Granulocyte-derived TNFα promotes vascular and hematopoietic regeneration in the bone marrow

Abstract

Endothelial cells are a critical component of the bone marrow (BM) stromal network, which maintains and regulates hematopoietic cells1,2,3,4,5,6,7,8,9. Vascular regeneration precedes, and is necessary for, successful hematopoietic stem cell (HSC) transplantation, the only cure for most hematopoietic diseases2,4. Recent data suggest that mature hematopoietic cells regulate BM stromal-cell function10,11,12,13. Whether a similar cross-talk regulates the BM vasculature is not known. Here we found that donor hematopoietic cells act on sinusoidal endothelial cells and induce host blood vessel and hematopoietic regeneration after BM transplantation in mice. Adoptive transfer of BM, but not peripheral, granulocytes prevented the death of mice transplanted with limited numbers of HSCs and accelerated recovery of host vessels and hematopoietic cells. Moreover, selective granulocyte ablation in vivo impaired vascular and hematopoietic regeneration after BM transplantation. Gene expression analyses indicated that granulocytes are the main source of the cytokine TNFα, whereas its receptor TNFR1 is selectively upregulated in regenerating blood vessels. In adoptive transfer experiments, wild type, but not Tnfa−/−, granulocytes induced vascular recovery, and wild-type granulocyte transfer did not prevent death or promote vascular regeneration in Tnfr1−/−; Tnfr2−/− mice. Thus, by delivering TNFα to endothelial cells, granulocytes promote blood vessel growth and hematopoietic regeneration. Manipulation of the cross-talk between granulocytes and endothelial cells may lead to new therapeutic approaches to improve blood vessel regeneration and increase survival and hematopoietic recovery after HSC transplantation.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Donor hematopoietic cells drive endogenous vascular regeneration after transplantation.
Figure 2: Granulocytes promote vascular regeneration.
Figure 3: Granulocytes are necessary for vascular regeneration.
Figure 4: Granulocytes promote vascular regeneration via TNFα.

References

  1. 1

    Butler, J.M. et al. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell 6, 251–264 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Hooper, A.T. et al. Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2-mediated regeneration of sinusoidal endothelial cells. Cell Stem Cell 4, 263–274 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Poulos, M.G. et al. Endothelial Jagged-1 is necessary for homeostatic and regenerative hematopoiesis. Cell Rep. 4, 1022–1034 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Doan, P.L. et al. Tie2+ bone marrow endothelial cells regulate hematopoietic stem cell regeneration following radiation injury. Stem Cells 31, 327–337 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Itkin, T. et al. Distinct bone marrow blood vessels differentially regulate haematopoiesis. Nature 532, 323–328 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Kusumbe, A.P. et al. Age-dependent modulation of vascular niches for haematopoietic stem cells. Nature 532, 380–384 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Greenbaum, A. et al. CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance. Nature 495, 227–230 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Ding, L., Saunders, T.L., Enikolopov, G. & Morrison, S.J. Endothelial and perivascular cells maintain haematopoietic stem cells. Nature 481, 457–462 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Himburg, H.A. et al. Pleiotrophin regulates the retention and self-renewal of hematopoietic stem cells in the bone marrow vascular niche. Cell Rep. 2, 964–975 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Casanova-Acebes, M. et al. Rhythmic modulation of the hematopoietic niche through neutrophil clearance. Cell 153, 1025–1035 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Chow, A. et al. Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche. J. Exp. Med. 208, 261–271 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Christopher, M.J., Rao, M., Liu, F., Woloszynek, J.R. & Link, D.C. Expression of the G-CSF receptor in monocytic cells is sufficient to mediate hematopoietic progenitor mobilization by G-CSF in mice. J. Exp. Med. 208, 251–260 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Winkler, I.G. et al. Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs. Blood 116, 4815–4828 (2010).

    CAS  Article  Google Scholar 

  14. 14

    Morrison, S.J. & Scadden, D.T. The bone marrow niche for haematopoietic stem cells. Nature 505, 327–334 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Winkler, I.G. et al. Vascular niche E-selectin regulates hematopoietic stem cell dormancy, self renewal and chemoresistance. Nat. Med. 18, 1651–1657 (2012).

    Article  CAS  Google Scholar 

  16. 16

    Himburg, H.A. et al. Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells. Nat. Med. 16, 475–482 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Himburg, H.A. et al. Pleiotrophin mediates hematopoietic regeneration via activation of RAS. J. Clin. Invest. 124, 4753–4758 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Kunisaki, Y. et al. Arteriolar niches maintain haematopoietic stem cell quiescence. Nature 502, 637–643 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Poulos, M.G. et al. Endothelial-specific inhibition of NF-κB enhances functional haematopoiesis. Nat. Commun. 7, 13829 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Radu, M. & Chernoff, J. An in vivo assay to test blood vessel permeability. J. Vis. Exp. e50062 (2013).

