IL-3 attenuates collagen-induced arthritis by modulating the development of Foxp3+ regulatory T cells

IL-3, a cytokine secreted by Th cells, functions as a link between the immune and the hematopoietic system. We previously demonstrated the potent inhibitory role of IL-3 on osteoclastogenesis, pathological bone resorption, and inflammatory arthritis. In this study, we investigated the novel role of IL-3 in development of regulatory T (Treg) cells. We found that IL-3 in a dose-dependent manner increases the percentage of Foxp3(+) Treg cells indirectly through secretion of IL-2 by non-Treg cells. These IL-3-expanded Treg cells are competent in suppressing effector T cell proliferation. Interestingly, IL-3 treatment significantly reduces the severity of arthritis and restores the loss of Foxp3(+) Treg cells in thymus, lymph nodes, and spleen in collagen-induced arthritis mice. Most significantly, we show that IL-3 decreases the production of proinflammatory cytokines IL-6, IL-17A, TNF-α, and IL-1 and increases the production of anti-inflammatory cytokines IFN-γ and IL-10 in collagen-induced arthritis mice. Thus, to our knowledge, we provide the first evidence that IL-3 play an important role in modulation of Treg cell development in both in vitro and in vivo conditions, and we suggest its therapeutic potential in the treatment of rheumatoid arthritis and other autoimmune diseases.     

Immature dendritic cells suppress collagen-induced arthritis by in vivo expansion of CD49b+ regulatory T cells

Dendritic cells (DCs) are specialized APCs with an important role in the initiation and regulation of immune responses. Immature DCs (iDCs) reportedly mediate tolerance in the absence of maturation/inflammatory stimuli, presumably by the induction of regulatory T cells. In this study, we show for the first time that repetitive iDC injections trigger the expansion of a novel regulatory population with high immunomodulatory properties, able to protect mice from collagen-induced arthritis. These regulatory T cells are characterized by the expression of the CD49b molecule and correspond to a CD4+ alpha-galactosylceramide/CD1d-nonrestricted T cell population producing IL-10. Adoptive transfer of < 10(5) TCRbeta+ CD49b+ cells isolated from the liver of iDCs-vaccinated mice, conferred a complete protection against arthritis. This protection was associated with an attenuation of the B and T cell response associated with a local secretion of IL-10. Thus, together these data demonstrate that iDCs can expand and activate a novel regulatory population of CD49b+ T cells, with high immunosuppressive potential able to mediate protection against a systemic autoimmune disease.     

Functions of metallothionein generating interleukin-10-producing regulatory CD4+ T cells potentiate suppression of collagen-induced arthritis

Metallothionein, a cysteine-rich stress response protein that is naturally induced by a variety of immunologic stressors, has been shown to suppress autoimmune disorders through mechanisms not yet fully defined. In the present study, we examined the underlying mechanisms by which metallothionein might mediate such regulation of autoimmunity. Na?ve CD4+ T cells from metallothionein-deficient mice differentiated to produce significantly less IL-10, TGF-gamma, and repressor of GATA, but more IFN-gamma and T-bet, when compared with those from wild-type mice. The levels of IL-4 and GATA-3 production were not different between the two groups of mice. Conversely, treatment with exogenous metallothionein during the priming phase drove na?ve wild-type CD4+ T cells to differentiate into cells producing more IL-10 and TGF-beta, but less IFN-gamma than untreated cells. Metallothionein-primed cells were hyporesponsive to restimulation, and suppressive to T cell proliferation in an IL-10-dependent manner. Lymphocytes from metallothionein-deficient mice displayed significantly elevated levels of AP-1 and JNK activities in response to stimulation compared with those from wild-type controls. Importantly, transgenic mice overexpressing metallothionein exhibited significantly reduced susceptibility to collagen-induced arthritis and enhanced IL-10 level in the serum, relative to their nontransgenic littermates. Taken together, these data suggest that metallothionein is able to promote the generation of IL-10- and TGF-beta-producing type 1 regulatory T-like cells by downregulating JNK-dependent AP-1 activity. Thus, metallothionein may play an important role in the regulation of Th1-dependent autoimmune arthritis, and may represent both a potential target for therapeutic manipulation and a critical element in the diagnostic assessment of disease potential.     

