Inflammatory cytokines and endogenous anti-oxidants are variables affecting disease progression in multiple sclerosis (MS). Here we demonstrate the dual capacity of triterpenoids to simultaneously repress production of IL-17 and other pro-inflammatory mediators while exerting neuroprotective effects directly through Nrf2-dependent induction of anti-oxidant genes. Derivatives of the natural triterpene oleanolic acid, namely CDDO-trifluoroethyl-amide (CDDO-TFEA), completely suppressed disease in a murine model of MS, experimental autoimmune encephalomyelitis (EAE), by inhibiting Th1 and Th17 mRNA and cytokine production. Encephalitogenic T cells recovered from treated mice were hypo-responsive to myelin antigen and failed to adoptively transfer the disease. Microarray analyses showed significant suppression of pro-inflammatory transcripts with concomitant induction of anti-inflammatory genes including Ptgds and Hsd11b1. Finally, triterpenoids induced oligodendrocyte maturation in vitro and enhanced myelin repair in an LPC-induced non-inflammatory model of demyelination in vivo. These results demonstrate the unique potential of triterpenoid derivatives for the treatment of neuroinflammatory disorders such as MS.
The synthetic triterpenoid, CDDO-Me, has potent antiproliferative and antioxidant properties. However, its immunomodulatory effects in the context of LPS challenge are incompletely defined. Pretreatment with oral CDDO-Me significantly improved survival following lethal-dose LPS challenge in mice. To define this protection further, we measured effects of CDDO-Me pretreatment on splenocyte populations and cytokine production following LPS challenge, using low-level LPS pretreatment as an in vivo control for reducing cytokine production. Despite similar decreases in levels of LPS-inducible, circulating proinflammatory cytokines (IL-12p70, IFN-γ, IL-6, IL-17, and IL-23) and increases in heme oxygenase 1 (HO-1) protein expression, low-dose LPS and CDDO-Me pretreatments markedly differed in their overall response profiles. Splenocytes from LPS-pretreated mice contained reduced numbers of dendritic cells, increased percentages of Th17 and T-regulatory cells, lower levels of TLR-inducible IL-6, and higher levels of TLR-inducible IL-10. In contrast, CDDO-Me protection against LPS challenge had no impact on absolute numbers or distribution of splenocyte subsets, despite attenuating in vivo induction of proinflammatory cytokines in an IL-10-independent manner. Together, these results suggest that CDDO-Me pretreatment uniquely confers protection against LPS challenge by modulating the in vivo immune response to LPS. Thus, CDDO-Me potentially represents a novel oral agent for use in LPS-mediated inflammatory diseases.
Bryonolic acid (BA) is a triterpenoid found in the Cucurbitaceae family of plants. Our interests in the immunomodulatory effects of this class of natural products led us to discover that BA induces a marked increase in the expression of a phase 2 response enzyme, heme oxygenase 1 (HO-1) in a dose dependent manner. This phenotype has translational implications in malarial disease progression, and consequently we developed a large scale isolation method for BA that will be enabling in terms of future in vitro and in vivo analysis. We have determined ideal growth conditions and time scale for maximizing BA content in the roots of Cucurbita pepo L., and analyzed BA production by HPLC. Large-scale extraction yielded 1.34% BA based on dry weight, allowing for the isolation of BA on a multi-gram scale.
Cyclin-dependent kinase 5 drives T cell activation and disease in EAE in part by regulating the actin binding protein coronin 1a.
