Background: We previously demonstrated that chronic alcohol ingestion augments TGFβ1 expression in the lung fibroblast and increases the risk of fibroproliferative disrepair in a mouse model of acute lung injury. The effect of alcohol on TGFβ1 is mitigated by treatment with sulforaphane (SFP), which can activate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, the mechanisms by which alcohol amplifies, or SFP attenuates, TGFβ1 expression in the fibroblast are not known. MicroRNA (miR)-21 has been shown to inhibit Smad7, a TGFβ1 signaling inhibitor. In this study, we hypothesized that alcohol augments TGFβ1 expression through up-regulation of miR-21, which subsequently inhibits Smad7. Methods: Primary mouse lung fibroblasts were cultured ± alcohol ± SFP and assessed for gene expression of miR-21, and gene and/or protein expression of Nrf2, Nrf2-regulated antioxidant enzymes, Smad7, STAT3, and TGFβ1. NIH 3T3 fibroblasts were transfected with a miR-21 inhibitor and cultured ± alcohol. α-SMA, Smad7, and TGFβ1 protein expression were then assessed. In parallel, NIH 3T3 lung fibroblasts were transfected with Nrf2 silencing RNA (siRNA) and cultured ± alcohol ± SFP. Gene expression of miR-21, Nrf2, Smad7, and TGFβ1 was assessed. Results: MiR-21 gene expression was increased by 12-fold at 48 hours, and Smad7 gene expression and protein expression were reduced by ~30% in alcohol-treated fibroblasts. In parallel, inhibition of miR-21 attenuated alcohol-mediated decrease in Smad7 and increase in TGFβ1 and α-SMA protein expression. Treatment with SFP mitigated the effect of alcohol on miR-21, Smad7 and total and phosphorylated STAT3, and restored Nrf2-regulated antioxidant gene expression. Silencing of Nrf2 prevented the effect of SFP on miR-21, Smad7, and TGFβ1 gene expression in alcohol-treated NIH 3T3 fibroblasts. Conclusions: Alcohol treatment increases TGFβ1 in fibroblasts, at least in part, through augmentation of miR-21, which then inhibits Smad7 expression. These effects can be attenuated by activation of Nrf2 with SFP.
Introduction
Idiopathic pulmonary fibrosis (IPF) is a devastating progressive lung disease with an average survival of only 3 to 5 years. The mechanisms underlying the initiation and progression of IPF are poorly understood, and treatments available have only modest effect on disease progression. Interestingly, the incidence of IPF is approximately 60 times more common in individuals aged 75 years and older, but the mechanism by which aging promotes fibrosis is unclear. The authors hypothesized that aged lungs have a profibrotic phenotype that render it susceptible to disrepair after injury.
Methods
Young and old mice were treated with bleomycin to examine disrepair in the aged lung. In addition, uninjured young and old mouse lungs were analyzed for transforming growth factor-beta 1 (TGF-β1) production, extracellular matrix composition and lung fibroblast phenotype. Lung fibroblasts were treated with a DNA methyltransferase inhibitor to examine the potential epigenetic mechanisms involved in age-associated phenotypic alterations.
Results
The lungs of old mice showed worse fibrosis after bleomycin-induced injury compared with the lungs from young mice. At baseline, aged lungs expressed a profibrotic phenotype characterized by increased mRNA expression for fibronectin extracellular domain A (Fn-EDA) and the matrix metalloproteinases (MMPs) MMP-2 and MMP-9. Old lungs also expressed higher levels of TGF-β receptor 1 and TGF-β1 mRNA, protein and activity as determined by increased Smad3 expression, protein phosphorylation and DNA binding. Lung fibroblasts harvested from aged lungs showed reduced expression of the surface molecule Thy-1, a finding also implicated in lung fibrosis; the latter did not seem related to Thy-1 gene methylation.
