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Expression and regulation of toll-like receptors (TLRs) in human intervertebral disc cells

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Abstract

Purpose

Although inflammatory processes play an essential role in painful intervertebral disc (IVD) degeneration, the underlying regulatory mechanisms are not well understood. This study was designed to investigate the expression, regulation and importance of specific toll-like receptors (TLRs)—which have been shown to play an essential role e.g. in osteoarthritis—during degenerative disc disease.

Methods

The expression of TLRs in human IVDs was measured in isolated cells as well as in normal or degenerated IVD tissue. The role of IL-1β or TNF-α in regulating TLRs (expression/activation) as well as in regulating activity of down-stream pathways (NF-κB) and expression of inflammation-related genes (IL-6, IL-8, HSP60, HSP70, HMGB1) was analyzed.

Results

Expression of TLR1/2/3/4/5/6/9/10 was detected in isolated human IVD cells, with TLR1/2/4/6 being dependent on the degree of IVD degeneration. Stimulation with IL-1β or TNF-α moderately increased TLR1/TLR4 mRNA expression (TNF-α only), and strongly increased TLR2 mRNA expression (IL-1β/TNF-α), with the latter being confirmed on the protein level. Stimulation with IL-1β, TNF-α or Pam3CSK4 (a TLR2-ligand) stimulated IL-6 and IL-8, which was inhibited by a TLR2 neutralizing antibody for Pam3CSK4; IL-1β and TNF-α caused NF-κB activation. HSP60, HSP70 and HMGB1 did not increase IL-6 or IL-8 and were not regulated by IL-1β/TNF-α.

Conclusion

We provide evidence that several TLRs are expressed in human IVD cells, with TLR2 possibly playing the most crucial role. As TLRs mediate catabolic and inflammatory processes, increased levels of TLRs may lead to aggravated disc degeneration, chronic inflammation and pain development. Especially with the identification of more endogenous TLR ligands, targeting these receptors may hold therapeutic promise.

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Acknowledgments

This study was made possible by grants from AOSpine (SRN 02/103) as well as by the Mäxi Foundation (CABMM). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of AOSpine or of the Mäxi Foundation. We thank Dr. Wojtal from the University Hospital Zurich for providing THP1 cells.

Conflict of interest

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Author information

Authors and Affiliations

  1. Competence Center for Applied Biotechnology and Molecular Medicine, University of Zurich, Zurich, Switzerland

    Marina Klawitter, Michiyuki Hakozaki, Lilian Quero, Caroline Ospelt, Steffen Gay, Oliver Hausmann, Norbert Boos, Stephen J. Ferguson & Karin Wuertz

  2. Department of Orthopaedic Surgery, Fukushima Medical University School of Medicine, Fukushima, Japan

    Michiyuki Hakozaki, Hiroshi Kobayashi, Miho Sekiguchi & Shin-ichi Konno

  3. Center of Experimental Rheumatology, University Hospital Zurich, Zurich, Switzerland

    Caroline Ospelt & Steffen Gay

  4. Center for Neurosurgery and Spine Surgery, St. Anna Clinic, Lucerne, Switzerland

    Oliver Hausmann

  5. Unfallkrankenhaus Berlin, Berlin, Germany

    Thomas Liebscher & Ullrich Meier

  6. AOSpine Research Network, Duebendorf, Switzerland

    Norbert Boos, Stephen J. Ferguson & Karin Wuertz

  7. Institute for Biomechanics, D-HEST, ETH Zurich, Schafmattstrasse 30 (HPP-O12), 8093, Zurich, Switzerland

    Michiyuki Hakozaki, Hiroshi Kobayashi, Olga Krupkova, Stephen J. Ferguson & Karin Wuertz

  8. Prodorso Centre for Spinal Medicine, Zurich, Switzerland

    Norbert Boos

Authors
  1. Marina Klawitter
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  2. Michiyuki Hakozaki
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  3. Hiroshi Kobayashi
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  4. Olga Krupkova
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  5. Lilian Quero
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  6. Caroline Ospelt
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  7. Steffen Gay
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  8. Oliver Hausmann
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  9. Thomas Liebscher
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  10. Ullrich Meier
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  11. Miho Sekiguchi
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  12. Shin-ichi Konno
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  15. Karin Wuertz
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Corresponding author

Correspondence to Karin Wuertz.

