The Role of IL-6 in Adenosine-Mediated Pulmonary Fibrosis

Adenosine is a purinergic signaling molecule that regulates various aspects of inflammation and has been implicated in the pathogenesis of chronic lung diseases. Previous studies have demonstrated that adenosine up-regulates IL-6 production through the engagement of the A2B adenosine receptor in various cell types, including alveolar macrophages. IL-6 is elevated in mouse models and humans with chronic lung disease, suggesting a potential role in disease progression. Furthermore, chronic elevation of adenosine in the lungs of adenosine deaminase deficient (Ada-/-) mice leads to the development of pulmonary inflammation, alveolar destruction, and fibrosis, in conjunction with IL-6 elevation. Thus, it was hypothesized that IL-6 contributes to pulmonary inflammation and fibrosis in this model. To test this hypothesis, Ada/IL-6 double knockout mice (Ada/IL-6-/-) were generated to assess the consequences of genetically removing IL-6 on adenosine-dependent pulmonary injury. Ada/IL-6-/- mice exhibited a significant reduction in inflammation, alveolar destruction, and pulmonary fibrosis. Next, Ada-/- mice were treated systematically with IL-6 neutralizing antibodies to test the efficacy of blocking IL-6 on chronic lung disease. These treatments were associated with decreased pulmonary inflammation, alveolar destruction, and fibrosis. To determine the role of IL-6 in a second model of pulmonary fibrosis, wild type mice and IL-6-/- mice were subjected to intraperitoneal injections of bleomycin twice a week for four weeks. Results demonstrated that IL-6-/- mice developed reduced pulmonary fibrosis. To examine a therapeutic approach in this model, wild type mice exposed to bleomycin were treated with IL-6 neutralizing antibodies. Similar results were observed as with Ada-/- mice, namely diminished pulmonary inflammation and fibrosis. In both models, elevations in IL-6 were associated with increased phosphorylated STAT-3 in the nuclei of numerous cell types in the airways, including type II alveolar epithelial cells (AEC). Genetic removal and neutralization of IL-6 in both models was associated with decreased STAT-3 activation in type II AEC. The mechanism of activation in these cells that lack the membrane bound IL-6Ra suggests IL-6 trans-signaling may play a role in regulating fibrosis. Characterization of this mechanism demonstrated that the soluble IL-6Ra (sIL-6Ra) is upregulated in both models during chronic conditions. In vitro studies in MLE-12 alveolar epithelial cells confirmed that IL-6, in combination with the sIL-6Ra, activates STAT-3 and TWIST in association with enhancement of epithelial-to-mesenchymal transition, which can contribute to fibrosis. Similarly, patients with idiopathic pulmonary fibrosis demonstrated a similar pattern of increased IL-6 expression, STAT-3 activation, and sIL-6Ra increases. These findings demonstrate that adenosine-dependent elevations in IL-6 contribute to the development and progression of pulmonary inflammation and fibrosis. The implications from these studies are that adenosine and/or IL-6 neutralizing agents represent novel therapeutic targets for the treatment of pulmonary disorders where fibrosis is a detrimental component

Interleukin-6 Contributes to Inflammation and Remodeling in a Model of Adenosine Mediated Lung Injury

Chronic lung diseases are the third leading cause of death in the United States due in part to an incomplete understanding of pathways that govern the progressive tissue remodeling that occurs in these disorders. Adenosine is elevated in the lungs of animal models and humans with chronic lung disease where it promotes air-space destruction and fibrosis. Adenosine signaling increases the production of the pro-fibrotic cytokine interleukin-6 (IL-6). Based on these observations, we hypothesized that IL-6 signaling contributes to tissue destruction and remodeling in a model of chronic lung disease where adenosine levels are elevated.We tested this hypothesis by neutralizing or genetically removing IL-6 in adenosine deaminase (ADA)-deficient mice that develop adenosine dependent pulmonary inflammation and remodeling. Results demonstrated that both pharmacologic blockade and genetic removal of IL-6 attenuated pulmonary inflammation, remodeling and fibrosis in this model. The pursuit of mechanisms involved revealed adenosine and IL-6 dependent activation of STAT-3 in airway epithelial cells.These findings demonstrate that adenosine enhances IL-6 signaling pathways to promote aspects of chronic lung disease. This suggests that blocking IL-6 signaling during chronic stages of disease may provide benefit in halting remodeling processes such as fibrosis and air-space destruction

Targeting of cadherin-11 decreases skin fibrosis in the tight skin-1 mouse model

<div><p>Objective</p><p>Systemic sclerosis (SSc) is an autoimmune disease clinically manifesting as progressive fibrosis of the skin and internal organs. Cadherin-11 (CDH11) expression is increased in fibrotic skin and lung tissue. Targeting CDH11 may be an effective approach to treating fibrosis. We hypothesize that targeting CDH11 will decrease fibrosis in the tight skin-1 (Tsk-1) mouse model.</p><p>Methods</p><p>CDH11 expression was determined in the Tsk-1 mouse model using quantitative real time PCR and immunofluorescence (IF). Inhibitory anti- CDH11 monoclonal antibodies were tested in Tsk-1 mice for their ability to decrease hypodermal fibrosis.</p><p>Results</p><p>Expression of CDH11 was increased in fibrotic skin from Tsk-1 mice compared to pallid controls. IF staining demonstrated that CDH11 expression localized to fibroblasts within the hypodermis of fibrotic skin. Treatment with inhibitory anti-CDH11 monoclonal antibodies decreased hypodermal thickness and fibrotic mediators in Tsk-1 mice compared to control antibodies.</p><p>Conclusions</p><p>These data demonstrate an important role for CDH11 in the development of skin fibrosis in Tsk-1 mice. These data add to the growing evidence for the important role of CDH11 in tissue fibrosis and fibrotic disease such as systemic sclerosis.</p></div

Anti-cadherin-11 antibody decreases mediators of fibrosis in Tsk-1 mice.

