Background. Metal-on-metal (MoM) hip arthroplasty has been associated with adverse reactions including pseudotumours, and osteolysis. Tissues surrounding failed MoM hip implants are often infiltrated by inflammatory cells such as monocytes and neutrophils. The mechanisms by which these cells are recruited to the tissues remain unclear. Cobalt from MoM implants activates Toll-like receptor 4 (TLR4), an immune cell surface receptor usually responsible for recognition of bacteria and prevention of sepsis. Activation by bacteria leads to secretion of pro-inflammatory cytokines which guide other immune cells to the site of inflammation. The effect of cobalt on this response is unknown and therefore this study aims to determine the effect of cobalt-mediated TLR4 activation on the migration of inflammatory cells. Methods. A human macrophage cell line (MonoMac 6) was stimulated with a physiologically-relevant range of cobalt ions for 24h with or without pre-treatment with a TLR4 antagonist. Conditioned media was collected and used in a trans-well migration assay to determine its effect on migration of primary monocytes and neutrophils isolated from whole human blood. Migrated cells were stained with haematoxylin and counted at ×40 magnification. Results. Conditioned media from cobalt-treated macrophages caused elevated monocyte and neutrophil migration across all concentrations. Pre-treatment of MonoMac 6 cells with a TLR4 antagonist significantly decreased the response. This suggests that the cytokine profile produced in response to cobalt-mediated TLR4 activation is pro-migratory for immune cells. Conclusions. Cobalt activation of TLR4 leads to secretion of inflammatory cytokines that attract monocytes and neutrophils. This work highlights a potential mechanism by which cobalt ions from failed MoM joints could be involved in inflammatory cell recruitment to the surrounding tissues. The TLR4 signalling pathway represents an exciting area for further investigation as a therapeutic target in the prevention of adverse reactions to cobalt ions. Disclosure. This work is funded by DePuy Synthes Ltd and the Newcastle NIHR Biomedical Research Centre

Aims. Pellino1 (Peli1) has been reported to regulate various inflammatory diseases. This study aims to explore the role of Peli1 in the occurrence and development of osteoarthritis (OA), so as to find new targets for the treatment of OA. Methods. After inhibiting Peli1 expression in chondrocytes with small interfering RNA (siRNA), interleukin (IL)-1β was used to simulate inflammation, and OA-related indicators such as synthesis, decomposition, inflammation, and apoptosis were detected. Toll-like receptor (TLR) and nuclear factor-kappa B (NF-κB) signalling pathway were detected. After inhibiting the expression of Peli1 in macrophages Raw 264.7 with siRNA and intervening with lipopolysaccharide (LPS), the polarization index of macrophages was detected, and the supernatant of macrophage medium was extracted as conditioned medium to act on chondrocytes and detect the apoptosis index. The OA model of mice was established by destabilized medial meniscus (DMM) surgery, and adenovirus was injected into the knee cavity to reduce the expression of Peli1. The degree of cartilage destruction and synovitis were evaluated by haematoxylin and eosin (H&E) staining, Safranin O/Fast Green staining, and immunohistochemistry. Results. In chondrocytes, knockdown of Peli1 produced anti-inflammatory and anti-apoptotic effects by targeting the TLR and NF-κB signalling pathways. We found that in macrophages, knockdown of Peli1 can inhibit M1-type polarization of macrophages. In addition, the corresponding conditioned culture medium of macrophages applied to chondrocytes can also produce an anti-apoptotic effect. During in vivo experiments, the results have also shown that knockdown Peli1 reduces cartilage destruction and synovial inflammation. Conclusion. Knockdown of Peli1 has a therapeutic effect on OA, which therefore makes it a potential therapeutic target for OA. Cite this article: Bone Joint Res 2023;12(2):121–132

Aims. This study examined the relationship between obesity (OB) and osteoporosis (OP), aiming to identify shared genetic markers and molecular mechanisms to facilitate the development of therapies that target both conditions simultaneously. Methods. Using weighted gene co-expression network analysis (WGCNA), we analyzed datasets from the Gene Expression Omnibus (GEO) database to identify co-expressed gene modules in OB and OP. These modules underwent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and protein-protein interaction analysis to discover Hub genes. Machine learning refined the gene selection, with further validation using additional datasets. Single-cell analysis emphasized specific cell subpopulations, and enzyme-linked immunosorbent assay (ELISA), protein blotting, and cellular staining were used to investigate key genes. Results. WGCNA revealed critical gene modules for OB and OP, identifying the Toll-like receptor (TLR) signalling pathway as a common factor. TLR2 was the most significant gene, with a pronounced expression in macrophages. Elevated TLR2 expression correlated with increased adipose accumulation, inflammation, and osteoclast differentiation, linking it to OP development. Conclusion. Our study underscores the pivotal role of TLR2 in connecting OP and OB. It highlights the influence of TLR2 in macrophages, driving both diseases through a pro-inflammatory mechanism. These insights propose TLR2 as a potential dual therapeutic target for treating OP and OB. Cite this article: Bone Joint Res 2024;13(10):573–587

