Extracellular vesicles secreted by primary human bronchial epithelial cells reduce Pseudomonas aeruginosa burden and inflammation in cystic fibrosis mouse lung.

Cystic fibrosis (CF) results in a reduction in the volume of airway surface liquid, increased accumulation of viscous mucus, persistent antibiotic-resistant lung infections that cause chronic inflammation, and a decline in lung function. More than 50% of adults with CF are chronically colonized by Pseudomonas aeruginosa (P. aeruginosa), the primary reason for morbidity and mortality in people with CF (pwCF). Although highly effective modulator therapy (HEMT) is an important part of disease management in CF, HEMT does not eliminate P. aeruginosa or lung inflammation. Thus, new treatments are required to reduce lung infection and inflammation in CF. In a previous in vitro study, we demonstrated that primary human bronchial epithelial cells (HBECs) secrete extracellular vesicles (EVs) that block the ability of P. aeruginosa to form biofilms by reducing the abundance of several proteins necessary for biofilm formation as well as enhancing the sensitivity of P. aeruginosa to ß-lactam antibiotics. In this study, using a CF mouse model of P. aeruginosa infection, we demonstrate that intratracheal administration of EVs secreted by HBEC reduced P. aeruginosa lung burden and several proinflammatory cytokines including IFN-γ, TNF-α, and MIP-1ß in bronchoalveolar lavage fluid (BALF), even in the absence of antibiotics. Moreover, EVs decreased neutrophils in BALF. Thus, EVs secreted by HBEC reduce the lung burden of P. aeruginosa, decrease inflammation, and reduce neutrophils in a CF mouse model. These results suggest that HBEC via the secretion of EVs may play an important role in the immune response to P. aeruginosa lung infection.NEW & NOTEWORTHY Our findings show that extracellular vesicles secreted by primary human bronchial epithelial cells significantly reduce Pseudomonas aeruginosa burden, inflammation, and weight loss in a cystic fibrosis mouse model of infection.

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in cystic fibrosis.

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lungs, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC (murine homolog of IL-8) secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection.NEW & NOTEWORTHY The experiments in this report identify a novel mechanism, whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet halves in OMVs secreted by P. aeruginosa, which reduced the OMV-LPS-induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF.

Histochemistry and Cell Biology-a glance into the past and a look ahead.

At the occasion of the 65th anniversary of Histochemistry and Cell Biology, we browse through its first ten years of publication and highlight a selection of papers from the early days of enzyme, protein, and carbohydrate histochemistry. In addition, we narrate recent progress to identify, quantify, and precisely determine the tissue localization of proteins and lipids, and small molecules by the combination of spectroscopic techniques and histology.

Novel post-acquisition image processing to attenuate red blood cell autofluorescence for quantitative image analysis.

Quantitative analysis of microscopy images from samples stained with fluorescent probes necessitates a very low fluorescence background signal. In tissues prepared by immersion in a chemical fixative, followed by conventional processing for paraffin embedding, red blood cell autofluorescence across several imaging channels can be a nuisance. Although many protocols have been proposed to suppress red blood cell autofluorescence prior to microscopy imaging, in many instances they may not prove totally effective. Moreover, in environments such as core facilities where control over tissue processing and staining may not be feasible, methods to address autofluorescence via post-image acquisition processing may be of some advantage. To this end, we have developed an image analysis algorithm using a commercially based software platform to remove contaminating red blood cell autofluorescence during quantitative evaluation of the fluorescence signal from an immunostaining protocol. The method is based upon the low autofluorescence signal of red blood cells exhibited in the blue channel (used to detect DAPI nuclear signal of all cells), which can be subtracted from the total channel signal by increasing the threshold for DAPI signal in the nuclear detection settings during nuclear segmentation. With the contributing signal from the red blood cells eliminated, the specific immunostained signal for the antigen of interest could be determined. We believe that this simple algorithm performed on post-acquisition microscopy images will be of use for quantitative fluorescence analyses whenever red blood cell autofluorescence is present, especially in amounts where creating regions of interest for evaluation is not possible.

Dysregulation of Pyruvate Kinase M2 Promotes Inflammation in a Mouse Model of Obese Allergic Asthma.

Obesity is a risk factor for the development of asthma and represents a difficult-to-treat disease phenotype. Aerobic glycolysis is emerging as a key feature of asthma, and changes in glucose metabolism are linked to leukocyte activation and adaptation to oxidative stress. Dysregulation of PKM2 (pyruvate kinase M2), the enzyme that catalyzes the last step of glycolysis, contributes to house dust mite (HDM)-induced airway inflammation and remodeling in lean mice. It remains unclear whether glycolytic reprogramming and dysregulation of PKM2 also contribute to obese asthma. The goal of the present study was to elucidate the functional role of PKM2 in a murine model of obese allergic asthma. We evaluated the small molecule activator of PKM2, TEPP46, and assessed the role of PKM2 using conditional ablation of the Pkm2 allele from airway epithelial cells. In obese C57BL/6NJ mice, parameters indicative of glycolytic reprogramming remained unchanged in the absence of stimulation with HDM. Obese mice that were subjected to HDM showed evidence of glycolytic reprogramming, and treatment with TEPP46 diminished airway inflammation, whereas parameters of airway remodeling were unaffected. Epithelial ablation of Pkm2 decreased central airway resistance in both lean and obese allergic mice in addition to decreasing inflammatory cytokines in the lung tissue. Lastly, we highlight a novel role for PKM2 in the regulation of glutathione-dependent protein oxidation in the lung tissue of obese allergic mice via a putative IFN-γ-glutaredoxin1 pathway. Overall, targeting metabolism and protein oxidation may be a novel treatment strategy for obese allergic asthma.

