The Interaction Between TLR2 And TNFR1 And Its Role And Mechanism In The Formation And Repair Of Osteomyelitis

Osteomyelitis is an inflammatory process accompanied by bone destruction and caused by an infecting microorganism.The infection can be limited to a single portion of the bone or can involve several regions, such as marrow, cortex, periosteum, and the surrounding soft Tissue.From a practical viewpoint, distinction of three types of osteomyelitis is useful.Osteomyelitis due to local spread from a contiguous contaminated source of infection follows trauma, bone surgery, or joint replacement.Osteomyelitis secondary to vascular insufficiency occurs predominantly in people with diabetes and in almost all cases follows a foot soft-tissue infection that spreads to bone. Haematogenous osteomyelitis is seen mostly in prepubertal children and in elderly patients and ischaracterised by nidation of bacteria within sometimes only slightly injured bone, presumably seeded by bacteria not apparent but present in the blood.Various pathogen cause infection of bone,and Staphylococcus aureus is the most common pathogen isolated in osteomyelitis. Adhesion and consecutive colonization of the host tissues are critical steps in the pathogenesis of S. aureus infections. The bone extracellular matrix(ECM) is rich in protein such as fibronectin, collagen, and bone sialoprotein, which are able to interact with bacterial adhesins. Biofilm formation is a critical pathogenetic mechanism that enables staphylococci to evade host immune defenses and systemic antibiotic therapies. In addition,vascular channels are compressed and obliterated by the inflammatory process.All these factors contribute to persistence of the infection. Therefore,Osteomyelitis is still a challenge for orthopedist.Animal models serve an integral role in exploring the pathogenesis of osteomyelitis, and aid in determining the efficacy of prophylactic and therapeutic treatments. Animal models should mimic the clinical scenarios seen in patients as closely as possible to permit the experimental results to be translated to the corresponding clinical care. Osteomyelitis is an inflammation of the bone caused by an invading organism, and it is characterized by progressive bone destruction. Bone remodeling requires the coordinated activities of osteoblasts and osteoclasts. Although certain factors such as inflammatory cytokines, which modulate osteoclast formation and activity have been identified, the exact mechanisms and factors mediating these effects remain unclear. In osteomyelitis, tissue damage is associated with bone cell death, which is important for bone remodeling. Toll-like receptors(TLRs) have been recognized as a system of innate immunity for the detection of invading microbes. TLR2 is essential for the recognition of microbial components, in particular lipoproteins lipopeptides, peptidoglycan and lipoteichoic acid from Gram-positive bacteria. The primary physiological functions of TNFR1 involve induction of antibody production and acute phase reactions by stimulating B cells. It also plays a role in antigen-specific immune responses and inflammatory reactions TNFR1 binding by SpA causes the activation of nuclear factor kappa B(NF-κB), which then translocated the nucleus and promotes the expression of IL-6, a promoter of inflammation.ObjectivesTo investigate the relationship between TNFR1 and TLR2 in S. aureus-infected osteoblasts,and suggesting novel targets for the treatment of osteomyelitis.To establish a mice model of osteomyelitis by the way of bacteria release,which is reliable for the study of osteomyelitis.To offer an animal foundation and clinical reference in the treatment of osteomyelitis by means of local inject SP600125 and evaluate its effectiveness.MethodsApoptosis was assessed in the osteoblastic cell line MC3T3-E1 by Annexin V-FITC/PI staining and flow cytometry. The expression of TLR2 and apoptosis-related and mitogen-activated protein kinase pathway proteins was assessed by RT-PCR and western blotting. Alkaline phosphatase(ALP) activity and calcium deposition were assessed by ALP activity assay and Alizarin red staining.Biodegradable chitosan-alginate microspheres containing Staphylococcus aureus was made with 1x105 CFU of S. Aureus by the way of two-step method. A bone hole of 1mm in diamete was made at the proximal tibia of the mice. Put the microspheres into the medullary cavity to induce the chronic osteomyelitis model. Some assessment methods were adopted for the effectiveness postoperatively, includ physical examination, microbiology test and histopathology test.An inhibitor of the JNK protein(25 μM of SP600125)was injected into infected tibia of mice and subjected to histopathological observation by HE staining.ResultsS. aureus induced apoptosis, upregulated TLR2 expression, and activated mitogen-activated protein kinase pathways in a time dependent manner. Inhibition of the c-Jun N-terminal kinase(JNK) pathway downregulated TLR2 and suppressed the S. aureus induced activation of pro-apoptotic pathways. Short-hairpin RNA mediated silencing of TLR2 reversed S. aureus induced apoptosis and decrease in ALP activity and calcium deposition, and inhibition of JNK had a similar effect.Cell viability decreased and apoptosis, expression of TLR2, and the secretion of inflammatory cytokines(TNF-a and IL-6) increased with increasing concentrations of S. aureus. The JNK pathway was also activated in response to S. aureus infection. Knockdown of TNFR1 inhibited the JNK pathway and reduced TLR2 protein levels in S. aureus-infected cells.Inhibition of the JNK pathway reduced the protein level of TLR2 and reduced inflammatory cytokine(TNF-a and IL-6) secretion in S. aureus-infected cells. Knockdown of TNFR1 also reduced RANKL levels in S. aureus infection cells.Two mice dead in one day after the operation without any inducement. Sinus tract were found at the operative site and positive cultures of Staphylococcus aureus in this group. Histopathology test demonstrated chronic osteomyelitis。As the result,all the animal recieved the expected goal after operation.HE staining showed that foci of infection exist in the treatment group(injection of SP600125),but the infection improved obviously compared with non-treatment group.Conclusion1、The elevated expression of TLR2 in response to infection was partially suppressed by TNFR1 knock down in cells exposed and fully suppressed by antibody against TLR2 itself. This same antibody to TLR2 also suppressed TNF-α and IL-6 expression, but not to pre-infection levels, again demonstrating the presence of alternative activating pathways. Inhibitor of the JNK protein suppressed TNF-α, IL-6 and TLR2 expression,but not to pre-infection as well. In summary, we demonstrated that TLR2 and TNFR1 expression are linked through a global cellular response to infection.2、Bacteria controlled-release can persistent infection of bone,is a effective and reliable method for establishing osteomyelitis animal model3、Osteoblast apoptosis and osteogenic differentiation in response to bacterial invasion are dependent on TLR2 expression and JNK activation, suggesting novel potential therapeutic targets for the treatment of osteomyelitis. The effectiveness of inhibitor of the JNK pathway in the treatment of osteomyelitis is true and reliable...