IntroductionSepsis survivors frequently develop long-term immune dysfunction, but the epigenetic mechanisms underlying persistent myeloid suppression remain unclear. Myeloid-derived suppressor cells (MDSCs), whose function is shaped by host age and sex, are key contributors to post-sepsis immune dysregulation.MethodsHere, we present a high-resolution epigenetic map targeting gene promoters of MDSCs after sepsis and daily chronic stress using MAPit-FENGC, a single-molecule assay that simultaneously profiles DNA methylation and chromatin accessibility. In a clinically relevant murine model, including young and older adult male and female mice, splenic MDSCs were isolated for MAPit-FENGC and single-cell RNA sequencing.ResultsUnsupervised clustering identified nine promoter classes reflecting chromatin dynamics: age- and sex-dependent sepsis-induced opening (Classes 1-4), persistent closure with varying levels of DNA methylation (Classes 5-7), and constitutive openness post-sepsis (Classes 8, 9). Transcriptomic profiling corroborated these promoter states, linking accessibility with gene expression.ConclusionsThese findings define promoter-level epigenetic classes across a targeted locus panel in splenic CD11b+Gr1+ cells within this murine sepsis model and generate mechanistic hypotheses regarding age- and sex-associated chromatin states.

Type 1 diabetes (T1D) results from dysfunction and loss of the insulin-producing pancreatic β cells. The body’s lipid metabolism is strongly regulated during this process but there is a need to understand how this regulation contributes to the β-cell death. Here, we investigated the role of free fatty acids (FFAs) in T1D development. Lipidomics data from the case-control study of The Determinants of Diabetes in the Young (TEDDY) consortium were re-analyzed to determine temporal changes in lipid profiles during T1D development. Fatty acid distribution across the pancreas was measured by mass spectrometry imaging. To model islet inflammation, FFAs were measured in human islets treated with the pro-inflammatory cytokines IL-1β + IFNγ by gas chromatography-mass spectrometry. Further, the effects of FFAs on MIN6 insulin-producing cells were measured by proteomics analysis along with biochemical and cell biology assays. We investigated if similar effects occur in vivo during T1D on the islets’ single-cell RNA sequencing data from Human Pancreas Analysis Program (HPAP). During islet autoimmunity, prior to T1D onset, plasma phosphatidylcholine and triacylglycerols are reduced with a simultaneous increase of FFAs. Similarly in human islets, IL-1β + IFNγ induce the increase of palmitate. Moreover, FFAs are abundantly detected across islets and surrounding exocrine tissue. FFAs synergistically enhanced cytokine-mediated apoptosis in MIN6 cells by downregulating the production of nicotinamide adenosine dinucleotide (NAD) via downregulating nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme of the NAD salvage pathway. The enhancement of cytokine-mediated apoptosis was reverted by supplementing cells with nicotinic acid and nicotinamide mononucleotide, metabolites that bypass NAMPT in NAD biosynthesis. NAMPT downregulation was further observed during T1D development, supporting that NAD production might be compromised in vivo. Our findings show that fatty acids are released during islet autoimmunity. These fatty acids enhance pro-inflammatory cytokine-mediated apoptosis through impaired NAD metabolism.

Melanoma differentiation-associated protein 5 (MDA5), encoded by IFIH1, is a cytosolic double-stranded RNA (dsRNA) sensor. Mutation of IFIH1 resulting in MDA5 deficiency causes immune dysfunction and predisposition to specific respiratory viral pathogens due to the inability of innate immune system to detect viral dsRNA. Additionally, gene variants in IFIH1 have been linked to autoimmunity, including type 1 diabetes. To understand structure-function, we integrate structural biology and signaling principles to explain how MDA5 architecture governs interactions with dsRNA and type 1 interferon (T1-IFN) outputs. MDA5 binds dsRNA via its helicase core and C-terminal domain, uses ATP-dependent conformational cycling to assemble filaments, and exposes N-terminal CARDs that nucleate mitochondrial antiviral signaling protein polymerization, activating TBK1/IKKε-IRF and NF-κB programs that amplify T1-IFN production and inflammatory gene expression. Risk-associated IFIH1 alleles are predicted to increase T1-IFN production/activation thresholds, whereas rare loss-of-function variants attenuate T1-IFN outputs and confer protection. Finally, we outline therapeutic entry points that preserve antiviral defense while constraining chronic T1-IFN signaling to restrain MHC class I expression, chemokine production, and autoreactive T-cell recruitment. Targeting downstream pathways with small molecule inhibitors may delay early autoimmunity and target tissue functions in genetically defined subgroups.

