During the acute liver injury, immune responses are provoked into eliciting inflammation in the acute phase. In the healing phase, the inflammation is terminated for wound healing and restoration of immune homeostasis. In this study, we sought to address how regulatory T cells (Tregs) are involved in the progression of liver injury and repair. In the acute phase, intrahepatic Tregs (CD4+FoxP3+Helios+) diminished promptly through apoptosis, which was followed by inflammation and tissue injury. In the healing phase, a new subset of Tregs (CD4+Foxp3+Helios−) was generated in correlation with the matrix metalloproteinase (MMP) cascade and transforming growth factor-beta (TGF-β) activation that were manifested mainly by hepatic stellate cells. Moreover, the induction of induced Tregs and wound healing were both impaired in mice lacking TGF-β signaling or MMPs. The depletion of induced Tregs also impeded wound healing for tissue repair. Together, this study demonstrates the mechanism that the loss of nTregs through apoptosis in the acute phase may facilitate inflammation, while regenerated Tregs through MMP9/13-dependent activation of TGF-β in the healing phase are critical to terminate inflammation and allow for wound healing.
liver injury; wound healing; regulatory T cells; TGF-β; IL-1; hepatic stellate cells; matrix metalloproteinase
TGF-β-induced CD4+Foxp3+ T cells (iTregs) have been identified as important prevention and treatment strategies for cell therapy in autoimmune diseases and other disorders. However, the potential use of iTregs as a treatment modality for acute graft-verse-host disease (GVHD) has not been realized because iTregs may be unstable and less suppressive in this disease. Here we restudied the ability of iTregs to prevent and treat acute GVHD in two different mouse models. Our results showed that so long as an appropriate iTreg-generation protocol is used, these iTregs consistently displayed a potent ability to control acute GVHD development and reduce mortality in the acute GVHD animal models. iTreg infusion markedly suppressed the engraftment of donor CD8+ cells and CD4+ cells, the expression of Granzyme A and B, the cytotoxic effect of donor CD8+ cells and the production of T cell cytokines in acute GVHD. We therefore conclude that so long as the right methods for generating iTreg cells have been employed, iTregs can indeed prevent and even treat acute GVHD.
Foxp3+ T regulatory cell (Treg) subsets play a crucial role in the maintenance of immune homeostasis against self-antigen. The lack or dysfunction of these cells is responsible for 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 characterizations of Treg subsets. A particular emphasis will be focused on the therapeutic role of TGF-β-induced CD4+Foxp3+ cells (iTregs) in established autoimmune disease. Moreover, the similarity and disparity of iTregs and naturally occurring, thymus-derived CD4+CD25+Foxp3+ regulatory T cells (nTregs) have also be discussed. While the proinflammatory cytokine IL-6 can convert nTregs to IL-17-producing cells, iTregs induced by TGF-β are resistant to the effects of this cytokine. Understanding this difference may play a key role in determining how Tregs can be used in the treatment of established autoimmune diseases.
Autoimmune diseases; Immunoregulation; Regulatory T cells; TGF-β; Foxp3; Th17 cells
Osteoclasts are responsible for bone destruction in rheumatoid arthritis (RA) and natural CD4+Foxp3+regulatory T cells (nTregs) can inhibit osteoclastogenesis. This study aims to determine whether TGF-β-induced CD4+Foxp3+regulatory T cells (iTregs) also suppress osteolastogenesis and bone erosion in collagen induced arthritis (CIA).
Osteoclasts were induced from bone-marrow CD11b+ cells with RANKL and macrophage colony-stimulating factor (M-CSF), and assessed with tartrate-resistant acid phosphatase (TRAP) staining. CD4+ iTregs were generated with TGF-β and added to cultures with different ratios with CD11b+ cells. Transwell and antibody blockade experiments were performed to define the mechanism of action. NF-kB activation was determined by western blot. 3×106 CD4+ iTregs, nTregs or control cells were adoptively transferred to DBA1/J mice on day 14 after immunization with CII/CFA. CIA onset and severity were monitored and bone erosion was examined by CT scan.
Both CD4+ Tregs almost completely suppressed osteoclastogenesis but only iTregs sustained the effect in the presence of IL-6 in vitro. CD4+ iTregs but not nTregs and control cells injected after immunization and before of onset of CIA significantly suppressed disease development. Of note, CT scan showed that the joints in CD4+ iTregs but not nTregs or control cells infused CIA had less bone erosion. Treatment with CD4+ iTregs but not other cells dramatically decreased the levels of NF-kB p65/p50 in osteoclasts in vitro and P65/50 and RANKL expression by synovial tissues in vivo.
