Due to their immunosuppressive properties and low immunogenicity, MSC have been proposed as thera- peutic agents for autoimmune diseases such as multiple sclerosis, autoimmune diabetes, systemic lupus erythe- matosus, and rheumatoid arthritis (RA) [11–13]. Re- cently, several studies have investigated the therapeutic role of MSC in the treatment of collageninduced- arthritis (CIA), a well-characterized model of chronic arthritis [14, 15]. One recent study has demonstrated that adult bone marrow allogeneic MSC migrate to the affected paws and reduce the severity of arthritis in CIA . It has also been shown that human umbilical cord- derived and adipose-tissue-derived MSC reduce the inci- dence and severity of arthritis in mice by inhibiting pro- inflammatory cytokines and inducing activation of regulatory T cells (Treg) [17–19]. Adult MSC are finite and their potential clinical use requires long-term ex- pansion protocols, which might be associated with in vitro acquired genomic instability and reactivation of im- munogenicity . Recently, through inhibition of Smad-2/3 signaling, MSC were reproducibly derived from human pluripotent stem cells (hPSC) spanning both embryonic stem cells (hESC) and induced pluripo- tent stem cells (iPSC) [6, 21]. Human PSC represent an infinite source for MSC, and hPSC-MSC display an identical phenotype, functional properties, and in vitro and in vivo immunosuppressive and anti-inflammatory features compared to adult bone-marrow-derived or cord-blood-derived MSC, as demonstrated in experi- mental models of acute inflammation in vivo when ad- ministered prophylactically . These findings prompted us to investigate the potential therapeutic use of hESC- MSC in the setting of the chronic inflammatory model
cells . To circumvent the side effects of c-Kit muta- tions, mice have been generated with a normal Kit sig- naling pathway, in which mast cell deficiency is more selective. Induction of arthritis by K/BxN serum transfer in, for example, Cpa3-cre mice induced clinical arthritis, which was comparable to mast cell-competent mice . Similarly, pharmacological stabilization of mast cells in the clinical phase of CIA with the drug nedocro- mil was unable to reduce the clinical score of DBA/1 mice compared to placebo-treated mice . In contrast, it was shown that the mast cell inducible knock-out Mcpt5-cre iDTr mouse developed reduced levels of collagen-inducedarthritis, when mast cells were de- pleted before induction of arthritis (i.e., before the first immunization with CII) . This depletion reduced both the number of immune cells in the draining lymph nodes and the amount of secreted inflammatory cyto- kines in response to collagen II. DT treatment of these Mcpt5-Cre iDTR mice results, however, in a reduction in the number of connective tissue-type mast cells (CTMC) only, not that of mucosal mast cells (MMC) . Interestingly, it is reported that mast cells in the in- flamed synovium express less Mcpt5 compared to
Abstract: Patients with rheumatoid arthritis (RA) suffer from joint pain, decreased physical capacity, muscle wasting, and fatigue. Physical exercise can improve these features, but the most appropriate training program is unclear. The objective of our study is to evaluate physical endurance and weight changes in collagen-inducedarthritis (CIA) mice subjected to an individualized moderate aerobic exercise protocol on an incline treadmill. Male DBA1/J mice were randomly divided into 3 groups: nonarthritic control with exercise (CO-EXE, n=4), CIA with exercise (CIA-EXE, n=5), and CIA without exercise (CIA, n=4). Endurance exercise performance testing was performed in all groups prior to booster injections every 15 days after protocol initiation. CO-EXE and CIA-EXE were made to train on an incline treadmill (θ=5°), 60 minutes/day, 5 days/week for 6 weeks, at 60% of their own maximum velocity that induced exhaustion. The variables of interest were disease score, change in body weight (g), and maximum velocity (m/min) as a measure of exercise performance. Statistical significance was accepted at P<0.05. Clinical arthritis scores did not differ between CIA-EXE and CIA. Body weight was significantly different at week 6 when comparing CIA (0.9±0.7 g) vs. CO-EXE (3.9±0.4 g) and CIA-EXE (2.6±1.6 g). Maximum velocity was significantly different at 4 weeks (CIA: 21±3 m/min; CIA-EXE: 28±4 m/min; CO-EXE: 35±2 m/min) and 6 weeks (CIA: 21±5 m/min; CIA-EXE: 28±4 m/min; CO-EXE: 35±2 m/min), demonstrating that CO-EXE and CIA had the highest and the lowest maximum velocity, re- spectively. Individualized moderate aerobic exercise on an incline treadmill appears to be an interesting intervention to treat decreased physical endurance without altered disease score in RA. This intervention had a positive impact on exercise endurance in CIA animals, although more limited than in non-arthritic controls.
