Deregulated TGF-β signaling in pancreatic cancer promotes tumor growth, invasion, metastasis, and a potent immunosuppressive network. A strategy for disrupting this tumor-promoting pathway is silencing TGF-β by siRNA. By introducing a triphosphate group at the 5' end of siRNA (ppp-siRNA), gene silencing can be combined with immune activation via the cytosolic helicase retinoic acid-inducible gene I (RIG-I), a ubiquitously expressed receptor recognizing viral RNA. We validated RIG-I as a therapeutic target by showing that activation of RIG-I in pancreatic carcinoma cells induced IRF-3 phosphorylation, production of type I IFN, the chemokine CXCL10, as well as caspase-9-mediated tumor cell apoptosis. Next, we generated a bifunctional ppp-siRNA that combines RIG-I activation with gene silencing of TGF-β1 (ppp-TGF-β) and studied its therapeutic efficacy in the orthotopic Panc02 mouse model of pancreatic cancer. Intravenous injection of ppp-TGF-β reduced systemic and tumor-associated TGF-β levels. In addition, it induced high levels of type I IFN and CXCL10 in serum and tumor tissue, systemic immune cell activation, and profound tumor cell apoptosis in vivo. Treatment of mice with established tumors with ppp-TGF-β significantly prolonged survival as compared with ppp-RNA or TGF-β siRNA alone. Furthermore, we observed the recruitment of activated CD8(+) T cells to the tumor and a reduced frequency of CD11b(+) Gr-1(+) myeloid cells. Therapeutic efficacy was dependent on CD8(+) T cells, whereas natural killer cells were dispensable. In conclusion, combing TGF-β gene silencing with RIG-I signaling confers potent antitumor efficacy against pancreatic cancer by breaking tumor-induced CD8(+) T cell suppression.

In the setting of myocardial infarction (MI), implanted stem cell viability is low and scar formation limits stem cell homing, viability, and integration. Thus, interventions that favorably remodel fibrotic healing may benefit stem cell therapies. However, it remains unclear whether it is feasible and safe to remodel fibrotic healing post-MI without compromising ventricular remodeling and dysfunction. This study, therefore, determined the anti-fibrotic and other effects of the hormone, relaxin in a mouse model of MI. Adult male mice underwent left coronary artery ligation-induced MI and were immediately treated with recombinant human relaxin (MI+RLX) or vehicle (MI+VEH) over 7 or 30 days, representing time points of early and mature fibrotic healing. Cardiac function was assessed by echocardiography and catheterization, while comprehensive immunohistochemistry, morphometry, and western blotting were performed to explore the relaxin-induced mechanisms of action post-MI. RLX significantly inhibited the MI-induced progression of cardiac fibrosis over 7 and 30 days, which was associated with a reduction in TGF-β1 expression, myofibroblast differentiation, and cardiomyocyte apoptosis in addition to a promotion of matrix metalloproteinase-13 levels and de novo blood vessel growth (all P<0.05 vs respective measurements from MI+VEH mice). Despite the evident fibrotic healing post-MI, relaxin did not adversely affect the incidence of ventricular free-wall rupture or the extent of LV remodeling and dysfunction. These combined findings demonstrate that RLX favorably remodels the process of fibrotic healing post-infarction by lowering the density of mature scar tissue in the infarcted myocardium, border zone, and non-infarcted myocardium, and may, therefore, facilitate cell-based therapies in the setting of ischemic heart disease.

The small G proteins Rac1 and RhoA regulate actin cytoskeleton, cell shape, adhesion, migration, and proliferation. Recent studies in our laboratory have shown that NADPH oxidase Nox4-derived ROS are involved in transforming growth factor (TGF)-β1-induced rat kidney myofibroblast differentiation assessed by the acquisition of an α-smooth muscle actin (α-SMA) phenotype and expression of an alternatively spliced fibronectin variant (Fn-EIIIA). Rac1 and RhoA are essential in signaling by some Nox homologs, but their role as effectors of Nox4 in kidney myofibroblast differentiation is not known. In the present study, we explored a link among Rac1 and RhoA and Nox4-dependent ROS generation in TGF-β1-induced kidney myofibroblast activation. TGF-β1 stimulated an increase in Nox4 protein expression, NADPH oxidase activity, and abundant α-SMA and Fn-EIIIA expression. RhoA but not Rac1 was involved in TGF-β1 induction of Nox4 signaling of kidney myofibroblast activation. TGF-β1 stimulated active RhoA-GTP and increased Rho kinase (ROCK). Inhibition of RhoA with small interfering RNA and ROCK using Y-27632 significantly reduced TGF-β1-induced stimulation of Nox4 protein, NADPH oxidase activity, and α-SMA and Fn-EIIIA expression. Treatment with diphenyleneiodonium, an inhibitor of NADPH oxidase, did not decrease RhoA activation but inhibited TGF-β1-induced α-SMA and Fn-EIIIA expression, indicating that RhoA is upstream of ROS generation. RhoA/ROCK also regulated polymerase (DNA-directed) δ-interacting protein 2 (Poldip2), a newly discovered Nox4 enhancer protein. Collectively, these data indicate that RhoA/ROCK is upstream of Poldip2-dependent Nox4 regulation and ROS production and induces redox signaling of kidney myofibroblast activation and may broader implications in the pathophysiology of renal fibrosis.

