Identification of CD64 as a marker for the destructive potential of synovitis in osteoarthritis.

OBJECTIVES: OA is characterized by cartilage degeneration and persistent pain. The majority of OA patients present with synovitis, which is associated with increased cartilage damage. Activated synovial macrophages are key contributors to joint destruction. Therefore, a marker that reflects the activation of these cells could be a valuable tool to characterize the destructive potential of synovitis and benefit monitoring of OA. Here, we aimed to investigate the use of CD64 (FcγRI) as a marker to characterize the damaging potential of synovitis in OA. METHODS: Synovial biopsies were obtained from end-stage OA patients that underwent joint replacement surgery. CD64 protein expression and localization was evaluated using immunohistochemistry and immunofluorescence and quantified using flow cytometry. qPCR was performed to measure the expression of FCGR1 and OA-related genes in synovial biopsies, and in primary chondrocytes and primary fibroblasts stimulated with OA conditioned medium (OAS-CM). RESULTS: Our data exposed a wide range of CD64 expression in OA synovium and showed positive correlations between FCGR1 and S100A8, S100A9, IL1B, IL6 and MMP1/2/3/9/13 expression. CD64 protein correlated with MMP1, MMP3, MMP9, MMP13 and S100A9. Furthermore, we observed that synovial CD64 protein levels in source tissue for OAS-CM significantly associated with the OAS-CM-induced expression of MMP1, MMP3 and especially ADAMTS4 in cultured fibroblasts, but not chondrocytes. CONCLUSION: Together, these results indicate that synovial CD64 expression is associated with the expression of proteolytic enzymes and inflammatory markers related to structural damage in OA. CD64 therefore holds promise as marker to characterize the damaging potential of synovitis.

S100A8/A9-alarmin promotes local myeloid-derived suppressor cell activation restricting severe autoimmune arthritis.

Immune-suppressive effects of myeloid-derived suppressor cells (MDSCs) are well characterized during anti-tumor immunity. The complex mechanisms promoting MDSC development and their regulatory effects during autoimmune diseases are less understood. We demonstrate that the endogenous alarmin S100A8/A9 reprograms myeloid cells to a T cell suppressing phenotype during autoimmune arthritis. Treatment of myeloid precursors with S100-alarmins during differentiation induces MDSCs in a Toll-like receptor 4-dependent manner. Consequently, knockout of S100A8/A9 aggravates disease activity in collagen-induced arthritis due to a deficit of MDSCs in local lymph nodes, which could be corrected by adoptive transfer of S100-induced MDSCs. Blockade of MDSC function in vivo aggravates disease severity in arthritis. Therapeutic application of S100A8 induces MDSCs in vivo and suppresses the inflammatory phenotype of S100A9ko mice. Accordingly, the interplay of T cell-mediated autoimmunity with a defective innate immune regulation is crucial for autoimmune arthritis, which should be considered for future innovative therapeutic options.

S100A8/A9 is not essential for the development of inflammation and joint pathology in interleukin-1 receptor antagonist knockout mice.