Manipulation of CD4+ iTregs may have therapeutic effects on rheumatoid arthritis and other bone erosion related diseases.
Interleukin-22 (IL-22) is an IL-10 family cytokine member that was recently discovered to be mainly produced by Th17 cells. Previous studies have indicated the importance of IL-22 in host defense against Gram-negative bacterial organisms (in gut and lung). Recently, there is emerging evidence that IL-22 is involved in the development and pathogenesis of several autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren’s syndrome (SS) and psoriasis. Therapeutics targeting IL-22 therefore may have promise for treating various autoimmune diseases. In this review, we discuss the recent progression of the involvement of IL-22 in the development and pathogenesis of autoimmune diseases, as well as its clinical implications and therapeutic potential.
IL-22; Th17; Th22; autoimmune; therapeutic
Follicular helper T cells (Tfh) have been referred as a lineage that provides a help for B cells to proliferate and undergo antibody affinity maturation in the germinal center. Evidence has supported that Tfh subset development, like other lineages, is dependent on microenvironment where a particular transcriptional program is initiated. It has been shown that Bcl-6 and IL-21 act as master regulators for the development and function of Tfh cells. Tfh dysregulation is involved in the development of autoimmune pathologies, such as systemic lupus erythematosus, rheumatoid arthritis and other autoimmune diseases. The present review highlights the recent advances in the field of Tfh cells and focus on their development and function.
autoimmune diseases; follicular helper T cells; systemic lupus erythematousus
Inteleurkin-22 (IL-22) is a IL-10 family cytokine member and is mainly produced by innate lymphoid cells (ILCs), Th17 cells, and Th22 cells. Previous studies have indicated that IL-23 and several transcription factors, including STAT3, RORγt, and the AhR are important stimulus. Recently, there is emerging evidence that Tregs can regulate IL-22 expression. In the review, we discuss the updated advancement on Tregs function and its regulatory role on IL-22 expression.
Interleukin-22; regulatory (Treg) cells; T helper cells; innate immune cells
The regulatory T (Treg) cells play an important role in the maintenance of homeostasis and the prevention of autoimmune diseases. Although most studies are focusing on the role of Treg cells in T cells and T cells-mediated diseases, these cells also directly affect B cells and other non-T cells. This manuscript updates the role of Treg cells on the B cells and B cell-mediated diseases. In addition, the mechanisms whereby Treg cells suppress B cell responses have been discussed.
Treg; Foxp3; B cells; antibodies; autoimmune diseases
Naive CD4+ cells differentiate into T helper (Th1, Th2, Th9, Th17) and regulatory T (Treg) cells to execute their immunologic function. Whereas TGF-β suppresses Th1 and Th2 cell differentiation, this cytokine promotes Th9, Th17 and Foxp3+ regulatory T cells depending upon the presence of other cytokines. IL-6 promotes Th17, but suppresses regulatory T cell differentiation. Moreover, natural but not TGF-β-induced regulatory T cells convert into Th17 cells in the inflammatory milieu. Here an update of T cell differentiation and conversion, as well as underlying mechanisms are given.
T helper cells; Th1; Th2; Th9; Th17; Tfh; Foxp3+ regulatory T cells; cytokine; differentiation; autoimmunity
For more than a decade now, the regulatory T (Treg) cell has widely been considered as a critical subpopulation of T cells which can suppress effector T cell responses as well as suppressing the activity of other immune cells, such as mast cell, dendritic cells, and B cells. Treg cells have been broadly characterized as comprising of two main populations: thymus-derived natural Treg (nTreg) cells, and peripherally generated induced Treg (iTreg) cells. Both subsets have similar phenotypic characteristics and comparable suppressive function against T cell-mediated immune response and diseases. However, both Foxp3 positive Treg subsets exhibit some specific differences such as different mRNA transcripts and protein expression, epigenetic modification, and stability. These subtle differences reinforce the notion that they represent unique and distinct subsets. Accurately distinguishing iTregs from nTregs will help to clarify the biological features and contributions of each Treg subsets in peripheral tolerance, autoimmunity and tumor immunity. One difficult problem is that it has not been possible to distinguish iTregs from nTregs using surface markers until two recent articles were published to address this possibility. This review will focus on very recent advances in using molecular markers to differentiate these Treg subsets.
