Implication of the deacetylase sirtuin-1 on synovial angiogenesis and persistence of experimental arthritis.

OBJECTIVES: To decipher the phenotype of endothelial cells (ECs) derived from circulating progenitors issued from patients with rheumatoid arthritis (RA). METHODS: RA and control ECs were compared according to their proliferative capacities, apoptotic profile, response to tumour necrosis factor (TNF)-α stimulation and angiogenic properties. Microarray experiments were performed to identify gene candidates relevant to pathological angiogenesis. Identified candidates were detected by RT-PCR and western blot analysis in ECs and by immunohistochemistry in the synovium. Their functional relevance was then evaluated in vitro after gene invalidation by small interfering RNA and adenoviral gene overexpression, and in vivo in the mouse model of methyl-bovine serum albumin-(mBSA)-induced arthritis. RESULTS: RA ECs displayed higher proliferation rate, greater sensitisation to TNF-α and enhanced in vitro and in vivo angiogenic capacities. Microarray analyses identified the NAD-dependent protein deacetylase sirtuin-1 (SIRT1) as a relevant gene candidate. Decreased SIRT1 expression was detected in RA ECs and synovial vessels. Deficient endothelial SIRT1 expression promoted a proliferative, proapoptotic and activated state of ECs through the acetylation of p53 and p65, and lead the development of proangiogenic capacities through the upregulation of the matricellular protein cysteine-rich angiogenic protein-61. Conditional deletion of SIRT1 in ECs delayed the resolution of experimental methyl-bovine serum albumin-(mBSA)-induced arthritis. Conversely, SIRT1 activation reversed the pathological phenotype of RA ECs and alleviates signs of experimental mBSA-induced arthritis. CONCLUSIONS: These results support a role of SIRT1 in RA and may have therapeutic implications, since targeting angiogenesis, and especially SIRT1, might be used as a complementary therapeutic approach in RA.

In Vivo Expansion of Activated Foxp3+ Regulatory T Cells and Establishment of a Type 2 Immune Response upon IL-33 Treatment Protect against Experimental Arthritis.

IL-33 is strongly involved in several inflammatory and autoimmune disorders with both pro- and anti-inflammatory properties. However, its contribution to chronic autoimmune inflammation, such as rheumatoid arthritis, is ill defined and probably requires tight regulation. In this study, we aimed at deciphering the complex role of IL-33 in a model of rheumatoid arthritis, namely, collagen-induced arthritis (CIA). We report that repeated injections of IL-33 during induction (early) and during development (late) of CIA strongly suppressed clinical and histological signs of arthritis. In contrast, a late IL-33 injection had no effect. The cellular mechanism involved in protection was related to an enhanced type 2 immune response, including the expansion of eosinophils, Th2 cells, and type 2 innate lymphoid cells, associated with an increase in type 2 cytokine levels in the serum of IL-33-treated mice. Moreover, our work strongly highlights the interplay between IL-33 and regulatory T cells (Tregs), demonstrated by the dramatic in vivo increase in Treg frequencies after IL-33 treatment of CIA. More importantly, Tregs from IL-33-treated mice displayed enhanced capacities to suppress IFN-γ production by effector T cells, suggesting that IL-33 not only favors Treg proliferation but also enhances their immunosuppressive properties. In concordance with these observations, we found that IL-33 induced the emergence of a CD39(high) Treg population in a ST2L-dependent manner. Our findings reveal a powerful anti-inflammatory mechanism by which IL-33 administration inhibits arthritis development.

Interplay between TNF and regulatory T cells in a TNF-driven murine model of arthritis.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) are involved in several autoimmune diseases, including rheumatoid arthritis. TNF-α blockers induce therapeutic benefits in rheumatoid arthritis via a variety of mechanisms. We aimed to characterize the impact on Treg of TNF-α overexpression in vivo and of TNF-α inhibiting treatments. We used human TNF-α transgenic mice as a model of strictly TNF-α-dependent arthritis. Our study showed that initial Treg frequency was lower in TNF-α transgenic mice than in wild-type mice. However, the course of arthritis was marked by elevation of Treg frequency and a dramatic increase in expression of TNFR2. Antagonizing TNF-α with either the anti-human TNF-α Ab (infliximab) or active immunotherapy (TNF-kinoid) increased the Treg frequency and upregulated CTLA-4, leading to enhancement of suppressor activity. Moreover, both anti-TNF-α strategies promoted the differentiation of a CD62L(-) Treg population. In conclusion, in an in vivo model of TNF-α-driven arthritis, Treg frequency increased with inflammation but failed to control the inflammatory process. Both passive and active TNF-α-inhibiting strategies restored the suppressor activity of Treg and induced the differentiation of a CD62L(-) Treg population.

