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Epidemiological studies have suggested that fine particulate matter (PM 2.5 ) and asthma have been independently associated with pulmonary fibrosis but rarely studied together. Furthermore, it is unknown whether airway fibrosis in asthma is more attributable to water-soluble ions of PM 2.5 . Our current study was to explore the potential mechanism of PM 2.5 and water-soluble components on airway fibrosis in ovalbumin (OVA)-sensitized asthmatic rats. Rats were intratracheally instilled with PM 2.5 and water-soluble components every 3 days for 4 times or 8 times. Histopathological examination demonstrated that lung inflammatory and airway fibrosis were induced after PM 2.5 and water-soluble components exposure. Meanwhile, PM 2.5 , in particular water-soluble extracts, increased expression of collagen 1 (COL-1), connective tissue growth factor (CTGF), interleukin-6 (IL-6), transforming growth factor-β1 (TGF-β1), Smad family member 3 (Smad3), and p-Smad3, whereas decreased secretion of heme oxygenase-1 (HO-1). However, pretreating asthmatic rats with SB432542, the inhibitor of TGF-β1, and SIS3 HCl, the antagonist of Smad3, both reversed the activation of airway fibrosis induced by water-soluble extracts. Therefore, TGF-β1/Smad3 signaling pathway may be responsible for the pathological process of airway fibrosis in asthmatic rats following PM 2.5 and water-soluble components exposure., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Treg are known to have a central role in orchestrating immune responses, but less is known about the destiny of Treg after being activated by specific Ags. This study aimed to investigate the role of superoxide dismutase, an active molecule in the regulation of oxidative stress in the body, in the prevention of Treg apoptosis induced by specific Ags. Ag-specific Tregs were isolated from the DO11.10 mouse intestine. A food allergy mouse model was developed with ovalbumin as the specific Ag and here, we observed that exposure to specific Ag induced Treg apoptosis through converting the precursor of TGF-β to its mature form inside the Tregs. Oxidative stress was induced in Tregs upon exposure to specific Ags, in which Smad3 bound the latency-associated peptide to induce its degradation, converting the TGF-β precursor to its mature form, TGF-β. Suppressing oxidative stress in Tregs alleviated the specific Ag-induced Treg apoptosis in in vitro experiments and suppressed experimental food allergy by preventing the specific Ag-induced Treg apoptosis in the intestine. In conclusion, exposure to specific Ags induces Treg apoptosis and it can be prevented by upregulating superoxide dismutase or suppressing reactive oxidative species in Tregs., (© 2021 Wiley-VCH GmbH.)

In this study, we investigated the role of tumor-associated macrophages (TAMs) in the progression of pancreatic ductal adenocarcinoma (PDAC). PDAC patients with higher levels of CD68 + TAMs exhibited shorter overall survival. In Transwell assays, PDAC cells incubated with TAMs or conditioned media from TAM cells (TAM-CM) showed higher migration and invasion rates than controls. PET/CT scan analysis of orthotopic PDAC model mice revealed greater primary tumor growth and liver metastasis in the TAM-CM treatment group than the controls. H&E staining of liver tissues showed significantly higher numbers of metastatic nodules in the TAM-CM treatment group. Heat inactivation of TAM-CM significantly reduced Transwell migration by PDAC cells, suggesting the involvement of one or more secreted proteins in PDAC progression. Transcriptome sequencing analysis of PDAC cells treated with TAM-CM revealed significant enrichment of transforming growth factor-β (TGF-β) signaling pathway genes. Western blot and qRT-PCR analysis showed that TAM-CM enhanced PDAC migration cells by inducing epithelial-to-mesenchymal transition through the TGF-β-Smad2/3/4-Snail signaling axis. The pro-tumorigenic effects of TAMs or TAM-CM were abolished by TGF-β signaling pathway inhibitors and neutralizing TGF-β antibody. These results demonstrate that TAMs promote PDAC progression through the TGF-β signaling pathway.

Objective: Cysteinyl leukotrienes (CysLTs), a group of inflammatory lipid mediators, are found elevated in obese-asthmatic patients. Leukotriene D 4 (LTD 4 ), a representative CysLT, is implicated in promoting lung inflammation and remodelling in allergic asthma, but its role in non-allergic asthma, especially in obese-asthmatic patients, is not known. Here, using primary human small airway epithelial cells (SAECs) we have investigated the mechanism of LTD 4 -induced inflammation and remodelling and assessed high proneness of obese mice to develop asthma upon challenge with allergen ovalbumin (OVA)., Methods: Primary human small airway epithelial cells (SAECs) were stimulated with different concentrations of LTD 4 for different time intervals and various inflammatory markers were measured through cytokine array, membrane-based ELISA and Western blotting. An air-liquid interface (ALI) model of SAECs was used to study the effects of LTD 4 -induced remodelling in SAECs using Western blotting, H&E staining and PAS staining. Further, OVA-based murine model was used to examine the propensity of high-fat diet (HFD)-fed obese mice to develop asthma symptoms by studying the infiltration of inflammatory cells (assessed by bronchioalveolar lavage (BAL) cytology) and airway remodelling (assessed by histopathology) upon allergen exposure., Results: The human primary small airway epithelial cells (SAECs) treated with LTD 4 showed significant alterations in the levels of inflammatory markers such as GM-CSF, TNF-α, IL-1β, EGF and eotaxin in dose- and time-dependent manner. Further, LTD 4 enhanced the activation of inflammasomes as evidenced by increased levels of NALP3, cleaved caspase-1 and IL-1β. LTD 4 also enhanced inflammation by increasing the expression of COX-2 in SAECs. The airway remodelling markers Vimentin and Muc5AC were found elevated in ALI culture of SAECs when stimulated with LTD 4 , as it also increased TGF-β levels and activation of Smad2/3 phosphorylation in SAECs. Last, sensitization and challenge of HFD-fed obese mice with OVA showed increased infiltration of inflammatory cells in BAL and enhanced levels of remodeling phenotypes like loss of cilia, mucus cell metaplasia and collagen deposition in mice lung tissues., Conclusion: The results suggest that LTD 4 could induce inflammatory response in human airway epithelial cell by activating NALP3 inflammasome. LTD 4 could further promote airway epithelial cells' remodelling through TGF-β/smad2/3-mediated pathway. Our in vivo results suggested that obesity predisposed the OVA challenged mice to develop lung inflammation and remodelling akin to asthma-like phenotypes during obesity.

MicroRNAs regulate gene expression of transcriptional factors, which influence Th17/Treg (regulatory T cells) balance, establishing the molecular mechanism of genetic and epigenetic regulation of Treg and Th17 cells is crucial for understanding rheumatoid arthritis (RA) pathogenesis. The study goal was to understand the potential impact of the selected microRNAs expression profiles on Treg/Th17 cells frequency, RA phenotype, the expression profile of selected microRNAs, and their correlation with the expression profiles of selected transcriptional factors: SOCS1, SMAD3, SMAD4, STAT3, STAT5 in RA; we used osteoarthritis (OA) and healthy controls (HCs) as controls. The study was conducted on 14 RA and 11 OA patients, and 15 HCs. Treg/Th17 frequency was established by flow cytometry. Gene expression analysis was estimated by qPCR. We noticed correlations in RA Th17 cells between miR-26 and SMAD3, STAT3, SOCS1; and miR-155 and STAT3-and in RA Treg cells between miR-26 and SOCS1; miR-31, -155 and SMAD3; and miR-155 and SMAD4. In RA Tregs, we found a negative correlation between miR-26, -126 and STAT5a. The expression level of miR-31 in Th17 cells from RA patients with DAS28 ≤ 5.1 is higher and that for miR-24 is greater in Tregs from patients with DAS28 > 5.1. MiR-146a in Tregs is higher in rheumatoid factor (RF) positive RA patients.