Conclusion
Altogether, aged lungs manifest a profibrotic phenotype characterized by enhanced fibronectin extracellular domain A and MMP expression and increased TGF-β1 expression and signaling and are populated by Thy-1–negative fibroblasts, all implicated in the pathogenesis of lung fibrosis.
The association of estrogen supplementation use and quality of life in women with cystic fibrosis (CF) is not well characterized. In this cross-sectional study, women with CF completed quality of life questionnaires during a routine CF clinic visit. The use of estrogen supplementation was associated with higher quality of life scores in all domains of the CF questionnaire-revised (CFQ-R) and was significant in the role limitations and respiratory domains. Most participants who were not currently using estrogen supplementation had previously used estrogen supplementation. Most participants had used estrogen to regulate menses, prevent pregnancy and control symptoms around menses. Use of estrogen supplementation was not associated with differences in life-space mobility or screening for sexual dysfunction. This is the largest study to date investigating the association of estrogen supplementation and quality of life in women with CF. Prospective randomized studies are needed to clarify the association of estrogen supplementation and quality of life in women with CF.
Fibrotic lung diseases increase with age. Previously we determined that senescence increases tissue expression of fibronectin EDA (Fn-EDA) and decreases fibroblast expression of Thy-1, and that fibrocytes contribute to fibrosis following bleomycin-induced lung injury in mice. In this study we hypothesized that fibroblasts lacking Thy-1 expression produce an extracellular matrix that promotes fibrocyte retention and myofibroblast transdifferentiation, thereby promoting fibrogenesis. Young and old mice were treated with bleomycin intratracheally; fibrocytes in the bone marrow, blood, and lungs were quantified, and lung fibroblast Thy-1 expression assessed. Bone marrow-derived fibrocytes were cultured on matrices derived from Thy-1(+) or Thy-1(−) fibroblasts ± the pro-fibrotic cytokine TGFβ1. Older mice had more fibrocytes in their bone marrows at baseline and more fibrocytes in their lungs following bleomycin treatment. In parallel, lung fibroblasts in older mice had lower expression of Thy-1 at baseline that increased transiently 7 days after bleomycin treatment but then rapidly waned such that 14 days after bleomycin treatment Thy-1 expression was again markedly lower. Fibrocytes cultured on matrices derived from Thy-1(−) fibroblasts + TGFβ1 had increased gene expression for collagen type 1, fibronectin, Fn-EDA, and α-smooth muscle actin. In parallel, whereas the matrices derived from Thy-1(−) fibroblasts stimulated phosphorylation of Akt in cultured fibrocytes, the matrices derived from Thy-1(+) fibroblasts induced apoptosis. These findings suggest that senescence increases fibrocyte recruitment to the lung following injury and that loss of Thy-1 expression by lung fibroblasts promotes fibrocyte retention and myofibroblast trans-differentiation that renders the “aging lung” susceptible to fibrosis.
Keywords: Lung Fibrosis, Thy-1, Fibrocytes, Extracellular Matrix, Fibronectin, TGF β1
Exaggerated transforming growth factor-beta 1 (TGFβ1) expression worsens fibroproliferation following bleomycin-induced lung injury in alcohol-fed mice. MicroRNA (miR)-1946a is predicted to bind to the TGFβ1 3′ untranslated region (UTR), thereby inhibiting its transcription. We hypothesize that alcohol suppresses miR-1946a and induces TGFβ1. Primary murine lung fibroblasts (PLFs) were cultured ± alcohol, miR-1946a mimic or inhibitor, and TGFβ1 signaling inhibitors. miR-1946a was analyzed after alcohol treatment in vitro and in vivo. TGFβ1 expression and TGFβ1 3′UTR-luciferase activity was quantified. We showed that alcohol suppressed miR-1946a in the alcohol-fed mouse lungs and PLFs. MiR-1946a inhibitor increased TGFβ1 expression in the fibroblast. MiR-1946a mimic treatment suppressed TGFβ1 gene expression and TGFβ1 3′UTR activity. Overexpression of miR1946a inhibited alcohol-induced TGFβ1 gene and protein expression as well as alcohol-induced TGFβ1 and α-smooth muscle actin (SMA) protein expression in PLFs. In conclusion, miR-1946a modulates TGFβ1 expression through direct interaction with TGFβ1 3′UTR. These findings identify a novel mechanism by which alcohol induces TGFβ1 in the lung.
