Extracellular Vesicles from Pseudomonas aeruginosa Suppress MHC-Related Molecules in Human Lung Macrophages.

Pseudomonas aeruginosa, a Gram-negative bacterium, is one of the most common pathogens colonizing the lungs of cystic fibrosis patients. P. aeruginosa secrete extracellular vesicles (EVs) that contain LPS and other virulence factors that modulate the host's innate immune response, leading to an increased local proinflammatory response and reduced pathogen clearance, resulting in chronic infection and ultimately poor patient outcomes. Lung macrophages are the first line of defense in the airway innate immune response to pathogens. Proper host response to bacterial infection requires communication between APC and T cells, ultimately leading to pathogen clearance. In this study, we investigate whether EVs secreted from P. aeruginosa alter MHC Ag expression in lung macrophages, thereby potentially contributing to decreased pathogen clearance. Primary lung macrophages from human subjects were collected via bronchoalveolar lavage and exposed to EVs isolated from P. aeruginosa in vitro. Gene expression was measured with the NanoString nCounter gene expression assay. DNA methylation was measured with the EPIC array platform to assess changes in methylation. P. aeruginosa EVs suppress the expression of 11 different MHC-associated molecules in lung macrophages. Additionally, we show reduced DNA methylation in a regulatory region of gene complement factor B (CFB) as the possible driving mechanism of widespread MHC gene suppression. Our results demonstrate MHC molecule downregulation by P. aeruginosa-derived EVs in lung macrophages, which is consistent with an immune evasion strategy employed by a prokaryote in a host-pathogen interaction, potentially leading to decreased pulmonary bacterial clearance.

Pre-Analytical Handling Conditions and Small RNA Recovery from Urine for miRNA Profiling.

There are currently no standardized protocols for pre-analytical handling of urine to best preserve small RNA for miRNA profiling studies. miRNA is an attractive candidate as a potential biomarker because of the high level of stability in body fluids and its ability to be quantified on multiple high-throughput platforms. We present a comparison of small RNA recovery and stability in urine under alternate pre-analytical handling conditions and extend recommendations on what conditions optimize yield of miRNA from cell-free urine and urine extracellular vesicles (EVs). Using an affinity slurry for isolation of small RNA from urine, we found that urine samples held at room temperature (20°C) for up to 8 hours before processing yield the highest amounts of intact small RNAs from EVs. Some miRNA is lost from urine samples when held 2°C to 4°C and/or frozen before EV isolation, likely because of EV entrapment in uromodulin precipitates. However, we found that a simple 5-minute incubation of urine containing cold-induced precipitate at 37°C resolubilizes much of this precipitate and results in an increased recovery of EVs and miRNAs. Finally, small RNA integrity can be compromised when whole urine is held at 37°C for as little as 4 hours and is not conducive to efficient miRNA profiling.

Effect of adenovirus infection on transgene expression under the adenoviral MLP/TPL and the CMVie promoter/enhancer in CHO cells.

The adenovirus major late promoter (MLP) and its translational regulator - the tripartite leader (TPL) sequence - can actively drive efficient gene expression during adenoviral infection. However, both elements have not been widely tested in transgene expression outside of the adenovirus genome context. In this study, we tested whether the combination of MLP and TPL would enhance transgene expression beyond that of the most widely used promoter in transgene expression in mammalian cells, the cytomegalovirus immediate early (CMVie) promoter/enhancer. The activity of these two regulatory elements was compared in Chinese hamster ovary (CHO) cells. Although transient expression was significantly higher under the control of the CMVie promoter/enhance compared to the MLP/TPL, this difference was greater at the level of transcription (30 folds) than translation (11 folds). Even with adenovirus infection to provide additional elements (in trans), CMVie promoter/enhancer exhibited significantly higher activity relative to MLP/TPL. Interestingly, the CMVie promoter/enhancer was 1.9 folds more active in adenovirus-infected cells than in non-infected cells. Our study shows that the MLP-TPL drives lower transgene expression than the CMVie promoter/enhancer particularly at the transcription level. The data also highlight the utility of the TPL sequence at the translation level and/or possible overwhelming of the cellular translational machinery by the high transcription activity of the CMVie promoter/enhancer. In addition, here we present data that show stimulation of the CMVie promoter/enhancer by adenovirus infection, which may prove interesting in future work to test the combination of CMVie/TPL sequence, and additional adenovirus elements, for transgene expression.