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Aims/hypothesis: Surviving beta cells in type 1 diabetes respond to inflammation by upregulating programmed death-ligand 1 (PD-L1) to engage immune cell programmed death protein 1 (PD-1) and limit destruction by self-reactive immune cells. Extracellular vesicles (EVs) and their cargo can serve as biomarkers of beta cell health and contribute to islet intercellular communication. We hypothesised that the inflammatory milieu of type 1 diabetes increases PD-L1 in beta cell EV cargo and that EV PD-L1 may protect beta cells against immune-mediated cell death. Methods: Beta cell lines and human islets were treated with proinflammatory cytokines to model the proinflammatory type 1 diabetes microenvironment. EVs were isolated using ultracentrifugation or size exclusion chromatography and analysed via immunoblot, flow cytometry and ELISA. EV PD-L1 binding to PD-1 was assessed using a competitive binding assay and in vitro functional assays testing the ability of EV PD-L1 to inhibit NOD CD8+ T cells. Plasma EV and soluble PD-L1 were assayed in the plasma of islet autoantibody-positive (Ab+) individuals or individuals with recent-onset type 1 diabetes and compared with levels in non-diabetic control individuals. Results: PD-L1 protein co-localised with tetraspanin-associated proteins intracellularly and was detected on the surface of beta cell EVs. Treatment with IFN-α or IFN-γ for 24 h induced a twofold increase in EV PD-L1 cargo without a corresponding increase in the number of EVs. IFN exposure predominantly increased PD-L1 expression on the surface of beta cell EVs and beta cell EV PD-L1 showed a dose-dependent capacity to bind PD-1. Functional experiments demonstrated specific effects of beta cell EV PD-L1 to suppress proliferation and cytotoxicity of murine CD8+ T cells. Plasma EV PD-L1 levels were increased in Ab+individuals, particularly in those positive for a single autoantibody. Additionally, in Ab+ individuals or those who had type 1 diabetes, but not in control individuals, plasma EV PD-L1 positively correlated with circulating C-peptide, suggesting that higher EV PD-L1 could be protective for residual beta cell function. Conclusions/interpretation: IFN exposure increases PD-L1 on the beta cell EV surface. Beta cell EV PD-L1 binds PD1 and inhibits CD8+ T cell proliferation and cytotoxicity. Circulating EV PD-L1 is higher in Ab+ individuals than in control individuals. Circulating EV PD-L1 levels correlate with residual C-peptide at different stages in type 1 diabetes progression. These findings suggest that EV PD-L1 could contribute to heterogeneity in type 1 diabetes progression and residual beta cell function and raise the possibility that EV PD-L1 could be exploited as a means to inhibit immune-mediated beta cell death. [ABSTRACT FROM AUTHOR]

The transcriptional activity of Pdx1 is modulated by a diverse array of coregulatory factors that govern chromatin accessibility, histone modifications, and nucleosome distribution. We previously identified the Chd4 subunit of the nucleosome remodeling and deacetylase complex as a Pdx1-interacting factor. To identify how loss of Chd4 impacts glucose homeostasis and gene expression programs in β-cells in vivo, we generated an inducible β-cell–specific Chd4 knockout mouse model. Removal of Chd4 from mature islet β-cells rendered mutant animals glucose intolerant, in part due to defects in insulin secretion. We observed an increased ratio of immature-to-mature insulin granules in Chd4-deficient β-cells that correlated with elevated levels of proinsulin both within isolated islets and from plasma following glucose stimulation in vivo. RNA sequencing and assay for transposase-accessible chromatin with sequencing showed that lineage-labeled Chd4-deficient β-cells have alterations in chromatin accessibility and altered expression of genes critical for β-cell function, including MafA, Slc2a2, Chga, and Chgb. Knockdown of CHD4 from a human β-cell line revealed similar defects in insulin secretion and alterations in several β-cell–enriched gene targets. These results illustrate how critical Chd4 activities are in controlling genes essential for maintaining β-cell function. Article Highlights: Pdx1–Chd4 interactions were previously shown to be compromised in β-cells from human donors with type 2 diabetes. β-Cell–specific removal of Chd4 impairs insulin secretion and leads to glucose intolerance in mice. Expression of key β-cell functional genes and chromatin accessibility are compromised in Chd4-deficient β-cells. Chromatin remodeling activities enacted by Chd4 are essential for β-cell function under normal physiological conditions. [ABSTRACT FROM AUTHOR]

