Better together: Alveolar macrophage and memory T cell cosignaling in ex vivo human lungs

Ex vivo lung perfusion enables assessment of the resident adaptive immune system and provides insight into the lung response to pathogen exposure.

Pathological features of COVID-19-associated lung injury: a preliminary proteomics report based on clinical samples

The COVID-19 pandemic has emerged as a global health emergency due to its association with severe pneumonia and relative high mortality. However, the molecular characteristics and pathological features underlying COVID-19 pneumonia remain largely unknown. To characterize molecular mechanisms underlying COVID-19 pathogenesis in the lung tissue using a proteomic approach, fresh lung tissues were obtained from newly deceased patients with COVID-19 pneumonia. After virus inactivation, a quantitative proteomic approach combined with bioinformatics analysis was used to detect proteomic changes in the SARS-CoV-2-infected lung tissues. We identified significant differentially expressed proteins involved in a variety of fundamental biological processes including cellular metabolism, blood coagulation, immune response, angiogenesis, and cell microenvironment regulation. Several inflammatory factors were upregulated, which was possibly caused by the activation of NF-κB signaling. Extensive dysregulation of the lung proteome in response to SARS-CoV-2 infection was discovered. Our results systematically outlined the molecular pathological features in terms of the lung response to SARS-CoV-2 infection, and provided the scientific basis for the therapeutic target that is urgently needed to control the COVID-19 pandemic.

Tobacco Smoke Exposure Exacerbated Crystalline Silica-Induced Lung Toxicity in Rats

Smoking may modify the lung response to silica exposure including cancer and silicosis. Nevertheless, the precise role of exposure to tobacco smoke (TS) on the lung response to crystalline silica (CS) exposure and the underlying mechanisms need further clarification. The objectives of the present study were to determine the role of TS on lung response to CS exposure and the underlying mechanism(s). Male Fischer 344 rats were exposed by inhalation to air, CS (15 mg/m3, 6 h/day, 5 days), TS (80 mg/m3, 3 h/day, twice weekly, 6 months), or CS (15 mg/m3, 6 h/day, 5 days) followed by TS (80 mg/m3, 3 h/day, twice weekly, 6 months). The rats were euthanized 6 months and 3 weeks following initiation of the first exposure and the lung response was assessed. Silica exposure resulted in significant lung toxicity as evidenced by lung histological changes, enhanced neutrophil infiltration, increased lactate dehydrogenase levels, enhanced oxidant production, and increased cytokine levels. The TS exposure alone had only a minimal effect on these toxicity parameters. However, the combined exposure to TS and CS exacerbated the lung response, compared with TS or CS exposure alone. Global gene expression changes in the lungs correlated with the lung toxicity severity. Bioinformatic analysis of the gene expression data demonstrated significant enrichment in functions, pathways, and networks relevant to the response to CS exposure which correlated with the lung toxicity detected. Collectively our data demonstrated an exacerbation of CS-induced lung toxicity by TS exposure and the molecular mechanisms underlying the exacerbated toxicity.

Quantification of regional murine ozone-induced lung inflammation using [18F]F-FDG microPET/CT imaging

Ozone (O3) is a highly potent and reactive air pollutant. It has been linked to acute and chronic respiratory diseases in humans by inducing inflammation. Our studies have found evidence that 0.05 ppm of O3, within the threshold of air quality standards, is capable of inducing acute lung injury. This study was undertaken to examine O3-induced lung damage using [18F]F-FDG (2-deoxy-2-[18F]fluoro-D-glucose) microPET/CT in wild-type mice. [18F]F-FDG is a known PET tracer for inflammation. Sequential [18F]F-FDG microPET/CT was performed at baseline (i.e. before O3 exposure), immediately (0 h), at 24 h and at 28 h following 2 h of 0.05 ppm O3 exposure. The images were quantified to determine O3 induced spatial standard uptake ratio of [18F]F-FDG in relation to lung tissue density and compared with baseline values. Immediately after O3 exposure, we detected a 72.21 ± 0.79% increase in lung [18F]F-FDG uptake ratio when compared to baseline measures. At 24 h post-O3 exposure, the [18F]F-FDG uptake becomes highly variable (S.D. in [18F]F-FDG = 5.174 × 10–4 units) with a 42.54 ± 0.33% increase in lung [18F]F-FDG compared to baseline. At 28 h time-point, [18F]F-FDG uptake ratio was similar to baseline values. However, the pattern of [18F]F-FDG distribution varied and was interspersed with zones of minimal uptake. Our microPET/CT imaging protocol can quantify and identify atypical regional lung uptake of [18F]F-FDG to understand the lung response to O3 exposure.