Abatacept decreases disease activity in a absence of CD4+ T cells in a collagen-induced arthritis model

IntroductionAbatacept is a fusion protein of human cytotoxic T-lymphocyte–associated protein (CTLA)-4 and the Fc portion of human immunoglobulin G1 (IgG1). It is believed to be effective in the treatment of rheumatoid arthritis by inhibiting costimulation of T cells via blocking CD28–B7 interactions as CTLA-4 binds to both B7.1 (CD80) and B7.2 (CD86). However, the interaction of CD28 with B7 molecules is crucial for activation of naive cells, whereas it is unclear whether the action of already activated CD4⁺ T cells, which are readily present in established disease, also depends on this interaction. The aim of this study was to determine whether the mode of action of abatacept depends solely on its ability to halt T cell activation in established disease.MethodsArthritis was induced in thymectomized male DBA/1 mice by immunisation with bovine collagen type II. The mice were subsequently depleted for CD4⁺ T cells. Abatacept or control treatment was started when 80 % of the mice showed signs of arthritis. Arthritis severity was monitored by clinical scoring of the paws, and anti-collagen antibody levels over time were determined by enzyme-linked immunosorbent assay.ResultsTreatment with abatacept in the absence of CD4⁺ T cells resulted in lower disease activity. This was associated with decreasing levels of collagen-specific IgG1 and IgG2a antibodies, whereas the antibody levels in control or CD4⁺ T cell–depleted mice increased over time.ConclusionsThese results show that abatacept decreased disease activity in the absence of CD4⁺ T cells, indicating that the mode of action of abatacept in established arthritis does not depend entirely on its effects on CD4⁺ T cell activation.     

IL-10 signaling in CD4+ T cells is critical for the pathogenesis of collagen-induced arthritis

Introduction

IL-10 is a very important anti-inflammatory cytokine. However, the role of this cytokine in T cells in the pathogenesis of collagen-induced arthritis is unclear. The purpose of this study was to define the role of IL-10 signaling in T cells in the pathogenesis of collagen-induced arthritis.

Methods

IL-10 receptor dominant-negative transgenic (Tg) and control mice were immunized with bovine type II collagen to induce arthritis. The severity of arthritis was monitored and examined histologically. T-cell activation and cytokine production were analyzed using flow cytometry. T-cell proliferation was examined by [³H]thymidine incorporation. Antigen-specific antibodies in serum were measured by ELISA. Foxp3 expression in CD4⁺ regulatory T cells (Tregs) was determined by intracellular staining or Foxp3-RFP reporter mice. The suppressive function of Foxp3⁺CD4⁺ Tregs was determined in vitro by performing a T-cell proliferation assay. The level of IL-17 mRNA in joints was measured by real-time PCR. A two-tailed nonparametric paired test (Wilcoxon signed-rank test) was used to calculate the arthritis and histological scores. Student's paired or unpaired t-test was used for all other statistical analyses (InStat version 2.03 software; GraphPad Software, San Diego, CA, USA).

Results

Blocking IL-10 signaling in T cells rendered mice, especially female mice, highly susceptible to collagen-induced arthritis. T-cell activation and proliferation were enhanced and produced more IFN-γ. The suppressive function of CD4⁺Foxp3⁺ regulatory T cells was significantly impaired in Tg mice because of the reduced ability of Tregs from Tg mice to maintain their levels of Foxp3. This was further confirmed by transferring Foxp3-RFP cells from Tg or wild-type (Wt) mice into a congenic Wt host. The higher level of IL-17 mRNA was detected in inflammatory joints of Tg mice, probably due to the recruitment of IL-17⁺γδ T cells into the arthritic joints.