This study was undertaken to evaluate the role of collagen matrix to enhance platelet-rich plasma (PRP) effects on pro-inflammatory cytokine-induced arthritic model. We have previously demonstrated the highly regenerative roles of PRP to restore disc degeneration and osteoporosis. In this study, PRP modulated by collagen matrix was used as a regenerative and anti-inflammatory mediator to rescue the chondrocyte degeneration induced by pro-inflammatory cytokines IL-1β (10 ng/ml)+TNF-α (20 ng/ml). First, the MTT result indicated that 1 ng/ml TGF-β1 in PRP showed an optimal dosage for chondrocytes proliferation. The chondrogenic-specific gene expressions were rescued by PRP from the inhibition of IL-1β+TNF-α, especially under the modulation of collagen matrix. The inflammatory molecules activated by IL-1β+TNF-α were also significantly diminished by PRP with collagen matrix. The membrane receptors integrin α1β1 and CD44 were strongly inhibited by IL-1β+TNF-α, while this inhibition was then recovered by PRP in collagen coating condition. In a 3D model encapsulated with collagen, PRP-induced chondrogenesis were highly enhanced, such as strong restoration of type II collagen and proteoglycan from the inhibition of IL-1β+TNF-α. The result indicated that collagen matrix enhances the effect of PRP on chondrogenesis in response to pro-inflammatory cytokines. The combination of PRP and collagen matrix might facilitate a physiological microenvironment beneficial for maintaining chondrocyte homeostasis and represents an advanced osteoarthritis therapy for clinical applications.

Increasing evidence shows that microRNAs play an important role in kidney disease. However, functions of long noncoding RNAs (lncRNAs) in kidney diseases remain undefined. We have previously shown that TGF-β1 plays a diverse role in renal inflammation and fibrosis and Smad3 is a key mediator in this process. In this study, we used RNA-sequencing to identify lncRNAs related to renal inflammation and fibrosis in obstructive nephropathy induced in Smad3 wild-type and knockout mice. We found that Arid2-IR was a Smad3-associated lncRNA as a Smad3 binding site was found in the promoter region of Arid2-IR and deletion of Smad3 abolished upregulation of Arid2-IR in the diseased kidney. In vitro knockdown of Arid2-IR from tubular epithelial cells produced no effect on TGF-β-induced Smad3 signaling and fibrosis but inhibited interleukin-1β-stimulated NF-κB-dependent inflammatory response. In contrast, overexpression of Arid2-IR promoted interleukin-1β-induced NF-κB signaling and inflammatory cytokine expression without alteration of TGF-β1-induced fibrotic response. Furthermore, treatment of obstructed kidney with Arid2-IR shRNA blunted NF-κB-driven renal inflammation without effect on TGF-β/Smad3-mediated renal fibrosis. Thus, Arid2-IR is a novel lncRNA that functions to promote NF-κB-dependent renal inflammation. Blockade of Arid2-IR may represent a novel and specific therapy for renal inflammatory disease.

Although the potential roles of endothelial cells in the microvascules of prostate cancer during angiogenesis have been documented, their direct impacts on the prostate cancer metastasis remain unclear. We found that the CD31-positive and CD34-positive endothelial cells are increased in prostate cancer compared with the normal tissues and that these endothelial cells were decreased upon castration, gradually recovered with time, and increased after prostate cancer progressed into the castration-resistant stage, suggesting a potential linkage of these endothelial cells with androgen deprivation therapy. The in vitro invasion assays showed that the coculture of endothelial cells with prostate cancer cells significantly enhanced the invasion ability of the prostate cancer cells. Mechanism dissection found that coculture of prostate cancer cells with endothelial cells led to increased interleukin (IL)-6 secretion from endothelial cells, which may result in downregulation of androgen receptor (AR) signaling in prostate cancer cells and then the activation of TGF-β/matrix metalloproteinase-9 (MMP-9) signaling. The consequences of the IL-6→AR→TGFβ→MMP-9 signaling pathway might then trigger the increased invasion of prostate cancer cells. Blocking the IL-6→AR→TGFβ→MMP-9 signaling pathway either by IL-6 antibody, AR-siRNA, or TGF-β1 inhibitor all interrupted the ability of endothelial cells to influence prostate cancer invasion. These results, for the first time, revealed the important roles of endothelial cells within the prostate cancer microenvironment to promote the prostate cancer metastasis and provide new potential targets of IL-6→AR→TGFβ→MMP-9 signals to battle the prostate cancer metastasis.

