Suppression of Rheumatoid Arthritis by Enhanced Lymph Node Trafficking of Engineered Interleukin-10 in Murine Models.

OBJECTIVE: Rheumatoid arthritis (RA) is a major autoimmune disease that causes synovitis and joint damage. Although clinical trials have been performed using interleukin-10 (IL-10), an antiinflammatory cytokine, as a potential treatment of RA, the therapeutic effects of IL-10 have been limited, potentially due to insufficient residence in lymphoid organs, where antigen recognition primarily occurs. This study was undertaken to engineer an IL-10-serum albumin (SA) fusion protein and evaluate its effects in 2 murine models of RA. METHODS: SA-fused IL-10 (SA-IL-10) was recombinantly expressed. Mice with collagen antibody-induced arthritis (n = 4-7 per group) or collagen-induced arthritis (n = 9-15 per group) were injected intravenously with wild-type IL-10 or SA-IL-10, and the retention of SA-IL-10 in the lymph nodes (LNs), immune cell composition in the paws, and therapeutic effect of SA-IL-10 on mice with arthritis were assessed. RESULTS: SA fusion to IL-10 led to enhanced accumulation in the mouse LNs compared with unmodified IL-10. Intravenous SA-IL-10 treatment restored immune cell composition in the paws to a normal status, elevated the frequency of suppressive alternatively activated macrophages, reduced IL-17A levels in the paw-draining LN, and protected joint morphology. Intravenous SA-IL-10 treatment showed similar efficacy as treatment with an anti-tumor necrosis factor antibody. SA-IL-10 was equally effective when administered intravenously, locally, or subcutaneously, which is a benefit for clinical translation of this molecule. CONCLUSION: SA fusion to IL-10 is a simple but effective engineering strategy for RA therapy and has potential for clinical translation.

Early supplemental α2-macroglobulin attenuates cartilage and bone damage by inhibiting inflammation in collagen II-induced arthritis model.

OBJECTIVE: To determine if early supplemental intra-articular α2-macroglobulin (A2M) has a chondroprotective effect in a collagen II-induced arthritis (CIA) mice model. METHODS: DBA/1 mice were randomized into four groups (n = 15/group): (a) CIA + 1.2 µg of A2M; (b) CIA + 0.8 µg of A2M; (c) CIA + 0.4 µg of A2M; (d) vehicle + phosphate-buffered saline (PBS). A2M was injected into right ankles and PBS was injected into the left ankles simultaneously as internal control at days 36, 43 and 50. The CIA inflammation clinical score and ankle thickness were recorded every other day starting on day 21 until sacrifice. Changes in inflammation were monitored by in vivo fluorescence molecular tomography (FMT). Inflammation, cartilage and bone damage were assessed with X-ray, histology and immunohistochemistry. Cartilage and inflammation-related gene expression was quantified by real-time polymerase chain reaction (PCR). RESULTS: All mice showed ankle inflammation on day 33. After day 43, lower clinical scores, ankle thickness and Sharp/van der Heijde method scores in A2M-treated ankles compared with PBS-treated ankles. FMT data indicated that the inflammation markers MMPSense and ProSense were significantly elevated in the PBS-treated ankles than A2M-treated ankles. Histology and X-ray analyses indicated that A2M administration resulted in lower levels of inflammatory infiltration and synovial hyperplasia, as well as more typical cartilage and bone organization with increased COL II and Aggrecan staining when compared with PBS-treated ankles. In addition, real-time PCR showed that,matrix metalloproteinase-3, -9, -13, COL X and Runx2 were significantly less expressed in A2M-treated groups than PBS-treated animals. CONCLUSION: Early supplemental intra-articular A2M exerts an anti-inflammatory effect and attenuates cartilage and bone damage in a CIA model.

Fluid distribution in ankle and midfoot joints: MR findings in asymptomatic volunteers.