Probing the unseen structure and function of liver cells through atomic force microscopy.

With the arrival of atomic force microscopy (AFM) about thirty years ago, this new imaging tool opened up a new area for the exploration of biological samples, ranging from the tissue and cellular level down to the supramolecular scale. Commercial instruments of this new imaging technique began to appear in the five years following its discovery in 1986 by Binnig, Quate & Gerber. From that point onwards the AFM has attracted many liver biologists, and the number of publications describing structure-function relationships on the diverse set of liver cells has grown steadily ever since. It is therefore timely to reflect on the achievements of AFM in disclosing the cellular architecture of hepatocytes, liver sinusoidal endothelial cells, Kupffer cells, stellate cells and liver-associated natural killer cells. In this thematic paper, we present new data and provide an in-depth overview of the current AFM literature on liver cell biology. We furthermore include a future outlook on how this scanning probe imaging tool and its latest developments can contribute to clarify various structural and functional aspects of cells in liver health and disease.

Nanoscale imaging using differential expansion microscopy.

Expensive and time-consuming approaches of immunoelectron microscopy of biopsy tissues continues to serve as the gold-standard for diagnostic pathology. The recent development of the new approach of expansion microscopy (ExM) capable of fourfold lateral expansion of biological specimens for their morphological examination at approximately 70 nm lateral resolution using ordinary diffraction limited optical microscopy, is a major advancement in cellular imaging. Here we report (1) an optimized fixation protocol for retention of cellular morphology while obtaining optimal expansion, (2) an ExM procedure for up to eightfold lateral and over 500-fold volumetric expansion, (3) demonstrate that ExM is anisotropic or differential between tissues, cellular organelles and domains within organelles themselves, and (4) apply image analysis and machine learning (ML) approaches to precisely assess differentially expanded cellular structures. We refer to this enhanced ExM approach combined with ML as differential expansion microscopy (DiExM), applicable to profiling biological specimens at the nanometer scale. DiExM holds great promise for the precise, rapid and inexpensive diagnosis of disease from pathological specimen slides.

Fluorine detection in the lung tissue of a worker with interstitial pulmonary fibrosis and long-term occupational exposure to polytetrafluoroethylene and perfluorooctanoic acid.

Exposure to polytetrafluoroethylene (PTFE), a compound used in nonstick cookware coating and a variety of other applications, is known to cause acute lung injury and granulomatous pneumonitis. It is uncertain whether PTFE and compounds used in its manufacture, such as perfluorooctanoic acid (PFOA), cause chronic lung disease. Here we report a case of interstitial pulmonary fibrosis in a 71-year-old man who died following a brief illness clinically suspected to be acute respiratory distress syndrome. He had a 25-year history of occupational exposure to PTFE and PFOA. At postmortem examination, the lungs demonstrated diffuse alveolar damage (DAD) superimposed on interstitial pulmonary fibrosis. The interstitial fibrosis lacked fibroblast foci and exhibited basilar and subpleural accentuation with focal microscopic honeycombing. Within the fibrotic lung parenchyma were scattered giant cells containing birefringent translucent particles. Scanning electron microscopy and energy-dispersive x-ray spectroscopy (SEM-EDS) were performed. A majority of the birefringent particles demonstrated a prominent peak for fluorine by EDS analysis. This is the first report to document the presence of fluorine, an elemental constituent of PTFE and PFOA, in fibrotic lung tissue. Careful evaluation of other individuals with long-term exposure to PTFE and/or PFOA appears warranted to better elucidate the spectrum of pulmonary disease associated with these compounds.

Quantitative pixel intensity- and color-based image analysis on minimally compressed files: implications for whole-slide imaging.

Current best practice in the quantitative analysis of microscopy images dictates that image files should be saved in a lossless format such as TIFF. Use of lossy files, including those processed with the JPEG algorithm, is highly discouraged due to effects of compression on pixel characteristics. However, with the growing popularity of whole-slide imaging (WSI) and its attendant large file sizes, compressed image files are becoming more prevelent. This prompted us to perform a color-based quantitative pixel analysis of minimally compressed WSI images. Sections from three tissues stained with one of three reagents representing the colors blue (hematoxylin), red (Oil-Red-O), and brown (immunoperoxidase) were scanned with a whole slide imager in triplicate at 20x, 40x, and 63x magnifications. The resulting files were in the form of a BigTIFF with a JPEG compression automatically applied during acquisition. Images were imported into analysis software, six regions of interest were applied to various morphological locations, and the areas assessed for the color of interest. Whereas the number of designated weakly or strongly positive pixels was variable across the triplicate scans for the individual regions of interest, the total number of positive pixels was consistent. These results suggest that total positivity for a specific color representing a histochemical or immunohistochemical stain can be adequately quantitated on compressed images, but degrees of positivity (e.g., weak vs. strong) may not be as reliable. However, it is important to assess individual whole-slide imagers, file compression level and algorithm, and analysis software for reproducibility.