Aims. Trained immunity confers non-specific protection against various types of infectious diseases, including bone and joint infection. Platelets are active participants in the immune response to pathogens and foreign substances, but their role in trained immunity remains elusive. Methods. We first trained the innate immune system of C57BL/6 mice via intravenous injection of two toll-like receptor agonists (zymosan and lipopolysaccharide). Two, four, and eight weeks later, we isolated platelets from immunity-trained and control mice, and then assessed whether immunity training altered platelet releasate. To better understand the role of immunity-trained platelets in bone and joint infection development, we transfused platelets from immunity-trained mice into naïve mice, and then challenged the recipient mice with Staphylococcus aureus or Escherichia coli. Results. After immunity training, the levels of pro-inflammatory cytokines (tumour necrosis factor alpha (TNF-α), interleukin (IL)-17A) and chemokines (CCL5, CXCL4, CXCL5, CXCL7, CXCL12) increased significantly in platelet releasate, while the levels of anti-inflammatory cytokines (IL-4, IL-13) decreased. Other platelet-secreted factors (e.g. platelet-derived growth factor (PDGF)-AA, PDGF-AB, PDGF-BB, cathepsin D, serotonin, and histamine) were statistically indistinguishable between the two groups. Transfusion of platelets from trained mice into naïve mice reduced infection risk and bacterial burden after local or systemic challenge with either S. aureus or E. coli. Conclusion. Immunity training altered platelet releasate by increasing the levels of inflammatory cytokines/chemokines and decreasing the levels of anti-inflammatory cytokines. Transfusion of platelets from immunity-trained mice conferred protection against bone and joint infection, suggesting that alteration of platelet releasate might be an important mechanism underlying trained immunity and may have clinical implications. Cite this article: Bone Joint Res 2022;11(2):73–81

This meta analysis address the relationship between infection developing after total hip arthroplasty (THA) and heterotopic ossification (HO). To identify the gaps in available knowledge, we screened for full-length peer-reviewed research articles listed in PubMed, Embase, and Web of Science over the past 20 years. The following search terms and Boolean operators were used: heterotopic ossification AND infection AND (hip replacement OR hip arthroplasty). The search resulted in the identification of as few as 14 articles describing periprosthetic joint infection (PJI) and HO after THA. Data summarized from 6 studies suitable for further meta-analysis yielded a cumulative sample size of 753 observations, with 186 recorded events of HO. The pooled RR was estimated at 2.22 (95% CI: 1.00 to 4.91, p = 0.0497), suggesting a more than twofold risk of HO compared to the group without PJI. In conclusion, there is a clear association between a higher risk of HO and PJI. Basic research findings support the hypothesis that bacterial pathogen-associated molecular patterns (PAMPs) can lead to osteogenesis through a toll-like receptor (TLR) and nuclear factor kappa B (NF-κB) pathway in the course of HO development. Together, these results suggest that HO prophylaxis should always be prescribed in PJI after THA. Moreover, during revisions following THA for presumed non-septic reasons, the presence of HO warrants consideration for infection, as there is a potential heightened risk of pathologic ossification induced by PAMPs. Keywords: heterotopic ossification; total hip arthroplasty; total hip replacement; periprosthetic joint infection; bacteria. Authors Ł. Pulik and P. Łęgosz contributed equally to this work

Introduction. Multiple studies have identified Cutibacterium acnes (C.acnes) and other microbes in intervertebral disc tissue using 16S DNA Sequencing and microbial cultures. However, it remains unclear whether these bacteria are native to the discs or result from perioperative contamination. Our study aimed to detect Gram-positive bacteria in non-herniated human disc samples and explore correlations with Toll-like receptors (TLR) 2, TLR4, NLRP3, and Gasdermin D. Methods. Immunohistochemical staining was conducted on 75 human IVD samples for Gram-positive bacteria, S. aureus, C.acnes, TLR2, TLR4, NLRP3, and Gasdermin D. Cell detection and classification were performed using QuPath. NP cells were treated with Lipopolysaccharide (LPS) and Peptidoglycan (PGN) in monolayer and alginate beads for up to 72 hours, followed by secretome analysis using Luminex. Statistical analysis included Kruskal-Wallis, Dunn's multiple comparison test, and Pearson correlation. Results. Immunohistochemical staining revealed Gram-positive bacteria exclusively within cells, with C. acnes positivity ranging from 5–99% and correlating with patient age (r=0.41, p= 0.007). TLR2 positivity ranged from 5–99% and TLR4 from 3–72%, showing a strong correlation (r= 0.62, p= 1.5e-006). Females with mid-degenerative grades exhibited significantly decreased TLR2 expression compared to those without degeneration signs. Treatment with LPS and PGN increased catabolic cyto- and chemokines associated with IVD degeneration. Conclusion. In conclusion, this study confirms Gram-positive bacteria presence in non-herniated human disc samples and highlights their role in triggering a catabolic response in disc cells. No conflicts of interest.  . Sources of funding. This project is part of the Disc4All Training network to advance integrated computational simulations in translational medicine, applies to intervertebral disc degeneration and funded by Horizon 2020 (H2020-MSCA-ITN-ETN-2020 GA: 955735)