PURPOSE: The purpose of this study is to assess the amount of fluid in the joints of the ankle and midfoot on MR imaging in asymptomatic volunteers. MATERIALS AND METHODS: Twenty-one healthy asymptomatic volunteers (42 ankles) were evaluated with MRI imaging. There were 13 men and 8 women. The mean age was 24.7 years (19-42 years). MR imaging was performed on a 3T MR system using proton density weighted images with fat saturation (TR 2969, TE 30 ms, NA 2, slice thickness 2.5 mm). Images were obtained in three orthogonal planes. The images were interpreted by two radiologists in two sessions. The maximum size of the joint effusion was measured in one plane. Descriptive statistics and variation between interpretation sessions were calculated. RESULTS: Fluid in the anterior tibiotalar joint had a mean size of 2.0 mm (0.0-5.5 mm), in the posterior tibiotalar joint 3.1 mm (0.0-6.3 mm), in the talonavicular joint 0.7 mm (0.0-2.9 mm), and in the anterolateral recess 2.0 mm (0.0-4.3 mm). Fluid in the posterior aspect of the posterior subtalar joint had a mean size of 2.6 mm (0.0-9.4 mm), in the anterior aspect of the posterior subtalar joint 1.9 mm (0.0-6.6 mm), at the middle subtalar joint 0.1 mm (0.0-1.7 mm), and at the anterior subtalar joint 1.6 mm (0.0-6.0 mm). Fluid in the tibiofibular joint had a mean height of 8.1 mm (0.0-16.4 mm). CONCLUSION: In asymptomatic volunteers, moderate to large amounts of fluid were common in all joint recesses of ankle and midfoot, and most pronounced in the anterior and posterior tibiotalar joint, anterolateral recess, and posterior subtalar joint. This should not be mistaken for evidence of a pathological condition.

Light and electron microscopic detection of inflammation-targeting liposomes encapsulating high-density colloidal gold in arthritic mice.

OBJECTIVE: We have previously demonstrated the efficient and time-dependent transvascular localization of Sialyl Lewis X (SLX)-liposomes to inflammatory sites, but the final target of the SLX-liposomes remained uncertain. The aim of this study was to identify the target cells of the liposomes within the inflamed joints of collagen antibody-induced arthritis (CAIA) model mice. METHODS: SLX-liposomes and unlabeled liposomes encapsulating high-density colloidal gold were administered intravenously into the caudal vein of CAIA mice on day 5 after induction of arthritis when the inflammatory score was maximal (n = 6 per group). Six hours or 24 h after liposome administration, animals were euthanized and hind limbs and ankles were excised without perfusion. After fixation, synovial tissues were examined by light microscopy after silver enhancement of colloidal gold or by transmission electron microscopy. RESULTS: Silver-enhanced signals were detected within the cells around E-selectin-positive blood vessels in the synovium of the SLX-liposome group. These cells were positive for the macrophage/monocyte marker F4/80 or neutrophil marker Ly-6G. Transmission electron microscopy detected the colloidal gold signals together with liposome-like structures within the phagosomes of synovial macrophages. Transmission electron microscopy and energy dispersive X-ray spectrometry could determine gold elements in the lysosomes of synovial macrophages. CONCLUSIONS: The results of the current study demonstrate that SLX-liposomes primarily targeting E-selectin in activated endothelial cells could potentially deliver their contents into inflammatory cells around synovial blood vessels in arthritic joints.

11ß-Hydroxysteroid dehydrogenase 1 inhibition attenuates collagen-induced arthritis.

