miR-221-3p Drives the Shift of M2-Macrophages to a Pro-Inflammatory Function by Suppressing JAK3/STAT3 Activation.

Objectives: Macrophages are conventionally classified as pro-inflammatory (M1) and anti-inflammatory (M2) functional types. There is evidence for a predominance of macrophages with an inflammatory phenotype (M1) in the rheumatoid arthritis (RA) synovium. MicroRNAs (miRs) play a pivotal role in regulating the inflammatory response in innate immune cells and are found at dysregulated levels in RA patients. Here we explored miRs that tune the inflammatory function of M2-macrophages. Methods: Expression profiles of miR-221-3p and miR-155-5p were analyzed in clinical samples from RA, other inflammatory arthritis (OIA), osteoarthritis (OA), and healthy donors (HD) by qPCR. In vitro generated macrophages were transfected with miR-mimics and inhibitors. Transcriptome profiling through RNA-sequencing was performed on M2-macrophages overexpressing miR-221-3p mimic with or without LPS treatment. Secretion of IL-6, IL-10, IL-12, IL-8, and CXCL13 was measured in M1- and M2-macrophages upon TLR2/TLR3/TLR4-stimulation using ELISA. Inflammatory pathways including NF-κB, IRF3, MAPKs, and JAK3/STAT3 were evaluated by immunoblotting. Direct target interaction of miR-221-3p and predicted target sites in 3'UTR of JAK3 were examined by luciferase reporter gene assay. Results: miR-221-3p in synovial tissue and fluid was increased in RA vs. OA or OIA. Endogenous expression levels of miR-221-3p and miR-155-5p were higher in M1- than M2-macrophages derived from RA patients or HD. TLR4-stimulation of M1- and M2-macrophages resulted in downregulation of miR-221-3p, but upregulation of miR-155-5p. M2-macrophages transfected with miR-221-3p mimics secreted less IL-10 and CXCL13 but more IL-6 and IL-8, exhibited downregulation of JAK3 protein and decreased pSTAT3 activation. JAK3 was identified as new direct target of miR-221-3p in macrophages. Co-transfection of miR-221-3p/miR-155-5p mimics in M2-macrophages increased M1-specific IL-12 secretion. Conclusions: miR-221-3p acts as a regulator of TLR4-induced inflammatory M2-macrophage function by directly targeting JAK3. Dysregulated miR-221-3p expression, as seen in synovium of RA patients, leads to a diminished anti-inflammatory response and drives M2-macrophages to exhibit a M1-cytokine profile.

TLR2 stimulation impairs anti-inflammatory activity of M2-like macrophages, generating a chimeric M1/M2 phenotype.

BACKGROUND: Toll-like receptors (TLRs) and macrophages play an important role in rheumatoid arthritis (RA). Currently, it is not clear whether inflammatory M1 or anti-inflammatory M2 predominate among the resident macrophages in the synovium. In the present study, we set out to investigate the impact of TLR stimulation on monocyte-derived M1 and M2 macrophage function and phenotype by mimicking the exposure to abundant TLR agonists as occurs in the context of RA. The response of macrophage subsets to TLR2 and TLR4 activation was evaluated on cluster of differentiation (CD) marker profile; cytokine secretion; gene expression; and NF-κB, interferon regulatory factors 3 and 7 (IRF3/7), and mitogen-activated protein kinase (MAPK) activation. METHODS: Human monocytes were isolated from peripheral blood of healthy individuals and patients with RA and differentiated into M1-like and M2-like macrophages by granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), respectively. Cells were either (1) stimulated with TLR ligands Pam3 or lipopolysaccharide (LPS) or (2) classically activated via interferon (IFN)-γ/LPS. Cytokine production was measured by enzyme-linked immunosorbent assay, and gene expression was measured by qPCR. Cells were stained for CD markers and analyzed by fluorescence-activated cell sorting. NF-κB, IRF3/7, and MAPKs were detected by Western blotting. RESULTS: Monocyte-derived macrophages of healthy donors (HD) or patients with RA displayed comparable subset-specific phenotypes upon exposure to TLR agonists. CD14 and CD163 marker expression on M2 macrophages did not change upon TLR2 and TLR4 engagement. By contrast, M2 gene markers HMOX1, FOLR2, and SLC40A1 were decreased. Importantly, M2 macrophages derived from HD or patients with RA showed both a decreased ratio of interleukin (IL)-10/IL-6 and IL-10/IL-8 upon stimulation with TLR2 ligand Pam3 compared with TLR4 ligand LPS. Gene expression of TLR2 was increased, whereas TLR4 expression was decreased, by TLR ligand stimulation. MAPKs p38, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase were activated more strongly in M2 than in M1 macrophages by Pam3 or LPS. CONCLUSIONS: We show that the anti-inflammatory activity of M2 macrophages is reduced in the presence of abundant TLR2 ligands without significant changes in cell surface markers. Thus, the classical M1/M2 paradigm based on cellular markers does not apply to macrophage functions in inflammatory conditions such as RA.

Expression and regulation of toll-like receptors (TLRs) in human intervertebral disc cells.

