In Type 1 diabetes (T1D), β-cell loss and autoimmunity initiate years before disease diagnosis. However, the mechanisms remain unknown. Here, we investigated the role and mechanisms of circulating extracellular vesicles (cEVs) on T1D pathogenesis and β-cell cytotoxicity. We used cEVs isolated from healthy donors (HD), T1D, multiple autoantibody-positive (AAb+), pre- and post-islet transplantation T1D subjects, NOD-T1D mice, and T1D PBMCs. Patient T1D-cEVs significantly induced apoptosis in vitro in human β-cells but not α-cells compared to HD-cEVs. cEVs from AAb+ subjects and pre-diabetic mice were cytotoxic, demonstrating that cEV-induced β-cell cytotoxicity precedes T1D onset and diagnosis. cEV reduction in prediabetic NOD-T1D mice improved β-cell health implying cEV contribution to T1D development. Proteomic analysis of patient T1D-cEVs found several proinflammatory proteins, including interferon gamma, which induced β-cell cytotoxicity. Our data using cEVs from pre- and post-islet transplant and PBMC-EVs from T1D patients implicated immune cells as a potential cellular source of cytotoxic cEVs in T1D. Collectively, our data using T1D patient samples coupled with studies in mice demonstrate that cEVs contribute to β-cell cytotoxicity and to the progression of T1D pathogenicity. Our study is one of the first to demonstrate a functional role of the proinflammatory cEV protein cargo on glucose homeostasis, β-cell health, and insulitis. Our studies on T1D-cEVs may lead to discovery of novel biomarkers and therapeutic targets for T1D.

Disclosure: N. Guthalu Kondegowda: None. Z. Cisneros: None. J. Do: None. J. Filipowska: None. R. Li: None. S. Zhang: None. S. Ogyaadu: None. C. Wasserfall: None. M. Atkinson: None. H. Reijonen: None. D. Roeth: None. M. Kalkum: None. C. Levy: None. F. Kandeel: None. S. Sahoo: None. S. Sahoo: None. R. Vasavada: None. Type 1 diabetes (T1D) results from diminished functional β-cell mass due to autoimmunity. Plasma-derived circulating extracellular vesicles (cEVs) contribute to multiple diseases. However, their impact on T1D remains unknown. Our finding that serum from T1D subjects is cytotoxic to human β-cells led to the hypothesis that cEVs mediate β-cell cytotoxicity through their distinct molecular cargo and thus contribute to T1D pathogenesis.cEVs isolated from T1D (<5 years since diagnosis), multiple autoantibody-positive (Aab+), age, sex, and ethnicity-matched non-diabetic healthy donor (HD), and T1D subjects pre- and post-islet transplantation, were characterized physically, and protein cargo identified. Human islet cells cultured with T1D or HD plasma (10% vol/vol) or cEVs (≥50 µg/ml) were examined for cell death by TUNEL staining. Glucose and β-cell homeostasis was assessed in NOD/ShiLtJj female mice, an autoimmune T1D mouse model, treated with GW4869, an EV secretion inhibitor. PBMC-derived EVs from T1D and HD subjects were evaluated for β-cell cytotoxicity. Plasma and cEVs from T1D but not HD subjects (n=10/group) significantly increased human β-cell but not α-cell death, mimicking disease pathology. Proteomic analysis (n=5/group) identified differential cargo in T1D vs HD cEVs. Proinflammatory cytokine, IFN-γ, was increased in T1D cEVs, mediating its β-cell cytotoxic effect (n=5/group). GW4869-treatment in NOD mice (n=8-9/group) reduced cEVs, improved glucose and β-cell homeostasis in vivo, and diminished β-cell cytotoxicity of serum ex vivo, supporting cEV contribution to T1D pathology. Of clinical relevance, serum and cEVs (n=7-8/group) from Aab+ subjects also induced human β-cell cytotoxicity, implying that humoral β-cell cytotoxicity precedes clinical disease-onset. cEVs from post-islet transplant T1D patients under immunosuppressive-regimen showed reduced β-cell cytotoxicity, suggesting that immune cells may be one source of cytotoxic cEVs in T1D. Supporting this, PBMC-derived EVs from T1D but not HD subjects (n=4/group), like cEVs, induced human β-cell cytotoxicity, which was reduced with in vitro immunosuppression.Our findings demonstrate a critical role of T1D-cEVs in disease pathology through their, selective cytotoxicity to human β-cells; distinctive proinflammatory protein cargo mediating the cytotoxicity; in vivo role in T1D pathogenesis; cytotoxic phenotype acquired prior to disease-onset; cytotoxicity modulated by immunosuppression; and potential source being PBMC. Presentation: Monday, July 14, 2025

