RGC-32 regulates reactive astrocytosis and extracellular matrix deposition in experimental autoimmune encephalomyelitis

Extracellular matrix (ECM) deposition in active demyelinating multiple sclerosis (MS) lesions may impede axonal regeneration and can modify immune reactions. Response gene to complement (RGC)-32 plays an important role in the mediation of TGF-β downstream effects, but its role in gliosis has not been investigated. To gain more insight into the role played by RGC-32 in gliosis, we investigated its involvement in TGF-β-induced ECM expression and the upregulation of the reactive astrocyte markers α-smooth muscle actin (α-SMA) and nestin. In cultured neonatal rat astrocytes, collagens I, IV, and V, fibronectin, α-SMA, and nestin were significantly induced by TGF-β stimulation, and RGC-32 silencing resulted in a significant reduction in their expression. Using astrocytes isolated from RGC-32 knock-out (KO) mice, we found that the expression of TGF-β-induced collagens I, IV, and V, fibronectin, and α-SMA was significantly reduced in RGC-32 KO mice when compared with wild-type (WT) mice. SIS3 inhibition of Smad3 phosphorylation was also associated with a significant reduction in RGC-32 nuclear translocation and TGF-β-induced collagen I expression. In addition, during experimental autoimmune encephalomyelitis (EAE), RGC-32 KO mouse astrocytes displayed an elongated, bipolar phenotype, resembling immature astrocytes and glial progenitors whereas those from WT mice had a reactive, hypertrophied phenotype. Taken together, our data demonstrate that RGC-32 plays an important role in mediating TGF-β-induced reactive astrogliosis in EAE. Therefore, RGC-32 may represent a new target for therapeutic intervention in MS.     

Dual role of Response gene to complement-32 in multiple sclerosis

Response gene to complement (RGC)-32 is a novel molecule that plays an important role in cell proliferation. We investigated the expression of RGC-32 in multiple sclerosis (MS) brain and in peripheral blood mononuclear cells (PBMCs) obtained from patients with relapsing–remitting multiple sclerosis. We found that CD3⁺, CD68⁺, and glial fibrillar acidic protein (GFAP)⁺ cells in MS plaques co-localized with RGC-32. Our results show a statistically significant decrease in RGC-32 mRNA expression in PBMCs during relapses when compared to the levels in stable MS patients. This decrease might be useful in predicting disease activity in patients with relapsing–remitting MS. RGC-32 expression was also correlated with that of FasL mRNA during relapses. FasL mRNA expression was significantly reduced after RGC-32 silencing, indicating a role for RGC-32 in the regulation of FasL expression. In addition, the expression of Akt1, cyclin D1, and IL-21 mRNA was significantly increased during MS relapses when compared to levels in healthy controls. Furthermore, we investigated the role of RGC-32 in TGF-β-induced extracellular matrix expression in astrocytes. Blockage of RGC-32 using small interfering RNA significantly inhibits TGF-β induction of procollagen I, fibronectin and of the reactive astrocyte marker α-smooth muscle actin (α-SMA). Our data suggest that RGC-32 plays a dual role in MS, both as a regulator of T-cells mediated apoptosis and as a promoter of TGF-β-mediated profibrotic effects in astrocytes.     

Role of response gene to complement 32 in diseases

The role of response gene to complement (RGC)-32 as a cell cycle regulator has been attributed to its ability to activate cdc2 kinases and to induce S-phase entry and mitosis. However, recent studies revealed novel functions for RGC-32 in diverse processes such as cellular differentiation, inflammation, and fibrosis. Besides responding to C5b-9 stimulation, RGC-32 expression is also induced by growth factors, hormones, and cytokines. Transforming growth factor β activates RGC-32 through Smad and RhoA signaling, thus initiating smooth muscle cell differentiation. Accumulating evidence has drawn attention to the deregulated expression of RGC-32 in human malignancies, hyper-immunoglobulin E syndrome, and fibrosis. RCG-32 expression is up-regulated in cutaneous T cell lymphoma and colon, ovarian, and breast cancer, but down-regulated in invasive prostate cancer, multiple myeloma, and drug-resistant glioblastoma. A better understanding of the mechanism by which RGC-32 contributes to the pathogenesis of these diseases will provide new insights into its therapeutic potential. In this review we provide an overview of this field and discuss the most recent research on RGC-32.     