11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) plays an important role in inflammation. However, the role of 11ß-HSD1 in rheumatoid arthritis (RA) remains unknown. The purpose of this study was to evaluate the therapeutic effects of a selective 11ß-HSD1 inhibitor BVT-2733 in collagen-induced arthritis (CIA) and its underlying mechanisms. CIA mice were treated with BVT-2733 (100 mg/kg, orally) or vehicle twice daily for 2 weeks. Arthritis score and joint histology were investigated. The levels of pro-inflammatory cytokines as well as anti-type II collagen antibody (anti-CII) were detected by ELISA. Western blot analysis was used to assess the activation of NF-κB and NLRP1 inflammasome in joint tissues and in human RA synovial cells. BVT-2733 treatment attenuated the arthritis severity and anti-CII level in CIA mice. BVT-2733 also decreased the levels of serum TNF-α, IL-1ß, IL-6 and IL-17. BVT-2733 treatment also significantly reduced synovial inflammation and joint destruction. NF-κB activation and NLRP1 inflammasome assembly were also inhibited in arthritic joints and human RA synovial cells. In conclusion, BVT-2733 exhibits an anti-inflammatory effect on CIA. This protective effect is, at least partly, mediated by inhibition of the NF-κB and NLRP1 inflammasome signaling pathways. 11ß-HSD1 inhibition may represent a potential therapeutic target for RA patients.

Joint TGF-ß type II receptor-expressing cells: ontogeny and characterization as joint progenitors.

TGF-ß type II receptor (Tgfbr2) signaling plays an essential role in joint-element development. The Tgfbr2(PRX-1KO) mouse, in which the Tgfbr2 is conditionally inactivated in developing limbs, lacks interphalangeal joints and tendons. In this study, we used the Tgfbr2-ß-Gal-GFP-BAC mouse as a LacZ/green fluorescent protein (GFP)-based read-out to determine: the spatial and temporally regulated expression pattern of Tgfbr2-expressing cells within joint elements; their expression profile; and their slow-cycling labeling with bromodeoxyuridine (BrdU). Tgfbr2-ß-Gal activity was first detected at embryonic day (E) 13.5 within the interphalangeal joint interzone. By E16.5, and throughout adulthood, Tgfbr2-expressing cells clustered in a contiguous niche that comprises the groove of Ranvier and the synovio-entheseal complex including part of the perichondrium, the synovium, the articular cartilage superficial layer, and the tendon's entheses. Tgfbr2-expressing cells were found in the synovio-entheseal complex niche with similar temporal pattern in the knee, where they were also detected in meniscal surface, ligaments, and the synovial lining of the infrapatellar fat pad. Tgfbr2-ß-Gal-positive cells were positive for phospho-Smad2, signifying that the Tgfbr2 reporter was accurate. Developmental-stage studies showed that Tgfbr2 expression was in synchrony with expression of joint-morphogenic genes such as Noggin, GDF5, Notch1, and Jagged1. Prenatal and postnatal BrdU-incorporation studies showed that within this synovio-entheseal-articular-cartilage niche most of the Tgfbr2-expressing cells labeled as slow-proliferating cells, namely, stem/progenitor cells. Tgfbr2-positive cells, isolated from embryonic limb mesenchyme, expressed joint progenitor markers in a time- and TGF-ß-dependent manner. Our studies provide evidence that joint Tgfbr2-expressing cells have anatomical, ontogenic, slow-cycling trait and in-vivo and ex-vivo expression profiles of progenitor joint cells.

[Functional metabolic characteristics of sodium hyaluronate in mammal organism].

In the review data concerning functional metabolic characteristics of sodium hyaluronate in mammal organism, as well as autologous results concerning the study of its intraarticular action on metabolism of conjunctive tissue in the conditions of experimental arthritis in rabbits are given.

Ultrasound imaging for the rheumatologist XXXVI. Sonographic assessment of the foot in gout patients.

OBJECTIVES: This study aims to investigate the relationship between clinical and US findings together with the prevalence and distribution of US findings indicative of monosodium urate (MSU) crystal deposition within the foot in patients with gout. METHODS: A total of 50 patients with gout attending the in-patient and the out-patient clinics of the Rheumatology Departments were prospectively enrolled in this multi-centre study. Multiplanar examination of the following 15 joints was performed: talo-navicular, navicular-cuneiform (medial, intermediate and lateral), calcaneo-cuboid, medial, intermediate and lateral cuneiform-metatarsal, cuboid-4th metatarsal, cuboid-5th metatarsal and all five metatarsophalangeal (MTP) joints. RESULTS: The following US findings were indicative of gout: enhancement of the superficial margin of the hyaline cartilage, intra-articular tophus, and extraarticular tophus. In 46 patients, a total of 1380 foot joints were investigated. In 1309 joints that were not clinically involved, US detected signs indicative of joint inflammation in 9% (121/1309). Talo-navicular joint and the first MTP joint were the joints in which the highest number of US findings were found at mid-foot and fore-foot, respectively. At MTP joint level, dorsal scans allowed the detection of a higher number of US findings indicative of joint inflammation, and MSU crystal deposits rather than on the volar plane. CONCLUSIONS: This study demonstrated that US detected a higher number of inflamed foot joints than clinical examination, and that the first MTP and the talo-navicular joints were the anatomic sites with the highest prevalence of US signs of MSU crystal aggregates.