Conclusion

IL-10 signaling in T cells is critical for dampening the pathogenesis of collagen-induced arthritis by maintaining the function of Tregs and the recruitment of IL-17⁺γδ T cells.     

The indispensable role of CCR5 for in vivo suppressor function of tumor-derived CD103+ effector/memory regulatory T cells

CD103 is a marker for identification of effector/memory regulatory T cells (Tregs). CD103(+) Tregs are potent suppressors of tissue inflammation in several infectious diseases, autoimmune diseases, and cancers. However, the underlying mechanisms for this potent suppression ability remain unclear. The current study was designed to clarify this issue. Unexpectedly, we found both CD103(+) and CD103(-) Tregs had similar suppression capacity in vitro. We then chose a murine tumor model for investigation of the in vivo behavior of these Tregs. The suppression ability in vivo against the anti-tumor ability of CD8(+) T cells was restricted to CD103(+) Tregs although both Tregs had equal in vitro suppression ability. In addition, CD103(+) Tregs expressed significantly higher levels of CCR5 than those of CD103(-) Tregs and accumulated more in tumors than did CD103(-) Tregs. Furthermore, blockade of CCR5 signaling, either by CCR5(-/-)CD103(+) Tregs or by CCL5 knockdown tumor, could reduce the migration of CD103(+) Tregs into tumors and impair their in vivo suppression ability. In conclusion, these results indicate that the potent in vivo suppression ability of CD103(+) Tregs is due to the tissue-migration ability through CCR5 expression.     

Timed ablation of regulatory CD4+ T cells can prevent murine AIDS progression

We describe successful immunotherapy of murine AIDS (MAIDS) in C57BL/6J mice based on the elimination of replicating CD4(+) regulator T cells. We demonstrate that a single injection of the antimitotic drug vinblastine (Vb) given 14 days postinfection (p.i.) with LP-BM5 can prevent MAIDS progression. Treatment with anti-CD4 mAb at 14 days p.i. is similarly able to prevent MAIDS. Treatment at other time points with Vb or anti-CD4 mAb is ineffective. The effect is based on ablation of a replicating dominantly suppressive CD4(+) T cell population, as indicated by adoptive transfer and in vivo depletion experiments using mAbs against CD4 as well as combinations of mAbs against the known regulatory cell surface markers CD25, GITR, and CTLA-4. Cell surface marker analysis shows a population of CD4(+)CD25(+) cells arising shortly before day 14 p.i. Cytokine analyses show a peak in IL-10 production from day 12 to day 16 p.i. MAIDS-infected mice also have CD4(+) T cells with significantly higher expression levels of CD38 and particularly CD69, which have been demonstrated to be regulator T cell markers in the Friend retroviral model. The immunotherapy appears to prevent disease progression, although no protection against reinfection with LP-BM5 is generated. These data define a new therapy for murine retroviral infection, which has potential for use in other diseases where T regulator cell-mediated immunosuppression plays a role in the disease process.     

Methionine aminopeptidase-2 blockade reduces chronic collagen-induced arthritis: potential role for angiogenesis inhibition

The enzyme methionine aminopeptidase-2 (MetAP-2) is thought to play an important function in human endothelial cell proliferation, and as such provides a valuable target in both inflammation and cancer. Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with increased synovial vascularity, and hence is a potential therapeutic target for angiogenesis inhibitors. We examined the use of PPI-2458, a selective non-reversible inhibitor of MetAP-2, in disease models of RA, namely acute and chronic collagen-induced arthritis (CIA) in mice. Whilst acute CIA is a monophasic disease, CIA induced with murine collagen type II manifests as a chronic relapsing arthritis and mimics more closely the disease course of RA. Our study showed PPI-2458 was able to reduce clinical signs of arthritis in both acute and chronic CIA models. This reduction in arthritis was paralleled by decreased joint inflammation and destruction. Detailed mechanism of action studies demonstrated that PPI-2458 inhibited human endothelial cell proliferation and angiogenesis in vitro, without affecting production of inflammatory cytokines. Furthermore, we also investigated release of inflammatory cytokines and chemokines from human RA synovial cell cultures, and observed no effect of PPI-2458 on spontaneous expression of cytokines and chemokines, or indeed on the angiogenic molecule vascular endothelial growth factor (VEGF). These results highlight MetAP-2 as a good candidate for therapeutic intervention in RA.     