Regulation of transforming growth factor-β production by interleukin-12

The induction of peripheral tolerance following oral antigen administration in several autoimmune disease and conventional animal models correlates with the production of transforming growth factor-β (TGF-β) and T helper type 2 (Th2) cytokines. The factors regulating TGF-β production and its relation to the Th2 response, however, have not been defined. We demonstrate that the systemic administration of antibodies to interleukin (IL)-12 to ovalbumin (OVA)-T cell receptor (TCR) transgenic mice fed high doses of OVA, followed by systemic OVA challenge, substantially enhances TGF-β, but not IL-4 production by peripheral T cells. Furthermore, we demonstrate in an in vitro T cell differentiation model that naive (CD4⁺/Mel-14^hi) OVA-TCR-T cells stimulated with OVA-pulsed dendritic cells (DC) produce four-to fivefold higher amounts of TGF-β when cultured with anti-IL-12 or anti-interferon-γ (IFN-γ). In this in vitro system, IL-4 was not required for TGF-β production by T cells; however, it appeared to enhance levels of TGF-β by promoting the growth of TGF-β-producing cells. Our findings demonstrate that IL-12 and IFN-γ are important negative regulators of TGF-β production both in vivo and in vitro, and that their modulation may be of benefit for the treatment of autoimmune disorders.     

Transcription factor Smad-independent T helper 17 cell induction by transforming-growth factor-β is mediated by suppression of eomesodermin

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Highlights? Suppression of Eomes by TGF-β is an important mechanism for Th17 cell differentiation ? Eomes expression is suppressed by TGF-β via a Smad-independent mechanism ? The JNK-c-Jun pathway suppresses Eomes expression and promotes Th17 cell differentiation ? Eomes directly inhibits RORγt and IL-17A expression through the T-box domain     

Adoptive transfer of all-trans-retinal-induced regulatory T cells ameliorates experimental autoimmune arthritis in an interferon-gamma knockout model

Maintaining an appropriate balance between subsets of CD4⁺ helper T cells and T regulatory cells (Tregs) is a critical process in immune homeostasis and a protective mechanism against autoimmunity and inflammation. To identify the role of vitamin A-related compounds, we investigated the regulation of interleukin (IL)-17-producing helper T cells (Th17 cells) and Tregs treated with all-trans-retinal (retinal). CD4⁺T cells or total cells from the spleens of C57BL/6 mice were stimulated under Treg-polarizing (anti-CD3/CD28 and TGF-β) or Th17-polarizing (anti-CD3/CD28, TGF-β, and IL-6) conditions in the presence or absence of retinal. To analyze their suppressive abilities, retinal-induced Tregs or TGF-β-induced Tregs were co-cultured with responder T cells. Collagen-induced arthritis (CIA) was established in interferon (IFN)-γ knockout mice. On day 13, retinal-induced Tregs were adoptively transferred to mice with established CIA after second immunizations. Compared with TGF-β-induced Treg cells, retinal-induced Tregs showed increased Foxp3 expression and mediated stronger suppressive activity. Under Th17-polarizing conditions, retinal inhibited the production of IL-17 and increased the expression of Foxp3.Retinal-induced Tregs showed therapeutic effects in IFN-γ knockout CIA mice. Thus, we demonstrated that retinal reciprocally regulates Foxp3⁺ Tregs and Th17 cells. These findings suggest that retinal, a vitamin A metabolite, can regulate the balance between pro- and anti-inflammatory immunity. A better understanding of the manipulation of Foxp3 and Tregs may enable the application of this tremendous therapeutic potential in various autoimmune diseases.     

Boswellic acids reduce Th17 differentiation via blockade of IL‐1β‐mediated IRAK1 signaling

Interferon‐gamma producing CD4⁺ T (Th1) cells and IL‐17‐producing CD4⁺ T (Th17) cells are involved in the pathogenesis of several autoimmune diseases including multiple sclerosis. Therefore, the development of treatment strategies controlling the generation and expansion of these effector cells is of high interest. Frankincense, the resin from trees of the genus Boswellia, and particularly its prominent bioactive compound acetyl‐11‐keto‐β‐boswellic acid (AKBA), have potent anti‐inflammatory properties. Here, we demonstrate that AKBA is able to reduce the differentiation of human CD4⁺ T cells to Th17 cells, while slightly increasing Th2‐ and Treg‐cell differentiation. Furthermore, AKBA reduces the IL‐1β‐triggered IL‐17A release of memory Th17 cells. AKBA may affect IL‐1β signaling by preventing IL‐1 receptor‐associated kinase 1 phosphorylation and subsequently decreasing STAT3 phosphorylation at Ser727, which is required for Th17‐cell differentiation. The effects of AKBA on Th17 differentiation and IL‐17A release make the compound a good candidate for potential treatment of Th17‐driven diseases.     

