Diabetes Volume 65, April 2016 823

Sarah J. Richardson1 and Marc S. Horwitz2 miR, miR in the Cell, Does the Virus Control Them All?

Diabetes 2016;65:823–825 | DOI: 10.2337/dbi15-0039

As if the pathway of regulation leading islet cells toward Previously, several groups have examined the impact dysfunction and diabetes was not difficult enough to ofcytokinesonmiRNAsinmouseandhumanb-cells grasp, here come viruses to further complicate matters. In (7,13,18), but the study by Kim et al. (19) in this issue of the past few years, we have come to recognize that a class Diabetes is the first study to examine the impact of an of small noncoding RNAs termed microRNAs (miRNAs) has enterovirus infection on miRNA expression in human pan- a powerful ability to regulate most, if not all, of the key creatic islets. Taking into consideration the important role processes in cell biology (1,2). In fact, there is evidence that of miRNAs in normal b-cellfunction,Kimetal.asked these key molecules can be transported from cell to cell, whether a virus infection could be in part responsible for thereby influencing gene expression not only within the changes in miRNA expression within the infected b-cell the host cell but also in neighboring cells and in distant that can subsequently contribute to a type 1 diabetes cell subsets when the miRNAs travel in vesicles via the phenotype. These changes could promote b-cell dysfunction – blood and lymph (3 5). and/or apoptosis, could alter the sensing and subsequent COMMENTARY The evidence that miRNAs play pivotal roles in b-cell antiviral host cell immune response, and could impact biology, including regulating their differentiation, glucose the ability of infiltrating and peripheral immune cells responsiveness, insulin production, apoptosis, and inflam- to respond to an infection, potentially promoting in- mation, is rapidly accumulating and was recently discussed creased inflammation, surveillance, or antigen presentation. by Filios and Shalev (4) in an issue of Diabetes.Asone Kim et al. (19) assessed the cellular expression of mightexpectgiventheirrangeoffunctionswithinthe miRNAs within human islets infected with CVB5. In total, b-cells, these same molecules are hypothesized to have key the expression of 754 miRNAs was assessed in the in- roles in the pathogenesis of type 1 diabetes (4,6–13). Yet, fected islets using a high-throughput quantitative PCR it is still not clear whether or not they are central to the array, with the expression of 33 miRNAs found to be induction of disease or if they are merely a biomarker of significantly altered when compared with the control is- active b-cell dysfunction. lets. Strikingly, these 33 miRNAs were predicted to target Type1diabetesisoftendescribedasachronicauto- 57 of the 72 known type 1 diabetes risk genes. The ma- immune disease characterized by the destruction of insulin- jority of miRNAs were downregulated postinfection but producing islet b-cells, and it is a widely held belief that 6 were upregulated (Fig. 1). Key immune modulatory a complex blend of genetic and environmental factors is miRNAs miR-155-5p (increased fourfold) and miR-181a-3p required to drive disease. In terms of clinically relevant (decreased 4.4-fold) were found to be dysregulated by 24 h environmental factors, the culprits that are most often after infection, and intriguingly, miR-34a-5p demonstrated mentioned and have the strongest evidentiary support are the greatest increase in expression (more than eightfold) of coxsackieviruses (CVBs). Evidence for CVB infection has any miRNA by 4 days postinfection. miR-34a-5p is plurip- often been described in recent-onset patients, and CVBs otent and has been shown to target genes resulting in have a clear propensity for infecting human islet b-cells diminishing insulin secretion (VAMP2) and increasing and can induce diabetes in mouse models (14–16). Whereas apoptosis (BclII) (18). Additionally, miR-155-5p has also many viruses encode their own miRNAs to control both viral been shown by others to affect several other diabetes risk and cellular processes, other viruses simply usurp cellular genes such as GLIS3 (b-cell survival) and SOCS1 (negative miRNAs to their own advantage (3,5,17). Small RNA viruses regulator of the type I interferon [antivirus] pathway) (8,9). such as CVBs seem to lack miRNAs, as none have been Other dysregulated miRNAs were predicted to target a identified in their genomes to date. number of pathogen recognition receptors, such as IFIHI,

