Delphine Lemeiter

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.

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.

Syngeneic fibroblasts transfected with a plasmid encoding interleukin-4 as non-viral vectors for anti-inflammatory gene therapy in collagen-induced arthritis.

No effective long‐term treatment is available for rheumatoid arthritis. Recent advances in gene therapy and cell therapy have demonstrated efficiency in collagen‐induced arthritis (CIA). Interleukin‐4 (IL‐4) is already known to be efficient in CIA in systemic injection or administered by gene therapy. This study was designed to evaluate the effect of a non‐viral gene therapy of CIA, involving injection of syngeneic fibroblasts transfected with a plasmid encoding for IL‐4.

Interleukin-4 Cellular Gene Therapy and Osteoprotegerin Decrease Inflammation-Associated Bone Resorption in Collagen-Induced Arthritis

To evaluate the respective action of IL-4, an anti-inflammatory cytokine, and OPG, an inhibitor of bone resorption, on the inflammatory process and the associated bone resorption in collagen-induced arthritis (CIA). After CIA induction, DBA/1 mice were treated with OPG or with IL-4 DBA/1 transfected fibroblasts or both OPG + IL-4. CIA significantly improved in IL-4 groups. OPG had no effect on arthritis clinical scores but histologic scores were reduced in OPG, IL-4, and OPG + IL-4 groups vs. nontreated CIA mice. OPG increased significantly BMD and decreased by 45% D-pyridinolin levels. Moreover association of IL-4 and OPG exerted an additive effect of BMD and resorption marker (−68%). Production of IFN-γ in the supernatants of spleen cells was reduced in IL-4 treated mice. OPG had a moderate effect on IFN-γ, but potentiated the inhibitory effect of IL-4. OPG and IL-4 prevent bone loss in CIA-mice model and could have additive effects on IFN-γ secretion.

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.

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 CD39high Treg population in a ST2L-dependent manner. Our findings reveal a powerful anti-inflammatory mechanism by which IL-33 administration inhibits arthritis development.

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.

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

BackgroundInterleukin (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.ResultsSeverity 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.ConclusionsDespite 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.