Background: Inducible CD4 + CD25 + regulatory T (iTreg) cells can become pathogenic effector cells, enhancing lung allergic responses., Objective: We aimed to define the underlying cellular and molecular pathways activated by TGF-β, which determine the suppressor or enhancing activities of iTreg cells., Methods: Sensitized wild-type and CD8-deficient (CD8 -/- ) mice were challenged with allergen. Isolated CD4 + CD25 - T cells were activated by using anti-CD3/anti-CD28. To generate suppressor iTreg cells, cells were then differentiated in the presence of TGF-β, whereas IL-17-producing effector T cells were additionally exposed to IL-6. After TGF-β, Smad3 and TGF-β-activated kinase 1 (TAK1) kinase levels were monitored. The consequences of inhibiting either kinase were determined in vitro and after transfer into CD8 -/- recipients. Quantitative PCR and chromatin immunoprecipitation were used to monitor gene expression and histone modifications at the retinoic acid-related orphan receptor γt (Rorγt) locus., Results: In wild-type mice, iTreg cells suppressed lung allergic responses linked to Smad3-dependent forkhead box P3 (Foxp3) expression and IL-10 production. In the presence of IL-6, iTreg cells converted to T H 17 cells, mediating a neutrophil-dependent enhancement of lung allergic responses in CD8 -/- mice. Conversion was regulated by TAK1. Inhibition or silencing of TAK1 prevented expression of Rorγt and T H 17 differentiation through histone modifications of Rorγt; Foxp3 expression and iTreg cell-mediated suppression remained intact. In the same cell, TGF-β induced coexpression of Smad3 and TAK1 proteins; in the presence of IL-6, expression of Smad3 and Foxp3 but not TAK1 decreased., Conclusion: TGF-β regulates iTreg cell outcomes through 2 distinct signal transduction pathways: one Smad3 dependent and the other TAK1 dependent. The balance of these pathways has important implications in T H 17-mediated autoimmune diseases and neutrophil-dependent asthma., (Copyright © 2019 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Although several studies confirmed that berberine may attenuate airway inflammation in mice with chronic obstructive pulmonary disease (COPD), its underlying mechanisms were not clear until now. We aimed to establish an experiment mouse model for COPD and to investigate the effects of berberine on airway inflammation and its possible mechanism in COPD model mice induced by cigarette smoke extract (CSE). Twenty SPF C57BL/6 mice were randomly divided into PBS control group, COPD model group, low-dose berberine group and high-dose berberine group, 5 mice in each group. The neutrophils and macrophages were examined by Wright's staining. The levels of inflammatory cytokines TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay. The expression levels of TGF-β1, Smad2 and Smad3 mRNA and proteins in lung tissues were respectively detected by quantitative real-time polymerase chain reaction and Western blotting. It was found that CSE increased the number of inflammation cells in BALF, elevated lung inflammation scores, and enhanced the TGF-β1/Smads signaling activity in mice. High-dose berberine restrained the alterations in the COPD mice induced by CSE. It was concluded that high-dose berberine ameliorated CSE-induced airway inflammation in COPD mice. TGF-β1/Smads signaling pathway might be involved in the mechanism. These findings suggested a therapeutic potential of high-dose berberine on the CSE-induced airway inflammation.

This study aimed to investigate the effect and underlying mechanism of Methyl helicterilate from Helicteres angustifolia (MHHA) on alcohol-induced hepatic fibrosis. The results showed that MHHA treatment markedly alleviated alcohol-induced liver injury and notably reduced collagen deposition in liver tissue. It significantly enhanced the activity of alcohol dehydrogenase and aldehyde dehydrogenase. Moreover, MHHA treatment markedly decreased the content of inflammatory cytokines, alleviated collagen accumulation, and inhibited the expression of TGF-β1 and Smad2/3 in liver tissue. The experiments in cells showed that MHHA significantly inhibited HSC activation by blocking TGF-β1/Smads signaling pathway. Additionally, it notably induced HSC apoptosis by modulating the mitochondria-dependent pathway. The present study demonstrates that MHHA treatment significantly ameliorates alcoholic hepatic fibrosis and the underlying mechanism may be ascribed to the inhibition of the TGF-β1/Smads pathway and regulation of the mitochondria-mediated apoptotic pathway., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Macrophages exhibit heterogeneity and plasticity and imbalance between pro-inflammatory and anti-inflammatory macrophages plays a critical role in atherosclerosis progression. Telomerase reverse transcriptase (TERT) in macrophages can be activated by nuclear factor-kappa B (NF-κB), but the regulation of telomerase activation on macrophages polarization remains unknown. We previously identified microRNA-216a (miR-216a) to promote inflammation through directly targeting the Smad3/NF-κB pathway. The present study aimed to assess whether miR-216a can regulate telomerase activity and promote macrophages polarization during atherosclerosis progression. The results verified that TERT was highly expressed in macrophages of human carotid atherosclerotic plaques. miR-216a was found to promote telomerase activation in macrophages by 4.5-fold (P = 0.002) through the Smad3/NF-κB pathway. miR-216a also induced macrophages senescence characterized by senescence-associated-β-galactosidase activity and p53 and p16 expression. TERT overexpression promoted the transformation of M2 to M1 while this conversion was suppressed once TERT was inhibited, and the related inflammatory factors and lipid uptake ability of M1 cells were also increased by TERT. In the carotid atherosclerotic plaques from miR-216a-treated apolipoprotein E -/- mice, the numbers of M1 macrophages were increased whereas M2 cells reduced, accompanying with inhibited Smad3 expression and upregulated inflammatory markers and TERT activity. Furthermore, plasma miR-216a level was specifically higher in patients with vulnerable mixed plaques (n = 181) than those with calcified plaques (n = 73) and controls (n = 264). In summary, our findings first revealed a new molecular mechanism of macrophage polarization involving telomerase activation induced by miR-216a through the Smad3/NF-κB signaling, which might serve as a potential therapeutic target for atherosclerosis progression., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Objective: Paeonol is a natural phenolic component isolated from the root bark of peony with multiple pharmacological activities. We investigated the anti-fibrotic effect and underlying mechanism of paeonol. Methods: Twenty-four male C57BL/6J mice were divided into 4 groups ( n  = 6 in each group), injected with CCl 4 to induce liver fibrosis and administrated with paeonol according to the regimen. The serum activity of ALT and AST, and H&E staining were to assess liver injury. Sirius and Masson staining, and hydroxyproline content were to evaluate the degree of liver fibrosis. TNF-α, IL-6, TGF-β, MDA, GSH-PX, SOD, and CAT were detected to reflect inflammation and oxidative stress. RT-qPCR and Western blot analysis to assess the activation of HSCs and TGF-β/Smad3 signaling. Results: Paeonol ameliorated liver injury and liver fibrosis, reflected by the decrease of ALT, AST, less lesion in H&E staining, mitigated fibrosis in Sirius and Masson staining, lessened content of hydroxyproline. Paeonol attenuated the level of IL-6 and TNF-α, and elevated the activity of GSH-PX, SOD, and CAT with reducing the level of MDA. The expression of col 1a, α-SMA, vimentin, and desmin were down-regulated and TGF-β/Smad3 signaling pathway was inhibited. Conclusion: These data demonstrated that paeonol could alleviate CCl 4 -induced liver fibrosis through suppression of hepatic stellate cells activation via inhibiting the TGF-β/Smad3 signaling.

CD4 + CD25 + Foxp3 + Tregs control the immune response and maintain immune homeostasis. This study examined whether Tregs can affect mouse enteritis and the Foxp3 (Forkhead transcription factor) transcriptional pathway. Mouse CD4 + CD25 + Treg cells were labelled using CFSE (5,6-carboxyfluorescein diacetate succinimidyl ester) and transferred to enteritis model mice. The mice were randomly divided into an enteritis group, a Treg-infusion group, a Treg-inhibiting group, and a control group. Histopathology, ELISA, flow cytometry, western blot, immunohistochemistry, and immunofluorescence were performed. Our results demonstrated that CD4 + CD25 + Tregs were successfully transferred. The disease activity index (DAI) scores in the Tregs-infusion group were lower than those of the enteritis and Tregs-inhibiting groups. The number of goblet cells and inflammatory cells was reduced, and the levels of IL-1 β , TNF- α , NO, and PGE2 were significantly decreased in the Tregs-infusion group compared to those in the enteritis group (p<0.05). The number of CD4 + CD25 + Foxp3 + Tregs and CD4 + IL-17A + Th17 cells in the mesenteric lymph nodes differed significantly from the enteritis and Tregs-inhibiting groups (p<0.05). There were more Foxp3 + Tregs and Smad3 and NFAT2 infiltrated into the duodenum after adoptive transfer of CD4 + CD25 + Tregs, which was a significant difference relative to the enteritis group (p<0.05). This study demonstrated that adoptive transfer of CD4 + CD25 + Tregs can decrease mouse enteritis. Foxp3 expression may be improved through the Smad3 and NFAT2 signalling pathways.