BACKGROUND: Excessive osteoclast activity, which is strongly stimulated by pro-inflammatory mediators, results in bone and cartilage degeneration as central features of many arthritides. Levels of the alarmin S100A8/A9 and interleukin (IL)-1ß are both increased in arthritis patients and correlate with disease activity and progression of tissue erosion. We previously presented S100A8/A9 as a good biomarker for joint inflammation and arthritis pathology under circumstances of high IL-1 signaling in mice that lack the gene encoding IL-1 receptor antagonist (Il1rn-/- mice). Here, we investigated whether S100A8/A9 is also actively involved in the development of joint inflammation and both cartilage and bone pathology under these conditions by comparing Il1rn-/- mice with mice that have an additional deficiency for S100a9 (Il1rn-/-XS100a9-/-). METHODS: Il1rn-/-XS100a9-/- on a BALB/c background were obtained by crossing S100a9-/- mice and Il1rn-/- mice. Arthritis incidence and severity were macroscopically scored. Myeloid cell populations in the bone marrow and spleen were determined using flow cytometry. In vitro osteoclastogenesis of bone marrow cells was evaluated with TRAP staining. Microscopic joint inflammation, cartilage degeneration, and bone destruction were evaluated using histology of ankle joints of 12- and 20-week-old mice. RESULTS: Macroscopically scored arthritis severity was comparable between Il1rn-/- and Il1rn-/-XS100a9-/- mice. Inflammation, cartilage erosion, and bone erosion were clearly present in 12-week-old mice of both strains lacking Il1rn-/-, but not significantly different between Il1rn-/-XS100a9-/- and Il1rn-/-. Moreover, we observed that the numbers of neutrophils and monocytes were increased by the absence of Il1rn, which was affected by the absence of S100a9 only in the spleen but not in the bone marrow. In line with our other findings, the absence of S100a9 did not affect the osteoclastogenic potential of osteoclast precursors in the absence of Il1rn. Finally, in agreement with the findings in early arthritis development in 12-week-old mice, cartilage and bone erosion in 20-week-old mice was significantly higher in both Il1rn-/- strains, but the additional absence of S100a9 did not further affect tissue pathology. CONCLUSION: S100A8/A9 deficiency does not significantly affect inflammation and joint destruction in mice with high IL1ß signaling suggesting that S100A8/A9 is not essential for the development of arthritis under these conditions.

Osteoarthritis-Related Inflammation Blocks TGF-ß's Protective Effect on Chondrocyte Hypertrophy via (de)Phosphorylation of the SMAD2/3 Linker Region.

Osteoarthritis (OA) is a degenerative joint disease characterized by irreversible cartilage damage, inflammation and altered chondrocyte phenotype. Transforming growth factor-ß (TGF-ß) signaling via SMAD2/3 is crucial for blocking hypertrophy. The post-translational modifications of these SMAD proteins in the linker domain regulate their function and these can be triggered by inflammation through the activation of kinases or phosphatases. Therefore, we investigated if OA-related inflammation affects TGF-ß signaling via SMAD2/3 linker-modifications in chondrocytes. We found that both Interleukin (IL)-1ß and OA-synovium conditioned medium negated SMAD2/3 transcriptional activity in chondrocytes. This inhibition of TGF-ß signaling was enhanced if SMAD3 could not be phosphorylated on Ser213 in the linker region and the inhibition by IL-1ß was less if the SMAD3 linker could not be phosphorylated at Ser204. Our study shows evidence that inflammation inhibits SMAD2/3 signaling in chondrocytes via SMAD linker (de)-phosphorylation. The involvement of linker region modifications may represent a new therapeutic target for OA.

The role of inflammation in mesenchymal stromal cell therapy in osteoarthritis, perspectives for post-traumatic osteoarthritis: a review.

OA is a complex and highly prevalent degenerative disease affecting the whole joint, in which factors like genetic predisposition, gender, age, obesity and traumas contribute to joint destruction. ∼50-80% of OA patients develop synovitis. OA-associated risk factors contribute to joint instability and the release of cartilage matrix fragments, activating the synovium to release pro-inflammatory factors and catabolic enzymes in turn damaging the cartilage and creating a vicious circle. Currently, no cure is available for OA. Mesenchymal stromal cells (MSCs) have been tested in OA for their chondrogenic and anti-inflammatory properties. Interestingly, MSCs are most effective when administered during synovitis. This review focusses on the interplay between joint inflammation and the immunomodulation by MSCs in OA. We discuss the potential of MSCs to break the vicious circle of inflammation and describe current perspectives and challenges for clinical application of MSCs in treatment and prevention of OA, focussing on preventing post-traumatic OA.

Systemic overexpression of interleukin-22 induces the negative immune-regulator SOCS3 and potently reduces experimental arthritis in mice.

