Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27

IL-27 is a recently defined family member of the long-chain, four-helix bundle cytokines, which consist of EBI3, an IL-12p40-related protein, and p28, an IL-12p35-related polypeptide. The role of IL-27 in the regulation of experimental autoimmune encephalomyelitis has never been studied. We show in this study that neutralizing the in vivo function of IL-27 by Abs against IL-27 p28 rapidly suppressed an ongoing long-lasting disease in C57BL/6 mice. These Abs were then used to determine the mechanistic basis of disease suppression. We show in this study that IL-27 is involved not only in the polarization of naive T cells undergoing Ag-specific T cell activation, but also in promoting the proliferation and IFN-gamma production by polarized T cells, including the long term Th1 line that has been previously selected against the target encephalitogenic determinant. This may explain in part why neutralizing IL-27 suppresses an already established disease in a very rapid and significant manner.     

Novel IL27p28/IL12p40 Cytokine Suppressed Experimental Autoimmune Uveitis by Inhibiting Autoreactive Th1/Th17 Cells and Promoting Expansion of Regulatory T Cells

IL-12 family cytokines are important in host immunity. Whereas some members (IL-12, IL-23) play crucial roles in pathogenesis of organ-specific autoimmune diseases by inducing the differentiation of Th1 and Th17 lymphocytes, others (IL-27 and IL-35) suppress inflammatory responses and limit tissue injury induced by these T cell subsets. In this study, we have genetically engineered a novel IL27p28/IL12p40 heterodimeric cytokine (p28/p40) that antagonizes signaling downstream of the gp130 receptor. We investigated whether p28/p40 can be used to ameliorate uveitis, a CNS inflammatory disease. Experimental autoimmune uveitis (EAU) is the mouse model of human uveitis and is mediated by Th1 and Th17 cells. We show here that p28/p40 suppressed EAU by inhibiting the differentiation and inflammatory responses of Th1 and Th17 cells while promoting expansion of IL-10⁺- and Foxp3⁺-expressing regulatory T cells. Lymph node cells from mice treated with p28/p40 blocked adoptive transfer of EAU to naïve syngeneic mice by immunopathogenic T cells and suppressive effects of p28/p40 derived in part from antagonizing STAT1 and STAT3 pathways induced by IL-27 and IL-6. Interestingly, IL27p28 also suppressed EAU, but to a lesser extent than p28/p40. The inhibition of uveitogenic lymphocyte proliferation and suppression of EAU by p28/p40 and IL27p28 establish efficacy of single chain and heterodimeric IL-12 family cytokines in treatment of a CNS autoimmune disease. Creation of the biologically active p28/p40 heterodimeric cytokine represents an important proof-of-concept experiment, suggesting that cytokines comprising unique IL-12 α- and β-subunit pairing may exist in nature and may constitute a new class of therapeutic cytokines.     

Coordination between TLR9 signaling in macrophages and CD3 signaling in T cells induces robust expression of IL-30

IL-30, the p28 subunit of IL-27, interacts with EBV-induced gene 3 to form IL-27, which modulates both proinflammatory and anti-inflammatory responses during autoimmune or infectious disease. It also acts as a natural antagonist of gp130, thereby attenuating the signals of other gp130-associated cytokines. IL-30 regulation via LPS has been reported by others, but the intercellular communication that induces IL-30 expression is unknown. In this study, we show that treatment with anti-CD3/CD28 Abs plus CpG oligodeoxynucleotides induces robust expression of IL-30, whereas either treatment alone induces only low expression of IL-30. This observation in vitro mirrors the murine model in which administration of CpG under inflammatory conditions in vivo induces IL-30 expression. This robust induction of IL-30 occurs through the coordination of helper CD4(+) T cells and innate immune cells (e.g., macrophages) and, to a lesser degree, B cells via the CD40/CD154 signaling pathway. These findings reveal a previously unrecognized mechanism that integrates signaling pathways from T cells and macrophages at the cellular level to induce IL-30 expression.     