The endogenous capacity to produce proinflammatory mediators by the ex vivo human perfused lung

Background The ex vivo human perfused lung model has enabled optimizing donor lungs for transplantation and delineating mechanisms of lung injury. Perfusate and airspace biomarkers are a proxy of the lung response to experimental conditions. However, there is a lack of studies evaluating biomarker kinetics during perfusion and after exposure to stimuli. In this study, we analyzed the ex vivo-perfused lung response to three key perturbations: exposure to the perfusion circuit, exogenous fresh whole blood, and bacteria. Results Ninety-nine lungs rejected for transplantation underwent ex vivo perfusion. One hour after reaching experimental conditions, fresh whole blood was added to the perfusate (n = 55). Two hours after reaching target temperature, Streptococcus pneumoniae was added to the perfusate (n = 42) or to the airspaces (n = 17). Perfusate and airspace samples were collected at baseline (once lungs were equilibrated for 1 h, but before blood or bacteria were added) and 4 h later. Interleukin (IL)-6, IL-8, angiopoietin (Ang)-2, and soluble tumor necrosis factor receptor (sTNFR)-1 were quantified. Baseline perfusate and airspace biomarker levels varied significantly, and this was not related to pre-procurement PaO2:FiO2 ratio, cold ischemia time, and baseline alveolar fluid clearance (AFC). After 4 h of ex vivo perfusion, the lung demonstrated a sustained production of proinflammatory mediators. The change in biomarker levels was not influenced by baseline donor lung characteristics (cold ischemia time, baseline AFC) nor was it associated with measures of experimental epithelial (final AFC) or endothelial (percent weight gain) injury. In the presence of exogenous blood, the rise in biomarkers was attenuated. Lungs exposed to intravenous (IV) bacteria relative to control lungs demonstrated a significantly higher rise in perfusate IL-6. Conclusions The ex vivo-perfused lung has a marked endogenous capacity to produce inflammatory mediators over the course of short-term perfusion that is not significantly influenced by donor lung characteristics or the presence of exogenous blood, and only minimally affected by the introduction of systemic bacteremia. The lack of association between biomarker change and donor lung cold ischemia time, final alveolar fluid clearance, and experimental percent weight gain suggests that the maintained ability of the human lung to produce biomarkers is not merely a marker of lung epithelial or endothelial injury, but may support the function of the lung as an immune cell reservoir.

The Endogenous Capacity to Produce Proinflammatory Mediators by the Ex Vivo Perfused Human Lung

Background: The ex vivo human perfused lung model has enabled optimizing donor lungs for transplantation and delineating mechanisms of lung injury. Perfusate and airspace biomarkers are a proxy of the lung response to experimental conditions. However, there is a lack of studies evaluating biomarker kinetics during perfusion and after exposure to stimuli. In this study we analyzed the ex vivo perfused lung response to three key perturbations: exposure to the perfusion circuit, exogenous fresh whole blood, and bacteria.Results: 99 lungs rejected for transplantation underwent ex vivo perfusion. One hour after reaching experimental conditions, fresh whole blood was added to the perfusate (n=55). Two hours after reaching target temperature, Streptococcus pneumoniae was added to the perfusate (n=42) or to the airspaces (n=17). Perfusate and airspace samples were collected at baseline (once lungs were equilibrated for 1 hour, but before blood or bacteria were added) and 4 hours later. Interleukin (IL)-6, IL-8, Angiopoietin (Ang)-2, and soluble tumor necrosis factor receptor (sTNFR)-1 were quantified. Baseline perfusate and airspace biomarker levels varied significantly, and this was not related to pre-procurement PaO2:FiO2 ratio, cold ischemia time, and baseline alveolar fluid clearance (AFC). After 4 hours of ex vivo perfusion, the lung demonstrated a sustained production of proinflammatory mediators. The change in biomarker levels was not influenced by baseline donor lung characteristics (cold ischemia time, baseline AFC) nor was it associated with measures of experimental epithelial (final AFC) or endothelial (percent weight gain) injury. In the presence of exogenous blood, the rise in biomarkers was attenuated. Lungs exposed to intravenous (IV) bacteria relative to control lungs demonstrated a significantly higher rise in perfusate IL-6.Conclusions: The ex vivo perfused lung has a marked endogenous capacity to produce inflammatory mediators over the course of short-term perfusion that is not significantly influenced by donor lung characteristics or the presence of exogenous blood, and only minimally affected by the introduction of systemic bacteremia. The lack of association between biomarker change and donor lung cold ischemia time, final alveolar fluid clearance, and experimental percent weight gain suggest that the maintained ability of the human lung to produce biomarkers is not merely a marker of lung epithelial or endothelial injury, but may support the function of the lung as an immune cell reservoir.