Cyclin-dependent kinase 5 (Cdk5) is a ubiquitously expressed serine/threonine kinase. However, a requirement for Cdk5 has been demonstrated only in postmitotic neurons where there is abundant expression of its activating partners p35 and/or p39. Although hyperactivation of the Cdk5–p35 complex has been found in a variety of inflammatory neurodegenerative disorders, the potential contribution of nonneuronal Cdk5–p35 activity has not been explored in this context. We describe a previously unknown function of the Cdk5–p35 complex in T cells that is required for induction of experimental autoimmune encephalomyelitis (EAE). T cell receptor (TCR) stimulation leads to a rapid induction of Cdk5–p35 expression that is required for T lymphocyte activation. Chimeric mice lacking Cdk5 gene expression in hematopoietic tissues (Cdk5−/−C) are resistant to induction of EAE, and adoptive transfer of either Cdk5−/−C or p35−/− encephalitogenic lymphocytes fails to transfer disease. Moreover, our data reveal a novel mechanism involving Cdk5-mediated phosphorylation of the actin modulator coronin 1a on threonine 418. Cdk5-deficient lymphocytes lack this posttranslational modification of coronin 1a and exhibit defective TCR-induced actin polarization and reduced migration toward CCL-19. These data define a distinct role for Cdk5 in lymphocyte biology and suggest that inhibition of this kinase may be beneficial in the treatment of T cell–mediated inflammatory disorders.
In naïve animals, γδ T cells are innate sources of IL-17, a potent proinflammatory cytokine mediating bacterial clearance as well as autoimmunity. However, mechanisms underlying the generation of these cells in vivo remain unclear. Here we show that TGFβ1 plays a key role in the generation of IL-17+ γδ T cells, and that it mainly occurs in the thymus particularly during the postnatal period. Interestingly, IL-17+ γδ TCR+ thymocytes were mainly CD44highCD25low cells, which seem to derive from DN4 γδ TCR+ cells that acquired CD44 and IL-17 expression. Our findings identify a novel developmental pathway during which IL-17-competent γδ T cells arise in the thymus by a TGFβ1-dependent mechanism.
γδ T cells; IL-17; TGFβ
It has been reported that retinoic acid (RA) enhances regulatory T (T reg) cell conversion by inhibiting the secretion of cytokines that interfere with conversion. This report shows that these conclusions provide a partial explanation at best. First, RA not only interfered with cytokine secretion but also with the ability of these cytokines to inhibit T reg cell conversion of naive T cells. Furthermore, RA enhanced conversion even in the absence of inhibitory cytokines. The latter effect depended on the RA receptor α (RARα) but did not require Smad3, despite the fact that RA enhanced Smad3 expression. The RARα1 isoform was not essential for RA-dependent enhancement of transforming growth factor β–driven conversion, suggesting that conversion can also be mediated by RARα2. Interleukin (IL)-6 strongly reduced RARα expression levels such that a deficiency of the predominant RARα1 isoform leaves too little RARα2 for RA to inhibit the generation of Th17 cells in the presence of IL-6.
Transforming growth factor (TGF)-β is the prototype in a family of secreted proteins that act in autocrine and paracrine pathways to regulate cell development and function. Normal cells typically coexpress TGF-β receptors and one or more isoforms of TGF-β, thus the synthesis and secretion of TGF-β as an inactive latent complex is considered an essential step in regula-ting the activity of this pathway. To determine whether intracellular activation of TGF-β results in TGF-β ligand–receptor interactions within the cell, we studied pristane-induced plasma cell tumors (PCTs). We now demonstrate that active TGF-β1 in the PCT binds to intracellular TGF-β type II receptor (TβRII). Disruption of the expression of TGF-β1 by antisense TGF-β1 mRNA restores localization of TβRII at the PCT cell surface, indicating a ligand-induced impediment in receptor trafficking. We also show that retroviral expression of a truncated, dominant-negative TβRII (dnTβRII) effectively competes for intracellular binding of active ligand in the PCT and restores cell surface expression of the endogenous TβRII. Analysis of TGF-β receptor–activated Smad2 suggests the intracellular ligand–receptor complex is not capable of signaling. These data are the first to demonstrate the formation of an intracellular TGF-β–receptor complex, and define a novel mechanism for modulating the TGF-β signaling pathway.
receptor; trafficking; intracellular; signal-transduction; plasmacytoma
In addition to classic Smad signaling pathways, the pleiotropic immunoregulatory cytokine TGF-β1 can activate MAP kinases, but a role for TGF-β1-MAP kinase pathways in T cells has not been defined heretofore. We have shown previously that TGF-β1 inhibits Th1 development by inhibiting IFN-γ’s induction of T-bet and other Th1 differentiation genes, and that TGF-β1 inhibits receptor-proximal IFN-γ-Jak-Stat signaling responses. We now show that these effects of TGF-β1 are independent of the canonical TGF-β1 signaling module Smad3, but involve a specific MAP kinase pathway. In primary T cells, TGF-β1 activated the MEK/ERK and p38 MAP kinase pathways, but not the JNK pathway. Inhibition of the MEK/ERK pathway completely eliminated the inhibitory effects of TGF-β1 on IFN-γ responses in T cells, whereas inhibition of the p38 pathway had no effect. Thus, TGF-β1’s inhibition of IFN-γ signaling in T cells is mediated through a highly specific Smad3-independent, MEK/ERK-dependent signaling pathway.
