Search

Career situation of first and presenting author Post-doctoral fellow. Introduction Recurrent activation of synovial macrophages is considered as intrinsic factor that precedes rheumatoid arthritis (RA)-related autonomous cytokine cascades. microRNAs (miRs) and Toll-like receptors (TLRs) play a pivotal role in regulating the inflammatory response in innate immune cells and are at dysregulated levels in RA patients. Objectives Here we explored TLR-regulated miRs that tune the inflammatory response of M2-type macrophages. Methods Based on a miR-screen in TLR-stimulated human M2-macrophages we selected miR-221-3p to analyze its expression profile in clinical samples from RA, other inflammatory arthritis (OIA), osteoarthritis (OA) and healthy donors (HD) by qPCR. We challenged M1- and M2-macrophages with elevated miR-221-3p and/or miR-155-5p expression thereby mimicking RA-related conditions. Cytokine/chemokine secretion of IL-6, IL-10, IL-12, IL-8 and CXCL13 was measured using ELISA. Changes of inflammatory pathways including JAK/STAT were evaluated by Western Blot. Results TLR4-stimulation of M2-macrophages results in suppression of miR-221-3p, while in vivo expression in synovial tissue and fluids is significantly increased in RA versus OA or OIA. M2-macrophages with high levels of miR-221-3p lose their anti-inflammatory response and support a M1-like profile. miR-221-3p in combination with miR-155-5p leads to the secretion of M1-specific IL-12 and lowered IL-10. miR-221-3p is affecting the JAK/STAT pathway in M2-macrophages by downregulating JAK3 protein leading to decreased STAT3 activation. Treatment of M2-macrophages using JAK3 inhibitors display the same shift in cytokine/chemokine pattern as increased miR-221-expression. Conclusions TLR4-activated M2-macrophages exposed to elevated miR-221-3p expression diminished anti-inflammatory response partly by suppressing JAK3/STAT3 pathway. Thus, miR-221-3p is a homeostatic regulator of M2-M1 inflammatory function, which is dysregulated in RA condition. Acknowledgements Tissue RNA from RA and OA patients was kindly provided by Caroline Ospelt, University of Zurich, Center of Experimental Rheumatology, Switzerland. Disclosure of Interest None declared.

Human HTRA1 is a highly conserved secreted serine protease that degrades numerous extracellular matrix proteins. We have previously identified HTRA1 as being up-regulated in osteoarthritic patients and as having the potential to regulate matrix metalloproteinase (MMP) expression in synovial fibroblasts through the generation of fibronectin fragments. In the present report, we have extended these studies and investigated the role of HTRA1 in the pathogenesis of intervertebral disc (IVD) degeneration. HTRA1 mRNA expression was significantly elevated in degenerated disc tissue and was associated with increased protein levels. However, these increases did not correlate with the appearance of rs11200638 single nucleotide polymorphism in the promoter region of the HTRA1 gene, as has previously been suggested. Recombinant HTRA1 induced MMP production in IVD cell cultures through a mechanism critically dependent on MEK but independent of IL-1β signaling. The use of a catalytically inactive mutant confirmed these effects to be primarily due to HTRA1 serine protease activity. HTRA1-induced fibronectin proteolysis resulted in the generation of various sized fragments, which when added to IVD cells in culture, caused a significant increase in MMP expression. Furthermore, one of these fragments was identified as being the amino-terminal fibrin- and heparin-binding domain and was also found to be increased within HTRA1-treated IVD cell cultures as well as in disc tissue from patients with IVD degeneration. Our results therefore support a scenario in which HTRA1 promotes IVD degeneration through the proteolytic cleavage of fibronectin and subsequent activation of resident disc cells.

Introduction Intervertebral disc (IVD) degeneration is characterized by extracellular matrix breakdown and is considered to be a primary cause of discogenic back pain. Although increases in pro-inflammatory cytokine levels within degenerating discs are associated with discogenic back pain, the mechanisms leading to their overproduction have not yet been elucidated. As fragmentation of matrix components occurs during IVD degeneration, we assessed the potential involvement of hyaluronic acid fragments (fHAs) in the induction of inflammatory and catabolic mediators. Methods Human IVD cells isolated from patient biopsies were stimulated with fHAs (6 to 12 disaccharides) and their effect on cytokine and matrix degrading enzyme production was assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). The involvement of specific cell surface receptors and signal transduction pathways in mediating the effects of fHAs was tested using small interfering RNA (siRNA) approaches and kinase inhibition assays. Results Treatment of IVD cells with fHAs significantly increased mRNA expression levels of interleukin (IL)-1β, IL-6, IL-8, cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-1 and -13. The stimulatory effects of fHAs on IL-6 protein production were significantly impaired when added to IVD cells in combination with either Toll-like receptor (TLR)-2 siRNA or a TLR2 neutralizing antibody. Furthermore, the ability of fHAs to enhance IL-6 and MMP-3 protein production was found to be dependent on the mitogen-activated protein (MAP) kinase signaling pathway. Conclusions These findings suggest that fHAs may have the potential to mediate IVD degeneration and discogenic back pain through activation of the TLR2 signaling pathway in resident IVD cells., Arthritis Research & Therapy, 15 (4), ISSN:1465-9905, ISSN:1465-9913, ISSN:1478-6362, ISSN:1478-6354