1University of Exeter Medical School, Exeter, U.K. © 2016 by the American Diabetes Association. Readers may use this article as 2The University of British Columbia, Vancouver, Canada long as the work is properly cited, the use is educational and not for profit, and Corresponding authors: Marc S. Horwitz, [email protected], and Sarah J. the work is not altered. Richardson, [email protected]. See accompanying article, p. 996. 824 Commentary Diabetes Volume 65, April 2016

TLR7, and TLR8; cytokines, including IL2, IL10, IL17D, has been shown to block pancreatic cell death in mouse and IL21; and regulators of cytokine apoptosis, such as models (12). BACH2. Many of these miRNA–target gene interactions Several miRNAs, including some identified by Kim have been validated in both adult pancreatic b-cells as et al. (19) (miR-21, miR-181, and miR-29a), were identi- well as b-cells from prediabetic mice (NOD and db/db) fied as dysregulated in the serum of children with type 1 (18), but others require further verification. Clearly though, diabetes (10,11). Additionally, multiple reports have iden- CVB5 infection alters cellular miRNA expression to focus tified exosomes containing miRNAs that are exchanged the cell’s material and energy to promote virus replica- between immune cells and result in changes in cellular ex- tion while at the same time attempting to downregulate pression, cytokine expression, and antigen presentation. pathways that would inhibit virus propagation, includ- An entire cluster of miRNA loci has been shown to target ing those affecting antivirus responses. The production the expression of type 1 diabetes autoantigens IA-2, of proteins that are not required for the virus life cycle, IA-2b,andGAD65(6). such as insulin and apoptosis pathways, appear to be It seems that virus infection reflects miRNA expression downregulated. An example of the latter is miR-21, back onto the cell in an effort to prioritize replication of which is increased more than sevenfold in the infected virus and on toward surrounding cells in an effort to cellsandtargetsthetumorsuppressorPDCD4,which minimize detection. In a susceptible individual, can this

