Nebulized platelet-derived extracellular vesicles attenuate chronic cigarette smoke-induced murine emphysema.

Chronic obstructive pulmonary disease (COPD) is a prevalent lung disease usually resulting from cigarette smoking (CS). Cigarette smoking induces oxidative stress, which causes inflammation and alveolar epithelial cell apoptosis and represents a compelling therapeutic target for COPD. Purified human platelet-derived exosome product (PEP) is endowed with antioxidant enzymes and immunomodulatory molecules that mediate tissue repair. In this study, a murine model of CS-induced emphysema was used to determine whether nebulized PEP can influence the development of CS-induced emphysema through the mitigation of oxidative stress and inflammation in the lung. Nebulization of PEP effectively delivered the PEP vesicles into the alveolar region, with evidence of their uptake by type I and type II alveolar epithelial cells and macrophages. Lung function testing and morphometric assessment showed a significant attenuation of CS-induced emphysema in mice treated with nebulized PEP thrice weekly for 4 weeks. Whole lung immuno-oncology RNA sequencing analysis revealed that PEP suppressed several CS-induced cell injuries and inflammatory pathways. Validation of inflammatory cytokines and apoptotic protein expression on the lung tissue revealed that mice treated with PEP had significantly lower levels of S100A8/A9 expressing macrophages, higher levels of CD4+/FOXP3+ Treg cells, and reduced NF-κB activation, inflammatory cytokine production, and apoptotic proteins expression. Further validation using in vitro cell culture showed that pretreatment of alveolar epithelial cells with PEP significantly attenuated CS extract-induced apoptotic cell death. These data show that nebulization of exosomes like PEP can effectively deliver exosome cargo into the lung, mitigate CS-induced emphysema in mice, and suppress oxidative lung injury, inflammation, and apoptotic alveolar epithelial cell death.

Glutamine restores mitochondrial respiration in bleomycin-injured epithelial cells.

Whether from known or unknown causes, loss of epithelial repair plays a central role in the pathogenesis of pulmonary fibrosis. Recently, diminished mitochondrial function has been implicated as a factor contributing to the loss of epithelial repair but the mechanisms mediating these changes have not been defined. Here, we investigated the factors contributing to mitochondrial respiratory dysfunction after bleomycin, a widely accepted agent for modeling pulmonary fibrosis in mice and in vitro systems. In agreement with previous reports, we found that mitochondrial respiration was decreased in lung epithelial cells exposed to bleomycin, but also observed that responses differed depending on the type of metabolic fuel available to cells. For example, we found that mitochondrial respiration was dramatically reduced when glucose served as the primary fuel. Moreover, this associated with a marked decrease in glucose uptake, expression of glucose uptake transport 1 and capacity to augment glycolysis to either glucose or oligomycin. Conversely, mitochondrial respiration was largely preserved if glutamine was present in culture medium. The addition of glutamine also led to increased intracellular metabolite levels, including multiple TCA cycle intermediates and the glycolytic intermediate lactate, as well as reduced DNA damage and cell death to bleomycin. Taken together, these findings indicate that glutamine, rather than glucose, supports mitochondrial respiration and metabolite production in injured lung epithelial cells, and suggest that this shift away from glucose utilization serves to protect the lung epithelium from injury.

A model of the aged lung epithelium in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an age-related disorder that carries a universally poor prognosis and is thought to arise from repetitive micro injuries to the alveolar epithelium. To date, a major factor limiting our understanding of IPF is a deficiency of disease models, particularly in vitro models that can recapitulate the full complement of molecular attributes in the human condition. In this study, we aimed to develop a model that more closely resembles the aberrant IPF lung epithelium. By exposing mouse alveolar epithelial cells to repeated, low doses of bleomycin, instead of usual one-time exposures, we uncovered changes strikingly similar to those in the IPF lung epithelium. This included the acquisition of multiple phenotypic and functional characteristics of senescent cells and the adoption of previously described changes in mitochondrial homeostasis, including alterations in redox balance, energy production and activity of the mitochondrial unfolded protein response. We also uncovered dramatic changes in cellular metabolism and detected a profound loss of proteostasis, as characterized by the accumulation of cytoplasmic protein aggregates, dysregulated expression of chaperone proteins and decreased activity of the ubiquitin proteasome system. In summary, we describe an in vitro model that closely resembles the aberrant lung epithelium in IPF. We propose that this simple yet powerful tool could help uncover new biological mechanisms and assist in developing new pharmacological tools to treat the disease.

