Extracellular vesicle miRNAs drive aberrant macrophage responses in NSAID-exacerbated respiratory disease.

BACKGROUND: Extracellular vesicles (EVs) have been implicated in the pathogenesis of asthma, however, how EVs contribute to immune dysfunction and type 2 airway inflammation remains incompletely understood. We aimed to elucidate roles of airway EVs and their miRNA cargo in the pathogenesis of NSAID-exacerbated respiratory disease (N-ERD), a severe type 2 inflammatory condition. METHODS: EVs were isolated from induced sputum or supernatants of cultured nasal polyp or turbinate tissues of N-ERD patients or healthy controls by size-exclusion chromatography and characterized by particle tracking, electron microscopy and miRNA sequencing. Functional effects of EV miRNAs on gene expression and mediator release by human macrophages or normal human bronchial epithelial cells (NHBEs) were studied by RNA sequencing, LC-MS/MS and multiplex cytokine assays. RESULTS: EVs were highly abundant in secretions from the upper and lower airways of N-ERD patients. N-ERD airway EVs displayed profoundly altered immunostimulatory capacities and miRNA profiles compared to airway EVs of healthy individuals. Airway EVs of N-ERD patients, but not of healthy individuals induced inflammatory cytokine (GM-CSF and IL-8) production by NHBEs. In macrophages, N-ERD airway EVs exhibited an impaired potential to induce cytokine and prostanoid production, while enhancing M2 macrophage activation. Let-7 family miRNAs were highly enriched in sputum EVs from N-ERD patients and mimicked suppressive effects of N-ERD EVs on macrophage activation. CONCLUSION: Aberrant airway EV miRNA profiles may contribute to immune dysfunction and chronic type 2 inflammation in N-ERD. Let-7 family miRNAs represent targets for correcting aberrant macrophage activation and mediator responses in N-ERD.

Inflammatory macrophage memory in nonsteroidal anti-inflammatory drug-exacerbated respiratory disease.

BACKGROUND: Nonsteroidal anti-inflammatory drug-exacerbated respiratory disease (N-ERD) is a chronic inflammatory condition, which is driven by an aberrant arachidonic acid metabolism. Macrophages are major producers of arachidonic acid metabolites and subject to metabolic reprogramming, but they have been neglected in N-ERD. OBJECTIVE: This study sought to elucidate a potential metabolic and epigenetic macrophage reprogramming in N-ERD. METHODS: Transcriptional, metabolic, and lipid mediator profiles in macrophages from patients with N-ERD and healthy controls were assessed by RNA sequencing, Seahorse assays, and LC-MS/MS. Metabolites in nasal lining fluid, sputum, and plasma from patients with N-ERD (n = 15) and healthy individuals (n = 10) were quantified by targeted metabolomics analyses. Genome-wide methylomics were deployed to define epigenetic mechanisms of macrophage reprogramming in N-ERD. RESULTS: This study shows that N-ERD monocytes/macrophages exhibit an overall reduction in DNA methylation, aberrant metabolic profiles, and an increased expression of chemokines, indicative of a persistent proinflammatory activation. Differentially methylated regions in N-ERD macrophages included genes involved in chemokine signaling and acylcarnitine metabolism. Acylcarnitines were increased in macrophages, sputum, nasal lining fluid, and plasma of patients with N-ERD. On inflammatory challenge, N-ERD macrophages produced increased levels of acylcarnitines, proinflammatory arachidonic acid metabolites, cytokines, and chemokines as compared to healthy macrophages. CONCLUSIONS: Together, these findings decipher a proinflammatory metabolic and epigenetic reprogramming of macrophages in N-ERD.

Functional impairment of microglia coincides with Beta-amyloid deposition in mice with Alzheimer-like pathology.

Microglial cells closely interact with senile plaques in Alzheimer's disease and acquire the morphological appearance of an activated phenotype. The significance of this microglial phenotype and the impact of microglia for disease progression have remained controversial. To uncover and characterize putative changes in the functionality of microglia during Alzheimer's disease, we directly assessed microglial behavior in two mouse models of Alzheimer's disease. Using in vivo two-photon microscopy and acute brain slice preparations, we found that important microglial functions - directed process motility and phagocytic activity - were strongly impaired in mice with Alzheimer's disease-like pathology compared to age-matched non-transgenic animals. Notably, impairment of microglial function temporally and spatially correlated with Aß plaque deposition, and phagocytic capacity of microglia could be restored by interventionally decreasing amyloid burden by Aß vaccination. These data suggest that major microglial functions progressively decline in Alzheimer's disease with the appearance of Aß plaques, and that this functional impairment is reversible by lowering Aß burden, e.g. by means of Aß vaccination.