Cutting Edge: Suppression of GM-CSF Expression in Murine and Human T Cells by IL-27

GM-CSF is a potent proinflammatory cytokine that plays a pathogenic role in the CNS inflammatory disease experimental autoimmune encephalomyelitis. As IL-27 alleviates experimental autoimmune encephalomyelitis, we hypothesized that IL-27 suppresses GM-CSF expression by T cells. We found that IL-27 suppressed GM-CSF expression in CD4(+) and CD8(+) T cells in splenocyte and purified T cell cultures. IL-27 suppressed GM-CSF in Th1, but not Th17, cells. IL-27 also suppressed GM-CSF expression by human T cells in nonpolarized and Th1- but not Th17-polarized PBMC cultures. In vivo, IL-27p28 deficiency resulted in increased GM-CSF expression by CNS-infiltrating T cells during Toxoplasma gondii infection. Although in vitro suppression of GM-CSF by IL-27 was independent of IL-2 suppression, IL-10 upregulation, or SOCS3 signaling, we observed that IL-27-driven suppression of GM-CSF was STAT1 dependent. Our findings demonstrate that IL-27 is a robust negative regulator of GM-CSF expression in T cells, which likely inhibits T cell pathogenicity in CNS inflammation.     

Treatment with IL-27 attenuates experimental colitis through the suppression of the development of IL-17-producing T helper cells

Inflammatory bowel disease (IBD) represents a group of chronic inflammatory diseases characterized by inflammation and relapsing gastrointestinal disorders. Recent studies have shown that Th17 cells, which are well known as key mediators of chronic inflammation, have a pivotal role in onset and development of IBD in humans and mice, alike. In recent years, it has been reported that IL-27, which is an IL-12-related heterodimeric cytokine consisting of EBI3 and p28 subunits, act directly on naive T cells to suppress the differentiation of Th17 cells. However, effects of exogenous IL-27 on the IBD are not well elucidated. To clarify the suppressive effect of IL-27 treatment on IBD, we applied the flexible linking method to EBI3 and p28 subunits and generated a single-chain human IL-27 (scIL-27). scIL-27 inhibited xenogenic mouse Th17 cell differentiation in vitro, indicating that scIL-27 also acts in mouse immune systems. In a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced mouse acute colitis model, subcutaneous scIL-27 treatment significantly improved the colon length, extent of necrosis, and ulceration and thickened epithelium and several pathological scores in a dose-dependent manner. scIL-27 clearly suppressed several inflammatory cytokines, including IL-17, in inflamed colon, except for anti-inflammatory cytokine IL-10. The mesenteric lymph node cells from scIL-27-treated mice also exhibited a reduced inflammatory response and, furthermore, a lower population of Th17 cells than those of PBS-treated mice. Finally, we showed the therapeutic efficacy of scIL-27 on TNBS-induced colitis even after active colitis was established. These results suggest new possible therapeutic approaches for IBD, including disorders such as Crohn's disease and ulcerative colitis.     

Suppressive effect of IL-27 on encephalitogenic Th17 cells and the effector phase of experimental autoimmune encephalomyelitis

IL-27 has been shown to play a suppressive role in experimental autoimmune encephalomyelitis (EAE) as demonstrated by more severe disease in IL-27R-deficient (WSX-1(-/-)) mice. However, whether IL-27 influences the induction or effector phase of EAE is unknown. This is an important question as therapies for autoimmune diseases are generally started after autoreactive T cells have been primed. In this study, we demonstrate maximal gene expression of IL-27 subunits and its receptor in the CNS at the effector phases of relapsing-remitting EAE including disease peak and onset of relapse. We also show that activated astrocyte cultures secrete IL-27p28 protein which is augmented by the endogenous factor, IFN-gamma. To investigate functional significance of a correlation between gene expression and disease activity, we examined the effect of IL-27 at the effector phase of disease using adoptive transfer EAE. Exogenous IL-27 potently suppressed the ability of encephalitogenic lymph node and spleen cells to transfer EAE. IL-27 significantly inhibited both nonpolarized and IL-23-driven IL-17 production by myelin-reactive T cells thereby suppressing their encephalitogenicity in adoptive transfer EAE. Furthermore, we demonstrate a strong suppressive effect of IL-27 on active EAE in vivo when delivered by s.c. osmotic pump. IL-27-treated mice had reduced CNS inflammatory infiltration and, notably, a lower proportion of Th17 cells. Together, these data demonstrate the suppressive effect of IL-27 on primed, autoreactive T cells, particularly, cells of the Th17 lineage. IL-27 can potently suppress the effector phase of EAE in vivo and, thus, may have therapeutic potential in autoimmune diseases such as multiple sclerosis.     

