Human adipose-derived mesenchymal stem cells attenuate collagen antibody-induced autoimmune arthritis by inducing expression of FCGIIB receptors.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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 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-1ß 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-1ß 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.

Stepwise combined cell transplantation using mesenchymal stem cells and induced pluripotent stem cell-derived motor neuron progenitor cells in spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is an intractable neurological disease in which functions cannot be permanently restored due to nerve damage. Stem cell therapy is a promising strategy for neuroregeneration after SCI. However, experimental evidence of its therapeutic effect in SCI is lacking. This study aimed to investigate the efficacy of transplanted cells using stepwise combined cell therapy with human mesenchymal stem cells (hMSC) and induced pluripotent stem cell (iPSC)-derived motor neuron progenitor cells (iMNP) in a rat model of SCI. METHODS: A contusive SCI model was developed in Sprague-Dawley rats using multicenter animal spinal cord injury study (MASCIS) impactor. Three protocols were designed and conducted as follows: (Subtopic 1) chronic SCI + iMNP, (Subtopic 2) acute SCI + multiple hMSC injections, and (Main topic) chronic SCI + stepwise combined cell therapy using multiple preemptive hMSC and iMNP. Neurite outgrowth was induced by coculturing hMSC and iPSC-derived motor neuron (iMN) on both two-dimensional (2D) and three-dimensional (3D) spheroid platforms during mature iMN differentiation in vitro. RESULTS: Stepwise combined cell therapy promoted mature motor neuron differentiation and axonal regeneration at the lesional site. In addition, stepwise combined cell therapy improved behavioral recovery and was more effective than single cell therapy alone. In vitro results showed that hMSC and iMN act synergistically and play a critical role in the induction of neurite outgrowth during iMN differentiation and maturation. CONCLUSIONS: Our findings show that stepwise combined cell therapy can induce alterations in the microenvironment for effective cell therapy in SCI. The in vitro results suggest that co-culturing hMSC and iMN can synergistically promote induction of MN neurite outgrowth.

Guidelines for Manufacturing and Application of Organoids: Skin.

To address the limitations of animal testing, scientific research is increasingly focused on developing alternative testing methods. These alternative tests utilize cells or tissues derived from animals or humans for in vitro testing, as well as artificial tissues and organoids. In western countries, animal testing for cosmetics has been banned, leading to the adoption of artificial skin for toxicity evaluation, such as skin corrosion and irritation assessments. Standard guidelines for skin organoid technology becomes necessary to ensure consistent data and evaluation in replacing animal testing with in vitro methods. These guidelines encompass aspects such as cell sourcing, culture techniques, quality requirements and assessment, storage and preservation, and organoid-based assays.

Combination of induced pluripotent stem cell-derived motor neuron progenitor cells with irradiated brain-derived neurotrophic factor over-expressing engineered mesenchymal stem cells enhanced restoration of axonal regeneration in a chronic spinal cord injury rat model.

BACKGROUND: Spinal cord injury (SCI) is a disease that causes permanent impairment of motor, sensory, and autonomic nervous system functions. Stem cell transplantation for neuron regeneration is a promising strategic treatment for SCI. However, selecting stem cell sources and cell transplantation based on experimental evidence is required. Therefore, this study aimed to investigate the efficacy of combination cell transplantation using the brain-derived neurotrophic factor (BDNF) over-expressing engineered mesenchymal stem cell (BDNF-eMSC) and induced pluripotent stem cell-derived motor neuron progenitor cell (iMNP) in a chronic SCI rat model. METHOD: A contusive chronic SCI was induced in Sprague-Dawley rats. At 6 weeks post-injury, BDNF-eMSC and iMNP were transplanted into the lesion site via the intralesional route. At 12 weeks post-injury, differentiation and growth factors were evaluated through immunofluorescence staining and western blot analysis. Motor neuron differentiation and neurite outgrowth were evaluated by co-culturing BDNF-eMSC and iMNP in vitro in 2-dimensional and 3-dimensional. RESULTS: Combination cell transplantation in the chronic SCI model improved behavioral recovery more than single-cell transplantation. Additionally, combination cell transplantation enhanced mature motor neuron differentiation and axonal regeneration at the injured spinal cord. Both BDNF-eMSC and iMNP played a critical role in neurite outgrowth and motor neuron maturation via BDNF expression. CONCLUSIONS: Our results suggest that the combined transplantation of BDNF- eMSC and iMNP in chronic SCI results in a significant clinical recovery. The transplanted iMNP cells predominantly differentiated into mature motor neurons. Additionally, BDNF-eMSC exerts a paracrine effect on neuron regeneration through BDNF expression in the injured spinal cord.

