Bacterial Outer Membrane Vesicles Promote Lung Inflammatory Responses and Macrophage Activation via Multi-Signaling Pathways.

Emerging evidence suggests that Gram-negative bacteria release bacterial outer membrane vesicles (OMVs) and that these play an important role in the pathogenesis of bacterial infection-mediated inflammatory responses and organ damage. Despite the fact that scattered reports have shown that OMVs released from Gram-negative bacteria may function via the TLR2/4-signaling pathway or induce pyroptosis in macrophages, our study reveals a more complex role of OMVs in the development of inflammatory lung responses and macrophage pro-inflammatory activation. We first confirmed that various types of Gram-negative bacteria release similar OMVs which prompt pro-inflammatory activation in both bone marrow-derived macrophages and lung alveolar macrophages. We further demonstrated that mice treated with OMVs via intratracheal instillation developed significant inflammatory lung responses. Using mouse inflammation and autoimmune arrays, we identified multiple altered cytokine/chemokines in both bone marrow-derived macrophages and alveolar macrophages, suggesting that OMVs have a broader spectrum of function compared to LPS. Using TLR4 knock-out cells, we found that OMVs exert more robust effects on activating macrophages compared to LPS. We next examined multiple signaling pathways, including not only cell surface antigens, but also intracellular receptors. Our results confirmed that bacterial OMVs trigger both surface protein-mediated signaling and intracellular signaling pathways, such as the S100-A8 protein-mediated pathway. In summary, our studies confirm that bacterial OMVs strongly induced macrophage pro-inflammatory activation and inflammatory lung responses via multi-signaling pathways. Bacterial OMVs should be viewed as a repertoire of pathogen-associated molecular patterns (PAMPs), exerting more robust effects than Gram-negative bacteria-derived LPS.

Microvesicle-Derived miRNAs Regulate Proinflammatory Macrophage Activation in the Lung Following Ozone Exposure.

Ozone is a ubiquitous air pollutant that causes lung damage and altered functioning. Evidence suggests that proinflammatory macrophages contribute to ozone toxicity. Herein, we analyzed the role of extracellular vesicles (EVs) and microRNA (miRNA) cargo in ozone-induced macrophage activation. Exposure of mice to ozone (0.8 ppm, 3 h) resulted in increases in bronchoalveolar lavage fluid EVs, which were comprised predominantly of microvesicles (MVs). NanoFACS analysis revealed that MVs generated following both air and ozone exposure was largely from CD45+ myeloid cells; these MVs were readily taken up by macrophages. Functionally, MVs from ozone, but not air treated mice, upregulated mRNA expression of inflammatory proteins in macrophages including inducible nitric oxide synthase (iNOS), CXCL-1, CXCL-2, and interleukin (IL)-1ß. The miRNA profile of MVs in bronchoalveolar lavage fluid (BALF) was altered after ozone exposure; thus, increases in miR-21, miR-145, miR320a, miR-155, let-7b, miR744, miR181, miR-17, miR-92a, and miR-199a-3p were observed, whereas miR-24-3p and miR-20 were reduced. Ingenuity pathway analysis revealed that these miRNAs regulate pathways that promote inflammatory macrophage activation, and predicted that let-7a-5p/let-7b, miR-24-3p, miR-21-5p, miR-17, and miR-181a-5p are key upstream regulators of inflammatory proteins. After ozone exposure, miR-199a-3p, but not precursor miR-199a-3p, was increased in lung macrophages, indicating that it is derived from MV-mediated delivery. Furthermore, lung macrophage mRNA expression of IL-1ß was upregulated after administration of MVs containing miR-199a-3p mimic but downregulated by miR-199a-3p inhibitor. Collectively, these data suggest that MVs generated following ozone exposure contribute to proinflammatory macrophage activation via MV-derived miRNAs including miR-199a-3p. These findings identify a novel pathway regulating macrophage inflammatory responses to inhaled ozone.

Extracellular Vesicle Associated Non-Coding RNAs in Lung Infections and Injury.

Extracellular vesicles (EVs) refer to a heterogenous population of membrane-bound vesicles that are released by cells under physiological and pathological conditions. The detection of EVs in the majority of the bodily fluids, coupled with their diverse cargo comprising of DNA, RNA, lipids, and proteins, have led to the accumulated interests in leveraging these nanoparticles for diagnostic and therapeutic purposes. In particular, emerging studies have identified enhanced levels of a wide range of specific subclasses of non-coding RNAs (ncRNAs) in EVs, thereby suggesting the existence of highly selective and regulated molecular processes governing the sorting of these RNAs into EVs. Recent studies have also illustrated the functional relevance of these enriched ncRNAs in a variety of human diseases. This review summarizes the current state of knowledge on EV-ncRNAs, as well as their functions and significance in lung infection and injury. As a majority of the studies on EV-ncRNAs in lung diseases have focused on EV-microRNAs, we will particularly highlight the relevance of these molecules in the pathophysiology of these conditions, as well as their potential as novel biomarkers therein. We also outline the current challenges in the EV field amidst the tremendous efforts to propel the clinical utility of EVs for human diseases. The lack of published literature on the functional roles of other EV-ncRNA subtypes may in turn provide new avenues for future research to exploit their feasibility as novel diagnostic and therapeutic targets in human diseases.

Low concentration of polyethylene glycol facilitates separation of extracellular vesicles from bronchoalveolar lavage fluid.

