Hyerin Jung

Temporal differential effects of proinflammatory cytokines on osteoclastogenesis

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.

Generation of disease-specific induced pluripotent stem cells from patients with rheumatoid arthritis and osteoarthritis

IntroductionSince 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.MethodsA 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.ResultsAfter 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.ConclusionFLSs 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.

A Dual Target-directed Agent against Interleukin-6 Receptor and Tumor Necrosis Factor α ameliorates experimental arthritis.

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.

A New Strategy to Deliver Synthetic Protein Drugs: Self-reproducible Biologics Using Minicircles

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.

Etanercept-Synthesising Mesenchymal Stem Cells Efficiently Ameliorate Collagen-Induced Arthritis

Mesenchymal stem cells (MSCs) have multiple properties including anti-inflammatory and immunomodulatory effects in various disease models and clinical treatments. These beneficial effects, however, are sometimes inconsistent and unpredictable. For wider and proper application, scientists sought to improve MSC functions by engineering. We aimed to invent a novel method to produce synthetic biological drugs from engineered MSCs. We investigated the anti-arthritic effect of engineered MSCs in a collagen-induced arthritis (CIA) model. Biologics such as etanercept are the most successful drugs used in anti-cytokine therapy. Biologics are made of protein components, and thus can be theoretically produced from cells including MSCs. MSCs were transfected with recombinant minicircles encoding etanercept (trade name, Enbrel), which is a tumour necrosis factor α blocker currently used to treat rheumatoid arthritis. We confirmed minicircle expression in MSCs in vitro based on GFP. Etanercept production was verified from the conditioned media. We confirmed that self-reproduced etanercept was biologically active in vitro. Arthritis subsided more efficiently in CIA mice injected with mcTNFR2MSCs than in those injected with conventional MSCs or etanercept only. Although this novel strategy is in a very early conceptual stage, it seems to represent a potential alternative method for the delivery of biologics and engineering MSCs.

Self in vivo production of a synthetic biological drug CTLA4Ig using a minicircle vector

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.

Induced production of anti-etanercept antibody in collagen-induced arthritis

Etanercept is a widespread biological drug for the treatment of rheumatoid arthritis, which inhibits tumor necrosis factor-α (TNF-α). Recently, the presence of antibodies targeting TNF-α inhibitors such as infliximab and adalimumab, was reported. However, few reports have studied etanercept in a mouse model of arthritis. We investigated the induction of anti-etanercept antibody production, along with the antibodys potential interfering effects on the biological function of etanercept, in mice with collagen-induced arthritis (CIA). CIA mice received an intraperitoneal injection of etanercept (25, 100 or 400 µg per mouse). The degree of inflammation and cartilage erosion was evaluated, and the number of osteoclasts in the ankle joints was assessed by TRAP staining. The level of pro-inflammatory cytokines in the serum was measured. To analyze the anti-osteoporotic effect of etanercept, microfocal computed tomography analyses of femurs and tibias were performed. Etanercept treatment decreased both the incidence and severity of arthritis in a dose-dependent manner, except for the highest dose of 400 µg. The mice that were treated with 25 and 100 µg etanercept showed an improvement in inflammation, cartilage damage, and even bone loss. However, mice treated with 400 µg etanercept showed no significant improvement in any of the tested parameters. Using a customized enzyme-linked immunosorbent assay (ELISA), the presence of the anti-etanercept antibody was detected in the serum in this treatment-refractory group. The therapeutic effect of etanercept was reduced in the CIA mice that developed the anti-etanercept antibody. In conclusion, the production of an anti-etanercept antibody can be induced in CIA mice, and this antibody can considerably reduce the anti-arthritic and anti-osteoporotic effects of etanercept.

Fulminant amoebic colitis mimicking intestinal vasculitis in a patient with systemic lupus erythematosus

Colitis in patients with systemic lupus erythematosus (SLE) is quite rare. It can be caused by intestinal vasculitis, mesenteric vascular thrombosis, concomitant inflammatory bowel disease or infectious colitis. It is important to make an accurate and early diagnosis as the treatments for each condition differ and a delayed diagnosis can result in life-threatening complications. However, non-specific gastrointestinal symptoms make a timely diagnosis challenging. Amoebic colitis is a rare condition in patients with SLE. Here we present a case of fulminant amoebic colitis in a patient with SLE which was initially misdiagnosed as ischemic colitis due to intestinal vasculitis. Her colitis was complicated with multiple intestinal perforations, disseminated intravascular coagulation and acute respiratory distress syndrome; but in the end, the patient was successfully treated with metronidazole and paromomycin.

Eupatilin Ameliorates Collagen Induced Arthritis

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. Graphical Abstract

Mesenchymal stem cells ameliorate experimental arthritis via expression of interleukin-1 receptor antagonist

Human bone marrow-derived mesenchymal stem cells (MSCs) have been observed to inhibit arthritis in experimental animal models such as collagen-induced arthritis. However, the exact anti-inflammatory mechanisms remain poorly understood. Interleukin-1 receptor antagonist (IL-1Ra) is an anti-inflammatory cytokine produced by immune and stromal cells. We postulated that MSCs could produce IL-1Ra and attenuate experimental arthritis. In this study, 5x106 MSCs were injected into the peritoneal cavity of IL-1Ra knockout (IL-1RaKO) mice. MSCs reduced the severity of the arthritis by histology and decreased pro-inflammatory cytokine levels in IL-1RaKO mice. The ratio of splenic T helper 17 (Th17) cells to regulatory T cells (Treg) was significantly decreased in MSC-injected IL-1RaKO mice. Purified splenic CD4+ T cells from mice in each of the treatment groups were cultured under Th17 polarizing conditions and analyzed by flow cytometry. Less expansion of the Th17 population was observed in the MSC-treated group. Interestingly, MSCs expressed inducible IL-1Ra against inflammatory environmental stimuli. Human recombinant IL-1Ra could suppress Th17 cells differentiation under Th17 polarizing conditions. These results indicate that IL-1Ra expressed by MSCs can inhibit Th17 polarization and decrease the immune response in IL-1RaKO mice. Therefore, MSC-derived IL-1Ra may inhibit inflammation in IL-1RaKO mice via effects on Th17 differentiation.