Transforming growth factor (TGF)-β1 is a major pluripotential cytokine with a pronounced immunosuppressive effect and its deficiency results in lethal autoimmunity in mice. However, mechanisms of its immunosuppressive action are not completely understood. Here, we report that TGF-β1 supports the maintenance of Foxp3 expression, regulatory function, and homeostasis in peripheral CD4+CD25+ regulatory T (T reg) cells, but is not required for their thymic development. We found that in 8–10-d-old TGF-β1–deficient mice, peripheral, but not thymic, T reg cells are significantly reduced in numbers. Moreover, our experiments suggest that a defect in TGF-β–mediated signaling in T reg cells is associated with a decrease in Foxp3 expression and suppressor activity. Thus, our results establish an essential link between TGF-β1 signaling in peripheral T reg cells and T reg cell maintenance in vivo.
Our previous work demonstrated that cytotoxic T lymphocyte (CTL)-mediated tumor immunosurveillance of the 15-12RM tumor could be suppressed by a CD1d-restricted lymphocyte, most likely a natural killer (NK) T cell, which produces interleukin (IL)-13. Here we present evidence for the effector elements in this suppressive pathway. T cell–reconstituted recombination activating gene (RAG)2 knockout (KO) and RAG2/IL-4 receptor α double KO mice showed that inhibition of immunosurveillance requires IL-13 responsiveness by a non–T non–B cell. Such nonlymphoid splenocytes from tumor-bearing mice produced more transforming growth factor (TGF)-β, a potent inhibitor of CTL, ex vivo than such cells from naive mice, and this TGF-β production was dependent on the presence in vivo of both IL-13 and CD1d-restricted T cells. Ex vivo TGF-β production was also abrogated by depleting either CD11b+ or Gr-1+ cells from the nonlymphoid cells of tumor-bearing mice. Further, blocking TGF-β or depleting Gr-1+ cells in vivo prevented the tumor recurrence, implying that TGF-β made by a CD11b+ Gr-1+ myeloid cell, in an IL-13 and CD1d-restricted T cell–dependent mechanism, is necessary for down-regulation of tumor immunosurveillance. Identification of this stepwise regulation of immunosurveillance, involving CD1-restricted T cells, IL-13, myeloid cells, and TGF-β, explains previous observations on myeloid suppressor cells or TGF-β and provides insights for targeted approaches for cancer immunotherapy, including synergistic blockade of TGF-β and IL-13.
TGF-β; NKT cells; immunologic surveillance; myeloid cells; IL-13
We have previously demonstrated that interleukin (IL)-10–deficient (IL-10 knockout [KO]) but not wild-type (WT) mice develop colitis after infection with Helicobacter hepaticus. Here, we show that infected recombination activating gene (RAG) KO mice develop intestinal inflammation after reconstitution with CD4+ T cells from IL-10 KO animals and that the cotransfer of CD4+ T cells from H. hepaticus–infected but not uninfected WT mice prevents this colitis. The disease-protective WT CD4+ cells are contained within the CD45RBlow fraction and unexpectedly were found in both the CD25+ and the CD25− subpopulations of these cells, their frequency being higher in the latter. The mechanism by which CD25+ and CD25− CD45RBlow CD4+ cells block colitis involves IL-10 and not transforming growth factor (TGF)-β, as treatment with anti–IL-10R but not anti–TGF-β monoclonal antibody abrogated their protective effect. In vitro, CD45RBlow CD4+ cells from infected WT mice were shown to produce IL-10 and suppress interferon-γ production by IL-10 KO CD4+ cells in an H. hepaticus antigen–specific manner. Together, our data support the concept that H. hepaticus infection results in the induction in WT mice of regulatory T cells that prevent bacteria-induced colitis. The induction of such cells in response to gut flora may be a mechanism protecting normal individuals against inflammatory bowel disease.