The human pancreas is a structurally and functionally complex organ in which endocrine islets are embedded within an exocrine matrix. Despite advances in spatial omics, three-dimensional (3D) proteomic map of the human islet microenvironment remains lacking. Here, we present a 3D spatial proteomics workflow that integrates immunofluorescence imaging, laser capture microdissection, nanoPOTS processing, and LC-MS/MS to map the islet microenvironment at 50 μm resolution, achieving ~3,000 protein identifications with spatial fidelity. Unsupervised clustering analyses revealed four molecularly distinct microenvironments spanning the acinar-to-islet axis, including a previously underappreciated peri-islet ductal-stromal niche enriched with canonical collagens and other extracellular matrix (ECM) proteins. Spatial correlation analysis (linking abundances to relative distance from the islet center) revealed proteins with interesting, reversed correlation patterns between islet and acinar compartments, which were enriched with ECM and cytoskeletal components. Finally, we provide a publicly accessible interactive web-based platform, enabling integrated 3D visualization of the spatial proteome alongside immunofluorescence imaging. Collectively, this work establishes a proof-of-principle framework for studying spatial tissue proteomic profiles, advances our understanding of the islet microenvironment, and lays a potential foundation for future applications in disease research.

Sepsis remains a leading cause of morbidity and mortality worldwide, with survivors often following divergent trajectories: rapid recovery (RAP) or progression to chronic critical illness (CCI). CCI is characterized by persistent organ dysfunction, recurrent infections, and immune dysregulation. Myeloid-derived suppressor cells (MDSCs), which expand in number after sepsis, are implicated in this maladaptive state, yet their epigenetic regulation remains poorly understood. Here, we applied an Omni-ATAC protocol optimized to profile chromatin accessibility in CD66b + MDSCs from healthy participants (HPs) and sepsis patients across time points (day 4, day 14-21, and 6 months) and clinical outcomes (RAP, CCI, and Deceased). Dimensionality reduction analyses of genome-wide chromatin accessibility showed clear separation of sepsis and HP samples. Furthermore, these analyses revealed distinct trajectories post-sepsis diagnosis: RAP samples progressively regained HP-like chromatin states, whereas CCI samples remained epigenetically "locked" in aberrant states. Differential accessibility analysis identified thousands of promoter regions with altered accessibility, including immune checkpoint and inflammatory genes (e.g., ARG1, CD274, S100A8 / 9 ). Pathway analyses predicted global suppression of immune, metabolic, and chromatin remodeling programs in CCI, contrasting with restoration in RAP. These findings from patient-derived CD66b + MDSCs suggest that epigenetic chromatin remodeling underlies divergent recovery trajectories and highlight chromatin-modifying pathways as potential therapeutic targets to restore immune competence in sepsis patients with CCI.

Sepsis induces profound immune dysregulation, often resulting in chronic critical illness characterized by persistent immunosuppression and poor outcomes. Myeloid-derived suppressor cells (MDSCs) are central mediators of this immunosuppressive phenotype, yet the influence of age and sex on their transcriptional and metabolic states remain poorly understood. Here, we employed single-cell RNA sequencing of splenic leukocytes from young (3-4 months) and older (18-24 months) adult male and female mice subjected to a clinically relevant murine sepsis model to define age- and sex-specific MDSC phenotypes. We identified significant differences regarding age and sex in MDSC expansion, transcriptome, canonical pathway activation, RNA velocity, mitochondrial metabolism, and predicted cell-cell communication after sepsis. Using drug2cell analysis of total leukocytes we also identified cohort-specific drug target profiles. These findings underscore the importance of age and sex in shaping sepsis-induced MDSC biology and suggest that personalized immunomodulatory strategies targeting MDSCs could improve sepsis outcomes.

