Is there a role for specialized pro-resolving mediators in pulmonary fibrosis?

Pulmonary fibrotic diseases are characterized by proliferation of lung fibroblasts and myofibroblasts and excessive deposition of extracellular matrix proteins. Depending on the specific form of lung fibrosis, there can be progressive scarring of the lung, leading in some cases to respiratory failure and/or death. Recent and ongoing research has demonstrated that resolution of inflammation is an active process regulated by families of small bioactive lipid mediators termed "specialized pro-resolving mediators." While there are many reports of beneficial effects of SPMs in animal and cell culture models of acute and chronic inflammatory and immune diseases, there have been fewer reports investigating SPMs and fibrosis, especially pulmonary fibrosis. Here, we will review evidence that resolution pathways are impaired in interstitial lung disease, and that SPMs and other similar bioactive lipid mediators can inhibit fibroblast proliferation, myofibroblast differentiation, and accumulation of excess extracellular matrix in cell culture and animal models of pulmonary fibrosis, and we will consider future therapeutic implications of SPMs in fibrosis.

Exhaled breath condensate identifies metabolic dysregulation in patients with radiation-induced lung injury.

Radiation-induced lung injury (RILI) is a consequence of therapeutic thoracic irradiation (TR) for many cancers, and there are no FDA-approved curative strategies. Studies report that 80% of patients who undergo TR will have CT-detectable interstitial lung abnormalities, and strategies to limit the risk of RILI may make radiotherapy less effective at treating cancer. Our lab and others have reported that lung tissue from patients with idiopathic pulmonary fibrosis (IPF) exhibits metabolic defects including increased glycolysis and lactate production. In this pilot study, we hypothesized that patients with radiation-induced lung damage will exhibit distinct changes in lung metabolism that may be associated with the incidence of fibrosis. Using liquid chromatography/tandem mass spectrometry to identify metabolic compounds, we analyzed exhaled breath condensate (EBC) in subjects with CT-confirmed lung lesions after TR for lung cancer, compared with healthy subjects, smokers, and cancer patients who had not yet received TR. The lung metabolomic profile of the irradiated group was significantly different from the three nonirradiated control groups, highlighted by increased levels of lactate. Pathway enrichment analysis revealed that EBC from the case patients exhibited concurrent alterations in lipid, amino acid, and carbohydrate energy metabolism associated with the energy-producing tricarboxylic acid (TCA) cycle. Radiation-induced glycolysis and diversion of lactate to the extracellular space suggests that pyruvate, a precursor metabolite, converts to lactate rather than acetyl-CoA, which contributes to the TCA cycle. This TCA cycle deficiency may be compensated by these alternate energy sources to meet the metabolic demands of chronic wound repair. Using an "omics" approach to probe lung disease in a noninvasive manner could inform future mechanistic investigations and the development of novel therapeutic targets.NEW & NOTEWORTHY We report that exhaled breath condensate (EBC) identifies cellular metabolic dysregulation in patients with radiation-induced lung injury. In this pilot study, untargeted metabolomics revealed a striking metabolic signature in EBC from patients with radiation-induced lung fibrosis compared to patients with lung cancer, at-risk smokers, and healthy volunteers. Patients with radiation-induced fibrosis exhibit specific changes in tricarboxylic acid (TCA) cycle energy metabolism that may be required to support the increased energy demands of fibroproliferation.

Dung biomass smoke exposure impairs resolution of inflammatory responses to influenza infection.

Epidemiological studies associate biomass smoke with an increased risk for respiratory infections in children and adults in the developing world, with 500,000 premature deaths each year attributed to biomass smoke-related acute respiratory infections including infections caused by respiratory viruses. Animal dung is a biomass fuel of particular concern because it generates more toxic compounds per amount burned than wood, and is a fuel of last resort for the poorest households. Currently, there is little biological evidence on the effects of dung biomass smoke exposure on immune responses to respiratory viral infections. Here, we investigated the impact of dung biomass exposure on respiratory infection using a mouse model of dung biomass smoke and cultured primary human small airway epithelial cells (SAECs). Mice infected with influenza A virus (IAV) after dung biomass smoke exposure had increased mortality, lung inflammation and virus mRNA levels, and suppressed expression of innate anti-viral mediators compared to air exposed mice. Importantly, there was still significant tissue inflammation 14 days after infection in dung biomass smoke-exposed mice even after inflammation had resolved in air-exposed mice. Dung biomass smoke exposure also suppressed the production of anti-viral cytokines and interferons in cultured SAECs treated with poly(I:C) or IAV. This study shows that dung biomass smoke exposure impairs the immune response to respiratory viruses and contributes to biomass smoke-related susceptibility to respiratory viral infections, likely due to a failure to resolve the inflammatory effects of biomass smoke exposure.

