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Neutrophil granulocytes recruited upon translocation of intestinal bacteria enhance graft-versus-host disease via tissue damage

Nature Medicine volume 20, pages 648–654 (2014)Cite this article

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Abstract

Acute graft-versus-host disease (GVHD) considerably limits wider usage of allogeneic hematopoietic cell transplantation (allo-HCT). Antigen-presenting cells and T cells are populations customarily associated with GVHD pathogenesis. Of note, neutrophils are the largest human white blood cell population. The cells cleave chemokines and produce reactive oxygen species, thereby promoting T cell activation1,2. Therefore, during an allogeneic immune response, neutrophils could amplify tissue damage caused by conditioning regimens. We analyzed neutrophil infiltration of the mouse ileum after allo-HCT by in vivo myeloperoxidase imaging and found that infiltration levels were dependent on the local microbial flora and were not detectable under germ-free conditions. Physical or genetic depletion of neutrophils reduced GVHD-related mortality. The contribution of neutrophils to GVHD severity required reactive oxygen species (ROS) because selective Cybb (encoding cytochrome b-245, beta polypeptide, also known as NOX2) deficiency in neutrophils impairing ROS production led to lower levels of tissue damage, GVHD-related mortality and effector phenotype T cells. Enhanced survival of Bcl-xL transgenic neutrophils increased GVHD severity. In contrast, when we transferred neutrophils lacking Toll-like receptor-2 (TLR2), TLR3, TLR4, TLR7 and TLR9, which are normally less strongly activated by translocating bacteria, into wild-type C57BL/6 mice, GVHD severity was reduced. In humans, severity of intestinal GVHD strongly correlated with levels of neutrophils present in GVHD lesions. This study describes a new potential role for neutrophils in the pathogenesis of GVHD in both mice and humans.

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Figure 1: Anti-Ly6G antibody treatment improves survival after MHC-mismatched allo-HCT.
Figure 2: Myeloperoxidase-positive cells accumulate on day 3 following allo-HCT in the terminal ileum.
Figure 3: NOX2 deficiency of the recipient delays GVHD.
Figure 4: Neutrophil recruitment into the intestines is dependent on commensal microflora, and neutrophil-mediated tissue damage requires neutrophil activation via TLRs.

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References

  1. Tester, A.M. et al. LPS responsiveness and neutrophil chemotaxis in vivo require PMN MMP-8 activity. PLoS ONE 2, e312 (2007).

    Article  Google Scholar 

  2. Sena, L.A. et al. Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling. Immunity 38, 225–236 (2013).

    Article  CAS  Google Scholar 

  3. 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 (2007).

    Article  Google Scholar 

  4. Giroux, M. et al. SMAD3 prevents graft-versus-host disease by restraining TH1 differentiation and granulocyte-mediated tissue damage. Blood 117, 1734–1744 (2011).

    Article  CAS  Google Scholar 

  5. Gross, S. et al. Bioluminescence imaging of myeloperoxidase activity in vivo. Nat. Med. 15, 455–461 (2009).

    Article  CAS  Google Scholar 

  6. Yasuda, M. et al. Potential role of the NADPH oxidase NOX1 in the pathogenesis of 5-fluorouracil-induced intestinal mucositis in mice. Am. J. Physiol. Gastrointest. Liver Physiol. 302, G1133–G1142 (2012).

    Article  CAS  Google Scholar 

  7. Dale, D.C., Boxer, L. & Liles, W.C. The phagocytes: neutrophils and monocytes. Blood 112, 935–945 (2008).

    Article  CAS  Google Scholar 

  8. Wang, G.G. et al. Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8. Nat. Methods 3, 287–293 (2006).

    Article  CAS  Google Scholar 

  9. Gautam, S. et al. Survival and differentiation defects contribute to neutropenia in glucose-6-phosphatase-β (G6PC3) deficiency in a model of mouse neutrophil granulocyte differentiation. Cell Death Differ. 20, 1068–1079 (2013).

    Article  CAS  Google Scholar 

  10. Robinson, K.M. et al. Selective fluorescent imaging of superoxide in vivo using ethidium-based probes. Proc. Natl. Acad. Sci. USA 103, 15038–15043 (2006).

    Article  CAS  Google Scholar 

  11. Ziegler, T.R. et al. Regulation of glutathione redox status in lung and liver by conditioning regimens and keratinocyte growth factor in murine allogeneic bone marrow transplantation. Transplantation 72, 1354–1362 (2001).

    Article  CAS  Google Scholar 

  12. Zhang, Y., Joe, G., Hexner, E., Zhu, J. & Emerson, S.G. Alloreactive memory T cells are responsible for the persistence of graft-versus-host disease. J. Immunol. 174, 3051–3058 (2005).

