Anne Denys

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.

Vaccination with cytokines in autoimmune diseases

Most autoimmune diseases have an unknown etiology, but all involve cytokines cascade in their development. At the present time, several cytokines have been identified as major targets in various autoimmune diseases, involving the development of monoclonal antibodies (MAbs) against those cytokines. Even if MAbs are indeed efficient, the passive immunotherapies also present some disadvantages and are expensive. To counter this, several strategies have been developed, including active immunotherapy, based on the vaccination principle. The aim of such a strategy is to induce a B cell response and to obtain autoantibodies able to neutralize the interaction of the self-cytokine with its receptor. To that purpose, cytokines (entire or peptide) are either coupled with a protein-carrier or virus-like particle, or modified with foreign Th cell epitopes. DNA vaccination can also be used with cytokine sequences. This review focuses on the different vaccination strategies with cytokines (including Tumor Necrosis Factor (TNF)α, Interleukin-1β (IL-1β), IL-17) in different autoimmune diseases in preclinical studies; the benefit/risk ratio of such a strategy and the present development of clinical trials in some autoimmune diseases are also discussed.

Blood vessels, a potential therapeutic target in rheumatoid arthritis?

New micro-vessels formation within synovium and macro-vessels endothelial damage with atheroma are two major features of rheumatoid arthritis, the former related to the articular involvement of the disease, the latter to its main systemic complication. The similarities between pannus development and solid tumors growth, and the efficacy of anti-angiogenic treatments in oncology, opened the perspective of directly targeting angiogenesis in arthritis. Nevertheless, despite the success of different anti-angiogenic therapeutic strategies in many arthritis experimental models, the application in human disease is still lacking. Recent data suggest that synovial neoangiogenesis and macro-vessels endothelial damage might be two linked phenomena. While synovial angiogenesis seems to be detrimental to endothelial damage repair, even anti-angiogenic treatments might paradoxically aggravate macro-vascular disease, especially in the context of uncontrolled inflammation. These elements induce to further explore the interconnections between inflammation and angiogenesis on one side and between micro- and macro-vascular diseases on the other, in order to establish the proper way to therapeutically target blood vessels in rheumatoid arthritis.

Interleukin-35 gene therapy exacerbates experimental rheumatoid arthritis in mice.

Interleukin (IL)-35 was initially described as an immunosuppressive cytokine specifically produced by CD4(+)FoxP3(+) regulatory T cells (Treg). Since Treg play a major role in autoimmunity control and protect from inflammation, we aimed at evaluating the role of IL-35 in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA), using a non-viral gene transfer strategy. The clinical and histological effect of IL-35 was assessed in mice with CIA receiving an injection of two distinct plasmids encoding IL-35 gene (pIGneo-mIL-35 or pORF-mIL-35) 3 and 18 days after CIA induction. Treg and Th17 were characterized by flow cytometry in the spleen and lymph nodes of treated mice. Our results showed that whatever the plasmid used, IL-35 gene transfer resulted in a statistically significant increase in clinical scores of CIA compared to results with empty plasmid. The underlying cellular mechanisms of this effect were shown to be related to an increased Th17/Treg ratio in the spleen of pORF-mIL-35 treated mice. In conclusion, we show an unexpected but clear exacerbating effect of IL-35 gene transfer in an autoimmune and inflammatory RA model, associated with a modification of the Th17/Treg balance. Altogether, these result shows that this cytokine can promote chronic inflammation.

Intra-articular electrotransfer of mouse soluble tumour necrosis factor receptor in a murine model of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes inflammation and destruction of the joints. In the collagen‐induced arthritis mouse model of RA, we developed a nonviral gene therapy method designed to block in situ the main cytokine tumour necrosis factor (TNF)‐α

A1.6 Vascular adhesion molecule VCAM-1 overexpression in collagen induced arthritis : a model for rheumatoid arthritis vascular dysfunction