Recent findings suggest that embryonic stem cells and stem cells derived from adult tissues, including bone marrow and umbilical cord blood, could be utilized in repair and regeneration of injured or diseased lungs. This is an exciting and rapidly moving field that holds promise as a therapeutic approach for variety of lung diseases. Although initial emphasis was on engraftment of stem cells in lung, more recent studies demonstrate that mesenchymal stem cells (MSCs) can modulate local inflammatory and immune responses in mouse lung disease models including acute lung injury and pulmonary fibrosis. Further, on the basis of initial reports of safety and efficacy following allogeneic administration of MSCs to patients with Crohn's disease or with graft-versus-host disease, a recent trial has been initiated to study the effect of MSCs in patients with chronic obstructive pulmonary disease. Notably, several recent clinical trials have demonstrated potential benefit of autologous stem cell administration in patient with pulmonary hypertension. In this review, we will describe recent advances in cell therapy with the focus on MSCs and their potential roles in lung development and repair.
Cell-based therapies with embryonic or adult stem cells, including induced pluripotent stem cells, have emerged as potential novel approaches for several devastating and otherwise incurable lung diseases, including emphysema, pulmonary fibrosis, pulmonary hypertension, and the acute respiratory distress syndrome. Although initial studies suggested engraftment of exogenously administered stem cells in lung, this is now generally felt to be a rare occurrence of uncertain physiologic significance. However, more recent studies have demonstrated paracrine effects of administered cells, including stimulation of angiogenesis and modulation of local inflammatory and immune responses in mouse lung disease models. Based on these studies and on safety and initial efficacy data from trials of adult stem cells in other diseases, groundbreaking clinical trials of cell-based therapy have been initiated for pulmonary hypertension and for chronic obstructive pulmonary disease. In parallel, the identity and role of endogenous lung progenitor cells in development and in repair from injury and potential contribution as lung cancer stem cells continue to be elucidated. Most recently, novel bioengineering approaches have been applied to develop functional lung tissue ex vivo. Advances in each of these areas will be described in this review with particular reference to animal models.
Background: Chronic alcohol ingestion induces the expression of transforming growth factor beta-1(TGFβ1), inhibits nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated activation of the antioxidant response element (ARE), depletes alveolar glutathione pools, and potentiates acute lung injury. In this study, we examined the mechanistic relationship between TGFβ1 and Nrf2-ARE signaling in the experimental alcoholic lung. Methods: Wild-type mice were treated ± alcohol in drinking water for 8 weeks and their lungs were assessed for Nrf2 expression. In parallel, mouse lung fibroblasts were cultured ± alcohol and treated ± sulforaphane (SFP; an activator of Nrf2), ±TGFβ1, ±TGFβ1 neutralizing antibody, and/or ±activin receptor-like kinase 5 inhibitors (to block TGβ1 receptor signaling) and then analyzed for the expression of Nrf2, Kelch-like ECH-associated protein 1 (Keap1) and TGFβ1, Nrf2-ARE activity, and the expression of the Nrf2-ARE-dependent antioxidants glutathione s-transferase theta 2 (GSTT2) and glutamate-cysteine ligase catalytic subunit (GCLC). Finally, silencing RNA (siRNA) of Nrf2 was then performed prior to alcohol exposure and subsequent analysis of TGFβ1 expression. Results: Alcohol treatment in vivo or in vitro decreased Nrf2 expression in murine whole lung and lung fibroblasts, respectively. In parallel, alcohol exposure in vitro decreased Keap1 gene and protein expression in lung fibroblasts. Furthermore, alcohol exposure increased TGFβ1 expression but decreased Nrf2-ARE activity and expression of the ARE-dependent genes for GSTT2 and GCLC. These effects of alcohol were prevented by treatment with SFP; in contrast, Nrf2 SiRNA expression exacerbated alcohol-induced TGFβ1 expression. Finally, TGFβ1 treatment directly suppressed Nrf2-ARE activity whereas blocking TGFβ1 signaling attenuated alcohol-induced suppression of Nrf2-ARE activity. Conclusions: Alcohol-induced oxidative stress is mediated by TGFβ1, which suppresses Nrf2-ARE-dependent expression of antioxidant defenses and creates a vicious cycle that feeds back to further increase TGFβ1 expression. These effects of alcohol can be mitigated by activation of Nrf2, suggesting a potential therapy in individuals at risk for lung injury due to alcohol abuse.