Regulatory T cells (Tregs) are essential to prevent autoimmunity, but excessive Treg function contributes to cancer progression by inhibiting antitumor immune responses. Tregs exert contact-dependent inhibition of immune cells through the production of active transforming growth factor-β1 (TGF-β1). On the Treg cell surface, TGF-β1 is in an inactive form bound to membrane protein GARP and then activated by an unknown mechanism. We demonstrate that GARP is involved in this activation mechanism. Two anti-GARP monoclonal antibodies were generated that block the production of active TGF-β1 by human Tregs. These antibodies recognize a conformational epitope that requires amino acids GARP137-139 within GARP/TGF-β1 complexes. A variety of antibodies recognizing other GARP epitopes did not block active TGF-β1 production by Tregs. In a model of xenogeneic graft-versus-host disease in NSG mice, the blocking antibodies inhibited the immunosuppressive activity of human Tregs. These antibodies may serve as therapeutic tools to boost immune responses to infection or cancer via a mechanism of action distinct from that of currently available immunomodulatory antibodies. Used alone or in combination with tumor vaccines or antibodies targeting the CTLA4 or PD1/PD-L1 pathways, blocking anti-GARP antibodies may improve the efficiency of cancer immunotherapy.

Transforming growth factor-beta (TGF-β), a key mediator of cardiac fibroblast activation, has a major influence on collagen type I production. However, the epigenetic mechanisms by which TGF-β induces collagen type I alpha 1 (COL1A1) expression are not fully understood. This study was designed to examine whether or not DNA methylation is involved in TGF-β-induced COL1A1 expression in cardiac fibroblasts. Cells isolated from neonatal Sprague-Dawley rats were cultured and stimulated with TGF-β1. The mRNA levels of COL1A1 and DNA methyltransferases (DNMTs) were determined via quantitative polymerase chain reaction and the protein levels of collagen type I were determined via Western blot as well as enzyme-linked immunosorbent assay. The quantitative methylation of the COL1A1 promoter region was analyzed using the MassARRAY platform of Sequenom. Results showed that TGF-β1 upregulated the mRNA expression of COL1A1 and induced the synthesis of cell-associated and secreted collagen type I in cardiac fibroblasts. DNMT1 and DNMT3a expressions were significantly downregulated and the global DNMT activity was inhibited when treated with 10 ng/mL of TGF-β1 for 48 h. TGF-β1 treatment resulted in a significant reduction of the DNA methylation percentage across multiple CpG sites in the rat COL1A1 promoter. Thus, TGF-β1 can induce collagen type I expression through the inhibition of DNMT1 and DNMT3a expressions as well as global DNMT activity, thereby resulting in DNA demethylation of the COL1A1 promoter. These findings suggested that the DNMT-mediated DNA methylation is an important mechanism in regulating the TGF-β1-induced COL1A1 gene expression.

To determine whether Th17/Treg balance was abnormal in adult patients with minimal change nephrotic syndrome (MCNS), we studied 25 patients with new-onset MCNS and 20 normal persons. The results showed that MCNS patients exhibited a significant increase in Th17 number, Th17-related cytokines (IL-17 and IL-23), and transcription factor (RORγt) levels, as well as an obvious decrease in Treg number, Treg-related cytokines (TGF-β1 and IL-10), and transcription factor (Foxp3) levels. The Th17/Treg ratios increased along with increased proteinuria and decreased albumin levels in patients with MCNS. IL-17 protein expression was also detected in the renal biopsy tissue of MCNS patients, particularly in patients with acute renal failure. Further, Th17/Treg balance returned to normal after effective corticosteroids therapy in 16 MCNS patients. These results indicated that Th17/Treg imbalance existed in MCNS patients, suggesting a potential role of Th17/Treg imbalance in the pathogenesis of MCNS.

