Simvastatin induces Foxp3+ T regulatory cells by modulation of transforming growth factor-�� signal transduction

Statins are widely used drugs for the treatment of hypercholesterolaemia. A number of recent studies have suggested that statins also have pleiotropic effects on immune responses and statins have proven to be effective in the treatment of autoimmune diseases in animal models. Foxp3⁺ T regulatory cells are a unique subset of CD4⁺ T cells that mediate immunosuppression. Foxp3⁺ T cells develop in the thymus, but can also be induced in peripheral sites in the presence of transforming growth factor‐β (TGF‐β). We demonstrate here that simvastatin blockade of the mevalonate pathway can mediate induction of mouse Foxp3⁺ T cells and that simvastatin can synergize with low levels of TGF‐β to induce Foxp3⁺ T cells. The effects of simvastatin are secondary to a blockade of protein geranylgeranylation, are mediated at late time‐points after T‐cell activation, and are associated with demethylation of the Foxp3 promoter. One major effect of simvastatin was inhibition of the induction of Smad6 and Smad7, inhibitory Smads that inhibit TGF‐β signalling. Our results suggest that one mechanism responsible for the immunosuppressive effects of statins is the ability to promote the generation of Foxp3⁺ T regulatory cells.     

Transforming growth factor-β regulates the growth of valve interstitial cells in vitro

Although valve interstitial cell (VIC) growth is an essential feature of injured and diseased valves, the regulation of VIC growth is poorly understood. Transforming growth factor (TGF)-β promotes VIC proliferation in early-stage wound repair; thus, herein, we tested the hypothesis that TGF-β regulates VIC proliferation under normal nonwound conditions using low-density porcine VIC monolayers. Cell numbers were counted during a 10-day period, whereas proliferation and apoptosis were quantified by bromodeoxyuridine staining and TUNEL, respectively. The extent of retinoblastoma protein phosphorylation and expression of cyclin D1, CDK 4, and p27 were compared using Western blot analysis. Adhesion was quantified using a trypsin adhesion assay, and morphological change was demonstrated by immunofluorescence localization of α-smooth muscle actin and vinculin. TGF-β-treated VICs were rhomboid; significantly decreased in number, proliferation, and retinoblastoma protein phosphorylation; and concomitantly had decreased expression of cyclin D1/CDK4 and increased expression of p27. TGF-β-treated VICs adhered better to substratum and had more vinculin plaques and α-smooth muscle actin stress fibers than did controls. Thus, the regulation of VIC growth by TGF-β is context dependent. TGF-β prevents excessive heart valve growth under normal physiological conditions while it promotes cell proliferation in the early stages of repair, when increased VICs are required.     

Transforming growth factor-β1 induces antigen-specific unresponsiveness in naive t cells

Transforming growth factor-β1 (TGF-β1) is a cytokine with complex immunomodulatory effects including the ability to inhibit the onset or seventy of autoimmune disease. This study was designed to test the possibility that one mechanism by which TGF-β1 exerts its immunosuppressive effects is by inducing antigen (Ag)-specific unresponsiveness in CD4⁺ cells. TGF-β1 was shown here to inhibit the Ag-specific proliferation of naive CD4⁺ cells from T cell receptor (TCR) transgenic mice. More importantly, the naive CD4⁺ cells exposed to TGF-β1 and Ag, but not to TGF-β1 alone, in primary cultures were unable to proliferate or secrete IL-2 in response to a subsequent Ag challenge following removal of TGF-β1 from the cultures. Anti-CD28 mAb partially blocked the Ag-specific inactivation induced by TGF-β1 in naive CD4⁺ cells. The inhibitory effects of TGF-β1 on CD4⁺ cells are not mediated by alterations in APC costimulation since TGF-β1 did not inhibit the Ag-induced expression of MHC class II molecules, CD80 or CD86 on splenic APC. Taken together, the results suggest that the immunosuppressive activities of TGF-β1 encompass direct induction of Ag-specific unresponsiveness in naive CD4⁺ cells.     