Treg; Foxp3; Helios; neuropilin 1
Interplay between Foxp3+ regulatory T cells (Treg) and dendritic cells (DCs) maintains immunologic tolerance, but the effects of each cell on the other are not well understood. We report that polyclonal CD4+Foxp3+ Treg cells induced ex vivo with transforming growth factor beta (TGFβ) (iTreg) suppress a lupus-like chronic graft-versus-host disease by preventing the expansion of immunogenic DCs and inducing protective DCs that generate additional recipient CD4+Foxp3+ cells. The protective effects of the transferred iTreg cells required both interleukin (IL)-10 and TGFβ, but the tolerogenic effects of the iTreg on DCs, and the immunosuppressive effects of these DCs were exclusively TGFβ-dependent. The iTreg were unable to tolerize Tgfbr2-deficient DCs. These results support the essential role of DCs in ‘infectious tolerance’ and emphasize the central role of TGFβ in protective iTreg/DC interactions in vivo.
regulatory T cells; dendritic cells; TGFβ; graft-versus-host disease
Recent studies have demonstrated that plasticity of naturally occurring CD4+Foxp3+ regulatory T cells (nTregs) may account for their inability to control chronic inflammation in established autoimmune diseases. All-trans retinoic acid (atRA), the active derivative of vitamin A, has been demonstrated to promote Foxp3+ Treg differentiation and suppress Th17 development. In this study, we report a vital role of atRA in sustaining the stability and functionality of nTregs in the presence of IL-6. We found that nTregs treated with atRA were resistant to Th17 and other Th cell conversion and maintained Foxp3 expression and suppressive activity in the presence of IL-6 in vitro. atRA decreased IL-6R expression and signaling by nTregs. Of interest, adoptive transfer of nTregs even from arthritic mice treated with atRA suppressed progression of established collagen-induced arthritis. We suggest that nTregs treated with atRA may represent a novel treatment strategy to control established chronic immune-mediated inflammatory diseases.
Whereas TGF-β is essential for the development of peripherally induced Foxp3+ regulatory T cells (iTreg cells) and Th17 cells, the intracellular signaling mechanism by which TGF-β regulates development of both cell subsets is less understood. In this study, we report that neither Smad2 nor Smad3 gene deficiency abrogates TGF-β–dependent iTreg induction by a deacetylase inhibitor trichostatin A in vivo, although the loss of the Smad2 or Smad3 gene partially reduces iTreg induction in vitro. Similarly, SMAD2 and SMAD3 have a redundant role in development of Th17 in vitro and in experimental autoimmune encephalomyelitis. In addition, ERK and/or JNK pathways were shown to be involved in regulating iTreg cells, whereas the p38 pathway predominately modulated Th17 and experimental autoimmune encephalomyelitis induction. Therefore, selective targeting of these intracellular TGF-β signaling pathways during iTreg and Th17 cell development might lead to the development of therapies in treating autoimmune and other chronic inflammatory diseases.
TGF-β plays an important role in the induction of regulatory T cells (Treg) and maintenance of immunologic tolerance, but whether other members of TGF-β superfamily act together or independently to achieve this effect is poorly understood. Although others have reported that the bone morphogenetic proteins (BMP) and TGF-β have similar effects on the development of thymocytes and T cells, in this study, we report that members of the BMP family, BMP-2 and BMP-4, are unable to induce non-regulatory T cells to become Foxp3+ Treg. Neutralization studies with Noggin have revealed that BMP-2/4 and the BMP receptor signaling pathway is not required for TGF-β to induce naive CD4+CD25- cells to express Foxp3; however, BMP-2/4 and TGF-β have a synergistic effect on the induction of Foxp3+ Treg. BMP-2/4 affects non-Smad signaling molecules including phosphorylated ERK and JNK, which could subsequently promote the differentiation of Foxp3+ Treg induced by TGF-β. Data further advocate that TGF-β is a key signaling factor for Foxp3+ Treg development. In addition, the synergistic effect of BMP-2/4 and TGF-β indicates that the simultaneous manipulation of TGF-β and BMP signaling might have considerable effects in the clinical setting for the enhancement of Treg purity and yield.
BMP; TGF-β; Foxp3; regulatory T cells
There are no ideal ways to identify and isolate viable and purified Foxp3+ regulatory T cells so far. Here we developed a novel procedure for the isolation of highly purified Foxp3+ cells using flow cytometry. This method relies on an identification and sorting of the lymphoblast cell population identified on a scatter plot using flow cytometry. We confirmed that greater than 98% of the cells sorted using this technique expressed Foxp3 and displayed a potent suppressive activity. This method provides a valuable tool for the study of the T regulatory cell biology and their therapeutic manipulation.