Posttranslational modifications can enhance immunogenicity of self-proteins. In several conditions, including hypertension, systemic lupus erythematosus, and heart failure, isolevuglandins (IsoLGs) are formed by lipid peroxidation and covalently bond with protein lysine residues. Here, we show that the murine class I major histocompatibility complex (MHC-I) variant H-2Db uniquely presents isoLG-modified peptides and developed a computational pipeline that identifies structural features for MHC-I accommodation of such peptides. We identified isoLG-adducted peptides from renal proteins, including sodium glucose transporter 2, cadherin 16, Kelch domain–containing protein 7A, and solute carrier family 23, that are recognized by CD8+ T cells in tissues of hypertensive mice, induce T cell proliferation in vitro, and prime hypertension after adoptive transfer. Finally, we find patterns of isoLG-adducted antigen restriction in class I human leukocyte antigens that are similar to those in murine analogs. Thus, we have used a combined computational and experimental approach to define likely antigenic peptides in hypertension.

Impaired angiogenesis in diabetes is a key process contributing to ischemic diseases such as peripheral arterial disease. Epigenetic mechanisms, including those mediated by long noncoding RNAs (lncRNAs), are crucial links connecting diabetes and the related chronic tissue ischemia. Here we identify the lncRNA that enhances endothelial nitric oxide synthase (eNOS) expression (LEENE) as a regulator of angiogenesis and ischemic response. LEENE expression was decreased in diabetic conditions in cultured endothelial cells (ECs), mouse hind limb muscles, and human arteries. Inhibition of LEENE in human microvascular ECs reduced their angiogenic capacity with a dysregulated angiogenic gene program. Diabetic mice deficient in Leene demonstrated impaired angiogenesis and perfusion following hind limb ischemia. Importantly, overexpression of human LEENE rescued the impaired ischemic response in Leene-knockout mice at tissue functional and single-cell transcriptomic levels. Mechanistically, LEENE RNA promoted transcription of proangiogenic genes in ECs, such as KDR (encoding VEGFR2) and NOS3 (encoding eNOS), potentially by interacting with LEO1, a key component of the RNA polymerase II-associated factor complex and MYC, a crucial transcription factor for angiogenesis. Taken together, our findings demonstrate an essential role for LEENE in the regulation of angiogenesis and tissue perfusion. Functional enhancement of LEENE to restore angiogenesis for tissue repair and regeneration may represent a potential strategy to tackle ischemic vascular diseases.

Post translational modifications can enhance immunogenicity of self-proteins. In several conditions including hypertension, systemic lupus, and heart failure, isolevuglandins (IsoLGs) are formed by lipid peroxidation and covalently bond with protein lysine residues. Here we show that the murine class-I major histocompatibility complex (MHC-I) variant H-2D b uniquely presents isoLG modified peptides and developed a computational pipeline that identifies structural features for MHC-I accommodation of such peptides. We identified isoLG-adducted peptides from renal proteins including the sodium glucose transporter 2, Cadherin 16, Kelch Domain containing protein 7A and solute carrier family 23, that are recognized by CD8 + T cells in tissues of hypertensive mice, induce T cell proliferation in vitro , and prime hypertension after adoptive transfer. Finally, we find similar patterns of isoLG-adducted antigen restriction in class-I human leukocyte antigens as in murine analogues. Thus, we have used a combined computational and experimental approach to define likely antigenic peptides in hypertension.

The C allele of rs11136000 variant in the clusterin (CLU) gene represents the third strongest known genetic risk factor for late-onset Alzheimer’s disease. However, whether this single-nucleotide polymorphism (SNP) is functional and what the underlying mechanisms are remain unclear. In this study, the CLU rs11136000 SNP is identified as a functional variant by a small-scale CRISPR-Cas9 screen. Astrocytes derived from isogenic induced pluripotent stem cells (iPSCs) carrying the “C” or “T” allele of the CLU rs11136000 SNP exhibit different CLU expression levels. TAR DNA-binding protein-43 (TDP-43) preferentially binds to the “C” allele to promote CLU expression and exacerbate inflammation. The interferon response and CXCL10 expression are elevated in cytokine-treated C/C astrocytes, leading to inhibition of oligodendrocyte progenitor cell (OPC) proliferation and myelination. Accordingly, elevated CLU and CXCL10 but reduced myelin basic protein (MBP) expression are detected in human brains of C/C carriers. Our study uncovers a mechanism underlying reduced white matter integrity observed in the CLU rs11136000 risk “C” allele carriers.