OBJECTIVE: High levels of IL-22 are present in serum and synovial fluid of patients with RA. As both pro- and anti-inflammatory roles for IL-22 have been described in studies using animal models of RA, its exact function in arthritis remains poorly defined. With this study we aimed to further unravel the mechanism by which IL-22 exerts its effects and to decipher its therapeutic potential by overexpression of IL-22 either locally or systemically during experimental arthritis. METHODS: CIA was induced in DBA-1 mice by immunization and booster injection with type II collagen (col II). Before arthritis onset, IL-22 was overexpressed either locally by intra-articular injection or systemically by i.v. injection using an adenoviral vector and clinical arthritis was scored for a period of 10 days. Subsequently, joints were isolated for histological analysis of arthritis severity and mRNA and protein expression of various inflammatory mediators was determined in the synovium, spleen and serum. RESULTS: Local IL-22 overexpression did not alter arthritis pathology, whereas systemic overexpression of IL-22 potently reduced disease incidence, severity and pathology during CIA. Mice systemically overexpressing IL-22 showed strongly reduced serum cytokine levels of TNF-α and macrophage inflammatory protein 1α that correlated significantly with the enhanced expression of the negative immune regulator SOCS3 in the spleen. CONCLUSION: With this study, we revealed clear anti-inflammatory effects of systemic IL-22 overexpression during CIA. Additionally, we are the first to show that the protective effect of systemic IL-22 during experimental arthritis is likely orchestrated via upregulation of the negative regulator SOCS3.

The alarmins S100A8 and S100A9 mediate acute pain in experimental synovitis.

BACKGROUND: Synovitis-associated pain is mediated by inflammatory factors that may include S100A8/9, which is able to stimulate nociceptive neurons via Toll-like receptor 4. In this study, we investigated the role of S100A9 in pain response during acute synovitis. METHODS: Acute synovitis was induced by streptococcal cell wall (SCW) injection in the knee joint of C57Bl/6 (WT) and S100A9-/- mice. The expression of S100A8/A9 was determined in serum and synovium by ELISA and immunohistochemistry. Inflammation was investigated by 99mTc accumulation, synovial cytokine release, and histology at days 1, 2, and 7. To assess pain, weight distribution, gait analysis, and mechanical allodynia were monitored. Activation markers in afferent neurons were determined by qPCR and immunohistochemistry in the dorsal root ganglia (DRG). Differences between groups were tested using a one-way or two-way analysis of variance (ANOVA). Differences in histology were tested with a non-parametric Mann-Whitney U test. p values lower than 0.05 were considered significant. RESULTS: Intra-articular SCW injection resulted in increased synovial expression and serum levels of S100A8/A9 at day 1. These increased levels, however, did not contribute to the development of inflammation, since this was equal in S100A9-/- mice. WT mice showed a significantly decreased percentage of weight bearing on the SCW hind paw on day 1, while S100A9-/- mice showed no reduction. Gait analysis showed increased "limping" behavior in WT, but not S100A9-/- mice. Mechanical allodynia was observed but not different between WT and S100A9-/- when measuring paw withdrawal threshold. The gene expression of neuron activation markers NAV1.7, ATF3, and GAP43 in DRG was significantly increased in arthritic WT mice at day 1 but not in S100A9-/- mice. CONCLUSIONS: S100A8/9, released from the synovium upon inflammation, is an important mediator of pain response in the knee during the acute phase of inflammation.

An optimized method for plasma extracellular vesicles isolation to exclude the copresence of biological drugs and plasma proteins which impairs their biological characterization.