A pivotal role for antigen-presenting cells overexpressing SOCS3 in controlling invariant NKT cell responses during collagen-induced arthritis.

OBJECTIVE: Suppressor of cytokine signalling (SOCS) proteins constitute a class of intracellular proteins that are key physiological regulators of immune cell function. It has previously been shown that antigen-presenting cells (APCs) overexpressing SOCS3 steer T helper immune responses and protect against experimental arthritis. A study was undertaken to investigate the contribution of SOCS3 in regulating invariant natural killer T (iNKT) cell responses during collagen-induced arthritis (CIA). METHODS: DBA/1 mice were immunised with type II collagen and adenoviruses encoding SOCS3 were administered intravenously before the clinical onset of arthritis. Murine APCs overexpressing SOCS3 were co-cultured with an iNKT cell hybridoma and interleukin 2 (IL-2) release was measured by Luminex multi-analyte technology. The frequency and activation of primary iNKT cells was assessed by flow cytometry. Murine APCs were analysed for cytokine and CD1d expression following viral SOCS3 gene transfer. RESULTS: Viral overexpression of SOCS3 in APCs resulted in reduced activation of the iNKT cell hybridoma. Importantly, during initiation of CIA, adenovirus-mediated overexpression of SOCS3 in hepatic and splenic APCs inhibited iNKT cell expansion in both organs. The iNKT cell population from SOCS3-treated mice showed low expression of the early activation marker CD69 and primary liver iNKT cells produced less interferon γ and IL-4 upon α-galactosylceramide stimulation. No differences in CD1d surface expression were observed, but SOCS3-transduced APCs produced decreased levels of proinflammatory cytokines and increased levels of IL-10. CONCLUSION: These results demonstrate a critical role for SOCS3 in controlling the immunostimulatory capacities of APCs, which has direct implications for the effector function of iNKT cells during arthritis.

Macroautophagy in lymphatic endothelial cells inhibits T cell-mediated autoimmunity.

Lymphatic endothelial cells (LECs) present peripheral tissue antigens to induce T cell tolerance. In addition, LECs are the main source of sphingosine-1-phosphate (S1P), promoting naive T cell survival and effector T cell exit from lymph nodes (LNs). Autophagy is a physiological process essential for cellular homeostasis. We investigated whether autophagy in LECs modulates T cell activation in experimental arthritis. Whereas genetic abrogation of autophagy in LECs does not alter immune homeostasis, it induces alterations of the regulatory T cell (T reg cell) population in LNs from arthritic mice, which might be linked to MHCII-mediated antigen presentation by LECs. Furthermore, inflammation-induced autophagy in LECs promotes the degradation of Sphingosine kinase 1 (SphK1), resulting in decreased S1P production. Consequently, in arthritic mice lacking autophagy in LECs, pathogenic Th17 cell migration toward LEC-derived S1P gradients and egress from LNs are enhanced, as well as infiltration of inflamed joints, resulting in exacerbated arthritis. Our results highlight the autophagy pathway as an important regulator of LEC immunomodulatory functions in inflammatory conditions.

Early activation of invariant natural killer T cells in a rheumatoid arthritis model and application to disease treatment.

