Change search
ReferencesLink to record
Permanent link

Direct link
Toll-like receptors (TLRs) and inflammatory bone modeling
Umeå University, Faculty of Medicine, Department of Odontology.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Toll-liknande receptorer och inflammatorisk benmodellering (Swedish)
Abstract [en]

Patients with inflammatory or infectious conditions such as periodontitis, peri-implantitis, osteomyelitis, rheumatoid arthritis, septic arthritis and loosened joint prosthesis display varying severity of destruction in the adjacent bone tissue. Bone loss in inflammatory diseases is considered a consequence of cytokine induced RANKL and subsequent enhanced osteoclast formation. Hence, osteotropic cytokines and their receptors have been suggested to be important for the pathogenesis of inflammation-induced osteolysis. It is, here, suggested that bacterial components, so called “pathogen associated molecular patterns=PAMPs”, may also be involved. Varieties of cells express receptors for PAMPs, including Toll-like receptors (TLRs) which are the first line of defence in the innate immune system. LPS (lipopolysaccharide), fimbria and lipoproteins from pathogenic bacteria such as P. gingivalis, S. aureus are ligands for TLR2 and flagellin from pathogenic flagellated bacteria like S. typhimurium is a ligand for TLR5.


Since the susceptibility to, or the severity of inflammation-associated bone diseases are likely related to differences in the tissue response, and the mechanisms by which PAMPs interact with bone cells are not fully understood, we aimed to elucidate the importance of different TLRs for inflammation induced bone loss by conducting in vitro and in vivo investigations.

Activation of TLR2 and TLR5 in organ cultured mouse parietal bones increased bone resorption in a time- and concentration-dependent manner by a process inhibited by OPG and bisphosphonate, showing the crucial role of RANKL-induced osteoclast formation. In addition, the number of osteoclasts, expression of osteoclastic genes and osteoclastogenic transcription factors were increased. In the bones and in osteoblasts isolated from the bones, TLR2 agonists increased the expression of RANKL without affecting OPG, while TLR5 activation resulted in enhanced RANKL and decreased OPG. Activation of both TLR2 and TLR5 stimulated the expression in both bones and osteoblasts of prostaglandins and pro-inflammatory cytokines, known to stimulate RANKL. By blocking the cytokines and prostaglandin, we showed that TLR2 and TLR5 induced bone resorption and RANKL expression are independent of these molecules.

Activation of TLR2, but not TLR5, in mouse bone marrow macrophage cultures inhibited RANKL-induced osteoclast formation, an effect not observed in committed pre-osteoclasts.

Local administration in vivo of TLR2 and TLR5 agonists on the top of mouse skull bones enhanced local and systemic osteoclast formation and bone resorption. Using knockout mice, we showed that the effects by LPS from P. gingivalis (used as TLR2 agonist) and flagellins (used as TLR5 agonists) are explicit for TLR2 and TLR5 ex vivo and in vivo, respectively.

These data show that stimulation of TLR2 and TLR5 results in bone resorption in vitro and in vivo mediated by increased RANKL in osteoblasts and thus may be one mechanism for developing inflammatory bone loss.

Interestingly, histological analyses of skull bones of mice treated locally with TLR2 and TLR5 agonists revealed that the bones not only reacted with locally increased osteoclastogenesis (osteoclast formation), but also with locally increased new bone formation. This was observed on both periosteal and endosteal sides of the bones, as well as in the bone marrow compartment. The formation of new bone was seen close to osteoclasts in some parts, but also in other areas, distant from these cells. The response was associated with active, cuboidal osteoblasts, extensive cell proliferation and increased expression of genes coding for bone matrix proteins and osteoblastic transcription factors.

In conclusion, activation of TLR2 and TLR5 in osteoblasts results in bone loss associated with enhanced osteoclast formation and bone resorption, as well as with increased osteoblast differentiation and new bone formation, indicating that inflammation causes bone modeling. The data provide an explanation why LPS from P. gingivalis and flagellin from flagella-expressing bacteria can stimulate bone loss. Since TLR2 and TLR5 can be activated not only by bacterial components, but also by endogenous ligands produced in inflammatory processes, the data also contribute to the understanding of inflammation induced bone loss in autoimmune diseases. Hopefully, these findings will contribute to the development of treatment strategies for inflammation induced bone loss.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2015. , 133 p.
Umeå University odontological dissertations, ISSN 0345-7532 ; 134
Keyword [en]
Toll-like receptor, osteoclast, osteoblast, bone resorption, bone formation, P. gingivalis, S. aureus, flagellin.
National Category
Cell and Molecular Biology Dentistry
URN: urn:nbn:se:umu:diva-110296ISBN: 978-91-7601-370-0OAI: diva2:861982
Public defence
2015-11-11, Hörsal B, Tandläkarhögskolan, plan 9, Norrlands universitetssjukhus§, Umeå, 09:00 (English)
Swedish Research Council
Available from: 2015-10-21 Created: 2015-10-19 Last updated: 2015-10-22Bibliographically approved
List of papers
1. Porphyromonas gingivalis Stimulates Bone Resorption by Enhancing RANKL (Receptor Activator of NF-kappa B Ligand) through Activation of Toll-like Receptor 2 in Osteoblasts
Open this publication in new window or tab >>Porphyromonas gingivalis Stimulates Bone Resorption by Enhancing RANKL (Receptor Activator of NF-kappa B Ligand) through Activation of Toll-like Receptor 2 in Osteoblasts
Show others...
2015 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 33, 20147-20158 p.Article in journal (Refereed) Published
Abstract [en]