    Article  CAS  Google Scholar 

  13. Seger, R.A. et al. Treatment of chronic granulomatous disease with myeloablative conditioning and an unmodified hemopoietic allograft: a survey of the European experience, 1985–2000. Blood 100, 4344–4350 (2002).

    Article  CAS  Google Scholar 

  14. Martinez, C.A. et al. Excellent survival after sibling or unrelated donor stem cell transplantation for chronic granulomatous disease. J. Allergy Clin. Immunol. 129, 176–183 (2012).

    Article  Google Scholar 

  15. Reichardt, W. et al. Impact of mammalian target of rapamycin inhibition on lymphoid homing and tolerogenic function of nanoparticle-labeled dendritic cells following allogeneic hematopoietic cell transplantation. J. Immunol. 181, 4770–4779 (2008).

    Article  CAS  Google Scholar 

  16. Jenq, R.R. et al. Regulation of intestinal inflammation by microbiota following allogeneic bone marrow transplantation. J. Exp. Med. 209, 903–911 (2012).

    Article  CAS  Google Scholar 

  17. Fischer, M.A. et al. CD11b+, Ly6G+ cells produce type I interferon and exhibit tissue protective properties following peripheral virus infection. PLoS Pathog. 7, e1002374 (2011).

    Article  CAS  Google Scholar 

  18. Dzhagalov, I., St. John, A. & He, Y.W. The antiapoptotic protein Mcl-1 is essential for the survival of neutrophils but not macrophages. Blood 109, 1620–1626 (2007).

    Article  CAS  Google Scholar 

  19. Kirschnek, S. et al. Molecular analysis of neutrophil spontaneous apoptosis reveals a strong role for the pro-apoptotic BH3-only protein Noxa. Cell Death Differ. 18, 1805–1814 (2011).

    Article  CAS  Google Scholar 

  20. Koedel, U. et al. Apoptosis is essential for neutrophil functional shutdown and determines tissue damage in experimental pneumococcal meningitis. PLoS Pathog. 5, e1000461 (2009).

    Article  Google Scholar 

  21. Li, H. et al. Graft-versus-host disease is independent of innate signaling pathways triggered by pathogens in host hematopoietic cells. J. Immunol. 186, 230–241 (2011).

    Article  CAS  Google Scholar 

  22. Luo, H.R. & Loison, F. Constitutive neutrophil apoptosis: mechanisms and regulation. Am. J. Hematol. 83, 288–295 (2008).

    Article  CAS  Google Scholar 

  23. Hill, G.R. et al. Differential roles of IL-1 and TNF-α on graft-versus-host disease and graft versus leukemia. J. Clin. Invest. 104, 459–467 (1999).

    Article  CAS  Google Scholar 

  24. Jankovic, D. et al. The Nlrp3-inflammasome regulates acute graft-versus-host disease. J. Exp. Med. 210, 1899–1910 (2013).

    Article  CAS  Google Scholar 

  25. Forlenza, M. et al. Differential contribution of neutrophilic granulocytes and macrophages to nitrosative stress in a host-parasite animal model. Mol. Immunol. 45, 3178–3189 (2008).

    Article  CAS  Google Scholar 

  26. Cooke, K.R. et al. Tumor necrosis factor-α production to lipopolysaccharide stimulation by donor cells predicts the severity of experimental acute graft-versus-host disease. J. Clin. Invest. 102, 1882–1891 (1998).

    Article  CAS  Google Scholar 

  27. Socié, G. et al. Prognostic value of apoptotic cells and infiltrating neutrophils in graft-versus-host disease of the gastrointestinal tract in humans: TNF and Fas expression. Blood 103, 50–57 (2004).

    Article  Google Scholar 

  28. Leonhardt, F. et al. Inflammatory neovascularization during graft-versus-host disease is regulated by αv integrin and miR-100. Blood 121, 3307–3318 (2013).

    Article  CAS  Google Scholar 

  29. Leonhardt, F. et al. Spleen tyrosine kinase (Syk) is a potent target for GVHD prevention at different cellular levels. Leukemia 26, 1617–1629 (2012).

    Article  CAS  Google Scholar 

  30. Wilhelm, K. et al. Graft-versus-host disease enhanced by extracellular adenosine triphosphate activating P2X7R. Nat. Med. 16, 1434–1438 (2010).

    Article  CAS  Google Scholar 

  31. Sitia, G. et al. Depletion of neutrophils blocks the recruitment of antigen-nonspecific cells into the liver without affecting the antiviral activity of hepatitis B virus-specific cytotoxic T lymphocytes. Proc. Natl. Acad. Sci. USA 99, 13717–13722 (2002).