Additional information

M. Klawitter, M. Hakozaki, and H. Kobayashi have equally contributed to the manuscript.

Electronic supplementary material

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586_2014_3442_MOESM1_ESM.jpg

Supplementary Figure S1: Cytotoxicity and bioactivity of Pam3CSK4. (a) Cytotoxicity after treatment with different concentrations of Pam3CSK4 for 18 h, measured by MTT. Mean ± SEM one donor. (b/c) Fold changes in gene expression of IL-6 (b) and IL-8 (c) after stimulation with different concentrations of Pam3CSK4 for 18 h, measured by Real-time RT-PCR and calculated by the 2−∆∆Ct method. Changes are calculated relative to untreated control cells. Mean ± SEM of one donor. (JPEG 190 kb)

586_2014_3442_MOESM2_ESM.jpg

Supplementary Figure S2: Correlation of TLR mRNA expression with the degree of IVD degeneration. Gene expression of TLR3 (a) and TLR10 (b) in IVD biopsies with different degrees of degeneration (1 = healthy; 2 = mild degeneration; 3 = moderate degeneration; 4 = severe degeneration), measured by Real-time RT-PCR and calculated by the 2−∆Ct method. Individual data points of five independent donors in each group. Asterisks indicate statistical significance between indicated groups (i.e. grades of degeneration) with p < 0.05. (JPEG 62 kb)

586_2014_3442_MOESM3_ESM.jpg

Supplementary Figure 3: Changes in TLR3 mRNA expression upon stimulation with TNF-α or IL-1β (time course & concentration dependency). Fold changes in gene expression of TLR3 after stimulation with 10 ng/ml TNF-α as time course (a) and concentration dependency (b) or 5 ng/ml IL-1β as time course (c) and concentration dependency (d) measured by Real-time RT-PCR and calculated by the 2−∆∆Ct method. Changes are calculated relative to untreated control cells. Mean ± SEM of five independent donors. Asterisks indicate statistical significance relative to untreated control with p < 0.05. (JPEG 111 kb)

586_2014_3442_MOESM4_ESM.jpg

Supplementary Figure 4: Changes in HSP60, HSP70 and HMGB1 mRNA expression upon stimulation with TNF-α or IL-1β. Fold changes in gene expression of HSP60 (a), HSP70 (b) and HMGB1 (c) after stimulation with 5 ng/ml IL-1β or 10 ng/ml TNF-α for 18 h, measured by Real-time RT-PCR and calculated by the 2−∆∆Ct method. Changes are calculated relative to untreated control cells. Mean ± SEM of five independent donors. Asterisks indicate statistical significance relative to untreated control with p < 0.05. (JPEG 95 kb)

586_2014_3442_MOESM5_ESM.jpg

Supplementary Figure 5: Changes in IL-6 and IL-8 mRNA expression upon stimulation with HSP60, HSP70 and HMGB1. Fold changes in gene expression of IL-6 (a-c) and IL-8 (d-f) after stimulation with HSP60 (a, d), HSP70 (b, e) or HMGB1 (c, f) for 18 h, measured by Real-time RT-PCR and calculated by the 2−∆∆Ct method. Changes are calculated relative to untreated control cells. Mean ± SEM of five independent donors. Asterisks indicate statistical significance relative to untreated control with p < 0.05. (JPEG 134 kb)

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Klawitter, M., Hakozaki, M., Kobayashi, H. et al. Expression and regulation of toll-like receptors (TLRs) in human intervertebral disc cells. Eur Spine J 23, 1878–1891 (2014). https://doi.org/10.1007/s00586-014-3442-4

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