Unlocking the
Mysteries of
Extracellular RNA
Communication

Once thought to exist only inside cells, RNA is
known to travel outside of cells and play a role in newly
discovered mechanisms of cell-to-cell communication.

Cell culture is a staple of modern biology, and Fetal Bovine Serum (FBS) is an essential component of many cell culture protocols. A specific use for FBS is to supply nutrients to cells and to stimulate their growth. Another role of FBS in cell culture research is to represent the complexities and functionality of endogenous biological environments; however, precisely this complexity has long been a potential confounding factor for researchers. For example, cytokines in FBS can lead to the stimulation of cells, thus producing unintended experimental environments. Despite these disadvantages, FBS retains a prominent role in modern cell culture, with estimated sales as high as 700,000 liters per year. Because of its ubiquity in cell culture research, it is critical to investigate how the components of FBS may be influencing experiments and downstream analysis.

Variability and uncertainty in the composition of FBS is especially problematic for studies that evaluate cellular secretions. For example, to successfully determine the array of RNA secreted by cultured cells, we need to know the extent to which the medium is contaminated by exogenous RNA. Additionally, extracellular RNA (exRNA) is not only found distributed freely throughout the liquid medium, but it is also often found packaged inside of extracellular vesicles (EVs) or lipoprotein complexes. Therefore, in a paper released online yesterday, Wei et al. evaluated how the RNA composition of FBS might be confounding research.

The authors first evaluated exogenous RNA contamination. They grew cultures of a cell type known not to express a particular RNA, then evaluated the presence of that RNA in the culture media. If that RNA was found, its origin was probably the media itself. For example, the authors demonstrated that miR-122, a liver-specific miRNA, is present in media from cultured glioma cells, suggesting that its source is likely FBS itself. They then attempted to deplete RNA from FBS via ultracentrifugation, but despite a 24 hour spin at 100,000g, about 75% of total RNA remained in the supernatant. This result has also been found by researchers attempting to deplete FBS of RNA-containing EVs and emphasizes the difficulty of producing media truly free from contaminating RNA.

These results led the authors to ask whether existing studies have wrongly attributed the presence of exRNA to a particular experimental procedure or cell type, when it should be recognized as a component of the FBS in the cell culture media. To answer this question, the authors first broadly profiled the RNA composition of FBS using RNA sequencing. They determined that between 9% and 22% of FBS RNA mapped to the human genome, depending on the stringency of the mapping algorithm and FBS preparation. They also checked for the presence of bovine-specific RNA in existing human cell culture exRNA datasets, finding levels as high as 17%, with samples from exosomes (a type of EV) containing particularly high levels. Finally, they demonstrated experimentally that bovine-specific transcripts are taken up into cells, interfering not only with exRNA analysis but also with intracellular RNA studies.

Moving forward, a significant remaining issue is deciding how to treat conserved RNA known to be present in both FBS and the cell line under study. Switching from FBS to purely chemically defined media can help with this problem, but it is not possible for all cell types and experimental conditions. Alternatively, a quantitative analysis of the chemical composition of the media might make it possible to estimate which RNAs are secreted by the cells of interest by filtering out known FBS RNAs from the total RNA pool.

This research cautions us to be careful in the design and interpretation of experiments to identify extracellular RNAs that use FBS in culture media. The paper, Fetal Bovine Serum RNA Interferes with the Cell Culture derived Extracellular RNA, released in Scientific Reports yesterday, is authored by Zhiyun Wei, Arsen O. Batagov, David R. F. Carter, and Anna M. Krichevsky.

Immunology 2016
Immunology 2016

Extracellular RNA was a hot topic of discussion at Immunology 2016, the annual meeting of the American Association of Immunologists (AAI), held at the Washington State Convention Center in Seattle, Washington May 13-17th, 2016. The National Cancer Institute (NCI) sponsored a symposium on “Extracellular RNA in the Immune System”, co-chaired by Dr. Kevin Howcroft (Division of Cancer Biology, Cancer Immunology, Hematology, and Etiology Branch, NCI) and K. Mark Ansel (University of California San Francisco – your faithful blogger). Four invited speakers presented and participated in lively discussion with an audience of gathered experts and curious newcomers to the field of extracellular RNA.

