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.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). Currently, magnetic resonance imaging (MRI) is the most commonly used method to diagnose and monitor MS, but there is a poor correlation between MRI disease measures and clinical disability or disease progression in MS. MRI is also an expensive tool that might carry potential risks due to brain accumulation of contrast material (Kanda et al., 2015). In the last few years, a lot of effort has been invested in the identification of biomarkers for MS; however, to date, few of these findings have proven clinically useful. Thus, there is a strong unmet clinical need for objective body fluid biomarkers to assist in early diagnosis, predicting long-term prognosis, monitoring treatment response, and predicting potential adverse effects in MS.

Circulating miRNAs have been detected in several body fluids (Cortez et al., 2011) where they are highly stable as they are resistant to circulating ribonucleases (Mitchell et al., 2008). Their stability, along with the development of sensitive methods for their detection and quantification (Guerau-de-Arellano M. et al., 2012), makes them ideal candidates for biomarkers. We previously reported changes in circulating plasma miRNAs in MS patients (Gandhi R. et al., 2013). In a new study, our group investigated serum miRNAs as biomarkers in MS as part of an NCATS-funded UH2 initiative. We found that several serum miRNAs were differentially expressed in MS, were associated with disease stage, and correlated with disability.

Study Design (Figure 1): Serum from 296 participants including patients with MS, other neurologic diseases (Alzheimer’s disease and amyotrophic lateral sclerosis), inflammatory diseases (rheumatoid arthritis and asthma), and healthy controls (HC) were tested. miRNA profiles were determined using LNA (locked nucleic acid) based qPCR. MS patients were categorized according to disease stage and disability. In the discovery phase, 652 miRNAs were measured from the serum of 26 MS patients and 20 healthy controls. Those miRNAs from the discovery set that were significantly differentially expressed (p <0.05) in cases vs controls were validated using qPCR in 58 MS patients and 30 healthy controls.

 

Serum miRNA biomarkers in MS - Figure 1

 

Note: Results in the current study were normalized to the four most stably expressed miRNA across all the subjects. We agree with other blogs posted on exRNA.org suggesting that there is an immediate need to identify reference miRNA/exRNA that could be used for data normalization.

 

Figure 2: Differentially expressed circulating miRNAs as biomarkers in Multiple Sclerosis (MS). Up to top five miRNAs with p<0.05 are represented for each group comparison; a) MS, b) relapsing remitting MS (RRMS) and secondary progressive (SPMS) compared to the healthy control (HC), c) RRMS vs. SPMS, and d) the correlation of miRNA with the expanded disease severity scale (EDSS).

 

Results: We found 7 miRNAs (p<0.05 in both discovery phase and validation) that differentiate MS patients from healthy controls; miR-320a up-regulation was the most significantly changing serum miRNA in MS patients. We found 8 miRNAs that differentiated relapsing-remitting MS (RRMS) from HC. Among these, miR-484 up-regulation in RRMS patients showed the strongest association. When comparing secondary progressive MS (SPMS) patients to HC, 34 miRNAs significantly differentiated between the groups in both phases, with miR-320a up-regulation showing the strongest link. We also identified two miRNAs linked to disease progression, with miR-27a-3p being the most significant. Ten miRNAs correlated with degree of disability according to the Kurtzke Expanded Disability Status Scale (EDSS), of which miR-199a-5p had the strongest correlation with disability. Of the 15 unique miRNAs we identified in the different group comparisons, 12 have previously been reported to be associated with MS, but not in serum. Kegg Pathway Analysis showed that significant and differentially expressed miRNAs target important immune functions and are related to the maintenance of neuronal homeostasis. For example, miR-27a-3p, the strongest miRNA to distinguish RRMS from SPMS and progressive MS (PMS) (up-regulated in the relapsing form as compared to the progressive forms) shows a strong link to both the neurotrophin signaling pathway and the T cell receptor signaling pathway. Other studies have shown that miR-27a-3p targets multiple proteins of intracellular signaling networks that regulate the activity of NF-κB and MAPKs 6. As a consequence, miR-27a inhibits differentiation of Th1 and Th17 cells and promotes the accumulation of Tr1 and Treg cells (Min S. et al., 2012). It has also been shown that miRa-27-3p is up-regulated in MS active brain lesions and that the level of miR-27a-3p in CSF is reduced in patients with dementia due to Alzheimer’s disease (AD) (Frigerio C.S. et al., 2013). Of all the miRNAs, miR-486-5p was identified in the largest number of comparisons. It correlates with EDSS and is up-regulated in MS compared to HC, to other neurological diseases, and to other inflammatory diseases. This particular miRNA was found to be associated with TGF-beta signaling pathways and is a known tumor suppressor (Oh H.K. et al., 2011). miR-320a has been previously described to be highly expressed in B cells of MS patients and was suggested to contribute to increased blood-brain barrier permeability due to regulation of MMP-9 (Aung L.L. et al., 2015). Pathway analysis links this miRNA to cell-to-cell adhesion pathways, another indication that it may be linked to blood-brain barrier permeability.

The current study is the most comprehensive evaluation to date of the role of serum miRNAs as biomarkers in MS, with the largest sample size and employing two independent cohort designs. One limitation of our study is that participant subject samples were collected from a single MS center. Further external validation of our results will require investigating samples from patients at other centers. We are currently performing such multicenter studies, which may also increase the power of our results. A second limitation of our study is the relatively small number of participants who contributed to each group comparison. Future work will require larger sample sizes to ensure that we have sufficient power to detect miRNAs with smaller effect sizes. Although miRNAs have been studied in cells and the CNS of MS patients, ours is the first comprehensive investigation of serum miRNAs.

Conclusions: Our findings identify circulating serum miRNAs (Figure 2) as potential biomarkers to diagnose and monitor disease status in MS. These findings are now being tested using patient samples obtained from other international MS centers. We are now investigating the role of miRNA as biomarkers for disease prognosis and treatment response in MS.

Acknowledgements: This study is a highly collaborative project, and I thank my whole team at the Ann Romney Center for Neurologic Diseases & MS Center for their contribution. The grant TR000890 is supported by the NIH Common Fund, through the Office of Strategic Coordination / Office of the NIH Director.

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.