Pseudouridine is the most frequent epitranscriptomic modification. However, its cellular functions remain largely unknown. Here, we show that pseudouridine synthase 7 (PUS7) is highly expressed in glioblastoma versus normal brain tissues, and high PUS7 expression levels are associated with worse survival in patients with glioblastoma. PUS7 expression and catalytic activity are required for glioblastoma stem cell (GSC) tumorigenesis. Mechanistically, we identify PUS7 targets in GSCs through small RNA pseudouridine sequencing and show that pseudouridylation of PUS7-regulated transfer RNA is critical for codon-specific translational control of key regulators of GSCs. Moreover, we identify chemical inhibitors for PUS7 and show that these compounds prevent PUS7-mediated pseudouridine modification, suppress tumorigenesis and extend the life span of tumor-bearing mice. Overall, we identify an epitranscriptomic regulatory mechanism in glioblastoma and provide preclinical evidence of a potential therapeutic strategy for glioblastoma. Shi and colleagues show that PUS7 controls tRNA pseudouridylation and codon-specific translation to fuel glioblastoma tumorigenesis, and discover a PUS7 inhibitor that delays tumor growth in glioblastoma models.

RNA splicing dysregulation is a hallmark of chronic lymphocytic leukemia (CLL). Although somatic mutations in SF3B1 or U1 snRNA present in >20% of CLL patients, general splicing defects cannot be fully explained by genetic alterations of spliceosome alone. We reported that splicing factors are upregulated at protein, but not RNA, level in CLL compared to normal B cells by an integrated transcriptomic and proteomic analysis. This highlights a post-transcriptional layer of regulation that controls the abundance of splicing factors and contributes to RNA splicing dysregulation in CLL, with mechanism that has yet to be elucidated.

Early work in rodents highlighted the gut microbiota’s importance in metabolic disease, including Type II Diabetes Mellitus (T2DM) and obesity. Glucagon-like peptide-1 (GLP-1), an incretin secreted by L-cells lining the gastrointestinal epithelium, has important functions: promoting insulin secretion, insulin sensitivity, and β-cell mass, while inhibiting gastric emptying and appetite. We set out to identify microbial strains with GLP-1 stimulatory activity as potential metabolic disease therapeutics. Over 1500 human-derived strains were isolated from healthy individuals and screened for GLP-1 modulation by incubating bacterial cell-free supernatants with NCI H716 L-cells. Approximately 45 strains capable of increasing GLP-1 were discovered. All GLP-1 positive strains were identified as Staphylococcus epidermidis by 16S rRNA sequencing. Mass spectrometry analysis identified a 3 kDa peptide, Hld (delta-toxin), present in GLP-1 positive supernatants but absent in GLP-1 neutral supernatants. Studies in NCI-H716 cells and human jejunal enteroids engineered to make more enteroendocrine cells demonstrated that Hld alone is sufficient to enhance GLP-1 secretion. When administered in high-fat-fed mice, Hld-producing S. epidermidis significantly reduced markers associated with obesity and T2DM. Further characterization of Hld suggests GLP-1 stimulatory action of Hld occurs via calcium signaling. The presented results identify a novel host-microbe interaction which may ultimately lead to the development of a microbial peptide-based therapeutic for metabolic disease.