Specialized pro-resolving mediators as modulators of immune responses.

Specialized pro-resolving mediators (SPMs) are endogenous small molecules produced mainly from dietary omega-3 polyunsaturated fatty acids by both structural cells and cells of the active and innate immune systems. Specialized pro-resolving mediators have been shown to both limit acute inflammation and promote resolution and return to homeostasis following infection or injury. There is growing evidence that chronic immune disorders are characterized by deficiencies in resolution and SPMs have significant potential as novel therapeutics to prevent and treat chronic inflammation and immune system disorders. This review focuses on important breakthroughs in understanding how SPMs are produced by, and act on, cells of the adaptive immune system, specifically macrophages, B cells and T cells. We also highlight recent evidence demonstrating the potential of SPMs as novel therapeutic agents in topics including immunization, autoimmune disease and transplantation.

Aryl hydrocarbon receptor deficiency causes the development of chronic obstructive pulmonary disease through the integration of multiple pathogenic mechanisms.

Emphysema, a component of chronic obstructive pulmonary disease (COPD), is characterized by irreversible alveolar destruction that results in a progressive decline in lung function. This alveolar destruction is caused by cigarette smoke, the most important risk factor for COPD. Only 15%-20% of smokers develop COPD, suggesting that unknown factors contribute to disease pathogenesis. We postulate that the aryl hydrocarbon receptor (AHR), a receptor/transcription factor highly expressed in the lungs, may be a new susceptibility factor whose expression protects against COPD. Here, we report that Ahr-deficient mice chronically exposed to cigarette smoke develop airspace enlargement concomitant with a decline in lung function. Chronic cigarette smoke exposure also increased cleaved caspase-3, lowered SOD2 expression, and altered MMP9 and TIMP-1 levels in Ahr-deficient mice. We also show that people with COPD have reduced expression of pulmonary and systemic AHR, with systemic AHR mRNA levels positively correlating with lung function. Systemic AHR was also lower in never-smokers with COPD. Thus, AHR expression protects against the development of COPD by controlling interrelated mechanisms involved in the pathogenesis of this disease. This study identifies the AHR as a new, central player in the homeostatic maintenance of lung health, providing a foundation for the AHR as a novel therapeutic target and/or predictive biomarker in chronic lung disease.

AT-RvD1 Mitigates Secondhand Smoke-Exacerbated Pulmonary Inflammation and Restores Secondhand Smoke-Suppressed Antibacterial Immunity.

Cigarette smoke is a potent proinflammatory trigger contributing to acute lung injury and the development of chronic lung diseases via mechanisms that include the impairment of inflammation resolution. We have previously demonstrated that secondhand smoke (SHS) exposure exacerbates bacterial infection-induced pulmonary inflammation and suppresses immune responses. It is now recognized that resolution of inflammation is a bioactive process mediated by lipid-derived specialized proresolving mediators that counterregulate proinflammatory signaling and promote resolution pathways. We therefore hypothesized that proresolving mediators could reduce the burden of inflammation due to chronic lung infection following SHS exposure and restore normal immune responses to respiratory pathogens. To address this question, we exposed mice to SHS followed by chronic infection with nontypeable Haemophilus influenzae (NTHI). Some groups of mice were treated with aspirin-triggered resolvin D1 (AT-RvD1) during the latter half of the smoke exposure period or during a period of smoking cessation and before infection. Treatment with AT-RvD1 markedly reduced the recruitment of neutrophils, macrophages, and T cells in lung tissue and bronchoalveolar lavage and levels of proinflammatory cytokines in the bronchoalveolar lavage. Additionally, treatment with AT-RvD1 improved Ab titers against the NTHI outer membrane lipoprotein Ag P6 following infection. Furthermore, treatment with AT-RvD1 prior to classically adjuvanted immunization with P6 increased Ag-specific Ab titers, resulting in rapid clearance of NTHI from the lungs after acute challenge. Collectively, we have demonstrated that AT-RvD1 potently reverses the detrimental effects of SHS on pulmonary inflammation and immunity and thus could be beneficial in reducing lung injury associated with smoke exposure and infection.

