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Osteoblasts participate in the innate immunity of the bone by producing human beta defensin-3

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Abstract

Gram-positive bacterial bone infections are an important cause of morbidity particularly in immunocompromised patients. Antimicrobial peptides (AP) are effectors of the innate immune system and directly kill microorganisms in the first hours after microbial infection. The aim of the present investigation was to study the expression and regulation of gram-positive specialized human β-defensin-3 (HBD-3) in bone. Samples of healthy and osteomyelitic human bone were assessed for the expression of HBD-3. Using primary and immortalized osteoblasts (SAOS-2 cells), release and regulation of HBD-3 was evaluated after exposure to Staphylococcus aureus supernatant and/or corticosteroids using PCR, immunohistochemistry, Western blot and ELISA. To determine the role of toll-like-receptors-2 and -4 (TLR-2/-4), shRNA was used to downregulate TLRs. An osteomyelitis mouse model was created performed to investigate the release of murine β-defensins using immunohistochemistry and RT-PCR. Cultured osteoblasts and human bone produce HBD-3 under standard conditions. The release increases within hours of bacterial supernatant exposure in cultured osteoblasts. This observation was not made in chronically infected bone samples. The shRNA-technology revealed the necessity of TLR-2 and -4 in HBD-3 induction in osteoblasts. Blocking protein synthesis with cycloheximide showed that the rapid release of HBD-3 is not dependent on a translational de novo synthesis and is not affected by glucocorticoids. The murine osteomyelitis model confirmed the in vivo release uptake of mouse β-defensins-4 (MBD-4) in bone. This report shows the bacterial induction of HBD-3 via TLR-2 and -4 in osteoblasts and suggests a central role of antimicrobial peptides in the prevention of bacterial bone infection. The rapid and effective induction of HBD-3 in osteoblasts incubated with conditioned media from bacteria is more likely a result of a rapid secretion of preformed HBD-3 by osteoblasts rather than a result of enhanced biosynthesis. The increased incidence of gram-positive bacterial bone infection in patients with regular intake of glucocorticoids does not seem to be caused by a deranged HBD-3 release in osteoblasts.

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References

  • Akira S, Takeda K, Kaisho T (2001) Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2:675–678

    Article  PubMed  CAS  Google Scholar 

  • Bals R, Wang X, Meegalla RL, Wattler S, Weiner DJ, Nehls MC, Wilson JM (1999) Mouse beta-defensin 3 is an inducible antimicrobial peptide expressed in the epithelia of multiple organs. Infect Immun 67:3542–3547

    PubMed  CAS  Google Scholar 

  • Burd RS, Furrer JL, Sullivan J, Smith AL (2002) Murine beta-defensin-3 is an inducible peptide with limited tissue expression and broad-spectrum antimicrobial activity. Shock 18:461–464

    Article  PubMed  Google Scholar 

  • Chertov O, Michiel DF, Xu L, Wang JM, Tani K, Murphy WJ, Longo DL, Taub DD, Oppenheim JJ (1996) Identification of defensin-1, defensin-2, and CAP37/azurocidin as T-cell chemoattractant proteins released from interleukin-8-stimulated neutrophils. J Biol Chem 271:2935–2940

    Article  PubMed  CAS  Google Scholar 

  • Cunningham R, Cockayne A, Humphreys H (1996) Clinical and molecular aspects of the pathogenesis of Staphylococcus aureus bone and joint infections. J Med Microbiol 44:157–164

    PubMed  CAS  Google Scholar 

  • Duits LA, Rademaker M, Ravensbergen B, van Sterkenburg MA, van Strijen E, Hiemstra PS, Nibbering PH (2001) Inhibition of hBD-3, but not hBD-1 and hBD-2, mRNA expression by corticosteroids. Biochem Biophys Res Commun 280(2):522–555

    Article  PubMed  CAS  Google Scholar 

  • Fukayama S, Tashjian AH (1989) Direct modulation by androgens of the response of human bone cells (SaOS-2) to human parathyroid hormone (PTH) and PTH-related protein. Endocrinology 125:1789–1794

    PubMed  CAS  Google Scholar 

  • Gläser R, Jürgen Harder J, Lange H, Joachim Bartels J, Christophers E, Schröder JM (2005) Antimicrobial psoriasin (S100A7) protects human skin from E. coli infection. Nat Immunol 6(1):57–64

    Article  PubMed  CAS  Google Scholar 

  • Harder J, Bartels J, Christophers E, Schröder JM (1997) A peptide antibiotic from human skin. Nature 387:861

    Article  PubMed  CAS  Google Scholar 

  • Harder J, Bartels J, Christophers E, Schröder JM (2001) Isolation and characterization of human β-defensin-3, a novel human inducible peptide antibiotic. J Biol Chem 276:5707–5713