This study combines live pancreas tissue slices with viral transduction of the Calcium Modulated Photoactivatable Ratiometric Integrator 2 (CaMPARI2) biosensor for high-throughput analysis of islet calcium secretagogue responses. A key challenge of the pancreas slice model has been efficient transgene delivery throughout the slice volume while maintaining viability and function. Here, we demonstrate a robust adenoviral gene delivery approach to transduce slices with CaMPARI2 and apply photoconverting light to permanently mark glucose-induced calcium activity across all islets. This approach demonstrates glucose responsive CaMPARI2 labeling that correlates with insulin secretion. Using this novel high-throughput approach, we examine the relationship between islet size and calcium response. Larger isolated islets exhibit greater CaMPARI2 photoconversion in high glucose, whereas no size-function correlation is observed in islets resident in live slices. We also observe that slices capture a substantially higher proportion of small islets than isolated islets. Integrating CaMPARI2 with live pancreas slice studies enables multiplexed analyses, linking functional readouts to spatial features.

NADPH oxidase (NOX) family members are major resources of intracellular reactive oxygen species (ROS). In the immune system, ROS derived from phagocytic NOX (NOX2) participate in both pathogen clearance and signaling transduction. The role of NOX2 in neutrophils and macrophages has been well studied as mutations in NOX2 subunits cause chronic granulomas disease (CGD). NOX2 is expressed across a wide range of immune cells and recent reports have demonstrated that NOX2-derived ROS play important roles in other immune cells during an immune response. In this review, we summarize current knowledge of functions of NADPH oxidase 2 in each subset of leukocytes, as well as associations of NOX2 deficiency with diseases associated specifically with autoimmunity and immune deficiency. We also discuss important knowledge gaps as well as potential future directions for NOX2 research.

Introduction and Objective: The PD-1/PD-L1 immune checkpoint pathway plays a key role in preventing type 1 diabetes (T1D). Plasma EV data from autoantibody-positive (AAB+) patients suggests, PD-L1 level correlates with beta cell function and T1D progression. Thus, restoring PD-1/PD-L1 function is considered one potential strategy for treating T1D at different stages. Here, we outline the novel cytokine-induced PD-L1 regulation by the neuronal pentraxin (NPTX) proteins. NPTX protein functions are known in the brain, where they regulate synaptic plasticity by interacting with glutamate receptors. Recent studies have reported elevated levels of NPTX2 in a subtype beta cells population from AAB+ patients. However, their function in beta cell biology and T1D pathogenesis is little known. Methods: We have implemented molecular biology, mass spectrometry, and advanced high-throughput data analysis tools to outline NPTX protein function in EndoC-betaH1 human beta cells. Results: We observed that NPTXR knockdown significantly reduces glucose-stimulated insulin release by ~30% and critical insulin secretion machinery proteins, including glutamate receptor (GRIA4) and its binding partner, GRIP proteins. In cytokine-treated (IL1-beta and IFN-gamma) conditions, NPTXR is proapoptotic, and its knockdown further increases PD-L1 expression by ~64% while significantly suppressing ~88% of enriched pro-inflammatory pathways. NPTXR regulates PD-L1 expression via ERK/c-JUN signaling, an indicator of glutamine deprivation. Conclusion: Overall, our study elucidates a novel role for NPTXR in regulating beta cell insulin secretion and, in cases of inflammation, PD-L1 expression. Targeting NPTX signaling components holds potential as an early intervention target for T1D due to its association with AAB+ patients and its role in modulating beta cell fitness. S. Sarkar: None. W. Qian: None. R. Kulkarni: Advisory Panel; Novo Nordisk, Biomea Fusion, REDD Pharma, Inversago Pharma. Research Support; Inversago Pharma. Stock/Shareholder; Biomea Fusion. D.F. De Jesus: None. M. Campbell-Thompson: None. C.E. Mathews: None. M.A. Gritsenko: None. National Institutes of Health (5R01DK122160-04, R01DK067536, R01DK123329)