Fig. 2 Gingival-derived mesenchymal stem cells (GMSCs) attenuate the inflammatory responses in collagen-inducedarthritis (CIA) mice via a Fas/FasL pathway. a Schema showing GMSC infusion in CIA mice. b Arthritis severity scores were determined at various time points after immunization. c Arthritis severity score system using a 5-point scale: representative images of swollen hind paws ranging from normal (left) to grossly swollen (right). d, e Histological analysis of paw joints of PBS (Control), GMSC derived from WT mice (GMSC-WT), FasL –/– GMSC- (FasL –/– ), and FasL TF GMSC (FasL TF)-treated mice using hematoxylin and eosin staining (original magnification × 40) and score. Serum concentrations of f tumor necrosis factor α (TNF α ) and g anti-CII IgG, analyzed by ELISA; n = 6 in each group. The animal experiments were performed in three independent experiments. *P < 0.05, **P < 0.01. Values are given as mean ± SEM. CFA complete Freund ’ s adjuvant, CII collagen type II, N.S. not significant
To evaluate the antioxidant activity of the glycosaminoglycans hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S), we used a rat model of collagen-inducedarthritis (CIA). Arthritis was induced in Lewis rats by multiple intradermal injections of 250 µ l of emulsion containing bovine type II collagen in complete Freund’s adjuvant at the base of the tail and into three to five other sites on the back. Rats were challenged again with the same antigen preparation 7 days later. Disease developed about 11 days after the second immunization. The effects of treatment in the rats were monitored by biochemical parameters and by macroscopic and histological evaluations in blood, synovial tissue and articular cartilage. Arthritis produced the following symptoms: severe periarticular erythema, edema and inflammation in the hindpaws; membrane peroxidation in the cartilage of the joints; endogenous antioxidant wasting;
Introduction: Antibodies towards type II collagen (CII) are detected in patients with rheumatoid arthritis (RA) and in non-human primates and rodents with collageninducedarthritis (CIA). We have previously shown that antibodies specific for several CII-epitopes are pathogenic using monoclonal antibodies from arthritic mice, although the role of different anti-CII epitopes has not been investigated in detail in other species. We therefore performed an inter-species comparative study of the autoantibody response to CII in patients with RA versus monkeys and mice with CIA. Methods: Analysis of the full epitope repertoire along the disease course of CIA was performed using a library of CII triple-helical peptides. The antibody responses to the major CII epitopes were analyzed in sera and synovial fluid from RA patients, and in sera from rhesus monkeys (Macaca mulatta), common marmosets (Callithrix jacchus) and mice. Results: Many CII epitopes including the major C1, U1, and J1 were associated with established CIA and arginine residues played an important role in the anti-CII antibody interactions. The major epitopes were also recognized in RA patients, both in sera and even more pronounced in synovial fluid: 77% of the patients had antibodies to the U1 epitope. The anti-CII immune response was not restricted to the anti-citrulline protein antibodies (ACPA) positive RA group.