Extracellular vesicles (EVs) in bodily fluids play an essential role in cell-cell cross talk and potentially serve as novel biomarkers in "liquid biopsy." It is crucial to have a consistent, efficient, and reliable method to separate EVs from bodily fluids. Currently, there is no universally accepted, "best" method to separate EVs. Besides differential ultracentrifugation (UC), polyethylene glycol (PEG) is among the commonly used methods for EV separation from bodily fluids. However, the optimal concentration of PEG to be used remains inadequately addressed. We initially observed that the concentration of PEG has a significant impact on the amount of separated EVs and EV-cargos, which are recovered from bronchoalveolar lavage fluid (BALF). To determine the optimal PEG concentration to be used in EV separation from BALF, we first separated the BALF and serum from wild-type C57BL/6 mice. Next, various concentrations of PEG (5%, 10%, and 15% PEG), a commercial kit, and UC were used to obtain EVs from BALF and serum. EVs were characterized, and EV-cargo protein, RNA, and miRNA levels were determined. We found that high concentration of PEG (10% and 15%) altered various EV parameters that are frequently used in EV studies, including EV yield, purity, and morphology. Using miR-15a, miR-142, and miR-223 as examples, we found that 10% and 15% PEG robustly reduced the detected levels of EV-cargo miRNAs compared with those in the EVs separated using UC or 5% PEG. Collectively, low concentration of PEG facilitates the optimal BALF EV separation.

The Evolving Role of Caveolin-1: A Critical Regulator of Extracellular Vesicles.

Emerging evidence suggests that extracellular vesicles (EVs) play an essential role in mediating intercellular communication and inter-organ crosstalk both at normal physiological conditions and in the pathogenesis of human diseases. EV cargos are made up of a broad spectrum of molecules including lipids, proteins, and nucleic acids such as DNA, RNA, and microRNAs. The complex EV cargo composition is cell type-specific. A dynamic change in EV cargos occurs along with extracellular stimuli and a change in the pathophysiological status of the host. Currently, the underlying mechanisms by which EVs are formed and EV cargos are selected in the absence and presence of noxious stimuli and pathogens remain incompletely explored. The term EVs refers to a heterogeneous group of vesicles generated via different mechanisms. Some EVs are formed via direct membrane budding, while the others are produced through multivesicular bodies (MVBs) or during apoptosis. Despite the complexity of EV formation and EV cargo selection, recent studies suggest that caveolin-1, a well-known structural protein of caveolae, regulates the formation and cargo selection of some EVs, such as microvesicles (MVs). In this article, we will review the current understanding of this emerging and novel role of cav-1.

Post-translational Modification Regulates Formation and Cargo-Loading of Extracellular Vesicles.

Accumulating evidence suggests that post-translational modifications (PTMs) regulate the selective encapsulation of non-coding RNA molecules into extracellular vesicles (EVs) and contribute to the downstream functions of EVs or EV-cargo non-coding RNAs. EVs are a newly studied mechanism of intercellular communication that involves the transfer of molecules, including but not limited to proteins, lipids, and non-coding RNAs, to induce functional changes in the recipient cells. In this present mini-review, we focus on the PTM-regulated protein and non-coding RNA selection into eukaryotic EVs.

Pretreated household materials carry similar filtration protection against pathogens when compared with surgical masks.

OBJECTIVE: The past 4 months, the emergence and spread of novel 2019 SARS-Cov-2 (COVID-19) has led to a global pandemic which is rapidly depleting supplies of personal protective equipment worldwide. There are currently over 1.6 million confirmed cases of COVID-19 worldwide which has resulted in more the 100,000 deaths. As these numbers grow daily, hospitals are being forced to reuse surgical masks in hopes of conserving their dwindling supply. Since COVID-19 will most likely have effects that last for many months, our nationwide shortage of masks poses a long term issue that must be addressed immediately. METHODS: Based on a previous study by Quan et al., a salt-based soaking strategy has been reported to enhance the filtration ability of surgical masks. We propose a similar soaking process which uses materials widely available in anyone's household. We tested this method of pretreating a variety of materials with a salt-based solution by a droplet test using fluorescently stained nanoparticles similar in size to the COVID-19 virus. RESULTS: In this study, we found that paper towels and surgical masks pretreated with the salt-based solution showed a noticeable increase in filtration of nanoparticles similar in size to the COVID-19 virus. We also show that the TWEEN20 used by Quan et al. is not a critical component for the solution, and using salt alone in solution still provides a dramatically increased level of protection. CONCLUSIONS: We believe this method will allow for healthcare workers to create a disposable added layer of protection to their surgical masks, N95s, or homemade masks by using household available products. Adoption of this method may play an essential role in ensuring the safety of healthcare workers during the COVID-19 pandemic and any pandemics that may arise in the future.

Extracellular Vesicle-Shuttling MicroRNAs Regulate the Development of Inflammatory Lung Responses.

MicroRNAs are small single-stranded, non-coding RNAs which have a known role in post-transcriptional regulation of gene expression. Recent studies have reported that extracellular vesicles are capable of specific delivery of miRNAs to a target cell or tissue from a host cell. MiRNAs are generated by host cells, selectively packaged into EVs, and then delivered to nearby target cells with full functionality. After delivery to the target cells, these EV-packaged miRNAs regulate the translation of their target genes. Thus, EV transported miRNAs have become a newly understood method for intercellular communication. In this review, we summarize the novel findings of EV-miRNA transfer in acute lung injury, chronic obstructive pulmonary disease, bronchopulmonary dysplasia, asthma, and idiopathic pulmonary fibrosis.