The loss of insulin secretory function associated with type 1 diabetes (T1D) is attributed to the immune-mediated destruction of beta cells. Yet, at onset of T1D, patients often retain a substantial beta cell mass, and T cell infiltration of pancreatic islets is typically sporadic. Here, we investigate the hypothesis that the remaining beta cells in T1D are dysfunctional, using live pancreas slices from organ donors recently diagnosed with T1D. Beta cells in slices from donors with T1D have significantly diminished Ca2+ mobilization and insulin secretion in response to glucose. Beta cell function is equally impaired in T-cell-infiltrated and non-infiltrated islets. Fixed tissue staining and gene expression profiling of laser-capture microdissected islets reveal significant reductions in proteins and genes involved in the glucose stimulus secretion coupling pathway. These findings support the notion that molecular changes evolve in beta cells during prediabetes and worsen to functional defects at human T1D diagnosis.

Severe burn injury induces long-lasting immune dysfunction, but the molecular mechanisms underlying this phenomenon remain unclear. We hypothesized that burn injury leads to epigenetic and transcriptional reprogramming of innate immune cells. Splenic F4/80⁺ macrophages were isolated from mice at days 2, 9, and 14 days post-20% contact burn injury. Targeted transcriptomics and MAPit single-molecule chromatin profiling were used to assess immune, metabolic, and epigenetic changes. Canonical pathway analysis was performed to infer functional shifts over time. Burn injury induced a biphasic response in macrophages. Early after injury (Day 2), there was broad transcriptional suppression and epigenetic silencing of inflammatory regulators, including Stat3, Traf6, and Nfkb1. Over time (Days 9 and 14), loci associated with anti-inflammatory mediators such as Il-10 and Socs3 exhibited progressive chromatin opening and transcriptional upregulation. Metabolic gene profiles revealed persistent suppression of mitochondrial and oxidative phosphorylation programs. Canonical pathway analysis demonstrated early IL-10 signaling activation with sustained suppression of classical macrophage activation pathways. Chromatin architecture changes included nucleosome sliding and ejection events, consistent with dynamic, locus-specific regulation. This work challenges the classical notion of burn-induced immune suppression as purely a consequence of systemic inflammation. Instead, we reveal a programmed and locus-specific epigenetic architecture that may shape macrophage immune and metabolic function long after the acute phase.

Introduction:Sepsis leads to expansion of myeloid-derived suppressor cells (MDSC) and their subtypes. These normally transitory MDSCs suppress T cell activation and alter T cell cytokine production while simultaneously promulgating systemic low-grade inflammation. Immune metabolism can shape cell responses, regulate immune suppression, and enhance effector activity. Although MDSC metabolism has been extensively studied in cancer, the metabolic phenotype of this heterogeneous population in sepsis remains unclear. Our goal was to assess metabolic flux in blood MDSCs during and after sepsis and to stratify these patients' clinical features and outcome with differences in metabolic flux that may guide treatment decisions. Methods:Peripheral blood mononuclear cells (PBMC) from healthy subjects and sepsis patients at 4 days, 2-3 weeks, and 6 months underwent CD66b+ or CD3+ enrichment, followed by assessment of metabolic flux, flow cytometry, mRNA sequencing, and chromatin accessibility. Results:Mitochondrial basal oxygen consumption rates (OCR) and maximal oxygen consumption rates (SRC, spare respiratory capacity) were decreased in MDSC from septic patients at 4 days after infection and persisted for up to 6 months after sepsis onset. Sepsis was not associated with differences in glycolysis. In contrast, oxidative metabolism in CD3+ T cells was similar between sepsis patients and healthy subjects. Reduced MDSC oxidative metabolism was linked to adverse clinical outcomes. The decline in oxygen consumption from MDSCs in septic patients was also associated with significant reductions in MDSC mitochondrial content. Transcriptomic analysis of CD66b+ cells isolated from PBMC of healthy participants and patients with sepsis at 4 days, 2-3 weeks, and 6 months revealed 19 differentially expressed genes and three long non-coding RNAs as potentially responsible for this decline in mitochondrial mass. Specifically, NR4A3, NR4A2, and TAMLIN/NR4A1 expression, all critical for mitochondrial biogenesis, were persistently decreased with reduced chromatin accessibility indicative of gene silencing. Discussion:After sepsis, blood CD66b+ cells present with reduced mitochondrial mass and oxidative metabolism that continue at least 6 months after sepsis. These changes in mitochondrial function result from a reduced content of these organelles. We have also identified gene silencing, reduced gene expression of key transcription factors that regulate mitochondrial biogenesis, as well as increased long non-coding RNA as potential drivers of this unique metabolic phenotype. These results highlight the potential benefit of targeting metabolism in sepsis to promote immune homeostasis and recovery.