The prevalence of serotonin syndrome increases over the past several years as more serotonergic medications are being used in clinical practice. It is a potentially lethal condition caused by excessive serotonergic activity. Common causes of serotonin syndrome are the use of prescription medications, illicit drugs, or a combination of substances, leading to an increase in the activity of serotonin in the central and peripheral nervous system. The clinical symptoms range from mild to severe. We report a case of a 25-year-old woman with polysubstance abuse, including cocaine, who presented with confusion, rigidity, high-grade fever, and reduced biventricular function on echocardiogram. Based on the combination of substance used history, clinical presentation, and echocardiogram findings, she was diagnosed with serotonin syndrome complicated by takotsubo cardiomyopathy. She improved after being treated in the intensive care unit and was discharged from the hospital. This patient demonstrates the importance of recognizing and promptly initiating management of serotonin syndrome in order to improve morbidity and mortality.
Background:
Alcohol exposure induces TGFβ1 and renders the lung susceptible to injury and disrepair. We determined that TGFβ1 regulates myofibroblast differentiation through the loss of Thy-1 expression and consequent induction of α-SMA. TGFβ1 is important for T helper 17 (Th17) differentiation and IL-17 secretion, which in turn participates in tissue repair. We hypothesized that alcohol induces Th17 differentiation via TGFβ1 and that IL-17 produced by these cells contributes to the development of pro-fibrotic lung myofibroblasts.
Methods:
Primary lung fibroblasts (PLFs) were treated with alcohol, TGFβ1, and IL-17 and then analyzed for Thy-1 expression and cell morphology. Naïve and Th17-polarized CD4+ T cells were exposed to alcohol and assessed for IL-17 expression. CD4+ T cells from alcohol-fed mice were analyzed for Th17 and IL-17 expression. Lungs of control-fed, bleomycin-treated and alcohol-fed, bleomycin-treated mice were analyzed for IL-17 protein expression.
Results:
Alcohol-treated PLFs expressed lower levels of Thy-1 than untreated cells. TGFβ1 or IL-17 exposure suppressed PLF Thy-1 expression. When administered together, TGFβ1 and IL-17 additively downregulated Thy-1 expression. Exposure of naïve and Th17-polarized CD4+ T cells to alcohol induced the Th17 phenotype and augmented their production of IL-17. CD4+ Th17+ levels are elevated in the peripheral compartment but not in the lungs of alcohol-fed animals. Treatment of the PLFs with IL-17 and alcohol induced α-SMA expression. Induction of α-SMA and myofibroblast morphology by IL-17 occurred selectively in a Thy-1− fibroblast subpopulation. Chronic alcohol ingestion augmented lung-specific IL-17 expression following bleomycin-induced lung injury.
Conclusion:
Alcohol exposure skews T cells toward a Th17 immune response that in turn primes the lung for fibroproliferative disrepair through loss of Thy-1 expression and induction of myofibroblast differentiation. These effects suggest that IL-17 and TGFβ1 contribute to fibroproliferative disrepair in the lung and targeting these proteins could limit morbidity and mortality following lung injury in alcoholic individuals.