    Article  PubMed  CAS  Google Scholar 

  • Jia HP, Wowk SA, Schutte BC, Lee SK, Vivado A, Tack BF, Bevins CL, McCray PB Jr (2000) A novel murine beta -defensin expressed in tongue, esophagus, and trachea. J Biol Chem 275:33314–33320

    Article  PubMed  CAS  Google Scholar 

  • Joly S, Maze C, McCray PB Jr, Guthmiller JM (2004) Human beta-defensins 2 and -3 demonstrate strain-selective activity against oral microorganisms. J Clin Microbiol 42(3):1024–1029

    Article  PubMed  CAS  Google Scholar 

  • Lew DP, Waldvogel FA (1997) Osteomyelitis. N Engl J Med 336:999–1007

    Article  PubMed  CAS  Google Scholar 

  • Lucke M, Schmidmaier G, Sadoni S, Wildemann B, Schiller R, Haas NP, Raschke M (2003) Gentamicin coating of metallic implants reduces implant-related osteomyelitis in rats. Bone 32(5):521–531

    Article  PubMed  CAS  Google Scholar 

  • Maisetta G, Batoni G, Esin S, Florio W, Bottai D, Favilli F, Campa M (2006) In vitro bactericidal activity of human beta-defensin 3 against multidrug resistant nosocomial strains. Antimicrob Agents Chemother 2:806–809

    Article  CAS  Google Scholar 

  • Marriott I, Gray DL, Tranguch SL, Fowler VG Jr, Stryjewski M, Scott Levin L, Hudson MC, Bost KL (2004) Osteoblasts express the inflammatory cytokine interleukin-6 in a murine model of Staphylococcus aureus osteomyelitis and infected human bone tissue. Am J Pathol 164(4):1399–1406

    PubMed  CAS  Google Scholar 

  • Marriott I, Gray DL, Rati DM, Fowler VG Jr, Stryjewski ME, Levin LS, Hudson MC, Bost KL (2005) Osteoblasts produce monocyte chemoattractant protein-1 in a murine model of Staphylococcus aureus osteomyelitis and infected human bone tissue. Bone 37(4):504–512

    Article  PubMed  CAS  Google Scholar 

  • Morrison GM, Davidson DJ, Dorin JR (1999) A novel mouse beta defensin, Defb2, which is upregulated in the airways by lipopolysaccharide. FEBS Lett 442:112–116

    Article  PubMed  CAS  Google Scholar 

  • Mushegian A, Medzhitov R (2001) Evolutionary perspective on innate immune recognition. J Cell Biol 155:705–711

    Article  PubMed  CAS  Google Scholar 

  • Muzio M, Polentarutti N, Bosisio D, Prahladan MK, Mantovani A (2000) Toll-like receptors: a growing family of immune receptors that are differentially expressed and regulated by different leukocytes. J Leukocyte Biol 67:450–455

    PubMed  CAS  Google Scholar 

  • Paulsen F, Pufe T, Petersen W, Tillmann B (2001) Expression of natural peptide antibiotics in human articular cartilage and synovial membrane. Clin Diagn Lab Immunol 8:1021–1023

    Article  PubMed  CAS  Google Scholar 

  • Paulsen F, Pufe T, Conradi L, Varoga D, Tsokos M, Papendieck J, Petersen W (2002) Antimicrobial peptides are expressed and produced in healthy and inflamed human synovial membranes. J Pathol 198:369–377

    Article  PubMed  CAS  Google Scholar 

  • Salzman NH, Ghosh D, Huttner KM, Paterson Y, Bevins CL (2003) Protection against enteric salmonellosis in transgenic mice expressing a human intestinal defensin. Nature 422:522–526

    Article  PubMed  CAS  Google Scholar 

  • Schon S, Prante C, Bahr C, Kuhn J, Kleesiek K, Gotting C (2006) Cloning and recombinant expression of active full-length xylosyltransferase I(XT-I) and characterization of subcellular localization of XT-I and XT-II. J Biol Chem 281(20):14224–14231

    Google Scholar 

  • Seitz M (2003) Toll-like receptors: sensors of the innate immune system. Allergy 58:1247–1249

    Article  PubMed  CAS  Google Scholar 

  • Singh PK, Jia HP, Wiles K, Hesselberth J, Liu L, Conway BA, Greenberg EP, Valore EV, Welsh MJ, Ganz T, Tack BF, McCray PB Jr (1998) Production of beta-defensins by human airway epithelia. Proc Natl Acad Sci USA 95:14961–14966

    Article  PubMed  CAS  Google Scholar 

  • Takeda K, Kaisho T, Akira S (2003) Toll-like receptors. Annu Rev Immunol 21:335–376