The most common reason for revision surgery of total hip replacements is aseptic loosening of implants secondary to osteolysis, which is caused by immune-mediated reactions to implant debris. These debris can cause pseudotumour formation. As revision surgery is associated with higher mortality and infection, it is important to understand the pro-inflammatory process to improve implant survival. Toll-like receptor 4 (TLR4) has been shown to mediate immune responses to cobalt ions. Statin use in epidemiological studies has been associated with reduced risk of revision surgery. In-vitro studies have demonstrated the potential for statins to reduce orthopaedic debris-induced immune responses and there is evidence that statins can modulate TLR4 activity. This study investigates simvastatin's effect on orthopaedic biomaterial-mediated changes in protein expression of key inflammatory markers and soluble-ICAM-1 (sICAM-1), an angiogenic factor implicated in pseudotumour formation. Human macrophage THP-1 cells were pre-incubated with 50µM simvastatin for 2-hours or a vehicle control (VC), before being exposed to 0.75mM cobalt chloride, 50μm3 per cell zirconium oxide or LPS as a positive control, in addition to a further 24-hour co-incubation with 50µM simvastatin or VC. Interleukin −8 (IL-8), sICAM-1, chemokine ligand 2 (CCL2), CCL3 and CCL4 protein secretion was measured by enzyme-linked immunosorbent assay (ELISA). GraphPad Prism 10 was used for statistical analysis including a one-way ANOVA. Pre-treatment with simvastatin significantly reduced LPS and cobalt-mediated IL-8 secretion (n=3) and sICAM-1 protein secretion (n=2) in THP-1 cells. Pre-treatment with simvastatin significantly reduced LPS-mediated but not cobalt ion-mediated CCL2 (n=3) and CCL3 protein (n=3) secretion in THP-1 cells. Simvastatin significantly reduced zirconium oxide-mediated CCL4 secretion (n=3). Simvastatin significantly reduced cobalt-ion mediated IL-8 and sICAM-1 protein secretion in THP-1 cells. This in-vitro finding demonstrates the potential for simvastatin to reduce recruitment of leukocytes which mediate the deleterious inflammatory processes driving implant failure

Summary. Metal-on-metal hip replacements have been associated with adverse reactions including inflammatory pseudotumours and soft tissue necrosis. We have shown that cobalt can directly activate toll-like receptor 4, an immune receptor causing pro-inflammatory interleukin-8 secretion. This may contribute to adverse reaction development. Introduction. Metal-on-metal hips have the highest failure rate of any joint arthroplasty material. Reasons for failure include the development of pseudotumours, soft tissue necrosis and pain around the affected joint. The adverse reactions appear to be inflammatory as failing joints are often infiltrated by immune cells such as lymphocytes. However the exact cellular and biological mechanisms underlying this inflammation are unknown. Toll-like receptor 4 (TLR4) is found on the surface of immune cells including macrophages and dendritic cells. It is activated by lipopolysaccharide (LPS) from Gram negative bacteria, inducing an immune response against the pathogen through increased secretion of pro-inflammatory cytokines. It has recently been shown that nickel can activate TLR4, causing inflammation. Cobalt, a component of many metal-on-metal joints, is adjacent to nickel in the periodic table and shares a number of nickel's properties. Consequently we hypothesised that cobalt ions from metal-on-metal joints can activate TLR4. Methods. An in vitro cell culture model was developed using human and murine TLR4 reporter cell lines to investigate the effects of metal ions, including cobalt, on TLR4. Real-time PCR was used to examine the effect of cobalt on inflammatory gene expression, including IL-8, CCL-2 and IRAK-2, while an ELISA assay was conducted to investigate IL-8 protein expression in a human macrophage cell line (MonoMac 6). The TLR4 agonist LPS was included as a positive control and as a negative control TLR4 activation was blocked using the chemical agonist CLI-095 (Invivogen, UK). Results. Using human TLR4 reporter cells we show that cobalt at clinically-relevant concentrations can activate human TLR4. This effect appears unique to humans as murine TLR4 is unresponsive to cobalt but still responds to LPS. We also demonstrate that in human macrophages physiologically-relevant concentrations of cobalt cause increased pro-inflammatory IL-8 secretion (p<0.001). IL-8 is involved in perpetuating the immune response by recruiting more inflammatory cells to the site of inflammation. Cobalt-induced IL-8 secretion can be blocked using a TLR4 antagonist (p<0.001) showing that the effect is due to cobalt activation. Cobalt ions also alter gene expression in human macrophages. Cobalt upregulates expression of IL-8 and IRAK2 genes; IRAK2 is a key component of the TLR4 signalling pathway. Interestingly, cobalt causes downregulation of the CCL2 gene whereas it is upregulated in response to LPS. Discussion. In this study we have demonstrated that cobalt ions can activate human TLR4 signalling and in human macrophages this can increase expression of pro-inflammatory IL-8. We have also developed a robust series of assays for determining the effects of metal ions and other orthopaedic materials on the TLR4 signalling pathway. These methods will be used to investigate the immunological effects of additional orthopaedic metals (e.g. chromium, titanium and molybdenum). This work has identified a key pathway involved in the immune response to metal ions which can now be investigated for genetic variability and as a potential therapeutic target