Peroxisome proliferator-activated receptor-alpha agonist fenofibrate regulates IL-12 family cytokine expression in the CNS: relevance to multiple sclerosis

The interleukin-12 (IL-12) family of cytokines which includes IL-12, IL-23, and IL-27 play critical roles in T cell differentiation and are important modulators of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Previously, we demonstrated that peroxisome proliferator-activated receptor (PPAR) -α agonists suppress the development of EAE. The present studies demonstrated that the PPAR-α agonist fenofibrate inhibited the secretion of IL-12p40, IL-12p70 (p35/p40), IL-23 (p19/p40), and IL-27p28 by lipopolysaccharide-stimulated microglia. The cytokines interferon-γ and tumor necrosis factor-α also stimulated IL-12 p40 and IL-27 p28 expression by microglia, which was suppressed by fenofibrate. Furthermore, fenofibrate inhibited microglial expression of CD14 which plays a critical role in TLR signaling, suggesting a mechanism by which this PPAR-α agonist regulates the production of these pro-inflammatory molecules. In addition, fenofibrate suppressed the secretion of IL-12p40, IL-23, and IL-27p28 by lipopolysaccharide-stimulated astrocytes. Importantly, fenofibrate suppression of EAE was associated with decreased expression of IL-12 family cytokine mRNAs as well as mRNAs encoding TLR4, CD14, and MyD88 known to play critical roles in MyD88-dependent TLR signaling. These novel observations suggest that PPAR-α agonists including fenofibrate may modulate the development of EAE, at least in part, by suppressing the production of IL-12 family cytokines and MyD88-dependent signaling.     

Dual role of the IL-12/IFN-gamma axis in the development of autoimmune myocarditis: induction by IL-12 and protection by IFN-gamma.

IL-12 and IFN-gamma positively regulate each other and type 1 inflammatory responses, which are believed to cause tissue damage in autoimmune diseases. We investigated the role of the IL-12/IFN-gamma (Th1) axis in the development of autoimmune myocarditis. IL-12p40-deficient mice on a susceptible background resisted myocarditis. In the absence of IL-12, autospecific CD4(+) T cells proliferated poorly and showed increased Th2 cytokine responses. However, IFN-gamma-deficient mice developed fatal autoimmune disease, and blockade of IL-4R signaling did not confer susceptibility to myocarditis in IL-12p40-deficient mice, demonstrating that IL-12 triggers autoimmunity by a mechanism independent of the effector cytokines IFN-gamma and IL-4. In conclusion, our results suggest that the IL-12/IFN-gamma axis is a double-edged sword for the development of autoimmune myocarditis. Although IL-12 mediates disease by induction/expansion of Th1-type cells, IFN-gamma production from these cells limits disease progression.     

Suppression of TNF-α and IL-1 signaling identifies a mechanism of homeostatic regulation of macrophages by IL-27

IL-27 is a pleiotropic cytokine with both activating and inhibitory functions on innate and acquired immunity. IL-27 is expressed at sites of inflammation in cytokine-driven autoimmune/inflammatory diseases, such as rheumatoid arthritis, psoriasis, inflammatory bowel disease, and sarcoidosis. However, its role in modulating disease pathogenesis is still unknown. In this study, we found that IL-27 production is induced by TNF-α in human macrophages (MΦ) and investigated the effects of IL-27 on the responses of primary human MΦ to the endogenous inflammatory cytokines TNF-α and IL-1. In striking contrast to IL-27-mediated augmentation of TLR-induced cytokine production, we found that IL-27 suppressed MΦ responses to TNF-α and IL-1β, thus identifying an anti-inflammatory function of IL-27. IL-27 blocked the proximal steps of TNF-α signaling by downregulating cell-surface expression of the signaling receptors p55 and p75. The mechanism of inhibition of IL-1 signaling was downregulation of the ligand-binding IL-1RI concomitant with increased expression of the receptor antagonist IL-1Ra and the decoy receptor IL-1RII. These findings provide a mechanism for suppressive effects of IL-27 on innate immune cells and suggest that IL-27 regulates inflammation by limiting activation of MΦ by inflammatory cytokines while preserving initial steps in host defense by augmenting responses to microbial products.     

