Sodium chloride (NaCl) has been proposed as a driving factor in autoimmune diseases through the induction of pathogenic CD4
T helper cells that produce interleukin-17 (Th17 cells). This study ...investigated the effects of NaCl on inflammatory arthritis in mice and humans.
Collagen-induced arthritis (CIA) mice were fed a normal or high-salt diet
, and clinical and histologic features of arthritis were evaluated. The proportion of Th17 cells in the spleens of CIA mice fed a normal or high-salt diet was evaluated by flow cytometry, and the expression of IL-17 in joints and intestines was determined by immunohistochemical staining. We also analyzed the effect of NaCl on Th17 differentiation from peripheral blood monocytes of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and evaluated the contents of sodium and IL-17 in the synovial fluid of RA and OA patients.
NaCl increased murine and human Th17 cell differentiation in a dose-dependent manner. Clinical and histological arthritis was more severe in the high-salt-fed CIA mice, compared to control CIA mice. The proportion of Th17 cells among splenocytes was higher in CIA mice fed a high-salt diet. Expression of synovial and intestinal IL-17 was also higher in high-salt-fed CIA mice. Comparison of synovial fluid between RA patients and OA patients revealed that Na
and IL-17 were more abundant in RA synovial fluid.
This study suggests that NaCl can aggravate arthritis by affecting Th17 differentiation. Accordingly, limiting salt intake may be helpful for treating inflammatory arthritis, such as RA.
Biologics are the most successful drugs used in anticytokine therapy. However, they remain partially unsuccessful because of the elevated cost of their synthesis and purification. Development of ...novel biologics has also been hampered by the high cost. Biologics are made of protein components; thus, theoretically, they can be produced in vivo. Here we tried to invent a novel strategy to allow the production of synthetic drugs in vivo by the host itself. The recombinant minicircles encoding etanercept or tocilizumab, which are synthesized currently by pharmaceutical companies, were injected intravenously into animal models. Self-reproduced etanercept and tocilizumab were detected in the serum of mice. Moreover, arthritis subsided in mice that were injected with minicircle vectors carrying biologics. Self-reproducible biologics need neither factory facilities for drug production nor clinical processes, such as frequent drug injection. Although this novel strategy is in its very early conceptual stage, it seems to represent a potential alternative method for the delivery of biologics.
Since the concept of reprogramming mature somatic cells to generate induced pluripotent stem cells (iPSCs) was demonstrated in 2006, iPSCs have become a potential substitute for embryonic stem cells ...(ESCs) given their pluripotency and "stemness" characteristics, which resemble those of ESCs. We investigated to reprogram fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) to generate iPSCs using a 4-in-1 lentiviral vector system.
A 4-in-1 lentiviral vector containing Oct4, Sox2, Klf4, and c-Myc was transduced into RA and OA FLSs isolated from the synovia of two RA patients and two OA patients. Immunohistochemical staining and real-time PCR studies were performed to demonstrate the pluripotency of iPSCs. Chromosomal abnormalities were determined based on the karyotype. SCID-beige mice were injected with iPSCs and sacrificed to test for teratoma formation.
After 14 days of transduction using the 4-in-1 lentiviral vector, RA FLSs and OA FLSs were transformed into spherical shapes that resembled embryonic stem cell colonies. Colonies were picked and cultivated on matrigel plates to produce iPSC lines. Real-time PCR of RA and OA iPSCs detected positive markers of pluripotency. Immunohistochemical staining tests with Nanog, Oct4, Sox2, Tra-1-80, Tra-1-60, and SSEA-4 were also positive. Teratomas that comprised three compartments of ectoderm, mesoderm, and endoderm were formed at the injection sites of iPSCs. Established iPSCs were shown to be compatible by karyotyping. Finally, we confirmed that the patient-derived iPSCs were able to differentiate into osteoblast, which was shown by an osteoimage mineralization assay.
FLSs derived from RA and OA could be cell resources for iPSC reprogramming. Disease- and patient-specific iPSCs have the potential to be applied in clinical settings as source materials for molecular diagnosis and regenerative therapy.