Alcohol-induced lipid dysregulation impairs glycolytic responses to LPS in alveolar macrophages.

Several conditions are marked by increased susceptibility to, and enhanced severity of, bacterial infections. Alcohol use disorder, one of these conditions, is known to predispose to bacterial pneumonia by suppressing the lung's innate immune system, and more specifically by disrupting critical alveolar macrophage (AM) functions. Recently, we established that chronic ethanol consumption also perturbs surfactant lipid homeostasis in the lung and that elevated concentrations of free fatty acids contribute to blocking essential AM functions, such as agonist-induced cytokine expression. In this study, we extend these observations by showing that elevated free fatty acid levels impair metabolic responses to lipopolysaccharide (LPS) in AMs. In particular, we show that the glycolytic reprogramming characteristic of LPS-stimulated AMs is blunted by the saturated fatty acid palmitate, whereas oleate, an unsaturated fatty acid, or ethanol alone, had no effect on this adaptive metabolic response. Additionally, we found that elevated concentrations of palmitate induced mitochondrial oxidative stress and that glycolytic reprogramming and cytokine production to LPS could be partially restored in AMs by either pharmacologically blocking palmitate entry into mitochondria or administering a mitochondrial-specific antioxidant. Taken together, these findings suggest that alcohol and elevated levels of saturated fatty acids conspire to impair pulmonary innate immunity by altering metabolic responses in AMs. Additionally, our findings suggest that targeting the mechanisms involved in fatty acid metabolism can restore pulmonary immunity and possibly limit bacterial pneumonia in individuals with alcohol use disorder.

Matrix metalloproteinase activity in the lung is increased in Hermansky-Pudlak syndrome.

BACKGROUND: Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized by oculocutaneous albinism and platelet dysfunction and can sometimes lead to a highly aggressive form of pulmonary fibrosis that mimics the fatal lung condition called idiopathic pulmonary fibrosis (IPF). Although the activities of various matrix metalloproteinases (MMPs) are known to be dysregulated in IPF, it remains to be determined whether similar changes in these enzymes can be detected in HPS. RESULTS: Here, we show that transcript and protein levels as well as enzymatic activities of MMP-2 and -9 are markedly increased in the lungs of mice carrying the HPS Ap3b1 gene mutation. Moreover, immunohistochemical staining localized this increase in MMP expression to the distal pulmonary epithelium, and shRNA knockdown of the Ap3b1 gene in cultured lung epithelial cells resulted in a similar upregulation in MMP-2 and -9 expression. Mechanistically, we found that upregulation in MMP expression associated with increased activity of the serine/threonine kinase Akt, and pharmacological inhibition of this enzyme resulted in a dramatic suppression of MMP expression in Ap3b1 deficient lung epithelial cells. Similarly, levels and activity of different MMPs were also found to be increased in the lungs of mice carrying the Bloc3 HPS gene mutation and in the bronchoalveolar lavage fluid of subjects with HPS. However, an association between MMP activity and disease severity was not detected in these individuals. CONCLUSIONS: In summary, our findings indicate that MMP activity is dysregulated in the HPS lung, suggesting a role for these proteases as biological markers or pathogenic players in HPS lung disease.

Metabolic Reprogramming as a Driver of Fibroblast Activation in PulmonaryFibrosis.

Pulmonary fibrosis refers to a heterogeneous group of disorders that scar the lung, most often irreversibly. To date, there are limited effective treatments for these conditions, despite decades of research in this area of investigation. In pulmonary fibrosis, the principle cell responsible for producing the vast majority of scar tissue is the fibroblast, making these cells ideally suited for drug targeting. For decades, the major experimental approach to blocking the activity of lung fibroblasts has been either to inhibit the interaction of fibroblast growth factors with their receptors or interfere with downstream effector molecules regulating extracellular matrix production. However, emerging evidence now indicates that lung fibroblasts also undergo dramatic metabolic reprogramming in the setting of growth factor stimulation. These discoveries, along with preclinical investigations showing marked reductions in lung fibrosis after targeting specific metabolic pathways, has led to a total rethinking of drug development in the pulmonary fibrosis field. Here, we review the major metabolic pathways and highlight some of the key metabolic events that occur in the transition of fibroblasts from quiescent to activated states. Moreover, we discuss the emerging evidence linking changes in fibroblast metabolism to pulmonary fibrosis and propose how targeting specific metabolic pathways could be employed in the treatment of fibrotic lung diseases.