Introduction and Objective. Total joint replacement is indicated for osteoarthritis where conservative treatment has failed, and in the UK the number of patients requiring hip and knee replacements is set to increase with an ageing population. Survival of total hip replacements is around 85% at 20 years with the most common reason for revision being aseptic loosening of the implant secondary to osteolysis, which is caused by immune-mediated reactions to implant debris. These debris can also cause pseudotumour formation. As revision surgery is associated with higher morbidity, mortality, infection rates, venous thromboembolism, resource demand and poorer subsequent function it is important to understand the mechanisms underlying the pro-inflammatory process to improve implant survival. Toll-like receptor 4 (TLR4), an innate immune receptor, has been demonstrated to mediate deleterious immune responses by the Tyson-Capper research group, including inflammatory cytokine interleukin-8 (IL-8) secretion. Statin use in epidemiological studies has been associated with reduced overall risk of revision surgery after hip replacement. In-vitro studies have demonstrated the potential for statins to reduce orthopaedic debris-induced immune responses which can lead to osteolysis and pseudotumour formation. As literature from cardiological investigations demonstrate that statins can reduce the expression and responsiveness of TLR4, this could be an exciting mechanism to exploit to reduce the host immune response to orthopaedic wear debris, thereby improving implant survival by reducing immune mediated osteolysis. This ongoing study investigates simvastatin's effect on cobalt ion-mediated changes in gene and protein expression of interleukin-8 and soluble-ICAM-1 (sICAM-1) which is an angiogenic factor implicated in pseudotumour formation. Materials and Methods. TLR4-expressing human monocyte/macrophage THP-1 cells were pre-incubated with 50μM simvastatin for 2-hours or a vehicle control, before being exposed to exposed to 0.75mM cobalt chloride, in addition to a further 24-hour co-incubation with 50μM simvastatin or vehicle control. IL-8 protein and sICAM-1 secretion was measured by enzyme-linked immunosorbent assay (ELISA). Gene expression changes were quantified by TaqMan-based real time polymerase chain reaction. Results. Pre-treatment with simvastatin significantly reduced cobalt-mediated IL-8 protein secretion (n=3) and sICAM-1 protein secretion (n=2) in THP-1 cells (p-value<0.0001). Work will be undertaken to determine changes in gene expression, the role of TLR4 in these responses and the effect of simvastatin on additional inflammatory markers. Conclusions. Simvastatin significantly reduces cobalt-ion mediated IL-8 and sICAM-1 protein secretion in THP-1 cells. This in-vitro finding demonstrates the potential for simvastatin to reduce recruitment of leukocytes which mediate the deleterious inflammatory processes driving aseptic loosening and pseudotumour formation

Introduction and Objective. Intervertebral disc (IVD) degeneration is one of the major contributors to low back pain, the leading cause of disability worldwide. This multifactorial pathological process involves the degradation of the extracellular matrix, inflammation, and cell loss due to apoptosis and senescence. While the deterioration of the extracellular matrix and cell loss lead to structural collapse of the IVD, increased levels of inflammation result in innervation and the development of pain. Amongst the known regulators of inflammation, toll-like receptors (TLRs) and more specifically TLR-2 have been shown to be specifically relevant in IVD degeneration. As strong post-transcriptional regulators, microRNAs (miRNAs) and their dysregulation has been connected to multiple pathologies, including degenerative diseases such as osteoarthritis and IVD degeneration. However, the role of miRNAs in TLR signalling in the IVD is still poorly understood and was hence investigated in this study. Materials and Methods. Human Nucleus pulposus (hNP) and Annulus fibrosus (hAF) cells (n=5) were treated with the TLR-2/6 specific agonist PAM2CSK4 (100 ng/mL for 6 hours) in order to activate the TLR2 signalling pathway. After the activation both miRNA and mRNA were isolated, followed by next-generation sequencing and qPCR analysis of proinflammatory cytokines respectively. Furthermore, cell supernatants were used to analyze the secretion of proinflammatory cytokines with enzyme-linked immunosorbent assay. TLR-2 knockdown (siRNA) cells were used as a control. Statistical analysis was conducted by performing Kolmogorov-Smirnov test and a two-tailed Student's t-test using GraphPad Prism version 9.0.2 for Windows (GraphPad Software, La Jolla California USA). Results. TLR-2 activation resulted in the induction of an inflammatory cell response, with a significant increase in gene expression of interleukin (IL)-6 (525 ± 180 fold change, p < 0.05) and IL-8 (7513 ± 1907 fold change, p < 0.05) and protein secretion of IL-6 (30.5 ± 8.1 pg/mL) and IL-8 (28.9 ± 5.4 pg/mL). TLR-2 activation was furthermore associated with changes in the miRNA profile of hNP and hAF cells. Specifically, we identified 10 differentially expressed miRNAs in response to TLR-2 activation, amongst which were miR-335–3p (1.45 log2 FC, p < 0.05), miR-125b-1–3p (0.55 log2 FC, p < 0.05), and miR-181a-3p (−1.05 log2 FC, p < 0.05). Conclusions. The identified miRNAs are known to be associated with osteoarthritis (miR-335-3p), inflammation and IVD degeneration (mir-125-1-3p and miR-181a-3p), but the link to TLR signalling has not been previously reported. Experiments to validate the identified miRNAs and elucidate their functional role are undergoing. The identification of these miRNAs provides an opportunity to further investigate miRNAs in the context of TLR activation and inflammation and to enhance our understanding of underlying molecular mechanisms behind disc degeneration, inflammation, and TLR dysregulation

