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

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

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

Introduction:. Wear particles cause aseptic loosening by stimulating macrophages to produce inflammatory cytokines. Recent studies indicate that Toll-like receptor 2 (TLR2) and TLR4 mediate macrophage responses to the wear particles [1–3]. TLR2 and TLR4 uniquely activate MyD88-dependent signaling via an additional adapter protein known as TIRAP/Mal [4]. Del Vescovo et al reported that three single nucleotide polymorphisms (SNPs) within the TIRAP/Mal gene associate with aseptic loosening in THA patients [5]. The goal of the current study was therefore to determine whether TIRAP/Mal mediates responses to orthopaedic wear particles. Methods:. Immortalized wild type (WT) and TIRAP/Mal knockout (KO) murine macrophages (Mfs) were incubated in the presence or absence of titanium (Ti) particles (1 × 10. 8. particles/cm. 2. [2]. Three types of particles were used as described previously [1,2]: Ti particles with adherent bacterial debris (38.3 Endotoxin Units/10. 9. particles), endotoxin-free Ti particles (<0.1 EU/10. 9. particles), and Ti particles with adherent lipopolysacharide (LPS, 32.8 EU/10. 9. particles). TNFa, IL-1b, and IL-6 mRNAs were measured by real-time PCR and the secreted cytokines were measured by ELISA. Particle-induced osteolysis in calvaria of TIRAP/Mal KO and WT mice was measured 7 days after particle implantation [1,2]. In vitro results are presented as mean ± SEM of 3–4 replicate experiments analyzed by two-way ANOVA with Bonferroni post-hoc corrections. In vivo results are presented as means of individual parietal bones ± SEM (n = 22) and analyzed by one-way ANOVA on ranks with Student Neuman-Keuls post-hoc corrections. * denotes p < 0.5, ** denotes p < 0.01, *** denotes p < 0. Results:. Ti particles with adherent bacterial debris induced substantial osteolysis and expression of TNFa, IL-1b, and IL-6 at both the mRNA and protein levels and all of those responses were significantly inhibited by TIRAP/Mal KO (Fig 1 & Fig 2). Endotoxin-free Ti particles had a small effect on osteolysis and cytokine mRNA expression that was not dependent on TIRAP/Mal (Fig 1 & data not shown). Adherence of highly purified LPS to the endotoxin-free particles reconstituted the stimulation of osteolysis and cytokine expression as well as the dependence on TIRAP/MAL (Fig 1 & data not shown). Specificity of the effects of TIRAP/Mal KO was demonstrated since responses induced by recombinant murine IL-1b were unaffected (data not shown). Discussion:. Our results are the first demonstration that TIRAP/Mal mediates effects of orthopaedic wear particles. TIRAP/Mal KO inhibited expression of TNFa by ∼50% and almost completely inhibited particle-induced osteolysis, as well as expression of IL-1b and IL-6. Our results, coupled with the genetic association of SNPs in human TIRAP/Mal with aseptic loosening [5], lead to two conclusions. First, activation of TIRAP/Mal likely contributes to aseptic loosening in patients. Second, pathogen-associated molecular patterns (PAMPs) also likely contribute to aseptic loosening since the results with endotoxin-free Ti particles demonstrate that adherent PAMPs are required for activation of TIRAP/Mal