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 the Notch signaling pathway disturbs the CD4+/CD8+, Th17/Treg balance in rats with experimental autoimmune uveitis

Objective and design

The present study aimed to investigate the relationship between the disturbed balance of CD4⁺/CD8⁺, Th17/Treg and the activation of the Notch signaling pathway in experimental autoimmune uveitis (EAU).

Methods

An EAU rat model was induced in Lewis rats, and pathology analysis was performed by hematoxylin and eosin (H&E) staining. CD4⁺, CD8⁺, Th17, and Treg levels in spleen, lymph nodes and eye tissues were determined by flow cytometry. Meanwhile, the expression of Notch1, DLL4, IL-10, and IL-17 was determined by quantitative polymerase chain reaction (Q-PCR) and enzyme-linked immunosorbent assay (ELISA). In addition, the inhibitory effect of N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT) on Th17 differentiation by Notch signaling in vitro was further investigated using T lymphocytes from EAU rats on day 12 post-immunization by flow cytometry.

Results

The pathological results showed that inflammatory cell infiltration occurred in ocular tissues in EAU rats. The CD4⁺/CD8⁺ and Th17/Treg ratios in EAU rats were apparently higher than those in normal control individuals. Q-PCR and ELISA analyses indicated the expression of Notch1, DLL4, IL-10, and IL-17 in EAU rats gradually increased on day 6 after immunization, peaked on day 12, and then gradually decreased. The dynamic trends in Notch1 and DLL4 expression in EAU rats were identical to those of CD4⁺/CD8⁺ and Th17/Treg levels. DAPT can significantly inhibit the activation of Notch signaling, decrease Th17 cell differentiation, and attenuate the level of the Th17 cell lineage, contributing to the balance of the Th17/Treg ratio.

Conclusion

The activation of the Notch signaling pathway can regulate Th17 and Treg cell differentiation, disrupt the CD4⁺/CD8⁺ and Th17/Treg balance, and aggravate the severity of EAU; inactivation of the Notch signaling pathway contributes to the CD4⁺/CD8⁺ and Th17/Treg balance in EAU rats. Our findings highlighted that the dynamic change in the CD4⁺/CD8⁺ and Th17/Treg ratio was consistent with the expression trend of Notch signaling in EAU rats, suggesting that Notch signaling may be a potentially important therapeutic target in clinical practice.

     

Response gene to complement 32 (RGC-32) expression on M2-polarized and tumor-associated macrophages is M-CSF-dependent and enhanced by tumor-derived IL-4

Response gene to complement 32 (RGC-32) is a cell cycle regulator involved in the proliferation, differentiation and migration of cells and has also been implicated in angiogenesis. Here we show that RGC-32 expression in macrophages is induced by IL-4 and reduced by LPS, indicating a link between RGC-32 expression and M2 polarization. We demonstrated that the increased expression of RGC-32 is characteristic of alternatively activated macrophages, in which this protein suppresses the production of pro-inflammatory cytokine IL-6 and promotes the production of the anti-inflammatory mediator TGF-β. Consistent with in vitro data, tumor-associated macrophages (TAMs) express high levels of RGC-32, and this expression is induced by tumor-derived ascitic fluid in an M-CSF- and/or IL-4-dependent manner. Collectively, these results establish RGC-32 as a marker for M2 macrophage polarization and indicate that this protein is a potential target for cancer immunotherapy, targeting tumor-associated macrophages.     

Immunomodulation of human CD19+CD25high regulatory B cells via Th17/Foxp3 regulatory T cells and Th1/Th2 cytokines

Regulatory B (Breg) cells are a special subset of immunoregulatory cells with unique phenotypes and functions. In this study, human CD19⁺CD25^high Breg cells were purified from human peripheral blood. Based on the coculture system of Breg cells and CD4⁺ T cells in vitro, Breg cells were found to promote the increase in regulatory T (Treg) cells while decreasing the number of Th17 cells. Breg cells regulate Treg cells through two processes: cell-cell contact and cytokines. TGF-βsRII, a blocker of transforming growth factor-β (TGF-β), can attenuate the effects of Treg elevation, suggesting that TGF-β is the main cytokine, while Breg cells rather than interleukin-10 (IL-10) regulate the differentiation of Treg cells. However, Th17 cells were mainly regulated by cytokines, without an obvious regulatory effect on cell-cell contacts. Breg cells may regulate Th17 cells by a pathway independent of TGF-β and IL-6. The coculture of Breg cells and CD4⁺ T cells led to changes in the cytokine spectrum, which included significant increases in IL-4, IL-6 and IL-10 but not obvious changes in IL-2, IFN-γ and TNF. The inhibitory effect of Breg cells was weakened by blocking cell-cell contacts in cultures separated with the Transwell chamber because IL-10 decreased while IL-6 increased when compared with cocultured Breg and CD4⁺ T cells. When the IL-10 inhibitor IL-10sRα was added, IL-6 and TNF levels significantly increased, while treatment with the TGF-β inhibitor TGF-βsRII did not result in similar changes, suggesting that IL-10 is an important molecule to inhibit the proinflammatory factors IL-6 and TNF in this culture system.     