Cardiovascular disease is a leading cause of morbidity in rheumatoid arthritis (RA) patients. This study aimed to generate and characterise cardiomyocytes from induced pluripotent stem cells (iPSCs) ...of RA patients. Fibroblast-like synoviocytes (FLSs) from patients with RA and osteoarthritis (OA) were successfully reprogrammed into RA-iPSCs and OA-iPSCs, respectively. The pluripotency of iPSCs was confirmed by quantitative reverse transcription-polymerase chain reaction and immunofluorescence staining. Established iPSCs were differentiated into cardiomyocytes using a small molecule-based monolayer differentiation protocol. Within 12 days of cardiac differentiation from patient-specific and control-iPSCs, spontaneously beating cardiomyocytes (iPSC-CMs) were observed. All iPSC-CMs exhibited a reliable sarcomeric structure stained with antibodies against cardiac markers and similar expression profiles of cardiac-specific genes. Intracellular calcium signalling was recorded to compare calcium-handling properties among cardiomyocytes differentiated from the three groups of iPSCs. RA-iPSC-CMs had a lower amplitude and a shorter duration of calcium transients than the control groups. Peak tangential stress and the maximum contractile rate were also decreased in RA-iPSC-CMs, suggesting that contractility was reduced. This study demonstrates the successful generation of functional cardiomyocytes from pathogenic synovial cells in RA patients through iPSC reprogramming. Research using RA-iPSC-CMs might provide an opportunity to investigate the pathophysiology of cardiac involvement in RA.
Cytotoxic T lymphocyte-associated antigen 4 immunoglobulin fusion protein (CTLA4Ig, abatacept) is a B7/CD28 costimulation inhibitor that can ward off the immune response by preventing the activation ...of naïve T cells. This therapeutic agent is administered to patients with autoimmune diseases such as rheumatoid arthritis. Its antiarthritic efficacy is satisfactory, but the limitations are the necessity for frequent injection and high cost. Minicircles can robustly express the target molecule and excrete it outside the cell as an indirect method to produce the protein of interest in vivo. We inserted the sequence of abatacept into the minicircle vector, and by successful in vivo injection the host was able to produce the synthetic protein drug. Intravenous infusion of the minicircle induced spontaneous production of CTLA4Ig in mice with collagen-induced arthritis. Self-produced CTLA4Ig significantly decreased the symptoms of arthritis. Injection of minicircle CTLA4Ig regulated Foxp3(+) T cells and Th17 cells. Parental and mock vectors did not ameliorate arthritis or modify the T cell population. We have developed a new concept of spontaneous protein drug delivery using a minicircle vector. Self in vivo production of a synthetic protein drug may be useful when biological drugs cannot be injected because of manufacturing or practical problems.
Adipose-derived stem cells (ASCs) are mesenchymal stem cells (MSCs) derived from adipose tissue. MSCs have multiple properties including anti-inflammatory and immunomodulatory effects in various ...disease models and human diseases. However, the mechanisms underlying this wide range of effects need to be explored.
Collagen antibody-induced arthritis (CAIA) is a unique model in which arthritis is rapidly and strongly induced. ASCs were intraperitoneally infused into CAIA mice before or after arthritis induction. The serum levels of various cytokines, adipokines, and chemokines were measured. The expression of FC gamma receptors (FCGRs) was investigated in peritoneal macrophages ex vivo. RAW264.7 cells and ASCs were co-cultured to elucidate the direct and indirect role of ASCs on FCGR expression.
ASCs attenuated arthritis in CAIA mice. Serum levels of tumor necrosis factor α, interleukin (IL)-15, resistin, and leptin were reduced in ASC-treated CAIA mice, whereas serum levels of IL-6 and adiponectin were not affected. In peritoneal macrophages isolated from ASC-treated mice, expression of FCGRIIB, which is immunoinhibitory, was higher than that of FCGRI. Co-culture of ASCs with RAW264.7 cells modulated the expression of FCGRs. The expression patterns and timings of peak expression differed among FCGRs. Expression of FCGRIIB was higher and peaked earlier than that of FCGRI. FCGRIII expression was not affected by this co-culture.
This is a study to show that ASCs have anti-arthritic effects in CAIA mice. Modulation of FCGRs by ASCs might be a therapeutic mechanism in this antibody-associated arthritis model.
Eupatilin is the main active component of DA-9601, an extract from Artemisia. Recently, eupatilin was reported to have anti-inflammatory properties. We investigated the anti-arthritic effect of ...eupatilin in a murine arthritis model and human rheumatoid synoviocytes. DA-9601 was injected into collagen-induced arthritis (CIA) mice. Arthritis score was regularly evaluated. Mouse monocytes were differentiated into osteoclasts when eupatilin was added simultaneously. Osteoclasts were stained with tartrate-resistant acid phosphatase and then manually counted. Rheumatoid synoviocytes were stimulated with TNF-α and then treated with eupatilin, and the levels of IL-6 and IL-1β mRNA expression in synoviocytes were measured by RT-PCR. Intraperitoneal injection of DA-9601 reduced arthritis scores in CIA mice. TNF-α treatment of synoviocytes increased the expression of IL-6 and IL-1β mRNAs, which was inhibited by eupatilin. Eupatilin decreased the number of osteoclasts in a concentration dependent manner. These findings, showing that eupatilin and DA-9601 inhibited the expression of inflammatory cytokines and the differentiation of osteoclasts, suggest that eupatilin and DA-9601 is a candidate anti-inflammatory agent.