Dr. Gyongyi Szabo (University of Massachusetts) opened the symposium with a presentation of her laboratory’s work on extracellular vesicles and miRNAs in innate immune cell communication in the liver. Alcohol exposure induces liver inflammation, marked by release of pro-inflammatory cytokines and activation of myeloid cells, including Kupffer cells, the resident macrophages of the liver. In a mouse model, alcohol consumption increased expression of miR-155 in both macrophages and hepatocytes via TLR4 and NFκB-driven transcription. Inhibition or genetic deletion of miR-155 in this model blunted macrophage activation and cytokine production. Exosomes loaded with miR-155 mimetics could be delivered to hepatocytes and other liver cells to correct some of the defects observed in miR-155-deficient animals. Remarkably, endogenous miR-155 and miR-122 were elevated in serum collected after controlled “binge-drinking” in human study subjects, and these exosomes also conveyed information to cultured monocytes, altering their production of TNF and IL-1. Together these data suggest that extracellular communication between hepatocytes and innate immune cells via exosomal miRNAs regulates inflammation in response to alcohol consumption.

The theme of regulation of inflammatory responses by miRNA-containing exosomes was extended by Dr. Ryan O’Connell (University of Utah). His pioneering work on miR-155 and miR-146 demonstrated their opposing roles in inflammatory processes mediated by various cell types in several tissues and disease settings. Recent work in his laboratory showed that both of these miRNAs are released by bone-marrow-derived dendritic cells in a fashion dependent on Rab27 and neutral sphingomyelinase (N-SMase) activity, and that these miRNAs could be exchanged between cells separated by a filter that prevents cell-cell contact. Transferred miR-146a reduced recipient cells’ response to bacterial lipopolysaccharide, a classical innate immune stimulant in vitro and in vivo. In addition, transferred miR-155 was found to directly repress the 3’ UTR of target genes in recipient cells, supporting the possibility that functional miRNA transfer via exosomes could be used as a therapeutic modality for regulating inflammation. Getting these miRNAs to the right cell types in vivo remains an important challenge to bringing this technology to the clinic.

In addition to exosomes, high density lipoprotein (HDL) particles carry miRNAs and other extracellular RNAs in blood. Abnormal pro-inflammatory HDL is associated with systemic lupus erythematosus (SLE). Dani Michell (Vanderbilt University), a postdoctoral fellow in Kasey Vickers’ laboratory, discussed her work, conducted in collaboration with Amy Major’s laboratory, on miRNAs in HDL in SLE. HDL from subjects with SLE contained increased levels of miR-22-3p and miR-192-5p compared with HDL from healthy control subjects. Blocking miR-22 with locked nucleic acid inhibitors in vivo reduced spleen size and interferon production, and affected some clinical features in a mouse model of lupus. Experiments aimed at defining source and recipient cells in this system indicated that monocytes are much better than T lymphocytes at taking up HDL-associated miRNAs. It will be interesting to learn how HDL-associated miRNAs regain gene regulatory function in recipient cells.

The final presentation focused on lymphocytes as source cells for naturally occurring exRNAs in body fluids. Immuno-compromised mice with a mutation that specifically blocks lymphocyte development exhibit altered serum extracellular miRNA profiles. In support of the idea that lymphocytes themselves are an important source of ex-miRNAs, the most reduced exRNA species detected was miR-150, a miRNA highly expressed by lymphocytes. Activated T lymphocytes secrete vesicles that are enriched for tRNA fragments and miRNAs including miR-150. Rigorous purification revealed that these vesicles have characteristics of exosomes, including defined density, size, and protein markers including the tetraspanin CD9. Cellular fractionation also revealed tRNA fragment and miRNA enrichment in membrane fractions containing multivesicular bodies. Whether these extracellular lymphocyte-derived RNAs mediate cell-to-cell communication or not, signal-mediated reduction of cellular miRNAs certainly alters gene regulation in activated T lymphocytes. Thus, exRNA secretion may have important roles in regulating inflammatory processes in both source and recipient cells.

