Jérôme Biton

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.

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.

TNFα: activator or inhibitor of regulatory T cells?

TNFα is a cytokine that is central to the pathogenesis of several autoimmune diseases. More specifically, the deleterious effects of TNFα in rheumatoid arthritis (RA) are well established. The proinflammatory influence of TNFα in RA is related both to direct effects mediated by the induction of other proinflammatory cytokines, metalloproteinases, and free radicals; and to modulation of the regulatory T cells (Tregs). Furthermore, the TNFα antagonists used to treat RA can induce the emergence of a distinctive Treg subpopulation. Nevertheless, a recent body of data suggests that TNFα may also exert anti-inflammatory effects, which may be mediated in part via Tregs. TNFα binds to the TNF receptor 2 expressed preferentially at the Treg surface, thereby activating and promoting the development of Tregs. Data from patients with RA and more recent evidence obtained in the absence of disease are consistent with a paradoxical effect of TNFα on Tregs. TNFα may have different effects on naturally occurring Tregs and induced Tregs.

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 α‐galactosylceramide (α‐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‐γ; 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.

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.

Protection from articular damage by passive or active anti‐tumour necrosis factor (TNF)‐α immunotherapy in human TNF‐α transgenic mice depends on anti‐TNF‐α antibody levels

Active anti‐tumour necrosis factor (TNF)‐α immunization with the kinoid of TNF‐α (TNF‐K) induces polyclonal anti‐TNF‐α antibodies and ameliorates arthritis in human TNF‐α (hTNF‐α) transgenic mice (TTg). We compared the efficacy of TNF‐K to that of infliximab (IFX) and of TNF‐K and IFX co‐administration, and evaluated whether the titres of anti‐hTNF‐α antibodies induced by immunization were a determinant of TNF‐K efficacy. Forty‐eight TTg mice received one of the following treatments: TNF‐K immunization (TNF‐K group); weekly IFX throughout the study duration (IFXw0–15); TNF‐K plus weekly IFX for 4 weeks (TNF‐K + IFX); and weekly IFX for 4 weeks (IFXw0–4); PBS. Animals were killed at week 16. Anti‐hTNF‐α antibody titres and clinical and histological scores were compared. All TNF‐K immunized mice (TNF‐K and TNF‐K + IFX) produced anti‐hTNF‐α antibodies. Titres were higher in TNF‐K versus TNF‐K + IFX (P < 0·001) and correlated inversely with histological inflammation (R = −0·78; P = 0·0001) and destruction (R = −0·67; P = 0·001). TNF‐K + IFX had higher histological inflammation and destruction versus TNF‐K (P < 0·05). A receiver operating characteristic (ROC) analysis of anti‐hTNF‐α antibody titres identified the criterion cut‐off value to discriminate most effectively between the TNF‐K and TNF‐K + IFX groups. Mice with high versus low titres had less histological inflammation and destruction (P < 0·05). In a model of TNF‐α‐dependent arthritis, protection from articular damage by TNF‐K correlates with the titres of induced anti‐hTNF‐α antibodies. The co‐administration of TNF‐K and a short course of infliximab does not result in less articular damage versus solely TNF‐K, due probably to lower anti‐hTNF‐α antibody production. These results are relevant for future development of active anti‐TNF‐α immunization in human disease.

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.

Reply: To PMID 24504799.

arthritis. Arthritis Rheumatol 2014;66:273–83. 2. Samson M, Audia S, Janikashvili N, Ciudad M, Trad M, Fraszczak J, et al. Inhibition of interleukin-6 function corrects Th17/Treg cell imbalance in patients with rheumatoid arthritis. Arthritis Rheum 2012;64:2499–503. 3. Van Gestel AM, Prevoo ML, van ’t Hof MA, van Rijswijk MH, van de Putte LB, van Riel PL. Development and validation of the European League Against Rheumatism response criteria for rheumatoid arthritis: comparison with the preliminary American College of Rheumatology and the World Health Organization/ International League Against Rheumatism criteria. Arthritis Rheum 1996;39:34–40. 4. Pesce B, Soto L, Sabugo F, Wurmann P, Cuchacovich M, Lopez MN, et al. Effect of interleukin-6 receptor blockade on the balance between regulatory T cells and T helper type 17 cells in rheumatoid arthritis patients. Clin Exp Immunol 2013;171:237–42. 5. Komatsu N, Okamoto K, Sawa S, Nakashima T, Oh-hora M, Kodama T, et al. Pathogenic conversion of Foxp3 T cells into TH17 cells in autoimmune arthritis. Nat Med 2014;20:62–8.