Transforming growth factor-β1 polymorphisms and the outcome of hematopoietic stem cell transplantation

Hematopoietic stem cell transplantation (HSCT) is a medical procedure used to treat malignant and nonmalignant haematological diseases, congenital immunodeficiency syndromes, solid tumours and metabolic diseases. Despite its usefulness, several major complications, such as graft-versus-host disease, can negatively affect patients treated with HSCT. Apart from clinical factors well known to affect the outcome of HSCT, patient and donor genetics have been shown to play an important role in the susceptibility to post-transplant complications. Histocompatibility as determined by the human leucocyte antigen (HLA) system has been a major genetic determinant of the success of HSCT. Non-HLA immunogenetics are increasingly recognized to play a part in the events related to transplantation. Cytokine genes, and their receptors, bear a considerable amount of polymorphism. One of the genes that may play an important role on the outcome of allogeneic HSCT is TGFB1, which encodes transforming growth factor, βeta 1 (TGF-β1). TGF-β1 is a pleiotropic cytokine, which plays a central role in the development, homeostasis and responses of the immune system. Several functional polymorphisms in TGFB1 have been identified, and these are known to cause alterations in cytokine secretion in several settings. The present review will focus on the current knowledge surrounding the effect of polymorphisms within TGFB1 on the outcome of HSCT.     

Transforming growth factor-β1 promotes homing of bone marrow mesenchymal stem cells in renal ischemia-reperfusion injury

Backgrounds: Acute ischemia reperfusion-induced kidney injury is a common cause of acute renal failure, and it is also an important cause of delayed recovery of transplanted kidney functions and even loss of function. However, there is no effective treatment method in clinical applications presently. Objective: The objective was to investigate effects of transforming growth factor-β1 on homing of bone marrow mesenchymal stem cells in renal ischemia-reperfusion injury. Methods: Effects of TGF-β1 over-expression in MSCs on expression of CXCR4 and chemotactic effect to SDF-1 were investigated by in vitro transmembrane chemotaxis. Anti-TGF-β1 antibody was incubated with ischemia reperfusion injury renal tissue homogenate and effects of anti-TGF-β1 antibody were observed. In addition, effects of TGF-β1 gene transfection and anti-CXCR4 antibody treatment in MSCs on expression of SDF-1/CXCR4 axis of renal tissues and damage repair were further explored. Results: Expression of TGF-β1 mRNA in the IRI group increased significantly, and MSCs transplantation could enhance expression of CXCR4 mRNA in rats of the IRI group, the expression of CXCR4 can be decreased by the anti-TGF-β1 antibody and the anti-CXCR4 antibody. TGF-β1 induced homing of MSCs in repair of renal ischemic reperfusion injury by regulating expression of CXCR4 on cell membranes. Blue fluorescence of DAPI-positive MSCs cells of renal parenchyma in the IRI+MSC group was enhanced significantly, which was significantly inhibited by anti-TGF-β1 and anti-CXCR4 antibody, and the inhibitory effect of anti-CXCR4 antibody was more obvious than that of anti-TGF-β1 antibody. Conclusion: Transforming growth factor-β1 promotes homing of bone marrow mesenchymal stem cells in renal ischemia-reperfusion injury, which will provide useful data on role of TGF-β1 in regulating SDF-1/CXCR4 axis-induced MSCs homing.     

TGF-β1 gene-modified,immature dendritic cells delay the development of inflammatory bowel disease by inducing CD4+Foxp3+ regulatory T cells

Inflammatory bowel disease (IBD) is caused by an uncontrolled immune response in the intestinal lumen, leading to inflammation in genetically predisposed individuals. Immunotherapy may be a promising approach to the treatment of IBD. Here, we show that transforming growth factor-β1 (TGF-β1) gene-modified immature dendritic cells (imDCs) could enhance the inhibitory function of imDCs and delay the progress of IBD induced by dextran sodium sulfate in mice. The results of fluorescence-activated cell sorter (FACS) demonstrated that this protective effect is mediated partially by inducing CD4⁺Foxp3⁺ regulatory T cells (Tregs) in mesentery lymph nodes to control inflammation. In vitro experiments also supported this hypothesis. In conclusion, we provide evidence that TGF-β1-modified bone marrow-derived imDCs may have a therapeutic effect to IBD.     