Myofibroblasts, specialized cells that play important roles in wound healing and fibrosis, can develop from epithelial cells through an epithelial-mesenchymal transition (EMT). During EMT, epithelial cells detach from neighboring cells and acquire an elongated, mesenchymal-like morphology. These phenotypic changes are accompanied by changes in gene expression patterns including upregulation of a variety of cytoskeletal associated proteins which contribute to the ability of myofibroblasts to exert large contractile forces. Here, the relationship between cell shape and cytoskeletal tension and the expression of cytoskeletal proteins in transforming growth factor (TGF)-β1-induced EMT is determined. We find that culturing cells in conditions which permit cell spreading and increased contractility promotes the increased expression of myofibroblast markers and cytoskeletal associated proteins. In contrast, blocking cell spreading prevents transdifferentiation to the myofibroblast phenotype. Furthermore, we find that cell shape regulates the expression of cytoskeletal proteins by controlling the subcellular localization of myocardin related transcription factor (MRTF)-A. Pharmacological inhibition of cytoskeletal tension or MRTF-A signaling blocks the acquisition of a myofibroblast phenotype in spread cells while overexpression of MRTF-A promotes the expression of cytoskeletal proteins for all cell shapes. These data suggest that cell shape is a critical determinant of myofibroblast development from epithelial cells.

Short-chain fatty acids (SCFAs), which are generated by the bacterial fermentation of dietary fibers, promote expansion of regulatory T cells (Tregs). Potential therapeutic value of SCFAs has been recently highlighted in the experimental models of T cell-mediated autoimmunity and allergic inflammation. These studies suggest that physiological intestinal concentrations of SCFAs within the millimolar range are crucial for dampening inflammation-mediated processes. Here, we describe opposing effects of SCFAs on T cell-mediated immune responses. In accordance with published data, lower butyrate concentrations facilitated differentiation of Tregs in vitro and in vivo under steady-state conditions. In contrast, higher concentrations of butyrate induced expression of the transcription factor T-bet in all investigated T cell subsets resulting in IFN-γ-producing Tregs or conventional T cells. This effect was mediated by the inhibition of histone deacetylase activity and was independent of SCFA-receptors FFA2 and FFA3 as well as of Na+-coupled SCFA transporter Slc5a8. Importantly, while butyrate was not able to induce the generation of Tregs in the absence of TGF-β1, the expression of T-bet and IFN-γ was triggered upon stimulation of CD4+ T cells with this SCFA alone. Moreover, the treatment of germ-free mice with butyrate enhanced the expression of T-bet and IFN-γ during acute colitis. Our data reveal that, depending on its concentration and immunological milieu, butyrate may exert either beneficial or detrimental effects on the mucosal immune system.

The epithelial-to-mesenchymal transition (EMT) plays crucial roles in embryonic development, wound healing, tissue repair, and cancer progression. Results of this study show how transforming growth factor β1 (TGF-β1) down-regulates expression of N-acetylglucosaminyltransferase III (GnT-III) during EMT-like changes. Treatment with TGF-β1 resulted in a decrease in E-cadherin expression and GnT-III expression, as well as its product, the bisected N-glycans, which was confirmed by erythro-agglutinating phytohemagglutinin lectin blot and HPLC analysis in human MCF-10A and mouse GE11 cells. In contrast with GnT-III, the expression of N-acetylglucosaminyltransferase V was slightly enhanced by TGF-β1 treatment. Changes in the N-glycan patterns on α3β1 integrin, one of the target proteins for GnT-III, were also confirmed by lectin blot analysis. To understand the roles of GnT-III expression in EMT-like changes, the MCF-10A cell was stably transfected with GnT-III. It is of particular interest that overexpression of GnT-III influenced EMT-like changes induced by TGF-β1, which was confirmed by cell morphological changes of phase contrast, immunochemical staining patterns of E-cadherin, and actin. In addition, GnT-III modified E-cadherin, which served to prolong E-cadherin turnover on the cell surface examined by biotinylation and pulse-chase experiments. GnT-III expression consistently inhibited β-catenin translocation from cell-cell contact into the cytoplasm and nucleus. Furthermore, the transwell assay showed that GnT-III expression suppressed TGF-β1-induced cell motility. Taken together, these observations are the first to clearly demonstrate that GnT-III affects cell properties, which in turn influence EMT-like changes, and to explain a molecular mechanism for the inhibitory effects of GnT-III on cancer metastasis.