The effects of antihypertensive drugs on bone mineral density in ovariectomized mice.

The effects of several antihypertensive drugs on bone mineral density (BMD) and micro-architectural changes in ovariectomized (OVX) mice were investigated. Eight-week-old female C57/BL6 mice were used for this study. Three days after ovariectomy, mice were treated intraperitoneally with nifedipine (15 mg/kg), telmisartan (5 mg/kg), enalapril (20 mg/kg), propranolol (1 mg/kg) or hydrochlorothiazide (12.5 mg/kg) for 35 consecutive days. Uterine atrophy of all mice was confirmed to evaluate estrogen deficiency state. BMD and micro-architectural analyses were performed on tibial proximal ends by micro-computed tomography (micro-CT). When OVX mice with uterine atrophy were compared with mice without atrophy, BMD decreased (P < 0.001). There were significant differences in BMD loss between different antihypertensive drugs (P = 0.005). Enalapril and propranolol increased BMD loss in mice with atrophied uteri compared with control mice. By contrast, thiazide increased BMD in mice with uterine atrophy compared with vehicle-treated mice (P = 0.048). Thiazide (P = 0.032) and telmisartan (P = 0.051) reduced bone loss and bone fraction in mice with uterine atrophy compared with the control. Thiazide affects BMD in OVX mice positively. The reduction in bone loss by thiazide and telmisartan suggest that these drugs may benefit menopausal women with hypertension and osteoporosis.

Intranasal administration of induced pluripotent stem cell-derived cortical neural stem cell-secretome as a treatment option for Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly, resulting in gradual destruction of cognitive abilities. Research on the development of various AD treatments is underway; however, no definitive treatment has been developed yet. Herein, we present induced pluripotent stem cell (iPSC)-derived cortical neural stem cell secretome (CNSC-SE) as a new treatment candidate for AD and explore its efficacy. METHODS: We first assessed the effects of CNSC-SE treatment on neural maturation and electromagnetic signal during cortical nerve cell differentiation. Then to confirm the efficacy in vivo, CNSC-SE was administered to the 5×FAD mouse model through the nasal cavity (5 µg/g, once a week, 4 weeks). The cell-mediated effects on nerve recovery, amyloid beta (Aß) plaque aggregation, microglial and astrocyte detection in the brain, and neuroinflammatory responses were investigated. Metabolomics analysis of iPSC-derived CNSC-SE revealed that it contained components that could exert neuro-protective effects or amplify cognitive restorative effects. RESULTS: Human iPSC-derived CNSC-SE increased neuronal proliferation and dendritic structure formation in vitro. Furthermore, CNSC-SE-treated iPSC-derived cortical neurons acquired electrical network activity and action potential bursts. The 5×FAD mice treated with CNSC-SE showed memory restoration and reduced Aß plaque accumulation. CONCLUSIONS: Our findings suggest that the iPSC-derived CNSC-SE may serve as a potential, non-invasive therapeutic option for AD in reducing amyloid infiltration and restoring memory.

Mitigating Effect of Estrogen in Alzheimer's Disease-Mimicking Cerebral Organoid.