Two-sided roles of IL-27: induction of Th1 differentiation on naive CD4+ T cells versus suppression of proinflammatory cytokine production including IL-23-induced IL-17 on activated CD4+ T cells partially through STAT3-dependent mechanism

Recent lines of evidence have demonstrated that IL-27, a newly identified IL-12-related cytokine, has two apparently conflicting roles in immune responses: one as an initiator of Th1 responses and the other as an attenuator of inflammatory cytokine production. Although the IL-27-mediated Th1 initiation mechanism has been elucidated, little is known about the molecular basis for the suppression of cytokine production. In the present study, we demonstrated that IL-27 suppressed the production of various proinflammatory cytokines by fully activated CD4+ T cells while it had no effect on the cytokine production by CD4+ T cells at early phases of activation. IL-27 also suppressed IL-17 production by activated CD4+ T cells, thereby counteracting IL-23, another IL-12-related cytokine with proinflammatory effects. In fully activated CD4+ T cells, STAT3 was preferentially activated by IL-27 stimulation, whereas both STAT1 and 3 were activated by IL-27 in early activated CD4+ T cells. Lack of STAT3 in fully activated cells impaired the suppressive effects of IL-27. These data indicated that the preferential activation of STAT3 in fully activated CD4+ T cells plays an important role in the cytokine suppression by IL-27/WSX-1.     

IL-4, IL-10, IL-13, and TGF-beta from an altered peptide ligand-specific Th2 cell clone down-regulate adoptive transfer of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is induced in the SJL/J mouse by adoptive transfer of activated proteolipid protein peptide (PLP) 139-151-specific Th1 cells. T cells responding to altered peptide ligands (APL) of PLP, previously shown to induce Th2 differentiation and regulate disease in PLP-immunized mice, do not transfer EAE. However, the exact mechanism of disease regulation by APL-specific T cells has not been elucidated. In this report, we show that 1F1, a Th2 clone specific for an APL of PLP139-151 can prevent adoptive transfer of EAE when cocultured with PLP-encephalitogenic spleen cells (PLP-spleen). Cytokines from activated 1F1 cells were detected by hybridization of mRNA to oligonucleotide arrays (DNA chip) and by ELISA. The Th2 cytokines found to be present at the highest protein and mRNA levels were evaluated for their role in suppression of adoptive transfer of EAE from PLP-activated spleen cell cultures. Abs to individual cytokines in 1F1 PLP-spleen cocultures suggested that IL-4, IL-13, and TGF-beta played a significant role in suppressing EAE. Abs to the combination of IL-4, IL-10, IL-13, and TGF-beta completely neutralized the protective effect of 1F1. Addition of Th2 cytokines to PLP-spleen cultures showed that IL-13 and TGF-beta were each individually effective and low levels of IL-4 synergized with IL-13 to inhibit disease transfer. IL-5, IL-9, and IL-10 had little or no effect whereas GM-CSF slightly enhanced EAE. Our results demonstrate that Th2 cytokines derived from APL-specific Th2 cells can effectively down-regulate the encephalitogenic potential of PLP-spleen cells if present during their reactivation in culture.     

Peroxisome proliferator-activated receptor-gamma agonists suppress the production of IL-12 family cytokines by activated glia