Mechanical Feed-Forward Loops Contribute to Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis is a progressive scarring disease characterized by extracellular matrix accumulation and altered mechanical properties of lung tissue. Recent studies support the hypothesis that these compositional and mechanical changes create a progressive feed-forward loop in which enhanced matrix deposition and tissue stiffening contribute to fibroblast and myofibroblast differentiation and activation, which further perpetuates matrix production and stiffening. The biomechanical properties of tissues are sensed and responded to by mechanotransduction pathways that facilitate sensing of changes in mechanical cues by tissue resident cells and convert the mechanical signals into downstream biochemical signals. Although our understanding of mechanotransduction pathways associated with pulmonary fibrosis remains incomplete, recent progress has allowed us to begin to elucidate the specific mechanisms supporting fibrotic feed-forward loops. The mechanosensors discussed here include integrins, Piezo channels, transient receptor potential channels, and nonselective ion channels. Also discussed are downstream transcription factors, including myocardin-related transcription factor and Yes-associated protein/transcriptional coactivator with PDZ-binding motif. This review describes mechanosensors and mechanotransduction pathways associated with fibrosis progression and highlights promising therapeutic insights.

Specialized Proresolving Mediators Overcome Immune Suppression Induced by Exposure to Secondhand Smoke.

Tobacco smoke exposure is associated with multiple diseases including, respiratory diseases like asthma and chronic obstructive pulmonary disease. Tobacco smoke is a potent inflammatory trigger and is immunosuppressive, contributing to increased susceptibility to pulmonary infections in smokers, ex-smokers, and vulnerable populations exposed to secondhand smoke. Tobacco smoke exposure also reduces vaccine efficacy. Therefore, mitigating the immunosuppressive effects of chronic smoke exposure and improving the efficacy of vaccinations in individuals exposed to tobacco smoke, is a critical unmet clinical problem. We hypothesized that specialized proresolving mediators (SPMs), a class of immune regulators promoting resolution of inflammation, without being immunosuppressive, and enhancing B cell Ab responses, could reverse the immunosuppressive effects resulting from tobacco smoke exposure. We exposed mice to secondhand smoke for 8 wk, followed by a period of smoke exposure cessation, and the mice were immunized with the P6 lipoprotein from nontypeable Haemophilus influenzae, using 17-HDHA and aspirin-triggered-resolvin D1 (AT-RvD1) as adjuvants. 17-HDHA and AT-RvD1 used as adjuvants resulted in elevated serum and bronchoalveolar lavage levels of anti-P6-specific IgG and IgA that were protective, with immunized mice exhibiting more rapid bacterial clearance upon challenge, reduced pulmonary immune cell infiltrates, reduced production of proinflammatory cytokines, and less lung-epithelial cell damage. Furthermore, the treatment of mice with AT-RvD1 during a period of smoke-cessation further enhanced the efficacy of SPM-adjuvanted P6 vaccination. Overall, SPMs show promise as novel vaccine adjuvants with the ability to overcome the tobacco smoke-induced immunosuppressive effects.

The self-fulfilling prophecy of pulmonary fibrosis: a selective inspection of pathological signalling loops.