Periodontitis has been associated with rheumatoid arthritis. In experimental arthritis, concomitant periodontitis caused by oral infection with Porphyromonas gingivalis enhances articular bone loss. The aim of this study was to investigate how lipopolysaccharide (LPS) from P. gingivalis stimulates bone resorption. The effects by LPS P. gingivalis and four other TLR2 ligands on bone resorption, osteoclast formation, and gene expression in wild type and Tlr2-deficient mice were assessed in ex vivo cultures of mouse parietal bones and in an in vivo model in which TLR2 agonists were injected subcutaneously over the skull bones. LPS P. gingivalis stimulated mineral release and matrix degradation in the parietal bone organ cultures by increasing differentiation and formation of mature osteoclasts, a response dependent on increased RANKL (receptor activator of NF-kappa B ligand). LPS P. gingivalis stimulated RANKL in parietal osteoblasts dependent on the presence of TLR2 and through a MyD88 and NF-kappa B-mediated mechanism. Similarly, the TLR2 agonists HKLM, FSL1, Pam2, and Pam3 stimulated RANKL in osteoblasts and parietal bone resorption. LPS P. gingivalis and Pam2 robustly enhanced osteoclast formation in periosteal/endosteal cell cultures by increasing RANKL. LPS P. gingivalis and Pam2 also up-regulated RANKL and osteoclastic genes in vivo, resulting in an increased number of periosteal osteoclasts and immense bone loss in wild type mice but not in Tlr2-deficient mice. These data demonstrate that LPS P. gingivalis stimulates periosteal osteoclast formation and bone resorption by stimulating RANKL in osteoblasts via TLR2. This effect might be important for periodontal bone loss and for the enhanced bone loss seen in rheumatoid arthritis patients with concomitant periodontal disease.

National Category
urn:nbn:se:umu:diva-108135 (URN)10.1074/jbc.M115.655787 (DOI)000359608900017 ()26085099 (PubMedID)
Available from: 2015-09-21 Created: 2015-09-04 Last updated: 2015-10-20Bibliographically approved
2. TLR5, a novel mediator of innate immunity-induced osteoclastogenesis and bone loss
Open this publication in new window or tab >>TLR5, a novel mediator of innate immunity-induced osteoclastogenesis and bone loss
Show others...
2015 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 29, no 11, 4449-4460 p.Article in journal (Refereed) Published
Abstract [en]

Accumulating evidence points to the importance of the innate immune system in inflammation-induced bone loss in infectious and autoimmune diseases. TLRs are well known for being activated by ligands expressed by bacteria, viruses, and fungi. Recent findings indicate that also endogenous ligands in inflammatory processes are important, one being a TLR5 agonist present in synovial fluid from patients with rheumatoid arthritis (RA). We found that activation of TLR5 by its specific ligand, flagellin, caused robust osteoclast formation and bone loss in cultured mouse neonatal parietal bones dependent on increased receptor activator of NF-κB ligand (RANKL):osteoprotegerin ratio, with half-maximal stimulation at 0.01 μg/ml. Flagellin enhanced Rankl mRNA in isolated osteoblasts by a myeloid differentiation primary response gene 88 and NF-κB-dependent mechanism. Injection of flagellin locally over skull bones in 5-wk-old mice resulted in increased mRNA expression of Rankl and osteoclastic genes, robust osteoclast formation, and bone loss. The effects in vitro and in vivo were absent in Tlr5(-/-) mice. These data show that TLR5 is a novel activator of RANKL and osteoclast formation and, therefore, a potential key factor in inflammation-induced bone erosions in diseases like RA, reactive arthritis, and periodontitis. TLR5 might be a promising novel treatment target for prevention of inflammatory bone loss.-Kassem, A., Henning, P., Kindlund, B., Lindholm, C., Lerner, U. H. TLR5, a novel mediator of innate immunity-induced osteoclastogenesis and bone loss.

inflammation, Toll-like receptors, osteoclasts, bone resorption, flagellin
National Category
Cell and Molecular Biology
urn:nbn:se:umu:diva-110286 (URN)10.1096/fj.15-272559 (DOI)000364514700006 ()26207027 (PubMedID)
Available from: 2015-10-19 Created: 2015-10-19 Last updated: 2015-12-08Bibliographically approved
3. Toll-Like Receptor 2 Stimulation on Osteoblasts Mediates Staphylococcus aureus-Induced Bone Resorption and Osteoclastogenesis through Enhanced RANKL
Open this publication in new window or tab >>Toll-Like Receptor 2 Stimulation on Osteoblasts Mediates Staphylococcus aureus-Induced Bone Resorption and Osteoclastogenesis through Enhanced RANKL
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Severe Staphylococcus aureus (S. aureus) infections pose an immense threat to population health and constitute a great burden for the health care worldwide. Inter alia, S. aureus septic arthritis is a disease with high mortality and morbidity caused by destruction of the infected joints and systemic bone loss, osteoporosis. Toll-Like receptors (TLRs) are innate immune cell receptors recognizing a variety of microbial molecules and structures. S. aureus recognition via TLR2 initiates a signaling cascade resulting in production of various cytokines, but the mechanisms by which S. aureus causes rapid and excessive bone loss are still unclear. Therefore, we investigated how S. aureus regulates periosteal/endosteal osteoclast formation and bone resorption.