Invariant NKT (iNKT) cells are a distinctive subtype of CD1d-restricted T cells involved in regulating autoimmunity and capable of producing various T helper type 1 (Th1), Th2 and Th17 cytokines. Activation of iNKT cells by their exogenous ligand alpha-galactosylceramide (alpha-GalCer) exerts therapeutic effects in autoimmune diseases such as rheumatoid arthritis (RA). However, the pathophysiological role of iNKT cells in RA, in the absence of exogenous stimulation, is incompletely understood. We investigated the potential pathophysiological effects of iNKT cells in mice with collagen-induced arthritis (CIA), a model of RA. We found that iNKT cells underwent activation only in the early phases of the disease (6 days post-induction). In the liver, but not the spleen or lymph nodes, this early activation led to the release of interleukins -4, -17A and -10 and of interferon-gamma; and an increased CD69 expression. Importantly, clinical and histological signs of arthritis were improved by the functional blockade of iNKT cells by a monoclonal antibody to CD1d at the early phase of the disease. This improvement was associated on day 6 post-induction with decreased expression of co-stimulatory molecules (CD80, CD86, CD40) on splenic dendritic cells and macrophages, whereas regulatory T-cell suppressive effects and proportions were not modified. Taken in concert, these findings suggest that iNKT cells are activated early in the course of CIA and contribute to the pathogenesis of arthritis. Therefore, iNKT-cell activation may be a valid treatment target in RA.

Origins of rheumatoid arthritis.

While the exact cause of rheumatoid arthritis is unknown, several mechanisms have been described extensively. The genetic predisposition for this autoimmune disease is largely attributed to MHC class II genes, especially the main polymorphism in the HLA shared epitope. Non-genetic factors account for the rest. The best known are autoantigens to citrullinated or carbmylated proteins, although there are many others. They are recognized by an immune system with defective control mechanisms, in which regulator T-cells are unable to prevent inflammation and the destruction of tissue, joint and vascular structures (among others). Polymorphonuclear neutrophils, which are very abundant at sites of inflammation, interfere with attempts at regulation. Cell metabolism, which typically participates in fighting against the autoantigen attack, does not respond correctly to the demands, making the inflammatory phenomenon worse. This is also the case for environmental factors such as atmospheric pollution, dust, diet (especially salt intake) and infections. Inflammatory cytokines such as TNF-α, IL-1 and IL-17, are certain implicated, but not initially. They appear as a common execution pathway for a lengthy sentence following an unfortunate encounter between genetic predisposition and a harmful environment.

Involvement of Tumor Necrosis Factor Receptor Type II in FoxP3 Stability and as a Marker of Treg Cells Specifically Expanded by Anti-Tumor Necrosis Factor Treatments in Rheumatoid Arthritis.

OBJECTIVE: To study the involvement of Treg cells expressing tumor necrosis factor receptor type II (TNFRII) in exerting control of inflammation in experimental models and in the response to anti-TNF treatments in patients with rheumatoid arthritis (RA) or spondyloarthritis (SpA). METHODS: The role of TNFRII in Treg cells was explored using a multilevel translational approach. Treg cell stability was evaluated by analyzing the methylation status of the Foxp3 locus using bisulfite sequencing. Two models of inflammation (imiquimod-induced skin inflammation and delayed-type hypersensitivity arthritis [DTHA]) were induced in TNFRII-/- mice, with or without transfer of purified CD4+CD25+ cells from wild-type (WT) mice. In patients with RA and those with SpA, the evolution of the TNFRII+ Treg cell population before and after targeted treatment was monitored. RESULTS: Foxp3 gene methylation in Treg cells was greater in TNFRII-/- mice than in WT mice (50% versus 36.7%). In cultured Treg cells, TNF enhanced the expression, maintenance, and proliferation of Foxp3 through TNFRII signaling. Imiquimod-induced skin inflammation and DTHA were aggravated in TNFRII-/- mice (P < 0.05 for mice with skin inflammation and P < 0.0001 for mice with ankle swelling during DTHA compared to WT mice). Adoptive transfer of WT mouse Treg cells into TNFRII-/- mice prevented aggravation of arthritis. In patients with RA receiving anti-TNF treatments, but not those receiving tocilizumab, the frequency of TNFRII+ Treg cells was increased at 3 months of treatment compared to baseline (mean ± SEM 65.2 ± 3.1% versus 49.1 ± 5.5%; P < 0.01). In contrast, in anti-TNF-treated patients with SpA, the frequency of TNFRII+ Treg cells was not modified. CONCLUSION: TNFRII expression identifies a subset of Treg cells that are characterized by stable expression of Foxp3 via gene hypomethylation, and adoptive transfer of TNFRII-expressing Treg cells ameliorates inflammation in experimental models. Expansion and activation of TNFRII+ Treg cells may be one of the mechanisms by which anti-TNF agents control inflammation in RA, but not in SpA.