    Article  CAS  Google Scholar 

  32. Lerner, K.G. et al. Histopathology of graft-vs.-host reaction (GvHR) in human recipients of marrow from HL-A-matched sibling donors. Transplant. Proc. 6, 367–371 (1974).

    CAS  PubMed  Google Scholar 

  33. Leonhardt, F. et al. Inflammatory neovascularization during graft-versus-host disease is regulated by αv integrin and miR-100. Blood 121, 3307–3318 (2013).

    Article  CAS  Google Scholar 

  34. Zeiser, R. et al. Early CD30 signaling is critical for adoptively transferred CD4+CD25+ regulatory T cells in prevention of acute graft versus host disease. Blood 109, 2225–2233 (2007).

    Article  CAS  Google Scholar 

  35. Kaplan, D.H. et al. Target antigens determine graft-versus-host disease phenotype. J. Immunol. 173, 5467–5475 (2004).

    Article  CAS  Google Scholar 

  36. Sadeghi, B. et al. GVHD after chemotherapy conditioning in allogeneic transplanted mice. Bone Marrow Transplant. 42, 807–818 (2008).

    Article  CAS  Google Scholar 

  37. Turner, B.E. et al. Reduced intensity conditioning for allogeneic hematopoietic stem-cell transplant determines the kinetics of acute graft-versus-host disease. Transplantation 86, 968–976 (2008).

    Article  Google Scholar 

  38. Wolf, P. et al. Three conformational antibodies specific for different PSMA epitopes are promising diagnostic and therapeutic tools for prostate cancer. Prostate 70, 562–569 (2010).

    CAS  PubMed  Google Scholar 

  39. Zeiser, R. et al. Differential impact of mTOR inhibition on CD4+CD25+Foxp3+ regulatory T cells as compared to conventional CD4+ T cells. Blood 111, 453–462 (2008).

    Article  CAS  Google Scholar 

  40. Gross, S. et al. Bioluminescence imaging of myeloperoxidase activity in vivo. Nat. Med. 15, 455–461 (2009).

    Article  CAS  Google Scholar 

  41. Wilhelm, K. et al. Graft-versus-host disease enhanced by extracellular adenosine triphosphate activating P2X7R. Nat. Med. 16, 1434–1438 (2010).

    Article  CAS  Google Scholar 

  42. Reichardt, W. et al. Impact of mammalian target of rapamycin inhibition on lymphoid homing and tolerogenic function of nanoparticle-labeled dendritic cells following allogeneic hematopoietic cell transplantation. J. Immunol. 181, 4770–4779 (2008).

    Article  CAS  Google Scholar 

  43. Hoerr, V. et al. Bacteria tracking by in vivo magnetic resonance imaging. BMC Biol. 11, 63 (2013).

    Article  Google Scholar 

  44. Haacke, E.M. et al. Susceptibility weighted imaging (SWI). Magn. Reson. Med. 52, 612–618 (2004).

    Article  Google Scholar 

  45. Wang, G.G. et al. Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8. Nat. Methods 3, 287–293 (2006).

    Article  CAS  Google Scholar 

  46. Weigmann, B. et al. Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue. Nat. Protoc. 2, 2307–2311 (2007).

    Article  CAS  Google Scholar 

  47. Irizarry, R.A. et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249–264 (2003).

    Article  Google Scholar 

  48. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Series B Stat. Methodol. 57, 289–300 (1995).

    Google Scholar 

  49. Davies, J.A., Anderson, G.K., Beveridge, T.J. & Clark, H.C. Chemical mechanism of the gram stain and synthesis of a new electron-opaque marker for electron microscopy which replaces the iodine mordant of the stain. J. Bacteriol. 156, 837–845 (1983).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Robinson, K.M. et al. Selective fluorescent imaging of superoxide in vivo using ethidium-based probes. Proc. Natl. Acad. Sci. USA 103, 15038–15043 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by the Deutsche Jose Carreras Leukämie Stiftung (DJCLS grant # R12/11), Deutsche Forschungsgemeinschaft (DFG), Germany, Heisenberg Professorship to R.Z. (DFG ZE 872/3-1) and DFG individual grant to R.Z. (DFG ZE 872/1-2), in part by SFB 850 (to R.Z.), Excellence Initiative of the German Research Foundation (GSC-4, Spemann Graduate School to R.Z. and G.H. and BIOSS II, project no. B13 to R.Z.), Deutsche Krebshilfe (to R.Z.), and Marie Curie FP7-IRG268390 (to A.T.) and also in part by the IDEA 2012 award (LSU Health Sciences Center, to G.C.H.). A.M. was funded by the European Research Council and the Wellcome Trust. H.H. was supported by the American Lebanese Syrian Associated Charities (ALSAC). We thank M. Follo for proofreading the manuscript.