Aim of the Study: The primary aim of the study was to test the effect of 2,4,5-trimethoxy-1-pro- penylbenzene (alpha asarone), a hypocholes- terolaemic drug, on the progression of collagen- inducedarthritis (CIA) in mice. Olive oil, the ve- hicle of alpha-asarone, and dexamethasone were used as control treatments. Set-Up: Four groups of DBA/1 mice were immunised with chicken type II collagen (CII) via the intradermal route and either left untreated or were treated with alpha asarone, olive oil, or dexamethasone. A non-immunised group was an additional con- trol. Follow-Up: The thicknesses of the rear and front footpads were continuously monitored, and the levels of anti-collagen antibodies were measured at the end of the experiment. The animals were then sacrificed, and their rear and front limbs were removed and processed for histological examination. Results: Alpha asarone had no anti-inflammatory effect on CIA, and in one-third of the animals, it showed a pro-inflam- matory effect that was characterised by a mark- ed accumulation of neutrophils. Olive oil did not show any obvious anti-inflammatory effect on CIA, but it lowered the level of CII anti-bodies by 50%, suggesting a potential long-term anti-in- flammatory effect. As expected, dexamethasone had a clear anti-inflammatory effect on CIA. Con- clusion: Alpha asarone did not show any anti- inflammatory effect on CIA in the mice under the above conditions; however, the accumulation of neutrophils in the CIA lesions of mice treated with alpha asarone and the effect of olive oil in downregulating the levels of anti-CII antibodies
BMD volumes in CIA-induced control group were significantly lower, compared to test volumes, in the left tibial metaphysis, the distal part of the calcaneous, and the distal part of the metatarsal bone, but did not improve after administration of MTX or brazilin. The BMD decreases in control group may be attributable to increased bone surface erosion and decreased trabecular thickness. However, both bone volume and surface area were greater in the control group. These results agree with those of a study on BMD and micro-CT joint pro- jection after administration of bisphosphonate, minodro- nic acid . High-level bone turnover rate near joints was also observed in a model of carrageenan-inducedarthritis . Both suppression and reduction of BMD loss near joints are measures of anti-arthritic efficacy [47,48]. Suppression of bone surface erosion, and main- tenance of trabecular thickness and the trabecular bone pattern, upon brazilin administration, reduced the extent of damage to both bone and joints. Therefore, brazilin from C. sappan maintained BMD and bone microstruc- ture, without inhibiting mineralization.
Regular X-ray images of the hindlimbs of rats were ob- tained, and no obvious osteoporosis was observed in the joints in the control group; furthermore, the joint space and the bone were intact. Rats in the CIA group may have had osteoporosis, and the joint space was markedly narrowed; furthermore, joint destruction and fusion were observed to a minor extent. Joint damage in the BMSC intervention group was lesser than that in the CIA control group (Fig. 2c). Micro-CT examination in- dicated that the trabeculae in the blank control group were neatly arranged, with a dense structure and complete bone microstructure. In the CIA group, the proximal femoral cortex was thinner; the trabeculae were sparse, thin, disordered, and fewer; and the inter- trabecular distance was increased. Cortical thickness in the BMSC group and MTX group was the same as that in the blank control group, and the number of trabecu- lae decreased slightly but increased significantly com- pared with that in the model group (Fig. 2d). The analysis results of bone structural parameters showed that, compared with those in the normal group, BV/TV, Tb. N, and Tb. Th of the CIA group were significantly reduced, while Tb. Sp was increased. The values of BV/ TV, Tb. N, and Tb. Th were dramatically higher in both the MTX and MSC groups than in the CIA group. The Tb. Sp of both intervention groups was decreased, but only the MSC group showed a statistical significance. There was no statistical difference in the Tb. Sp value between the MSC and normal control groups (Fig. 2e).
In this study, we have analyzed the modulation of the mRNA levels of TF, TFPI, PAR1, EGR1, VEGF, UPA, and PAI1 in the inflamed joint in two murine models of arthritis, namely AIA and collagen-inducedarthritis (CIA). These two models recapitulate many features of RA, especially fibrin deposition in arthritic joints (for AIA, see ; for CIA, Marty et al, submitted). We have used a novel multiprobe RNase protection assay that was developed to facilitate the simultaneous analysis of all the genes studied from small amounts of tissue. Functional assays of procoagu- lant activity (PCA) and TF activity were also performed on these tissues. Evidence of ongoing coagulation was sought in arthritic mice by measuring the plasma concen- tration of TAT.