Background and Objective Rheumatoid Arthritis (RA) patients are at risk of developing early coronary heart disease. Cardiovascular risk in RA is attributed to both traditional risk factors and to uncontrolled systemic in ammation over a long period. The aim of this study was to develop a model that would mimic both vascular dysfunction and articular inflammation in RA, using collagen induced arthritis (CIA). We mainly focused on VCAM-1 expression, early marker of vascular activation and dysfunction. Materials and Methods Arthritis was induced in 11 weeks old C57BL/6 mice with 2 injections (at day 0 and 21) of chicken type II collagen (cCII) emulsioned in complete Freund adjuvant (CFA). Half of mice were fed a fat-enriched diet (Western Diet (WD)) (n = 12) while the other half were fed a standard diet. Control mice (not immunised with cCII) were fed either WD (n = 12) or standard diet (n = 12). The aorta and synovial membrane were removed at day 105 after the first cCII immunisation. We analysed VCAM-1, IL-6 and IL-17 mRNA level in both the aorta and the synovium by real time quantitative PCR (qRT-PCR). VCAM-1 localisation in the aortic sinus layers (intima, media and adventitia) was determined by immunohistochemistry (IHC). Results Immunised mice fed either WD or standard diet had higher qRT-PCR VCAM-1 expression in aorta vs. non immunised mice (p<0.05). Although all mice groups, constitutively expressed VCAM-1 on aortic sinus endothelial cell monolayer, the whole intimal layer stained for VCAM-1 at IHC in both immunised mice (fed standard or western diet) as well as in non immunised mice fed WD. Conversely, the expression of IL-6 and IL-17 in the aorta was similar in all groups. Conclusion In CIA, C57BL6 mice display aortic inflammation characterised by VCAM-1 overexpression, independently from the diet (WD or standard diet). C57BL6 CIA might be a pertinent model to study the early mechanisms of large blood vessels vascular dysfunction in RA.

Aortic VCAM-1: an early marker of vascular inflammation in collagen-induced arthritis.

Cardiovascular disease (CVD) is a major cause of morbidity and mortality in rheumatoid arthritis (RA). There are limited experimental data on vascular involvement in arthritis models. To study the link between CVD and inflammation in RA, we developed a model of vascular dysfunction and articular inflammation by collagen‐induced arthritis (CIA) in C57Bl/6 (B6) mice. We studied the expression of vascular inflammatory markers in CIA with and without concomitant hyperlipidic diet (HD). Collagen‐induced arthritis was induced with intradermal injection of chicken type‐II collagen followed by a boost 21 days later. Mice with and without CIA were fed a standard diet or an HD for 12 weeks starting from the day of the boost. Arthritis severity was evaluated with a validated clinical score. Aortic mRNA levels of vascular cell adhesion molecule‐1 (VCAM‐1), inducible nitric oxide synthase (iNOS) and interleukin‐17 were analysed by quantitative RT‐PCR. Vascular cell adhesion molecule‐1 localization in the aortic sinus was determined by immunohistochemistry. Atherosclerotic plaque presence was assessed in aortas. Collagen‐induced arthritis was associated with increased expression of VCAM‐1, independent of diet. VCAM‐1 overexpression was detectable as early as 4 weeks after collagen immunization and persisted after 15 weeks. The HD induced atheroma plaque formation and aortic iNOS expression regardless of CIA. Concomitant CIA and HD had no additive effect on atheroma or VCAM‐1 or iNOS expression. CIA and an HD diet induced a distinct and independent expression of large‐vessel inflammation markers in B6 mice. This model may be relevant for the study of CVD in RA.