Therapeutic potential of TGF-β-induced CD4+ Foxp3+ regulatory T cells in autoimmune diseases

Foxp3⁺ T regulatory cell (Treg) subsets play a crucial role in the maintenance of immune homeostasis against self-antigens. The lack or dysfunction of these cells contributes to the pathogenesis and development of many autoimmune diseases. Therefore, manipulation of these cells may provide a novel therapeutic approach to treat autoimmune diseases. In this review, we provide current opinions concerning the classification, developmental, and functional characterization of Treg subsets. Particular emphasis will be focused on the therapeutic role of TGF-β-induced CD4M⁺ Foxp3⁺ cells (iTregs) in established autoimmune disease. Moreover, the similarity and diversity of iTregs and naturally occurring, thymus-derived CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells (nTregs) will be discussed, including the finding that the pro-inflammatory cytokine IL-6 can convert nTregs to IL-17-producing cells, whereas iTregs induced by TGF-β are resistant to the effects of this cytokine. Understanding these aspects may help to determine how Tregs can be used in the treatment of autoimmune diseases.     

Activation of CD4⁺ Foxp3⁺ regulatory T cells proceeds normally in the absence of B cells during EAE

B cells and regulatory T (Treg) cells can both facilitate remission from experimental auto immune encephalomyelitis (EAE), a disease of the central nervous system (CNS) used as a model for multiple sclerosis (MS). Considering that B-cell-depletion therapy (BCDT) is used to treat MS patients, we asked whether Treg-cell activation depended on B cells during EAE. Treg-cell proliferation, accumulation in CNS, and augmentation of suppressive activity in the CNS were normal in B-cell-deficient mice, indicating that B cells are not essential for activation of the protective Treg-cell response and thus provide an independent layer of regulation. This function of B cells involved early suppression of the encephalitogenic CD4(+) T-cell response, which was enhanced in B-cell-deficient mice. CD4(+) T-cell depletion was sufficient to intercept the transition from acute-to-chronic EAE when applied to B-cell-deficient animals that just reached the peak of disease severity. Intriguingly, this treatment did not improve disease when applied later, implying that chronic disability was ultimately maintained independently of pathogenic CD4(+) T cells. Collectively, our data indicate that BCDT is unlikely to impair Treg-cell function, yet it might produce undesirable effects on T-cell-mediated autoimmune pathogenesis.     

Transforming growth factor-β induces nerve growth factor expression in pancreatic stellate cells by activation of the ALK-5 pathway

Nerve growth factor (NGF), a survival factor for neurons enforces pain by sensitizing nociceptors. Also in the pancreas, NGF was associated with pain and it can stimulate the proliferation of pancreatic cancer cells. Hepatic stellate cells (HSC) respond to NGF with apoptosis.

Transforming growth factor (TGF)-β, one of the strongest pro-fibrogenic activators of pancreatic stellate cells (PSC) induced NGF and its two receptors in an immortalized human cell line (ihPSC) and primary rat PSC (prPSC) as determined by RT-PCR, western blot, and immunofluorescence. In contrast to HSC, PSC expressed both NGF receptors, although p75^NTR expression was weak in prPSC. In contrast to ihPSC TGF-β activated both Smad signaling cascades in prPSC. NGF secretion was diminished by the activin-like kinase (ALK)-5 inhibitor SB431542, indicating the predominant role of ALK5 in activating the NGF system in PSC. While NGF did not affect proliferation or survival of PSC it induced expression of Inhibitor of Differentiation-1.