Purpose Although inflammatory processes play an essential role in painful intervertebral disc (IVD) degeneration, the underlying regulatory mechanisms are not well understood. This study was designed to investigate the expression, regulation and importance of specific toll-like receptors (TLRs)—which have been shown to play an essential role e.g. in osteoarthritis—during degenerative disc disease. Methods The expression of TLRs in human IVDs was measured in isolated cells as well as in normal or degenerated IVD tissue. The role of IL-1β or TNF-α in regulating TLRs (expression/activation) as well as in regulating activity of down-stream pathways (NF-κB) and expression of inflammation-related genes (IL-6, IL-8, HSP60, HSP70, HMGB1) was analyzed. Results Expression of TLR1/2/3/4/5/6/9/10 was detected in isolated human IVD cells, with TLR1/2/4/6 being dependent on the degree of IVD degeneration. Stimulation with IL-1β or TNF-α moderately increased TLR1/TLR4 mRNA expression (TNF-α only), and strongly increased TLR2 mRNA expression (IL-1β/TNF-α), with the latter being confirmed on the protein level. Stimulation with IL-1β, TNF-α or Pam3CSK4 (a TLR2-ligand) stimulated IL-6 and IL-8, which was inhibited by a TLR2 neutralizing antibody for Pam3CSK4; IL-1β and TNF-α caused NF-κB activation. HSP60, HSP70 and HMGB1 did not increase IL-6 or IL-8 and were not regulated by IL-1β/TNF-α. Conclusion We provide evidence that several TLRs are expressed in human IVD cells, with TLR2 possibly playing the most crucial role. As TLRs mediate catabolic and inflammatory processes, increased levels of TLRs may lead to aggravated disc degeneration, chronic inflammation and pain development. Especially with the identification of more endogenous TLR ligands, targeting these receptors may hold therapeutic promise., European Spine Journal, 23 (9), ISSN:0940-6719, ISSN:1432-0932, ISSN:001991155

European Spine Journal, 21 (Supplement 6), ISSN:0940-6719, ISSN:1432-0932, ISSN:001991155

Background: MMP28 (epilysin) is a recently discovered member of the MMP (matrix metalloproteinase) family that is, amongst others, expressed in osteoarthritic cartilage and intervertebral disc (IVD) tissue. In this study the hypothesis that increased expression of MMP28 correlates with higher grades of degeneration and is stimulated by the presence of proinflammatory molecules was tested. Gene expression levels of MMP28 were investigated in traumatic and degenerative human IVD tissue and correlated to the type of disease and the degree of degeneration (Thompson grade). Quantification of MMP28 gene expression in human IVD tissue or in isolated cells after stimulation with the inflammatory mediators lipopolysaccharide (LPS), interleukin (IL)-1β, tumor necrosis factor (TNF)-α or the histondeacetylase inhibitor trichostatin A was performed by real-time RT PCR. Results: While MMP28 expression was increased in individual cases with trauma or disc degeneration, there was no significant correlation between the grade of disease and MMP28 expression. Stimulation with LPS, IL-1β, TNF-α or trichostatin A did not alter MMP28 gene expression at any investigated time point or any concentration. Conclusions: Our results demonstrate that gene expression of MMP28 in the IVD is not regulated by inflammatory mechanisms, is donor-dependent and cannot be positively or negatively linked to the grade of degeneration and only weakly to the occurrence of trauma. New hypotheses and future studies are needed to find the role of MMP28 in the intervertebral disc., Journal of Negative Results in Biomedicine, 10, ISSN:1477-5751

