Bone Marrow Granulocytes Drive Vascular and Hematopoietic Regeneration
Authors: Bowers Emily , Anastasiya Slaughter , Lucas-Alcaraz Daniel 
Published: October 2019

Abstract
In addition to eliminating host hematopoietic cells myeloablation also disrupts the blood vessels that sustain hematopoiesis. Regeneration of the bone marrow (BM) vasculature is necessary for hematopoietic recovery and survival after transplantation (Cell Stem Cell. 2009 Mar 6;4(3):263-74) but the mechanisms that drive vascular regeneration are not clear.
We found that, fourteen days after lethal irradiation and transplantation, mice transplanted with 20x106 bone marrow nucleated cells (BMNC) had ~6-fold more CD45-Ter119-CD31+CD105+ endothelial cells (6.9x103 vs 0.96x103 EC/femur, p<0.001), 2-fold more blood vessels (195 vs 87 blood vessels/sternum, p<0.05) and ~2-fold less vascular leakage (4.8 vs 9.3 ng of Evans Blue/ml of BM extracellular fluid, p<0.001) than mice transplanted with 105 BMNC. Transplant experiments using GFP+donor BMNC revealed that all endothelial cells after transplantation were host derived.
Because hematopoietic progenitors inhibit vascular regeneration via angiopoietin 1 (Elife2015 Mar 30;4:e05521) we hypothesized that mature hematopoietic cells mediated vascular recovery. To test this we adoptively transferred, B and T cells, monocytes and macrophages (MO), granulocytes and erythroid cells into lethally irradiated recipients previously transplanted with 105 donor BMNC. Only CD115-Gr1+ granulocytes promoted endothelial cell regeneration (2.5x103 for granulocyte treated mice vs 0.9x103 for PBS, 0.3x103 for B- and T-cell, 0.7 1x103 for MO and 0.3x103 EC/femur for erythroid cell-treated mice; p<0.01). Granulocyte transfer also promoted survival (granulocytes=100%, PBS=50% p<0.05), probably due to faster host platelets and red blood cells recovery (granulocytes= 4.5x107, PBS=2.1x107 platelets/ml of blood, p<0.001; granulocytes=4x109, PBS=6.2x109 RBC/ml of blood, p<0.01). Importantly, competitive BM transplants showed that granulocytes did not exhaust donor HSC. These demonstrate that granulocyte transfer is sufficient to promote survival and drive vascular and hematopoietic recovery after transplantation. We then generated Mrp8-cre:iDTR mice which allowed us to specifically ablate BM granulocytes via diphtheria toxin (DT) injection. We transplanted lethally irradiated WT recipients with 106 BMNC purified from C67BL/6 WT or Mrp8-cre:iDTR mice followed by DT treatment for 7 days. This led to granulocyte depletion (1.6x106 vs 0.4x106 p<0.001) and impaired endothelial cell recovery (5.7x103 vs 2.4.x103 p<0.05) in mice transplanted with Mrp8-cre:iDTR BMNC. These results demonstrate that donor granulocytes are necessary for vascular regeneration.
We found that granulocytes produced high levels of the angiogenic cytokine TNFα. This cytokine signals via Tnfrsf1aand Tnfrsf1b. Tnfrsf1a was upregulated specifically in BM endothelial cells. After myeloablation with 5-fluorouracil Tnfa-/-mice have reduced survival (Tnfa-/-= 13% vs WT= 93%; p<0.001) and reduced endothelial cell numbers (WT=9x103, Tnfa-/-=4.1x103 EC/femur; p<0.05) indicating that TNFα is necessary for survival and vascular regeneration after myeloablation. To test whether granulocytes promoted vascular regeneration via TNFα we lethally irradiated and transplanted C57BL/6 recipients followed by treatment with PBS or adoptive transfer of 106 WT or Tnfa-/- granulocytes. Only WT granulocytes induced vascular recovery as demonstrated by quantification of endothelial cells (PBS=0.9 x103, WT granulocytes=5.24x103 and Tnfa-/- granulocytes=3.0x103 cells/femur, p<0.05) and blood vessel numbers (PBS=126, WT granulocytes=186 and Tnfa-/- granulocytes=84 vessels per sternum BM; p<0.05). Further, adoptive transfer of WT granulocytes promoted survival and vascular regeneration (WT+PBS=1.4x103 vs WT+granulocytes=2.6x103, p<0.05; Tnfrsf1a-/-:Tnfrsf1b-/- +PBS=0.8x103 vs Tnfrsf1a-/-:Tnfrsf1b-/-+granulocytes=0.7x103 EC/femur p=0.83) in WT but not Tnfrsf1a-/-:Tnfrsf1b-/-recipients after transplantation. These experiments demonstrate that granulocytes crosstalk directly with stromal cells (likely endothelial cells) via TNFα to drive vascular regeneration.
We have identified a new type of cellular crosstalk in the microenvironment that drives regeneration. Our research also provides proof of principle for studies targeting BM granulocytes to enhance vascular recovery and survival after transplantation in patients.

Disclosures
No relevant conflicts of interest to declare.