Alcohol-Induced Interleukin-17 Expression Causes Murine Lung Fibroblast-to-Myofibroblast Transdifferentiation via Thy-1 Down-Regulation.

BACKGROUND: Alcohol exposure induces TGFß1 and renders the lung susceptible to injury and disrepair. We determined that TGFß1 regulates myofibroblast differentiation through the loss of Thy-1 expression and consequent induction of α-SMA. TGFß1 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 TGFß1 and that IL-17 produced by these cells contributes to the development of profibrotic lung myofibroblasts. METHODS: Primary lung fibroblasts (PLFs) were treated with alcohol, TGFß1, 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. TGFß1 or IL-17 exposure suppressed PLF Thy-1 expression. When administered together, TGFß1 and IL-17 additively down-regulated 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. CONCLUSIONS: 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 TGFß1 contribute to fibroproliferative disrepair in the lung and targeting these proteins could limit morbidity and mortality following lung injury in alcoholic individuals.

HIV-1 decreases Nrf2/ARE activity and phagocytic function in alveolar macrophages.

Respiratory complications occur frequently in individuals living with human immunodeficiency-1 virus (HIV) infection, and there is evidence that HIV-related oxidative stress impairs alveolar macrophage immune function. We hypothesized that nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a master transcription factor that activates the antioxidant response element (ARE) and regulates antioxidant defenses, has an important role in alveolar macrophage (AMs) immune dysfunction in individuals with HIV infections. To test that hypothesis, we analyzed human monocyte-derived macrophages (MDMs) that were either infected with HIV-1 or were exposed to the HIV-related proteins gp120 and Tat ex vivo and determined that either stress affected the expression of Nrf2 and the Nrf2-ARE-dependent genes for NAD(P)H dehydrogenase, quinone 1 (NQO1) and glutamate-cysteine ligase, catalytic subunit (GCLC). We then determined that the expression of Nrf2, NQO1, and GCLC was significantly decreased in primary AMs isolated from HIV-1 transgenic rats. In parallel, treating a rat macrophage cell line (NR8383 cells) with the HIV-related proteins gp120 or Tat similarly decreased the gene and protein expression of Nrf2, NQO1, and GCLC. Further, phagocytic function was decreased in both human MDMs infected with HIV-1 and primary AMs from HIV-1 transgenic rats. Importantly, treating HIV-1-infected human MDMs or AMs from HIV-1 transgenic rats with sulforaphane (SFN, an Nrf2 activator) significantly improved their phagocytic function. The salutary effects of SFN were abrogated by silencing RNA to Nrf2 in wild-type rat macrophages. Our findings demonstrate that HIV-1 infection and exposure to HIV-1-related proteins inhibit Nrf2-ARE activity in the AMs and impair their phagocytic function. Treatments targeted at increasing Nrf2-ARE activity could, therefore, enhance lung innate immunity in people living with HIV-1.

TGF-ß1 epigenetically modifies Thy-1 expression in primary lung fibroblasts.

Idiopathic pulmonary fibrosis is a progressive lung disease that increases in incidence with age. We identified a profibrotic lung phenotype in aging mice characterized by an increase in the number of fibroblasts lacking the expression of thymocyte differentiation antigen 1 (Thy-1) and an increase in transforming growth factor (TGF)-ß1 expression. It has been shown that Thy-1 expression can be epigenetically modified. Lung fibroblasts (PLFs) were treated with TGF-ß1 ± DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-AZA) and analyzed for Thy-1 gene and protein expression, DNMT protein expression, and activity. α-Smooth muscle actin (α-SMA) and collagen type 1 (Col1A1) gene and protein expression was assessed. PLFs were transfected with DNMT1 silencing RNA ± TGF-ß1. TGF-ß1 inhibited Thy-1 gene and protein expression in PLFs, and cotreatment with 5-AZA ameliorated this effect and appeared to inhibit DNMT1 activation. TGF-ß1 induced Thy-1 promoter methylation as assessed by quantitative methyl PCR. Treatment with 5-AZA attenuated TGF-ß1-induced Col1A1 gene and protein expression and α-SMA gene expression (but not α-SMA protein expression). Inhibiting DNMT1 with silencing RNA attenuated TGF-ß1-induced DNMT activity and its downstream suppression of Thy-1 mRNA and protein expression as well as inhibited α-SMA mRNA and Col1A1 mRNA and protein expression, and showed a decreased trend in Thy-1 promoter methylation. Immunofluorescence for α-SMA suggested that 5-AZA inhibited stress fiber formation. These findings suggest that TGF-ß1 epigenetically regulates lung fibroblast phenotype through methylation of the Thy-1 promoter. Targeted inhibition of DNMT in the right clinical context might prevent fibroblast to myofibroblast transdifferentiation and collagen deposition, which in turn could prevent fibrogenesis in the lung and other organs.