γδT cells have been reported to exert immunosuppressive functions in multiple solid malignant diseases, but their immunosuppressive functional subpopulation in breast cancer (BC) is still undetermined. Here, we collected 40 paired BC and normal tissue samples from Chinese patients for analysis. First, we showed that γδT1 cells comprise the majority of CD3+ T cells in BC; next, we found that CD73+γδT1 cells were the predominant regulatory T-cell (Treg) population in BC, and that their prevalence in peripheral blood was also related to tumour burden. In addition, CD73+γδT1 cells exert an immunosuppressive effect via adenosine generation. We also found that BC could modulate CD73 expression on γδT cells in a non-contact manner. The microarray analysis and functional experiments indicated that breast tumour cell-derived exosomes (TDEs) could transmit lncRNA SNHG16, which upregulates CD73 expression, to Vδ1 T cells. Regarding the mechanism, SNHG16 served as a ceRNA by sponging miR-16-5p, which led to the derepression of its target gene SMAD5 and resulted in potentiation of the TGF-β1/SMAD5 pathway to upregulate CD73 expression in Vδ1 T cells. Our results showed that the BC-derived exosomal SNHG16/miR-16-5p/SMAD5-regulatory axis potentiates TGF-β1/SMAD5 pathway activation, thus inducing CD73 expression in Vδ1 T cells. Our results first identify the significance of CD73+Vδ1 Tregs in BC, and therapy targeting this subpopulation or blocking TDEs might have potential for BC treatment in the future.

BACKGROUND

Based on some well-documented reports, we attempted to clarify the antifibrotic mechanisms of human Wharton's-jelly-derived mesenchymal stromal cells (WJ-MSCs) from the perspective of induction of hepatocyte growth factor (HGF) expression in tubular epithelial cells (TECs).

METHODS

A rat model of acute kidney injury (AKI) was established through unilateral renal ischemia for 1 hour. Two days later, a single intravenous cell or vehicle injection, or contralateral nephrectomy, was performed. Rats were sacrificed at 1 day, 1 week, 4 weeks, or 6 weeks after the intervention. Renal fibrosis was evaluated by Masson trichrome staining and Sircol collagen assay. The upregulation of α-smooth muscle actin (α-SMA) versus E-cadherin expression was adopted as an indicator of tubular epithelial-mesenchymal transition (EMT). Gene and protein expression of HGF or transforming growth factor-beta1 (TGF-β1) was determined by real-time polymerase chain reaction (RT-PCR) and Western blot, respectively. HGF expression in TECs was detected with immunostaining. In vitro, rat TECs subjected to hypoxia injury were incubated with or without conditioned medium (CM) from WJ-MSCs for 1, 3, 24, or 48 hours. Rat or human HGF synthesis in TECs was assessed with immunostaining, RT-PCR, or ELISA.

RESULTS

Cell delivery or nephrectomy led to abrogation of renal scarring. At the incipient period of AKI, through induction of HGF expression, either of them remarkably promoted the upregulation of HGF versus TGF-β1 expression in damaged kidney. Rat TECs were not only the principal cells expressing HGF but also exhibited human HGF expression after cell infusion. During fibrogenesis, the downregulation of HGF versus TGF-β1 expression was greatly prevented by WJ-MSCs or kidney removal, thereby resulting in tubular EMT delay. In vitro, after 24 or 48 hours of incubation, CM not only robustly induced the upregulation of rat HGF gene expression in TECs but substantially amplified the release of rat HGF. Under the induction of CM, human HGF mRNA and protein were detected in rat TECs.

CONCLUSIONS

WJ-MSCs contribute to tubular EMT delay and the alleviation of renal fibrosis. Induction of native and foreign HGF synthesis in damaged TECs at the initial stage of AKI leads to recovery of the disturbed balance of HGF/TGF-β1 during scar formation, being one of the vital mechanisms.

BACKGROUND

Fibroblast-to-myofibroblast transition is a key event during wound healing and hypertrophic scar formation. Previous studies suggested Wnt/β-catenin signaling might be involved in the wound healing. However, its specific role in skin fibroblast-to-myofibroblast transition remains unclear.

OBJECTIVE

To investigate the specific role of β-catenin during the transforming growth factor-β1 induced normal skin myofibroblasts transition.

METHODS

By real-time quantitative polymerase chain reaction, Western-blot and immunocytochemistry, the activation of Wnt/β-catenin pathway in cultured human normal skin fibroblasts during TGF-β1 induced fibroblast-to-myofibroblast transition was investigated. The effects of β-catenin on myofibroblasts transition were also investigated when SB-216763, over-expression and siRNA of β-catenin were utilized. In addition, fibroblasts populated collagen lattices contraction assays were conducted to examine the effects of β-catenin on the contractility of the fibroblasts induced by TGF-β1. Furthermore, the effects of β-catenin on the expression of α-smooth muscle actin and collagen types I and III in hypertrophic scar derived fibroblasts were studied.

RESULTS

The expression of Wnts mRNA and β-catenin protein was up-regulated by TGF-β1 stimulation during the myofibroblasts transition. Both of SB-216763 and β-catenin over-expression was paralleled with decreased expression of α-smooth muscle actin, collagen types I and III, while siRNA targeting β-catenin leads to up-regulation of α-smooth muscle actin, collagen types I and III. The increased contractility and α-smooth muscle actin expression of the fibroblasts in the collagen lattices induced by TGF-β1 was inhibited by SB-216763. In addition, the expression levels of α-smooth muscle actin, collagen types I and III in hypertrophic scar derived fibroblasts were also down-regulated by SB-216763.

