Ann Harvey

Interleukin-10 regulates the inflammasome-driven augmentation of inflammatory arthritis and joint destruction

IntroductionActivation of the inflammasome has been implicated in the pathology of various autoinflammatory and autoimmune diseases. While the NLRP3 inflammasome has been linked to arthritis progression, little is known about its synovial regulation or contribution to joint histopathology. Regulators of inflammation activation, such as interleukin (IL)-10, may have the potential to limit the inflammasome-driven arthritic disease course and associated structural damage. Hence, we used IL-10-deficient (IL-10KO) mice to assess NLRP3 inflammasome-driven arthritic pathology.MethodsAntigen-induced arthritis (AIA) was established in IL-10KO mice and wild-type controls. Using histological and radiographic approaches together with quantitative real-time PCR of synovial mRNA studies, we explored the regulation of inflammasome components. These were combined with selective blocking agents and ex vivo investigative studies in osteoclast differentiation assays.ResultsIn AIA, IL-10KO mice display severe disease with increased histological and radiographic joint scores. Here, focal bone erosions were associated with increased tartrate-resistant acid phosphatase (TRAP)-positive cells and a localized expression of IL-1β. When compared to controls, IL-10KO synovium showed increased expression of Il1b, Il33 and NLRP3 inflammasome components. Synovial Nlrp3 and Casp1 expression further correlated with Acp5 (encoding TRAP), while neutralization of IL-10 receptor signaling in control mice caused increased expression of Nlrp3 and Casp1. In ex vivo osteoclast differentiation assays, addition of exogenous IL-10 or selective blockade of the NLRP3 inflammasome inhibited osteoclastogenesis.ConclusionsThese data provide a link between IL-10, synovial regulation of the NLRP3 inflammasome and the degree of bone erosions observed in inflammatory arthritis.

AMPA/kainate glutamate receptors contribute to inflammation, degeneration and pain related behaviour in inflammatory stages of arthritis

Objectives Synovial fluid glutamate concentrations increase in arthritis. Activation of kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (GluRs) increase interleukin-6 (IL-6) release and cause arthritic pain, respectively. We hypothesised that AMPA and KA GluRs are expressed in human arthritis, and that intra-articular NBQX (AMPA/KA GluR antagonist) prevents pain and pathology in antigen-induced arthritis (AIA). Methods GluR immunohistochemistry was related to synovial inflammation and degradation in osteoarthritis (OA) and rheumatoid arthritis (RA). A single intra-articular NBQX injection was given at induction, and knee swelling and gait of AIA and AIA+NBQX rats compared over 21 days, before imaging, RT-qPCR, histology and immunohistochemistry of joints. Effects of NBQX on human primary osteoblast (HOB) activity were determined. Results AMPAR2 and KA1 immunolocalised to remodelling bone, cartilage and synovial cells in human OA and RA, and rat AIA. All arthritic tissues showed degradation and synovial inflammation. NBQX reduced GluR abundance, knee swelling (p<0.001, days 1–21), gait abnormalities (days 1–2), end-stage joint destruction (p<0.001), synovial inflammation (p<0.001), and messenger RNA expression of meniscal IL-6 (p<0.05) and whole joint cathepsin K (p<0.01). X-ray and MRI revealed fewer cartilage and bone erosions, and less inflammation after NBQX treatment. NBQX reduced HOB number and prevented mineralisation. Conclusions AMPA/KA GluRs are expressed in human OA and RA, and in AIA, where a single intra-articular injection of NBQX reduced swelling by 33%, and inflammation and degeneration scores by 34% and 27%, respectively, exceeding the efficacy of approved drugs in the same model. AMPA/KA GluR antagonists represent a potential treatment for arthritis.

Imaging Pain in Arthritis: Advances in Structural and Functional Neuroimaging

Arthritis is a heterogeneous disease characterized by joint stiffness, swelling, and pain. Although primarily considered a peripheral joint disease, the severity of pain reported by arthritis patients does not always reflect the extent of joint pathology detectable by conventional means. Using structural and functional brain imaging techniques, a growing number of evolving neuroimaging methods are providing insight into these observed discrepancies at different time-scales. Of these methods, functional magnetic resonance imaging is exploited for short-term evoked pain examination and treatment evaluation; ‘resting-state’ approaches provide insight into fluctuations in pain; perfusion imaging captures elements of on-going clinical pain; and morphological brain assessment provides evidence for long-term structural changes in the brain associated with chronic pain. Further insight into arthritic pain processing at the brain-systems level could in the future be provided by combined neuroimaging approaches, specifically investigating the interactions between functional and structural alterations.