Extracellular vesicles (EVs) are cell membrane-derived phospholipid bilayer nanostructures that contain bioactive proteins, enzymes, lipids and polymers of nucleotides. They play a role in intercellular communication and are present in body fluids. EVs can be isolated by methods like ultracentrifugation (UC), polyethylene-glycol-precipitation (PEG) or size exclusion chromatography (SEC). The co-presence of immunoglobulins (Ig) in EV samples isolated from plasma (pEVs) is often reported and this may influence the assessment of the biological function and phenotype of EVs in bio- and immunoassay. Here, we studied the presence of an Ig-based therapeutic (etanercept) in pEV samples isolated from rheumatoid arthritis (RA) patients and improved the isolation method to obtain purer pEVs. From plasma of etanercept (Tumor-necrosis-factor (TNF)-α antibodies)-treated RA patients pEVs were isolated by either UC, PEG or SEC. SEC isolated pEVs showed the highest particle-to-protein ratio. Strong TNF-α inhibition determined in a TNF-α sensitive reporter assay was observed by pEVs isolated by UC and PEG, and to a lesser extent by SEC, suggesting the presence of functional etanercept. SEC isolation of etanercept or labelled immunoglobulin G (IgG) showed co-isolation of these antibodies in the pEV fraction in the presence of plasma or a high protein (albumin) concentration. To minimize the presence of etanercept or immunoglobulins, we extended SEC (eSEC) column length from 56mm to 222mm (total stacking volume unchanged). No effect on the amount of isolated pEVs was observed while protein and IgG content were markedly reduced (90%). Next, from six etanercept- treated RA patients, pEVs were isolated on a eSEC or standard SEC column, in parallel. TNF-α inhibition was again observed in pEVs isolated by conventional SEC but not by eSEC. To confirm the purer pEVs isolated by eSEC the basal IL-8 promoter activation in human monocytes was determined and in 4 out of 5 SEC isolated pEVs activation was observed while eSEC isolated pEVs did not. This study shows that extended SEC columns yielded pEVs without detectable biologicals and with low protein and IgG levels. This isolation method will improve the characterization of pEVs as potential biomarkers and mediators of disease.

Increase in the Number of Bone Marrow Osteoclast Precursors at Different Skeletal Sites, Particularly in Long Bone and Jaw Marrow in Mice Lacking IL-1RA.

Recently, it was shown that interleukin-1ß (IL-1ß) has diverse stimulatory effects on different murine long bone marrow osteoclast precursors (OCPs) in vitro. In this study, interleukin-1 receptor antagonist deficient (Il1rn-/-) and wild-type (WT) mice were compared to investigate the effects of enhanced IL-1 signaling on the composition of OCPs in long bone, calvaria, vertebra, and jaw. Bone marrow cells were isolated from these sites and the percentage of early blast (CD31hi Ly-6C-), myeloid blast (CD31+ Ly-6C+), and monocyte (CD31- Ly-6Chi) OCPs was assessed by flow cytometry. At the time-point of cell isolation, Il1rn-/- mice showed no inflammation or bone destruction yet as determined by histology and microcomputed tomography. However, Il1rn-/- mice had an approximately two-fold higher percentage of OCPs in long bone and jaw marrow compared to WT. Conversely, vertebrae and calvaria marrow contained a similar composition of OCPs in both strains. Bone marrow cells were cultured with macrophage colony stimulating factor (M-CSF) and receptor of NfκB ligand (RANKL) on bone slices to assess osteoclastogenesis and on calcium phosphate-coated plates to analyze mineral dissolution. Deletion of Il1rn increased osteoclastogenesis from long bone, calvaria, and jaw marrows, and all Il1rn-/- cultures showed increased mineral dissolution compared to WT. However, osteoclast markers increased exclusively in Il1rn-/- osteoclasts from long bone and jaw. Collectively, these findings indicate that a lack of IL-1RA increases the numbers of OCPs in vivo, particularly in long bone and jaw, where rheumatoid arthritis and periodontitis develop. Thus, increased bone loss at these sites may be triggered by a larger pool of OCPs due to the disruption of IL-1 inhibitors.

Elevation of inflammatory S100A8/S100A9 complexes in intracranial aneurysms.