The present study aimed to investigate the effect of epigallocatechin gallate (EGCG) on airway inflammation in mice with bronchial asthma, and the regulatory mechanism of transforming growth factor (TGF)‑β1 signaling pathway, so as to provide theoretical basis for research and development of a novel drug for clinical treatment. Mouse models of bronchial asthma were established and injected with dexamethasone and EGCG via the caudal vein. 7 days later, bronchoalveolar tissue was collected for hematoxylin and eosin staining. Determination of airway resistance (AWR) and lung function in mice was detected. Serum was separated for cytometric bead array to detect the changes in inflammatory factors. Bronchoalveolar lavage fluid was collected for eosinophil and neutrophil counts. Fresh blood was obtained for flow cytometry to determine the percentages of Th17 and Treg cells. Bronchovesicular tissue was utilized for western blot assay and reverse transcription‑quantitative polymerase chain reaction to determine the proteins and transcription factors in the TGF‑β1 pathway. EGCG 20 mg/kg significantly reduced asthmatic symptoms, lung inflammatory cell infiltration, and the inflammatory factor levels of interleukin (IL)‑2, IL‑6 and tumor necrosis factor (TNF)‑α. In addition, it increased the levels of inflammatory factors, including IL‑10, diminished the percentage of Th17 cells, increased the percentage of Treg cells, and decreased the expression of TGF‑β1 and phosphorylated (p)‑Smad2/3 expression. Following the inhibition of the TGF‑β1 receptor, the anti‑inflammatory effect of EGCG disappeared, and the expression of TGF‑β1 and p‑Smad2/3 increased. EGCG attenuated airway inflammation in asthmatic mice, decreased the percentage of Th17 cells and increased the percentage of Treg cells. The anti‑inflammatory effect of EGCG is achieved via the TGF‑β1 signaling pathway.

Natural killer (NK) cells, early effectors in anticancer immunity, are paralyzed by TGFβ1, an immunosuppressive cytokine produced by cancer cells. Development and activity of NK cells are largely inhibited in the Smad3-dependent tumor microenvironment. Here, we used genetic engineering to generate a stable SMAD3-silencing human NK cell line, NK-92-S3KD, whose cancer-killing activity and cytokine production were significantly enhanced under TGFβ1-rich condition compared with the parental cell line. Interestingly, we identified that the IFNG gene is a direct E4BP4 target gene. Thus, silencing of SMAD3 allows upregulation of E4BP4 that subsequently promoting interferon-γ (IFNγ) production in the NK-92-S3KD cells. More importantly, NK-92-S3KD immunotherapy increases the production of not only IFNγ, but also granzyme B and perforin in tumors; therefore, inhibiting cancer progression in two xenograft mouse models with human hepatoma (HepG2) and melanoma (A375). Thus, the NK-92-S3KD cell line may be useful for the clinical immunotherapy of cancer. Cancer Immunol Res; 6(8); 965-77. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Depending on the duration and severity, psychological tension and physical stress can enhance or suppress the immune system in both humans and animals. Although it has been established that chronic stress exerts a significant suppressive effect on immune function, the mechanisms by which affects immune responses remain elusive. By employing an in vivo murine system, we revealed that TGF-β1/Smad2/3/Foxp3 axis was remarkably activated following chronic stress. Furthermore, TLR9 and p38 MAPK played a critical role in the activation of TGF-β1/Smad2/3/Foxp3 signaling cascade. Moreover, inhibition of TGF-β1/Smad2/3/Foxp3 or p38 significantly attenuated chronic stress-induced lymphocyte apoptosis and apoptosis-related proteins, as well as the differentiation of T regulatory cells in spleen. Interestingly, disequilibrium of pro-inflammatory and anti-inflammatory cytokines balance caused by chronic stress was also rescued by blocking TGF-β1/Smad2/3/Foxp3 axis. These findings yield insight into a novel mechanism by which chronic stress modulates immune functions and identifies new targets for the development of novel anti-immune suppressant medications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Radiation-induced lung injury (RILI) is one of the most common and fatal complications of thoracic radiotherapy. It is characterized with two main features including early radiation pneumonitis and fibrosis in later phase. This study was to investigate the potential radioprotective effects of polydatin (PD), which was shown to exert anti-inflammation and anti-oxidative capacities in other diseases. In this study, we demonstrated that PD-mitigated acute inflammation and late fibrosis caused by irradiation. PD treatment inhibited TGF-β1-Smad3 signalling pathway and epithelial-mesenchymal transition. Moreover, radiation-induced imbalance of Th1/Th2 was also alleviated by PD treatment. Besides its free radical scavenging capacity, PD induced a huge increase of Sirt3 in culture cells and lung tissues. The level of Nrf2 and PGC1α in lung tissues was also elevated. In conclusion, our data showed that PD attenuated radiation-induced lung injury through inhibiting epithelial-mesenchymal transition and increased the expression of Sirt3, suggesting PD as a novel potential radioprotector for RILI., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Genetic studies in complex diseases have been highly successful, but have also been largely one-dimensional: predominantly focusing on the genetic contribution to disease susceptibility. While this is undoubtedly important-indeed it is a pre-requisite for understanding the mechanisms underlying disease development-there are many other important aspects of disease biology that have received comparatively little attention. In this review, I will discuss how existing genetic data can be leveraged to provide new insights into other aspects of disease biology, why such insights could change the way we think about complex disease, and how this could provide opportunities for better therapies and/or facilitate personalised medicine. To do this, I will use the example of Crohn's disease-a chronic form of inflammatory bowel disease that has been one of the main success stories in complex disease genetics. Indeed, thanks to genetic studies, we now have a much more detailed understanding of the processes involved in Crohn's disease development, but still know relatively little about what determines the subsequent disease course (prognosis) and why this differs so considerably between individuals. I will discuss how we came to realise that genetic variation plays an important role in determining disease prognosis and how this has changed the way we think about Crohn's disease genetics. This will illustrate how phenotypic data can be used to leverage new insights from genetic data and will provide a broadly applicable framework that could yield new insights into the biology of multiple diseases., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Introduction: Interleukin (IL)-37 is a newly identified member of the IL-1 family, and shows a growing role in a variety of diseases. This review aims at summarizing and discussing the role of IL-37 in cardiovascular diseases., Methods: Data for this review were identified by searches of MEDLINE, Embase, and PubMed using appropriate search terms., Results: IL-37 is a newly identified cytokine belonging to the IL-1 family and is expressed in inflammatory immune cells and several parenchymal cells. It has potent anti-inflammatory and immunosuppressive properties, with two mechanisms underlying this function. IL-37 is produced as a precursor and then cleaved into mature form in the cytoplasm by caspase-1, translocating to nucleus and suppressing the transcription of several pro-inflammatory genes by binding SMAD-3. Besides, IL-37 can be secreted extracellularly, and binds to IL-18Ra chain and recruits Toll/IL-1R (TIR)-8 for transducing anti-inflammatory signaling. IL-37 is upregulated in an inducible manner and negatively regulates signaling mediated by TLR agonists and pro-inflammatory cytokines. The cytokine has been shown to inhibit both innate and adaptive immunological responses, exert antitumor effects, and act as a prognostic marker in a variety of autoimmune diseases., Conclusions: Recent studies have suggested that IL-37 plays a role in cardiovascular diseases. In this review, we provide an overview of the cytokine biology, discuss recent advances made in unraveling its cardio-protective effects, and suggest guidelines for future research., (© 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.)