We conclude that under conditions of upregulated TGF-β, like fibrosis, NGF levels will also increase in PSC which might contribute to pancreatic wound healing responses.     

Sub-optimal CD4+ T-cell activation triggers autonomous TGF-β-dependent conversion to Foxp3+ regulatory T cells

Classical in vitro Treg conversion assays, which rely on optimal T-cell activation in the presence of exogenous TGF-β, induce Foxp3 expression at a frequency far above that which is observed in vivo in Treg-dependent models of oral or transplantation tolerance. We have found that suboptimal murine T-cell activation in vitro results in induction of Foxp3 expression, in the absence of exogenous TGF-β, at a frequency similar to that which we found in vivo upon anti-CD4-induced transplantation tolerance. We show that TCR triggering with either low-dose anti-CD3 or low-dose agonist peptide, as well as down-modulation of the TCR signal with non-depleting anti-CD4, promotes TGF-β production by T cells, an event that precedes Foxp3 expression and is Foxp3 independent. These findings support the view that sub-immunogenic regimens lead to dominant tolerance as a result of T-cell intrinsic properties.     

Integrated T-cell receptor and costimulatory signals determine TGF-β-dependent differentiation and maintenance of Foxp3+ regulatory T cells

Foxp3‐expressing Tregs play a non‐redundant role in protecting against immune pathologies. Foxp3⁺ Tregs can arise intra‐ and extra‐thymically, however, the signals directing their differentiation and maintenance in the periphery are not well understood. We show that stimulation of mouse naïve CD4⁺ T cells in vitro with optimal doses of anti‐CD3/anti‐CD28 resulted in high frequencies of Foxp3⁺ T cells via a TGF‐β‐dependent mechanism. Addition of TGF‐β and retinoic acid overcame the inhibition of Foxp3 expression observed during high‐strength anti‐CD3/anti‐CD28 stimulation. Reducing the strength of TCR or costimulatory signals with inhibitors of mammalian target of rapamycin (mTOR) or MEK/ERK signalling also enhanced expression of Foxp3 in a TGF‐β‐dependent manner. Addition of TGF‐β was further required to maintain Foxp3 expression in ex vivo derived Foxp3⁺ Tregs upon prolonged anti‐CD3/anti‐CD28 signalling. Thus, induction/maintenance of Foxp3 expression by TGF‐β is modulated by the integrated strength of TCR/costimulatory signals.     

Transforming growth factor-β1 mediated up-regulation of lysyl oxidase in the kidneys of hereditary nephrotic mouse with chronic renal fibrosis

Lysyl oxidase (LOX), an extracellular emzyme, plays a key role in the post-translational modification of collagens and elastin, catalyzing inter- and intra-crosslinking reactions. Because the crosslinked extracellular matrices (ECMs) are highly resistant to degradative enzymes, it is considered that the over-expression of LOX may cause severe fibrotic degeneration. In the present study, we addressed the role of LOX-mediated crosslinking in chronic renal tubulointerstitial fibrosis using an animal model of hereditary nephrotic syndrome, the Institute of Cancer Research (ICR)-derived glomerulonephritis (ICGN) mouse. Ribonuclease protection assay (RPA) revealed that LOX mRNA expression was up-regulated in the kidneys of ICGN mice as compared with control ICR mice. High-level expression of LOX and transforming growth factor (TGF)-β1 (an up-regulator of LOX) mRNA was detected in tubular epithelial cells of ICGN mouse kidneys by in situ hybridization. Type-I and -III collagens, major substrates for LOX, were accumulated in tubulointerstitium of ICGN mouse kidneys. The present findings imply that TGF-β1 up-regulates the production of LOX in tubular epithelial cells of ICGN mouse kidneys, and the excessive LOX acts on interstitial collagens and catalyzes crosslinking reactions. As a result, the highly crosslinked collagens induce an irreversible progression of chronic renal tubulointerstitial fibrosis in the kidneys of ICGN mice.     

