ABSTRACT
The balance between pro- and anti-inflammatory signals maintains tissue homeostasis and defines the outcome of chronic inflammatory diseases such as periodontitis, a condition that afflicts the tooth-supporting tissues and exerts an impact on systemic health. The induction of tissue inflammation relies heavily on Toll-like receptor (TLR) signaling, which drives a proinflammatory pathway through recruiting myeloid differentiation primary response gene 88 (MyD88) and activating nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). TLR-induced production of proinflammatory cytokines and chemokines is reined in by anti-inflammatory cytokines, including the transforming growth factor ß (TGFß) family of cytokines. Although Smad6 is a key mediator of TGFß-induced anti-inflammatory signaling, the exact mechanism by which TGFß regulates TLR proinflammatory signaling in the periodontal tissue has not been addressed to date. In this study, we demonstrate for the first time that the ability of TGFß to inhibit TLR-NFκB signaling is mediated by protein arginine methyltransferase 1 (PRMT1)-induced Smad6 methylation. Upon methylation, Smad6 recruited MyD88 and promoted MyD88 degradation, thereby inhibiting NFκB activation. Most important, Smad6 is expressed and methylated in the gingival epithelium, and PRMT1-Smad6 signaling promotes tissue homeostasis by limiting inflammation. Consistent with this, disturbance of Smad6 methylation exacerbates inflammation and bone loss in experimental periodontitis. The dissected mechanism is therapeutically important, as it highlights the manipulation of PRMT1-Smad6 signaling as a novel promising strategy to modulate the host immune response in periodontitis.
Subject(s)
NF-kappa B/immunology , Periodontitis/immunology , Smad6 Protein/immunology , Arginine/immunology , Cells, Cultured , Gingiva/cytology , Humans , Inflammation/immunology , Methylation , Myeloid Differentiation Factor 88/immunology , Protein Interaction Domains and Motifs , Protein-Arginine N-Methyltransferases/immunology , Repressor Proteins/immunology , Signal Transduction , Transforming Growth Factor beta/immunology , Ubiquitin-Protein Ligases/immunologyABSTRACT
BACKGROUND: Breast milk is known to protect the infant against infectious and immuno-inflammatory diseases, but the mechanisms of this protection are poorly understood. OBJECTIVES: We hypothesized that transforming growth factor-ß2 (TGF-ß2), an immunoregulatory cytokine abundant in breast milk, may have a direct anti-inflammatory effect on immature human intestinal epithelial cells (IECs). METHODS: Human fetal ileal organ culture, primary human fetal IECs, and the human fetal small intestinal epithelial cell line H4 were stimulated with interleukin 1ß (IL-1ß) with or without TGF-ß2. Pro-inflammatory cytokine secretion and mRNA expression were measured by ELISA and quantitative real-time polymerase chain reaction, respectively. Alterations in ERK signalling were detected from IECs by immunoblotting and in fetal ileal tissue culture by immunohistochemistry. SMAD6 knockdown was performed by transfecting the cells with SMAD6 siRNA. RESULTS: TGF-ß2 significantly attenuated IL-1ß-induced pro-inflammatory cytokine production in fetal intestinal organ culture and the cell culture models. In addition, TGF-ß2 reduced the IL-1ß-induced IL-8 and IL-6 mRNA response in H4 cells. TGF-ß2 markedly inhibited IL-1ß-induced phosphorylation of ERK, which was necessary for the cytokine response. The inhibitory effect of TGF-ß2 on IL-1ß-induced cytokine production was completely abrogated by SMAD6 siRNA knockdown. CONCLUSIONS: TGF-ß2 attenuates IL-1ß-induced pro-inflammatory cytokine production in immature human IECs by inhibiting ERK signalling. The anti-inflammatory effect of TGF-ß2 is dependent on SMAD6. Breast milk TGF-ß2 may provide the neonate with important immunoregulatory support. TGF-ß2 might provide a novel means to improve intestinal immunophysiology in premature neonates.
Subject(s)
Extracellular Signal-Regulated MAP Kinases/immunology , Interleukin-1beta/immunology , Intestine, Small/immunology , Milk, Human/immunology , Smad6 Protein/immunology , Transforming Growth Factor beta2/pharmacology , Cell Line , Epithelial Cells/cytology , Epithelial Cells/immunology , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Fetus , Flavonoids/pharmacology , Humans , In Vitro Techniques , Infant, Newborn , Interleukin-6/genetics , Interleukin-6/immunology , Interleukin-8/genetics , Interleukin-8/immunology , Intestine, Small/cytology , Protein Kinase Inhibitors/pharmacology , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Smad6 Protein/antagonists & inhibitorsABSTRACT
Statins are widely used drugs for the treatment of hypercholesterolaemia. A number of recent studies have suggested that statins also have pleiotropic effects on immune responses and statins have proven to be effective in the treatment of autoimmune diseases in animal models. Foxp3(+) T regulatory cells are a unique subset of CD4(+) T cells that mediate immunosuppression. Foxp3(+) T cells develop in the thymus, but can also be induced in peripheral sites in the presence of transforming growth factor-beta (TGF-beta). We demonstrate here that simvastatin blockade of the mevalonate pathway can mediate induction of mouse Foxp3(+) T cells and that simvastatin can synergize with low levels of TGF-beta to induce Foxp3(+) T cells. The effects of simvastatin are secondary to a blockade of protein geranylgeranylation, are mediated at late time-points after T-cell activation, and are associated with demethylation of the Foxp3 promoter. One major effect of simvastatin was inhibition of the induction of Smad6 and Smad7, inhibitory Smads that inhibit TGF-beta signalling. Our results suggest that one mechanism responsible for the immunosuppressive effects of statins is the ability to promote the generation of Foxp3(+) T regulatory cells.