CONCLUSIONS

Specifically in normal skin fibroblasts, β-catenin might be involved in the myofibroblasts transition and negatively regulate the TGF-β1-induced myofibroblast transition.

Dysregulated amphiregulin (AR) expression and EGR receptor (EGFR) activation have been described in animal models of pulmonary fibrosis and in patients with idiopathic pulmonary fibrosis. However, the exact role of AR in the pathogenesis of pulmonary fibrosis has not been clearly defined. Here, we show that a potent profibrogenic cytokine TGF-β1 significantly induced the expression of AR in lung fibroblasts in vitro and in murine lungs in vivo. AR stimulated NIH3T3 fibroblast cell proliferation in a dose-dependent manner. Silencing of AR expression by siRNA or chemical inhibition of EGFR signaling, utilizing AG1478 and gefitinib, significantly reduced the ability of TGF-β1 to stimulate fibroblast proliferation and expression of α-smooth muscle actin, collagen, and other extracellular matrix-associated genes. TGF-β1-stimulated activation of Akt, ERK, and Smad signaling was also significantly inhibited by these interventions. Consistent with these in vitro findings, AR expression was impressively increased in the lungs of TGF-β1 transgenic mice, and either siRNA silencing of AR or chemical inhibition of EGFR signaling significantly reduced TGF-β1-stimulated collagen accumulation in the lung. These studies showed a novel regulatory role for AR in the pathogenesis of TGF-β1-induced pulmonary fibrosis. In addition, these studies suggest that AR, or AR-activated EGFR signaling, is a potential therapeutic target for idiopathic pulmonary fibrosis associated with TGF-β1 activation.

Cells of the immune system that reside in barrier epithelia provide a first line of defense against pathogens. Langerhans cells (LCs) and CD8(+) tissue-resident memory T cells (TRM cells) require active transforming growth factor-β1 (TGF-β) for epidermal residence. Here we found that integrins αvβ6 and αvβ8 were expressed in non-overlapping patterns by keratinocytes (KCs) and maintained the epidermal residence of LCs and TRM cells by activating latent TGF-β. Similarly, the residence of dendritic cells and TRM cells in the small intestine epithelium also required αvβ6. Treatment of the skin with ultraviolet irradiation decreased integrin expression on KCs and reduced the availability of active TGF-β, which resulted in LC migration. Our data demonstrated that regulated activation of TGF-β by stromal cells was able to directly control epithelial residence of cells of the immune system through a novel mechanism of intercellular communication.

Cervical cancer is a worldwide disease that constitutes a significant public health problem, especially in developing countries, not only due to its high incidence but also because the most affected population comprises women who belong to marginalized socio-economic classes. Clinical and molecular research has identified immunological impairment in squamous intraepithelial cervical lesions and cervical cancer patients. Human Papillomavirus (HPV) has several mechanisms for avoiding the immune system: it down-regulates the expression of interferon and upregulates interleukin (IL)-10 and transforming growth factor (TGF)-β1 to produce a local immunosuppressive environment, which, along with altered tumor surface antigens, forms an immunosuppressive network that inhibits the antitumor immune response. In this review we analyzed the available data on several deregulated cellular immune functions in patients with NIC I, NIC II and NIC III and cervical cancer. The effects of immunosuppressive cytokines on innate immune response, T-cell activation and cellular factors that promote tumor cell proliferation in cervical cancer patients are summarized. We discuss the functional consequences of HPV E2, E6, and E7 protein interactions with IL-10 and TGF-β1 promoters in the induction of these cytokines and postulate its effect on the cellular immune response in squamous intraepithelial cervical lesions and cervical cancer patients. This review provides a comprehensive picture of the immunological functions of IL-10 and TGF-β1 in response to HPV in humans.

Current hypotheses suggest that aberrant wound healing has a critical role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). In these hypotheses, continuous TGF-β1 secretion by alveolar epithelial cells (AECs) in abnormal wound healing has a critical role in promoting fibroblast differentiation into myofibroblasts. Mesenchymal stem cells (MSCs) home to the injury site and reduce fibrosis by secreting multifunctional antifibrotic humoral factors in IPF. In this study, we show that MSCs can correct the inadequate-communication between epithelial and mesenchymal cells through STC1 (Stanniocalcin-1) secretion in a bleomycin-induced IPF model. Inhalation of recombinant STC1 shows the same effects as the injection of MSCs. Using STC1 plasmid, it was possible to enhance the ability of MSCs to ameliorate the fibrosis. MSCs secrete large amounts of STC1 in response to TGF-β1 in comparison to AECs and fibroblasts. The antifibrotic effects of STC1 include reducing oxidative stress, endoplasmic reticulum (ER) stress, and TGF-β1 production in AECs. The STC1 effects can be controlled by blocking uncoupling protein 2 (UCP2) and the secretion is affected by the PI3/AKT/mTORC1 inhibitors. Our findings suggest that STC1 tends to correct the inappropriate epithelial-mesenchymal relationships and that STC1 plasmid transfected to MSCs or STC1 inhalation could become promising treatments for IPF.