Dendritic cells modulated by innate immunity improve collagen-induced arthritis and induce regulatory T cells in vivo.

Dendritic cells (DCs) mediate interactions between innate and specific immunity and may induce regulatory mechanisms. We investigated the effects of modulated DCs in mice with collagen-induced arthritis (CIA) and tested the responses of cells to induced naturally occurring regulatory T cells. DCs were stimulated or not with DNA or lipopolysaccharide (LPS) for 24 hr. DC maturation was assayed, and then modulated DCs were intraperitoneally injected on day 14 into DBA/1 mice to treat CIA. In addition to arthritis scores and type 2 collagen (CII) response, the induction of CD4(+) CD25(+) T cells was analysed by flow cytometry in peripheral blood and the expression of Foxp3, transforming growth factor (TGF)-beta, interleukin (IL)-10 and cytotoxic T-lymphocyte antigen (CTLA)-4 was quantified. Finally, the expression of indoleamine-2,3-dioxygenase (IDO) was assayed in DCs. In comparison with LPS-stimulated DCs, plasmid-stimulated DCs expressed lower levels of major histocompatibility complex (MHC) class II, CD40, CD80 and CD86 molecules and secreted less IL-12p70, interferon (IFN)-gamma, IL-10 and TNF-alpha, displaying a semi-mature phenotype. Compared with non-stimulated DCs, stimulated DCs improved arthritis scores when injected after immunization, without modifying the T helper type 1 (Th1)/Th2 balance of the immune response against collagen. Stimulated DCs induced markers for regulatory T cells (Foxp3, TGF-beta1 and CTLA-4) in vivo. Only LPS-stimulated DCs expressed IDO, which may explain their better therapeutic efficacy. Regulatory mechanisms were induced using DCs modulated by innate immunity stimulators. Innate immunity mechanisms do not require the presence of the disease-causing antigen, even in T- and B-cell specific diseases. Our results have implications for the treatment of rheumatoid arthritis, an autoimmune disease whose triggering antigen has not been identified, and substantially clarify the role of regulatory T cells in CIA.

Salt, inflammatory joint disease, and autoimmunity.

Salt is a vital nutrient. Excess salt intake, however, has recently been blamed for triggering and/or worsening certain autoimmune diseases. In vitro, the cells involved in innate and adaptive immune responses exhibit an inflammatory profile when placed in hypertonic saline. More specifically, macrophages release increased amounts of proinflammatory cytokines, produce reactive oxygen species, and become capable of activating the inflammasome. T helper cells, via activation of serum and glucocorticoid-regulated kinase 1 (SGK1), overexpress IL-17A and IL-23R and differentiate into Th17 cells; whereas regulatory T cells lose the inhibitory capabilities needed to preserve self-tolerance. The data from animal models of autoimmune diseases and human patients are less consistent. SGK1 has been implicated in polarization toward the Th17 phenotype, which worsens conditions such as multiple sclerosis, systemic lupus erythematosus, autoimmune colitis, and transplant rejection. Observational epidemiological studies of patients with multiple sclerosis have demonstrated an association between excessive salt intake and a higher number of flares. Excessive salt intake is associated with a higher risk of developing rheumatoid arthritis, particularly in smokers. These data suggest that salt may stimulate certain immunological processes. Studies are therefore needed to assess the potential influence of dietary habits on the development and progression of autoimmune diseases.

Arthritis models: usefulness and interpretation.

Animal models of arthritis are used to better understand pathophysiology of a disease or to seek potential therapeutic targets or strategies. Focusing on models currently used for studying rheumatoid arthritis, we show here in which extent models were invaluable to enlighten different mechanisms such as the role of innate immunity, T and B cells, vessels, or microbiota. Moreover, models were the starting point of in vivo application of cytokine-blocking strategies such as anti-TNF or anti-IL-6 treatments. The most popular models are the different types of collagen-induced arthritis and arthritis in KBN mice. As spontaneous arthritides, human TNF-α transgenic mice are a reliable model. It is mandatory to use animal models in the respect of ethical procedure, particularly regarding the number of animals and the control of pain. Moreover, design of experiments should be of the highest level, animal models of arthritis being dedicated to exploration of well-based novelties, and never used for confirmation or replication of already proven concepts. The best interpretations of data in animal models of arthritis suppose integrated research, including translational studies from animals to humans.