  21. 21

    Zhou, B.O., Ding, L. & Morrison, S.J. Hematopoietic stem and progenitor cells regulate the regeneration of their niche by secreting Angiopoietin-1. eLife 4, e05521 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  22. 22

    Na Nakorn, T., Traver, D., Weissman, I.L. & Akashi, K. Myeloerythroid-restricted progenitors are sufficient to confer radioprotection and provide the majority of day 8 CFU-S. J. Clin. Invest. 109, 1579–1585 (2002).

    Article  PubMed  PubMed Central  Google Scholar 

  23. 23

    Tsuchiya, Y., Nakabayashi, O. & Nakano, H. FLIP the switch: regulation of apoptosis and necroptosis by cFLIP. Int. J. Mol. Sci. 16, 30321–30341 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Daley, J.M., Thomay, A.A., Connolly, M.D., Reichner, J.S. & Albina, J.E. Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice. J. Leukoc. Biol. 83, 64–70 (2008).

    Article  CAS  PubMed  Google Scholar 

  25. 25

    Brenner, D., Blaser, H. & Mak, T.W. Regulation of tumour necrosis factor signalling: live or let die. Nat. Rev. Immunol. 15, 362–374 (2015).

    Article  CAS  PubMed  Google Scholar 

  26. 26

    Leibovich, S.J. et al. Macrophage-induced angiogenesis is mediated by tumour necrosis factor-alpha. Nature 329, 630–632 (1987).

    Article  CAS  PubMed  Google Scholar 

  27. 27

    Baluk, P. et al. TNF-alpha drives remodeling of blood vessels and lymphatics in sustained airway inflammation in mice. J. Clin. Invest. 119, 2954–2964 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Espín, R. et al. TNF receptors regulate vascular homeostasis in zebrafish through a caspase-8, caspase-2 and P53 apoptotic program that bypasses caspase-3. Dis. Model. Mech. 6, 383–396 (2013).

    Article  CAS  PubMed  Google Scholar 

  29. 29

    Asada, N. et al. Differential cytokine contributions of perivascular haematopoietic stem cell niches. Nat. Cell Biol. 19, 214–223 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Eash, K.J., Greenbaum, A.M., Gopalan, P.K. & Link, D.C. CXCR2 and CXCR4 antagonistically regulate neutrophil trafficking from murine bone marrow. J. Clin. Invest. 120, 2423–2431 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Rezzoug, F. et al. TNF-alpha is critical to facilitate hemopoietic stem cell engraftment and function. J. Immunol. 180, 49–57 (2008).

    Article  CAS  PubMed  Google Scholar 

  32. 32

    Grunewald, M. et al. VEGF-induced adult neovascularization: recruitment, retention, and role of accessory cells. Cell 124, 175–189 (2006).

    Article  CAS  PubMed  Google Scholar 

  33. 33

    Doan, P.L. et al. Epidermal growth factor regulates hematopoietic regeneration after radiation injury. Nat. Med. 19, 295–304 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Pietras, E.M. et al. Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions. Cell Stem Cell 17, 35–46 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Pearl-Yafe, M. et al. Tumor necrosis factor receptors support murine hematopoietic progenitor function in the early stages of engraftment. Stem Cells 28, 1270–1280 (2010).

    CAS  PubMed  Google Scholar 

  36. 36

    Rebel, V.I. et al. Essential role for the p55 tumor necrosis factor receptor in regulating hematopoiesis at a stem cell level. J. Exp. Med. 190, 1493–1504 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Pronk, C.J., Veiby, O.P., Bryder, D. & Jacobsen, S.E. Tumor necrosis factor restricts hematopoietic stem cell activity in mice: involvement of two distinct receptors. J. Exp. Med. 208, 1563–1570 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Ishida, T. et al. Pre-transplantation blockade of TNF-α-mediated oxygen species accumulation protects hematopoietic stem cells. Stem Cells 35, 989–1002 (2017).

    Article  CAS  PubMed  Google Scholar 

  39. 39

    Lucas, D. et al. Chemotherapy-induced bone marrow nerve injury impairs hematopoietic regeneration. Nat. Med. 19, 695–703 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Galotto, M. et al. Stromal damage as consequence of high-dose chemo/radiotherapy in bone marrow transplant recipients. Exp. Hematol. 27, 1460–1466 (1999).