All types of regenerative materials, including metal implants, porous scaffolds and cell-laden hydrogels, interact with the living tissue and cells. Such interaction is key to the settlement and regenerative outcomes of the biomaterials. Notably, the immune reactions from the host body crucially mediate the tissue-biomaterials interactions. Macrophages (as well as monocytes and neutrophils), traditionally best known as defenders, accumulate at the tissue-biomaterials interface and secrete abundant cytokines to create a microenvironment that benefits or inhibits regeneration. Because the phenotype of these cells is highly plastic in response to varying stimuli, it may be feasible to manipulate their activity at the interface and harness their power to mediate bone regeneration. Towards this goal, our team have been working on macrophage-driven bone regeneration in two aspects. First, targeting the abundant, glucan/mannan-recognising receptors on macrophages, we have devised a series of glucomannan polymers that can stimulate macrophages to secrete pro-osteogenic cytokines, and applied them as coating polymer of mesenchymal stem cells-laden hydrogels. Second, targeting the toll-like receptors (TLRs) on macrophages, we have screened TLR-activating polysaccharides and picked up zymosan (beta-glucan) to be modified onto titanium and glass implants. We evaluated both the efficacy of integration and safety of immune stimulation in both in vitro and in vivo models. Our future exploration lies in further elaborating the different roles and mechanisms of macrophages of various types and origins in the regenerative process

Increased revision rates and early failure of Metal-on-Metal (MoM) hip replacements are often due to adverse reaction to metal debris (ARMD). Cobalt is a major component of MoM joints and can initiate an immune response via activation of the innate immune receptor Toll-like receptor 4 (TLR4). This leads to increased secretion of inflammatory cytokines/chemokines e.g. CCL3 and CCL4. The aim of this study was to evaluate whether TLR4-specific neutralising antibodies can prevent cobalt-mediated activation of TLR4. MonoMac 6 (MM6) cells, a human macrophage cell line, were treated with two different TLR4-specific monoclonal antibodies followed by 0.75mM of cobalt chloride (CoCl2). Lipopolysaccharide (LPS), a known TLR4 agonist was used as a positive control. Enzyme-linked immunosorbent assay (ELISA) was used to assess CCL3/CCL4 protein secretion and real time- polymerase chain reaction (RT-PCR) allowed quantification of CCL3/CCL4 gene expression. MM6 cells treated with cobalt and LPS up-regulate CCL3 and CCL4 gene expression and protein secretion. MM6 cells pre-treated with both monoclonal antibodies prior to stimulation with 0.75mM CoCl2 for 16 hours demonstrated significant inhibition of both CCL3 and CCL4 secretion as well as gene expression (both p=<0.0001). One of the antibodies failed to inhibit chemokine expression and secretion in LPS treated cells. This study identifies for the first time the use of TLR4-specific monoclonal antibodies to prevent cobalt activation of TLR4 and subsequent inflammatory response. This finding demonstrates the potential to exploit TLR4 inhibition in the context of MoM joint replacements by contributing to the development of novel therapeutics designed to reduce the incidence of ARMD