Relationship between angiogenesis and inflammation in experimental arthritis.

Background. Angiogenesis is involved in rheumatoid arthritis (RA) leading to leucocyte recruitment and inflammation in the synovium. Furthermore, synovial inflammation itself further potentiates endothelial proliferation and angiogenesis. In this study, we aimed at evaluating the reciprocical relationship between synovial inflammation and angiogenesis in a RA model, namely collagen-induced arthritis (CIA). Methods. CIA was induced by immunization of DBA/1 mice with collagen type II in adjuvant. Endothelial cells were detected using a GSL-1 lectin-specific immunohistochemical staining on knee joint sections. Angiogenesis, clinical scores and histological signs of arthritis were evaluated from the induction of CIA until the end of the experiment. Angiogenesis was quantified by counting both the isolated endothelial cells and vessels stained on each section. To evaluate the effect of increased angiogenesis on CIA, VEGF gene transfer was performed using an adeno-associated virus encoding VEGF (AAV-VEGF), by intra-muscular or intra-articular injection in mice with CIA. Results. We showed an increase in synovial angiogenesis from day 6 to day 55 after CIA induction, and, moreover, joint vascularization and clinical scores of arthritis were correlated (p < 0.0001, r = 0.61). Vascularization and histological scores were also correlated (p = 0.0006, r = 0.51). Systemic VEGF overexpression in mice with CIA was followed by an aggravation of arthritis as compared to AAV-lacZ control group (p < 0.0001). In contrast, there was no difference in clinical scores between control mice and mice injected within the knee with AAV-VEGF, even if joint vascularization was higher in this group than in all other groups (p = 0,05 versus non-injected group). Intra-articular AAV-VEGF injections induced more severe signs of histological inflammation and bone destruction than AAV-Lac Z or no injection. Conclusion. Angiogenesis and joint inflammation evolve in parallel during collagen-induced arthritis. Furthermore, this work shows that exogenous VEGF can aggravate CIA. It is direct evidence that the increase in joint vascularization leads to an exacerbation of arthritis. Taken together, these results emphasize the role of angiogenesis in inflammatory arthritis. It also suggests an early involvement of angiogenesis in joint inflammation.

Gene therapy for patients with rheumatoid arthritis.

Gene therapy seeks either to supply a missing or dysfunctional gene or to ensure continuous long-lasting production of a therapeutic protein. Rheumatoid arthritis is a candidate for gene therapy, as the mechanisms leading to joint inflammation and destruction have been partly elucidated. Nevertheless, several crucial questions need to be addressed. Knowledge of the underlying pathophysiological mechanisms is needed to guide selection of the candidate gene. In the light of current data, TNF and IL-1 antagonists are generating interest. A choice must be made between a viral vector (adenovirus, retrovirus, adeno-associated virus) and a nonviral vector (naked DNA, administered by electrotransfer or in liposomes). Finally, the relative merits of intraarticular and systemic administration need to be considered. Safety is a primary concern. The transgene and/or vector may induce adverse effects. For instance, a transgene inserted within the host genome (when a retroviral vector is used) may induce a mutation. A number of vectors and transgenes induce immune responses. Numerous studies are ongoing to investigate the safety and efficacy of gene therapy strategies in experimental models of rheumatoid arthritis. These studies will have to be completed before further clinical trials of gene therapy in rheumatoid arthritis are considered.

Novel Immunotherapeutic Avenues for Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is the most common inflammatory rheumatic disease. It leads to irreversible joint damage, physical handicap, and reduced life expectancy. The past two decades have seen considerable therapeutic advances with the development of biologic treatments to block proinflammatory cytokines or modulate lymphocyte function, followed by the development of small molecules to target intracellular signaling. Nevertheless, only a minority of patients can achieve disease remission, especially long term, warranting further investigation into newer therapeutic options. Targeting single proinflammatory pathways may not be sufficient, as suggested by variable results with T helper (Th)-17-related cytokine blockade. Multilevel information from 'omics' techniques along with data from mechanistic studies might facilitate the identification of pivotal checkpoints in RA disease pathogenesis and the subsequent development of new effective treatments.

Personality modulates proportions of CD4+ regulatory and effector T cells in response to socially induced stress in a rodent of wild origin.