OBJECTIVE

To review recent advances in the use of transforming growth factor (TGF)-beta in acute lung injury and to apply this knowledge to understanding the pathophysiology of this syndrome.

METHODS

Published research and review articles in the English language related to the role of TGF-beta in acute lung injury.

METHODS

The cytokine TGF-beta plays a critical role in the resolution of tissue injury in multiple organs, including the lung. Following injury, TGF-beta has been most thoroughly evaluated during the late phases of tissue repair, where it plays a critical role in the development of pulmonary fibrosis. In contrast, recent animal studies showed that expression levels of several TGF-beta-inducible genes were dramatically increased as early as 2 days after the induction of injury. The integrin alpha(v)beta(6) activates latent TGF-beta in the lungs. Mice lacking this integrin were completely protected from pulmonary edema in a model of bleomycin-induced acute lung injury. Pharmacologic inhibition of TGF-beta also protected wild-type mice from pulmonary edema induced by bleomycin or Escherichia coli endotoxin. Similar findings also have been reported in patients in a clinical study evaluating TGF-beta in the bronchoalveolar lavage fluid during the course of acute respiratory distress syndrome (ARDS). Indeed, the bronchoalveolar lavage concentrations were dramatically increased as early as 1 day after the initiation of ARDS criteria and were correlated with decreases in the Pao(2)/Fio(2) ratio, suggesting an important role for TGF-b1 in the development of ARDS in humans.

CONCLUSIONS

These studies suggest that TGF-beta not only participates in the late phase of acute lung injury, but also might be active early in acute lung injury and potentially could contribute to the development of pulmonary edema. Integrin-mediated local activation of TGF-beta is critical to the development of pulmonary edema in ARDS, and blocking TGF-beta or its activation could be an effective treatment for this disorder.

BACKGROUND

Chronic rhinosinusitis (CRS) with nasal polyps (NPs) in Western populations is associated with TH2 cytokine polarization. IL-25, an IL-17 family cytokine, was recently reported to induce TH2-type immune responses and to contribute to several allergic diseases, such as atopic dermatitis and asthma. However, the role of IL-25 in Asian patients with nasal polyposis remains unclear.

OBJECTIVE

We sought to determine the role of IL-25 in Asian patients with nasal polyposis and CRS.

METHODS

We investigated IL-25 expression and its cellular origins in NPs of human subjects using immunohistochemistry (IHC), quantitative RT-PCR, and ELISA of NP tissues. Correlations between IL-25 expression and expression of other inflammatory markers in NP tissues were also explored. Anti-IL-25 neutralizing antibody was administered in an ovalbumin- and staphylococcal enterotoxin B-induced murine NP model to confirm the function of IL-25 during nasal polypogenesis.

RESULTS

IL-25 expression was upregulated in NP mucosa from patients with CRS with NPs compared with uncinate process tissue from control subjects and those with CRS without NPs. Overexpression of epithelial IL-25 was confirmed by using IHC, and double IHC staining showed that tryptase-positive cells were one of the main sources of IL-25 among immune cells. Furthermore, IL-17 receptor B levels were also increased in immune cells of patients with NPs compared with those in control subjects. In NPs IL-25 mRNA expression positively correlated with the expression of several inflammatory markers, including T-box transcription factor, RAR-related orphan receptor C, GATA3, eosinophil cationic protein, TGF-β1, and TGF-β2. IL-25 was more abundant in the murine NP model compared with control mice, and similar correlations between IL-25 and inflammatory markers were observed in murine models. Anti-IL-25 treatment reduced the number of polyps, mucosal edema thickness, collagen deposition, and infiltration of inflammatory cells, such as eosinophils and neutrophils. This treatment also inhibited expression of local inflammatory cytokines, such as IL-4 and IFN-γ. Furthermore, expression of CCL11, CXCL2, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 in the nasal mucosa was suppressed in the anti-IL-25-treated group.