Pulmonary fibrosis is a devastating, progressive disease and carries a prognosis worse than most cancers. Despite ongoing research, the mechanisms that underlie disease pathogenesis remain only partially understood. However, the self-perpetuating nature of pulmonary fibrosis has led several researchers to propose the existence of pathological signalling loops. According to this hypothesis, the normal wound-healing process becomes corrupted and results in the progressive accumulation of scar tissue in the lung. In addition, several negative regulators of pulmonary fibrosis are downregulated and, therefore, are no longer capable of inhibiting these feed-forward loops. The combination of pathological signalling loops and loss of a checks and balances system ultimately culminates in a process of unregulated scar formation. This review details specific signalling pathways demonstrated to play a role in the pathogenesis of pulmonary fibrosis. The evidence of detrimental signalling loops is elucidated with regard to epithelial cell injury, cellular senescence and the activation of developmental and ageing pathways. We demonstrate where these loops intersect each other, as well as common mediators that may drive these responses and how the loss of pro-resolving mediators may contribute to the propagation of disease. By focusing on the overlapping signalling mediators among the many pro-fibrotic pathways, it is our hope that the pulmonary fibrosis community will be better equipped to design future trials that incorporate the redundant nature of these pathways as we move towards finding a cure for this unrelenting disease.

Cigarette smoke increases susceptibility to infection in lung epithelial cells by upregulating caveolin-dependent endocytosis.

Cigarette smoke exposure is a risk factor for many pulmonary diseases, including Chronic Obstructive Pulmonary Disease (COPD). Cigarette smokers are more prone to respiratory infections with more severe symptoms. In those with COPD, viral infections can lead to acute exacerbations resulting in lung function decline and death. Epithelial cells in the lung are the first line of defense against inhaled insults such as tobacco smoke and are the target for many respiratory pathogens. Endocytosis is an essential cell function involved in nutrient uptake, cell signaling, and sensing of the extracellular environment, yet, the effect of cigarette smoke on epithelial cell endocytosis is not known. Here, we report for the first time that cigarette smoke alters the function of several important endocytic pathways in primary human small airway epithelial cells. Cigarette smoke exposure impairs clathrin-mediated endocytosis and fluid phase macropinocytosis while increasing caveolin mediated endocytosis. We also show that influenza virus uptake is enhanced by cigarette smoke exposure. These results support the concept that cigarette smoke-induced dysregulation of endocytosis contributes to lung infection in smokers. Targeting endocytosis pathways to restore normal epithelial cell function may be a new therapeutic approach to reduce respiratory infections in current and former smokers.

Advances in Comprehensive Exposure Assessment: Opportunities for the US Military.

OBJECTIVE: Review advances in exposure assessment offered by the exposome concept and new -omics and sensor technologies. METHODS: Narrative review of advances, including current efforts and potential future applications by the US military. RESULTS: Exposure assessment methods from both bottom-up and top-down exposomics approaches are advancing at a rapid pace, and the US military is engaged in developing both approaches. Top-down approaches employ various -omics technologies to identify biomarkers of internal exposure and biological effect. Bottom-up approaches use new sensor technology to better measure external dose. Key challenges of both approaches are largely centered around how to integrate, analyze, and interpret large datasets that are multidimensional and disparate. CONCLUSIONS: Advances in -omics and sensor technologies may dramatically enhance exposure assessment and improve our ability to characterize health risks related to occupational and environmental exposures, including for the US military.

Associations of Benzo(ghi)perylene and Heptachlorodibenzo-p-dioxin in Serum of Service Personnel Deployed to Balad, Iraq, and Bagram, Afghanistan Correlates With Perturbed Amino Acid Metabolism in Human Lung Fibroblasts.

OBJECTIVE: A study was conducted to identify metabolic-related effects of benzo(ghi)perylene (BghiP) and 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD), on primary human fibroblasts to verify biological associations previously found in occupational health research. METHODS: Human lung fibroblasts were exposed to BghiP or HpCDD and extracts were analyzed with a metabolome-wide association study to test for pathways and metabolites altered relative to controls. Gene expression was measured by quantitative-real time polymerase chain reaction. RESULTS: Metabolic perturbations in amino-acid, oxidative stress, and fatty-acid pathways were observed for BghiP and HpCDD. HpCDD but not BghiP exposure increased gene expression of the amino acid transporters SLC7A5 and SLC7A11. CONCLUSIONS: Exposure to polycyclic aromatic hydrocarbons (PAH) or dioxins perturbs amino acid pathways at physiologically relevant concentrations with different mechanisms. These findings imply an effect on central homeostatic systems by environmental exposures which could have implications on disease susceptibility.