Foxp3; TGF-β; regulatory T cells
Like natural CD4+CD25+ Treg cells, TGF-β-induced Treg cells also prevent allograft rejection in MHC-mismatched organ transplantation models. In analyzing this effect with greater detail, we determined that injection of TGF-β-induced, alloactivated CD4+CD25+ cells induces antigen-specific immune tolerance in vivo. Increased CD4+CD25+ cells in recipients contribute to this immune tolerance. In addition, adoptive transfer of TGF-β-induced CD4+CD25+ cells did not result in significant toxic and side effects in recipients. These results indicate that TGF-β-induced, alloactivated CD4+CD25+ cells may provide a safe and effective approach to protect MHC-mismatched organ grafts from rejection in a clinical setting.
TGF-β; Foxp3; regulatory T cells; Immunoregulation; transplant tolerance
Foxp3+T regulatory cell (Treg) subsets play a crucial role in the maintenance of immune homeostasis against self-antigen. The lack or dysfunction of these cells is responsible for the pathogenesis and development of many autoimmune diseases. Therefore, manipulation of these cells may provide a novel therapeutic approach to treat autoimmune diseases and prevent allograft rejection during organ transplantation. In the article, we will provide current opinions concerning the classification, developmental and functional characterizations of Treg subsets. A particular emphasis will be focused on transforming cell growth factor beta (TGF-β) and its role in the differentiation and development of induced regulatory T cells (iTregs) in the periphery. Moreover, the similarity and disparity of iTregs and naturally occurring, thymus-derived CD4+CD25+Foxp3+ regulatory T cells (nTregs) will also be discussed. While proinflammatory cytokine IL-6 can convert nTregs to IL-17-producing cells, peripheral Tregs induced by TGF-β are resistant to this cytokine. This difference may affect the role of each in the adaptive immune response.
Immunoregulation; regulatory T cells; TGF-β; Foxp3; Th17 cells
It has been well recognized that TGF-β is able to induce CD4+CD25+Foxp3+ suppressor/regulatory T (iTreg) cells and IL-2 facilitates iTreg induction and expansion, however, only half of TGF-β-induced CD4+CD25+ cells express Foxp3 and remaining CD4+CD25+Foxp3- cells may represent effector cells. Whether other factor(s) can increase Foxp3 expression by CD4+CD25+ cells induced with TGF-β is still unclear. Here we show that addition of exogenous IFN-γ or IL-4 diminished the ability of TGF-β to induce Foxp3 expression and IL-2 failed to rescue this decreased Foxp3 expression. Conversely, neutralization of IFN-γ and IL-4 significantly enhanced the ability of TGF-β to induce Foxp3 and develop the suppressive activity, indicating that different cytokine profiles affect the differentiation of CD4+CD25+Foxp3+ subset induced by TGF-β. These results show that combination of antibodies against IFN-γ and IL-4 and TGF-β enhances the efficacy of generation and function of iTreg cells and may therefore provide a novel therapeutic strategy for the treatment of autoimmune and other chronic inflammatory diseases.
suppressor/regulatory T cells; cytokine; Foxp3; autoimmunity
To evaluate the biological functions of myogenic regulatory factors, we have examined the effects of ectopic expression of MyoD and Cx43 genes in the fibroblasts on the differentiation of myoblast in vitro. The expression of MyoD and Cx43 in the transfectants was confirmed by RT-PCR and Western blot. More than 50% of fibroblasts transfected with MyoD or both MyoD and Cx43 genes displayed typical morphological features of myoblast-like cells at 20 days following gene transfection, including cell elongation, cytoplasm enrichment and granule manifold. Moreover, these myoblast-like cells also expressed both desmin and α-actin. These results demonstrate that direct exogenous expression of the myogenic regulatory factors is sufficient to induce transdifferentiation of fibroblasts into a myoblast-like lineage and provide new insights into the trauma repair after myocardial infraction.