Alzheimer's disease (AD) is the most common condition in patients with dementia and affects a large population worldwide. The incidence of AD is expected to increase in future owing to the rapid expansion of the aged population globally. Researchers have shown that women are twice more likely to be affected by AD than men. This phenomenon has been attributed to the postmenopausal state, during which the level of estrogen declines significantly. Estrogen is known to alleviate neurotoxicity in the brain and protect neurons. While the effects of estrogen have been investigated in AD models, to our knowledge, they have not been investigated in a stem cell-based three-dimensional in vitro system. Here, we designed a new model for AD using induced pluripotent stem cells (iPSCs) in a three-dimensional, in vitro culture system. We used 5xFAD mice to confirm the potential of estrogen in alleviating the effects of AD pathogenesis. Next, we confirmed a similar trend in an AD model developed using iPSC-derived cerebral organoids, in which the key characteristics of AD were recapitulated. The findings emphasized the potential of estrogen as a treatment agent for AD and also showed the suitability of AD-recapitulating cerebral organoids as a reliable platform for disease modeling and drug screening.

Metabolomic profiles of induced pluripotent stem cells derived from patients with rheumatoid arthritis and osteoarthritis.

BACKGROUND: Metabolomics is the systemic study of the unique fingerprints of metabolites involved in cellular processes and biochemical reactions. The metabolomic approach is useful in diagnosing and predicting the development of rheumatoid arthritis (RA) and osteoarthritis (OA) and is emerging as a useful tool for identifying disease biomarkers. The aim of this study was to compare the metabolic blueprint of fibroblast-like synoviocyte (FLS) cells and induced pluripotent stem cells (iPSCs) derived from RA and OA patients. METHODS: Somatic cells of RA patients (n = 3) and OA patients (n = 3) were isolated, transduced with a lentiviral plasmid, and reprogrammed into iPSCs displaying pluripotency. Metabolic profiling of RA and OA patient-derived FLS cells and iPSCs was performed using liquid chromatography/mass spectrometry and statistical analysis. After normalization by the sum of the peak intensities through LC/MS, 37 metabolites were detected across RA and OA patients. RESULTS: The metabolites of RA and OA were distinguishable according to the PLS-DA analysis. LysoPC (20:4), 4-methoxychalcone, phosphorylcholine, and nicotinamide (NAM) were significantly higher in RA iPSCs than in OA iPSCs (p < 0.05). The NMNAT-3 enzyme, which catalyzes an important step in the biosynthesis of NAD+ from adenosine triphosphate, was also upregulated in RA iPSCs. Interestingly, the proliferation of RA iPSCs was significantly greater than OA iPSC proliferation (p < 0.05). NAM played a critical role in the proliferation of RA iPSCs but not in OA iPSCs. When iPSCs were treated with 100 nM of the NAM inhibitor tannic acid (TA), the proliferation of RA iPSCs was significantly reduced (p < 0.001). CONCLUSIONS: The metabolites of RA and OA FLS cells and RA and OA iPSCs were all clearly distinguishable from each other. NAM played a critical role in the proliferation of RA iPSCs but not in OA iPSCs. TA effectively inhibited the expression of NAM in RA iPSCs and is a possible effective treatment for RA patients.

Sodium Chloride Aggravates Arthritis via Th17 Polarization.

PURPOSE: 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. MATERIALS AND METHODS: Collagen-induced arthritis (CIA) mice were fed a normal or high-salt diet ad libitum, 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. RESULTS: 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. CONCLUSION: 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.

Intraperitoneal infusion of mesenchymal stem cell attenuates severity of collagen antibody induced arthritis.