Analysis of Postdeployment Serum Samples Identifies Potential Biomarkers of Exposure to Burn Pits and Other Environmental Hazards.

OBJECTIVE: The potential health risks of deployment to sites with open burn pits remain poorly understood, in part, because personal exposure monitoring was not performed. Here, we investigated whether postdeployment serum samples contain biomarkers associated with exposure to burn pits. METHODS: A total of 237 biomarkers were measured in 800 serum samples from deployed and never-deployed subjects. We used a regression model and a supervised vector machine to identify serum biomarkers with significant associations with exposures and deployment. RESULTS: We identified 101 serum biomarkers associated with polycyclic aromatic hydrocarbons, dioxins or furans, and 54 biomarkers associated with deployment. Twenty-six of these biomarkers were shared in common by the exposure and deployment groups. CONCLUSIONS: We identify a potential signature of exposure to open burn pits, and provide a framework for using postexposure sera to identify exposures when contemporaneous monitoring was inadequate.

Machine Learning Approach for Predicting Past Environmental Exposures From Molecular Profiling of Post-Exposure Human Serum Samples.

OBJECTIVE: To develop an approach for a retrospective analysis of post-exposure serum samples using diverse molecular profiles. METHODS: The 236 molecular profiles from 800 de-identified human serum samples from the Department of Defense Serum Repository were classified as smokers or non-smokers based on direct measurement of serum cotinine levels. A machine-learning pipeline was used to classify smokers and non-smokers from their molecular profiles. RESULTS: The refined supervised support vector machines with recursive feature elimination predicted smokers and non-smokers with 78% accuracy on the independent held-out set. Several of the identified classifiers of smoking status have previously been reported and four additional miRNAs were validated with experimental tobacco smoke exposure in mice, supporting the computational approach. CONCLUSIONS: We developed and validated a pipeline that shows retrospective analysis of post-exposure serum samples can identify environmental exposures.

Integrative Network Analysis Linking Clinical Outcomes With Environmental Exposures and Molecular Variations in Service Personnel Deployed to Balad and Bagram.

OBJECTIVE: To develop a computational approach to link clinical outcomes with environmental exposures and molecular variations measured in Department of Defense (DOD) serum-repository samples. METHODS: International Classification of Diseases, Ninth Division codes which corresponded to cardiopulmonary symptoms for service personnel were selected to test for associations with deployment-related inhalation hazards and metabolomics, micro-RNA, cytokine, plasma markers, and environmental exposure analyses for corresponding samples. xMWAS and Mummichog were used for integrative network and pathway analysis. RESULTS: Comparison between 41 personnel exhibiting new cardio-pulmonary diagnoses after deployment start-date to 25 personnel exhibiting no symptoms identified biomarkers associated with cardiopulmonary conditions. Integrative network and pathway analysis showed communities of clinical, molecular, and environmental markers associated with fatty acid, lipid, nucleotide, and amino acid metabolism pathways. CONCLUSIONS: The current proof of principle study establishes a computational framework for integrative analysis of deployment-related exposures, molecular responses, and health outcomes.

Exposure to Heptachlorodibenzo-p-dioxin (HpCDD) Regulates microRNA Expression in Human Lung Fibroblasts.

OBJECTIVE: Benzo(ghi)perylene (BghiP) and 1,2,3,4,6,7,8-Heptachlorodibenzo-p-dioxin (HpCDD) were elevated in serum from personnel deployed to sites with open burn pits. Here, we investigated the ability of BghiP and HpCDD to regulate microRNA (miRNA) expression through the aryl hydrocarbon receptor (AHR). METHODS: Human lung fibroblasts (HLFs) were exposed to BghiP and HpCDD. AHR activity was measured by reporter assay and gene expression. Deployment related miRNA were measured by quantitative polymerase chain reaction. AHR expression was depleted using siRNA. RESULTS: BghiP displayed weak AHR agonist activity. HpCDD induced AHR activity in a dose-dependent manner. Let-7d-5p, miR-103-3p, miR-107, and miR-144-3p levels were significantly altered by HpCDD. AHR knockdown attenuated these effects. CONCLUSIONS: These studies reveal that miRNAs previously identified in sera from personnel deployed to sites with open burn pits are altered by HpCDD exposure in HLFs.