IL-35 gene therapy in a model of rheumatoid arthritis

Backgroundand objectives Interleukin (IL)-35 is a novel interleukin described as an immunosuppressive cytokine specifically produced by CD4+FoxP3+ regulatory T (Tregs) cells but not by conventional T cells. Since Tregs play a major role in autoimmunity maintenance and protects from inflammation, the authors aimed at evaluating the role of IL-35 in a model of rheumatoid arthritis in mice using a non viral gene transfer strategy. Methods Two plasmids were used and compared: pIGneo-mouse (m)IL-35-GFP (gift from Dario Vignali) and pORF-mIL-35, where IL-35 is expressed under CMV or HTLV promoter, respectively. pIGneo-mouse (m)IL-35-GFP contains a ‘native’ form of IL-35 made of Ebi3 and p35 linked subunits, while pORF-mIL-35contains a ‘single chain’ construct. In vitro, IL-35 expression was detected in lysate of transfected HEK cells by western blot (p35 or Ebi3) and immunoprecipitation (IP). The in vivo trangene expression was detected by fluorescence analysis of GFP on muscle slides after intra-muscular electrotransfer (ET) of 60 µg pIGneo-mIL-35-GFP plasmid. The clinical effect of IL-35 was assessed in collagen-induced arthritis in mice receiving two im ET of 60 µg pIGneo-mIL-35-GFP or pORF-IL-35, 3 and 18 days after CIA induction. Control groups received the corresponding empty plasmids or PBS. Tregs and Th17 frequencies and phenotypes were performed by flow cytometry in the spleen and lymph nodes (LN) of pORF-IL-35 treated mice at day 34. Results Both subunits p35 and Ebi3 were expressed in lysates from either pIGneo-mIL-35-GFP or pORF-IL-35 HEK transfected cells, and formed the IL-35 heterodimer as shown by IP. At least 3 days after intramuscular ET of pIGneo-mIL-35-GFP plasmid, the authors were able to detect GFP expression, revealing in vivo IL-35 expression, on muscle histological slides. Surprisingly, whatever the plasmid used, IL-35 gene transfer resulted in a statistically significant increase in clinical scores of collagen-induced arthritis as compared to empty plasmid. The underlying cellular mechanisms of this effect were shown to be related to an increased in number and frequency of Th17 cells in the spleen of pORF-mIL-35 treated mice, leading to an increased Th17/Tregs ratio. Interestingly, CD39 expression on Tregs increased (frequency and intensity) in the LN of pORF-mIL-35 treated mice as compared to control groups, but CD62L expression also increased (spleen and LN) on these cells. Conclusion The authors show an unexpected but clear exacerbating effect of IL-35 gene transfer in an autoimmune and inflammatory model of RA. The authors can hypothesise that IL-35, as already known, induced an activation of Tregs by up-regulating CD39 expression on these cells, but that the concomitant up-regulation of CD62L prevent their migration into the inflammatory joint by retaining them in secondary lymphoid organs, thus impeding them from exerting their immunosuppressive role.

AB0125 Differential Expression of Vascular Inflammation Markers in Collagen-Induced Arthritis and Experimental Atherosclerosis

Background Cardiovascular disease (CVD) is a major cause of morbidity and mortality in rheumatoid arthritis (RA). Increased cardiovascular risk in RA is attributed to both traditional risk factors and systemic inflammation over a long period. Limited experimental data exist on vascular involvement in arthritis models1,2,3. Thus, we developed a model that would mimic both vascular dysfunction and articular inflammation in RA, using collagen-induced arthritis (CIA) in in C57Bl/6 (B6) mice. Objectives To study the expression of vascular inflammatory markers and atheroma formation in CIA with and without concomitant hyperlipidic diet (HD). Methods CIA was induced in B6 mice with chicken type-II collagen (cCII) followed by a boost 21 days later. These mice were fed with a standard (chow) diet or with HD given for 12 weeks starting from the day of the boost. The control mice were not immunized (NI) but were fed either the standard diet or HD. Arthritis severity was evaluated using a validated clinical score. Aorta and synovial membranes were removed 15 weeks after the first cCII immunization. We analysed vascular cell adhesion molecule-1 (VCAM-1), inducible NO-synthase (iNOS) and interleukin-17 mRNA level in aorta with real-time quantitative PCR. VCAM-1 localisation in the aortic sinus was determined by immunohistochemistry. In addition, atherosclerotic plaques were assessed in the aorta. Results CIA was associated with high expression of VCAM-1, independently of the fed diet. VCAM-1 overexpression was detectable 4 weeks after cCII immunization and persisted at 15 weeks. HD induced atheroma plaque formation and aortic iNOS expression which were both independent from CIA. Concomitant CIA and HD had no additive effect on atheroma, VCAM-1 or iNOS expression. Conclusions In B6 mice, HD and CIA induce distinct and independent expression of large vessel inflammation markers. This model, sensitive to both CIA and experimental atherosclerosis may be relevant for the study of CVD in RA. References Asquith, D. L., Miller A. M., Hueber A. J., et al. Apolipoprotein E-deficient mice are resistant to the development of collagen-induced arthritis. Arthritis Rheum 2010.62:472-7. Postigo, J., Genre F., Iglesias M., et al. Exacerbation of type II collagen-induced arthritis in apolipoprotein E-deficient mice in association with the expansion of Th1 and Th17 cells. Arthritis Rheum 2011.63:971-80. Rose, S., Eren M., Murphy S., et al. A novel mouse model that develops spontaneous arthritis and is predisposed towards atherosclerosis. Ann Rheum Dis 2013.72:89-95. Disclosure of Interest None declared