DNA methylation governs the dynamic regulation of inflammation by apoptotic cells during efferocytosis.

Efficient clearance of apoptotic cells (AC) is pivotal in preventing autoimmunity and is a potent immunosuppressive stimulus. However, activation of cells prior to apoptosis abolishes their immunoregulatory properties. Here we show using the antigen-induced model of arthritis that the degree of DNA methylation within AC confers their immunomodulatory plasticity. DNA isolated from resting and activated AC mimicked their respective immune effects. Demethylation of DNA abrogated the protective effect of AC whereas remethylation of AC DNA reversed the effects of activation and restored the ability to inhibit inflammation. Disease suppression or lack thereof was associated with TGFß and IL-6 production respectively. Apoptotic CD4+ T cells from patients with rheumatoid arthritis and systemic lupus erythematosus were demethylated compared to healthy controls and favoured production of IL-6 when cultured with healthy macrophages, in contrast to the TGFß produced in response to healthy AC. Our data implicate AC DNA methylation as the molecular switch that imprints their regulatory properties.

Engulfment of activated apoptotic cells abolishes TGF-ß-mediated immunoregulation via the induction of IL-6.

Phagocytosis of apoptotic cells (ACs) is usually a potent immunoregulatory signal but can also promote inflammation. In this article, we show that administration of apoptotic dendritic cells (DCs) inhibited inflammation in vivo through increasing production of TGF-ß from intrinsic DCs and B cells. However, ACs derived from LPS-activated DCs failed to restrain inflammation because of a short-lived but marked IL-6 response, which abolished the increase in TGF-ß. Inhibition of IL-6 restored the protective anti-inflammatory properties of aACs and the TGF-ß response. DCs isolated from mice that had received resting but not activated ACs could transfer the suppression of inflammation to recipient mice. These transferred DCs stimulated B cell TGF-ß production and relied on an intact B cell compartment to limit inflammation. These results highlight how the activation state of AC governs their ability to control inflammation through reciprocal regulation of IL-6 and TGF-ß.

Induced CD8+FoxP3+ Treg cells in rheumatoid arthritis are modulated by p38 phosphorylation and monocytes expressing membrane tumor necrosis factor α and CD86.

OBJECTIVE: Limiting the severity of inflammation and promoting its eventual resolution are vital for protecting host tissues both in autoimmunity and chronic infection. The aim of this study was to determine the suitability of repurposing anti-CD3 monoclonal antibody (mAb) therapy for rheumatoid arthritis (RA) by analyzing its ability to induce CD8+FoxP3+ Treg cells from peripheral blood mononuclear cells (PBMCs). METHODS: Anti-CD3 mAb was cultured with RA PBMCs to induce CD8+FoxP3+ Treg cells, which were analyzed by flow cytometry to determine their phenotype. Treg cell induction was investigated via neutralization or blocking antibodies, cellular depletion, or ImageStream technology. Blotting was used to determine the signaling pathways involved in CD8+FoxP3+ Treg cell induction. Suppression of CD4+ T cell effector responses was assessed by Treg cell suppression assays and Mosaic enzyme-linked immunosorbent assay. RESULTS: Potent CD8+FoxP3+ Treg cells were induced from RA PBMCs by anti-CD3 mAb. Unlike their CD4+ counterparts, CD8+FoxP3+ Treg cells inhibited Th17 responses in a contact-dependent manner, thereby functioning to limit a wider range of inflammatory pathways. CD8+FoxP3+ Treg cell induction was supported both by p38 phosphorylation intrinsic to naive CD8+ T cells and by monocytes via CD86 and membrane tumor necrosis factor α (TNFα). Artificially increasing monocyte membrane TNFα or inhibiting CD8+ T cell p38 phosphorylation drove FoxP3 expression in a subset of initially unresponsive CD8+ T cells. CONCLUSION: These data define an unknown mechanism of CD8+FoxP3+ Treg cell induction by anti-CD3 mAb, which could be combined with a p38 inhibitor to improve therapeutic efficacy in RA patients and resolve chronic inflammation via the restoration of tolerance.