BACKGROUND: Inflammation-related factors might give further insight into the pathophysiology of vessel wall inflammation and intracranial aneurysm (IA) rupture. One of these factors is the protein complex S100A8/A9, which is released by neutrophils, monocytes, and activated macrophages and is known for its role in cardiovascular disease. OBJECTIVE: To determine if venous S100A8/A9 levels in patients with a ruptured IA (rIA) or unruptured IA (uIA) are elevated compared with a control group. Second, to assess differences between venous and intra-aneurysmal S100A8/A9 levels of rIA and uIA patients. METHODS: A prospective case study was performed between June 2016 and May 2017 in patients harboring a ruptured or unruptured saccular IA. Primary outcome measures were individual S100A8/A9 serum concentrations as measured in venous and intra-aneurysmal blood samples during endovascular treatment. Venous serum S100A8/A9 concentrations from a healthy control group served as a reference. RESULTS: We included 16 patients with either a rIA or uIA and 47 healthy controls. Venous S100A8/A9 concentrations were higher in aneurysm patients (rIA and uIA) than those of healthy controls (P≤0.001). S100A8/A9 concentrations were higher in intra-aneurysmal samples than in venous samples of rIA patients (P=0.011). This difference was not found in uIA patients (P=0.054). Intra-aneurysmal S100A8/A9 levels were higher in rIAs than in uIAs (P=0.04). CONCLUSIONS: Venous S100A8/A9 levels are elevated in patients with both rIAs and uIAs compared with healthy controls and likely represents aneurysm wall inflammation. S100A8/A9 causes macrophage-induced inflammation and degeneration of the vessel wall which might explain higher intra-aneurysmal S100A8/A9 levels found in rIAs than in uIAs.

Galectin 3 Deficiency Alters Chondrocyte Primary Cilium Formation and Exacerbates Cartilage Destruction via Mitochondrial Apoptosis.

Mechanical overload and aging are the main risk factors of osteoarthritis (OA). Galectin 3 (GAL3) is important in the formation of primary cilia, organelles that are able to sense mechanical stress. The objectives were to evaluate the role of GAL3 in chondrocyte primary cilium formation and in OA in mice. Chondrocyte primary cilium was detected in vitro by confocal microscopy. OA was induced by aging and partial meniscectomy of wild-type (WT) and Gal3-null 129SvEV mice (Gal3-/-). Primary chondrocytes were isolated from joints of new-born mice. Chondrocyte apoptosis was assessed by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), caspase 3 activity and cytochrome c release. Gene expression was assessed by qRT-PCR. GAL3 was localized at the basal body of the chondrocyte primary cilium. Primary cilia of Gal3-/- chondrocytes were frequently abnormal and misshapen. Deletion of Gal3 triggered premature OA during aging and exacerbated joint instability-induced OA. In both aging and surgery-induced OA cartilage, levels of chondrocyte catabolism and hypertrophy markers and apoptosis were more severe in Gal3-/- than WT samples. In vitro, Gal3 knockout favored chondrocyte apoptosis via the mitochondrial pathway. GAL3 is a key regulator of cartilage homeostasis and chondrocyte primary cilium formation in mice. Gal3 deletion promotes OA development.

Macrophage-Derived Extracellular Vesicles as Carriers of Alarmins and Their Potential Involvement in Bone Homeostasis.

Extracellular vesicles are a heterogeneous group of cell-derived membranous structures, which facilitate intercellular communication. Recent studies have highlighted the importance of extracellular vesicles in bone homeostasis, as mediators of crosstalk between different bone-resident cells. Osteoblasts and osteoclasts are capable of releasing various types of extracellular vesicles that promote both osteogenesis, as well as, osteoclastogenesis, maintaining bone homeostasis. However, the contribution of immune cell-derived extracellular vesicles in bone homeostasis remains largely unknown. Recent proteomic studies showed that alarmins are abundantly present in/on macrophage-derived EVs. In this review we will describe these alarmins in the context of bone matrix regulation and discuss the potential contribution macrophage-derived EVs may have in this process.

The alarmin S100A9 hampers osteoclast differentiation from human circulating precursors by reducing the expression of RANK.

