The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
Cryptosporidium parvum is a protozoan parasite that infects the gastrointestinal epithelium and causes diarrheal disease worldwide. Innate epithelial immune responses are key mediators of the host's defense to C. parvum. MicroRNAs (miRNAs) regulate gene expression at the posttranscriptional level and are involved in regulation of both innate and adaptive immune responses. Using an in vitro model of human cryptosporidiosis, we analyzed C. parvum-induced miRNA expression in biliary epithelial cells (i.e., cholangiocytes). Our results demonstrated differential alterations in the mature miRNA expression profile in cholangiocytes following C. parvum infection or lipopolysaccharide stimulation. Database analysis of C. parvum-upregulated miRNAs revealed potential NF-κB binding sites in the promoter elements of a subset of miRNA genes. We demonstrated that mir-125b-1, mir-21, mir-30b, and mir-23b-27b-24-1 cluster genes were transactivated through promoter binding of the NF-κB p65 subunit following C. parvum infection. In contrast, C. parvum transactivated mir-30c and mir-16 genes in cholangiocytes in a p65-independent manner. Importantly, functional inhibition of selected p65-dependent miRNAs in cholangiocytes increased C. parvum burden. Thus, we have identified a panel of miRNAs regulated through promoter binding of the NF-κB p65 subunit in human cholangiocytes in response to C. parvum infection, a process that may be relevant to the regulation of epithelial anti-microbial defense in general.
MicroRNAs (miRNAs), small non-coding regulatory RNAs that regulate gene expression at the post-transcriptional level, are master regulators of a wide array of cellular processes. Altered miRNA expression could be a determinant of disease development and/or progression and manipulation of miRNA expression represents a potential avenue of therapy. Exosomes are cell-derived extracellular vesicles that promote cell–cell communication and immunoregulatory functions. These “bioactive vesicles” shuttle various molecules, including miRNAs, to recipient cells. Inappropriate release of miRNAs from exosomes may cause significant alterations in biological pathways that affect disease development, supporting the concept that miRNA-containing exosomes could serve as targeted therapies for particular diseases. This review briefly summarizes recent advances in the biology, function, and therapeutic potential of exosomal miRNAs.
LincRNAs are long non-coding transcripts (>200 nt) from the intergenic regions of annotated protein-coding genes. One of the most highly induced lincRNAs in macrophages upon TLR ligation is lincRNA-Cox2, which has recently been shown to mediate both the activation and repression of distinct classes of immune genes in innate immune cells. We report here that lincRNA-Cox2 located at chromosome 1 proximal to the prostaglandin-endoperoxide synthase 2 (Ptgs2/Cox2) gene is an early-primary inflammatory gene controlled by NF-κB signaling in murine macrophages. Functionally, lincRNA-Cox2 is required for the transcription of NF-κB-regulated late-primary inflammatory response genes stimulated by bacterial lipopolysaccharide. Specifically, lincRNA-Cox2 is assembled into the SWI/SNF (SWItch/Sucrose NonFermentable) complex in cells after lipopolysaccharide stimulation. This resulting lincRNA-Cox2/SWI/SNF complex can modulate the assembly of NF-κB subunits to the SWI/SNF complex, and ultimately, SWI/SNF-associated chromatin remodeling and transactivation of the late-primary inflammatory response genes in macrophages in response to microbial challenge. Therefore, our data indicate a new regulatory role of NF-κB-induced lincRNA-Cox2 to act as a co-activator of NF-κB for the transcription of late-primary response genes in innate immune cells through modulation of epigenetic chromatin remodeling.
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