Advances on regulatory T cells from the 15th International Congress of Immunology.

The International Congress of Immunology is the largest congregation of immunologists and meets every three years. This year, the congress was held in Milan and included talks on both basic and translational aspects of immunology. Talks on the field of regulatory T cell biology and function are highlighted in this report.

Germinal center B cells govern their own fate via antibody feedback.

Affinity maturation of B cells in germinal centers (GCs) is a process of evolution, involving random mutation of immunoglobulin genes followed by natural selection by T cells. Only B cells that have acquired antigen are able to interact with T cells. Antigen acquisition is dependent on the interaction of B cells with immune complexes inside GCs. It is not clear how efficient selection of B cells is maintained while their affinity matures. Here we show that the B cells' own secreted products, antibodies, regulate GC selection by limiting antigen access. By manipulating the GC response with monoclonal antibodies of defined affinities, we show that antibodies in GCs are in affinity-dependent equilibrium with antibodies produced outside and that restriction of antigen access influences B cell selection, seen as variations in apoptosis, plasma cell output, T cell interaction, and antibody affinity. Feedback through antibodies produced by GC-derived plasma cells can explain how GCs maintain an adequate directional selection pressure over a large range of affinities throughout the course of an immune response, accelerating the emergence of B cells of highest affinities. Furthermore, this mechanism may explain how spatially separated GCs communicate and how the GC reaction terminates.

Th17 cells are restrained by Treg cells via the inhibition of interleukin-6 in patients with rheumatoid arthritis responding to anti-tumor necrosis factor antibody therapy.

OBJECTIVE: The importance of interleukin-17 (IL-17) is underscored both by its resistance to control by Treg cells and the propensity of Treg cells to produce this highly inflammatory cytokine. This study sought to address whether Th17 cells are inhibited by Treg cells in rheumatoid arthritis (RA) patients responding to anti-tumor necrosis factor (anti-TNF) therapy, and if so defining the underlying mechanisms of suppression. METHODS: Inhibition of Th17 cell responses was determined by Treg cell suppression assays. The Treg cell phenotype was analyzed using flow cytometry and enzyme-linked immunosorbent assay. Mechanisms of suppression were tested by cytokine addition or antibody blockade. RESULTS: Th17 responses were inhibited by Treg cells from RA patients responding to the anti-TNF antibody adalimumab (Treg(ada) ), but not by Treg cells from healthy individuals or patients with active RA. Furthermore, Treg(ada) cells secreted less IL-17, even when exposed to proinflammatory monocytes from patients with active RA. Treg(ada) cells suppressed Th17 cells through the inhibition of monocyte-derived IL-6, but this effect was independent of IL-10 and transforming growth factor ß, which mediated the suppression of Th1 responses. Adalimumab therapy led to a reduction in retinoic acid receptor-related orphan nuclear receptor C-positive Th17 cells and an increase in FoxP3+ Treg cells lacking expression of the transcription factor Helios. However, this acquisition of IL-17-suppressor function was not observed in RA patients responding to treatment with etanercept, a modified TNF receptor-Fc fusion protein. Indeed, there was no alteration in Treg cell number, function, or phenotype in etanercept-treated patients, and Th17 responses remained unchecked. CONCLUSION: Th1 and Th17 responses are controlled through distinct mechanisms by Treg cells from patients responding to anti-TNF antibody therapy. Adalimumab therapy, but not etanercept therapy, induces a potent and stable Treg cell population with the potential to restrain the progression of IL-17-associated inflammation in RA via regulation of monocyte-derived IL-6.

Production of IL-17: What's STAT got to do with it?