Cholera toxin B subunit fusion to autoantigens such as proinsulin (CTB-INS) down regulate dendritic cell (DC) activation and stimulate synthesis of DC immunosuppressive cytokines. Recent studies of CTB-INS induction of immune tolerance in human DCs indicate that increased biosynthesis of indoleamine 2,3-dioxygenase (IDO1) may play an important role in CTB-INS vaccine suppression of DC activation. Studies in murine models suggest a role for transforming growth factor beta (TGF-β) in the stimulation of IDO1 biosynthesis, for the induction of tolerance in DCs. Here, we investigated the contribution of TGF-β superfamily proteins to CTB-INS induction of IDO1 biosynthesis in human monocyte-derived DCs (moDCs). We show that CTB-INS upregulates the level of TGF-β1, activin-A and the TGF-β activator, integrin αvβ8 in human DCs. However, inhibition of endogenous TGF-β, activin-A or addition of biologically active TGF-β1, and activin-A, did not inhibit or stimulate IDO1 biosynthesis in human DCs treated with CTB-INS. While inhibition with the kinase inhibitor, RepSox, blocked SMAD2/3 phosphorylation and diminished IDO1 biosynthesis in a concentration dependent manner. Specific blocking of the TGF-β type 1 kinase receptor with SB-431542 did not arrest IDO1 biosynthesis, suggesting the involvement of a different kinase pathway other than TGF-β type 1 receptor kinase in CTB-INS induction of IDO1 in human moDCs. Together, our experimental findings identify additional immunoregulatory proteins induced by the CTB-INS fusion protein, suggesting CTB-INS may utilize multiple mechanisms in the induction of tolerance in human moDCs., (Copyright © 2017. Published by Elsevier Inc.)

Regulatory T (Treg) cells are important for the maintenance of immune homoeostasis and prevention of autoimmune diseases. Epigenetic modifications have been reported to modulate autoimmunity by altering Treg cell fate. Here we show that the H3K4 methyltransferase Ash1l facilitates TGF-β-induced Treg cell polarization in vitro and protects mice from T cell-mediated colitis in vivo. Ash1l upregulates Smad3 expression by directly targeting Smad3 promoter to increase local H3K4 trimethylation. Furthermore, we identify an lncRNA, namely lnc-Smad3, which interacts with the histone deacetylase HDAC1 and silences Smad3 transcription. After TGF-β stimulation, activated Smad3 suppresses lnc-Smad3 transcription, thereby recovering the Smad3 promoter accessibility to Ash1l. By revealing the opposite regulatory functions of Ash1l and lnc-Smad3 in Smad3 expression, our data provide insights for the epigenetic control of Treg cell fate to potentially aid in the development of therapeutic intervention for autoimmune diseases.

Chronic mucocutaneous candidiasis, characterized by persistent or recurrent fungal infections, represents the clinical hallmark in gain-of-function (GOF) signal transducer and activator of transcription 1 (STAT1) mutation carriers. Several cases of intracranial aneurysms have been reported in patients with GOF STAT1 mutation but the paucity of reported cases likely suggested this association still as serendipity. In order to endorse this association, we link the development of intracranial aneurysms with STAT1 GOF mutation by presenting the two different cases of a patient and her mother, and demonstrate upregulated phosphorylated STAT4 and IL-12 receptor β1 upon stimulation in patient's blood cells. We also detected increased transforming growth factor (TGF)-β type 2 receptor expression, particularly in CD14 + cells, and a slightly higher phosphorylation rate of SMAD3. In addition, the mother of the patient developed disseminated bacille Calmette-Guérin disease after vaccination, speculating that GOF STAT1 mutations may confer a predisposition to weakly virulent mycobacteria., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Epithelial-mesenchymal transition (EMT) has been recognized to accelerate peritoneal membrane dysfunction. 1,25(OH)2D3/vitamin D receptor (VDR) is important for preventing various types of EMT in vivo. However, its function on EMT and inflammation of human peritoneal mesothelial cells (HPMCs) remains to be elucidated. Therefore, the present study investigated the effects of 1,25(OH)2D3/VDR on high glucose (HG)‑induced EMT and inflammation in HPMCs and the underlying molecular mechanism. It was determined that HG reduced VDR expression, increased inflammatory cytokine expression, including transforming growth factor β (TGFβ) and interleukin‑6 (IL‑6) and phosphorylated‑SMAD family member 3 (p‑Smad3) expression. EMT was promoted as the expression level of the epithelial marker E‑cadherin was reduced, whereas expression levels of the mesenchymal markers α‑SMA and FN were increased. 1,25(OH)2D3 pretreatment inhibited the expression of inflammatory cytokines in HPMCs and attenuated HG‑induced EMT, possibly through inhibition of the TGFβ/Smad pathway by binding to its receptor VDR.

Objective. Reprogramming of M1 macrophage phenotype with inhibited M2 phenotype transcription factors, such as STAT3, STAT6 and SMAD and assess their impact on the development of Ehrlich carcinoma (EC) in vitro and in vivo. Methods. Tumor growth in vitro was initiated by addition of EC cells in RPMI-1640 culture medium and in vivo by intraperitoneal of EC cell injection into mice. Results. It was found that M1-STAT3/6- SMAD3 macrophages have a pronounced anti-tumor effect in vitro, and in vivo, which was greater than anti-tumor effects of M1, M1-STAT 3/6, M1-SMAD3 macrophages and cisplatin. Conclusion. M1 macrophages with inhibited STAT3, STAT6 and/or SMAD3 effectively restrict tumor growth. The findings justify the development of new anti-tumor cell therapy technology.

In this research, compound Maqin decoction (CMD) has been shown to positively affect in airway inflammation of asthma models. We evaluated the effects of CMD on the expression of transforming growth factor (TGF)-β1/Smad proteins, interleukin (IL)-17, and IL-10 in lung tissue of asthmatic rats. Asthma was induced in a rat model using ovalbumin. After a 4-week treatment with CMD, rats were killed to evaluate the expression of TGF-β1 and Smad proteins in lung tissue. IL-10 and IL-17 levels in lung tissue homogenates were determined by ELISA. The expression of TGF-β1 and Smad3 protein increased, whereas expression of Smad7 protein decreased upon high-dose or low-dose treatment with CMD or by intervention with dexamethasone, compared to the control. There was a significant difference between treatment with a high dose CMD and the control treatment, but no significant difference was found between high-dose CMD treatment and dexamethasone intervention. The expression of TGF-β1 and Smad7 protein increased, whereas the expression of Smad3 protein decreased in the model group compared to other groups. In the CMD high-dose group, low-dose group, and dexamethasone intervention group, the IL-17 concentrations in lung tissue homogenates were decreased, while IL-10 levels were increased. Again, there was a significant difference between CMD high-dose and control treatment, but not between CMD high-dose treatment and dexamethasone intervention. Thus, positive effects of CMD against asthmatic airway remodeling may be due to its regulatory effect on TGF-β1, Smad3, and Smad7 protein levels and on cytokines such as IL-10 and IL-17., Competing Interests: The authors declare no conflict of interest.

Pulmonary fibrosis (PF) can severely disrupt lung function, leading to fatal consequences. Salidroside is a principal active ingredient of Rhodiola rosea and has recently been reported to protect against lung injures. The present study was aimed at exploring its therapeutic effects on PF. Lung fibrotic injuries were induced in SD rats by a single intratracheal instillation of 5 mg/kg bleomycin (BLM). Then, these rats were administrated with 50, 100, or 200 mg/kg salidroside for 28 days. BLM-triggered structure distortion, collagen overproduction, excessive inflammatory infiltration, and pro-inflammatory cytokine release, and oxidative stress damages in lung tissues were attenuated by salidroside in a dose-dependent manner. Furthermore, salidroside was noted to inhibit IκBα phosphorylation and nuclear factor kappa B (NF-κB) p65 nuclear accumulation while activating Nrf2-antioxidant signaling in BLM-treated lungs. Downregulation of E-cadherin and upregulation of vimentin, fibronectin, and α-smooth muscle actin (α-SMA) indicated an epithelial-mesenchymal transition (EMT)-like shift in BLM-treated lungs. These changes were suppressed by salidroside. The expression of TGF-β1 and the phosphorylation of its downstream targets, Smad-2/-3, were enhanced by BLM, but weakened by salidroside. Additionally, salidroside was capable of reversing the recombinant TGF-β1-induced EMT-like changes in alveolar epithelial cells in vitro. Our study reveals that salidroside's protective effects against fibrotic lung injuries are correlated to its anti-inflammatory, antioxidative, and antifibrotic properties.