inflammatory bowel disease; regulatory T cells; intestinal flora; CD25; immunoregulation
CD4+CD25+ regulatory T cells inhibit organ-specific autoimmune diseases induced by CD4+CD25−T cells and are potent suppressors of T cell activation in vitro. Their mechanism of suppression remains unknown, but most in vitro studies suggest that it is cell contact–dependent and cytokine independent. The role of TGF-β1 in CD4+CD25+ suppressor function remains unclear. While most studies have failed to reverse suppression with anti–transforming growth factor (TGF)-β1 in vitro, one recent study has reported that CD4+CD25+ T cells express cell surface TGF-β1 and that suppression can be completely abrogated by high concentrations of anti–TGF-β suggesting that cell-associated TGF-β1 was the primary effector of CD4+CD25+-mediated suppression. Here, we have reevaluated the role of TGF-β1 in CD4+CD25+-mediated suppression. Neutralization of TGF-β1 with either monoclonal antibody (mAb) or soluble TGF-βRII-Fc did not reverse in vitro suppression mediated by resting or activated CD4+CD25+ T cells. Responder T cells from Smad3−/− or dominant-negative TGF-β type RII transgenic (DNRIITg) mice, that are both unresponsive to TGF-β1–induced growth arrest, were as susceptible to CD4+CD25+-mediated suppression as T cells from wild-type mice. Furthermore, CD4+CD25+ T cells from neonatal TGF-β1−/− mice were as suppressive as CD4+CD25+ from TGF-β1+/+ mice. Collectively, these results demonstrate that CD4+CD25+ suppressor function can occur independently of TGF-β1.
CD4+ suppressor T cells; autoimmunity; tolerance; immunoregulation; IL-2 receptor
TGF-βs play diverse and complex roles in many biological processes. In tumorigenesis, they can function either as tumor suppressors or as pro-oncogenic factors, depending on the stage of the disease. We have developed transgenic mice expressing a TGF-β antagonist of the soluble type II TGF-β receptor:Fc fusion protein class, under the regulation of the mammary-selective MMTV-LTR promoter/enhancer. Biologically significant levels of antagonist were detectable in the serum and most tissues of this mouse line. The mice were resistant to the development of metastases at multiple organ sites when compared with wild-type controls, both in a tail vein metastasis assay using isogenic melanoma cells and in crosses with the MMTV-neu transgenic mouse model of metastatic breast cancer. Importantly, metastasis from endogenous mammary tumors was suppressed without any enhancement of primary tumorigenesis. Furthermore, aged transgenic mice did not exhibit the severe pathology characteristic of TGF-β null mice, despite lifetime exposure to the antagonist. The data suggest that in vivo the antagonist may selectively neutralize the undesirable TGF-β associated with metastasis, while sparing the regulatory roles of TGF-βs in normal tissues. Thus this soluble TGF-β antagonist has potential for long-term clinical use in the prevention of metastasis.
Transforming growth factor β (TGFβ) can inhibit the in vitro proliferation, survival and differentiation of B cell progenitors, mature B lymphocytes and plasma cells. Here we demonstrate unexpected, age-dependent reductions in the bone marrow (BM) B cell progenitors and immature B cells in TGFβ1-/- mice. To evaluate TGFβ responsiveness during normal B lineage development, cells were cultured in interleukin 7 (IL7)±TGFβ. Picomolar doses of TGFβ1 reduced pro-B cell recoveries at every timepoint. By contrast, the pre-B cells were initially reduced in number, but subsequently increased compared to IL7 alone, resulting in a 4-fold increase in the growth rate for the pre-B cell population. Analysis of purified BM sub-populations indicated that pro-B cells and the earliest BP1- pre-B cells were sensitive to the inhibitory effects of TGFβ1. However, the large BP1+ pre-B cells, although initially reduced, were increased in number at days 5 and 7 of culture. These results indicate that TGFβ1 is important for normal B cell development in vivo, and that B cell progenitors are differentially affected by the cytokine according to their stage of differentiation.