The way how individuals respond to chronic challenges can vary tremendously, and such differences are closely linked to personality. The few available studies on individual differences in stress-related immunosuppression in non-human mammals have been mainly carried out with laboratory strains. We conducted a study in male mound-building mice (Mus spicilegus) of wild origin. We distinguished between high (HAN) and low anxious/neophobic (LAN) personality types, quantified by subjects' consistent and associated behavioral responses in repeated elevated plus maze and novel object tests. After reaching maturity, parts of the subjects were regularly confronted to different resident pairs over a period of 5days to provoke a condition of chronic social stress, while others were used as untreated controls. We measured fecal corticosteroid metabolite (FCM) concentrations and different cellular immune parameters from blood and spleen. Socially confronted HAN showed higher increases in FCM concentrations than LAN, indicating a more pronounced physiological stress response in the former personality type. HAN of the experimental group also showed lower percentages of effector T cells (Teff) and higher regulatory T cells (Treg) in the spleen; the latter are known for their immunosuppressive activity. Considering the ratio of Teff/Treg, animals with higher increases in FCM concentrations during the late period of the experiment showed a stronger shift towards Treg cells, supporting immunosuppressive effects of chronically elevated corticosteroid levels. Summarizing, our results strongly suggest that immunomodulatory effects of socially induced stress were altered by individual differences in anxiety/neophobia, emphasizing the significance of personality in shaping physiological responses to challenge.

Collagen-induced arthritis and imiquimod-induced psoriasis develop independently of interleukin-33.

BACKGROUND: Interleukin (IL)-33 is a dual cytokine with both an alarmin role and a T helper 2 cell (Th2)-like inducing effect. It is involved in the pathogenesis of rheumatoid arthritis (RA) and its models; we recently demonstrated that exogenous IL-33 could inhibit collagen-induced arthritis (CIA) in C57BL/6 mice. However, its pathophysiological role in RA is unclear. Indeed, mice deficient in the IL-33 receptor ST2 show reduced susceptibility to arthritis, and the disease is not modified in IL-33-deficient mice. We examined the immune response in wild-type (WT) and IL-33-deficient mice with CIA. To further understand the role of endogenous IL-33 in inflammatory diseases, we studied its role in a skin psoriasis model. Mice on a C57BL/6 background were deficient in IL-33 but expressed lacZ under the IL-33 promoter. Therefore, IL-33 promotor activity could be analyzed by lacZ detection and IL-33 gene expression was analyzed by X-Gal staining in various mice compartments. Frequencies of CD4(+)FoxP3(+) regulatory T cells (Tregs) and Th1 and Th17 cells were evaluated by flow cytometry in WT and IL-33(-/-) mice. Bone resorption was studied by evaluating osteoclast activity on a synthetic mineral matrix. Psoriasis-like dermatitis was induced by application of imiquimod to the skin of mice. RESULTS: Severity of CIA was similar in IL-33(-/-) and WT littermates. Joints of IL-33(-/-) mice with CIA showed IL-33 promotor activity. In mice with CIA, frequencies of Tregs, Th1 and Th17 in the spleen or lymph nodes did not differ between the genotypes; osteoclast activity was higher but not significantly in IL-33(-/-) than WT mice. Psoriasis development did not differ between the genotypes. CONCLUSIONS: Despite its expression in the synovium of arthritic mice and normal keratinocytes, IL-33 is not required for CIA development in arthritis or psoriasis. Its absence does not induce a T cell shift toward Th1, Th17 or Treg subpopulations. Altogether, these data and our previous ones, showing that exogenous IL-33 can almost completely inhibit CIA development, suggest that this cytokine is not crucial for development of chronic inflammation. Studies of RA patients are needed to determine whether treatment targeting the IL-33/ST2 axis would be effective.

Gene therapy of collagen-induced arthritis by electrotransfer of human tumor necrosis factor-alpha soluble receptor I variants.