Rationale: The etiology of schistosomiasis-associated pulmonary arterial hypertension (PAH), a major cause of PAH worldwide, is poorly understood. Schistosoma mansoni exposure results in prototypical type-2 inflammation. Furthermore, transforming growth factor (TGF)-β signaling is required for experimental pulmonary hypertension (PH) caused by Schistosoma exposure., Objectives: We hypothesized type-2 inflammation driven by IL-4 and IL-13 is necessary for Schistosoma-induced TGF-β-dependent vascular remodeling., Methods: Wild-type, IL-4(-/-), IL-13(-/-), and IL-4(-/-)IL-13(-/-) mice (C57BL6/J background) were intraperitoneally sensitized and intravenously challenged with S. mansoni eggs to induce experimental PH. Right ventricular catheterization was then performed, followed by quantitative analysis of the lung tissue. Lung tissue from patients with schistosomiasis-associated and connective tissue disease-associated PAH was also systematically analyzed., Measurements and Main Results: Mice with experimental Schistosoma-induced PH had evidence of increased IL-4 and IL-13 signaling. IL-4(-/-)IL-13(-/-) mice, but not single knockout IL-4(-/-) or IL-13(-/-) mice, were protected from Schistosoma-induced PH, with decreased right ventricular pressures, pulmonary vascular remodeling, and right ventricular hypertrophy. IL-4(-/-)IL-13(-/-) mice had less pulmonary vascular phospho-signal transducer and activator of transcription 6 (STAT6) and phospho-Smad2/3 activity, potentially caused by decreased TGF-β activation by macrophages. In vivo treatment with a STAT6 inhibitor and IL-4(-/-)IL-13(-/-) bone marrow transplantation also protected against Schistosoma-PH. Lung tissue from patients with schistosomiasis-associated and connective tissue disease-associated PAH had evidence of type-2 inflammation., Conclusions: Combined IL-4 and IL-13 deficiency is required for protection against TGF-β-induced pulmonary vascular disease after Schistosoma exposure, and targeted inhibition of this pathway is a potential novel therapeutic approach for patients with schistosomiasis-associated PAH.

T cells play a critical role in acute allograft rejection. TGF-β/Smad3 signaling is a key pathway in regulating T cell development. We report here that Smad3 is a key transcriptional factor of TGF-β signaling that differentially regulates T cell immune responses in a mouse model of cardiac allograft rejection in which donor hearts from BALB/c mice were transplanted into Smad3 knockout (KO) and wild type (WT) mice. Results showed that the cardiac allograft survival was prolonged in Smad3 KO recipients. This allograft protection was associated with a significant inhibition of proinflammatory cytokines (IL-1β, TNF-α, and MCP-1) and infiltration of neutrophils, CD3+ T cells, and F4/80+ macrophages. Importantly, deletion of Smad3 markedly suppressed T-bet and IFN-γ while enhancing GATA3 and IL-4 expression, resulting in a shift from the Th1 to Th2 immune responses. Furthermore, mice lacking Smad3 were also protected from the Th17-mediated cardiac injury, although the regulatory T cell (Treg) response was also suppressed. In conclusion, Smad3 is an immune regulator in T cell-mediated cardiac allograft rejection. Loss of Smad3 results in a shift from Th1 to Th2 but suppressing Th17 immune responses. Thus, modulation of TGF-β/Smad3 signaling may be a novel therapy for acute allograft rejection.

Macrophages are responsible for the control of inflammation and healing, and their malfunction results in cardiometabolic disorders. TGF-β is a pleiotropic growth factor with dual (protective and detrimental) roles in atherogenesis. We have previously shown that in human macrophages, TGF-β1 activates Smad2/3 signaling and induces a complex gene expression program. However, activated genes were not limited to known Smad2/3-dependent ones, which prompted us to study TGF-β1-induced signaling in macrophages in detail. Analysis of Id3 regulatory sequences revealed a novel enhancer, located between +4517 and 4662 bp, but the luciferase reporter assay demonstrated that this enhancer is not Smad2/3 dependent. Because Id3 expression is regulated by Smad1/5 in endothelial cells, we analyzed activation of Smad1/5 in macrophages. We demonstrate here for the first time, to our knowledge, that TGF-β1, but not BMPs, activates Smad1/5 in macrophages. We show that an ALK5/ALK1 heterodimer is responsible for the induction of Smad1/5 signaling by TGF-β1 in mature human macrophages. Activation of Smad1/5 by TGF-β1 induces not only Id3, but also HAMP and PLAUR, which contribute to atherosclerotic plaque vulnerability. We suggest that the balance between Smad1/5- and Smad2/3-dependent signaling defines the outcome of the effect of TGF-β on atherosclerosis where Smad1/5 is responsible for proatherogenic effects, whereas Smad2/3 regulate atheroprotective effects of TGF-β., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Clinical studies showed that renal expression and serum levels of nerve growth factor (NGF) are increased in renal diseases characterized by progressive fibrosis, a pathologic process in which TGF-β1 mediates most of the key events leading to tubular epithelial-mesenchymal transition (EMT). However, the pathogenic role of high NGF levels has not yet been elucidated. In this study, we found that in tubular renal cells, HK-2, NGF transcriptionally up-regulated TGF-β1 expression and secretion and enhanced cell motility by activating EMT markers via its receptors, TrkA and p75(NTR). Interestingly, we observed that TGF-β1-SMAD pathway activation and the up-regulation of EMT markers NGF-induced were both prevented when knockdown of TGF-β1 gene occurred and that the pretreatment with an antibody anti-NGF reversed the nuclear translocation of pSMAD3/SMAD4 complex. Collectively, our results demonstrated that NGF promotes renal fibrosis via TGF-β1-signaling activation, suggesting that in kidney diseases high NGF serum levels could contribute to worsen renal fibrosis.

Tumor-reactive T cells become unresponsive in advanced tumors. Here we have characterized a common mechanism of T cell unresponsiveness in cancer driven by the upregulation of the transcription factor Forkhead box protein P1 (Foxp1), which prevents CD8⁺ T cells from proliferating and upregulating Granzyme-B and interferon-γ in response to tumor antigens. Accordingly, Foxp1-deficient lymphocytes induced rejection of incurable tumors and promoted protection against tumor rechallenge. Mechanistically, Foxp1 interacted with the transcription factors Smad2 and Smad3 in preactivated CD8⁺ T cells in response to microenvironmental transforming growth factor-β (TGF-β), and was essential for its suppressive activity. Therefore, Smad2 and Smad3-mediated c-Myc repression requires Foxp1 expression in T cells. Furthermore, Foxp1 directly mediated TGF-β-induced c-Jun transcriptional repression, which abrogated T cell activity. Our results unveil a fundamental mechanism of T cell unresponsiveness different from anergy or exhaustion, driven by TGF-β signaling on tumor-associated lymphocytes undergoing Foxp1-dependent transcriptional regulation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The β-galactoside-binding protein galectin-9 is critical in regulating the immune response, but the mechanism by which it functions remains unclear. We have demonstrated that galectin-9 is highly expressed by induced regulatory T cells (iTreg) and was crucial for the generation and function of iTreg cells, but not natural regulatory T (nTreg) cells. Galectin-9 expression within iTreg cells was driven by the transcription factor Smad3, forming a feed-forward loop, which further promoted Foxp3 expression. Galectin-9 increased iTreg cell stability and function by directly binding to its receptor CD44, which formed a complex with transforming growth factor-β (TGF-β) receptor I (TGF-βRI), and activated Smad3. Galectin-9 signaling was further found to regulate iTreg cell induction by dominantly acting through the CNS1 region of the Foxp3 locus. Our data suggest that exogenous galectin-9, in addition to being an effector molecule for Treg cells, acts synergistically with TGF-β to enforce iTreg cell differentiation and maintenance., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Inducible regulatory T-cells (iTReg) can be generated from CD4(+)Foxp3(-) naïve conventional T-cells by a combination of TGF-β and T-cell receptor (TCR) signaling. It is of enormous clinical importance to identify agents that can promote the generation and differentiation of functional iTreg cells. We have established a phenotypic screening platform to identify new compounds that can promote the TGFβ-mediated iTreg differentiation. We have found Kenpaullone, a potent CDK1, CDK2 and CDK5 inhibitor, as new enhancer for iTreg cell differentiation. Kenpaullone promotes iTreg cell differentiation through increased and prolonged transcription of foxp3 gene by enhancing TGFβ-Smad3 signaling pathway. Thus, we have demonstrated that CDK2 is the biological target of Kenpaullone and proven that CDK2 is a novel negative regulator of iTreg cell differentiation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cytokines play a very important role in the regulation of immune homeostasis. Regulatory T cells (Tregs) responsible for the generation of peripheral tolerance are under the tight regulation of the cytokine milieu. In this study, we report a novel role of a bipyridyl compound, Caerulomycin A (CaeA), in inducing the generation of Tregs. It was observed that CaeA substantially up-regulated the pool of Tregs, as evidenced by an increased frequency of CD4(+) Foxp3(+) cells. In addition, CaeA significantly suppressed the number of Th1 and Th17 cells, as supported by a decreased percentage of CD4(+)/IFN-γ(+) and CD4(+)/IL-17(+) cells, respectively. Furthermore, we established the mechanism and observed that CaeA interfered with IFN-γ-induced STAT1 signaling by augmenting SOCS1 expression. An increase in the TGF-β-mediated Smad3 activity was also noted. Furthermore, CaeA rescued Tregs from IFN-γ-induced inhibition. These results were corroborated by blocking Smad3 activity, which abolished the CaeA-facilitated generation of Tregs. In essence, our results indicate a novel role of CaeA in inducing the generation of Tregs. This finding suggests that CaeA has enough potential to be considered as a potent future drug for the treatment of autoimmunity., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mesenchymal stem cells (MSCs) have been suggested to participate in immune regulation and airway repair/remodeling. TGF-β1 is critical in the recruitment of stem/progenitor cells for tissue repair, remodeling, and cell differentiation. In this study, we sought to investigate the role of TGF-β1 in MSC migration in allergic asthma. We examined nestin expression (a marker for MSCs) and TGF-β1 signaling activation in airways in cockroach allergen extract (CRE)-induced mouse models. Compared with control mice, there were increased nestin(+) cells in airways and higher levels of active TGF-β1 in serum and p-Smad2/3 expression in lungs of CRE-treated mice. Increased activation of TGF-β1 signaling was also found in CRE-treated MSCs. We then assessed MSC migration induced by conditioned medium from CRE-challenged human epithelium in air/liquid interface culture in Transwell assays. MSC migration was stimulated by epithelial-conditioned medium, but was significantly inhibited by either TGF-β1-neutralizing Ab or TβR1 inhibitor. Intriguingly, increased migration of MSCs from blood and bone marrow to the airway was also observed after systemic injection of GFP(+) MSCs and from bone marrow of Nes-GFP mice following CRE challenge. Furthermore, TGF-β1-neutralizing Ab inhibited the CRE-induced MSC recruitment, but promoted airway inflammation. Finally, we investigated the role of MSCs in modulating CRE-induced T cell response and found that MSCs significantly inhibited CRE-induced inflammatory cytokine secretion (IL-4, IL-13, IL-17, and IFN-γ) by CD4(+) T cells. These results suggest that TGF-β1 may be a key promigratory factor in recruiting MSCs to the airways in mouse models of asthma.