Dendritic trafficking and translation of brain-derived neurotrophic factor (BDNF) transcripts play a key role in mediating synaptic plasticity. Recently, we demonstrated that siRNAmediated knockdown of translin, an RNA-binding protein, impairs KCl-induced dendritic trafficking of BDNF mRNA in cultured hippocampal neurons. We have now assessed whether translin deletion impairs dendritic trafficking of BDNF mRNA in hippocampal neurons in vivo. We have found that translin and its partner protein, trax, undergo dendritic translocation in response to treatment with pilocarpine, a pro-convulsant muscarinic agonist that increases dendritic trafficking of BDNF mRNA in hippocampal neurons. In translin knockout mice, the basal level of dendritic BDNF mRNA is decreased in CA1 pyramidal neurons. However, translin deletion does not block pilocarpine's ability to increase dendritic trafficking of BDNF mRNA indicating that the requirement for translin in this process varies with the stimulus employed to drive it. Consistent with this inference, we found that dendritic trafficking of BDNF mRNA induced by bath application of recombinant BDNF in cultured hippocampal neurons, is not blocked by siRNA-mediated knockdown of translin. Taken together, these in vivo and in vitro findings indicate that dendritic trafficking of BDNF mRNA can be mediated by both translin-dependent and -independent mechanisms. [ABSTRACT FROM AUTHOR]

The biosynthesis of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) requires the removal of the covalently linked viral polymerase from the 5= end of the minus strand [()strand] of viral relaxed circular DNA (rcDNA), which generates a deproteinated rcDNA (DP-rcDNA) intermediate. In the present study, we systematically characterized the four termini of cytoplasmic HBV DP-rcDNA by 5=/3= rapid amplification of cDNA ends (RACE), 5= radiolabeling, and exonuclease digestion, which revealed the following observations: (i) DP-rcDNA and rcDNA possess an identical 3= end of ()strand DNA; (ii) compared to rcDNA, DP-rcDNA has an extended but variable 3= end of plus strand [()strand] DNA, most of which is in close proximity to direct repeat 2 (DR2); (iii) DP-rcDNA exhibits an RNA primer-free 5= terminus of ()strand DNA with either a phosphate or hydroxyl group; and (iv) the 5= end of the DP-rcDNA ()strand is unblocked at nucleotide G1828, bearing a phosphate moiety, indicating the complete removal of polymerase from rcDNA via unlinking the tyrosyl-DNA phosphodiester bond during rcDNA deproteination. However, knockout of cellular 5= tyrosyl-DNA phosphodiesterase 2 (TDP2) did not markedly affect rcDNA deproteination or cccDNA formation. Thus, our work sheds new light on the molecular mechanisms of rcDNA deproteination and cccDNA biogenesis. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Surviving beta cells in type 1 diabetes respond to inflammation by upregulating programmed death-ligand 1 (PD-L1) to engage immune cell programmed death-1 (PD-1) and limit destruction by self-reactive immune cells. Extracellular vesicles (EVs) and their cargo can serve as biomarkers of beta cell health and contribute to islet intercellular communication. We hypothesized that the inflammatory milieu of type 1 diabetes increases PD-L1 in beta cell EV cargo and that EV PD-L1 may protect beta cells against immune-mediated cell death., Methods: Beta cell lines and human islets were treated with proinflammatory cytokines to model the proinflammatory type 1 diabetes microenvironment. EVs were isolated using ultracentrifugation or size exclusion chromatography and analysed via immunoblot, flow cytometry, and ELISA. EV PD-L1: PD-1 binding was assessed using