Adolescent idiopathic scoliosis (AIS) is a poorly understood progressive curvature of the spine. The 3-dimmensionnal spinal deformation brings abnormal biomechanical stresses on the load-bearing organs. We have recently reported for the first time the presence of facet joint cartilage degeneration comparable to age-related osteoarthritis in scoliotic adolescents. To better understand the degenerative mechanisms and explore new therapeutic possibilities, we focused on Toll-like receptors (TLRs) which are germline-encoded pattern recognition receptors that recognize pathogens and endogenous proteins such as fragmented extracellular matrix components (alarmins) present in intervertebral discs (IVD) and articular cartilage. Once activated, they regulate the production pro-inflammatory cytokines, proteases and neurotrophins which can lead to matrix catabolism, inflammation and potentially pain. These mechanisms have however not been studied in the context of AIS or facet joints. Facet joints of AIS patients undergoing corrective surgery and of cadaveric donors (non-scoliotic) were collected from consenting patients or organ donors with ethical approval. Cartilage biopsies and chondrocytes were isolated using 3mm biopsy punches and collagenase type 2 digestion respectively. qPCR was used to assess gene expression of the degenerative factors (MMP3, MMP13, IL-1ß, IL-6, IL-8) The biopsies were cut into two equal halves, one was treated for 4 days with a TLR2 agonist (Pam2CSK4, Invivogen) in serum-free chondrocyte media while the other one was cultured in media alone. MMP3, MMP13, IL-6 and IL-8 ELISAs and DMMB assays were performed on the biopsy cultured media. The ex vivo cartilage was then fixed, cryosectionned and also stained with SafraninO-Fast Green dyes. Baseline gene expression levels of TLR1,−2,−4,−6 were all upregulated in scoliotic chondodryctes compared to non-scoliotic. Pearson correlation analysis revealed that all TLR1,−2,−4,−6 gene expression correlated strongly and significantly with degenerative markers (MMP3, MMP13, IL-6, IL-8) in scoliotic chondrocytes but not in non-scoliotic. (Figure 1) When monolayer facet joint chondrocytes were activated with Pam2CSk4, there was a significant upregulation in previously described degenerative markers, TLR2 and NGF, a potent neurotrophin. These findings were strengthened by protein secretion analysis of select markers such as MMP-3, −13, IL-6 and IL-8 which were all upregulated after TLR2 activation. The scoliotic biopsies which were treated with Pam2CSK4 had a significant loss of proteoglycan content as shown by histology, was reflected in the proteoglycan content found in the media by DMMB. TLR gene expression levels were upregulated and correlated with proteases and pro-inflammatory cytokines in degenerating scoliotic cartilage, suggesting they promote cartilage degradation, especially considering the lack of correlations in non-scoliotic healthy cartilage. Furthermore, when TLRs are activated by Pam2CSK4 it triggers the release of the same proteases and pro-inflammatory cytokines in our ex vivo experiment. All this exacerbates the loss of proteoglycan in the cartilage ex vivo model after four days of insult with a TLR2 specific agonist. These results suggest that TLRs are an important pathway partaking in the cartilage degeneration of scoliotic facet joints and potentially all cartilage beyond our scope. Future studies aim at blocking TLRs to alleviate proteolysis and inflammation. For any figures or tables, please contact the authors directly

Background. Increased revision rates and early failure of Metal-on-Metal (MoM) hip replacements are often due to adverse reaction to metal debris (ARMD). ARMD describes numerous symptoms in patients such as pain, osteolysis and soft tissue damage. Cobalt is a major component of MoM joints and can initiate an immune response via activation of the innate immune receptor Toll-like receptor 4 (TLR4). This leads to increased secretion of inflammatory cytokines e.g. interleukin-8 (IL-8). This study investigates whether TLR4-specific antagonists inhibit the inflammatory response to cobalt using IL-8 gene expression and protein secretion as a marker of TLR4 activation. Methods. MonoMac 6 (MM6) cells, a human macrophage cell line, were treated with TLR4-specific antagonists followed by 0.75mM of cobalt chloride. Lipopolysaccharide (LPS), a known TLR4 agonist was used as a positive control. Enzyme-linked immunosorbent assay (ELISA) was used to assess IL-8 protein secretion and real time- polymerase chain reaction (RT-PCR) allowed quantification of IL-8 gene expression. Results. MM6 cells treated with cobalt and LPS up-regulate IL-8 gene expression and protein secretion (n=3). The addition of TLR4-specific antagonists significantly inhibits this up-regulation suggesting the observed effects are TLR4-mediated. MM6 cells stimulated with cobalt (0.75mM) for 16 hours demonstrated a 27-fold increase in IL-8 gene expression (p-value = < 0.0001). When pre-treated with 10μg/ml of a TLR4-specific antagonist fold increase decreased to 6-fold (p-value = < 0.0001). IL-8 secretion decreased from 5000pg/ml to 3000pg/ml (p-value = < 0.0001). Conclusion. TLR4-specific antagonists inhibit cobalt-mediated IL-8 gene expression and protein secretion in MM6 cells. This finding demonstrates the potential to exploit this inhibition in the context of MoM joint replacements by contributing to the development of novel therapeutics designed to improve MoM implant longevity, reduce the incidence of ARMD and prevent subsequent revision surgery