The interaction of integrin αE(CD103)β7, often expressed on tumor-infiltrating T lymphocytes, with its cognate ligand, the epithelial cell marker E-cadherin on tumor cells, plays a major role in antitumor CTL responses. CD103 is induced on CD8 T cells upon TCR engagement and exposure to TGF-β1, abundant within the tumor microenvironment. However, the transcriptional mechanisms underlying the cooperative role of these two signaling pathways in inducing CD103 expression in CD8 T lymphocytes remain unknown. Using a human CTL system model based on a CD8(+)/CD103(-) T cell clone specific of a lung tumor-associated Ag, we demonstrated that the transcription factors Smad2/3 and NFAT-1 are two critical regulators of this process. We also identified promoter and enhancer elements of the human ITGAE gene, encoding CD103, involved in its induction by these transcriptional regulators. Overall, our results explain how TGF-β1 can participate in CD103 expression on locally TCR-engaged Ag-specific CD8 T cells, thus contributing to antitumor CTL responses and cancer cell destruction.

Low-dose tolerance therapy with nucleosomal histone peptide epitopes blocks lupus disease in mouse models, but effect in humans is unknown. Herein, we found that CD4(+)CD25(high)FoxP3(+) or CD4(+)CD45RA(+)FoxP3(low) T-cells, and CD8(+)CD25(+)FoxP3(+) T-cells were all induced durably in PBMCs from inactive lupus patients and healthy subjects by the histone peptide/s themselves, but in active lupus, dexamethasone or hydroxychloroquine unmasked Treg-induction by the peptides. The peptide-induced Treg depended on TGFβ/ALK-5/pSmad 2/3 signaling, and they expressed TGF-β precursor LAP. Lupus patients' sera did not inhibit Treg induction. The peptide epitope-induced T cells markedly suppressed type I IFN related gene expression in lupus PBMC. Finally, the peptide epitopes suppressed pathogenic autoantibody production by PBMC from active lupus patients to baseline levels by additional mechanisms besides Treg induction, and as potently as anti-IL6 antibody. Thus, low-dose histone peptide epitopes block pathogenic autoimmune response in human lupus by multiple mechanisms to restore a stable immunoregulatory state., (© 2013.)

Leprosy is a chronic human disease that results from infection of Mycobacterium leprae. T reg cells have been shown to have important implications in various diseases. However, in leprosy, it is still unclear whether T regs can mediate immune suppression during progression of the disease. In the present study, we have proposed the putative mechanism leading to high proportion of T reg cells and investigated its significance in human leprosy. High levels of TGF-β followed by adaptation of FoxP3(+) naive and memory (CD4(+)CD45RA(+)/RO(+)) T cells were observed as the principal underlying factors leading to higher generation of T reg cells during disease progression. Furthermore, TGF-β was found to be associated with increased phosphorylation-mediated-nuclear-import of SMAD3 and NFAT towards BL/LL pole to facilitate FoxP3 expression in these cells, the same as justified after using nuclear inhibitors of SMAD3 (SIS3) and NFAT (cyclosporin A) in CD4(+)CD25(+) cells in the presence of TGF-β and IL-2. Interestingly, low ubiquitination of FoxP3 in T reg cells of BL/LL patients was revealed to be a major driving force in conferring stability to FoxP3 which in turn is linked to suppressive potential of T regs. The present study has also pinpointed the presence of CD4(+)CD25(+)IL-10(+) sub class of T regs (Tr1) in leprosy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Montelukast, a leukotriene receptor antagonist, is used commercially as a maintenance treatment for asthma and to relieve allergic symptoms. In this study, we evaluated the protective effects of montelukast against the airway inflammation and fibrosis using a murine model of ovalbumin (OVA) induced chronic asthma. The animals received OVA challenge three times a week for 4 weeks. Montelukast (30 mg/kg) was administrated orally once a day for 4 weeks. The administration of montelukast caused a reduction in elevated interleukin (IL)-4, IL-13, eotaxin, immunoglobulin (Ig), inflammatory cell infiltration into the airways, and mucus production after repeated OVA challenges. To investigate the antifibrotic mechanism of montelukast, we examined the expression of profibrotic mediators, including vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β1, and Smad3 proteins in the lung tissue using western blotting and immunohistochemistry. The administration of montelukast reduced the overexpression of profibrotic proteins in the lung tissue, which was confirmed by immnunohistochemistry. These results are consistent with a histopathological examination of lung tissue with Masson's trichrome stain. In conclusion, the administration of montelukast reduced airway inflammation and pulmonary fibrosis by reducing the release of Th2 cytokines and the expression of VEGF, TGF-β1/Smad3 in the lung tissue., (© 2013.)

IL-9 is a pleiotropic cytokine that can regulate autoimmune and allergic responses. Th9 cells can develop from naive T cells or Th2 cells through stimulation by TGF-β in vitro. In this study, we demonstrated that Smad2 and Smad3 are necessary for IL-9 production from T cells in an OVA-induced asthma model using T cell-specific Smad2- and Smad3-deficient mice. Smad2 and Smad3 were also redundantly essential for TGF-β signaling to induce histone modifications for Il9 transcription. Although Smad2/3 was recruited to the Il9 promoter by TGF-β stimulation, they are not sufficient to activate the Il9 promoter. By the screening the transcription factors, we found that IFN regulatory factor 4 (IRF4) was essential for the Smad2/3-mediated Il9 promoter activation. In addition, Smad2/3 physically interacted with IRF4, and Smad2/3 did not bind to the Il9 promoter and could not induce Th9 in IRF4-deficient T cells. Similarly, IRF4 could not stimulate Il9 transcription in the absence of Smad2/3, and TGF-β enhanced IRF4 recruitment to the Il9 promoter in a Smad2/3-dependent manner. We propose that Smad2/3 and IRF4 cooperatively transactivate the Il9 promoter and play an important role in regulating allergic immune responses by inducing Th9 cells.