Inhibition of CCL3 abrogated precursor cell fusion and bone erosions in human osteoclast cultures and murine collagen-induced arthritis

Abstract Objective Macrophage inflammatory protein 1-alpha (CCL3) is a chemokine that regulates macrophage trafficking to the inflamed joint. The agonistic effect of CCL3 on osteolytic lesions in patients with multiple myeloma is recognized; however, its role in skeletal damage during inflammatory arthritis has not been established. The aim of the study was to explore the role of osteoclast-associated CCL3 upon bone resorption, and to test its pharmacological blockade for protecting against bone pathology during inflammatory arthritis. Methods CCL3 production was studied during osteoclast differentiation from osteoclast precursor cells: human CD14-positive mononuclear cells. Mice with CIA were treated with an anti-CCL3 antibody. The effect of CCL3 blockade through mAb was studied through osteoclast number, cytokine production and bone resorption on ivory disks, and in vivo through CIA progression (clinical score, paw diameter, synovial inflammation and bone damage). Results Over time, CCL3 increased in parallel with the number of osteoclasts in culture. Anti-CCL3 treatment achieved a concentration-dependent inhibition of osteoclast fusion and reduced pit formation on ivory disks (P ⩽ 0.05). In CIA, anti-CCL3 treatment reduced joint damage and significantly decreased multinucleated tartrate-resistant acid phosphatase-positive osteoclasts and erosions in the wrists (P < 0.05) and elbows (P < 0.05), while also reducing joint erosions in the hind (P < 0.01) and fore paws (P < 0.01) as confirmed by X-ray. Conclusion Inhibition of osteoclast-associated CCL3 reduced osteoclast formation and function whilst attenuating arthritis-associated bone loss and controlling development of erosion in murine joints, thus uncoupling bone damage from inflammation. Our findings may help future innovations for the diagnosis and treatment of inflammatory arthritis.

02.31 Targeted inhibition of macrophage inflammatory protein 1-alpha (ccl3) prevents pit formation by human osteoclasts and potently attenuates the erosion of bone in collagen-induced arthritis

Background In patients with rheumatoid arthritis (RA), osteolysis, osteopenia and osteoporosis are predictive of a severe disease phenotype. CCL3 is a chemokine associated with inflammatory osteolytic lesions and bone resorption by osteoclasts in patients with multiple myeloma. CCL3’s role in modulating bone destruction during inflammatory arthritis has not been established. The purpose of this study was to assess pharmacological blockade of CCL3 as a strategy to prevent or inhibit osteoclast-associated bone damage during inflammatory arthritis. Method Fully differentiated osteoclasts were formed from human CD14-positive mononuclear cell precursors cultured with macrophage-colony stimulating factor (m-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Collagen-induced arthritis (CIA) was selected for in vivo studies. Antibodies (anti-CCL3 and isotype control) were tested for their potency to modulate; (i) osteoclast differentiation and resorption of bone substrate in vitro and (ii) arthritis severity, synovial inflammation by histology and bone erosion by x-ray. Results The concentration of CCL3 in supernatants increased in a time-dependent manner over the 14-day osteoclast culture period (p<0.05). There was a strong, specific correlation between osteoclast number and CCL3 level (p<0.01). Significant concentration-dependent inhibition of osteoclastogenesis was observed by neutralising CCL3 (p≤0.05). The erosion of ivory discs by osteoclasts was reduced significantly (p≤0.05) by ant-CCL3. Mean area of disc resorbed was 0.2% (anti-CCL3), 0.1% (osteoprotegerin) and 1.5% (Isotype control). Synovial inflammation and joint erosion by histology were both significantly reduced by CCL3 blockade in CIA. The composite histology score was 3.8±0.36 (anti-CCL3) versus 6.8±0.6 (isotype control). Osteoclast number was significantly reduced in the wrist (p<0.05) and elbow (p<0.05) joints by anti-CCL3 blockade. Bone erosion by x-ray in the hind paws (p<0.05) and fore paws (p<0.01) was also significantly reduced by anti-CCL3. Conclusion Targeted inhibition of CCL3 protect the synovial joint against arthritis-associated pathology by potent inhibition osteoclast formation and bone erosion. Our data may help guide future innovations for the diagnosis and treatment of inflammatory arthritis.

