Wanda Niedbala

IL-35 is a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory T cells and suppression of Th17 cells

Epstein‐Barr virus‐induced gene 3 (EBI3) and the p35 subunit of IL‐12 have been reported to form a heterodimeric hematopoietin in human and mouse. We have constructed a heterodimeric protein covalently linking EBI3 and p35, to form a novel cytokine which we now call IL‐35. The Fc fusion protein of IL‐35 induced proliferation of murine CD4+CD25+ and CD4+CD25– T cells when stimulated with immobilized anti‐CD3 and anti‐CD28 antibodies in vitro. The IL‐35‐expanded CD4+CD25+ T cell population expressed Foxp3 and produced elevated levels of IL‐10, whereas the IL‐35‐induced CD4+CD25– T cells produced IFN‐γ but not IL‐4. The in vitro expanded CD4+CD25+ T cells retained their suppressive functions against CD4+CD25– effector cells. Furthermore, when cultured with soluble anti‐CD3 antibody and antigen‐presenting cells, IL‐35 suppressed the proliferation of CD4+CD25– effector cells. Moreover, IL‐35 inhibited the differentiation of Th17 cells in vitro. In vivo, IL‐35 effectively attenuated established collagen‐induced arthritis in mice, with concomitant suppression of IL‐17 production but enhanced IFN‐γ synthesis. Thus, IL‐35 is a novel anti‐inflammatory cytokine suppressing the immune response through the expansion of regulatory T cells and suppression of Th17 cell development.

Nitric oxide regulates Th1 cell development through the inhibition of IL-12 synthesis by macrophages

We have previously reported that mice lacking inducible nitric oxide synthase (NOS2) developed enhanced Th1 cell responses. We now investigated the mechanism by which NO modulates Th1 cells differentiation. Peritoneal macrophages from NOS2‐deficient mice infected with Leishmania major in vivo or stimulated with IFN‐γ or lipopolysaccharide (LPS) in vitro produced significantly higher levels of IL‐12 than those from heterozygous or wild‐type mice. A macrophage cell line, J774, produced significant amounts of IL‐12 following activation with LPS, or LPS plus IFN‐γ. This could be markedly enhanced by the NOS inhibitor L‐NG monomethyl arginine (L‐NMMA), but profoundly inhibited by the NO‐generating compound S‐nitroso‐N‐acetyl‐penicillamine (SNAP). The effect of NO in this system is selective, since SNAP enhanced and L‐NMMA decreased TNF‐α synthesis by LPS‐activated J774 cells. The differential effect of NO on IL‐12 and TNF‐α is at the transcriptional level and is activation dependent. Since IL‐12 is a major inducer of Th1 cells which produce IFN‐γ that can activate macrophages to produce IL‐12, our data demonstrate that NO can be an inhibitor of this feedback loop, preventing the excessive amplification of Th1 cells which are implicated in a range of immunopathologies.

IL-33 attenuates EAE by suppressing IL-17 and IFN-γ production and inducing alternatively activated macrophages

Interleukin (IL)‐33, a member of the IL‐1 cytokine family, is an important modulator of the immune system associated with several immune‐mediated disorders. High levels of IL‐33 are expressed by the central nervous system (CNS) suggesting a potential role of IL‐33 in autoimmune CNS diseases. We have investigated the expression and function of IL‐33 in the development of experimental autoimmune encephalomyelitis (EAE) in mice. We report here that IL‐33 and its receptor ST2 (IL‐33Rα) are highly expressed in spinal cord tissue, and ST2 expression is markedly increased in the spinal cords of mice with EAE. Furthermore, ST2‐deficient (ST2−/−) mice developed exacerbated EAE compared with wild‐type (WT) mice while WT, but not ST2−/− EAE mice treated with IL‐33 developed significantly attenuated disease. IL‐33‐treated mice had reduced levels of IL‐17 and IFN‐γ but produced increased amounts of IL‐5 and IL‐13. Lymph node and splenic macrophages of IL‐33‐treated mice showed polarization toward an alternatively activated macrophage (M2) phenotype with significantly increased frequency of MR+PD‐L2+ cells. Importantly, adoptive transfer of these IL‐33‐treated macrophages attenuated EAE development. Our data therefore demonstrate that IL‐33 plays a therapeutic role in autoimmune CNS disease by switching a predominantly pathogenic Th17/Th1 response to Th2 activity, and by polarization of anti‐inflammatory M2 macrophages.