The alarmin S100A8/A9 is implicated in sterile inflammation-induced bone resorption and has been shown to increase the bone-resorptive capacity of mature osteoclasts. Here, we investigated the effects of S100A9 on osteoclast differentiation from human CD14+ circulating precursors. Hereto, human CD14+ monocytes were isolated and differentiated toward osteoclasts with M-CSF and receptor activator of NF-κB (RANK) ligand (RANKL) in the presence or absence of S100A9. Tartrate-resistant acid phosphatase staining showed that exposure to S100A9 during monocyte-to-osteoclast differentiation strongly decreased the numbers of multinucleated osteoclasts. This was underlined by a decreased resorption of a hydroxyapatite-like coating. The thus differentiated cells showed a high mRNA and protein production of proinflammatory factors after 16 h of exposure. In contrast, at d 4, the cells showed a decreased production of the osteoclast-promoting protein TNF-α. Interestingly, S100A9 exposure during the first 16 h of culture only was sufficient to reduce osteoclastogenesis. Using fluorescently labeled RANKL, we showed that, within this time frame, S100A9 inhibited the M-CSF-mediated induction of RANK. Chromatin immunoprecipitation showed that this was associated with changes in various histone marks at the epigenetic level. This S100A9-induced reduction in RANK was in part recovered by blocking TNF-α but not IL-1. Together, our data show that S100A9 impedes monocyte-to-osteoclast differentiation, probably via a reduction in RANK expression.-Di Ceglie, I., Blom, A. B., Davar, R., Logie, C., Martens, J. H. A., Habibi, E., Böttcher, L.-M., Roth, J., Vogl, T., Goodyear, C. S., van der Kraan, P. M., van Lent, P. L., van den Bosch, M. H. The alarmin S100A9 hampers osteoclast differentiation from human circulating precursors by reducing the expression of RANK.

Fc-gamma receptors and S100A8/A9 cause bone erosion during rheumatoid arthritis. Do they act as partners in crime?

Bone erosion is one of the central hallmarks of RA and is caused by excessive differentiation and activation of osteoclasts. Presence of autoantibodies in seropositive arthritis is associated with radiographic disease progression. ICs, formed by autoantibodies and their antigens, activate Fcγ-receptor signalling in immune cells, and as such stimulate inflammation-mediated bone erosion. Interestingly, ICs can also directly activate osteoclasts by binding to FcγRs on their surface. Next to autoantibodies, high levels of alarmins, among which is S100A8/A9, are typical for RA and they can further activate the immune system but also directly promote osteoclast function. Therefore, IC-activated FcγRs and S100A8/A9 might act as partners in crime to stimulate inflammation and osteoclasts differentiation and function, thereby stimulating bone erosion. This review discusses the separate roles of ICs, FcγRs and alarmins in bone erosion and sheds new light on the possible interplay between them, which could fuel bone erosion.

IL-1ß-Mediated Activation of Adipose-Derived Mesenchymal Stromal Cells Results in PMN Reallocation and Enhanced Phagocytosis: A Possible Mechanism for the Reduction of Osteoarthritis Pathology.