Male DBA/1 mice at 8–10 weeks of age were obtained from the Shanghai Laboratory Animal Center and were maintained in a specific pathogen-free animal facility at Soochow University. All animal experiments conducted in this study were approved by the University Committee on the Use of Live Animals in Teaching and Research at our institution. Male DBA/1 mice were immunized intra- dermally at the base of the tail with 200 μg of bovine collagen type II (CII; Chondrex, Redmond, WA, USA) dis- solved in 100 μl of 0.05 M acetic acid and mixed with an equal volume of complete Freund’ s adjuvant containing heat-killed Mycobacterium tuberculosis (H37Ra strain, 4 mg/ml; Chondrex). Three weeks later, animals were reimmunized with 200 μg of CII emulsified in incomplete Freund’s adjuvant (Chondrex). Mice were scored for clin- ical signs as follows (per paw): 0, paws with no swelling; 1, paws with swelling of finger joints or focal redness; 2, paws with mild swelling of the wrist or ankle joints; 3, paws with severe swelling of the entire paw; and 4, paws with deformity or ankylosis. CIA mice were grouped into acute collagen-inducedarthritis (A-CIA) and chronic collagen-inducedarthritis (C-CIA) stages according to the criteria described by Thornton et al. . On day 35 after the first immunization, dexamethasone (Dex; Tianjin Pharmaceutical Jiaozuo Co., Tianjin, China) was intraperi- toneally injected for 7 days, including low dose (L-dose, 0.5 mg/kg/day), high dose (H-dose, 2 mg/kg/day), and a PBS control.
Our results show that FSTL1 activates the NF-κB path- way upon binding to its unidentified receptor in OA FLS. We provide evidence that the active transcriptional elem- ent p65 is recruited to the promoter region of TNF-α and IL-6, and as a result directly enhances the expression level of these cytokines. Since TNF-α and IL-1β also can be in- duced by activation of p38 and c-Jun N-terminal kinase (JNK) and their downstream transcriptional factor AP1 , it may be an explanation of the not fully abolished level of the cytokines after inhibition of NF-κB signaling by BAY 11–7082. Furthermore, it was reported that FSTL1 contributes to arthritis as a proinflammatory medi- ator by enhancing the IFN-γ signaling pathway in a collagen-inducedarthritis (CIA) mouse model . Overall, FSTL1 can activate multi-proinflammatory pathways to participate in OA pathophysiology.
The therapeutic activity of CpdA was extensively tested in comparison with Gcs by us and other research groups in various Th1/Th17 and Th2 in vivo models of inflammation (Table 1). In the Th1/Th17 models, which included zymozan paw inflammation, collagen-inducedarthritis (CIA), experimental autoimmune neuritis (EAN) and encephalomyelitis (EAE), type 1 diabetes and acute colitis, CpdA inhibited the development of clinical symptoms and morphological manifestations of disease, such as paw swelling, inflammation in joints, colon, pancreas, central nerve system and peripheral nerves [37, 38, 62–66]. CpdA also reduced neuronal damage and demyelination, as well as progression of neuropathic pain . Furthermore, in the predominantly Th2-driven mouse asthma model, CpdA reduced inflammation and airway hyper-responsiveness . In all the aforementioned models, CpdA decreased both the severity of localized clinical symptoms and the systemic signs of inflammation. However, CpdA doses in most studies were ~10-fold higher compared to Gcs, possibly due to a lower GR affinity and the non-steroidal nature of the compound [26, 33, 37].
C-X-C motif chemokine 10 (CXCL10), also known as interferon-gamma-inducible protein 10 (IP-10), was ini- tially identified as a chemokine secreted by several cell types including macrophages, endothelial cells, and fi- broblasts in response to interferon gamma (IFN-γ) . CXCL10 binds to its receptor CXCR3 and regulates im- mune responses through recruitment of leukocytes, including T cells and monocytes/macrophages . In addition to its binding to CXCR3, CXCL10 has been shown to bind with and activate toll-like receptor 4 (TLR4) . The crucial role of CXCL10 has been well characterized in chronic Th1 inflammatory diseases. In inflamed tissues, CXCL10 recruits Th1 cells via CXCR3 where IFN-γ is secreted, which in turn stimulates CXCL10 expression in various cell types, resulting in positive feedback to amplify CXCL10 and Th1 responses . It has been shown that CXCL10 levels in synovial fluid and serum are elevated in human RA patients and in mice with collagen-inducedarthritis (CIA) [6, 15, 16]. Blockade of the CXCL10–CXCR3 axis has been shown to inhibit the infiltration of inflammatory cells, including T cells and macrophages, into inflamed joints and to decrease the severity of arthritis and bone and cartil- age destruction in animal models of RA [6, 17]. In addition to its chemotactic effect, CXCL10 has been shown to increase RANKL expression in CD4 + T cells [6, 18]. However, the exact mechanisms by which CXCL10 regulates inflammatory cell infiltration and osteoclastogenic cytokine production in RA are not fully understood.