Altered GLUT4 trafficking in adipocytes in the absence of the GTPase Arfrp1.

The GTPase ADP-ribosylation factor related protein 1 (ARFRP1) controls the recruitment of proteins such as golgin-245 to the trans-Golgi. ARFRP1 is highly expressed in adipose tissues in which the insulin-sensitive glucose transporter GLUT4 is processed through the Golgi to a specialized endosomal compartment, the insulin-responsive storage compartment from which it is translocated to the plasma membrane in response to a stimulation of cells by insulin. In order to examine the role of ARFRP1 for GLUT4 targeting, subcellular distribution of GLUT4 was investigated in adipose tissue specific Arfrp1 knockout (Arfrp1(ad)(-/-)) mice. Immunohistochemical and ultrastructural studies of brown adipocytes demonstrated an abnormal trans-Golgi in Arfrp1(ad)(-/-) adipocytes. In addition, in Arfrp1(ad)(-/-) adipocytes GLUT4 protein accumulated at the plasma membrane rather than being sequestered in an intracellular compartment. A similar missorting of GLUT4 was produced by siRNA-mediated knockdown of Arfrp1 in 3T3-L1 adipocytes which was associated with significantly elevated uptake of deoxyglucose under basal conditions. Thus, Arfrp1 appears to be involved in sorting of GLUT4.

A dual role of the N-terminal FQQI motif in GLUT4 trafficking.

In adipocytes, the glucose transporter GLUT4 recycles between intracellular storage vesicles and the plasma membrane. GLUT4 is internalized by a clathrin- and dynamin-dependent mechanism, and sorted into an insulin-sensitive storage compartment. Insulin stimulation leads to GLUT4 accumulation on the cell surface. The N-terminal F5QQI motif in GLUT4 has been shown previously to be required for sorting of the protein in the basal state. Here, we show that the FQQI motif is a binding site for the medium chain adaptin micro1, a subunit of the AP-1 adaptor complex that plays a role in post-Golgi/endosomal trafficking events. In order to investigate the role of AP-1 and AP-2 in GLUT4 trafficking, we generated 3T3-L1 adipocytes expressing HA-GLUT4-GFP and knocked down the AP-1 and AP-2 complex by RNAi, respectively. In AP-1 and AP-2 knockdown adipocytes, GLUT4 accumulates at the cell surface in the basal state, consistent with a role of AP-1 in post-endosomal sorting of GLUT4 to the insulin-sensitive storage compartment, and of AP-2 in clathrin-mediated endocytosis. Our data demonstrate a dual role of the F5QQI motif and support the conclusion that the AP complexes direct GLUT4 trafficking and endocytosis.

Tbc1d1 mutation in lean mouse strain confers leanness and protects from diet-induced obesity.

We previously identified Nob1 as a quantitative trait locus for high-fat diet-induced obesity and diabetes in genome-wide scans of outcross populations of obese and lean mouse strains. Additional crossbreeding experiments indicated that Nob1 represents an obesity suppressor from the lean Swiss Jim Lambert (SJL) strain. Here we identify a SJL-specific mutation in the Tbc1d1 gene that results in a truncated protein lacking the TBC Rab-GTPase-activating protein domain. TBC1D1, which has been recently linked to human obesity, is related to the insulin signaling protein AS160 and is predominantly expressed in skeletal muscle. Knockdown of TBC1D1 in skeletal muscle cells increased fatty acid uptake and oxidation, whereas overexpression of TBC1D1 had the opposite effect. Recombinant congenic mice lacking TBC1D1 showed reduced body weight, decreased respiratory quotient, increased fatty acid oxidation and reduced glucose uptake in isolated skeletal muscle. Our data strongly suggest that mutation of Tbc1d1 suppresses high-fat diet-induced obesity by increasing lipid use in skeletal muscle.