A considerable proportion of patients with rheumatoid arthritis (RA) do not respond to monospecific agents. The purpose of our study was to generate a hybrid form of biologics, targeting ...tumor-necrosis factor alpha (TNFα) and interleukin-6 receptor (IL-6R), and determine its anti-arthritic properties in vitro and in vivo. A novel dual target-directed agent (DTA(A7/sTNFR2)) was generated by conjugating soluble TNF receptor 2 (sTNFR2) to the Fc region of A7, a new anti-IL-6R antibody obtained by screening the phage display human antibody library. DTA(A7/sTNFR2) inhibited the proliferation and migration of fibroblast-like synoviocytes from patients with RA (RA-FLS) more efficiently than single target-directed agents. DTA(A7/sTNFR2) also blocked osteoclastogenesis from bone marrow cells. The arthritis severity scores of the experimental arthritis mice with DTA(A7/sTNFR2) tended to be lower than those of mice with IgG, A7, or sTNFR2. Histological data suggested that DTA(A7/sTNFR2) is more efficient than single-target drugs in preventing joint destruction and bone loss. These results were confirmed in vivo using the minicircle system. Taken together, the results show that DTA(A7/sTNFR2) may be a promising therapeutic agent for the treatment of RA.
Bone destruction and inflammation are closely linked. Cytokines play an important role in inflammatory bone destruction by upregulating the receptor activator of nuclear factor-κB (NF-κB) ligand ...(RANKL). The direct role of cytokines that act in a non-RANKL-dependent manner has yet to be elucidated. The aim of this study was to investigate the direct osteoclastogenic properties of inflammatory cytokines at different time-points of osteoclastogenesis. Mouse bone marrow macrophages were stimulated with the macrophage colony-stimulating factor (M-CSF) and various concentrations of RANKL. Inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-17 and IL-23, were added to the culture system of osteoclastogenesis. Two time-points of cytokine treatment were set. The 'early' effect of each cytokine was investigated at the time of first RANKL treatment, whereas the 'late' effect was investigated 48 h after the first RANKL challenge. Osteoclast differentiation and function were assessed using an osteoclast marker tartrate-resistant acid phosphatase (TRAP) and by visualization of pit formation. A permissive level of RANKL was required for cytokine-associated osteoclastogenesis in all experiments. In the M-CSF/RANKL monocellular culture system, IL-1β enhanced and IL-6 decreased osteoclast formation in a dose-dependent manner, regardless of temporal differences. Other cytokines showed various responses according to the phase of osteoclast maturation and the concentration of each cytokine and RANKL. Furthermore, luciferase assays showed that both IL-1β and RANKL activated the NF-κB signaling pathway. Collectively, our data revealed that targeting IL-1β may be a promising strategy to inhibit inflammation-associated bone destruction and osteoporosis.
Two-dimensional (2D) materials and van der Waals heterostructures with atomic-scale thickness provide enormous potential for advanced science and technology. However, insufficient knowledge of ...compatible synthesis impedes wafer-scale production. PdSe2 and Pd2Se3 are two of the noble transition-metal chalcogenides with excellent physical properties that have recently emerged as promising materials for electronics, optoelectronics, catalyst, and sensors. This research presents a feasible approach to synthesize PdSe2 and Pd2Se3 with inherently asymmetric structure on honeycomb lattice 2D monolayer substrates of graphene and MoS2. We directly deposit a molecular transition-metal precursor complex on the surface of the 2D substrates, followed by low-temperature selenization by chemical vapor flow. Parameter control leads to tuning of the material from monolayer nanocrystals with Pd2Se3 phase, to continuous few-layer PdSe2 films. Annular dark-field scanning transmission electron microscopy (ADF-STEM) reveals the structure, phase variations, and heteroepitaxy at the atomic level. PdSe2 with unconventional interlayer stacking shifts appeared as the kinetic product, whereas the bilayer PdSe2 and monolayer Pd2Se3 are the thermodynamic product. The epitaxial alignment of interlayer rotation and translation between the PdSe2 and underlying 2D substrate was also revealed by ADF-STEM. These results offer both nanoscale and atomic-level insights into direct growth of van der Waals heterostructures, as well as an innovative method for 2D synthesis by predetermined nucleation.
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