Tumor-associated macrophages promote pancreatic ductal adenocarcinoma progression by inducing epithelial-to-mesenchymal transition.

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-ß (TGF-ß) 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-ß-Smad2/3/4-Snail signaling axis. The pro-tumorigenic effects of TAMs or TAM-CM were abolished by TGF-ß signaling pathway inhibitors and neutralizing TGF-ß antibody. These results demonstrate that TAMs promote PDAC progression through the TGF-ß signaling pathway.

Cysteinyl leukotriene D4 (LTD4) promotes airway epithelial cell inflammation and remodelling.

OBJECTIVE: Cysteinyl leukotrienes (CysLTs), a group of inflammatory lipid mediators, are found elevated in obese-asthmatic patients. Leukotriene D4 (LTD4), 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 LTD4-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 LTD4 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 LTD4-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 LTD4 showed significant alterations in the levels of inflammatory markers such as GM-CSF, TNF-α, IL-1ß, EGF and eotaxin in dose- and time-dependent manner. Further, LTD4 enhanced the activation of inflammasomes as evidenced by increased levels of NALP3, cleaved caspase-1 and IL-1ß. LTD4 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 LTD4, as it also increased TGF-ß 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 LTD4 could induce inflammatory response in human airway epithelial cell by activating NALP3 inflammasome. LTD4 could further promote airway epithelial cells' remodelling through TGF-ß/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.

Berberine Attenuates Cigarette Smoke Extract-induced Airway Inflammation in Mice: Involvement of TGF-ß1/Smads Signaling Pathway.

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-ß1, 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-ß1/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-ß1/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.

Enhanced Cancer Immunotherapy with Smad3-Silenced NK-92 Cells.

Natural killer (NK) cells, early effectors in anticancer immunity, are paralyzed by TGFß1, 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 TGFß1-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.

Epigallocatechin gallate improves airway inflammation through TGF­ß1 signaling pathway in asthmatic mice.

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)­ß1 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 transcription­quantitative polymerase chain reaction to determine the proteins and transcription factors in the TGF­ß1 pathway. EGCG 20 mg/kg significantly reduced asthmatic symptoms, lung inflammatory cell infiltration, and the inflammatory factor levels of interleukin (IL)­2, IL­6 and tumor necrosis factor (TNF)­α. In addition, it increased the levels of inflammatory factors, including IL­10, diminished the percentage of Th17 cells, increased the percentage of Treg cells, and decreased the expression of TGF­ß1 and phosphorylated (p)­Smad2/3 expression. Following the inhibition of the TGF­ß1 receptor, the anti­inflammatory effect of EGCG disappeared, and the expression of TGF­ß1 and p­Smad2/3 increased. EGCG attenuated airway inflammation in asthmatic mice, decreased the percentage of Th17 cells and increased the percentage of Treg cells. The anti­inflammatory effect of EGCG is achieved via the TGF­ß1 signaling pathway.

[Reprogrammed M1 macrophages with inhibited STAT3, STAT6 and/or SMAD3 extends lifespan of mice with experimental carcinoma].

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.

Beyond disease susceptibility-Leveraging genome-wide association studies for new insights into complex disease biology.

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.

Polydatin alleviated radiation-induced lung injury through activation of Sirt3 and inhibition of epithelial-mesenchymal transition.

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-ß1-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.

The emerging role of interleukin-37 in cardiovascular diseases.

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.

1,25(OH)2D3/VDR attenuates high glucose­induced epithelial­mesenchymal transition in human peritoneal mesothelial cells via the TGFß/Smad3 pathway.

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 ß (TGFß) and interleukin­6 (IL­6) and phosphorylated­SMAD family member 3 (p­Smad3) expression. EMT was promoted as the expression level of the epithelial marker E­cadherin was reduced, whereas expression levels of the mesenchymal markers α­SMA and FN were increased. 1,25(OH)2D3 pretreatment inhibited the expression of inflammatory cytokines in HPMCs and attenuated HG­induced EMT, possibly through inhibition of the TGFß/Smad pathway by binding to its receptor VDR.

The Causal Role of IL-4 and IL-13 in Schistosoma mansoni Pulmonary Hypertension.

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)-ß 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-ß-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-ß 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-ß-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.

Nerve growth factor exposure promotes tubular epithelial-mesenchymal transition via TGF-ß1 signaling activation.

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-ß1 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-ß1 expression and secretion and enhanced cell motility by activating EMT markers via its receptors, TrkA and p75(NTR). Interestingly, we observed that TGF-ß1-SMAD pathway activation and the up-regulation of EMT markers NGF-induced were both prevented when knockdown of TGF-ß1 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-ß1-signaling activation, suggesting that in kidney diseases high NGF serum levels could contribute to worsen renal fibrosis.

TGF-ß1, but not bone morphogenetic proteins, activates Smad1/5 pathway in primary human macrophages and induces expression of proatherogenic genes.

Macrophages are responsible for the control of inflammation and healing, and their malfunction results in cardiometabolic disorders. TGF-ß is a pleiotropic growth factor with dual (protective and detrimental) roles in atherogenesis. We have previously shown that in human macrophages, TGF-ß1 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-ß1-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-ß1, 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-ß1 in mature human macrophages. Activation of Smad1/5 by TGF-ß1 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-ß on atherosclerosis where Smad1/5 is responsible for proatherogenic effects, whereas Smad2/3 regulate atheroprotective effects of TGF-ß.

Transforming growth factor ß-mediated suppression of antitumor T cells requires FoxP1 transcription factor expression.

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-ß (TGF-ß), 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-ß-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-ß signaling on tumor-associated lymphocytes undergoing Foxp1-dependent transcriptional regulation.

Functional effects of TGF-ß1 on mesenchymal stem cell mobilization in cockroach allergen-induced asthma.

Mesenchymal stem cells (MSCs) have been suggested to participate in immune regulation and airway repair/remodeling. TGF-ß1 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-ß1 in MSC migration in allergic asthma. We examined nestin expression (a marker for MSCs) and TGF-ß1 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-ß1 in serum and p-Smad2/3 expression in lungs of CRE-treated mice. Increased activation of TGF-ß1 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-ß1-neutralizing Ab or TßR1 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-ß1-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-ß1 may be a key promigratory factor in recruiting MSCs to the airways in mouse models of asthma.

Smad and NFAT pathways cooperate to induce CD103 expression in human CD8 T lymphocytes.

The interaction of integrin αE(CD103)ß7, 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-ß1, 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-ß1 can participate in CD103 expression on locally TCR-engaged Ag-specific CD8 T cells, thus contributing to antitumor CTL responses and cancer cell destruction.

Major pathogenic steps in human lupus can be effectively suppressed by nucleosomal histone peptide epitope-induced regulatory immunity.

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 TGFß/ALK-5/pSmad 2/3 signaling, and they expressed TGF-ß 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.

Effects of montelukast on subepithelial/peribronchial fibrosis in a murine model of ovalbumin induced chronic asthma.

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)-ß1, 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-ß1/Smad3 in the lung tissue.

TGF-ß enhanced IL-21-induced differentiation of human IL-21-producing CD4+ T cells via Smad3.

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-1ß, IL-6 or IL-21 induced differentiation of human IL-21-producing CD4(+) T cells, and TGF-ß 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-ß 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-ß. Further study indicated that IL-21 induced phosphorylation of transcriptional factors of STAT1, STAT3 and STAT5, and TGF-ß induced phosphorylation of Smad3 in CD4(+) T cells. Taken together, our data indicated that TGF-ß 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.

Evaluation of the immunosuppressive activity of artesunate in vitro and in vivo.

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-ß, 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.