Novel application of behavioral assays allows dissociation of joint pathology from systemic extra-articular alterations induced by inflammatory arthritis

Introduction: Although rheumatoid arthritis (RA) is a disease of articular joints, patients often suffer from co-morbid neuropsychiatric changes, such as anxiety, that may reflect links between heightened systemic inflammation and abnormal regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Here, we apply behavioral neuroscience methods to assess the impact of antigeninduced arthritis (AIA) on behavioral performance in wild type (WT) and interleukin-10 deficient (Il10-/-) mice. Our aim was to identify limb-specific motor impairments, as well as neuropsychological responses to inflammatory arthritis. Methods: Behavioral testing was performed longitudinally in WT and Il10-/- mice before and after the induction of arthritic joint pathology. Footprint analysis, beam walking and open field assessment determined a range of motor, exploratory and anxietyrelated parameters. Specific gene changes in HPA axis tissues were analyzed using qPCR. Results: Behavioral assessment revealed transient motor and exploratory impairments in mice receiving AIA, coinciding with joint swelling. Hind limb coordination deficits were independent of joint pathology. Behavioral impairments returned to baseline by 10 days post-AIA in WT mice. Il10-/- mice demonstrated comparable levels of swelling and joint pathology as WT mice up to 15 days post-AIA, but systemic differences were evident in mRNA expression in HPA axis tissues from Il10-/- mice post-AIA. Interestingly, the behavioral profile of Il10-/- mice revealed a significantly longer time post-AIA for activity and anxiety-related behaviors to recover. Conclusions: The novel application of sensitive behavioral tasks has enabled dissociation between behaviors that occur due to transient joint-specific pathology and those generated by more subtle systemic alterations that manifest post-AIA.

Erratum to: Imaging pain in arthritis: Advances in structural and functional neuroimaging

The original version of this article unfortunately contained a mistake. The citation [10•, 15, 18, 31] should read [10•, 31]. In the sentence beginning with “ASL is suitable for studying clinical pain [47], for example in arthritis [19],” the citation [19] should be changed to [22•]. In the sentence “Furthermore, a barcode system comprising within-subject co-variation of grey matter density has been used to classify CBP and knee OA [17•],” the citation [17•] should be changed to [29•]. The citation [65] in Table 2 should read [29•]. Additionally, references [26] and [35] are duplicates.

Intra-articular AMPA/kainate glutamate receptor antagonists alleviate inflammation, pain and pathology in rat antigen induced arthritis [Abstract]

Introduction: Concentrations of the neurotransmitter glutamate are greatly increased in synovial fluids of RA and OA patients, where they correlate with cytokine concentrations. Previously, we demonstrated that human synoviocytes express functional glutamate receptors (GluRs) and that activation of NMDA GluRs decreases proMMP2 expression, whilst activation of kainate GluRs increases IL-6 release (Flood et al. 2007). High glutamate concentrations in the joint cause arthritic pain which is alleviated by intra-articular injection of GluR antagonists. However, the effects of such inhibitors on arthritis progression via GluRs on other joint tissues have not been considered. We are investigating the hypothesis that specific GluR subunits in the arthritic synovium mediate proinflammatory, degradative and proliferative responses, and may be therapeutically targeted to reduce disease rogression and pain. Materials and Methods: Using the mono-articular antigen induced arthritis (AIA) rat model, we used intra-articular injection of NBQX to inhibit AMPA/kainate receptors at the time of arthritis induction, prior to peak IL-6 levels. Over a 21 day period, we measured knee swelling and gait patterns from AIA, AIA + NBQX and normal rats (n = 6 per group). A combination of motion analysis, using Qualisys Pro-Reflex MCU-1000 cameras, and footprint characteristics revealed limping and abnormal movements as an indirect measure of pain. On day 21, joint tissues were taken for reverse transcription PCR, x-ray, immunohistochemistry and histology to examine GluR expression and joint destruction. Results: Less knee swelling (ANOVA with Fisher’s post hoc test, P < 0.0006) was found in NBQX treated rats compared to AIA rats. Using footprints to indicate abnormal loading and movements, NBQX treated rats displayed less pain related behaviour during the initial flare of arthritis compared to AIA rats (Mann–Whitney test, P = 0.0002). Radiological pathology appeared reduced in NBQX rats compared to AIA rats (8.33 and 12, mean x-ray erosion/osteophyte score respectively). Ionotropic and metabotropic GluRs mRNA was differentially expressed in cartilage, synovium, meniscus,fat pad, patella, femoral head and shaft of rat knees. Discussion: We have shown that intra-articular NBQX treatment alleviates inflammation, pain and pathology in arthritis in vivo and that GluRs are differentially expressed in knee joint tissues. This supports our hypothesis that kainate GluRs may be specifically targeted to ease pain, inflammation and pathology in arthritis.