a competitive binding assay and in vitro functional assays testing the ability of EV PD-L1 to inhibit NOD CD8 T cells. Plasma EV and soluble PD-L1 were assayed in plasma of individuals with islet autoantibody positivity (Ab+) or recent-onset type 1 diabetes and compared to non-diabetic controls., Results: PD-L1 protein colocalized with tetraspanin-associated proteins intracellularly and was detected on the surface of beta cell EVs. 24-h IFN-α or IFN-γ treatment induced a two-fold increase in EV PD-L1 cargo without a corresponding increase in number of EVs. IFN exposure predominantly increased PD-L1 expression on the surface of beta cell EVs and beta cell EV PD-L1 showed a dose-dependent capacity to bind PD-1. Functional experiments demonstrated specific effects of beta cell EV PD-L1 to suppress proliferation and cytotoxicity of murine CD8 T cells. Plasma EV PD-L1 levels were increased in islet Ab+ individuals, particularly in those with single Ab+, Additionally, in from individuals with either Ab+ or type 1 diabetes, but not in controls, plasma EV PD-L1 positively correlated with circulating C-peptide, suggesting that higher EV-PD-L1 could be protective for residual beta cell function., Conclusions/interpretation: IFN exposure increases PD-L1 on the beta cell EV surface. Beta cell EV PD-L1 binds PD1 and inhibits CD8 T cell proliferation and cytotoxicity. Circulating EV PD-L1 is higher in islet autoantibody positive patients compared to controls. Circulating EV PD-L1 levels correlate with residual C-peptide at different stages in type 1 diabetes progression. These findings suggest that EV PD-L1 could contribute to heterogeneity in type 1 diabetes progression and residual beta cell function and raise the possibility that EV PD-L1 could be exploited as a means to inhibit immune-mediated beta cell death.

β cell extracellular vesicles (EVs) play a role as paracrine effectors in islet health, yet mechanisms connecting β cell stress to changes in EV cargo and potential impacts on diabetes remain poorly defined. We hypothesized that β cell inflammatory stress engages neutral sphingomyelinase 2 (nSMase2)-dependent EV formation pathways, generating ceramide-enriched EVs that could impact surrounding β cells. Consistent with this, proinflammatory cytokine treatment of INS-1 β cells and human islets concurrently increased β cell nSMase2 and ceramide expression, as well as EV ceramide staining. Direct chemical activation or genetic knockdown of nSMase2, or treatment with a GLP-1 receptor agonist also modulated cellular and EV ceramide. Small RNA sequencing of ceramide-enriched EVs identified a distinct set of miRNAs linked to β cell function and identity. Coculture experiments using CD9-GFP tagged INS-1 cell EVs demonstrated that either cytokine treatment or chemical nSMase2 activation increased EV transfer to recipient cells. Children with recent-onset T1D showed no abnormalities in circulating ceramide-enriched EVs, suggesting a localized, rather than systemic phenomenon. These findings highlight nSMase2 as a regulator of β cell EV cargo and identify ceramide-enriched EV populations as a contributor to EV-related paracrine signaling under conditions of β cell inflammatory stress., Article Highlights: a. Why did we undertake this study?: Mechanisms connecting β cell stress to changes in extracellular vesicle (EV) cargo and potential impacts on diabetes are poorly defined. b. What is the specific question we wanted to answer?: Does β cell inflammatory stress engage neutral sphingomyelinase 2 (nSMase2)-dependent EV formation pathways to generate ceramide-enriched EVs. c. What did we find?: Proinflammatory cytokine treatment of β cells increased β cell ceramide expression, along with EV ceramide in part via increases in nSMase2. Ceramide-enriched EVs housed a distinct set of miRNAs linked to insulin signaling. Both cytokine treatment and nSMase2 activation increase EV transfer to other β cells. d. What are the implications of our findings?: Our findings highlight nSMase2 as a regulator of β cell EV cargo and identify ceramide-enriched EV populations as a contributor to EV-related paracrine signaling under conditions of β cell inflammatory stress.