CONCLUSIONS

Our results suggest that IL-25 secreted from the sinonasal epithelia and infiltrating mast cells plays a crucial role in the pathogenesis of CRS with NPs in Asian patients. In addition, our results suggest the novel possibility of treating nasal polyposis with anti-IL-25 therapy.

In highly purified human polymorphonuclear leukocyte (PMN) preparations containing less than 0.1% contaminating monocytes, significant amounts of interleukin-8 (IL-8) and small amounts of IL-1 alpha, IL-1 beta, and tumor necrosis factor-alpha (TNF-alpha) were produced by lipopolysaccharide (LPS) stimulation. Contrary to published reports, IL-6 production could not be detected. IL-10 inhibited the production of IL-1 alpha, IL-1 beta, IL-8, and TNF-alpha in LPS-stimulated PMNs, as it did in human blood mononuclear cell (MNC) preparations enriched in monocytes. Subsequent investigation of cytokine synthesis inhibitory effect of IL-10 on PMNs was focused on IL-8. IL-10 inhibited IL-8 synthesis in a dose-dependent manner and, in this regard, it was more potent than IL-4 and transforming growth factor-beta 1 (TGF-B1). In both MNCs and PMNs, degradation of LPS-induced IL-8 mRNA was enhanced by IL-10. Furthermore, as determined by nuclear run-on assays, IL-10 inhibited LPS-induced transcription of IL-8 gene in MNCs. However, in PMNs, run-on assays could not reliably detect IL-8 gene transcription. These results provide the first evidence that the human peripheral neutrophil is a target for inhibition of cytokine synthesis by IL-10, and that IL-10 acts by affecting both gene transcription and mRNA stability.

Patients with prolonged ulcerative colitis (UC) frequently develop colorectal adenocarcinoma for reasons that are not fully clear. To analyze inflammation-associated colonic tumorigenesis, we developed a chronic form of oxazolone-induced colitis in mice that, similar to UC, was distinguished by the presence of IL-13-producing NKT cells. In this model, the induction of tumors using azoxymethane was accompanied by the coappearance of F4/80+CD11b(high)Gr1(low) M2 macrophages, cells that undergo polarization by IL-13 and are absent in tumors that lack high level IL-13 production. Importantly, this subset of macrophages was a source of tumor-promoting factors, including IL-6. Similar to dextran sodium sulfate-induced colitis, F4/80+CD11b(high)Gr1(intermediate) macrophages were present in the mouse model of chronic oxazolone-induced colitis and may influence tumor development through production of TGF-β1, a cytokine that inhibits tumor immunosurveillance. Finally, while robust chronic oxazolone-induced colitis developed in myeloid differentiation primary response gene 88-deficient (Myd88-/-) mice, these mice did not support tumor development. The inhibition of tumor development in Myd88-/- mice correlated with cessation of IL-6 and TGF-β1 production by M2 and F4/80+CD11b(high)Gr1(intermediate) macrophages, respectively, and was reversed by exogenous IL-6. These data show that an UC-like inflammation may facilitate tumor development by providing a milieu favoring development of MyD88-dependent tumor-supporting macrophages.

OBJECTIVE

Human thoracic aortic aneurysms (TAAs) are characterized by extracellular matrix breakdown associated with progressive smooth muscle cell (SMC) rarefaction. These features are present in all types of TAA: monogenic forms [mainly Marfan syndrome (MFS)], forms associated with bicuspid aortic valve (BAV), and degenerative forms. Initially described in a mouse model of MFS, the transforming growth factor-β1 (TGF-β1)/Smad2 signalling pathway is now assumed to play a role in TAA of various aetiologies. However, the relation between the aetiological diversity and the common cell phenotype with respect to TGF-β signalling remains unexplained.

RESULTS

This study was performed on human aortic samples, including TAA [MFS, n = 14; BAV, n = 15; and degenerative, n = 19] and normal aortas (n = 10) from which tissue extracts and human SMCs and fibroblasts were obtained. We show that all types of TAA share a complex dysregulation of Smad2 signalling, independent of TGF-β1 in TAA-derived SMCs (pharmacological study, qPCR). The Smad2 dysregulation is characterized by an SMC-specific, heritable activation and overexpression of Smad2, compared with normal aortas. The cell specificity and heritability of this overexpression strongly suggest the implication of epigenetic control of Smad2 expression. By chromatin immunoprecipitation, we demonstrate that the increases in H3K9/14 acetylation and H3K4 methylation are involved in Smad2 overexpression in TAA, in a cell-specific and transcription start site-specific manner.

CONCLUSIONS

Our results demonstrate the heritability, the cell specificity, and the independence with regard to TGF-β1 and genetic backgrounds of the Smad2 dysregulation in human thoracic aneurysms and the involvement of epigenetic mechanisms regulating histone marks in this process.