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

Toll-like receptors (TLRs) are crucial components of the immune system that recognize microbial infection and trigger anti-microbial host defense responses. Gram positive bacteria are causative factors of bone infections, as they alter the balance of coordinated activities during bone remodeling, stimulating osteoclastogenesis. The aim of the study was to investigate whether genetic variation in TLR2 and TLR4 genes predisposes to bone infections’ susceptibility. One hundred and twenty patients with bone infections (osteomyelitis) and 200 healthy controls were genotyped for two single nucleotide polymorphisms (SNPs), R753Q [A/G] in TLR2 gene and T399I [C/T] in TLR4 gene. DNA was extracted from whole blood and the above SNPs were typed with PCR-RFLP (Polymerase Chain Reaction- Restriction Fragment Length Polymorphism) method for genotype identification. All patients were infected by Gram-positive bacteria, predominantly Staphylococcus aureus. Statistical analysis was carried out using the chi-square test. We observed a significantly increased frequency in patients carrying the GA genotype of TLR2 R753Q polymorphism compared to controls (p<0.05). We also found that the A allele was more common in patients than in controls. All individuals carrying the A allele were heterozygous for this variant, while homozygous mutant individuals were not detected in the patients and the control group. In contrast, we found that the TLR4 T399I [C/T] SNP was similarly distributed among the two groups (patients and controls). The mechanism through which TLR2 mediates its effect in bone infections is under investigation. A significant difference was observed in the genotype frequency of TLR2 R753Q [A/G] polymorphism in patients, suggesting that genetic variability in TLR2 gene may be associated with susceptibility to osteomyelitis in response to bacterial invasion in the bone

Aim. The diagnosis of periprosthetic joint infection (PJI) in total joint arthroplasty (TJA) remains a serious clinical challenge. Nowadays, limited biomarkers associated with PJI are available. We investigated therefore the utility of gene expression pattern of Toll-like receptors (TLR) and members of interleukin (IL)1/IL1R family, molecules critically involved in the innate immune response to invading pathogens, for detecting PJI in periprosthetic tissues around TJA. Method. Periprosthetic tissues were collected from 37 patients presenting with PJI and 39 patients having an aseptic failure of TJA. mRNA expression of known TLR receptors (TLR1–10) and 21 members of IL-1/IL-1R family was investigated using an innovative Smartchip Real-Time RT-PCR System. *. ; the data were normalized relative to the housekeeping gene GAPDH. Statistical tests were performed using GraphPad Prism. **. and bio-data mining methods. Results. In PJI, elevated mRNA expression levels of TLR1 (P=0.03), TLR4 (P=0.01) and TLR6 (P=0.01) were detected when compared to tissues from aseptic cases. On the contrary, lower mRNA expression of TLR3 (P=0.04) and TLR7 (P=0.047) were detected in PJI than in aseptic loosening. From IL1/IL-1R family, PJI was associated with elevated levels of IL1β (P=0.0004), IL1RN (P=0.05), IL1R1 (P=0.04), IL1R2 (P=0.01), and IL18RAP (P=0.02) comparing to aseptic failure. Multivariate analysis and sophisticated bio-data mining analysis are ongoing to determine the potential of TLRs and IL1/IL1R family as biomarkers of PJI in TJA. Conclusions. Tissue expression of TLRs and IL1/IL-1R family differ in terms of pattern and expression level between septic and aseptic failure of TJA. Our data support the potential of “innate gene” expression panel as candidate biomarker for assessment of PJI. Further studies are required to replicate our data and also to enable valid interpretation of our findings. Grant support: AZV MZ CR VES15-27726A, VES16-131852A, IGA LF UP_2016_011

Alarmins- also referred to as damage associated molecular patterns (DAMPS)- are endogenous molecules mobilized in response to tissue damage known to activate the innate immune system and regulate tissue repair and remodelling. The molecular mechanisms that regulate inflammatory and remodelling pathways in tendinopathy are largely unknown therefore identifying early immune effectors is essential to understanding the pathology. S100A8 and S100A9 are low molecular weight calcium binding proteins primarily released by activated phagocytes in an inflammatory setting and also secreted as a heterodimeric complex that exhibits cytokine like functions. Based on our previous investigations we sought evidence of S100A8/A9 expression in human tendinopathy and thereafter, to explore mechanisms whereby S100 proteins may regulate inflammatory mediators and matrix regulation in human tenocytes. Torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from patients undergoing arthroscopic stabilisation surgery. S100A8/A9 expression was analysed at transcript and protein level using quantitative RT-PCR and immunohistochemistry, respectively. Primary human tenocytes were cultured from hamstring tendon tissue obtained during hamstring tendon ACL reconstruction. The in vitro effect of recombinant human S100 A8/A9 on primary human tenocytes was measured using quantitative RT-PCR and ELISA. Immunohistochemistry of tendinopathic tissues demonstrated the presence of S100 A8/A9 in diseased tissues compared to control tissue. In addition, early pathological diseased tissue indicated greater S100A9 expression compared with established diseased pathology. These findings were reflected by data obtained at transcript level from diseased tissues. Recombinant human S100A8, A9 and A8/A9 complex led to significant increase in expression of inflammatory mediators, including IL-6 in vitro. Further analysis via quantitative RT-PCR demonstrated recombinant S100A8, A9 and A8/A9 complex treatment on tenocytes, in vitro, had no direct effect on the expression of genes involved in matrix remodelling. The presence of S100A8 and S100A9 in early tendinopathic lesions suggests expression is upregulated in response to cellular damage. S100A8 and S100A9 are endogenous ligands of Toll-like receptors (TLRs) and receptor for advanced glycation end products (RAGE). These receptors have known regulatory effects on immune mediated cytokine production. We propose S100A8 and S100A9 as active alarmins in the early stages of tendinopathy and thus targeting of its downstream signalling may offer novel therapeutic approaches in the management of human tendon disorders