Quenching the fires: Pro-resolving mediators, air pollution, and smoking.

Exposure to air pollution and other environmental inhalation hazards, such as occupational exposures to dusts and fumes, aeroallergens, and tobacco smoke, is a significant cause of chronic lung inflammation leading to respiratory disease. It is now recognized that resolution of inflammation is an active process controlled by a novel family of small lipid mediators termed "specialized pro-resolving mediators" or SPMs, derived mainly from dietary omega-3 polyunsaturated fatty acids. Chronic inflammation results from an imbalance between pro-inflammatory and pro-resolution pathways. Research is ongoing to develop SPMs, and the pro-resolution pathway more generally, as a novel therapeutic approach to diseases characterized by chronic inflammation. Here, we will review evidence that the resolution pathway is dysregulated in chronic lung inflammatory diseases, and that SPMs and related molecules have exciting therapeutic potential to reverse or prevent chronic lung inflammation, with a focus on lung inflammation due to inhalation of environmental hazards including urban particulate matter, organic dusts and tobacco smoke.

Activated Human Lung Fibroblasts Produce Extracellular Vesicles with Antifibrotic Prostaglandins.

The differentiation of interstitial lung fibroblasts into contractile myofibroblasts that proliferate and secrete excessive extracellular matrix is critical for the pathogenesis of pulmonary fibrosis. Certain lipid signaling molecules, such as prostaglandins (PGs), can inhibit myofibroblast differentiation. However, the sources and delivery mechanisms of endogenous PGs are undefined. Activated primary human lung fibroblasts (HLFs) produce PGs such as PGE2. We report that activation of primary HLFs with IL-1ß inhibited transforming growth factor ß-induced myofibroblast differentiation in both the IL-1ß-treated cells themselves (autocrine signal) and adjacent naive HLFs in cocultures (paracrine signal). Additionally, we demonstrate for the first time that at least some of the antifibrotic effect of activated fibroblasts on nearby naive fibroblasts is carried by exosomes and other extracellular vesicles that contain several PGs, including high levels of the antifibrotic PGE2. Thus, activated fibroblasts communicate with surrounding cells to limit myofibroblast differentiation and maintain homeostasis. This work opens the way for future research into extracellular vesicle-mediated intercellular signaling in the lung and may inform the development of novel therapies for fibrotic lung diseases.

Lipoxin B4 Enhances Human Memory B Cell Antibody Production via Upregulating Cyclooxygenase-2 Expression.

Vaccination has been the most effective way to prevent or reduce infectious diseases; examples include the eradication of smallpox and attenuation of tetanus and measles. However, there is a large segment of the population that responds poorly to vaccines, in part because they are immunocompromised because of disease, age, or pharmacologic therapy and are unable to generate long-term protection. Specialized proresolving mediators are endogenously produced lipids that have potent proresolving and anti-inflammatory activities. Lipoxin B4 (LXB4) is a member of the lipoxin family, with its proresolving effects shown in allergic airway inflammation. However, its effects on the adaptive immune system, especially on human B cells, are not known. In this study, we investigated the effects of LXB4 on human B cells using cells from healthy donors and donors vaccinated against influenza virus in vitro. LXB4 promoted IgG Ab production in memory B cells and also increased the number of IgG-secreting B cells. LXB4 enhanced expression of two key transcription factors involved in plasma cell differentiation, BLIMP1 and XBP1. Interestingly, LXB4 increased expression of cyclooxygenase-2 (COX2), an enzyme that is required for efficient B cell Ab production. The effects of LXB4 are at least partially COX2-dependent as COX2 inhibitors attenuated LXB4-stimulated BLIMP1 and Xpb-1 expression as well as IgG production. Thus, our study reveals for the first time, to our knowledge, that LXB4 boosts memory B cell activation through COX2 and suggests that LXB4 can serve as a new vaccine adjuvant.