lentivirus; fibroblast; myogenesis; MyoD; Cx43
We report that polyclonal CD8regs generated in one week ex-vivo with anti-CD3/28 beads and cytokines rapidly developed suppressive activity in vitro sustained by TGF-β. In immunodeficient mice, these CD8regs demonstrated a markedly protective, IL-10 dependent activity against a xeno-GVHD. They expressed IL-2Rα/β, Foxp3, TNFR2, and the negative co-stimulatory receptors CTLA-4, PD-1, PD-L1 and Tim-3. Suppressive activity in vitro correlated better with TNFR2 and PD-L1 than Foxp3. Blocking studies suggested that TNF enhanced PD-L1 expression and the suppressive activity of the CD8regs generated. Unlike other polyclonal CD4 and CD8 Tregs, these CD8regs preferentially targeted allogeneic T cells, but they lacked cytotoxic activity against them even after sensitization. Unlike CD4regs, these CD8regs could produce IL-2 and proliferate while inhibiting target cells. If these CD8regs can persist in foreign hosts without impairing immune surveillance, they could serve as a practical remission-inducing product for the treatment of autoimmune diseases, graft-versus-host disease, and allograft rejection.
Regulatory T (Treg) cells suppress inflammatory immune responses and autoimmunity caused by self-reactive T cells. The key Treg cell transcription factor Foxp3 is downregulated during inflammation to allow for the acquisition of effector T cell-like functions. Here, we demonstrate that stress signals elicited by proinflammatory cytokines and lipopolysaccharide lead to the degradation of Foxp3 through the action of the E3 ubiquitin ligase Stub1. Stub1 interacted with Foxp3 to promote its K48-linked polyubiquitination in an Hsp70-dependent manner. Knockdown of endogenous Stub1 or Hsp70 prevented Foxp3 degradation. Furthermore, the overexpression of Stub1 in Treg cells abrogated their ability to suppress inflammatory immune responses in vitro and in vivo, and conferred a T helper 1 (Th1) cell-like phenotype. Our results demonstrate the critical role of the stress-activated Stub1-Hsp70 complex in promoting Treg cell inactivation, thus providing a potential therapeutic target for the intervention against autoimmune disease, infection and cancer.
In vitrodata and transgenic mouse models suggest a role for TGFβ signaling in dendritic cells (DC) to prevent autoimmunity primarily through maintenance of DCs in their immature and tolerogenic state characterized by low expression of MHCII and co-stimulatory molecules, and increased expression of indoleamine 2,3-dioxygenase (IDO), among others. To test whether a complete lack of TGFβ signaling in DCs predisposes mice to spontaneous autoimmunity, and to verify the mechanisms implicated previously in vitro, we generated conditional knock-out mice with Cre-mediated DC-specific deletion of Tgfbr2 (DC-Tgfbr2 KO). DC-Tgfbr2 KO mice die before 15 weeks of age with multi-organ autoimmune inflammation and spontaneous activation of T and B cells. Interestingly, there were no significant differences in the expression of MHCII, co-stimulatory molecules, or IDO in secondary lymphoid organ DCs, although Tgfbr2-deficient DCs were more pro-inflammatory in vitro and in vivo. DC-Tgfbr2 KO showed attenuated FoxP3 expression in regulatory T cells (Tregs) and abnormal expansion of CD25−FoxP3+ Tregs in vivo. Tgfbr2-deficient DCs secreted elevated levels of IFNγ and were not capable of directing antigen-specific Treg conversion unless in the presence of anti-IFNγ blocking antibody. Adoptive transfer of iTregs into DC-Tgfbr2 KO mice partially rescued the phenotype. Therefore, in vivo, TGFβ signaling in DCs is critical in the control of autoimmunity through both Treg dependent and independent mechanisms, but it does not affect MHCII and co-stimulatory molecule expression.
Interleukin 1 is a critical inflammatory mediator and involved in host defense to several pathogens. Oral T. gondii infection causes lethal ileitis in C57BL/6 (BL6) mice and serves to investigate the mechanisms of acute intestinal inflammation. Here we show that IL-1 is expressed upon oral T. gondii (76K strain) infection in the small intestine and mediates ileitis as IL-1R1 deficient mice have reduced neutrophil recruitment in the lamina propria, parasite invasion, inflammatory lesions and enhanced survival as compared to BL6 infected control mice. Protection in the absence of IL-1R1 signaling was associated with reduced IFN-γ expression and preserved Paneth cells, while these cells were eliminated in infected BL6 mice. Furthermore, blockade of IL-1 by IL-1β antibody attenuated inflammation in BL6 mice. In conclusion, IL-1 signaling contributes to the inflammatory response with increase IFN-γ expression and Paneth cell depletion upon oral T. gondii infection.
Toxoplasma gondii; IL-1R1 receptor signaling; Paneth neutralizing antibody; inflammation; innate immunity