Portable flat-panel detector for low-dose imaging in a pediatric intensive care unit: comparison with an asymmetric film-screen system.

OBJECTIVE: We sought to evaluate the diagnostic performance of a portable indirect flat-panel detector for low-dose imaging as compared with an asymmetric film-screen system in a pediatric intensive care unit. MATERIALS AND METHODS: A total of 120 neonates underwent chest radiographs using a portable flat-panel detector (digital speed 800) and an asymmetric film-screen system (400 speed). Four readers evaluated the detection of 11 anatomic and 5 pathologic landmarks and 4 support devices. Statistical analysis was performed using repeated analysis of variance. The level of statistical significance was P = 0.05. RESULTS: The detection of 4 anatomic/4 pathologic landmarks and 2 support devices was significantly better using the flat-panel detector as compared with the asymmetric film-screen system (P < 0.05). Another 8 anatomic and one pathologic landmarks were detected equally well or slightly better with the flat-panel detector (P > 0.05). CONCLUSIONS: The portable flat-panel detector offers the potential of a 50% dose reduction with equal or significantly better detection of clinically important structures.

Experimental evaluation of a portable indirect flat-panel detector for the pediatric chest: comparison with storage phosphor radiography at different exposures by using a chest phantom.

PURPOSE: To compare the exposure dose requirements and performance of a portable indirect flat-panel detector for pediatric use in the depiction of catheters, simulated pulmonary nodules, and simulated interstitial lung disease with those of storage phosphor radiography. MATERIALS AND METHODS: Catheters and simulated nodules and subtle interstitial lung disease (miliary, reticular, linear, and ground-glass patterns) were superimposed over an anthropomorphic chest phantom. Images were obtained with different exposures corresponding to simulated speeds of 400 and 800 with a portable flat-panel detector and printed on hard copies. These images were compared with those from storage phosphor radiography at a simulated speed of 400, which is typically used in pediatric radiology. Four independent readers recorded 7200 observations per pattern (for a total of 600 statistically independent observations), and these observations were subjected to receiver operating characteristic (ROC) analysis. Differences were considered significant at a P value of .05. RESULTS: Catheters over obscured chest areas, nodules 10 mm or smaller and larger than 10 mm over lucent lung, nodules 10 mm or smaller over obscured chest areas, and miliary and linear patterns over lucent lung showed higher areas under the ROC curve (A(z)) with the flat-panel detector at 400 and 800 digital speed compared with storage phosphor radiography. A(z) values for reticular and ground-glass patterns with the flat-panel detector were equal to or less than those with storage phosphor radiography. These differences, however, were not statistically significant. CONCLUSION: In the detection of catheters, nodules, and almost all interstitial lung disease, A(z) values were higher with the portable flat-panel detector than with storage phosphor radiography at equivalent and reduced speeds. These results suggest that the portable flat-panel detector could be used with reduced exposure dose in pediatric patients.

Low-voltage digital selenium radiography: detection of simulated interstitial lung disease, nodules, and catheters--a phantom study.

PURPOSE: To compare three tube voltages in digital selenium radiography for the detection of simulated interstitial lung disease, nodules, and catheters. MATERIALS AND METHODS: Simulated catheters, nodules, and ground-glass, linear, miliary, and reticular patterns were superimposed over an anthropomorphic chest phantom. Digital selenium radiography was performed with different tube voltages (70, 90, and 150 kVp). Hard-copy images were generated. Detection performance of five radiologists was compared by using receiver operating characteristic (ROC) analysis involving 54,000 observations. RESULTS: The detection of ground-glass, linear, miliary, and reticular patterns over lucent lung and of nodules equal to, smaller than, and larger than 10 mm increased when 70 kVp and/or 90 kVp was used. However, only the reticular pattern was significantly better detected at lower peak voltage (P <.05). Simulated catheters and nodules over the mediastinum showed smaller areas under the ROC curve at lower peak voltage. These results were not statistically significant (P >.05). CONCLUSION: The diagnostic performance of digital selenium radiography at lower peak voltage is at least as good as that at higher peak voltage for interstitial lung disease over lucent lung. Performance is equivalent for nodules and catheters over obscured chest regions at lower peak voltages compared with that at 150 kVp. Our results implicate that the use of high-voltage technique in digital selenium radiography should be reassessed.

