Human and Murine β-Defensin-Derived Peptides Induce Rapid Mobilization Of Murine Hematopoietic Stem and Progenitor Cells Via Activation Of CXCR4 Signaling and CXCL12 Release
Authors: Schajnovitz Amir , Kalinkovich Alexander , Lapid Kfir , Berchanski Alexander , Itkin Tomer , Ludin Aya , Gur-Cohen Shiri , Golan Karin , Saez Borja , Kollet Orit , Ahrens Kerstin , Proksch Ehrahrdt , Lapidot Aviva , Fridkin Matityahu , Scadden David T. , Lapidot Tsvee 
Published: October 2019

Abstract

Background
Rapid mobilization of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) to the peripheral blood by anti-CXCR4 agents such as AMD3100 is a complex process, which requires CXCL12 secretion, activation of proteolytic enzymes and supporting cellular compartments (Dar et. al, Leukemia 2011). Notably, components of innate immune system were also shown to be involved (Ratajczak et. al, Leukemia 2010). Human β-defensin-3 (hBD3) is an antimicrobial peptide possessing also anti-CXCR4 effects on human T cells in vitro (Feng et. al, JI 2006), suggesting its HSPC mobilizing potential. Here, we describe a novel approach for targeting CXCR4 in vivo by utilizing β-defensin-derived peptides, resulting in rapid HSPC mobilization.

Results
While AMD3100 blocked CXCL12-mediated signaling and migration of enriched BM mononuclear cells (MNCs) in vitro, we unexpectedly detected rapid phosphorylation of AKT, p38 and ERK1/2 in BM stromal cells (BMSCs). Interestingly, single administration of AMD3100 to mice resulted in enhancement of CXCR4 phosphorylation within minutes in both BM residing mesenchymal stem/progenitor cells (MSCs) and HSPCs, thus suggesting a CXCR4 agonistic activity. Aiming to test HSPC mobilizing potential of hBD3 and avoiding potential toxicity of systemic administration, we synthesized short linear peptides, comprising the C-terminal parts of hBD3 and the murine ortholog β-defensin-14 (mBD14), as well as a cyclic peptide of hBD3, comprising the same amino acids as the linear one, to serve as a control. All full-length β-defensins and tested peptides (both linear and cyclic) specifically bound CXCR4 (demonstrated by docking approach and anti-CXCR4 antibody competition assay) and efficiently blocked CXCL12-mediated activity of enriched BM MNCs in vitro including cell migration and CXCR4-dependent HIV infection. Intriguingly, full-length β-defensins and derived linear peptides (but not cyclic) revealed a strong stimulatory effect on BMSCs in vitro: triggering phosphorylation of AKT, p38 and ERK1/2 along with enhancing secretion of functional CXCL12. Administration of linear peptides to mice led to a fast activation of CXCR4 signaling in BMSCs and MSCs as well as in HSPCs accompanied by CXCL12 release to the circulation, increased activity of proteolytic enzymes and consequent rapid mobilization of progenitors as well as long-term repopulating stem cells. In addition, linear peptides augmented AMD3100-induced rapid mobilization. Importantly, the control cyclic peptide, which bound CXCR4 but failed to activate BMSCs in vitro, did not induce HSPC mobilization in vivo. Moreover, it inhibited both steady-state egress and AMD3100-induced mobilization of HSPCs. A series of in vivo inhibitory analyses confirmed dependence of hBD3- and mBD14-derived peptide-induced HSPC mobilization on the activation of CXCL12/CXCR4 axis and revealed involvement of uPA and JNK signaling as well as ROS generation.

Conclusions
Our study demonstrated for the first time the capability of β-defensin-derived peptides to activate in vivo CXCL12/CXCR4 signaling in both hematopoietic and non-hematopoietic BM cells, leading to rapid HSPC mobilization. We suggest that activation of CXCR4 signaling in non-hematopoietic BM cells is crucial for inducing HSPC mobilization. Accordingly, CXCR4-binding agents capable of triggering CXCR4 signaling in non-hematopoietic BM cells in vitro, would induce rapid HSPC mobilization. The results presented here help to better understand the mechanisms of rapid HSPC mobilization and have the potential of improving existing clinical protocols to increase the yield of HSPC harvest for transplantation.

Disclosures:
Scadden: Fate Therapeutics: Consultancy, Equity Ownership.