It is unclear how systemic administration of mesenchymal stem cells (MSCs) controls local inflammation. The aim of this study was to evaluate the therapeutic effects of human MSCs on inflammatory arthritis and to identify the underlying mechanisms. Mice with collagen antibody-induced arthritis (CAIA) received two intraperitoneal injections of human bone marrow-derived MSCs. The clinical and histological features of injected CAIA were then compared with those of non-injected mice. The effect of MSCs on induction of regulatory T cells was examined both in vitro and in vivo. We also examined multiple cytokines secreted by peritoneal mononuclear cells, along with migration of MSCs in the presence of stromal cell-derived factor-1 alpha (SDF-1α) and/or regulated on activation, normal T cell expressed and secreted (RANTES). Sections of CAIA mouse joints and spleen were stained for human anti-nuclear antibodies (ANAs) to confirm migration of injected human MSCs. The results showed that MSCs alleviated the clinical and histological signs of synovitis in CAIA mice. Peritoneal lavage cells from mice treated with MSCs expressed higher levels of SDF-1α and RANTES than those from mice not treated with MSCs. MSC migration was more prevalent in the presence of SDF-1α and/or RANTES. MSCs induced CD4+ T cells to differentiate into regulatory T cells in vitro, and expression of FOXP3 mRNA was upregulated in the forepaws of MSC-treated CAIA mice. Synovial and splenic tissues from CAIA mice receiving human MSCs were positive for human ANA, suggesting recruitment of MSCs. Taken together, these results suggest that MSCs migrate into inflamed tissues and directly induce the differentiation of CD4+ T cells into regulatory T cells, which then suppress inflammation. Thus, systemic administration of MSCs may be a therapeutic option for rheumatoid arthritis.

Recapitulation of methotrexate hepatotoxicity with induced pluripotent stem cell-derived hepatocytes from patients with rheumatoid arthritis.

BACKGROUND: Methotrexate (MTX) is widely used for the treatment of rheumatoid arthritis (RA). The drug is cost-effective, but sometimes causes hepatotoxicity, requiring a physician's attention. In this study, we simulated hepatotoxicity by treating hepatocytes derived from RA patient-derived induced pluripotent stem cells (RA-iPSCs) with MTX. METHODS: RA-iPSCs and healthy control iPSCs (HC-iPSCs) were established successfully. RA-iPSCs were differentiated into hepatocytes in two-dimensional (2D) monolayers and three-dimensional (3D) hepatocyte spheroid cultures; this process required growth factors such as BMP4, bFGF, HGF, and OSM. Immunofluorescence staining and flow cytometry were performed to confirm that the mature hepatocytes expressed cytokeratin 18, anti-alpha-1 antitrypsin, and albumin. MTX toxicity was evaluated via monitoring of cell viability, alanine aminotransferase, and mitochondrial status after MTX treatment in 2D and 3D cultures. RESULTS: RA-iPSCs generated from three RA patients suffering from MTX-induced hepatotoxicity differentiated into the endoderm lineage, hepatoblasts, and hepatocytes. In 2D culture, RA-iPSC-derived hepatocytes were more sensitive to MTX than healthy controls. A 3D culture system using hepatocyte spheroids also successfully recapitulated MTX-induced hepatotoxicity. The 3D culture system had several advantages, including longer culture periods under more complex conditions. CONCLUSIONS: A patient-derived iPSC platform could recapitulate MTX toxicity. Simulation of drug toxicity in vitro may help clinicians choose safer drugs or less toxic doses.

Interrupting oral infection of Porphyromonas gingivalis with anti-FimA antibody attenuates bacterial dissemination to the arthritic joint and improves experimental arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that typically results in strong inflammation and bone destruction in the joints. It is generally known that the pathogenesis of RA is linked to cardiovascular and periodontal diseases. Though rheumatoid arthritis and periodontitis share many pathologic features such as a perpetual inflammation and bone destruction, the precise mechanism underlying a link between these two diseases has not been fully elucidated. Collagen-induced arthritis (CIA) mice were orally infected with Porphyromonas gingivalis (Pg) or Pg preincubated with an anti-FimA antibody (FimA Ab) specific for fimbriae that are flexible appendages on the cell surface. Pg-infected CIA mice showed oral microbiota disruption and increased alveolar bone loss and had synovitis and joint bone destruction. However, preincubation with FimA Ab led to a significant reduction in the severity of both oral disease and arthritis. Moreover, FimA Ab attenuated bacterial attachment and aggregation on human gingival and rheumatoid arthritis synovial fibroblasts. In addition, we discovered bacteria may utilize dendritic cells, macrophages and neutrophils to migrate into the joints of CIA mice. These results suggest that disrupting Pg fimbriae function by FimA Ab ameliorates RA.