    Article  PubMed  CAS  Google Scholar 

  • Varoga D, Pufe T, Harder J, Meyer-Hoffert U, Schröder JM, Mentlein R, Petersen W, Tillmann B, Proksch E, Goldring MB, Paulsen F (2004) Production of endogenous antibiotics in articular cartilage. Arthritis Rheum 50:3526–3534

    Article  PubMed  CAS  Google Scholar 

  • Varoga D, Pufe T, Harder J, Schröder JM, Meyer-Hoffert U, Mentlein R, Tillmann B, Goldring MB, Hassenpflug J, Paulsen F (2005a) Human β-defensin-3 mediates tissue remodelling processes in articular cartilage by increasing metalloproteinases and reducing their endogenous inhibitors. Arthritis Rheum 52:1736–1745

    Article  PubMed  CAS  Google Scholar 

  • Varoga D, Pufe T, Mentlein R, Kohrs S, Grohmann S, Tillmann B, Hassenpflug J, Paulsen F (2005b) Expression and regulation of antimicrobial peptides in articular joints. Ann Anat 187:499–508

    Article  PubMed  CAS  Google Scholar 

  • Warnke PH, Springer IN, Russo PA, Wiltfang J, Essig H, Kosmahl M, Sherry E, Acil Y (2006) Innate immunity in human bone. Bone 38(3):400–408

    Article  PubMed  CAS  Google Scholar 

  • Wu Z, Hoover DM, Yang D, Boulege C, Santamaria F, Oppenheim J, Lubkowski J, Lu W (2003) Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human beta-defensin 3. Proc Natl Acad Sci USA 100:8880–8885

    Article  PubMed  CAS  Google Scholar 

  • Yamaguchi Y, Nagase T, Makita R, Fukuhara S, Tomita T, Tominaga T, Kurihara H, Ouchi Y (2002) Identification of multiple novel epididymis-specific beta-defensin isoforms in humans and mice. J Immunol 169:2516–2523

    PubMed  CAS  Google Scholar 

  • Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, Anderson M, Schröder JM, Wang JM, Howard OM, Oppenheim JJ (1999) Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 286:525–528

    Article  PubMed  CAS  Google Scholar 

  • Yang D, Chen Q, Chertov O, Oppenheim JJ (2000) Human neutrophil defensins selectively chemoattract naive T and immature dendritic cells. J Leukocyte Biol 68:9–14

    PubMed  CAS  Google Scholar 

  • Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415:389–395

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We thank K. Brabänder, S. Echterhagen, I. Geurink, C. Jaeschke, P. Kozak, U. Mundt, M. Nicolau, A. Rüben, S. Seiter and S. Sepke for their excellent technical assistance, C. Franke for the drawing and A. Drapkin for the proofreading. Part of this work was supported by grants from the German Research Community (Deutsche Forschungsgemeinschaft) (SFB 617, project A22, DFG Va 220/2-1, Pu 214/3-2 and Pu 214/4-2 and Pu 214/5-2) and from the “Endowment for the Advancement of Medical Research” (Stiftung zur Förderung der Medizinischen Forschung) of the Medical Faculty of the University of Kiel (to D.V. and T.P) and by the Association for the Advancement of Research and the Fight Against Rheumatic Diseases of Bad Bramstedt (Verein zur Förderung der Erforschung und Bekämpfung rheumatischer Erkrankungen Bad Bramstedt e.V.).

Conflict of interest statement

The authors have no conflict of interest.

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Authors and Affiliations

  1. Department of Anatomy, Christian-Albrechts-University, Kiel, Germany

    D. Varoga, C. J. Wruck, L. Brandenburg, R. Mentlein & T. Pufe

  2. Department of Trauma Surgery, UKSH Campus Kiel, Kiel, Germany

    D. Varoga, A. Seekamp & L. Besch

  3. Department of Anatomy and Cell Biology, RWTH Aachen University, Aachen, Germany

    C. J. Wruck, M. Tohidnezhad, L. Brandenburg & T. Pufe

  4. Department of Anatomy and Cell Biology, Martin-Luther-University, Halle, Germany

    F. Paulsen

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  1. D. Varoga
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  2. C. J. Wruck
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  4. L. Brandenburg
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  7. A. Seekamp
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  8. L. Besch
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  9. T. Pufe
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Correspondence to T. Pufe.

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The experiments comply with current German law.

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Varoga, D., Wruck, C.J., Tohidnezhad, M. et al. Osteoblasts participate in the innate immunity of the bone by producing human beta defensin-3. Histochem Cell Biol 131, 207–218 (2009). https://doi.org/10.1007/s00418-008-0522-8

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  • DOI: https://doi.org/10.1007/s00418-008-0522-8

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