Introduction. Macrophages phagocytes implant wear debris and produce various cytokines to evoke inflammation and periprosthetic osteolysis of aseptic loosening. It had been reported that expression of Toll-like receptor (TLR) 2 and other TLRs increased in periprosthetic tissues of aseptic loosening. Pathogen-associated molecular patterns (PAMPs) and damaged-associated molecular patterns (DAMPs) have been known as ligands of TLRs and considered to be involved in the osteolytic reactions via TLRs. Another type of immune sensors, nucleotide-binding and oligomerization domain (NOD)-like receptors (NLR) with a pyrin domain 3 (NLRP3) can also recognize PAMPs and DAMPs as their lignds, which has been presumed to participate in the local host response of macrophage cascade via phagocytosis of implant wear particles. However, the contribution of NLRP3 in periprosthetic tissues of aseptic loosening and the correlation between TLR2 and NLRP3 are still unclear. Materials and methods. TLR1, TLR2, TLR6, NLRP3, TNF-α and IL-1β of macrophages in aseptic loose periprosthetic tissues were immnohistorically evaluated and compared to osteoarthritic synovium. RAW264.7 cells, macrophagic cell line, were stimulated by titanium particles (Ti) and lipoteichoic acid (LTA)-coated Ti. The celluar reaction associated with TLR2 and NLRP3 and the correlation of them were analyzed at mRNA expression levels with small-interfering RNA of Irak2, one of adaptor molecules in TLR2 cascades. Results. Macrophages, which expressed abundant TLR2, NLRP3, TNF-α and IL-1β, were observed dominantly in foreign body granuloma of aseptic periprosthetic tissues. The features of abundant expression were quite different from osteoarthritic synovium. In vitro experiment of RAW264.7, mRNA levels of NLRP3 and TNF-α increased after stimulation of Ti. mRNA levels of TLR2, NLRP3, TNF-α and IL-1β were enhanced by LTA-coated Ti. mRNA expression level of NLRP3 were suppressed by silencing Irak2. Discussion and conclusion. This study indicated that innate immune sensors, TLR2 and NLRP3, could respond to foreign body particles in aseptic loose periprosthetic connective tissues. In this process, mRNA expression levels of TLR2 and NLRP3 in RAW264.7 were increased by phagocytosis of Ti particles, especially by LTA-coated Ti stimulation. Suppressed mRNA expression level of NLRP3 by knocked down of Irak2 indicated that TLR2 cascade could enhance activation NLRP3 cascade and/or free LTA may stimulate NLRP3 cascade directly. It may be possible that TLR2 and NLRP3 cascades in macrophages recognising PAMPs and/or DAMPs are activated each other and they play an important role of the pathogenesis of wear debris around loose hip joints

Implant-associated osteomyelitis is caused by persistent bacterial infections, predominantly by staphylococci species forming biofilms on implants or osteosynthesis – materials. In the majority of patients the systemic immune response appears to be inconspicous with only minor upregulation of activation-associated receptors on the polymorphonuclear neutrophils (PMN). We found, however, evidence the activation of T cells, apparent as the expansion of CD4+ and CD8+ T cells bearing the activation-associated receptor CD11b. These cells also lacked the co-stimulatory molecule CD28, which is a further indicator for T cell activation. Moreover, small populations of T cells expressing Toll-like receptors (TLR)1, TLR2 or TLR4 were detected in the patients, while in healthy donors less than 1 % of T cells express TLR. A preferential association of TLR1- and TLR2-expression with CD28-CD11b+ cells was seen, compatible with the fact these cells represent an activated phenotype. In addition to the peripheral blood we also analysed leukocytes recovered from the infected site during surgery for removal of the implant. Predominantly PMN were found, highly activated as judged from their surace recpetor pattern, but also CD4+ and CD8+ T cells. As expected, these T cells represented an activated phenotype, and particularly the CD8+ cells expressed CD57, a receptor identifying end-differentiated T cells. The T cells recovered from the infected site, but not the peripheral blood T cells, produced interferon gamma, a cytokine known to support the function of phagocytic cells. In conclusion our data provide evidence that in response to local, persistent bacterial infections T cells are activated to acquire – among others – receptors selective for bacterial products, which in turn might modulate the T cell function and hence the host defence