Interaction of Endogenous Proinflammatory Mediators on the Function of the Ex Vivo Perfused Human lung

Background The ex vivo human perfused lung model has enabled optimizing donor lungs for transplantation and delineating mechanisms of lung injury. Perfusate and airspace biomarkers are a proxy of the lung response to experimental conditions. However, there is a lack of studies evaluating biomarker kinetics during perfusion and after exposure to stimuli. In this study we analyzed the ex vivo perfused lung response to three key perturbations: exposure to the perfusion circuit, exogenous fresh whole blood, and bacteria. Results 99 lungs rejected for transplantation underwent ex vivo perfusion. One hour after reaching experimental conditions, fresh whole blood was added to the perfusate (n=55). Two hours after reaching target temperature, Streptococcus pneumoniae was added to the perfusate (n=42) or to the airspaces (n=17). Perfusate and airspace samples were collected at baseline (once lungs were equilibrated for 1 hour, but before blood or bacteria were added) and 4 hours later. Interleukin (IL)-6, IL-8, Angiopoietin (Ang)-2, and soluble tumor necrosis factor receptor (sTNFR)-1 were quantified. Baseline perfusate and airspace biomarker levels varied significantly, and this was not related to pre-procurement PaO2:FiO2 ratio, cold ischemia time, and baseline alveolar fluid clearance (AFC). After 4 hours of ex vivo perfusion, the lung demonstrated a sustained production of proinflammatory mediators. The change in biomarker levels was not influenced by baseline donor lung characteristics (cold ischemia time, baseline AFC) nor was it associated with measures of experimental epithelial (final AFC) or endothelial (percent weight gain) injury. In the presence of exogenous blood, the rise in biomarkers was attenuated. Lungs exposed to intravenous (IV) bacteria relative to control lungs demonstrated a significantly higher rise in perfusate IL-6. Conclusions The ex vivo perfused lung has a marked endogenous capacity to generate inflammatory responses over the course of short-term perfusion. The lack of association between biomarker change and donor lung cold ischemia time as well as final alveolar fluid clearance and experimental percent weight gain suggests that the maintained ability to produce biomarkers is not merely a marker of lung epithelial or endothelial injury but may support the lung’s role as an immune cell reservoir.

Quantification of Regional Murine Ozone-Induced Lung Inflammation Using [18F]FDG MicroPET/CT Imaging

Purpose Ozone (O3) is a highly potent and reactive air pollutant, which is formed from human-related activities. It has been linked to acute and chronic respiratory diseases in humans by inducing inflammation. Our studies have found evidence that 0.05 ppm of O3, within the threshold of air quality standards, is capable of inducing acute lung injury. This study was undertaken to examine O3-induced lung damage using [18F]FDG (Fluorodeoxyglucose) microPET/CT imaging in wild-type mice. [18F]FDG is a known PET tracer for inflammation.Methods Sequential [18F]FDG microPET/CT imaging was performed at baseline (before O3 exposure), immediately following 2 h of 0.05 ppm O3 exposure (0 h), at 24 h and at 28 h post-exposure. The images were quantified to determine spatial standard uptake values of [18F]FDG in relation to tissue density and compared with baseline values.Results Immediately after O3 exposure, we detected a 72.21 ± 0.79% increase in lung [18F]FDG uptake ratio when compared to baseline measures. At 24 h post-O3 exposure, the [18F]FDG uptake becomes highly variable (S.D. in FDG = 0.0005174 units) with a 42.54 ± 0.33% increase in lung [18F]FDG compared to baseline.Conclusion We have developed a microPET/CT imaging protocol to quantify regional lung uptake of [18F]FDG to understand lung response to O3 exposure.