Aberrant mRNA processing is known to drive the pathogenesis of chronic lymphocytic leukemia (CLL). Recurrent gene mutations in the RNA splicing factor SF3B1 and widespread RNA intronic polyadenylation impact genome-wide gene expression and inactivate tumor suppressors, respectively. Nevertheless, how mRNA processing is regulated and exerts its function in CLL remain elusive. To comprehensively characterize the role of mRNA processing in CLL, we performed RNA sequencing (RNA-seq) and Tandem Mass Tag (TMT) proteomics using normal and CLL B cells derived from healthy donors (n=5) and untreated CLL patients (n=22). We detected 328 proteins differentially expressed between normal and CLL B cells (|Log2FC|>0.58, q<0.05). Gene set enrichment analysis (GSEA) revealed that proteins involved in RNA metabolism (transcription, splicing, modification, 3'end processing, nuclear export, decay) were upregulated in CLL, while those impacting translation were downregulated. These findings were validated by immunoblotting in an independent set of samples (n=10). However, we observed no significant gene expression changes of RNA metabolism at the transcript level, indicating that regulation of these proteins occurred post-transcriptionally. Since N6-methyladenosine (m6A) is the most abundant RNA internal modification and has emerged as a key regulator for RNA metabolism, we sought to determine whether m6A is dysregulated in CLL cells. With an m6A dotblot assay and HPLC-MS, we consistently detected increased level of m6A in mRNA from CLL cells compared with normal B cells. As one of the most upregulated proteins in CLL, METTL3 writes m6A and promotes translation efficiency through its writer and reader functions, respectively. When we knocked down (KD) METTL3 in CLL cell lines (HG3, MEC1) as well as in primary CLL cells, we observed significant cell death and growth disadvantage in CLL compared to control cells, highlighting METTL3 is essential for CLL survival. We next examined whether KD of METTL3 affects m6A and RNA translation using m6A dotblot and O-propargyl-puromycin run on assays. Loss of METTL3 had subtle impact on m6A levels but it significantly decreased protein translation (t test, p<0.01) in all the cell lines tested (HG3, MEC1, JeKo-1, Mino). To define the target protein that METTL3 affects, we performed an integrated Ribosome profiling and RNA-seq analysis using HG3 and Mino cells with or without METTL3. At both transcriptome and translatome levels, loss of METTL3 significantly decreased genes enriched in the mTORC1 pathway, which has an essential role in translation (Metascape, hypergeometric test, q<0.05). Furthermore, it also decreased the translation efficiencies of genes involved in mRNA processing, DNA synthesis, and cell cycle pathways. This observation suggests that upregulation of METTL3 in CLL cells may regulate protein translation of the RNA metabolism pathway. Since m6A at the stop codon region is critical for METTL3 regulating protein translation, we performed MAZTER sequencing to determine m6A modification sites in normal and CLL B cells derived from healthy donors (n=5) and untreated CLL patients (n=11). We identified 214 genes with significant differential m6A modification at the stop codon region (delta cleavage efficiency>0.1, Wilcoxon rank-sum test, p<0.1, within DRACA motif) between normal and CLL B cells. These genes were highly enriched for mRNA processing (Metascape, q=0.017), supporting our notion that METTL3 may modulate protein expression of mRNA processing genes by recognizing m6A modification via its reader function in CLL. Consistent with its role in regulating protein expression, we detected downregulation of splicing factors (SF3A1, SF3A2, SF3B1, U2AF1) in various METTL3 KD cell lines (HG3, MEC1, JeKo-1, Mino) at only protein level but not transcription level. These data link METTL3 upregulation with RNA metabolism protein enrichment in CLL. Altogether, our integrated analysis uncovered a novel regulatory axis of METTL3 in CLL biology. We demonstrated that CLL cells have an increased m6A modification and upregulation of METTL3 at the protein level, resulting in translation of RNA metabolism related genes through its reader function by the recognition of m6A modification. Our results collectively suggest METTL3 as a central regulator for mRNA processing in CLL and provide a rationale for targeting METTL3 in this disease. Disclosures Brown: Janssen, Teva: Speakers Bureau; Gilead, Loxo, Sun, Verastem: Research Funding; Abbvie, Acerta, AstraZeneca, Beigene, Invectys, Juno/Celgene, Kite, Morphosys, Novartis, Octapharma, Pharmacyclics, Sunesis, TG Therapeutics, Verastem: Consultancy. Rosen:Seattle Genetics: Consultancy; NeoGenomics: Consultancy; Aileron Therapeutics: Consultancy; Novartis: Consultancy; Pebromene: Consultancy; Celgene: Speakers Bureau; paradigm Medical Communications: Speakers Bureau; Abbvie: Speakers Bureau. Siddiqi:TG Therapeutics: Research Funding; Janssen: Speakers Bureau; Seattle Genetics: Speakers Bureau; Oncternal: Research Funding; BeiGene: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Juno: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Research Funding, Speakers Bureau.

The “free radical theory of aging” suggests that reactive oxygen species (ROS) are responsible for age‐related loss of cellular functions and, therefore, represent the main cause of aging. Redox regulation by thioredoxin‐1 (TRX) plays a crucial role in responses to oxidative stress. We show that thioredoxin‐interacting protein (TXNIP), a negative regulator of TRX, plays a major role in maintaining the redox status and, thereby, influences aging processes. This role of TXNIP is conserved from flies to humans. Age‐dependent upregulation of TXNIP results in decreased stress resistance to oxidative challenge in primary human cells and in Drosophila. Experimental overexpression of TXNIP in flies shortens lifespan due to elevated oxidative DNA damage, whereas downregulation of TXNIP enhances oxidative stress resistance and extends lifespan.

