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Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_3 | Pages 35 - 35
1 Apr 2018
Hägele Y Rapp A Ignatius A
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Complement C5a receptor 1 (C5aR1) has crucial functions in host defense against danger molecules, as does toll-like receptor 2 (TLR2). Both innate immunity receptors interact in immune cells in the context of infectious inflammatory diseases often associated with bone loss, such as periodontitis. C5aR1 plays an important role in bone, as it is expressed on bone cells and strongly upregulated due to bone injury. Importantly, C5aR1-ko mice are protected against arthritis and C5aR1 contributes to bone loss in periodontitis. In contrast, less data exist on the role of TLR2 on osteoblasts, however, it is known that TLR2 is expressed on osteoblasts and contributes to bacterial-induced bone resorption. The aim of this study was to evaluate the interaction of C5aR1 and TLR2 in osteoblasts, including intracellular signaling pathways and gene expression patterns.

Primary osteoblasts were isolated from 8–12 week-old WT mice and differentiated for 14 days. Osteoblasts were assessed for expression of C5aR1 and TLR2. Phosphorylation of mitogen-activated protein kinases (MAPK) in response to C5a and Pam3CSK4 (TLR2 agonist) was analyzed by immunoblotting. Gene expression profiling after 30 min and 4 h stimulation of C5a was performed by microarray and candidate genes were validated by quantitative Real-Time PCR (qRT-PCR). Immunoprecipitation was performed using a C5aR1-antibody and C5aR1 and TLR2 were subsequently detected by immunoblotting. Statistics: One way ANOVA p<0.05, n=4–6.

We showed that C5aR1 and TLR2 are expressed on osteoblasts and strongly upregulated during differentiation. Via immunoprecipitation, we could show that C5aR1 and TLR2 do physically interact in osteoblasts. We then examined if C5aR1 and TLR2, besides their physical interaction, also act via the same intracellular signaling pathways. Gene expression profiling upon C5a stimulation revealed that the top regulated pathways are related to MAPK and transforming growth factor beta (TGF-β). Respective genes, such as TGF-β (Tgfb1) and its receptor (Tgfbr) were found to be upregulated, and negative MAPK regulators were found to be downregulated, both by microarray analysis and qRT-PCR. Accordingly, we saw a C5aR1- and TLR2-dependent phosphorylation of p38 MAPK. Interestingly, this effect was enhanced and prolonged by costimulation of both receptors. An additive effect of C5aR1 and TLR2 was also seen regarding Cxcl10 levels, which were enhanced compared to C5aR1 or TLR2 stimulation alone.

This study shows that C5aR1 and TLR2 interact in osteoblasts, not only physically but also functionally, regarding downstream signaling and target genes. Those data strongly imply a synergistic interplay between the receptors, through which osteoblasts possibly contribute to inflammatory reactions affecting bone.


Orthopaedic Proceedings
Vol. 95-B, Issue SUPP_16 | Pages 21 - 21
1 Apr 2013
Bindl R Recknagel S Rapp A Erbacher A Mueller I Ignatius A
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There is evidence that fracture healing is impaired in patients with chronic immune disorders the reasons remaining unclear so far. To further elucidate the role of the immune system in bone healing, this study investigated the hypothesis that fracture healing would be considerably disturbed in a mouse model with severe defects of the innate as well as adaptive immune system.

Immune deficient Nod-scidIL2Rγnull and immune competent BALBcByJ mice were used (12 weeks, male, each n=24). The mice received a femur osteotomy stabilized by an external fixator and were sacrificed at d 21, 28, and 35. The calli were evaluated by three-point-bending testing, μCT and histomorphometry.

The flexural rigidity of the callus did not significantly differ between both genotypes after 21 and 28 days but was significantly lower in Nod-scidIL2Rγnull mice after 35 days (31%). The maximum moment of inertia was significantly increased after 21 days (by 34%), and the callus cross section area after 21, 28 and 35 days in Nod-scidIL2Rγnull mice. BV/TV of the callus of Nod-scidIL2Rγnull mice was significantly decreased after 28 and 35 days (by 32% and 41%). The histological evaluation showed a significantly enhanced amount of cartilage in the fracture gap of Nod-scidIL2Rγnull mice.

These data indicate an only moderate delay in fracture healing in Nod-scidIL2Rγnull mice suffering on severe defects in innate and adaptive immune response.


Orthopaedic Proceedings
Vol. 95-B, Issue SUPP_16 | Pages 59 - 59
1 Apr 2013
Ehrnthaller C Huber-Lang M Recknagel S Bindl R Redeker S Rapp A Gebhard F Ignatius A
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Since osteoimmunology is gaining increasingly interest and evidence for involvement of complement in bone biology was found, the role of complement in bone biology and fracture healing was evaluated.

After characterizing the bone phenotype, a fracture healing experiment with C3- and C5- deficient mice was performed. After osteotomy of the right femur and external fixation, healing was analyzed after 1, 3, 7 and 21 days. Bone characterization revealed a reduced number of osteoclasts in C5-deficient animals with a significantly reduced resorption activity. While bone mineral density was significantly higher in complement-deficient strains, stiffness was significantly reduced. After 21 days of fracture healing, C5-deficient animals showed reduced stiffness and a smaller callus volume compared to controls. Interestingly, C3- more than C5-deficient animals showed reduced bone formation. Altogether, bone phenotype of complement-deficient animals resembles a mild form of osteopetrosis.

This might be due to the resorption defect seen in C5-deficient mice. A reason for the minor involvement of C3-deficient mice compared to the C5-deficient animals could be the cross-talk between the coagulation cascade with side activation of complement factor C5 by thrombin.

These results indicate for the first time an essential role of complement in bone biology and fracture healing. Future studies should focus on the molecular basis of complement involvement and the osteoclastic resorption defect.