Electrotransfer is a simple and efficient strategy of nonviral gene delivery. We have used this method to deliver plasmids encoding three human tumor necrosis factor-alpha soluble receptor I variants (hTNFR-Is) a monomeric hTNFR-Is, a chimeric hTNFR-Is/mIgG1, and a dimeric (hTNFR-Is)(2) form. Electrotransfer parameters were studied and because anti-TNF strategies have proven efficient for the treatment of rheumatoid arthritis in clinics, we used a collagen-induced arthritis (CIA) mouse model to assess the efficacy of our constructs in the treatment of the disease. All proteins were proven bioactive, both in vitro and ex vivo. Plasmid intramuscular electrotransfer in mice resulted in a local expression of the three variants for at least 6 months; systemic expression lasted also more than 6 months for the hTNFR-Is/mIgG1 form, while it was shorter for the two other forms. This expression was plasmid dose-dependent. Electrotransfer of 50 microg of hTNFR-Is/mIgG1 at the onset of a CIA induced a clear-cut decrease in both clinical and histologic signs of the disease; the dimeric form also showed some efficacy. Moreover, the long-lasting protective effect was observed for more than 5 weeks. Comparison of this electrotransfer approach with repeated recombinant protein (etanercept) injections highlighted the potential practical interest of gene therapy approach for CIA, which leads to sustained therapeutic effect after single treatment. These results show that electrotransfer may be a useful method to deliver cytokine or anticytokine therapy in rheumatoid arthritis and also illustrate the potentiality of plasmid intramuscular electrotransfer for the rapid screening and assessment of different variant forms of secreted proteins.

Osteoprotegerin and inflammation.

RANK, RANKL, and OPG have well established regulatory effects on bone metabolism. RANK is expressed at very high levels on osteoclastic precursors and on mature osteoclasts, and is required for differentiation and activation of the osteoclast. The ligand, RANKL binds to its receptor RANK to induce bone resorption. RANKL is a transmembrane protein expressed in various cells type and particularly on osteoblast and activated T cells. RANKL can be cleaved and the soluble form is active. Osteoprotegerin decoy receptor (OPG), a member of the TNF receptor family expressed by osteoblasts, strongly inhibits bone resorption by binding with high affinity to its ligand RANKL, thereby preventing RANKL from engaging its receptor RANK. This system is regulated by the calciotropic hormones. Conversely, the effects of RANKL, RANK, and OPG on inflammatory processes, most notably on the bone resorption associated with inflammation, remain to be defined. The RANK system seems to play a major role in modulating the immune system. Activated T cells express RANKL messenger RNA, and knock-out mice for RANKL acquire severe immunological abnormalities and osteopetrosis. RANKL secretion by activated T cells can induce osteoclastogenesis. These mechanisms are enhanced by cytokines such as TNF-alpha, IL-1, and IL-17, which promote both inflammation and bone resorption. Conversely, this system is blocked by OPG, IL-4, and IL-10, which inhibit both inflammation and osteoclastogenesis. These data may explain part of the abnormal phenomena in diseases such as rheumatoid arthritis characterized by both inflammation and destruction. Activated T cells within the rheumatoid synovium express RANKL. Synovial cells are capable of differentiating to osteoclast-like cells under some conditions, including culturing with M-CSF and RANKL. This suggests that the bone erosion seen in rheumatoid arthritis may result from RANKL/RANK system activation by activated T cells. This opens up the possibility that OPG may have therapeutic effects mediated by blockade of the RANKL/RANK system.

Recent data on the role for angiogenesis in rheumatoid arthritis.

Angiogenesis is central to the development and perpetuation of rheumatoid synovitis. Vascular endothelial growth factor (VEGF), the main mediator of angiogenesis, is found in the synovial fluid and serum of patients with rheumatoid arthritis (RA), and its expression is correlated with disease severity. Compelling evidence that VEGF is involved in synovitis has been obtained from experimental models of RA. In particular, VEGF inhibition by synthetic compounds (e.g. TNP-470) or by naturally occurring factors (e.g., the soluble VEGF receptor) produce therapeutic effects. Angiopoietin-1, a recently discovered growth factor specific for neovascularization, is expressed within the rheumatoid synovium and may be stimulated by TNF-alpha. Other compounds, including integrins, fibroblast growth factor, and proinflammatory cytokines contribute to joint angiogenesis and, therefore, to the development of rheumatoid synovitis. Assessing vascularity may prove useful for evaluating or even predicting bone destruction. Furthermore, inhibition of angiogenesis may prove useful as an adjunct to current anti-inflammatory treatments.