Beta cell extracellular vesicle miR-21-5p cargo is increased in response to inflammatory cytokines and serves as a biomarker of type 1 diabetes
Improved biomarkers are acutely needed for the detection of developing type 1 diabetes, prior to critical loss of beta cell mass. We previously demonstrated that elevated beta cell microRNA 21-5p (miR-21-5p) in rodent and human models of type 1 diabetes increased beta cell apoptosis. We hypothesised that the inflammatory milieu of developing diabetes may also increase miR-21-5p in beta cell extracellular vesicle (EV) cargo and that circulating EV miR-21-5p would be increased during type 1 diabetes development.
MIN6 and EndoC-βH1 beta cell lines and human islets were treated with IL-1β, IFN-γ and TNF-α to mimic the inflammatory milieu of early type 1 diabetes. Serum was collected weekly from 8-week-old female NOD mice until diabetes onset. Sera from a cross-section of 19 children at the time of type 1 diabetes diagnosis and 16 healthy children were also analysed. EVs were isolated from cell culture media or serum using sequential ultracentrifugation or ExoQuick precipitation and EV miRNAs were assayed.
Cytokine treatment in beta cell lines and human islets resulted in a 1.5- to threefold increase in miR-21-5p. However, corresponding EVs were further enriched for this miRNA, with a three- to sixfold EV miR-21-5p increase in response to cytokine treatment. This difference was only partially reduced by pre-treatment of beta cells with Z-VAD-FMK to inhibit cytokine-induced caspase activity. Nanoparticle tracking analysis showed cytokines to have no effect on the number of EVs, implicating specific changes within EV cargo as being responsible for the increase in beta cell EV miR-21-5p. Sequential ultracentrifugation to separate EVs by size suggested that this effect was mostly due to cytokine-induced increases in exosome miR-21-5p. Longitudinal serum collections from NOD mice showed that EVs displayed progressive increases in miR-21-5p beginning 3 weeks prior to diabetes onset. To validate the relevance to human diabetes, we assayed serum from children with new-onset type 1 diabetes compared with healthy children. While total serum miR-21-5p and total serum EVs were reduced in diabetic participants, serum EV miR-21-5p was increased threefold compared with non-diabetic individuals. By contrast, both serum and EV miR-375-5p were increased in parallel among diabetic participants.
We propose that circulating EV miR-21-5p may be a promising marker of developing type 1 diabetes. Additionally, our findings highlight that, for certain miRNAs, total circulating miRNA levels are distinct from circulating EV miRNA content.
KeywordsBeta cell signal transduction Cell lines Human Prediction and prevention of type 1 diabetes
Nanoparticle tracking analysis
Quantitative real-time PCR
Transmission electron microscopy
We thank C. Evans-Molina and R. Mirmira (Departments of Medicine and Pediatrics, Indiana University School of Medicine) for their support in developing this project. We thank the Indiana University Islet and Physiology core for assistance with serum collection and islet isolation and the University of Nebraska College of Medicine Electron Microscopy Core for assistance with TEM imaging. This work has been partially presented in oral abstract form and in poster presentations at the 2015–2017 American Diabetes Association Scientific Sessions, 2017 Human Islet Research Network, Pediatric Endocrine Society, Endocrine Society, 2015–2016 Midwest Islet Club, 2016 Central Society for Clinical and Translational Research, 2017 Extracellular RNA Communication Consortium and the 2017 International Society for Extracellular Vesicles meeting.
AJL, REP, REM, and KKD performed the experiments, acquired the data and revised the manuscript. BFM made substantial contributions to study design and analysis of data and revised the manuscript. LAD made substantial contributions to acquisition of data and revised the manuscript. AJL and EKS designed the experiments, acquired and interpreted data, and drafted the manuscript. EKS is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors provided final approval of the version to be published.
This work was supported by NIDDK K08DK103983 (EKS), Pediatric Endocrine Society Clinical Scholar Award (EKS), a pilot and feasibility award within the Center for Diabetes and Metabolic Diseases NIH/NIDDK grant number P30 DK097512, funding by Indiana University Health and the Indiana Clinical and Translational Sciences Institute (EKS), Grant no. UL1TR001108, a Ralph W. and Grace M. Showalter research award (EKS), JDRF Pioneer Award and Strategic Research Agreement (LAD) and NIH grant no. 32DK064466 (AJL). This study utilised core services provided by the Diabetes Research Center grant P30 DK097512 to Indiana University School of Medicine. Human pancreatic islets were provided by the NIDDK-funded Integrated Islet Distribution Program (IIDP) at City of Hope, NIH grant no. 2UC4DK098085-02.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
- 5.Heninger A-K, Eugster A, Kuehn D, et al. (2017) A divergent population of autoantigen-responsive CD4+ T cells in infants prior to β cell autoimmunity. Science Translational Medicine 9Google Scholar
- 8.Gould SJ, Raposo G (2013) As we wait: coping with an imperfect nomenclature for extracellular vesicles. J Extracell Vesicles. https://doi.org/10.3402/jev.v2i0.20389
- 24.Thery C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol 3:3.22.1–3.22.29Google Scholar
- 27.Stull ND, Breite A, McCarthy R, Tersey SA, Mirmira RG (2012) Mouse islet of Langerhans isolation using a combination of purified collagenase and neutral protease. J Vis Exp 67:4137Google Scholar
- 28.Crescitelli R, Lasser C, Szabo TG et al (2013) Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes. J Extracell Vesicles. https://doi.org/10.3402/jev.v2i0.20677
- 43.Nielsen LB, Wang C, Sørensen K et al (2012) Circulating levels of microRNA from children with newly diagnosed type 1 diabetes and healthy controls: evidence that miR-25 associates to residual beta-cell function and glycaemic control during disease progression. Exp Diabetes Res 2012:896362PubMedPubMedCentralGoogle Scholar