Th17 cells are important mediators of autoimmunity, yet the mechanisms by which they are controlled are not fully understood. Studies in mice, including a recent article in Nature Immunology by Yang et al., show that IL-2 is an important inhibitory factor for the differentiation of Th17 cells, inducing phosphorylation of STAT5, which outcompetes STAT3 binding at the IL-17 locus. In humans however, IL-2 appears to be crucial for Th17 differentiation, yet inhibits the expansion of antigen-specific Th17 clones, again via a STAT5 mechanism. Here we discuss how the article by Yang et al. offers a novel mechanism to explain how changes in the balance of different cytokines in the inflammatory environment may alter the stability or phenotype of regulatory T cells and T helper cell subsets.

Natural IgM is required for suppression of inflammatory arthritis by apoptotic cells.

The clearance of dying cells is vital for re-establishing tolerance during inflammation and has potent immunoregulatory consequences. Because natural IgM plays a key role in the removal of apoptotic cells, we investigated whether the immune modulatory properties of apoptotic cells depended on its presence. Using an Ab-independent, Ag-induced model of inflammatory arthritis, we tested whether natural IgM is essential for the arthritis-suppressing properties of apoptotic cells. Whereas administration of apoptotic cells reduced joint inflammation and damage in normal mice accompanied by suppression of the Th17 response, no protection was afforded in secreted IgM-deficient (Sµ(-)) mice. The enhanced production of IL-10 by T cells from draining lymph nodes and splenic marginal zone B cells, driven by the infusion of apoptotic cells, was abrogated in the absence of natural IgM. Apoptotic cells were present shortly after administration in the splenic marginal zone, but their removal was substantially delayed in the absence of natural IgM. Incubation of apoptotic cells with natural IgM in vitro restored their arthritis-suppressing properties in Sµ(-) mice. Moreover, these IgM-coated apoptotic cells were cleared rapidly after injection from the spleens of Sµ(-) mice. Our results demonstrate that natural IgM is a critical factor in a chain of events triggered by the administration of apoptotic cells that promote IL-10-secreting B and T cells and restrain the development of inflammation.

The importance of natural IgM: scavenger, protector and regulator.

The existence of IgM has been known for more than a century, but its importance in immunity and autoimmunity continues to emerge. Studies of mice deficient in secreted IgM have provided unexpected insights into its role in several diverse processes, from B cell survival to atherosclerosis, as well as in autoimmunity and protection against infection. Among the various distinct properties that underlie the functions of IgM, two stand out: its polyreactivity and its ability to facilitate the removal of apoptotic cells. In addition, new B cell-targeted therapies for the treatment of autoimmunity have been shown to cause a reduction in serum IgM, potentially disrupting the functions of this immunoregulatory molecule and increasing susceptibility to infection.

The yin and yang of regulatory T cells and inflammation in RA.

Rheumatoid arthritis (RA) is characterized by chronic inflammation leading to joint destruction. Regulatory T (T(REG)) cells are potent suppressors of autoimmunity, but are not capable of controlling every aspect of the inflammatory reaction. We have found that T(REG)-cell function is abnormal in patients with RA, and that a distinct population of T(REG) cells with potent suppressive properties is induced after therapy with inhibitors of tumor necrosis factor. In this Review, we discuss the mutual interactions between the opposing forces of T(REG) cells and inflammation in the context of RA. Therapeutic approaches that enhance T(REG)-cell function whilst controlling inflammation are likely to be the most effective strategies for restoring immune tolerance in patients with this disease.

Secreted IgM enhances B cell receptor signaling and promotes splenic but impairs peritoneal B cell survival.

B cell survival has a central role in maintaining immune responses to foreign organisms while curbing autoimmunity. In this study, we show that mature B cell survival is impaired and B cell turnover is accelerated in the spleen of mice lacking secreted IgM. Although in vitro responses to BCR cross-linking were normal, there was a marked reduction in basal ERK and global tyrosine phosphorylation in splenic B cells from serum IgM-deficient mice, suggesting diminished interaction with cognate Ag in vivo. The provision of BAFF either in vitro or in vivo reversed the increase in B cell apoptosis, demonstrating that other survival signals can compensate for the loss of secreted IgM in the spleen. In striking contrast to the splenic compartment, peritoneal B cell survival was enhanced in secreted IgM-deficient mice, despite a similar reduction in basal BCR signaling compared with wild type mice. These results suggest that secreted IgM acts as an adjuvant, boosting BCR signals to maintain survival and maintenance of mature splenic B cells while increasing B cell apoptosis in the peritoneum. BAFF administration mitigated the consequences of secreted IgM deficiency on B cell survival in the spleen but not in the peritoneum. This work provides new insight into the regulation of B cell signaling and homeostasis in different peripheral compartments by secreted IgM.