NKG2D receptor signaling enhances cytolytic activity by virus-specific CD8+ T cells: evidence for a protective role in virus-induced encephalitis

Inoculation with the neurotropic JHM strain of mouse hepatitis virus (JHMV) into the central nervous system (CNS) of mice results in an acute encephalitis associated with an immune-mediated demyelinating disease. During acute disease, infiltrating CD8⁺ T cells secrete gamma interferon (IFN-γ) that controls replication in oligodendrocytes, while infected astrocytes and microglia are susceptible to perforin-mediated lysis. The present study was undertaken to reveal the functional contributions of the activating NKG2D receptor in host defense and disease following JHMV infection. NKG2D ligands RAE-1, MULT1, and H60 were expressed within the CNS following JHMV infection. The immunophenotyping of infiltrating cells revealed that NKG2D was expressed on ~90% of infiltrating CD8⁺ T cells during acute and chronic disease. Blocking NKG2D following JHMV infection resulted in increased mortality that correlated with increased viral titers within the CNS. Anti-NKG2D treatment did not alter T-cell infiltration into the CNS or the generation of virus-specific CD8⁺ T cells, and the expression of IFN-γ was not affected. However, cytotoxic T-lymphocyte (CTL) activity was dependent on NKG2D expression, because anti-NKG2D treatment resulted in a dramatic reduction in lytic activity by virus-specific CD8⁺ T cells. Blocking NKG2D during chronic disease did not affect either T-cell or macrophage infiltration or the severity of demyelination, indicating that NKG2D does not contribute to virus-induced demyelination. These findings demonstrate a functional role for NKG2D in host defense during acute viral encephalitis by selectively enhancing CTL activity by infiltrating virus-specific CD8⁺ T cells.     

Human peripheral blood T regulatory cells (Tregs), functionally primed CCR4+ Tregs and unprimed CCR4- Tregs, regulate effector T cells using FasL

Regulatory CD25(+)CD4(+) T cells (Tregs) play an important role in the control of peripheral tolerance. In this study we demonstrate that human peripheral blood Tregs can be divided into two distinct populations based on the expression of CCR4. The majority ( approximately 75%) of freshly isolated Tregs express CCR4 and presumably represent memory-type Tregs. Interestingly, CCR4(-) Tregs require anti-CD3 Ab-mediated activation to acquire a regulatory activity, while CCR4(+) Tregs appear to be already primed to suppress the proliferation of CD8(+) T cells. CCR4 is also expressed on CD25(low)CD4(+) T cells (CCR4(+) non-Tregs) that mostly suppress Th1-type polarization without affecting T cell proliferation, presumably via the production of immunomodulatory cytokines like IL-10. In contrast, CCR4(+) Tregs express FasL to primarily regulate T cell proliferation via a contact-mediated process involving FasL/Fas signaling, a major regulatory pathway of T cell homeostasis. Finally, we also demonstrate that the depletion of CCR4(+) T cells leads to Th1-type polarization of CD4(+) T cells and augmentation of CD8(+) T cell responses to tumor Ags.     