Author Correction: Interrupting oral infection of Porphyromonas gingivalis with anti-FimA antibody attenuates bacterial dissemination to the arthritic joint and improves experimental arthritis.

After online publication of this article, the authors noticed an error in the Figure section. The correct statement of this article should have read as below.

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.

Centrifugal gravity-induced BMP4 induces chondrogenic differentiation of adipose-derived stem cells via SOX9 upregulation.

BACKGROUND: Cartilage does not have the capability to regenerate itself. Therefore, stem cell transplantation is a promising therapeutic approach for impaired cartilage. For stem cell transplantation, in vitro enrichment is required; however, stem cells not only become senescent but also lose their differentiation potency during this process. In addition, cytokines are normally used for chondrogenic differentiation induction of stem cells, which is highly expensive and needs an additional step to culture. In this study, we introduced a novel method to induce chondrogenic differentiation of adipose-derived stem cells (ASCs), which are more readily available than bone marrow-derived mesenchymal stem cells(bMSCs), using centrifugal gravity (CG). METHODS: ASCs were stimulated by loading different degrees of CG (0, 300, 600, 1200, 2400, and 3600 g) to induce chondrogenic differentiation. The expression of chondrogenic differentiation-related genes was examined by RT-PCR, real-time PCR, and western blot analyses. The chondrogenic differentiation of ASCs stimulated with CG was evaluated by comparing the expression of positive markers [aggrecan (ACAN) and collagen type II alpha 1 (COL2A1)] and negative markers (COL1 and COL10) with that in ASCs stimulated with transforming growth factor (TGF)-ß1 using micromass culture, immunofluorescence, and staining (Alcian Blue and Safranin O). RESULTS: Expression of SOX9 and SOX5 was upregulated by CG (2400 g for 30 min). Increased expression of ACAN and COL2A1 (positive markers) was detected in monolayer-cultured ASCs after CG stimulation, whereas that of COL10 (a negative marker) was not. Expression of bone morphogenetic protein (BMP) 4, an upstream stimulator of SOX9, was upregulated by CG, which was inhibited by Dorsomorphin (an inhibitor of BMP4). Increased expression of proteoglycan, a major component of cartilage, was confirmed in the micromass culture of ASCs stimulated with CG by Alcian Blue and Safranin O staining. CONCLUSIONS: Chondrogenic differentiation of ASCs can be induced by optimized CG (2400 g for 30 min). Expression of SOX9 is upregulated by CG via increased expression of BMP4. CG has a similar ability to induce SOX9 expression as TGF-ß1.

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.

Phosphorylation status determines the opposing functions of Smad2/Smad3 as STAT3 cofactors in TH17 differentiation.

Transforming growth factor-ß (TGF-ß) and interleukin-6 (IL-6) are the pivotal cytokines to induce IL-17-producing CD4(+) T helper cells (TH17); yet their signalling network remains largely unknown. Here we show that the highly homologous TGF-ß receptor-regulated Smads (R-Smads): Smad2 and Smad3 oppositely modify STAT3-induced transcription of IL-17A and retinoic acid receptor-related orphan nuclear receptor, RORγt encoded by Rorc, by acting as a co-activator and co-repressor of STAT3, respectively. Smad2 linker phosphorylated by extracellular signal-regulated kinase (ERK) at the serine 255 residue interacts with STAT3 and p300 to transactivate, whereas carboxy-terminal unphosphorylated Smad3 interacts with STAT3 and protein inhibitor of activated STAT3 (PIAS3) to repress the Rorc and Il17a genes. Our work uncovers carboxy-terminal phosphorylation-independent noncanonical R-Smad-STAT3 signalling network in TH17 differentiation.

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 antibody's 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.

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.