ANTI-CD3 therapy expands the numbers of CD4+ and CD8+ Treg cells and induces sustained amelioration of collagen-induced arthritis.

OBJECTIVE: To assess the therapeutic potential of anti-CD3 monoclonal antibodies (mAb) for rheumatoid arthritis, using collagen-induced arthritis as an animal model. METHODS: Arthritis was induced in DBA/1 mice by immunization with type II collagen. After disease onset, a single injection of anti-CD3 mAb (20 microg/mouse) was administered, and arthritis severity was monitored over a 10-day period. RESULTS: Anti-CD3 mAb treatment resulted in a sustained reduction in disease activity, which was associated with an increase in the proportion of naturally occurring CD4+CD25+FoxP3+ regulatory T (Treg) cells and the generation of a population of CD8+CD25+FoxP3+ Treg cells. Anti-CD3 mAb treatment did not alter the capacity of CD4+ Treg cells to suppress effector T cell proliferation and interferon-gamma (IFNgamma) production in vitro. However, CD4+ Treg cells from both anti-CD3 mAb-treated and control mice were unable to suppress interleukin-17 (IL-17) production. In contrast, CD8+ Treg cells induced by anti-CD3 therapy suppressed IL-17 production as well as CD4+ T cell proliferation and IFNgamma production. CONCLUSION: These results show that anti-CD3 mAb treatment has important therapeutic potential for rheumatoid arthritis and has the capacity to generate antiarthritic CD8+ Treg cells and expand the relative numbers of CD4+ Treg cells.

Adoptive therapy with redirected primary regulatory T cells results in antigen-specific suppression of arthritis.

Regulatory T cells (Tregs) can suppress a wide range of immune cells, making them an ideal candidate for the treatment of autoimmunity. The potential clinical translation of targeted therapy with antigen-specific Tregs is hampered by the difficulties of isolating rare specificities from the natural polyclonal T cell repertoire. Moreover, the initiating antigen is often unknown in autoimmune disease. Here we tested the ability of antigen-specific Tregs generated by retroviral gene transfer to ameliorate arthritis through linked suppression and therefore without cognate recognition of the disease-initiating antigen. We explored two distinct strategies: T cell receptor (TCR) gene transfer into purified CD4+CD25+ T cells was used to redirect the specificity of naturally occurring Tregs; and co-transfer of FoxP3 and TCR genes served to convert conventional CD4(+) T cells into antigen-specific regulators. Following adoptive transfer into recipient mice, the gene-modified T cells engrafted efficiently and retained TCR and FoxP3 expression. Using an established arthritis model, we demonstrate antigen-driven accumulation of the gene modified T cells at the site of joint inflammation, which resulted in a local reduction in the number of inflammatory Th17 cells and a significant decrease in arthritic bone destruction. Together, we describe a robust strategy to rapidly generate antigen-specific regulatory T cells capable of highly targeted inhibition of tissue damage in the absence of systemic immune suppression. This opens the possibility to target Tregs to tissue-specific antigens for the treatment of autoimmune tissue damage without the knowledge of the disease-causing autoantigens recognized by pathogenic T cells.

Blockade of tumor necrosis factor in collagen-induced arthritis reveals a novel immunoregulatory pathway for Th1 and Th17 cells.