Interleukin 27 attenuates collagen-induced arthritis

Objective: To investigate the potential role of interleukin (IL) 27 in rheumatoid arthritis (RA) by examining the expression of IL27 in the articular joints of patients with RA and the effect of recombinant IL27 in vivo in a murine model of collagen-induced arthritis (CIA). Methods: Synovial membranes from patients with RA were examined for the presence of IL27 by immunohistochemistry and by western blot. Mice developing CIA were treated with IL27 and the ensuing disease progression and immunological profile determined. The effect of IL27 on T-cell response in vitro was also ascertained. Results: IL27 was clearly detected in the RA synovial membranes. Short-term administration of IL27 at the onset of the disease significantly attenuated disease severity compared with untreated controls. Histological examination showed that while untreated mice developed severe cellular infiltration in the joints, synovial hyperplasia and joint erosion, this pathology was profoundly reduced in IL27-treated animals. Treatment of mice with IL27 also decreased the amounts of serum IL6 and collagen-specific IgG2a. Spleen and lymph node cells from the IL27-treated mice produced significantly less interferon γ and IL17 than cells from the control mice when cultured with collagen in vitro. Conclusion: These results demonstrate that IL27 may be a potential therapeutic agent against RA at the onset of the disease.

Effects of nitric oxide on the induction and differentiation of Th1 cells

We have previously shown that mice lacking inducible NO synthase are markedly more susceptible to Leishmania major infection but developed a significantly enhanced Th1 cell response compared with wild‐type mice. Furthermore, at high concentrations, NO inhibited IL‐12 synthesis by activated macrophages, thereby indirectly suppressing the expansion of Th1 cells. We report here that at low concentrations, NO selectively enhanced the induction of Th1 cells and had no effect on Th2 cells. NO exerted this effect in synergy with IL‐12 during Th1 cell differentiation and had no effect on fully committed Th1 cells. NO appears to affect CD4+ T cells directly and not at the antigen‐presenting cells. These results therefore provide an additional pathway by which NO modulates the immune response and contributes to the homeostasis of the immune system.

Nitric oxide induces CD4+CD25+ Foxp3− regulatory T cells from CD4+CD25− T cells via p53, IL-2, and OX40

The principal aim of the immune system is to establish a balance between defense against pathogens and avoidance of autoimmune disease. This balance is achieved partly through regulatory T cells (Tregs). CD4+CD25+ Tregs are either naturally occurring or induced by antigens and are characterized by the expression of the X-linked forkhead/winged helix transcription factor, Foxp3. Here we report a previously unrecognized subset of CD4+CD25+ Tregs derived from CD4+CD25− T cells induced by nitric oxide (NO). The induction of Tregs (NO-Tregs) is independent of cGMP but depends on p53, IL-2, and OX40. NO-Tregs produced IL-4 and IL-10, but not IL-2, IFNγ, or TGFβ. The cells were GITR+, CD27+, T-betlow, GATA3high, and Foxp3−. NO-Tregs suppressed the proliferation of CD4+CD25− T cells in vitro and attenuated colitis- and collagen-induced arthritis in vivo in an IL-10-dependent manner. NO-Tregs also were induced in vivo in SCID mice adoptively transferred with CD4+CD25− T cells in the presence of LPS and IFNγ, and the induction was completely inhibited by NG-monomethyl-l-arginine, a pan NO synthase inhibitor. Therefore, our findings uncovered a previously unrecognized function of NO via the NO–p53–IL-2–OX40–survivin signaling pathway for T cell differentiation and development.

Nitric oxide preferentially induces type 1 T cell differentiation by selectively up-regulating IL-12 receptor β2 expression via cGMP

Nitric oxide plays an important role in immune regulation. We have shown that although high concentrations of NO generally were immune-suppressive, low concentrations of NO selectively enhanced the differentiation of T helper (Th)1 cells but not Th2 cells. This finding provided an explanation for the crucial role of NO in defense against intracellular pathogens. However, the mechanism for the selective induction of Th1 cells was unknown. We report here that at low concentrations, NO activates soluble guanylyl cyclase, leading to the up-regulation of cGMP, which selectively induces the expression of IL-12 receptor β2 but has no effect on IL-4 receptor. Because IL-12 and IL-4 are the key cytokines for induction of Th1 and Th2 cells, respectively, these results, therefore, provide the mechanism for the selective action of NO on T cell subset differentiation. Furthermore, this selectivity also applies to CD8+ cytotoxic and human T cells and, thus, demonstrates the general implication of this observation in immune regulation. Our results also provide an example of the regulation of cytokine receptor expression by NO. The selectivity of such action via cGMP suggests that it is amenable to therapeutic intervention.