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-1ß 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-1ß or TNF-α moderately increased TLR1/TLR4 mRNA expression (TNF-α only), and strongly increased TLR2 mRNA expression (IL-1ß/TNF-α), with the latter being confirmed on the protein level. Stimulation with IL-1ß, TNF-α or Pam3CSK4 (a TLR2-ligand) stimulated IL-6 and IL-8, which was inhibited by a TLR2 neutralizing antibody for Pam3CSK4; IL-1ß 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-1ß/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.

Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways.

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)-1ß, 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.

Curcuma DMSO extracts and curcumin exhibit an anti-inflammatory and anti-catabolic effect on human intervertebral disc cells, possibly by influencing TLR2 expression and JNK activity.

BACKGROUND: As proinflammatory cytokines seem to play a role in discogenic back pain, substances exhibiting anti-inflammatory effects on intervertebral disc cells may be used as minimal-invasive therapeutics for intradiscal/epidural injection. The purpose of this study was to investigate the anti-inflammatory and anti-catabolic potential of curcuma, which has been used in the Indian Ayurvedic medicine to treat multiple ailments for a long time. METHODS: Human disc cells were treated with IL-1ß to induce an inflammatory/catabolic cascade. Different extracts of curcuma as well as curcumin (= a component selected based on results with curcuma extracts and HPLC/MS analysis) were tested for their ability to reduce mRNA expression of proinflammatory cytokines and matrix degrading enzymes after 6 hours (real-time RT-PCR), followed by analysis of typical inflammatory signaling mechanisms such as NF-κB (Western Blot, Transcription Factor Assay), MAP kinases (Western Blot) and Toll-like receptors (real-time RT-PCR). Quantitative data was statistically analyzed using a Mann Whitney U test with a significance level of p < 0.05 (two-tailed). RESULTS: Results indicate that the curcuma DMSO extract significantly reduced levels of IL-6, MMP1, MMP3 and MMP13. The DMSO-soluble component curcumin, whose occurrence within the DMSO extract was verified by HPLC/MS, reduced levels of IL-1ß, IL-6, IL-8, MMP1, MMP3 and MMP13 and both caused an up-regulation of TNF-α. Pathway analysis indicated that curcumin did not show involvement of NF-κB, but down-regulated TLR2 expression and inhibited the MAP kinase JNK while activating p38 and ERK. CONCLUSIONS: Based on its anti-inflammatory and anti-catabolic effects, intradiscal injection of curcumin may be an attractive treatment alternative. However, whether the anti-inflammatory properties in vitro lead to analgesia in vivo will need to be confirmed in an appropriate animal model.

Detrimental role for human high temperature requirement serine protease A1 (HTRA1) in the pathogenesis of intervertebral disc (IVD) degeneration.

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-1ß 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.

Human MMP28 expression is unresponsive to inflammatory stimuli and does not correlate to the grade of intervertebral disc degeneration.

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)-1ß, 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-1ß, 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.

The red wine polyphenol resveratrol shows promising potential for the treatment of nucleus pulposus-mediated pain in vitro and in vivo.

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 1ß (IL-1ß) 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 < 0.05). 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.

Bupivacaine--the deadly friend of intervertebral disc cells?

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-1ß) (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-1ß, 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<.05. 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-1ß 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-1ß. 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.

Analysis of Ewing sarcoma (EWS)-binding proteins: interaction with hnRNP M, U, and RNA-helicases p68/72 within protein-RNA complexes.

The human Ewing Sarcoma (EWS) protein belongs to the TET family of RNA-binding proteins and consists of an N-terminal transcriptional activation domain (EAD) and a C-terminal RNA-binding domain (RBD), which is extensively methylated at arginine residues. This multifunctional protein acts in transcriptional co-activation, DNA-recombination, -pairing and -repair, in splicing, and mRNA transport. The role of arginine methylation in these processes as well as the time and place of methylation within cells is still unclear. In this study, we show that methylation of recombinant EWS protein in HEK cells occurs immediately after or even during translation. Pull-down experiments with recombinant EWS protein as bait, followed by mass spectrometric analysis identified more than 30 interacting proteins independent of whether the EWS protein was methylated or not. The EWS protein interacts via its RBD with RNase-sensitive protein complexes consisting of mainly heterogeneous nuclear ribonucleoproteins (hnRNPs) and RNA helicases. HnRNP M and U, the RNA-helicases p68 and p72, but also actin and tubulin were found to interact directly with the EWS protein. Co-precipitation experiments with recombinant proteins confirmed the interaction of the EWS protein with p68 via its RBD. Colocalization of the EWS protein and the RNA-helicases in the nucleus of HEK cells was visualized by expressing labeled EWS protein and p68 or p72. When co-expressed, the labeled proteins relocated from the nucleoplasm to nucleolar capping structures. As arginine methylation within the RBD of the EWS protein are neither needed for its subcellular localization nor for its protein-protein interaction, a role of EWS protein methylation in RNA-binding and affecting the activation/repression activity or even in the stabilization of the EWS protein seems very likely.

Dynamic subcellular localization of the Ewing sarcoma proto-oncoprotein and its association with and stabilization of microtubules.

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.