SOCS3 is a suppressor of γc cytokine signaling and constrains generation of murine Foxp3+ regulatory T cells

SOCS3 is a cytosolic inhibitor of cytokine signaling that suppresses the activation of cytokine receptor-associated JAK kinases. Mechanistically, SOCS3 is recruited to a site in the cytokine receptors known as the SOCS3-interaction motif, and then binds JAK molecules to inhibit their kinase activity. The SOCS3-interaction motif is found in receptors of the gp130 cytokine family but mostly absent from other cytokine receptors, including γc. Thus, SOCS3 has been considered a selective suppressor of gp130 family cytokines, but not γc cytokines. Considering that γc signaling induces SOCS3 expression in T cells, here we revisited the role of SOCS3 on γc signaling. Using SOCS3 transgenic mice, we found that increased abundance of SOCS3 not only suppressed signaling of the gp130 family cytokine IL-6, but also signaling of the γc family cytokine IL-7. Consequently, SOCS3 transgenic mice were impaired in IL-7-dependent T cell development in the thymus and the homeostasis of mature T cells in peripheral tissues. Moreover, enforced SOCS3 expression interfered with the generation of Foxp3⁺ regulatory T cells that requires signaling by the γc family cytokine IL-2. Collectively, we report an underappreciated role for SOCS3 in suppressing γc cytokine signaling, effectively expanding its scope of target cytokines in T cell immunity.     

Controlled release of transforming growth factor-β3 from cartilage-extra-cellular-matrix-derived scaffolds to promote chondrogenesis of human-joint-tissue-derived stem cells

The objective of this study was to develop a scaffold derived from cartilaginous extracellular matrix (ECM) that could be used as a growth factor delivery system to promote chondrogenesis of stem cells. Dehydrothermal crosslinked scaffolds were fabricated using a slurry of homogenized porcine articular cartilage, which was then seeded with human infrapatellar-fat-pad-derived stem cells (FPSCs). It was found that these ECM-derived scaffolds promoted superior chondrogenesis of FPSCs when the constructs were additionally stimulated with transforming growth factor (TGF)-β3. Cell-mediated contraction of the scaffold was observed, which could be limited by the additional use of 1-ethyl-3-3dimethyl aminopropyl carbodiimide (EDAC) crosslinking without suppressing cartilage-specific matrix accumulation within the construct. To further validate the utility of the ECM-derived scaffold, we next compared its chondro-permissive properties to a biomimetic collagen–hyaluronic acid (HA) scaffold optimized for cartilage tissue engineering (TE) applications. The cartilage-ECM-derived scaffold supported at least comparable chondrogenesis to the collagen–HA scaffold, underwent less contraction and retained a greater proportion of synthesized sulfated glycosaminoglycans. Having developed a promising scaffold for TE, with superior chondrogenesis observed in the presence of exogenously supplied TGF-β3, the final phase of the study explored whether this scaffold could be used as a TGF-β3 delivery system to promote chondrogenesis of FPSCs. It was found that the majority of TGF-β3 that was loaded onto the scaffold was released in a controlled manner over the first 10 days of culture, with comparable long-term chondrogenesis observed in these TGF-β3-loaded constructs compared to scaffolds where the TGF-β3 was continuously added to the media. The results of this study support the use of cartilage-ECM-derived scaffolds as a growth factor delivery system for use in articular cartilage regeneration.     

CD4+ Foxp3+ regulatory T cells suppress γδ T-cell effector functions in a model of T-cell-induced mucosal inflammation