S. aureus stimulation of neonatal mouse parietal bone induced ex vivo bone resorption and osteoclastic gene expression. This effect was associated with increased mRNA and protein expression of receptor activator of NF-kB ligand (RANKL) without significant change in osteoprotegerin (OPG) expression. S. aureus also increased the expression of proinflammatory cytokines and prostaglandins in parietal bones but the stimulatory effect of S. aureus on bone resorption and Tnfsf11 mRNA expression was independent of these cytokines and prostaglandins. Stimulation of isolated periosteal osteoblasts with S. aureus also resulted in increased expression of Tnfsf11 mRNA, an effect lost in osteoblasts from Tlr2 knockout mice. S. aureus stimulated osteoclastogenesis in isolated periosteal cells without affecting RANKL-stimulated resorption.  In contrast, S. aureus inhibited RANKL-induced osteoclast formation in bone marrow macrophages. These data show that S. aureus enhances bone resorption and periosteal osteoclast formation by increasing osteoblast RANKL production through TLR2. In contrast, S. aureus inhibits osteoclastogenesis from bone marrow derived precursors. Our study indicates the importance of using different in vitro approaches for studies of regulation of osteoclastogenesis by S. aureus to obtain better understanding of the complex mechanisms of S. aureus bone destruction in vivo.

National Category
urn:nbn:se:umu:diva-110287 (URN)
Available from: 2015-10-19 Created: 2015-10-19 Last updated: 2015-10-20
4. Toll-like receptor induced inflammation causes local bone formation
Open this publication in new window or tab >>Toll-like receptor induced inflammation causes local bone formation
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

It is well established that inflammatory processes in the vicinity of bone often induce osteoclast formation and bone resorption. Effects on bone formation by inflammatory processes are much less studied and available information is partly contradictory. In the present study, we have assessed the effect on bone formation by locally induced inflammation. LPS from Porphyromonas gingivalis and Pam2, used as Toll-like receptor (TLR) 2 agonists, and flagellin from Salmonella typhimurium, used as TLR5 agonist, were injected subcutaneously on the top of mouse skull bones. After 1-5 days, the calvarial bones were dissected and processed either for histological or gene expression analyses. Femur was dissected for analysis with microCT and histology. At day 5, all three agonists induced bone formation on periosteal and endosteal sites, as well as in the bone marrow compartment of the calvaria. This response was seen both in close vicinity to, but also apart from, osteoclasts and bone resorption cavities. In areas close to new bone formation, abundance of proliferating cells was observed as assessed by Ki67 labelling. Gene expression analyses showed that Pam2 treatment resulted in increased mRNA expression at day 5 of genes encoding bone matrix proteins, alkaline phosphatase and of the osteoblastic transcription factors Runx2 and osterix. Robust Runx2 protein was observed in osteoblasts in areas with new bone formation. Pam2 treatment also increased the mRNA expression of cytokines in the IL-6 family, as well as of their cognate receptors and common signaling transduction subunit gp130. At day 5, the mRNA expression of Bmp2, Bmp4, Tgfb1, Lrp5, Lrp6 and Wnt7b was increased, whereas Sost was decreased. In the femur, excessive osteoclast formation and trabecular bone loss was found at day 5, but new bone formation was not observed. In conclusion, these data show that inflammatory processes not only induce osteoclastogenesis but also have the capacity to activate osteoblasts and stimulate new bone formation distinct from bone remodeling sites. Stimulation of inflammation- induced new bone formation may be due to enhanced gp130 signaling. Osteoblast activation in the inflammatory processes may also involve the BMP and WNT signaling systems.

National Category
Cell and Molecular Biology
urn:nbn:se:umu:diva-110288 (URN)
Toll-like receptors, osteoclasts, osteoblasts, bone formation
Available from: 2015-10-19 Created: 2015-10-19 Last updated: 2015-10-20

Open Access in DiVA

fulltext(12488 kB)1064 downloads
File information
File name FULLTEXT01.pdfFile size 12488 kBChecksum SHA-512
Type fulltextMimetype application/pdf
spikblad(117 kB)32 downloads
File information
File name FULLTEXT02.pdfFile size 117 kBChecksum SHA-512
Type spikbladMimetype application/pdf

Search in DiVA

By author/editor
Kassem, Ali
By organisation
Department of Odontology
Cell and Molecular BiologyDentistry

Search outside of DiVA

GoogleGoogle Scholar
Total: 1096 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Total: 1212 hits
ReferencesLink to record
Permanent link

Direct link