Background: Injection of adipose-derived mesenchymal stromal cells (ASCs) into murine knee joints after induction of inflammatory collagenase-induced osteoarthritis (CiOA) reduces development of joint pathology. This protection is only achieved when ASCs are applied in early CiOA, which is characterized by synovitis and high S100A8/A9 and IL-1ß levels, suggesting that inflammation is a prerequisite for the protective effect of ASCs. Our objective was to gain more insight into the interplay between synovitis and ASC-mediated amelioration of CiOA pathology. Methods: CiOA was induced by intra-articular collagenase injection. Knee joint sections were stained with hematoxylin/eosin and immunolocalization of polymorphonuclear cells (PMNs) and ASCs was performed using antibodies for NIMP-R14 and CD271, respectively. Chemokine expression induced by IL-1ß or S100A8/A9 was assessed with qPCR and Luminex. ASC-PMN co-cultures were analyzed microscopically and with Luminex for inflammatory mediators. Migration of PMNs through transwell membranes toward conditioned medium of non-stimulated ASCs (ASCNS-CM) or IL-1ß-stimulated ASCs (ASCIL-1ß-CM) was examined using flow cytometry. Phagocytic capacity of PMNs was measured with labeled zymosan particles. Results: Intra-articular saline injection on day 7 of CiOA increased synovitis after 6 h, characterized by PMNs scattered throughout the joint cavity and the synovium. ASC injection resulted in comparable numbers of PMNs which clustered around ASCs in close interaction with the synovial lining. IL-1ß-stimulation of ASCs in vitro strongly increased expression of PMN-attracting chemokines CXCL5, CXCL7, and KC, whereas S100A8/A9-stimulation did not. In agreement, the number of clustered PMNs per ASC was significantly increased after 6 h of co-culturing with IL-1ß-stimulated ASCs. Also migration of PMNs toward ASCIL-1ß-CM was significantly enhanced (287%) when compared to ASCNS-CM. Interestingly, association of PMNs with ASCs significantly diminished KC protein release by ASCs (69% lower after 24 h), accompanied by reduced release of S100A8/A9 protein by the PMNs. Moreover, phagocytic capacity of PMNs was strongly enhanced after priming with ASCIL-1ß-CM. Conclusions: Local application of ASCs in inflamed CiOA knee joints results in clustering of attracted PMNs with ASCs in the synovium, which is likely mediated by IL-1ß-induced up-regulation of chemokine release by ASCs. This results in enhanced phagocytic capacity of PMNs, enabling the clearance of debris to attenuate synovitis.

Increased WISP1 expression in human osteoarthritic articular cartilage is epigenetically regulated and decreases cartilage matrix production.

OBJECTIVES: Previously, we have shown the involvement of Wnt-activated protein Wnt-1-induced signaling protein 1 (WISP1) in the development of OA in mice. Here, we aimed to characterize the relation between WISP1 expression and human OA and its regulatory epigenetic determinants. METHODS: Preserved and lesioned articular cartilage from end-stage OA patients and non-OA-diagnosed individuals was collected. WISP1 expression was determined using immunohistochemistry and damage was classified using Mankin scoring. RNA expression and DNA methylation were assessed in silico from genome-wide datasets (microarray analysis and RNA sequencing, and 450 k-methylationarrays, respectively). Effects of WISP1 were tested in pellet cultures of primary human chondrocytes. RESULTS: WISP1 expression in cartilage of OA patients was increased compared with non-OA-diagnosed controls and, within OA patients, WISP1 was even higher in lesioned compared with preserved regions, with expression strongly correlating with Mankin score. In early symptomatic OA patients with disease progression, higher synovial WISP1 expression was observed as compared with non-progressors. Notably, increased WISP1 expression was inversely correlated with methylation levels of a positional CpG-dinucleotide (cg10191240), with lesioned areas showing strong hypomethylation for this CpG as compared with preserved cartilage. Additionally, we observed that methylation levels were allele-dependent for an intronic single-nucleotide polymorphism nearby cg10191240. Finally, addition of recombinant WISP1 to pellets of primary chondrocytes strongly inhibited deposition of extracellular matrix as reflected by decreased pellet circumference, proteoglycan content and decreased expression of matrix components. CONCLUSION: Increased WISP1 expression is found in lesioned human articular cartilage, and appears epigenetically regulated via DNA methylation. In vitro assays suggest that increased WISP1 is detrimental for cartilage integrity.

Presence of inflammatory proteins S100A8 and S100A9 in a giant intracranial aneurysm after flow diverter treatment.

We demonstrate the presence of S100A8 and S100A9 proteins in the wall and thrombosed lumen of an enlarged intracranial aneurysm after flow diverter treatment. These proteins have shown to play an important role in vascular inflammation and may serve as a biomarker and potential therapeutic target for intracranial aneurysms.