The IL-12 family of cytokines, which include IL-12, IL-23, and IL-27, play critical roles in the differentiation of Th1 cells and are believed to contribute to the development of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Relatively little is known concerning the expression of IL-12 family cytokines by cells of the CNS, the affected tissue in MS. Previously, we and others demonstrated that peroxisome proliferator-activated receptor (PPAR)-gamma agonists suppress the development of EAE, alter T cell proliferation and phenotype, and suppress the activation of APCs. The present studies demonstrated that PPAR-gamma agonists, including the naturally occurring 15-deoxy-Delta(12,14)-PGJ(2) and the synthetic thiazoladinedione rosiglitazone, inhibited the induction of IL-12p40, IL-12p70 (p35/p40), IL-23 (p19/p40), and IL-27p28 proteins by LPS-stimulated primary microglia. In primary astrocytes, LPS induced the production of IL-12p40, IL-23, and IL-27p28 proteins. However, IL-12p70 production was not detected in these cells. The 15-deoxy-Delta(12,14)-PGJ(2) potently suppressed IL-12p40, IL-23, and IL-27p28 production by primary astrocytes, whereas rosiglitazone suppressed IL-23 and IL-27p28, but not IL-12p40 in these cells. These novel observations suggest that PPAR-gamma agonists modulate the development of EAE, at least in part, by inhibiting the production of IL-12 family cytokines by CNS glia. In addition, we demonstrate that PPAR-gamma agonists inhibit TLR2, MyD88, and CD14 expression in glia, suggesting a possible mechanism by which these agonists modulate IL-12 family cytokine expression. Collectively, these studies suggest that PPAR-gamma agonists may be beneficial in the treatment of MS.     

Paradoxical anti-inflammatory actions of TNF-alpha: inhibition of IL-12 and IL-23 via TNF receptor 1 in macrophages and dendritic cells

IL-12 and TNF-alpha are central proinflammatory cytokines produced by macrophages and dendritic cells. Disregulation of TNF-alpha is associated with sepsis and autoimmune diseases such as rheumatoid arthritis. However, new evidence suggests an anti-inflammatory role for TNF-alpha. TNF-alpha-treated murine macrophages produced less IL-12p70 and IL-23, after stimulation with IFN-gamma and LPS. Frequency of IL-12p40-producing macrophages correspondingly decreased as measured by intracellular cytokine staining. IL-12p40 production was also inhibited in dendritic cells. TNFR1 was established as the main receptor involved in IL-12p40 regulation, because IL-12p40 levels were not affected by TNF-alpha in TNFR1(-/-)-derived macrophages. Macrophages activated during Listeria monocytogenes infection were more susceptible to inhibition by TNF-alpha than cells from naive animals, which suggests a regulatory role for TNF-alpha in later stages of infection. This nonapoptotic anti-inflammatory regulation of IL-12 and IL-23 is an important addition to the multitude of TNF-alpha-induced responses determined by cell-specific receptor signaling.     

白介素-33在疾病中的作用

白介素-33(IL-33)是近年来新发现的IL-1家族的新成员,通过结合其受体ST2诱导Th2型细胞因子的产生。IL-33既可以调节Th2型免疫反应、刺激肥大细胞产生前炎性因子,又可以作为核因子调控基因转录。IL-33在血管性疾病、变态反应性疾病、自身免疫性疾病和炎症性疾病中均发挥重要作用。对IL-33功能及机制的研究将有助于进一步了解这些疾病的致病机制,为疾病治疗提供新的策略。     

Coadministration of plasmid DNA constructs encoding an encephalitogenic determinant and IL-10 elicits regulatory T cell-mediated protective immunity in the central nervous system

We have previously shown that Ag-specific IL-10-producing regulatory T cells (Tr1) participate in the regulation of experimental autoimmune encephalomyelitis and that their specificity undergoes determinant spread in a reciprocal manner to effector T cell specificity. The current study shows that coadministration of plasmid DNA vaccines encoding IL-10 together with a plasmid encoding a myelin basic protein (MBP) encephalitogenic determinant during an ongoing disease rapidly amplifies this Tr1-mediated response, in a disease-specific manner. Thus, coadministration of both plasmids, but not the plasmid DNA encoding MBP alone, rapidly suppresses an ongoing disease. Tolerance included elevation in Ag-specific T cells producing IL-10 and an increase in apoptosis of cells around high endothelial venules in the CNS after successful therapy. Tolerance could be transferred by MBP-specific primary T cells isolated from protected donors and reversed by neutralizing Abs to IL-10 but not to IL-4. Due to the nature of determinant spread in this model, we could bring about evidence implying that rapid and effective induction of Tr1-induced active tolerance is dependent on redirecting the Tr1 response to the epitope to which the effector function dominates the response at a given time. The consequences of these findings to multiple sclerosis, and possibly other inflammatory autoimmune diseases are discussed.