Diagnostic performance of a flat-panel detector at low tube voltage in chest radiography: a phantom study.

RATIONALE AND OBJECTIVES: To evaluate a large area, cesium iodide amorphous silicon flat-panel detector (CsI/a-Si) at 3 tube voltages to detect simulated interstitial lung disease, nodules, and catheters. METHODS: Simulated interstitial lung disease, nodules, and catheters were superimposed over a chest phantom. Images were generated at 125 kVp, 90 kVp, and 70 kVp at the same surface dose and reduced effective dose equivalent for 90 kVp and 70 kVp and printed on hard copies. Fifty-four thousand observations were analyzed by receiver operating characteristic (ROC). RESULTS: Detectability of linear, miliary, reticular pattern, and nodules over lucent lung as well as of catheters and nodules over obscured chest areas increased at 90 and/or 70 kVp with higher Az values; however, only it was statistically significant for reticular pattern at 70 kVp and nodules at 90 kVp compared with 125 kVp (P < 0.05). The detection of ground-glass pattern was worse at lower kVp (P > 0.05). CONCLUSION: For most simulated patterns, differences in diagnostic performance at 70 kVp/90 kVp and 125 kVp were not significant, except for reticular pattern and nodules over lucent lung.

Flat-panel x-ray detector based on amorphous silicon versus asymmetric screen-film system: phantom study of dose reduction and depiction of simulated findings.

PURPOSE: To compare a large-area amorphous silicon flat-panel detector with an asymmetric screen-film system for the depiction of simulated patterns of interstitial lung disease, nodules, and catheters, as well as for evaluation of dose reduction. MATERIALS AND METHODS: Ground-glass, linear, miliary, and reticular patterns; nodules; and catheters were superimposed over an anthropomorphic chest phantom. Hard copies were generated at different dose levels (speeds: 400, 800, and 1,600) with a flat-panel detector and were compared with copies generated with an asymmetric screen-film system (speed, 400). Detection performance of eight radiologists was compared with a receiver operating characteristic analysis of 19,200 observations per pattern. A difference was significant with a P value of.05. RESULTS: There was no statistically significant difference between the flat-panel detector and the asymmetric screen-film system at the same speed (P >.05) and between the flat-panel detector at a speed of 800 and the asymmetric screen-film system at a speed of 400 (P >.05). The visibility of linear, miliary, and reticular patterns over lucent lung and of nodules smaller than 10 mm and catheters over obscured chest regions on copies generated at a speed of 1,600 with the flat-panel detector decreased, compared with the visibility of these features on copies generated with the asymmetric screen-film system (P <.05). CONCLUSION: The diagnostic performance of the flat-panel detector is comparable to that of the asymmetric screen-film system for depiction of all simulated patterns of interstitial lung diseases, nodules, and catheters at the same speed and offers the potential of dose reduction to a speed of 800.

Diameters of the thoracic aorta throughout life as measured with helical computed tomography.

OBJECTIVE: The use of helical computed tomography is well established in the evaluation of the thoracic aorta. Nevertheless, normal diameters and their changes during adult life according to this method are not available. We planned to set up normal diameters for the thoracic aorta of adults obtained by helical computed tomography. METHODS: Seventy adults, 17 to 89 years old, without any signs of cardiovascular disease were investigated with helical computed tomography. Aortic diameters were measured at seven predefined thoracic levels. RESULTS: Aortic diameters (mean +/- SD) were 2.98 +/- 0.46 cm at the aortic valve sinus, 3.09 +/- 0.41 cm at the ascending aorta, 2.94 +/- 0.42 cm proximal to the innominate artery, 2.77 +/- 0.37 cm at the proximal transverse arch, 2.61 +/- 0.41 cm at the distal transverse arch, 2.47 +/- 0.40 cm at the isthmus, and 2.43 +/- 0.35 cm at the diaphragm. Men had slightly longer diameters than did women. All diameters increased with age. There was no influence of weight, height, or body surface area. After normalization to the diameter at diaphragmatic level, no statistically significantly influential factor could be detected. CONCLUSIONS: This study delineates normal intrathoracic aortic diameters for helical computed tomography, including relationships with sex and age. Pathologic dimensions of the aorta should preferably be provided as percentiles or z scores.