment. DisclosuresNo relevant conflicts of interest to declare.