These topics will certainly remain on the mind of immunologists that attended the exRNA symposium — at least until Immunology 2017, to be held in Washington DC next May.

Secreted RNAs leave the intracellular environment by associating with diverse vesicular and protein components. Secreted vesicles are heterogeneous and follow various routes of egress from the cell (1). Subclasses of such vesicles contain distinct cell surface proteins (2). In order to fully understand the diversity of vesicles that contain RNA, it is necessary to analyze and sort vesicle populations (3). One way to do this is by flow sorting such vesicles based on the presence of distinct vesicular surface proteins.

The ability to perform flow cytometric analysis and sorting of exosomes has been an ongoing area of controversy due to the small size of exosomes, which range in size from 40-130nm, near or below the diffraction limit of light. Nevertheless, a variety of groups have used this technique to analyze different subsets of small vesicles successfully (4-14), including proteomic analyses (4, 15-17). The efficacy of these flow-sorting experiments has been cross-validated by a variety of means, including western blots and co-localization of coincidently expressed factors. Fluorescence-Activated Vesicle Sorting (FAVS) uses light scattering properties of vesicles to analyze and sort individual exosomes using fluorescent labels. (See a previous blog on FAVS here.)

In the paper, “Identification and Characterization of EGF Receptor in Individual Exosomes by Fluorescence-Activated Vesicle Sorting (FAVS)”, published in the Journal of Extracellular Vesicles (JEV), Higginbotham and colleagues have used FAVS to analyze exosomal subsets that express varying amounts of EGFR in different cell-culture and in vivo contexts. This was done using DiFi cells, a human colorectal cancer (CRC) cell line, and A431, an epidermoid cancer cell line, which express approximately 5×106 and 2.5×106 EGFRs per cell, respectively (18, 19). The FAVS results showed that DiFi exosomes contain far more EGFR than do A431 exosomes, far exceeding the two-fold difference in EGFR levels present in these cell lines. Furthermore, using an antibody that recognizes an active form of EGFR, mAb806 (20-22), the amount of active EGFR was also found to be dramatically higher in DiFi exosomes than in A431 exosomes.

FAVS was also used to sort EGFR/CD9 double-positive and double-negative exosome populations, allowing enrichment of both subsets by post-sort analysis as well as western blot validation of the sorted exosomes (see Figure). Using human-specific reagents, FAVS was able to detect DiFi exosomes in the plasma of mice bearing DiFi xenografts. FAVS was also used to demonstrate that EGFR and one of its ligands, amphiregulin (AREG) are present in the plasma of normal individuals.

 

Results from the JEV paper derived from Fig 2. DiFi exosomes were flow sorted using antibodies to EGFR and CD9. Sorted purified double-negative vesicles (blue box/arrow) and double-positive vesicles (red box/arrow) were probed by western blot for markers as shown. These results validate the flow sorting enrichment of these different classes of vesicles.  Also shown is a STORM image of an individual flow sorted double-positive vesicle.

Results from the JEV paper derived from Fig 2. DiFi exosomes were flow sorted using antibodies to EGFR and CD9. Sorted purified double-negative vesicles (blue box/arrow) and double-positive vesicles (red box/arrow) were probed by western blot for markers as shown. These results validate the flow sorting enrichment of these different classes of vesicles. Also shown is a STORM image of an individual flow sorted double-positive vesicle.

 

This work joins flow-sorting work done by other labs using somewhat different techniques (6-14) and has implications for similar kinds of work done by other members of this consortium (23-25). Common to all these techniques was the use of lipid and/or specific extracellular vesicle markers to identify classes of secreted vesicles. Unlike FAVS, many sorting methods trigger vesicular events based on fluorescence rather than scatter. In all of these cases, analysis of secreted vesicle populations was performed. In some cases vesicle sorting was also achieved.

Thus, FAVS appears to be a promising technique to identify and purify distinct subsets of exosomes for discovery studies. It also holds promise for the detection of biomarkers in disease states including subsets of associated secreted RNAs.

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