Chemical changes demonstrated in cartilage by synchrotron infrared microspectroscopy in an antibody-induced murine model of rheumatoid arthritis.

Collagen antibody-induced arthritis develops in mice following passive transfer of monoclonal antibodies (mAbs) to type II collagen (CII) and is attributed to effects of proinflammatory immune complexes, but transferred mAbs may react directly and damagingly with CII. To determine whether such mAbs cause cartilage damage in vivo in the absence of inflammation, mice lacking complement factor 5 that do not develop joint inflammation were injected intravenously with two arthritogenic mAbs to CII, M2139 and CIIC1. Paws were collected at day 3, decalcified, paraffin embedded, and 5-µm sections were examined using standard histology and synchrotron Fourier-transform infrared microspectroscopy (FTIRM). None of the mice injected with mAb showed visual or histological evidence of inflammation but there were histological changes in the articular cartilage including loss of proteoglycan and altered chondrocyte morphology. Findings using FTIRM at high lateral resolution revealed loss of collagen and the appearance of a new peak at 1635 cm(-1) at the surface of the cartilage interpreted as cellular activation. Thus, we demonstrate the utility of synchrotron FTIRM for examining chemical changes in diseased cartilage at the microscopic level and establish that arthritogenic mAbs to CII do cause cartilage damage in vivo in the absence of inflammation.

Genetic sphingosine kinase 1 deficiency significantly decreases synovial inflammation and joint erosions in murine TNF-alpha-induced arthritis.

Sphingosine kinase 1 (SphK1) is an enzyme that converts sphingosine to bioactive sphingosine-1-phosphate. Recent in vitro data suggest a potential role of SphK1 in TNF-alpha-mediated inflammation. Our aims in this study were to determine the in vivo significance of SphK1 in TNF-alpha-mediated chronic inflammation and to define which pathogenic mechanisms induced by TNF-alpha are SphK1 dependent. To pursue these aims, we studied the effect of SphK1 deficiency in an in vivo model of TNF-alpha-induced chronic inflammatory arthritis. Transgenic hTNF-alpha mice, which develop spontaneous inflammatory erosive arthritis beginning at 14-16 wk, were crossed with SphK1 null mice (SphK1(-/-)), on the C57BL6 genetic background. Beginning at 4 mo of age, hTNF/SphK1(-/-) mice had significantly less severe clinically evident paw swelling and deformity, less synovial and periarticular inflammation, and markedly decreased bone erosions as measured quantitatively through micro-CT images. Mechanistically, the mice lacking SphK1 had less articular cyclooxygenase 2 protein and fewer synovial Th17 cells than did hTNF/SphK1(+/+) littermates. Microarray analysis and real-time RT-PCR of the ankle synovial tissue demonstrated that hTNF/SphK1(-/-) mice had increased transcript levels of suppressor of cytokine signaling 3 compared with hTNF/SphK1(+/+) mice, likely also contributing to the decreased inflammation in the SphK1-deficient mice. Finally, significantly fewer mature osteoclasts were detected in the ankle joints of hTNF/SphK1(-/-) mice compared with hTNF/SphK1(+/+) mice. These data indicate that SphK1 plays a key role in hTNF-alpha-induced inflammatory arthritis via impacting synovial inflammation and osteoclast number.