Uncoupling of IL-2 signaling from cell cycle progression in naive CD4+ T cells by regulatory CD4+CD25+ T lymphocytes

Prior reports have shown that CD4(+)CD25(+) regulatory T cells suppress naive T cell responses by inhibiting IL-2 production. In this report, using an Ag-specific TCR transgenic system, we show that naive T cells stimulated with cognate Ag in the presence of preactivated CD4(+)CD25(+) T cells also become refractory to the mitogenic effects of IL-2. T cells stimulated in the presence of regulatory T cells up-regulated high affinity IL-2R, but failed to produce IL-2, express cyclins or c-Myc, or exit G(0)-G(1). Exogenous IL-2 failed to break the mitotic block, demonstrating that the IL-2 production failure was not wholly responsible for the proliferation defect. This IL-2 unresponsiveness did not require the continuous presence of CD4(+)CD25(+) regulatory T cells. The majority of responder T cells reisolated after coculture with regulatory cells failed to proliferate in response to IL-2, but were not anergic and proliferated in response to Ag. The mitotic block was also dissociated from the antiapoptotic effects of IL-2, because IL-2 still promoted the survival of T cells that had been cocultured with CD4(+)CD25(+) T cells. IL-2-induced STAT5 phosphorylation in the cocultured responder cells was intact, implying that the effects of the regulatory cells were downstream of receptor activation. Our results therefore show that T cell activation in the presence of CD4(+)CD25(+) regulatory T cells can induce an alternative stimulation program characterized by up-regulation of high affinity IL-2R, but a failure to produce IL-2, and uncoupling of the mitogenic and antiapoptotic effects of IL-2.     

Phenotypic and functional characterisation of CCR7+ and CCR7- CD4+ memory T cells homing to the joints in juvenile idiopathic arthritis

The aim of the study was to characterise CCR7+ and CCR7- memory T cells infiltrating the inflamed joints of patients with juvenile idiopathic arthritis (JIA) and to investigate the functional and anatomical heterogeneity of these cell subsets in relation to the expression of the inflammatory chemokine receptors CXCR3 and CCR5. Memory T cells freshly isolated from the peripheral blood and synovial fluid (SF) of 25 patients with JIA were tested for the expression of CCR7, CCR5, CXCR3 and interferon-gamma by flow cytometry. The chemotactic activity of CD4 SF memory T cells from eight patients with JIA to inflammatory (CXCL11 and CCL3) and homeostatic (CCL19, CCL21) chemokines was also evaluated. Paired serum and SF samples from 28 patients with JIA were tested for CCL21 concentrations. CCR7, CXCR3, CCR5 and CCL21 expression in synovial tissue from six patients with JIA was investigated by immunohistochemistry. Enrichment of CD4+, CCR7- memory T cells was demonstrated in SF in comparison with paired blood from patients with JIA. SF CD4+CCR7- memory T cells were enriched for CCR5+ and interferon-gamma+ cells, whereas CD4+CCR7+ memory T cells showed higher coexpression of CXCR3. Expression of CCL21 was detected in both SF and synovial membranes. SF CD4+ memory T cells displayed significant migration to both inflammatory and homeostatic chemokines. CCR7+ T cells were detected in the synovial tissue in either diffuse perivascular lymphocytic infiltrates or organised lymphoid aggregates. In synovial tissue, a large fraction of CCR7+ cells co-localised with CXCR3, especially inside lymphoid aggregates, whereas CCR5+ cells were enriched in the sublining of the superficial subintima. In conclusion, CCR7 may have a role in the synovial recruitment of memory T cells in JIA, irrespective of the pattern of lymphoid organisation. Moreover, discrete patterns of chemokine receptor expression are detected in the synovial tissue.     

CD4+CD25+ T regulatory cells limit effector T cells and favor the progression of brucellosis in BALB/c mice

Brucellosis is one of the most common bacterial zoonoses worldwide. Infection is usually chronic and sometimes lifelong. Different mechanisms can be postulated as to the basis for the induction of the chronic status of brucellosis, but a comprehensive knowledge is still lacking. Here, we carried out a series of experiments in order to assess if the persistence of Brucella abortus could be ascribed to the effect of a down regulation of the immune response due to activity of regulatory T cells. We demonstrate that CD4 + CD25 + T regulatory cells are able to limit the effectiveness of CD4 + T cells and are able to favor the maintenance and the progression of B. abortus infection.     