Aging promotes pro-fibrotic matrix production and increases fibrocyte recruitment during acute lung injury.

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 TGFß1. 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 + TGFß1 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.

Fungal allergen ß-glucans trigger p38 mitogen-activated protein kinase-mediated IL-6 translation in lung epithelial cells.

In addition to immune cells, airway epithelial cells can contribute to and shape the immune response in the lung by secreting specific cytokines. IL-6 is a key factor in determining the effector fate of CD4(+) T cells. Here we show that under basal conditions, the IL-6 gene is already highly expressed in lung epithelial cells, but not in immune cells resident in the lung. However, upon exposure of the lungs to fungal allergens, the direct contact of ß-glucans present in the fungus cell wall with lung epithelial cells is sufficient to trigger the rapid synthesis and secretion of IL-6 protein. This posttranscriptional regulation of IL-6 in response to fungal extracts is mediated by the p38 mitogen-activated protein kinase pathway. The inhalation of ß-glucans with a nonallergenic antigen is sufficient to provide an adjuvant effect that leads to mucous hyperplasia in the airways. Thus, ß-glucans may constitute a common determinant of the fungal and plant-derived allergens responsible for some of the pathological features in allergic asthma.

Elevation of IL-6 in the allergic asthmatic airway is independent of inflammation but associates with loss of central airway function.

BACKGROUND: Asthma is a chronic inflammatory disease of the airway that is characterized by a Th2-type of immune response with increasing evidence for involvement of Th17 cells. The role of IL-6 in promoting effector T cell subsets suggest that IL-6 may play a functional role in asthma. Classically IL-6 has been viewed as an inflammatory marker, along with TNFalpha and IL-1beta, rather than as regulatory cytokine. OBJECTIVE: To investigate the potential relationship between IL-6 and other proinflammatory cytokines, Th2/Th17 cytokines and lung function in allergic asthma, and thus evaluate the potential role of IL-6 in this disease. METHODS: Cytokine levels in induced sputum and lung function were measured in 16 healthy control and 18 mild-moderate allergic asthmatic subjects. RESULTS: The levels of the proinflammatory biomarkers TNFalpha and IL-1beta were not different between the control and asthmatic group. In contrast, IL-6 levels were specifically elevated in asthmatic subjects compared with healthy controls (p < 0.01). Hierarchical regression analysis in the total study cohort indicates that the relationship between asthma and lung function could be mediated by IL-6. Among Th2 cytokines only IL-13 (p < 0.05) was also elevated in the asthmatic group, and positively correlated with IL-6 levels (rS = 0.53, p < 0.05). CONCLUSIONS: In mild-moderate asthma, IL-6 dissociates from other proinflammatory biomarkers, but correlates with IL-13 levels. Furthermore, IL-6 may contribute to impaired lung function in allergic asthma.

IL-6 is required for airway mucus production induced by inhaled fungal allergens.

Allergic asthma is caused by inhaled allergens and is characterized by airway eosinophilia, as well as mucus hypersecretion, which can lead to airflow obstruction. Despite the association of increased IL-6 levels with human atopic asthma, the contribution of IL-6 to the development of allergic airway inflammation triggered by inhaled allergens remains unclear. In this study, we examined the role of IL-6 in a mouse model of allergic airway inflammation induced by direct airway exposure to extracts of Aspergillus fumigatus, a common allergen in humans. We show that inhaled A. fumigatus extracts rapidly trigger the production of IL-6 in the airways. IL-6 appears to be dispensable for the recruitment of eosinophils to the lung during the development of allergic airway inflammation. However, IL-6 is essential for mucus hypersecretion by airway epithelial cells triggered in response to inhaled A. fumigatus Ags. Impaired mucus production caused by IL-6 deficiency correlates with a severe reduction in the levels of IL-13, a major inducer of mucin glycoproteins. Thus, IL-6 is a key regulator of specific hallmark features of allergic airway inflammation and it could be a potential target for pulmonary diseases that are associated with goblet cell metaplasia and mucus hypersecretion.