Natacha Bessis

Adeno-associated virus-mediated delivery of IL-4 prevents collagen-induced arthritis

Immunomodulation of autoimmune inflammatory diseases like rheumatoid arthritis can be achieved by anti-inflammatory T2 cytokines such as interleukin (IL)-4 administered by gene therapy. In this study we investigated the efficiency of adeno-associated viruses (AAV) vectors in collagen-induced arthritis (CIA). After injection of AAV-LacZ in the tarsus area of mice, the expression of the transgene was localized in the deep muscles cells near the bone. LacZ expression was found in liver, heart and lung after i.m. injection of AAV-LacZ, showing a spread of the vector over the body. Anti-AAV neutralizing antibodies were detected in the serum after i.m. injection of AAV-LacZ, but they did not alter the transgene expression after re-administration of AAV-LacZ. Long-term IL-4 expression persisted 129 days after intra-muscular injection of 3.7 × 1010 or 11.2 × 1010AAV-IL-4 p.p. (average 7.7 or 17.5 pg IL-4/mg proteins, respectively). More importantly, the treatment of CIA with AAV-IL-4 vector in mice produced a therapeutic benefit, since we show a diminished prevalence of the disease, a significant reduction in paw swelling, attenuated histological synovitis and a 10 days delayed onset of arthritis. This is the first evidence that AAV vector-mediated gene therapy using a T2 cytokine is efficient in an animal model of rheumatoid arthritis.

Regulatory T cells (Treg) in rheumatoid arthritis

Modulation of the T-cell response depends chiefly on regulatory T cells (Treg), which express CD4 and CD25. Some Treg cells are present naturally, whereas others are induced in response to antigens. The immunomodulating effects of Treg cells are mediated by membrane molecules (e.g., CTLA4, GITR, and OX40) and cytokines. IL-35 seems to be a crucial mediator, although IL-10 and TGFbeta are also important. The role for Treg cells in rheumatoid arthritis (RA) has been established in both patients and animal models. Treg function is deficient in RA, whereas Treg counts vary. Treg counts increase in patients who are responding to TNFalpha antagonist therapy. Among current hypotheses, Treg expansion or transfer may hold promise for the treatment of RA.

The type II decoy receptor of IL‐1 inhibits murine collagen‐induced arthritis

IL‐1 is a key cytokine involved in the inflammatory response. The type II receptor of IL‐1 (IL‐1RII) acts as a decoy receptor, binding and inhibiting the effect of IL‐1. This study was undertaken to establish whether IL‐1RII can ameliorate collagen‐induced arthritis, a model of inflammatory arthritis in mice. We used human keratinocytes transfected with the human (h)IL‐1RII gene as a source of hIL‐1RII protein. We showed that these cells expressed both the membrane and soluble form of receptor. In vitro, IL‐1‐stimulated murine macrophage cells showed a decreased expression of TNF‐α in the presence of hIL‐1RII. We engrafted the hIL‐1RII‐transfected cells in the back of mice developing collagen‐induced arthritis. We found that clinical and histological parameters of arthritis were significantly decreased in mice treated with cells producing hIL‐1RII. In addition, hIL‐1RII administration was able to reduce the expression of mRNA for IL‐6 and myeloperoxidase in the joints of treated animals. These data show that hIL‐1RII anti‐inflammatory properties in the model of collagen‐induced arthritis in mice and could have a regulatory role in rheumatoid arthritis.

TNFalpha kinoid vaccination-induced neutralizing antibodies to TNFalpha protect mice from autologous TNFalpha-driven chronic and acute inflammation.

The proinflammatory cytokine TNFα is a potent mediator of septic shock and a therapeutic target for chronic inflammatory pathologies including rheumatoid arthritis and Crohns disease. As an alternative to anti-human TNFα (hTNFα) mAbs and other hTNFα blocker approved drugs, we developed an active anti-hTNFα immunotherapy, based on a vaccine comprised of a keyhole limpet hemocyanin-hTNFα heterocomplex immunogen (hTNFα kinoid) adjuvanted in incomplete Freunds adjuvant. In mice transgenic for hTNFα (TTg mice), hTNFα kinoid vaccination elicited high titers of Abs that neutralized hTNFα bioactivities but did not result in a cellular response to hTNFα. The vaccine was safe and effective in two experimental models. Kinoid-immunized but not control TTg mice resisted hTNFα-driven shock in one model and were prevented from spontaneous arthritis, inflammatory synovitis, and articular destruction in a second model. These data demonstrate an anti-cytokine induction of autoimmune protection against both acute and chronic hTNFα exposure. They show that active vaccination against a human cytokine can be achieved, and that the immune response can be effective and safe.

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.

Gene therapy for rheumatoid arthritis

Rheumatoid arthritis (RA) is a severe autoimmune systemic disease. Chronic synovial inflammation results in destruction of the joints. No conventional treatment is efficient in RA. Gene therapy of RA targets mainly the players of inflammation or articular destruction: TNF‐α or IL‐1 blocking agents (such as anti‐TNF‐α monoclonal antibodies, soluble TNF‐α receptor, type II soluble receptor of IL‐1, IL‐1 receptor antagonist), antiinflammatory cytokines (such as IL‐4, IL‐10, IL‐1), and growth factors. In this polyarticular disease, the vector expressing the therapeutic protein can be administered as a local (intra‐articular injection) or a systemic treatment (extra‐articular injection). All the main vectors have been used in experimental models, including the more recent lentivirus and adeno‐associated virus. Ex vivo gene transfer was performed with synovial cells, fibroblasts, T cells, dendritic cells, and different cells from xenogeneic origin. In vivo gene therapy is simpler, although a less controlled method. Clinical trials in human RA have started with ex vivo retrovirus‐expressing IL‐1 receptor antagonists and have demonstrated the feasibility of the strategy of gene therapy. The best target remains to be determined and extensive research has to be conducted in preclinical studies. Copyright

Gene Therapy of Collagen-Induced Arthritis by Electrotransfer of Human Tumor Necrosis Factor-α 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.

Interleukin‐6 Receptor Blockade Enhances CD39+ Regulatory T Cell Development in Rheumatoid Arthritis and in Experimental Arthritis

The rationale for blocking interleukin‐6 (IL‐6) in rheumatoid arthritis (RA) lies chiefly in the proinflammatory effect of this cytokine. Few studies have evaluated the consequences of anti–IL‐6 receptor (IL‐6R) antibody treatment on Treg cells. This study was undertaken to elucidate the mechanism of action of anti–IL‐6R antibody treatment by studying the effects on Treg cells in an experimental arthritis model and in patients with RA.

Efficacy of interleukin-10 gene electrotransfer into skeletal muscle in mice with collagen-induced arthritis.

Gene therapy is very promising in the treatment of rheumatoid arthritis (RA). Electrotransfer is a recent method reported to enhance in vivo intramuscular DNA transfection. Interleukin‐10 (IL‐10) has antiinflammatory effects in RA and in collagen‐induced arthritis (CIA), a murine model of RA. In order to improve our strategy of gene therapy, we used electrotransfer to enhance penetration into skeletal muscle with CIA of plasmids encoding IL‐10.

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)‐β, 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)‐γ, IL‐10 and TNF‐α, 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‐β1 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.