We and others have shown an important role CD8+ cells in both experimental and human hypertension. CD8+ T cells are activated by antigens presented by major histocompatibility complex 1 (MHC1). C57BL/6 mice express two MHC1, referred to as H2-Kb and H2-Db. To identify antigenic peptides responsible for hypertension, we made two transgenic mice lacking the transmembrane domains of MHC1 and an added His-tag to the modified MHC1. These transgenes are driven by CD11c promoter, allowing expression in antigen presenting cells. The soluble Kb and Db (sKb and sDb) mice received 2 week infusions of sham or angiotensin II and their splenocytes placed in culture for two days. Ni-NTA beads were then used to bind the shed MHC-1 and these beads mixed with 10 6 T cells from other ang II infused mice (Figure panel A). CFSE dilution was used to monitor T cell proliferation. We found that sDb from ang II infused male mice, but not sham infused mice, potently stimulated proliferation of CD8+ T cells from ang II infused male mice. This degree of stimulation was significantly greater than that observed by sKb (Figure panel B). The shed MHC1 from female ang II-treated mice caused significantly less stimulation of female CD8+ T cells compared to male MHC1, in keeping with prior observations that female mice have less immune activation in hypertension. In summary, these data strongly suggest that unique antigens, presented in the context of H2-Db, are generated in hypertension. These data also suggest that there may be unique human lymphocyte antigens (HLAs), the analog of mouse MHC, that predispose to hypertension and related end-organ damage.

Bacterial‐derived compounds from the intestinal microbiome modulate host mucosal immunity. Identification and mechanistic studies of these compounds provide insights into host–microbial mutualism. Specific Lactobacillus reuteri strains suppress production of the proinflammatory cytokine, tumor necrosis factor (TNF), and are protective in a mouse model of colitis. Human‐derived L. reuteri strain ATCC PTA 6475 suppresses intestinal inflammation and produces 5,10‐methenyltetrahydrofolic acid polyglutamates. Insertional mutagenesis identified the bifunctional dihydrofolate synthase/folylpolyglutamate synthase type 2 ( folC2 ) gene as essential for 5,10‐methenyltetrahydrofolic acid polyglutamate biosynthesis, as well as for suppression of TNF production by activated human monocytes, and for the anti‐inflammatory effect of L. reuteri 6475 in a trinitrobenzene sulfonic acid‐induced mouse model of acute colitis. In contrast, folC encodes the enzyme responsible for folate polyglutamylation but does not impact TNF suppression by L. reuteri . Comparative transcriptomics between wild‐type and mutant L. reuteri strains revealed additional genes involved in immunomodulation, including previously identified hdc genes involved in histidine to histamine conversion. The folC2 mutant yielded diminished hdc gene cluster expression and diminished histamine production, suggesting a link between folate and histadine/histamine metabolism. The identification of genes and gene networks regulating production of bacterial‐derived immunoregulatory molecules may lead to improved anti‐inflammatory strategies for digestive diseases.

Mitochondrial reactive oxygen species (ROS) are indispensible for T cell activation-induced expression of interleukin 2 (IL-2) and CD95 ligand (CD95L, FasL/Apo-1L) genes, and in turn, for CD95L-mediated activation-induced cell death (AICD). Here, we show that manganese superoxide dismutase (MnSOD/SOD2), a major mitochondrial antioxidative enzyme, constitutes an important control switch in the process of activation-induced oxidative signal generation in T cells. Analysis of the kinetics of T cell receptor (TCR)-triggered ROS production revealed a temporal association between higher MnSOD abundance/activity and a shut-down phase of oxidative signal generation. Transient or inducible MnSOD overexpression abrogated T cell activation-triggered mitochondrial ROS production as well as NF-κB- and AP-1-mediated transcription. Consequently, lowered expression of IL-2 and CD95L genes resulted in decreased IL-2 secretion and CD95L-dependent AICD. Moreover, upregulation of the mitochondrial MnSOD level is dependent on oxidation-sensitive transcription and not on the increase of mitochondrial mass. Thus, MnSOD-mediated negative feedback regulation of activation-induced mitochondrial ROS generation exemplifies a process of retrograde mitochondria-to-nucleus communication. Our finding underlines the critical role for MnSOD and mitochondria in the regulation of human T cell activation.