Study design Descriptive and mechanistic investigation of the anti-inflammatory and anticatabolic effect of resveratrol in intervertebral discs (IVDs) in vitro and of the analgetic effect in vivo. Objective To determine whether resveratrol may be useful in treating nucleus pulposus (NP)-mediated pain. Summary of background data Proinflammatory cytokines seem to be key mediators in the development of NP-mediated pain. Patients with discogenic or radiculopathic pain may substantially benefit from anti-inflammatory substances that could be used in a minimal-invasive treatment approach. Resveratrol, a polyphenolic phytoalexin found in red wine exhibits anti-inflammatory effects in various cell types and tissues, but no data exists so far with regards to the IVD in the context of low back and leg pain. Methods In part 1, the anti-inflammatory and anticatabolic effect of resveratrol was investigated in a cell culture model on interleukin 1β (IL-1β) prestimulated human IVD cells on the gene and protein expression level. In part 2, the molecular mechanisms underlying the effects observed upon resveratrol treatment were investigated (toll-like receptors, nuclear factor κB, sirtuin 1 (SIRT1), mitogen-activated protein (MAP) kinases p38/ERK/JNK). In part 3, the analgetic effects of resveratrol were investigated in vivo using a rodent model of radiculopathy and von Frey filament testing. All quantitative data were statistically evaluated either by Mann-Whitney U test or by one-way analysis of variance and Bonferroni post hoc testing (P Results In vitro, resveratrol exhibited an anti-inflammatory and anticatabolic effect on the messenger RNA and protein level for IL-6, IL-8, MMP1, MMP3 and MMP13. This effect does not seem to be mediated via the MAP kinase pathways (p38, ERK, JNK) or via the NF-κB/SIRT1 pathway, although toll-like receptor 2 was regulated to a minor extent. In vivo, resveratrol significantly reduced pain behavior triggered by application of NP tissue on the dorsal root ganglion for up to 14 days. Conclusion Resveratrol was able to reduce levels of proinflammatory cytokines in vitro and showed analgetic potential in vivo. A decrease in proinflammatory cytokines may possibly be the underlying mechanism of pain reduction observed in vivo. Resveratrol seems to have considerable potential for the treatment of NP-mediated pain and may thus be an alternative to other currently discussed (biological) treatment options.

Background context Bupivacaine is commonly used as an adjunct during provocative discography and is administered intradiscally in patients with discogenic back pain. Recent studies demonstrated that bupivacaine is cytotoxic for articular chondrocytes in vitro at clinically used concentrations (0.25%–0.5%). Purpose To analyze a concentration-dependent effect of bupivacaine on cell viability and gene expression of human intervertebral disc (IVD) cells in an in vitro model. Study design In vitro cell culture study. Patient sample Disc cells were isolated from human disc biopsies from 11 patients undergoing surgery because of degenerative disc disease or disc herniation. Outcome measures Cell viability and gene expression after exposure to bupivacaine. Methods Human IVD cells were treated with different concentrations of bupivacaine for 2 (n=5) or 18 hours (n=5) and analyzed for cell viability and proliferation (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay). Additionally, cells were prestimulated with interleukin-1 beta (IL-1β) (5 ng/mL) to increase the levels of proinflammatory cytokines and matrix-degrading enzymes and thereafter treated with 0.75 mmol bupivacaine (as determined in the cell viability test) for 2 (n=5) or 18 hours (n=5). Prestimulated cells with or without bupivacaine treatment were analyzed for gene expression of IL-1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), matrix metalloproteinase-3 (MMP3), MMP9, MMP13, and a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) using real-time reverse transcription-polymerase chain reaction. Statistical analysis was performed by using the Mann-Whitney U test with a significance level of p Results After 18 hours, bupivacaine exhibited either a cytotoxic or a proliferative effect on human IVD cells, depending on the concentration. Similar but lower effects could be observed already after 2 hours. With a concentration of 0.75 mmol (proliferative effect), bupivacaine significantly decreased messenger RNA levels of TNF-α, COX-2, MMP13, and ADAMTS4 after 18 hours. In contrast, expression of IL-6, IL-8, and MMP9 did not differ; expression of IL-1β and MMP3 was stimulated with 0.75 mmol. After 2 hours, we observed a reduction in the expression of COX-2, MMP3, MMP13, and ADAMTS4, without any effect regarding IL-1β. Conclusions Application of bupivacaine in clinically relevant concentrations was toxic for IVD cells in vitro. A low concentration stimulated cell proliferation and reduced gene expression of certain matrix-degrading enzymes and proinflammatory cytokines. If these results can be corroborated in tissue explant models or animal studies, caution regarding provocative discography with bupivacaine is prompted.

The Ewing sarcoma (EWS) protein is a member of a large family of RNA-binding proteins. Chimeric EWS oncoproteins generated by chromosomal translocations between the EWS protein and several transcription factors cause various malignant tumors. Due to its multifunctional properties, the EWS protein is involved in such processes as meiotic DNA pairing/recombination, cellular senescence, gene expression, RNA processing and transport, and cell signaling. The EWS protein is predominantly located in the nucleus. It was found in the cytoplasm and associated with the cell membrane. In this study, analysis of the localization of endogenous and fluorescently labeled recombinant EWS protein in different phases of the cell cycle in different cell lines revealed a very dynamic subcellular distribution of the EWS protein. In Cos7 and HeLa cells, an association of the EWS protein with the centrosomal compartments was shown. Furthermore, in HEK (human embryonic kidney)-293 (T) cells, an interaction of the overexpressed recombinant EWS-yellow fluorescent protein fusion protein with microtubules, leading to their stabilization and cell cycle arrest, was demonstrated. As an outlook, the present findings provide an important insight into temporally and spatially regulated functions of the EWS protein and, particularly, into its role in the regulation of the cell cycle and possibly cell differentiation.