Figure 1—How a viral infection could impact on the development of autoimmunity by altering host miRNA expression. ① The enterovirus infects b-cells within an islet. ② The enterovirus replicates, and viral proteins are produced. ③ The virus induces upregulation and downregulation of specific host miRNAs. ④ miR-155-5p at early time points and miR-21-3p at later time points impact on the host cell apoptotic responses. ⑤ Several of the miRNAs impact on the expression levels of key pathogen recognition receptors (PRRs) and SOCS1, which could alter host response to infection. ⑥ miR-34a and miR-21-3p alter expression of key genes involved in the regulation of b-cell exocytosis (VAMP2, OC2), thus potentially impacting on host cell insulin secretion. ⑦ Furthermore, miRNAs can be packaged into microvesicles and/or endosomes and released from the infected cells. ⑧ Neighboring islet cells can take up secreted miRNAs, which can then subsequently impact on their gene expression, either making them more or less susceptible to infection or apoptosis, which can alter their insulin secretion. ⑨ Secreted miRNAs can also be taken up by infiltrating and peripheral immune cells, which could then significantly impact on their regulatory or effector cell functions. diabetes.diabetesjournals.org Richardson and Horwitz 825 induce islet cell dysfunction and diabetes? This is the 2. Yamakuchi M, Ferlito M, Lowenstein CJ. miR-34a repression of SIRT1 premise of the study by Kim et al. (19), yet a number of regulates apoptosis. Proc Natl Acad Sci U S A 2008;105:13421–13426 questions are raised. Prior work has demonstrated that 3. Cullen BR. Five questions about viruses and microRNAs. PLoS Pathog 2010; different strains and substrains of CVB are associated 6:e1000787 4. Filios SR, Shalev A. b-Cell microRNAs: small but powerful. Diabetes 2015; with diabetes induction and protection (20). As such, 64:3631–3644 the most strongly associated diabetogenic strains are 5. Kincaid RP, Sullivan CS. Virus-encoded microRNAs: an overview and a look CVB1 and CVB4, and their miRNA expression pattern to the future. PLoS Pathog 2012;8:e1003018 fi may reveal further speci c diabetes-associated miRNAs 6. Abuhatzira L, Xu H, Tahhan G, Boulougoura A, Schäffer AA, Notkins AL. and targets. Likewise, the virus-mediated mechanism Multiple microRNAs within the 14q32 cluster target the mRNAs of major type 1 for the induction of diabetes remains elusive, yet two diabetes autoantigens IA-2, IA-2b, and GAD65. FASEB J 2015;29:4374–4383 viable hypotheses endure and suggest that the disease 7. Fernandez-Valverde SL, Taft RJ, Mattick JS. microRNAs in b-cell biology, could either be induced following an acute infection that insulin resistance, diabetes and its complications. Diabetes 2011;60:1825– is subsequently resolved or via a more chronic, persistent 1831 infection of b-cells. The expression of miRNA during 8. Kohlhaas S, Garden OA, Scudamore C, Turner M, Okkenhaug K, Vigorito E. acute and latent herpesvirus infection has been well de- Cutting edge: the Foxp3 target miR-155 contributes to the development of regulatory T cells. J Immunol 2009;182:2578–2582 scribed to change over the life cycle of the virus, and it 9. Lu LF, Thai TH, Calado DP, et al. Foxp3-dependent microRNA155 confers wouldbeexpectedthatsimilarresultswouldoccurwith competitive fitness to regulatory T cells by targeting SOCS1 protein. Immunity CVB (5). Kim et al. describe the early events of acute 2009;30:80–91 infection with exhibited changes in miRNA expression 10. Nielsen LB, Wang C, Sørensen K, et al. Circulating levels of microRNA from within the first 7 days. Later time points during persistent children with newly diagnosed type 1 diabetes and healthy controls: evidence infection would likely reveal a novel pattern of miRNA that miR-25 associates to residual beta-cell function and glycaemic control expression. Furthermore, exosome analysis of the in- during disease progression. Exp Diabetes Res 2012;2012:896362 tercommunication between cells could uncover com- 11. Osipova J, Fischer DC, Dangwal S, et al. Diabetes-associated microRNAs in munication within the islet to neighboring endocrine pediatric patients with type 1 diabetes mellitus: a cross-sectional cohort study. cells as well as with the immune cells responding to J Clin Endocrinol Metab 2014;99:E1661–E1665 infection. Clearly, longitudinal studies of in vivo tis- 12. Ruan Q, Wang T, Kameswaran V, et al. The microRNA-21-PDCD4 axis prevents type 1 diabetes by blocking pancreatic beta cell death. Proc Natl Acad suefrommiceandhumanswilladdtotheconfirma- Sci U S A 2011;108:12030–12035 tion of potential viral etiology. 13. Ventriglia G, Nigi L, Sebastiani G, Dotta F. microRNAs: novel players in the In summary, CVB infection of human islets dramat- dialogue between pancreatic islets and immune system in autoimmune diabetes. fi ically alters the pro le of miRNA expression. Many of BioMed Res Int 2015;2015:749734 the affected miRNAs target the type 1 diabetes risk 14. Drescher KM, von Herrath M, Tracy S. 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This work was partially funded by a JDRF Career Development effects exerted by proin ammatory cytokines on pancreatic beta-cells. Diabetes 2010; – Award (5-CDA-2014-221-A-N) to S.J.R. 59:978 986 Duality of Interest. No potential conflicts of interest relevant to this article 19. Kim KW, Ho A, Alshabee-Akil A, et al. Coxsackievirus B5 infection induces were reported. dysregulation of microRNAs predicted to target known type 1 diabetes risk genes in human pancreatic islets. Diabetes 2016;65:996–1003 References 20. Laitinen OH, Honkanen H, Pakkanen O, et al. Coxsackievirus B1 is associated 1. Melton C, Judson RL, Blelloch R. Opposing microRNA families regulate self- with induction of b-cell autoimmunity that portends type 1 diabetes. Di- renewal in mouse embryonic stem cells. Nature 2010;463:621–626 abetes 2014;63:446–455