Indoleamine 2,3-dioxygenase-expressing dendritic cells are involved in the generation of CD4+CD25+ regulatory T cells in Peyer's patches in an orally tolerized, collagen-induced arthritis mouse model

Introduction

The present study was devised to understand the role of systemic indoleamine 2,3-dioxygenase (IDO) in the tolerance induction for orally tolerized mice in collagen-induced arthritis (CIA). We examined whether IDO-expressing dendritic cells (DCs) are involved in the generation of CD4⁺CD25⁺ regulatory T cells during the induction of oral tolerance in a murine CIA model.

Methods

Type II collagen was fed six times to DBA/1 mice beginning 2 weeks before immunization, and the effect on arthritis was assessed. To examine the IDO expression, the DCs of messenger RNA and protein were analyzed by RT-PCR and Flow cytometry. In addition, a proliferative response assay was also carried out to determine the suppressive effects of DCs through IDO. The ability of DCs expressing IDO to induce CD4⁺CD25⁺ T regulatory cells was examined.

Results

CD11c⁺ DCs in Peyer's patches from orally tolerized mice expressed a higher level of IDO than DCs from nontolerized CIA mice. IDO-expressing CD11c⁺ DCs were involved in the suppression of type II collagen-specific T-cell proliferation and in the downregulation of proinflammatory T helper 1 cytokine production. The suppressive effect of IDO-expressing CD11c⁺ DCs was mediated by Foxp3⁺CD4⁺CD25⁺ regulatory T cells.

Conclusion

Our data suggest that tolerogenic CD11c⁺ DCs are closely linked with the induction of oral tolerance through an IDO-dependent mechanism and that this pathway may provide a new therapeutic modality to treat autoimmune arthritis.     

CXC chemokine receptor 4 expressed in T cells plays an important role in the development of collagen-induced arthritis

Introduction  

Chemokines and their receptors are potential therapeutic targets in rheumatoid arthritis (RA). Among these, several studies suggested the involvement of CXC chemokine 4 (CXCR4) and its ligand CXC ligand 12 (SDF-1) in RA pathogenesis. However, the role of these molecules in T-cell function is not known completely because of embryonic lethality of Cxcr4- and Cxcl12- deficient mice. In this report, we generated T cell-specific Cxcr4 -deficient mice and showed that the CXCR4 in T cells is important for the development of collagen-induced arthritis (CIA).     

Experience-driven development: effector/memory-like alphaE+Foxp3+ regulatory T cells originate from both naive T cells and naturally occurring naive-like regulatory T cells

Naturally occurring Foxp3+CD25+CD4+ regulatory T cells (Treg) have initially been described as anergic cells; however, more recent in vivo studies suggest that Tregs vigorously proliferate under both homeostatic as well as inflammatory conditions. We have previously identified a subset of murine CD4+ Tregs, which is characterized by expression of the integrin alphaEbeta7 and which displays an effector/memory-like phenotype indicative of Ag-specific expansion and differentiation. In the present study, the alphaE+ Treg subset was found to contain a large fraction of cycling cells under homeostatic conditions in healthy mice. Using an adoptive transfer system of Ag-specific T cells, we could demonstrate that the vast majority of transferred natural, naive-like CD25+CD4+ Tregs acquired expression of the integrin alphaEbeta7 upon tolerogenic application of Ag via the oral route. In addition, using the same system, Foxp3+ Tregs could be de novo induced from conventional naive CD25-CD4+ T cells, and this conversion was associated with concomitant expression of alphaE. These findings suggest that Tregs expressing the integrin alphaE are effector/memory Tregs with a high turnover rate that can develop in the periphery upon Ag contact under tolerogenic conditions, both from thymic-derived CD25+CD4+ Tregs with a naive-like phenotype as well as from conventional naive T cells.