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

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.

Keywords: Beta cell signal transduction; Cell lines; Human; Prediction and prevention of type 1 diabetes.

Fig. 1

Inflammatory cytokines increase beta cell EV miR-21-5p cargo. MIN6 and EndoC beta cells and human islets were treated with vehicle or a mix of IL-1β, INF-γ and TNF-α for 24 h. (a–c) miR-21-5p expression was assessed in MIN6 cells (n=3) (a), EndoC cells(n=6) (b) and human islets (n-10)(c);. (d–f) miR-21-5p levels were assessed in EVs from MIN6 cells (n=3) (d), EndoC cells (n=3) (e) and human islets (n=6) (f);. (g–i) NTA was performed to profile EV particle concentration and size distribution in MIN6 cells (n=3) (g), EndoC beta cells (n=3)(h) and human islets (n=3) (i). (j, k) MIN6 cells (n=3) (j) and EndoC cells (n=3) (k) were treated with inflammatory cytokines or cytokines in combination with the pan-caspase inhibitor Z-VAD-FMK. Effects on relative levels of EV miR-21-5p were assessed by qPCR. Results are displayed as mean ± SD; *p≤0.05, **p≤0.01 and ***p≤0.001. White circles, vehicle; black circles, cytokines. Cyto, cytokines; Veh, vehicle; RQ, relative quantity

Fig. 2

Cytokine-induced increase in beta cell EV miR-21-5p is predominantly due to changes in exosome cargo. Media from vehicle- and cytokine-treated MIN6 cells, EndoC cells and human islets was separated by sequential centrifugation into large EV (apoptotic bodies), intermediate-sized EV (microvesicles) and small EV (exosomes) fractions. EV-depleted media was also retained. (a, b) Immunoblot (a) and TEM (b) analysis of EndoC cell-derived EVs was used to validate EV isolation by serial ultracentrifugation. TEM images display representative data from three independent experiments. Scale bars, 400 nm. (c–e) Relative levels of miR-21-5p were assessed by qPCR. (n=3 in MIN6 and EndoC cells; n=5 in human islets) (f–h) NTA of each ultracentrifugation fraction from EndoC cells was used to establish EV quantity and size distribution; (n=3 in MIN6 and EndoC cells; n=5 in human islets). White bars, vehicle-treated media; black bars, cytokine-treated media. Results are displayed as mean ± SD; **p≤0.01 and ***p≤0.001. EXO, exosomes; MV, microvesicles; RQ, relative quantity

Fig. 3

Elevation in circulating EV miR-21-5p precedes onset of diabetes in NOD mice. (a, b) qPCR was performed to quantify relative levels of EV miR-21-5p in islets (a) and terminal serum (b) of diabetic NOD mice (n=4) compared with NOR controls (n=5). (c) Specificity of EV isolation was validated by western blotting of total serum and circulating EVs, probed for EV markers CD63 and CD9 and the endoplasmic reticulum marker calreticulin. (d) Longitudinal weekly serum collection and blood glucose assessment of NOD (n=7–9) and control NOR (n=5) mice were performed, starting at 8 weeks (wks) of age until either development of diabetes or 20 weeks of age. (e) qPCR was performed to quantify serum EV miR-21-5p in NOD mice vs NOR control mice according to age (n=3–9/group). (f) Serum EV miR-21-5p in NOD mice and age-matched NOR control mice was analysed in relation to diabetes onset (defined as first glucose >4.4 mmol/l). (n=3–9/group) (g, h) NTA of serum EVs in NOD mice showed no significant changes with relation to age (g) or diabetes onset (n=3–9/group) (h). Results are displayed as mean ± SD; ^†p=0.053, *p≤0.05, **p≤0.01 and ***p≤0.001. White circles, NOR; black circles, NOD. BG, blood glucose; RQ, relative quantity, T1D, type 1 diabetes

Fig. 4

Circulating EV miR-21-5p is elevated in children with new-onset type 1 diabetes. (a, b) Serum samples from healthy control children and children with new-onset type 1 diabetes were assessed for relative levels of circulating miR-21-5p in whole serum (a) and in circulating EVs (b). (c) Circulating EV miR-21-5p normalised to total serum miR-21-5p. (d–f) NTA was performed to quantify serum EV concentration and size distribution. (e) Quantification of total particle concentration by group. (f) Quantification of particle size by group. (g) Serum EV miR-21-5p level normalised to total serum particle number. (h, i) Relative levels of total serum miR-375-5p (h) and circulating EV miR-375-5p (i) were also quantified. (j) Relationship between serum EV miR-21-5p and EV miR-375-5p in samples from children with type 1 diabetes. (k) EV isolation from serum was validated by TEM; a representative image from three independent experiments is shown. Scale bar, 400 nm. Results are shown as median ± interquartile range (a–c, e–j) or mean ± SEM (d); *p≤0.05, **p≤0.01 and ***p≤0.001. White circles, healthy controls, n=16; black circles, type 1 diabetes, n=19. HC, healthy controls; RQ, relative quantity; T1D, type 1 diabetes