IL-17 is implicated in the pathogenesis of rheumatoid arthritis (RA) and has previously been shown to be induced by tumor necrosis factor (TNF) in vitro. The aim of this study was to assess the impact of TNF inhibition on IL-17 production in collagen-induced arthritis, a model of RA. TNF blockade using TNFR-Fc fusion protein or anti-TNF monoclonal antibody reduced arthritis severity but, unexpectedly, expanded populations of Th1 and Th17 cells, which were shown by adoptive transfer to be pathogenic. Th1 and Th17 cell populations were also expanded in collagen-immunized TNFR p55(-/-) but not p75(-/-) mice. The expression of IL-12/IL-23 p40 was up-regulated in lymph nodes (LN) from p55(-/-) mice, and the expansion of Th1/Th17 cells was abrogated by blockade of p40. Treatment of macrophages with rTNF also inhibited p40 production in vitro. These findings indicate that at least one of the ways in which TNF regulates Th1/Th17 responses in arthritis is by down-regulating the expression of p40. Finally, although TNF blockade increased numbers of Th1 and Th17 cells in LN, it inhibited their accumulation in the joint, thereby providing an explanation for the paradox that anti-TNF therapy ameliorates arthritis despite increasing numbers of pathogenic T cells.

Collagen-induced arthritis as a model of hyperalgesia: functional and cellular analysis of the analgesic actions of tumor necrosis factor blockade.

OBJECTIVE: There is a disparity in the animal models used to study pain in rheumatoid arthritis (RA), which tends to be acute in nature, and models used to assess the pathogenesis of RA. The latter models, like human RA, are lymphocyte-driven and polyarthritic. We assessed pain behavior and mechanisms in collagen-induced arthritis (CIA), the model of preclinical arthritis used most commonly in the field of immunology. We then validated the model using anti-tumor necrosis factor (anti-TNF) therapy, which has analgesic effects in models of inflammation as well as in human RA. METHODS: CIA was induced in DBA/1 mice by immunization with type II collagen at the base of the tail. Swelling and mechanical and thermal hyperalgesia were assessed before and for 28 days after the onset of arthritis. Spontaneous behavior was assessed using an automated activity monitor. Glial activity was assessed by glial fibrillary acidic protein expression, and nerve damage was evaluated by activating transcription factor 3 expression. The actions of anti-TNF therapy on nociception were then evaluated. RESULTS: Arthritis resulted in a decrease in the threshold for thermal and mechanical stimuli, beginning on the day of onset. Decreased spontaneous activity was also observed. A significant increase in the number of hyperplasic spinal cord astrocytes was observed beginning 10 days after the onset of arthritis. Anti-TNF therapy was profoundly analgesic, with an efficacy similar to that of cyclooxygenase 2 inhibition, and reduced astrocyte activity in CIA. CONCLUSION: This study shows that the CIA model is suitable for testing not only antiinflammatory but also analgesic drugs for potential use in RA, and highlights the importance of using appropriate disease models to assess relevant pain pathways.

Protection against the early acute phase of Cryptosporidium parvum infection conferred by interleukin-4-induced expression of T helper 1 cytokines.

Immunity to Cryptosporidium parvum infection involves a T helper (Th) 1 response with interferon (IFN)- gamma and interleukin (IL)-12 activity, but the role of Th2 cytokines, such as IL-4, is unclear. Around the peak of infection, production of oocysts in IL-4-deficient and IL-4 receptor alpha -deficient neonatal BALB/c mice was greater than that in wild-type (wt) mice. Susceptibility to infection was increased or decreased, respectively, in wt mice treated with anti-IL-4 neutralizing antibodies or recombinant IL-4. Excretion of oocysts by IFN- gamma -deficient mice was unaffected by treatment with anti-IL-4, indicating that IL-4 stimulated IFN- gamma activity. Early during infection, wt mice had increased intestinal expression of IFN- gamma and IL-12 mRNA, compared with IL-4-deficient mice. Intestinal IL-4 was detected by Western blotting in wt mice 24 h after infection but not in uninfected control mice. These findings suggest that, early during C. parvum infection of BALB/c mice, there is production of IL-4 that promotes Th1-mediated immunity.