Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease with progressive fibrosis and death within 2–3 y of diagnosis. IPF incidence and prevalence rates are increasing annually with few effective treatments available. Inhibition of IL-6 results in the attenuation of pulmonary fibrosis in mice. It is unclear whether this is due to blockade of classical signaling, mediated by membrane-bound IL-6Rα, or trans signaling, mediated by soluble IL-6Rα (sIL-6Rα). Our study assessed the role of sIL-6Rα in IPF. We demonstrated elevations of sIL-6Rα in IPF patients and in mice during the onset and progression of fibrosis. We demonstrated that protease-mediated cleavage from lung macrophages was important in production of sIL-6Rα. In vivo neutralization of sIL-6Rα attenuated pulmonary fibrosis in mice as seen by reductions in myofibroblasts, fibronectin, and collagen in the lung. In vitro activation of IL-6 trans signaling enhanced fibroblast proliferation and extracellular matrix protein production, effects relevant in the progression of pulmonary fibrosis. Taken together, these findings demonstrate that the production of sIL-6Rα from macrophages in the diseased lung contributes to IL-6 trans signaling that in turn influences events crucial in pulmonary fibrosis.

Lung fibrosis is the hallmark of the interstitial lung diseases. Alveolar epithelial cell (AEC) injury is akey step that contributes to a profibrotic microenvironment. Fibroblasts and myofibroblasts subsequently accumulate and deposit excessive extracellular matrix. In addition to TGF‐β, the IL‐6 family of cytokines, which signal through STAT‐3, may also contribute to lung fibrosis. In the current manuscript, the extent to which STAT‐3 inhibition decreases lung fibrosis is investigated. Phosphorylated STAT‐3 was elevated in lung biopsies from patients with idiopathic pulmonary fibrosis and bleomycin (BLM)‐induced fibrotic murine lungs. C‐188‐9, a small molecule STAT‐3 inhibitor, decreased pulmonary fibrosis in the intraperitoneal BLM model as assessed by arterial oxygen saturation (control, 84.4 ± 1.3%; C‐188‐9, 94.4 ± 0.8%), histology (Ashcroft score: untreated, 5.4 ± 0.25; C‐188‐9, 3.3 ± 0.14), and attenuated fibrotic markers such as diminished α‐smooth muscle actin, reduced collagen deposition. In addition, C‐188‐9 decreased the expression of epithelial injury markers, including hypoxia‐inducible factor‐1α (HIF‐1α) and plasminogen activator inhibitor‐1 (PAI‐1). In vitro studies show that inhibition of STAT‐3 decreased IL‐6‐ and TGF‐β‐induced expression of multiple genes, including HIF‐1α and PAI‐1, in AECs. Furthermore, C‐188‐9 decreased fibroblast‐to‐myofibroblast differentiation. Finally, TGF‐β stimulation of lung fibroblasts resulted in SMAD2/SMAD3‐dependent phosphorylation of STAT‐3. These findings demonstrate that STAT‐3 contributes to the development of lung fibrosis and suggest that STAT‐3 may be a therapeutic target in pulmonary fibrosis.—Pedroza, M., Le, T. T., Lewis, K., Karmouty‐Quintana, H., To, S., George, A. T., Blackburn, M. R., Tweardy, D. J., Agarwal, S. K. STAT‐3 contributes to pulmonary fibrosis through epithelial injury and fibroblast‐myofibroblast differentiation. FASEB J. 30, 129‐140 (2016). www.fasebj.org

Background: High altitude is a challenging condition caused by insufficient oxygen supply. Inability to adjust to hypoxia may lead to pulmonary edema, stroke, cardiovascular dysfunction, and even death. Thus, understanding the molecular basis of adaptation to high altitude may reveal novel therapeutics to counteract the detrimental consequences of hypoxia.Methods: Using high-throughput, unbiased metabolomic profiling, we report that the metabolic pathway responsible for production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O₂binding affinity, was significantly induced in 21 healthy humans within 2 hours of arrival at 5260 m and further increased after 16 days at 5260 m.Results: This finding led us to discover that plasma adenosine concentrations and soluble CD73 activity rapidly increased at high altitude and were associated with elevated erythrocyte 2,3-BPG levels and O₂releasing capacity. Mouse genetic studies demonstrated that elevated CD73 contributed to hypoxia-induced adenosine accumulation and that elevated adenosine-mediated erythrocyte A2B adenosine receptor activation was beneficial by inducing 2,3-BPG production and triggering O₂release to prevent multiple tissue hypoxia, inflammation, and pulmonary vascular leakage. Mechanistically, we demonstrated that erythrocyte AMP-activated protein kinase was activated in humans at high altitude and that AMP-activated protein kinase is a key protein functioning downstream of the A2B adenosine receptor, phosphorylating and activating BPG mutase and thus inducing 2,3-BPG production and O₂release from erythrocytes. Significantly, preclinical studies demonstrated that activation of AMP-activated protein kinase enhanced BPG mutase activation, 2,3-BPG production, and O₂release capacity in CD73-deficient mice, in erythrocyte-specific A2B adenosine receptor knockouts, and in wild-type mice and in turn reduced tissue hypoxia and inflammation.Conclusions: Together, human and mouse studies reveal novel mechanisms of hypoxia adaptation and potential therapeutic approaches for counteracting hypoxia-induced tissue damage.

Adenosine is an extracellular signaling molecule that is generated in response to cell injury where it orchestrates tissue protection and repair. Whereas adenosine is best known for promoting anti-inflammatory activities during acute injury responses, prolonged elevations can enhance destructive tissue remodeling processes associated with chronic disease states. The generation of adenosine and the subsequent activation of the adenosine 2B receptor (A_2BR) is an important processes in the regulation of both acute and chronic lung disease. The goal of this study was to examine the contribution of the A_2BR in models of bleomycin-induced lung injury that exhibit varying degrees of acute and chronic injury. Intratracheal bleomycin exposure results in substantial acute lung injury followed by progressive fibrosis. In this model, genetic removal of the A_2BR resulted in enhanced loss of barrier function and increased pulmonary inflammation, with few differences in indexes of pulmonary fibrosis. These results support an anti-inflammatory role for this receptor in this model of acute lung injury. In contrast, systemic exposure of mice to bleomycin resulted in modest acute lung injury together with progressive pulmonary fibrosis. In this model, the effects of A_2BR removal on acute lung injury were negligible; however, there were substantial reductions in pulmonary fibrosis, supporting a profibrotic role for this receptor. A_2BR-dependent regulation of IL-6 production was identified as a potential mechanism involved in the diminished pulmonary fibrosis seen in A_2BR knockout mice exposed to i.p. bleomycin. These studies highlight the distinct roles of A_2BR signaling during acute and chronic stages of lung injury.