Browsing by Author "Xu, Jerry"

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    Beta cell extracellular vesicle PD-L1 as a novel regulator of CD8+ T cell activity and biomarker during the evolution of Type 1 Diabetes
    (bioRxiv, 2024-09-19) Rao, Chaitra; Cater, Daniel T.; Roy, Saptarshi; Xu, Jerry; Olivera, Andre De G.; Evans-Molina, Carmella; Piganelli, Jon D.; Eizirik, Decio L.; Mirmira, Raghavendra G.; Sims, Emily K.; Pediatrics, School of Medicine
    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.
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    Dynamic regulation of pancreatic β cell function and gene expression by the SND1 coregulator in vitro
    (Taylor & Francis, 2023) Kanojia, Sukrati; Davidson, Rebecca K.; Conley, Jason M.; Xu, Jerry; Osmulski, Meredith; Sims, Emily K.; Ren, Hongxia; Spaeth, Jason M.; Biochemistry and Molecular Biology, School of Medicine
    The pancreatic β cell synthesizes, packages, and secretes insulin in response to glucose-stimulation to maintain blood glucose homeostasis. Under diabetic conditions, a subset of β cells fail and lose expression of key transcription factors (TFs) required for insulin secretion. Among these TFs is Pancreatic and duodenal homeobox 1 (PDX1), which recruits a unique subset of transcriptional coregulators to modulate its activity. Here we describe a novel interacting partner of PDX1, the Staphylococcal Nuclease and Tudor domain-containing protein (SND1), which has been shown to facilitate protein-protein interactions and transcriptional control through diverse mechanisms in a variety of tissues. PDX1:SND1 interactions were confirmed in rodent β cell lines, mouse islets, and human islets. Utilizing CRISPR-Cas9 gene editing technology, we deleted Snd1 from the mouse β cell lines, which revealed numerous differentially expressed genes linked to insulin secretion and cell proliferation, including limited expression of Glp1r. We observed Snd1 deficient β cell lines had reduced cell expansion rates, GLP1R protein levels, and limited cAMP accumulation under stimulatory conditions, and further show that acute ablation of Snd1 impaired insulin secretion in rodent and human β cell lines. Lastly, we discovered that PDX1:SND1 interactions were profoundly reduced in human β cells from donors with type 2 diabetes (T2D). These observations suggest the PDX1:SND1 complex formation is critical for controlling a subset of genes important for β cell function and is targeted in diabetes pathogenesis.
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    The Chd4 Helicase Regulates Chromatin Accessibility and Gene Expression Critical for β-Cell Function In Vivo
    (American Diabetes Association, 2023) Davidson, Rebecca K.; Kanojia, Sukrati; Wu, Wenting; Kono, Tatsuyoshi; Xu, Jerry; Osmulski, Meredith; Bone, Robert N.; Casey, Nolan; Evans-Molina, Carmella; Sims, Emily K.; Spaeth, Jason M.; Biochemistry and Molecular Biology, School of Medicine
    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.
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    β-Cell pre-mir-21 induces dysfunction and loss of cellular identity by targeting transforming growth factor beta 2 (Tgfb2) and Smad family member 2 (Smad2) mRNAs
    (Elsevier, 2021-11) Ibrahim, Sara; Johnson, Macey; Hernandez Stephens, Clarissa; Xu, Jerry; Moore, Rachel; Mariani, Andrea; Contreras, Christopher; Syed, Farooq; Mirmira, Raghavendra G.; Anderson, Ryan M.; Sims, Emily K.; Biochemistry and Molecular Biology, School of Medicine
    Objective: β-cell microRNA-21 (miR-21) is increased by islet inflammatory stress but it decreases glucose-stimulated insulin secretion (GSIS). Thus, we sought to define the effects of miR-21 on β-cell function using in vitro and in vivo systems. Methods: We developed a tetracycline-on system of pre-miR-21 induction in clonal β-cells and human islets, along with transgenic zebrafish and mouse models of β-cell-specific pre-miR-21 overexpression. Results: β-cell miR-21 induction markedly reduced GSIS and led to reductions in transcription factors associated with β-cell identity and increased markers of dedifferentiation, which led us to hypothesize that miR-21 induces β-cell dysfunction by loss of cell identity. In silico analysis identified transforming growth factor-beta 2 (Tgfb2) and Smad family member 2 (Smad2) mRNAs as predicted miR-21 targets associated with the maintenance of β-cell identity. Tgfb2 and Smad2 were confirmed as direct miR-21 targets through RT-PCR, immunoblot, pulldown, and luciferase assays. In vivo zebrafish and mouse models exhibited glucose intolerance, decreased peak GSIS, decreased expression of β-cell identity markers, increased insulin and glucagon co-staining cells, and reduced Tgfb2 and Smad2 expression. Conclusions: These findings implicate miR-21-mediated reduction of mRNAs specifying β-cell identity as a contributor to β-cell dysfunction by the loss of cellular differentiation.