The aetiology of both Crohn's disease and ulcerative colitis, the major forms of inflammatory bowel diseases in human beings, remains unknown. However, compelling evidence suggests that the associated pathological process in inflammatory bowel disease is driven by an excessive immune response directed against normal components of the bacterial microflora and marked by defects in counter-regulatory mechanisms, such as those involving transforming growth factor-β1. Indeed, a diminished activity of transforming growth factor-β1, as indicated by a reduced phosphorylation of Smad3, a signalling molecule associated with the activated transforming growth factor-β receptor, is evident in the inflamed gut of inflammatory bowel disease patients and this alteration is due to high Smad7, an intracellular inhibitor of Smad3 phosphorylation. Consistently, silencing of Smad7 with a specific antisense oligonucleotide restores transforming growth factor-β1/Smad3 signalling, thereby leading to inhibition of inflammatory cytokine production and attenuation of experimental colitis in mice. These findings together with the demonstration that Smad7 antisense oligonucleotide is safe and well-tolerated in patients with Crohn's disease indicate that Smad7 antisense oligonucleotide-based pharmaceutical compounds could enter the therapeutic armamentarium of these disorders. In this article we review the available data supporting the pathogenic role of Smad7 in the gut and discuss why Smad7 antisense therapy could help dampen the mucosal inflammation in inflammatory bowel disease., (Copyright © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Artemisinin and its derivatives have been reported to have immunosuppressive activity in some laboratory studies. However, the detail of mechanism remains to be demonstrated. The objective of this study is to clarify the immunosuppressive activity of artesunate (AST), one kind of artemisinin derivatives, and to find its unexplored mode of action. In vitro, the proliferation of T lymphocytes and its cytotoxicity were measured by WST-1 and MTT assay. In vivo, the immunomodulatory effect of AST was evaluated in a mouse model of delayed type hypersensitivity reaction (DTH), which was based on a T cell-mediated immune response. The data displayed that AST had a relatively high immunosuppressive activity with low toxicity, and could inhibit T lymphocyte proliferation induced by mitogen and alloantigen. Meanwhile, topical administration of AST could suppress DTH response significantly. Moreover, AST could also increase the secretion of TFG-β, coupling with the striking enhance of NF-κB/p65 and Smad2/3 signaling. The promotion of CD4(+)CD25(+) regulatory T cells (Tregs) was shown to be a possible mechanism involved in AST-mediated regulation. Taken together, these observations exhibit the potential of developing AST as a novel safe remedy for the treatment of T cell-mediated immune disorders., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

IL-21 has pleiotropic effects on innate and adaptive immune response, and plays an important role in the development of autoimmune disease and antitumor activity. It has been reported that IL-21 is produced by CD4(+) T cells and NKT cells. However, the differentiation of IL-21-producing CD4(+) T cells in humans remains largely unclear. In the present study, we showed that cytokines of IL-1β, IL-6 or IL-21 induced differentiation of human IL-21-producing CD4(+) T cells, and TGF-β enhanced the effect of inflammatory cytokines on the development of IL-21-producing CD4(+) T cells. Furthermore, we found that the majority of IL-21-producing cells were distinct from Th17 cells and Th1 cells since they did not co-express IL-17 and IFN-γ. TGF-β significantly inhibited the production of IFN-γ and enhanced the effect of IL-21 on the development of IL-21-producing CD4(+) T cells. In addition, we found that IL-21 inhibited the differentiation of CD4(+) Foxp3(+) T cells induced by TGF-β. Further study indicated that IL-21 induced phosphorylation of transcriptional factors of STAT1, STAT3 and STAT5, and TGF-β induced phosphorylation of Smad3 in CD4(+) T cells. Taken together, our data indicated that TGF-β enhanced IL-21-induced differentiation of IL-21-producing CD4(+) T cells, and the majority of IL-21-producing cells were different from Th17 and Th1 cells. Our results provide a new sight regarding the differentiation of human CD4(+) T cells.

The responsiveness of DCs and their precursors to transforming growth factor beta1 (TGF-β1) affects the nature of differentiating DC subsets, which are essential for the severity of atopic dermatitis (AD). To evaluate TGF-β signaling in monocytes and monocyte-derived DCs of AD patients compared with that of controls, in vitro generated Langerhans cell (LC) like DCs, expression of TGF-β receptors, phospho-Smad2/3 and Smad7 were evaluated. Furthermore, TNF-α expression and synergistic effects of TNF-α upon TGF-β signaling and DC generation were evaluated. We found LC-like DC differentiation of monocytes from AD patients in response to TGF-β1 was remarkably reduced and TGF-β1 receptor expression was significantly lower compared with that of healthy controls. Attenuated TGF-β1 responsiveness mirrored by lower phospho-Smad2/3 expression after TGF-β1 stimulation and higher expression of inhibitory Smad7 was observed in monocytes from AD patients. During DC generation, mRNA expression of Smad7 was relatively higher in LC-like DCs of AD patients. Lower TNF-α expression of monocytes from AD patients might further contribute to attenuated TGF-β signaling in the disease since TNF-α had synergistic effects on TGF-β1 signaling and LC generation through mediating the degradation of Smad7. Our results demonstrate alleviated TGF-β1 signaling together with the amount of soluble co-factors might direct the nature of differentiating DCs., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Activated T cells infiltrate a renal allograft during rejection and can respond to TGF-β within the tubules, causing local differentiation and expression of the αE(CD103)β7 integrin. This study was performed to examine the expression of latent TGF-β within renal allograft tissues and to define a mechanism by which T cells can activate and respond to this latent factor. Rejecting renal allograft biopsy tissues showed increased expression of the latent TGF-β complex, which was localized around the tubules by a mechanism that might involve interaction with heparan sulfate in the basement membrane. A cultured renal TEC line also expressed the latent complex, but these cells did not respond to this form of TGF-β by pSmad 3. However, coculture of these cells with activated T cells induced the expression of CD103, suggesting that T cells can activate and respond to the latent TGF-β associated with TEC. Although activated T cells expressed little cell-surface TSP-1, this was increased by culture with fibronectin or fibronectin-expressing renal TEC. Blockade of TSP-1 using LSKL peptides reduced the potential of activated T cells to differentiate in response to latent TGF-β. This study suggests that penetration of renal tubules by activated T cells leads to increased expression of T cell-surface TSP-1, allowing activation of latent TGF-β sequestered on heparan sulfate within the microenvironment. This mechanism may be important for localized phenotypic maturation of T cells that have infiltrated the kidney during allograft rejection.

A number of studies have implicated tumor-induced T(reg) cell activity in the sub-optimal response to therapeutic vaccines. Development of neo-adjuvant strategies targeting T(reg) cells is therefore imperative. Scutellaria extracts or constituent flavonoids have shown encouraging efficacy against various tumors, including gliomas, in both pre-clinical and clinical studies. We report here, for the first time, that Scutellaria ocmulgee leaf extract (SocL) and flavonoid wogonin could inhibit TGF-β1-induced T(reg) activity in malignant gliomas. F344 rats, subcutaneously transplanted with F98 gliomas, were treated with SocL. There was a significant inhibition of intra-tumoral TGF-β1 and T(reg) cell frequency as well as peripheral blood TGF-β1 levels in SocL-treated animals compared to the controls. SocL extract and wogonin also inhibited glioma-induced, TGF-β1-mediated T(reg) activity in vitro. SocL extract and wogonin also inhibited the secretion of IL-10 in T(reg) culture; whereas the level of IL-2 was either unchanged or marginally enhanced. We also observed an inhibition of Smad-3, GSK-3β and ERK1/2 signaling by SocL and wogonin in T(reg) cells, while phosphorylation of P38 MAPK was considerably enhanced, indicating that SocL or wogonin could inhibit the T cells' response to TGF-β1 via modulation of both Smad and non-Smad signaling pathways. Overall, this study suggests that Scutellaria can potentially reverse tumor-mediated immune suppression via inhibition of TGF-β1 secretion as well as via inhibition of T cells' response to TGF-β1. This may provide an opportunity for developing a novel adjuvant therapeutic strategy for malignant gliomas, combining Scutellaria with immunotherapy and chemo/radio-therapeutic regimen, which could potentially improve the disease outcome.

