Beta cell extracellular vesicle miR-21-5p cargo is increased in response to inflammatory cytokines and serves as a biomarker of type 1 diabetes

Alexander J. Lakhter, Rachel E. Pratt, Rachel E. Moore, Kaitlin K. Doucette, Bernhard Maier, Linda DiMeglio, Emily K. Sims

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Aims/hypothesis: 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. Methods: 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. Results: 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. Conclusions/interpretation: 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.

Original languageEnglish (US)
Pages (from-to)1-11
Number of pages11
JournalDiabetologia
DOIs
StateAccepted/In press - Feb 14 2018

Fingerprint

MicroRNAs
Type 1 Diabetes Mellitus
Biomarkers
Cytokines
Serum
Inbred NOD Mouse
Extracellular Vesicles
Ultracentrifugation
Exosomes
Cell Line
Caspases
Interleukin-1
Nanoparticles
Culture Media
Rodentia

Keywords

  • Beta cell signal transduction
  • Cell lines
  • Human
  • Prediction and prevention of type 1 diabetes

ASJC Scopus subject areas

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism

Cite this

Beta cell extracellular vesicle miR-21-5p cargo is increased in response to inflammatory cytokines and serves as a biomarker of type 1 diabetes. / Lakhter, Alexander J.; Pratt, Rachel E.; Moore, Rachel E.; Doucette, Kaitlin K.; Maier, Bernhard; DiMeglio, Linda; Sims, Emily K.

In: Diabetologia, 14.02.2018, p. 1-11.

Research output: Contribution to journalArticle

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abstract = "Aims/hypothesis: 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. Methods: 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. Results: 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. Conclusions/interpretation: 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.",
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AU - Pratt, Rachel E.

AU - Moore, Rachel E.

AU - Doucette, Kaitlin K.

AU - Maier, Bernhard

AU - DiMeglio, Linda

AU - Sims, Emily K.

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