The level of synovial AXL expression determines the outcome of inflammatory arthritis, possibly depending on the upstream role of TGF-ß1.

OBJECTIVE: To investigate the role of AXL, a member of the anti-inflammatory TYRO3, AXL MER (TAM) receptor family, in arthritis. METHODS: KRN serum transfer arthritis was induced in Axl-/- and wild-type mice. Knee and ankle joints were scored macro- and microscopically. Synovial gene and protein expression of Axl was determined in naïve and TGF-ß1-overexpressing joints. AXL expression was determined in M1-like or M2-like macrophages and RA synovium. Human macrophages, fibroblasts and synovial micromasses were stimulated with TGF-ß1 or the AXL inhibitor R428. RESULTS: Ankle joints of Axl-/- mice showed exacerbated arthritis pathology, whereas no effect of Axl gene deletion was observed on gonarthritis pathology. To explain this spatial difference, we examined the synovium of naïve mice. In contrast to the knee, the ankle synovial cells prominently expressed AXL. Moreover, the M2-like macrophage phenotype was the dominant cell type in the naïve ankle joint. Human M2-like macrophages expressed higher levels of AXL and blocking AXL increased their inflammatory response. In the murine ankle synovium, gene expression of Tgfb1 was increased and Tgb1 correlated with Axl. Moreover, TGFB1 and AXL expression also correlated in human RA synovium. In human macrophages and synovial micromasses, TGF-ß1 enhanced AXL expression. Moreover, TGF-ß1 overexpression in naïve murine knee joints induced synovial AXL expression. CONCLUSION: Differences in synovial AXL expression are in accordance with the observation that AXL dampens arthritis in ankle, but not in knee joints. We provide evidence that the local differences in AXL expression could be due to TGF-ß1, and suggest similar pathways operate in RA synovium.

Interleukin-37 Protects Stem Cell-Based Cartilage Formation in an Inflammatory Osteoarthritis-Like Microenvironment.

IMPACT STATEMENT: Catabolic factors present in a damaged joint inhibit chondrogenic differentiation of mesenchymal stem cells, thereby reducing the chance for successful cartilage formation. By improving stem cell-based cartilage repair with interleukin-37 (IL37), we might be able to inhibit the worsening progression of focal cartilage defects and prevent further development of joint diseases such as osteoarthritis. This will avoid chronic pain and impaired joint mobility for patients and reduce costs for society.

A three-dimensional model to study human synovial pathology.

Therapeutic agents that are used by patients with rheumatic and musculoskeletal diseases were originally developed and tested in animal models, and although retrospective studies show a limited predictive value, it could be explained by the fact that there are no good in vitro alternatives. In this study, we developed a 3-dimensional synovial membrane model made of either human primary synovial cell suspensions or a mix of primary fibroblast-like synoviocytes and CD14+ mononuclear cells. We analyzed the composition of the mature micromasses by immunohistochemical staining and flow cytometry and show that the outer surface forms a lining layer consisting out of fibroblast-like and macrophage-like cells, reflecting the in vivo naïve synovial membrane. To recreate the affected synovial membrane in rheumatoid arthritis (RA), the micromasses were exposed to the pro-inflammatory cytokine Tumor Necrosis Factor Alpha (TNF-α). This led to increased pro-inflammatory cytokine expression and production and to hyperplasia of the membrane. To recreate the synovial membrane in osteoarthritis (OA), the micromasses were exposed to Transforming Growth Factor Beta (TGF-ß). This led to fibrosis-like changes of the membrane, including increased Alpha Smooth Muscle Actin and increased expression of fibrosis-related genes PLOD2 and COL1A1. Interestingly, the macrophages in the micromass showed phenotypic plasticity as prolonged TNF-α or TGF-ß stimulation strongly reduced the occurrence of Cluster of Differentiation 163-positive M2-like macrophages. We showed the plasticity of the micromasses as a synovial model for studying RA and OA pathology and propose that the synovial lining micromass system can be a good alternative for testing drugs.