    Article  CAS  Google Scholar 

  41. 41

    Schaefer, B.C., Schaefer, M.L., Kappler, J.W., Marrack, P. & Kedl, R.M. Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions in vivo. Cell. Immunol. 214, 110–122 (2001).

    Article  CAS  PubMed  Google Scholar 

  42. 42

    Passegué, E., Wagner, E.F. & Weissman, I.L. JunB deficiency leads to a myeloproliferative disorder arising from hematopoietic stem cells. Cell 119, 431–443 (2004).

    Article  PubMed  Google Scholar 

  43. 43

    Buch, T. et al. A Cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration. Nat. Methods 2, 419–426 (2005).

    Article  CAS  Google Scholar 

  44. 44

    Pasparakis, M., Alexopoulou, L., Episkopou, V. & Kollias, G. Immune and inflammatory responses in TNF alpha-deficient mice: a critical requirement for TNF alpha in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response. J. Exp. Med. 184, 1397–1411 (1996).

    Article  CAS  PubMed  Google Scholar 

  45. 45

    Peschon, J.J. et al. TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation. J. Immunol. 160, 943–952 (1998).

    CAS  PubMed  Google Scholar 

  46. 46

    Lucas, D., Battista, M., Shi, P.A., Isola, L. & Frenette, P.S. Mobilized hematopoietic stem cell yield depends on species-specific circadian timing. Cell Stem Cell 3, 364–366 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Méndez-Ferrer, S., Lucas, D., Battista, M. & Frenette, P.S. Haematopoietic stem cell release is regulated by circadian oscillations. Nature 452, 442–447 (2008).

    Article  CAS  Google Scholar 

  48. 48

    Puram, R.V. et al. Core circadian clock genes regulate leukemia stem cells in AML. Cell 165, 303–316 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Suire, C., Brouard, N., Hirschi, K. & Simmons, P.J. Isolation of the stromal-vascular fraction of mouse bone marrow markedly enhances the yield of clonogenic stromal progenitors. Blood 119, e86–e95 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank M. May for excellent technical support. This work was supported by the Pardee Foundation (D.L.). E.B. was funded through a T32 training grant (2T32HD007505-21) from the Center of Organogenesis at the University of Michigan. We thank M. Hoenerhoff and the rest of the University of Michigan in vivo core facility for performing pathology analyses. We thank the mouse imaging laboratory and the flow cytometry core facility at the University of Michigan for help with imaging and FACS experiments. R.K. and flow cytometry and whole-mount immunofluorescence analyses were partially supported by a core grant from the NIH to the University of Michigan Cancer Center (P30-CA46592).

Author information

Affiliations

Authors

Contributions

E.B. and D.L. designed the study; E.B., A.S. and D.L. designed, performed and analyzed experiments. O.M.P. suggested and designed experiments. R.K. performed statistical analyses. P.S.F. provided Nestin-gfp, Tnfa−/−, Tnfr1−/− and Tnfr2−/− mice and designed experiments. E.B. and D.L. wrote the manuscript with help from all coauthors. D.L. supervised the manuscript preparation.

Corresponding author

Correspondence to Daniel Lucas.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Table 1 (PDF 2606 kb)

Life Sciences Reporting Summary (PDF 251 kb)

41591_2018_BFnm4448_MOESM3_ESM.mov

True vessels (with lumen) are indicated by blue arrows and can be easily distinguished from vascular sheets that appear after irradiation (yellow arrows). (MOV 1032 kb)

3D reconstruction of endothelial cells (CD31/CD144+, white) in the sternum of a lethally irradiated mouse fourteen days after transplantation of 20×106 BMNC.

True vessels (with lumen) are indicated by blue arrows and can be easily distinguished from vascular sheets that appear after irradiation (yellow arrows). (MOV 1032 kb)

41591_2018_BFnm4448_MOESM4_ESM.mov

True vessels (with lumen) are indicated by blue arrows and can be easily distinguished from vascular sheets that appear after irradiation (yellow arrows). (MOV 1007 kb)

3D reconstruction of endothelial cells (CD31/CD144+, white) in the sternum of a lethally irradiated mouse fourteen days after transplantation of 0.1×106 BMNC.

True vessels (with lumen) are indicated by blue arrows and can be easily distinguished from vascular sheets that appear after irradiation (yellow arrows). (MOV 1007 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bowers, E., Slaughter, A., Frenette, P. et al. Granulocyte-derived TNFα promotes vascular and hematopoietic regeneration in the bone marrow. Nat Med 24, 95–102 (2018). https://doi.org/10.1038/nm.4448

Download citation

Further reading