Candida albicans is an opportunistic fungal pathogen and a major cause of morbidity and mortality in patients with compromised immune function. The cytokine response to tissue invasion by C. albicans can influence the differentiation and function of lymphocytes and other mononuclear cells that are critical components of the host response. While the production of transforming growth factor β (TGF-β) has been documented in mice infected with C. albicans and is known to suppress phagocyte function, the cellular source and role of this cytokine in the pathogenesis of systemic candidiasis are not well understood. We have investigated the source of production of TGF-β by immunohistochemical studies in tissue samples from patients with an uncommon complication of lymphoreticular malignancy, chronic disseminated candidiasis (CDC), and from a neutropenic-rabbit model of CDC. Liver biopsy specimens from patients with documented CDC demonstrated intense staining for extracellular matrix-associated TGF-β1 within inflammatory granulomas, as well as staining for TGF-β1 and TGF-β3 within adjacent hepatocytes. These results correlate with the immunolocalization of TGF-β observed in livers of infected neutropenic rabbits, using a neutralizing antibody that recognizes the mature TGF-β protein. Human peripheral blood monocytes incubated with C. albicans in vitro release large amounts of biologically active TGF-β1. The data demonstrate that local production of active TGF-βs by hepatocytes and by infected mononuclear cells is a component of the response to C. albicans infection that most probably contributes to disease progression in the immunocompromised host.
Purpose. Production of active transforming growth factor-β (TGF-β )
by human osteosarcoma may contribute to malignant progression through mechanisms
that include induction of angiogenesis, immune suppression and autocrine growth
stimulation of tumor cell growth.To study events associated with induction of cell proliferation
by TGF-β , we have evaluated the TGF-β pathway in two murine osteosarcoma cell lines, K7
Results. Northern and immunohistochemical analyses show that each cell
line expressesTGF-β1 and TGF-β3 mRNA and protein. Both cell lines secrete activeTGF-β 1
and display a 30–50% reduction in growth when cultured in the presence of a TGF-β blocking
antibody. Expression of TGF-β receptors TβRI, TβRII and TβRIII is demonstrated by affinity
labeling with 125
-TGF-β 1, and the intermediates, Smads 2, 3 and 4, are uniformly expressed.
Smads 2 and 3 are phosphorylated in response toTGF-β , while pRb phosphorylation in each
osteosarcoma cell line is not affected by either exogenousTGF-β or TGF-β antibody.
Conclusions. The data implicate events downstream of Smad activation,
including impaired regulation of pRb, in the lack of a growth inhibitory response toTGF-β ,
and indicate that this murine model of osteosarcoma is valid for investigating the roles of
autocrineTGF-β in vivo.
Transforming growth factor β1 (TGF-β1) regulates leukocytes and epithelial cells. To determine whether the pleiotropic effects of TGF-β1, a cytokine that is produced by both keratinocytes and Langerhans cells (LC), extend to epidermal leukocytes, we characterized LC (the epidermal contingent of the dendritic cell [DC] lineage) and dendritic epidermal T cells (DETC) in TGF-β1 null (TGF-β1 −/−) mice. I-A+ LC were not detected in epidermal cell suspensions or epidermal sheets prepared from TGF-β1 −/− mice, and epidermal cell suspensions were devoid of allostimulatory activity. In contrast, TCR-γδ+ DETC were normal in number and appearance in TGF-β1 −/− mice and, importantly, DETC represented the only leukocytes in the epidermis. Immunolocalization studies revealed CD11c+ DC in lymph nodes from TGF-β1 −/− mice, although gp40+ DC were absent. Treatment of TGF-β1 −/− mice with rapamycin abrogated the characteristic inflammatory wasting syndrome and prolonged survival indefinitely, but did not result in population of the epidermis with LC. Thus, the LC abnormality in TGF-β1 −/− mice is not a consequence of inflammation in skin or other organs, and LC development is not simply delayed in these animals. We conclude that endogenous TGF-β1 is essential for normal murine LC development or epidermal localization.