MicroRNAs in Infectious Disease: Emerging Clinical Applications

MicroRNAs (miRNAs) are short, endogenous, single-stranded RNA sequences that derive from noncoding DNA. Because they do not encode proteins, scientists originally believed that miRNAs are not relevant to the functioning of the human body. However, despite their “humble” genetic origins, emerging evidence points to a key role of miRNAs in the host response to infection. As a consequence, miRNAs are currently being explored as biomarkers¹ and potential new therapies² for infectious diseases. What was once considered transcriptomic “junk” is now getting a second, closer look.

A recent review pointed to the role of miRNAs in the regulation of cellular processes affecting homeostasis, neurobiology, immunobiology, and organismal development.¹ MiRNAs have also been found to exert profound effects on innate and adaptive immune pathways² by posttranscriptionally regulating up to 60% of protein-encoding genes.³ By binding to target sequences in messenger RNA, miRNAs can interfere with the translation process and prevent or alter the production of proteins.⁴

In an interview with Infectious Disease Advisor, David MacHugh, PhD, professor in genomics at University College Dublin, Ireland, and Timothy D. Minogue, PhD, chief of the Molecular Diagnostics Department at the US Army Medical Research Institute of Infectious Diseases (USAMRIID) in Frederick, Maryland, discussed the emerging clinical applications of miRNAs in infectious disease.

Infectious Disease Advisor: What are some of the characteristics of circulating miRNAs that make them suitable for use as biomarkers of infectious disease? 

David MacHugh, PhD: First discovered in 1993,⁵ miRNAs are small, noncoding, regulatory RNA molecules that can exhibit high evolutionary conservation across divergent metazoan groups, highlighting their role in the control of gene regulatory networks that govern diverse and important cellular processes.

More recently, miRNA-based gene regulation has been shown to be an important component of the molecular fine-tuning that underpins vertebrate host immune responses during microbial infections. MiRNAs have also been shown to be highly stable in extracellular fluids of mammals, such as blood plasma, serum, urine, saliva, and semen, which, coupled with active release of specific extracellular miRNAs from immune cells and infected cells, makes them useful biomarkers of infectious disease.

Infectious Disease Advisor: In which infectious conditions/diseases has the role of miRNAs as biomarkers of disease been demonstrated? 

Dr MacHugh: Circulating miRNAs have been studied as biomarkers of infection and disease state for human tuberculosis disease caused by infection with Mycobacterium tuberculosis. For example, circulating serum miRNAs have been used to distinguish latent and active tuberculosis. Circulating miRNAs have also been studied intensively to generate an early-warning diagnostic or prognostic biosignature for sepsis caused by a range of microbial pathogens. They have also been studied as biomarkers of disease severity for viral hepatitis caused by hepatitis B virus and hepatitis C virus, and for malaria caused by infection with protozoan Plasmodium spp. parasites.

Infectious Disease Advisor: In which areas of miRNA biomarker applications is improvement still necessary before their widespread use in the clinical setting?

Dr MacHugh: Technical improvements are needed in the laboratory setting and in data standardization, as summarized in our recently published review paper.¹ MiRNAs represent only a very small proportion of total RNA in biological fluids. The quality of miRNA extraction and purification from serum and plasma samples is influenced by the protocols used in pre- and poststorage sample processing. In addition, precautionary measures are needed to avoid potential contamination with intracellular miRNAs derived from platelets and erythrocytes, which can alter the results of circulating miRNA expression profiles.¹

Furthermore, as transcriptomic assays using circulating miRNAs are typically “noisy,” it is critical to apply data normalization techniques to minimize any variation that may adversely influence the interpretation of results. The most appropriate methods for data normalization of circulating miRNA data (eg, from high-throughput sequencing) still need to be fully evaluated and standardized. There are also issues with detecting, cataloguing, and quantifying subtlety different miRNA sequence variants such as specific isomiRs or particular miRNA family members.

It is important that rigorous independent benchmarking studies are undertaken to evaluate the wide range of methods available for circulating miRNA purification, identification, quantification, and comparative data analysis and interpretation.

Infectious Disease Advisor: What are some potential clinical applications of miRNAs in infectious disease?

Timothy D. Minogue, PhD: Clinically, miRNAs are used as a cancer diagnostic and as disease-specific biomarkers for detection and prognosis. At this time, the field is segueing from cancer applications to an infectious disease context, in which we find very similar utility, but with a slightly more complex implementation. For infectious diseases, miRNA classifiers can be used to determine reactivity of the host to specific pathogens. Beyond this application, both viruses and bacteria can encode their own miRNAs that can affect pathogenesis and, more important, could be targeted in clinical diagnostic assays. Specific to our group, we are trying to determine the relevance of both sources of miRNAs to provide a capability for clinical detection and prediction of disease outcomes for high-consequence pathogens.

Infectious Disease Advisor: Your research team recently evaluated the utility of circulating miRNAs in diagnostics of presymptomatic and asymptomatic Ebola virus infection.⁶ What did you find?

Dr Minogue: Our Ebola virus research, as well as unpublished work with other bacteria and viruses, shows that a defined host-derived miRNA profile can be used to stratify acutely infected patients based on the overall abundance of specific miRNA species. In these studies, we query longitudinal time-courses that included acute, presymptomatic, and asymptomatic samples. Predicting acute phase Ebola virus infection is interesting; however, our findings, in which we determined Ebola virus infection presymptomatically in more than 50% of the appropriate samples queried, may have a greater effect on the ability to diagnose and treat these diseases. With further refinement of the classifier, we hope to improve that percentage to a range that would be more impactful for patient care.

Infectious Disease Advisor: How do you envision the field of miRNA applications in infectious disease developing in the future?

Dr Minogue: In my mind, miRNAs show the most promise as indicators of host response and as potential diagnostic targets. I am perhaps slightly biased in my perspective, coming from a molecular diagnostic laboratory, but I think the relative structural integrity of miRNAs in clinical matrix, along with the pleiotropic regulatory function miRNAs serve, make these molecules great diagnostic targets. In the future, host-derived miRNA profiles, combined with pathogen-specific miRNA species, could provide a complete diagnosis without necessarily having a priori knowledge of the etiologic agent. One of the primary intents for host biomarker research is to move the diagnostic window into the early presymptomatic phase of disease progression. I think miRNAs will ultimately provide this capability of early detection, as well as disease prognosis, to the clinician.

References

1. Correia CN, Nalpas NC, McLoughlin KE, et al. Circulating microRNAs as potential biomarkers of infectious disease. Front Immunol. 2017;8:118.
2. Drury RE, O’Connor D, Pollard AJ. The clinical application of microRNAs in infectious disease. Front Immunol. 2017;8:1182.
3. Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 2009;19:92-105.
4. Bhaskaran M, Mohan M. MicroRNAs: history, biogenesis, and their evolving role in animal development and disease. Vet Pathol. 2014;51:759-774.
5. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993;75:843-854.
6. Duy J, Koehler JW, Honko AN, et al. Circulating microRNA profiles of Ebola virus infection. Sci Rep. 2016;6:24496.