Figure 5. MPs and Exos suppress inflammation in the DTH and CIA models. (A) Inhibition of inflammation in the Delayed-Type Hypersensitivity (DTH) model with increasing doses of MPs or Exos from IFN-γ primed MSCs, as measured by swelling of hind paws at day 6 (n=7 biological replicates). (B) Incidence of mice with inflammation in the collagen-inducedarthritis (CIA) model until day 37 at euthanasia (n=15 biological replicates). (C) Inhibition of inflammation as measured by clinical score in the same mice as in (B). (D) Representative 3D reconstruction images of hind paws by µCT analysis showing bone degradation in feet (top) or ankles (bottom) from a Ctrl mouse (left), MPs-treated mouse (middle) and Exos-treated mouse (right). (E) Mean thickness of cuneiform bone from Ctrl mice or mice treated with MPs or Exos as evaluated by histomorphometric µCT analysis. (F) Mean bone degradation of cuneiform bone as evaluated by area/volume parameter by µCT. Statistical analysis used a nonparametric Kruskal-Wallis test followed by Dunn’s multiple comparison test (A, E, F) or a two-way ANOVA followed by Tukey’s multiple comparison test (C). *: p<0.05 or **: p<0.01 compared to Ctrl.
patient . Th ese data, together with the biological similari ties between LTα and TNFα, suggest that resis- tance to TNFα blockage may occur when TNFα is not the dominant inﬂ ammatory cytokine and that LTα may play a role in the disease. An important advancement in the understanding of the participation of LTα in RA came from a study using the collagen-inducedarthritis (CIA) mouse model, the most commonly used animal model for arthritis . In this model, the blocking of LTα with a monoclonal antibody signiﬁ cantly improved the disease . Th e main mechanism responsible for this improve- ment in the CIA model appears not to be the blocking of soluble LTα but the depletion of LTα expressing Th 1 and Figure 1. Proposed model for the action of lymphotoxin alpha (LTα) in rheumatoid arthritis (RA) fi broblast-like synoviocytes (FLSs). RA FLSs express all LTα receptors (TNFR1, TNFR2, and HVEM). TNFR1 contains a cytoplasmic death domain (DD). Although the specifi c contribution of each receptor for LTα signaling remains to be clarifi ed, RA FLSs are activated upon LTα binding through the phosphorylation of the mitogen- activated protein kinases p38 and ERK1/2 and of the phosphatidylinositol 3-kinase (PI3K) Akt. Transcription factors such as nuclear factor-kappa-B (NF-κB), in turn, are activated. These events lead to cell responses involved in the pathogenesis of RA, such as proliferation, survival, and secretion of proinfl ammatory cytokines, chemokines, and matrix metalloproteinases (MMPs). Based on . ERK, extracellular signal-regulated kinase; HVEM, herpesvirus entry mediator; IL, interleukin; JNK, c-jun N-terminal kinase; RANTES, regulated upon activation, normal T cell expressed and secreted; TNFR, tumor necrosis factor receptor.
The involvement of prostaglandin pathways in the pathogenesis of arthritis has been shown in animal mod- els by using mice lacking genes, such as cycolooxygen- ase-2 (COX-2), prostaglandin E synthase, or prostacyclin receptor [13-15]. As COX-2 knockout mice normally develop autoreactive T cells in collagen-inducedarthritis (CIA) , prostaglandin pathways appear to be involved mainly in the effector phase of arthritis. How- ever, treatment with celecoxib, a prototype drug belong- ing to a new generation of highly specific COX-2 inhibitors has been reported to have only mild suppres- sive effects on animal models of arthritis, and strong inhibition of arthritis was achieved only when mice were treated in the combination of celecoxib with leukotriene inhibitors [16-19]. In humans, although celecoxib is widely used as an analgesic agent in patients with RA or osteoarthritis, there is no evidence that celecoxib ther- apy modulates the clinical course of RA. In addition, recently it has been shown that celecoxib enhances TNFa production by RA synovial membrane cultures and human monocytes .