CD4(+) CD25(+) Foxp3(+) regulatory T (T(REG) ) cells are critical mediators of peripheral immune tolerance, and abrogation of their function provokes a variety of autoimmune and inflammatory states including inflammatory bowel disease. In this study, we investigate the functional dynamics of T(REG) -cell responses in a CD4(+) T-cell-induced model of intestinal inflammation in αβ T-cell-deficient (TCR-β(-/-) ) hosts to gain insights into the mechanism and cellular targets of suppression in vivo. We show that CD4(+) T effector cell transfer into T-cell-deficient mice rapidly induces mucosal inflammation and colitis development, which is associated with prominent Th1 and Th17 responses. Interestingly, we unveil a prominent role for resident γδ T cells in mucosal inflammation as they promote Th1 and particularly Th17 responses in the early phase of inflammation, thus exacerbating colitis development. We further demonstrate that CD4(+) CD25(+) Foxp3(+) T(REG) cells readily inhibit these responses and mediate disease protection, which correlates with their accumulation in the draining LN and lamina propria. Moreover, T(REG) cells can directly suppress γδ T-cell expansion and cytokine production in vitro and in vivo, suggesting a pathogenic role of γδ T cells in intestinal inflammation. Thus, functional alterations in T(REG) cells provoke dysregulated CD4(+) and γδ T-cell responses to commensal antigens in the intestine.     

2-Gy whole-body irradiation significantly alters the balance of CD4+CD25? T effector cells and CD4+CD25+Foxp3+ T regulatory cells in mice

CD4⁺CD25⁺ T regulatory (Treg) cells are critical in inducing and maintaining immunological self-tolerance as well as transplant tolerance. The effect of low doses of whole-body irradiation (WBI) on CD4⁺CD25⁺Foxp3⁺ Treg cells has not been determined. The proportion, phenotypes and function of CD4⁺CD25⁺ Treg cells were investigated 0.5, 5 and 15 days after euthymic, thymectomized or allogeneic bone marrow transplanted C57BL/6 mice received 2-Gy γ-rays of WBI. The 2-Gy WBI significantly enhanced the ratios of CD4⁺CD25⁺ Treg cells and CD4⁺CD25⁺Foxp3⁺ Treg cells to CD4⁺ T cells in peripheral blood, lymph nodes, spleens and thymi of mice. The CD4⁺CD25⁺ Treg cells of the WBI-treated mice showed immunosuppressive activities on the immune response of CD4⁺CD25⁻ T effector cells to alloantigens or mitogens as efficiently as the control mice. Furthermore, 2-Gy γ-ray WBI significantly increased the percentage of CD4⁺CD25⁺Foxp3⁺ Treg cells in the periphery of either thymectomized mice or allogeneic bone marrow transplanted mice. The in vitro assay showed that ionizing irradiation induced less cell death in CD4⁺CD25⁺Foxp3⁺ Treg cells than in CD4⁺CD25⁻ T cells. Thus, a low dose of WBI could significantly enhance the level of functional CD4⁺CD25⁺Foxp3⁺ Treg cells in the periphery of naive or immunized mice. The enhanced proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells in the periphery by a low dose of WBI may make hosts more susceptible to immune tolerance induction.     

Induction of latency-associated peptide (transforming growth factor-β(1)) expression on CD4+ T cells reduces Toll-like receptor 4 ligand-induced tumour necrosis factor-α production in a transforming growth factor-β-dependent manner

CD4(+) T cells expressing the latent form of transforming growth factor-β [latency-associated peptide (LAP) (TGF-β(1))] play an important role in the modulation of immune responses. Here, we identified a novel peptide ligand (GPC(81-95) ) with an intrinsic ability to induce membrane-bound LAP (TGF-β(1)) expression on a subpopulation of human CD4(+) T cells (using flow cytometry; ranging from 0·8% to 2·6%) and stimulate peripheral blood mononuclear cells to release LAP (TGF-β(1) ) (using ELISPOT assay; ranging from 0·03% to 0·16%). In spite of this low percentage of responding cells, GPC(81-95) significantly reduced Toll-like receptor 4 ligand-induced tumour necrosis factor-α production in a TGF-β(1) - and CD4(+) T-cell-dependent manner. The results demonstrate that GPC(81-95) is a useful tool to study the functional properties of a subpopulation of LAP (TGF-β(1))(+) CD4(+) T cells and suggest a pathway that can be exploited to suppress inflammatory response.