Aim: To identify and characterize drosophila mothers against decapentaplegic (SMAD)3-dependent changes in immune cell populations following infection with Helicobacter hepaticus (H. hepaticus)., Methods: SMAD3(-/-) (n = 19) and colitis-resistant SMAD3(+/-) (n = 24) mice (8-10 wk of age) were infected with H. hepaticus and changes in immune cell populations [T lymphocytes, natural killer (NK) cells, T regulatory cells] were measured in the spleen and mesenteric lymph nodes (MsLNs) at 0 d, 3 d, 7 d and 28 d post-infection using flow cytometry. Genotype-dependent changes in T lymphocytes and granzyme B(+) cells were also assessed after 28 d in proximal colon tissue using immunohistochemistry., Results: As previously observed, SMAD3(-/-), but not SMAD3(+/-) mice, developed colitis, peaking at 4 wk post-infection. No significant changes in T cell subsets were observed in the spleen or in the MsLNs between genotypes at any time point. However, CD4(+) and CD8(+)/CD62L(lo) cells, an effector T lymphocyte population, as well as NK cells (NKp46/DX5(+)) were significantly higher in the MsLNs of SMAD3(-/-) mice at 7 d and 28 d post-infection. In the colon, a higher number of CD3(+) cells were present in SMAD3(-/-) compared to SMAD3(+/-) mice at baseline, which did not significantly change during infection. However, the number of granzyme B(+) cells, a marker of cytolytic lymphocytes, significantly increased in SMAD3(-/-) mice 28 d post-infection compared to both SMAD3(+/-) mice and to baseline values. This was consistent with more severe colitis development in these animals., Conclusion: Data suggest that defects in SMAD3 signaling increase susceptibility to H. hepaticus-induced colitis through aberrant activation and/or dysregulation of effector lymphocytes.

Tuberculosis (TB) is associated with excessive production and bioactivation of transforming growth factor bets (TGF-β) in situ. Here, modification of expression of components of plasminogen/plasmin pathway in human monocytes (MN) by inhibitors of TGF-β signalling was examined. Smad3 siRNA effectively inhibited TGF-β-induced urokinase plasminogen activator receptor (uPAR). Agents known to interfere with TGF-β signalling, including the Smad inhibitors SIS3 and erythromycin derivatives, and ALK5 receptor inhibitor (SB 431542) in inhibition of uPAR expression in response to Mycobacterium tuberculosis (MTB) were examined. Inhibition by SIS3 only inhibited uPAR mRNA significantly. SIS3 may prove to be an effective adjunct to TB therapy., (© 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.)

The maintenance of T cell memory is critical for the development of rapid recall responses to pathogens, but may also have the undesired side effect of clonal expansion of T effector memory (T(EM)) cells in chronic autoimmune diseases. The mechanisms by which lineage differentiation of T cells is controlled have been investigated, but are not completely understood. Our previous work demonstrated a role of the voltage-gated potassium channel Kv1.3 in effector T cell function in autoimmune disease. In the present study, we have identified a mechanism by which Kv1.3 regulates the conversion of T central memory cells (T(CM)) into T(EM). Using a lentiviral-dominant negative approach, we show that loss of function of Kv1.3 mediates reversion of T(EM) into T(CM), via a delay in cell cycle progression at the G2/M stage. The inhibition of Kv1.3 signaling caused an up-regulation of SMAD3 phosphorylation and induction of nuclear p21(cip1) with resulting suppression of Cdk1 and cyclin B1. These data highlight a novel role for Kv1.3 in T cell differentiation and memory responses, and provide further support for the therapeutic potential of Kv1.3 specific channel blockers in T(EM)-mediated autoimmune diseases.

Background: Ets-1 controls osteoblast differentiation and bone development; however, its downstream mechanism of action in osteoblasts remains largely undetermined. CCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF-β1 and acts as a mediator of TGF-β1 induced matrix production in osteoblasts; however, the molecular mechanisms that control CCN2 induction are poorly understood. In this study, we investigated the role of Ets-1 for CCN2 induction by TGF-β1 in primary osteoblasts., Results: We demonstrated that Ets-1 is expressed and induced by TGF-β1 treatment in osteoblasts, and that Ets-1 over-expression induces CCN2 protein expression and promoter activity at a level similar to TGF-β1 treatment alone. Additionally, we found that simultaneous Ets-1 over-expression and TGF-β1 treatment synergize to enhance CCN2 induction, and that CCN2 induction by TGF-β1 treatment was impaired using Ets-1 siRNA, demonstrating the requirement of Ets-1 for CCN2 induction by TGF-β1. Site-directed mutagenesis of eight putative Ets-1 motifs (EBE) in the CCN2 promoter demonstrated that specific EBE sites are required for CCN2 induction, and that mutation of EBE sites in closer proximity to TRE or SBE (two sites previously shown to regulate CCN2 induction by TGF-β1) had a greater effect on CCN2 induction, suggesting potential synergetic interaction among these sites for CCN2 induction. In addition, mutation of EBE sites prevented protein complex binding, and this protein complex formation was also inhibited by addition of Ets-1 antibody or Smad 3 antibody, demonstrating that protein binding to EBE motifs as a result of TGF-β1 treatment require synergy between Ets-1 and Smad 3., Conclusions: This study demonstrates that Ets-1 is an essential downstream signaling component for CCN2 induction by TGF-β1 in osteoblasts, and that specific EBE sites in the CCN2 promoter are required for CCN2 promoter transactivation in osteoblasts.

Transforming growth factor β (TGFβ) plays a central role in maintaining immune homeostasis by regulating the initiation and termination of immune responses and thus preventing the development of autoimmune diseases. In this study, we describe an essential mechanism by which the actin regulatory protein Coronin 1A (Coro1A) ensures the proper response of Th17 CD4(+) T cells to TGFβ. Coro1A has been established as a key player in T cell survival, migration, activation, and Ca(2+) regulation in naive T cells. We show that mice lacking Coro1a developed less severe experimental autoimmune encephalomyelitis (EAE). Unexpectedly, upon the re-induction of EAE, Coro1a(-/-) mice exhibited enhanced EAE signs that correlated with increased numbers of IL-17 producing CD4(+) cells in the central nervous system (CNS) compared to wild-type mice. In vitro differentiated Coro1a(-/-) Th17 CD4(+) T cells consistently produced more IL-17 than wild-type cells and displayed a Th17/Th1-like phenotype in regard to the expression of the Th1 markers T-bet and IFNγ. Mechanistically, the Coro1a(-/-) Th17 cell phenotype correlated with a severe defect in TGFβR-mediated SMAD3 activation. Taken together, these data provide experimental evidence of a non-redundant role of Coro1A in the regulation of Th17 CD4(+) cell effector functions and, subsequently, in the development of autoimmunity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Suppression of IL-2 βproduction from T cells is an important process for the immune regulation by TGF-β. However, the mechanism by which this suppression occurs remains to be established. Here, we demonstrate that Smad2 and Smad3, two major TGF-β-downstream transcription factors, are redundantly essential for TGF-β-mediated suppression of IL-2 production in CD4(+) T cells using Smad2- and Smad3-deficient T cells. Both Smad2 and Smad3 were recruited into the proximal region of the IL-2 promoter in response to TGF-β. We then investigated the histone methylation status of the IL-2 promoter. Although both histone H3 lysine 9 (H3K9) and H3K27 trimethylation have been implicated in gene silencing, only H3K9 trimethylation was increased in the proximal region of the IL-2 promoter in a Smad2/3-dependent manner, whereas H3K27 trimethylation was not. The H3K9 methyltransferases Setdb1 and Suv39h1 bound to Smad3 and suppressed IL-2 promoter activity in collaboration with Smad3. Overexpression of Suv39h1 in 68-41 T cells strongly inhibited IL-2 production in response to T cell receptor stimulation irrespective of the presence or absence of TGF-β, whereas Setdb1 overexpression only slightly suppressed IL-2 production. Silencing of Suv39h1 by shRNA reverted the suppressive effect of TGF-β on IL-2 production. Furthermore, TGF-β induced Suv39h1 recruitment to the proximal region of the IL-2 promoter in wild type primary T cells; however, this was not observed in Smad2(-/-)Smad3(+/-) T cells. Thus, we propose that Smads recruit H3K9 methyltransferases Suv39h1 to the IL-2 promoter, thereby inducing suppressive histone methylation and inhibiting T cell receptor-mediated IL-2 transcription.