Author information

Author notes

  1. Lukas Schwab, Luise Goroncy and Senthilnathan Palaniyandi: These authors contributed equally to this work.

  2. Gerhard C Hildebrandt, Georg Häcker and Robert Zeiser: These authors jointly supervised this work.

Authors and Affiliations

  1. Department of Hematology and Oncology, University Medical Center, Freiburg, Germany

    Lukas Schwab, Luise Goroncy, Kathrin Hanke, Franziska Leonhardt, Dietmar Pfeifer, Oliver Schmah, Anne Schönle, Roland Mertelsmann, Justus Duyster, Jürgen Finke & Robert Zeiser

  2. Faculty of Biology, Albert-Ludwigs-University, Freiburg, Germany

    Luise Goroncy, Sanjivan Gautam, Kathrin Hanke & Franziska Leonhardt

  3. Division of Hematology and Oncology, Louisiana State University Health Sciences Center Shreveport, Shreveport, Louisiana, USA

    Senthilnathan Palaniyandi, Fridrik J Karlsson, Sabarinath V Radhakrishnan & Gerhard C Hildebrandt

  4. Division of Bone Marrow Transplantation, University of Utah School of Medicine, Huntsman Cancer Institute, Salt Lake City, Utah, USA

    Senthilnathan Palaniyandi, Sabarinath V Radhakrishnan & Gerhard C Hildebrandt

  5. Department of Medical Microbiology and Hygiene, University Medical Center, Freiburg, Germany

    Sanjivan Gautam, Friederike D von Loewenich & Georg Häcker

  6. Spemann Graduate School of Biology and Medicine, Albert-Ludwigs-University Freiburg, Germany

    Sanjivan Gautam, Marco Prinz, Georg Häcker & Robert Zeiser

  7. Center of Chronic Immunodeficiency, Albert-Ludwigs-University Freiburg, Germany

    Antigoni Triantafyllopoulou & Philipp Henneke

  8. Department of Physiology, Semmelweis University School of Medicine, Budapest, Hungary

    Attila Mocsai

  9. Department of Radiology Medical Physics, University Medical Center, Freiburg, Germany

    Wilfried Reichardt & Nicoleta Baxan

  10. Department of Pathology, University Medical Center, Albert-Ludwigs-University, Freiburg, Germany

    Annette Schmitt-Graeff

  11. Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany

    Marina Freudenberg

  12. Centre for Biological Signaling Studies BIOSS, Albert-Ludwigs-University Freiburg, Germany

    Marina Freudenberg & Robert Zeiser

  13. Department of Urology, University Medical Center, Freiburg, Germany

    Philipp Wolf

  14. Department of Dermatology, Allergy Research Group, University Medical Center, University Freiburg, Germany

    Stefan F Martin

  15. Department of Neuropathology, University Medical Center, Freiburg, Germany

    Marco Prinz

  16. Center for Pediatrics and Adolescent Medicine, University Medical Center, Freiburg, Germany

    Philipp Henneke

  17. Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

    Hans Häcker

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  1. Lukas Schwab
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Contributions

L.S., L.G., S.P., F.J.K, S.V.R., A.T., S.G., M.F., F.L., A.S. and K.H. helped design the experiments and performed experiments. S.F.M., R.M., J.F., P.H., M.P., A.M., F.D.v.L. and J.D. helped design the experiments, provided essential reagents and discussed the data. O.S. helped collect patient samples and provided clinical data, H.H. helped design experiments with HoxB8 cells and discussed data. W.R. and N.B. performed and analyzed magnetic resonance imaging experiments. P.W. performed anti-Ly6G antibody measurement in serum of mice, analyzed data and helped write the manuscript. A.S.-G. analyzed GVHD histopathology and neutrophil infiltration. D.P. performed and analyzed microarray experiments. G.C.H., G.H. and R.Z. developed the overall concept, supervised the experiments, discussed the data and wrote the manuscript.

Corresponding author

Correspondence to Robert Zeiser.

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The authors declare no competing financial interests.

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Schwab, L., Goroncy, L., Palaniyandi, S. et al. Neutrophil granulocytes recruited upon translocation of intestinal bacteria enhance graft-versus-host disease via tissue damage. Nat Med 20, 648–654 (2014). https://doi.org/10.1038/nm.3517

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