Role of nitric oxide in the regulation of T cell functions

There is a close relation between T helper (Th) 1 cells and nitric oxide in disease. Thus it is possible that a reciprocal regulatory mechanism exists between them. This paper briefly describes the experimental studies which have helped elucidate the mechanism by which nitric oxide selectively enhances Th 1 cell proliferation and the potential effect of nitric oxide on regulatory T (Treg) cells. On the basis of the results the authors propose that nitric oxide represents an additional signal for the induction of T cell subset response, contributing to the increasingly complex network of immune regulation essential for health and disease.

The sushi domain of soluble IL-15 receptor alpha is essential for binding IL-15 and inhibiting inflammatory and allogenic responses in vitro and in vivo

IL-15 is a pleiotropic cytokine that plays important roles in both innate and adaptive immunity. It is associated with a range of immunopathology, including rheumatoid arthritis and allograft rejection. IL-15 functions through the trimeric IL-15R complex, which consists of a high affinity binding α-chain and the common IL-2R β- and γ-chains. Characterization of IL-15/IL-15R interactions may facilitate the development of improved IL-15 antagonists for therapeutic interventions. We previously constructed soluble murine IL-15Rα (sIL-15Rα) by deleting the cytoplasmic and transmembrane domains. To localize the functional domain of IL-15Rα, we have now constructed various truncated versions of sIL-15Rα. The shortest region retaining IL-15 binding activity is a 65-aa sequence spanning the Sushi domain of IL-15Rα. Sushi domains, common motifs in protein-protein interactions, contain four cysteines forming two disulfide bonds in a 1-3 and 2-4 pattern. Amino acid substitution of the first or fourth cysteine in sIL-15Rα completely abolished its IL-15 binding activity. This also abrogated the ability of sIL-15Rα to neutralize IL-15-induced proinflammatory cytokine production and anti-apoptotic response in vitro. Furthermore, the mutant sIL-15Rα lost its ability to inhibit carrageenan-induced local inflammation and allogenic cell-induced T cell proliferation and cytokine production in vivo. Thus, the Sushi domain is critical for the functional activity of sIL-15Rα.

IL-15 induces type 1 and type 2 CD4+ and CD8+ T cells proliferation but is unable to drive cytokine production in the absence of TCR activation or IL-12/IL-4 stimulation in vitro

Interleukin 15 (IL‐15) is a pleiotropic cytokine produced principally by monocytes and affects both innate and acquired immunity. It has been shown that IL‐15 is essential for the proliferation and maintenance of CD8+ memory cells but has little or no effect on naive CD8+ cells or CD4+ T cells. We report here, using an in vitro culture system of antigen‐specific OVA TCR transgenic T cells as well as normal mouse T cell activated with anti‐CD3 antibody that IL‐15, at high concentrations, induced proliferation of both naive and memory CD4+and CD8+ cells. IL‐15 also enhanced the differentiation of type 1 (IFN‐γ‐producing) and type 2 (IL‐5‐producing) CD4+ and CD8+ T cells under IL‐12 and IL‐4 driving conditions, respectively. However, IL‐15 alone was not efficient in stimulating cytokine production of these cells in the absence of T cell subset driving cytokines (IL‐12 or IL‐4) and / or simultaneous TCR activation. Together, these results demonstrate that IL‐15, at high dose, is a pan‐T cell growth factor. The apparent requirement of IL‐15 for the maintenance of memory CD8+ cell in vivo may reflect the exceptionally restricted nature of this subpopulation of cells for IL‐15. The inability of IL‐15 alone to stimulate cytokine synthesis also suggests that IL‐15 on its own doesnot drive antigen‐specific T cells to exhaustion. The levels of these cells are maintained by IL‐15 and they are only mobilized to carry out effector functions when subsequently confronted with specific pathogens.