Th9 cells and IL-9 in autoimmune disorders: Pathogenesis and therapeutic potentials

Naïve CD4⁺ T cells are pleiotropically divided into various T helper (Th) cell subsets, according to their pivotal roles in the regulation of immune responses. The differentiation of Th9 cells, an interleukin (IL)-9 producing subset, can be impacted by specific environmental cues, co-stimulation with transforming growth factor β (TGF-β) and IL-4, and other regulatory factors. Although IL-9 has been recognized as a classical Th2-related cytokine, recent studies have indicated that IL-9-producing cells contribute to a group of autoimmune disorders including systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel diseases (IBD), rheumatoid arthritis (RA) and psoriasis. Studies of Th9 cells in autoimmune diseases, although in their infancy, are expected to be of growing interest in the study of potential mechanisms of cytokine regulatory pathways and autoimmune pathogenesis. Several in vitro and in vivo pre-clinical trials have been conducted to explore potential therapeutic strategies by targeting the IL-9 pathway. Specifically, anti-IL-9 monoclonal antibodies (mAbs) and IL-9 inhibitors may potentially be used for the clinical treatment of allergic diseases, autoimmune diseases or cancers. Here, we review recent research on Th9 cells and IL-9 pertaining to cell differentiation, biological characteristics and pivotal cellular inter-relationships implicated in the development of various diseases.     

The p38 mitogen‐activated protein kinase cascade modulates T helper type 17 differentiation and functionality in multiple sclerosis

The p38 mitogen‐activated protein kinase cascade is required for the induction of a T helper type 17 (Th17) ‐mediated autoimmune response, which underlies the development and progression of several autoimmune diseases, such as experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis (MS). However, the contribution of p38 phosphorylation to human Th cell differentiation has not been clarified. Here we demonstrate that the p38 signalling pathway is implicated in the generation of Th17 lymphocytes from human CD4⁺ CD27⁺ CD45RA⁺ naive T cells, both in healthy donors and in patients affected by the relapsing–remitting form of MS. Our data also indicate that p38 activation is essential for interleukin‐17 release from central memory lymphocytes and committed Th17 cell clones. Furthermore, CD4⁺ T cells isolated from individuals with relapsing–remitting MS display an altered responsiveness of the p38 cascade, resulting in increased p38 phosphorylation upon stimulation. These findings suggest that the p38 signalling pathway, by modulating the Th17 differentiation and response, is involved in the pathogenesis of MS, and open new perspectives for the use of p38 inhibitors in the treatment of Th17‐mediated autoimmune diseases.     

Th17 cells generated in the absence of TGF-β induce experimental allergic encephalitis upon adoptive transfer

The discovery of IL-17-producing helper T cells (Th17) has led to new concepts of T-cell differentiation and immunity. Importantly, Th17 cells are thought to be important drivers of autoimmunity. TGF-β and IL-6 in combination were shown to induce differentiation of murine naive CD4 T cells into IL-17-producing cells in vitro. By contrast, human Th17 differentiation was shown to be independent of TGF-β and could be induced by IL-6 in conjunction with IL-21 or with IL-1. Ghoreschi et al. have elegantly demonstrated that mouse Th17 cell differentiation can also occur in the absence of TGF-β. A combination of IL-23, IL-1 and IL-6 can give rise to IL-17-producing cells, which are characterized by the expression of T-bet. Intriguingly, the adoptive transfer of such in vitro differentiated Th17 cells into lymphocyte-deficient mice resulted in the induction of experimental allergic encephalitis, which was more severe than in mice receiving Th17 cells differentiated in the presence of TGF-β. Collectively, the results suggest that a subpopulation of Th17 cells differentiated in the absence of TGF-β, expressing T-bet and RORγt, occur in vivo and may be responsible for autoimmunity.