<p>Total RNA was isolated from skin biopsies from pa/pa control mice, Tsk-1 mice treated with isotype antibodies, and Tsk-1 mice treated with antibodies against CDH11 (13C2). Transcripts were determined for <i>Col1</i><b>α</b><i>1</i> (a), <b>α</b><i>SMA</i> (b), <i>CCN2</i> (c), fibronectin (d), <i>TGF-</i><b>β</b> (e), <i>IL-6</i> (f) in parallel with <i>18S</i> rRNA. Data are presented as mean of fold change transcripts ± SEM, n≥10 (*p ≤ 0.05 pa/pa vs. Tsk-1 isotype; #p ≤ 0.05 Tsk-1 isotype vs. Tsk-1 anti-CDH11).</p

Characterization of Cadherin-11 expression in the Tsk-1 mouse model of skin fibrosis.

<p>(a) Skin biopsies from pa/pa and Tsk-1 mice were stained with H&E demonstrating increased thickness of the hypodermis (arrow) in Tsk-1 mice compared to pa/pa mice. (10x magnification, scale bar 50 μm). These data were quantified in (b) (n = 10; * p ≤ 0.05 pa/pa vs. Tsk-1). (c) Tsk-1 mice have increased deposition of collagen using Sircol assay (n = 10; * p ≤ 0.05 pa/pa vs. Tsk-1). (d) Skin biopsies were used to isolate total RNA and transcripts were determined for <i>Cdh11</i>. Transcripts were measured in parallel with <i>18S</i> RNA and values are presented as mean of fold change transcripts ± SEM, n = 10 (* p ≤ 0.05 pa/pa vs. Tsk-1). (e) Immunofluorescence of CDH11 expression was determined in the hypodermal layer (arrows) in skin sections from pa/pa and Tsk-1 mice. Images are representative of 10 mice from each group. Top panels, Low magnification 20X, scale bars: 50 μm. Middle and lower panel, high magnification 100x, scale bars: 10 μm. (f) Dual color IF of hypodermis from pa/pa and Tsk-1 mice stained for <b>α</b>SMA (red) and CDH11 (green) or F4/80 (red) and CDH11 (green) demonstrate that CDH11 expression primarily localizes to <b>α</b>SMA myofibroblasts (100x, scale bars: 10 μm). (g, h) Quantification of <b>α</b>SMA and F4/80 positive cells that express CDH11 per high power field in the hypodermis of pa/pa and Tsk-1 mice (* p ≤ 0.05 pa/pa vs. Tsk-1).</p

Hypoxia-induced deoxycytidine kinase expression contributes to apoptosis in chronic lung disease

Chronic obstructive pulmonary disease (COPD) is characterized by persistent inflammation and tissue remodeling and is a leading cause of death in the United States. Increased apoptosis of pulmonary epithelial cells is thought to play a role in COPD development and progression. Identification of signaling pathways resulting in increased apoptosis in COPD can be used in the development of novel therapeutic interventions. Deoxyadenosine (dAdo) is a DNA breakdown product that amplifies lymphocyte apoptosis by being phosphorylated to deoxyadenosine triphosphate (dATP). dAdo is maintained at low levels by adenosine deaminase (ADA). This study demonstrated that mice lacking ADA developed COPD manifestations in association with elevated dAdo and dATP levels and increased apoptosis in the lung. Deoxycitidine kinase (DCK), a major enzyme for dAdo phosphorylation, was up-regulated in mouse and human airway epithelial cells in association with air-space enlargement. Hypoxia was identified as a novel regulator of DCK, and inhibition of DCK resulted in diminished dAdo-mediated apoptosis in the lungs. Our results suggest that activating the dAdo-DCK-dATP pathway directly results in increased apoptosis in the lungs of mice with air-space enlargement and suggests a novel therapeutic target for the treatment of COPD.—Weng, T., Karmouty-Quintana, H., Garcia-Morales, L. J., Molina, J. G., Pedroza, M., Bunge, R. R., Bruckner, B. A., Loebe, M., Seethamraju, H., and Blackburn, M. R. Hypoxia-induced deoxycytidine kinase expression contributes to apoptosis in chronic lung disease

Adenosine A2B Receptor and Hyaluronan Modulate Pulmonary Hypertension Associated with Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide. The development of pulmonary hypertension (PH) in patients with COPD is strongly associated with increased mortality. Chronic inflammation and changes to the lung extracellular matrix (ECM) have been implicated in the pathogenesis of COPD, yet the mechanisms that lead to PH secondary to COPD remain unknown. Our experiments using human lung tissue show increased expression levels of the adenosine A 2B receptor (ADORA2B) and a heightened deposition of hyaluronan (HA; a component of the ECM) in remodeled vessels of patients with PH associated with COPD. We also demonstrate that the expression of HA synthase 2 correlates with mean pulmonary arterial pressures in patients with COPD, with and without a secondary diagnosis of PH. Using an animal model of airspace enlargement and PH, we show that the blockade of ADORA2B is able to attenuate the development of a PH phenotype that correlates with reduced levels of HA deposition in the vessels and the down-regulation of genes involved in the synthesis of HA