Exosomes: from bins to biomarkers

Exosomes are microscopic particles which bud off from the cell membrane and circulate in the blood. Once regarded as useless ‘slough’, they are now recognised to be immensely important and ubiquitous signalling beacons which can co-ordinate and control the function of distant cells. They exert their complex effects on cell function by delivering ‘cargo’, such as non-coding riboxynucleic acid (RNA), to distant cells. We don’t fully understand what exosomes do or how their release is controlled. But there is now lots of exciting evidence to suggest that they play important roles in cell function and dysfunction in health and disease. Here is a cartoon of what they look like:

In an ambitious study published this week in Annals of Neurology, Mycko and friends describe their needle-in-a-haystack efforts to find biomarkers for MS in the contents of exosomes. To do this, they took blood samples from 3 groups: pwRRMS who were relapse-free, pwRRMS who were undergoing a relapse, and people without MS. They extracted the exosomal content of these blood samples, and then extracted the entire RNA content of these exosomes. Using next-generation sequencing they identified all the RNAs present in their cohort.
They decided to focus on microRNAs (miRNAs), which are a particularly well-studied group of non-coding RNAs thought to be involved in various disease states. In a subset of 29 patients (10 people with active MS, 10 people with quiescent MS, and 9 people without MS), there were between 350 and 400 miRNAs per subject. Comparing the profile of individual miRNAs across these three groups, the authors found that 4 particular miRNAs were present with different frequencies in the three groups to a statistically significant extent (False discovery rate < 5%). These are catchily named hsa-miR-196b-5p, hsa-miR-532-5p, hsa-miR-122-5p, and hsa-miR-301a-3p.
To validate these findings in a bigger cohort, the authors measured the absolute quantities of each of these 4 miRNAs in 95 people, split roughly into 1/3 controls, 1/3 active MS, and 1/3 quiescent MS. Interestingly, all 4 miRNAs were down-regulated in active MS vs controls. 3 of the 4 miRNAs were down-regulated in active MS vs quiescent disease, but only miR-122-5p was expressed at a different level in quiescient disease vs control.
The headline is that these 4 miRNAs are good ways of distinguishing between people with active MS and people with no MS. This finding was borne out by MRI evidence that people with Gadolinium-enhancing lesions on MRI – a sign of active disease – had lower levels of all 4 miRNAs than pwMS without them.
These results are nowhere near being clinically useful just yet. Distinguishing between people without MS and people with active relapsing MS is not a clinical problem. I think the most important areas in which blood biomarkers could help are early diagnosis, prognosis, and monitoring disease activity. For these purposes it would be more useful to know if biomarkers can distinguish between, for example, people who will never develop MS vs people who will, people who will only ever have a CIS vs people who will develop clinically-definite MS, and people who will have aggressive disease vs people who will have a more indolent course. Also, if we do develop robust exosome biomarkers, they could become part of treatment targets down the line alongside our current ‘NEDA’ targets.
For now though, while it’s not particularly useful, this study is amazing because of the questions it raises about the pathobiology of MS. Do these exosomal signals have a role to play in perpetuating inflammation? Are they just epiphenomena? Do they have consequences outside the nervous system? Are they important in CSF as well as blood? The questions, as always, are even more interesting than the answers.  
Accumulating evidence supports a role for exosomes in immune regulation. In this study, we investigated the total circulating exosome transcriptome in relapsing–remitting multiple sclerosis (RRMS) patients and healthy controls (HC).
Next generation sequencing (NGS) was used to define the global RNA profile of serum exosomes in 19 RRMS patients (9 in relapse, 10 in remission) and 10 HC. We analyzed 5 million reads and >50,000 transcripts per sample, including a detailed analysis of microRNAs (miRNAs) differentially expressed in RRMS. The discovery set data were validated by quantification using digital quantitative polymerase chain reaction with an independent cohort of 63 RRMS patients (33 in relapse, 30 in remission) and 32 HC.
Exosomal RNA NGS revealed that of 15 different classes of transcripts detected, 4 circulating exosomal sequences within the miRNA category were differentially expressed in RRMS patients versus HC: hsa-miR-122-5p, hsa-miR-196b-5p, hsa-miR-301a-3p, and hsa-miR-532-5p. Serum exosomal expression of these miRNAs was significantly decreased during relapse in RRMS. These miRNAs were also decreased in patients with a gadolinium enhancement on brain magnetic resonance imaging. In vitro secretion of these miRNAs by peripheral blood mononuclear cells was also significantly impaired in RRMS.
These data show that circulating exosomes have a distinct RNA profile in RRMS. Because putative targets for these miRNAs include the signal transducer and activator of transcription 3 and the cell cycle regulator aryl hydrocarbon receptor, the data suggest a disturbed cell-to-cell communication in this disease. Thus, exosomal miRNAs might represent a useful biomarker to distinguish multiple sclerosis relapse. Ann Neurol 2017;81:703–717

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