Genome-wide association studies (GWAS) have linked dozens of genetic loci to type 1 diabetes (T1D). The IFIH1 gene, which encodes the double-stranded RNA sensor MDA5, is one such locus. The E627* single nucleotide polymorphism (SNP) in IFIH1 is associated with protection against T1D, while the A946T variant is linked to increased risk. While the E627* variant has been shown to result in a truncated protein and dampen type I interferon (IFN) signaling, its specific role in human pancreatic islet health and function remains unclear. We hypothesized that MDA5627* would protect islet cells from stress-induced dysfunction, identity loss, and cell death. Using CRISPR-Cas9 technology, we introduced the E627* and A946T variants into human pluripotent stem cells (hPSCs) derived from a T1D patient. We differentiated these hPSCs into stem cell-derived islets (SC-islets) and treated them with IFNα, poly(I:C), and coxsackievirus B3, an enterovirus implicated in T1D pathogenesis. Using single-cell RNA sequencing and an array of functional assays, we investigated the variant impact on both whole SC-islets and their individual cell populations. Our analysis revealed that SC-islets, and their β, α, and δ cell subpopulations, harboring the MDA5627* variant exhibit an attenuated immune response to the various stressors compared to MDA5946T cells. We also report unique, cell-type-specific transcriptional responses that vary across variants. Notably, MDA5627* SC-islets showed reduced apoptosis rates and viral genome expression, as well as attenuated negative effects on mitochondrial function and insulin secretion in response to stress. Overall, our findings demonstrate that a clinically relevant MDA5 variant confers protection by dampening stress-mediated transcriptional responses, reducing cell dysfunction, and preventing apoptosis. These insights provide a mechanistic framework for understanding T1D pathogenesis and offer new avenues for developing preventative therapies.

Progression to type 1 diabetes is associated with genetic factors, the presence of autoantibodies and a decline in beta cell insulin secretion in response to glucose. Very little is known regarding the molecular changes that occur in human insulin-secreting beta cells prior to the onset of type 1 diabetes. Herein, we applied an unbiased proteomics approach to identify changes in proteins and potential mechanisms of islet dysfunction in islet-autoantibody-positive organ donors with pre-symptomatic stage 1 type 1 diabetes (HbA1c ≤42 mmol/mol [6.0 https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp ) with accession no. MSV000090212.

Progressive β-cell dysfunction precedes the onset of type 1 diabetes (T1D), yet the molecular mechanisms driving early T1D development remain poorly understood. Although single-cell RNA-sequencing has uncovered transcript-level changes in human islet cells, it offers limited insight into the heterogeneity of distinct islet microenvironments. Here, we applied a single-islet proteomics workflow to profile intra-donor islet heterogeneity in three stage 1 T1D cases with matched non-diabetic controls and define in situ protein signatures of pseudo-temporal islet dysfunction. Intra-donor analyses of ~100 individual islets per donor revealed highly consistent proteomic patterns reflecting pseudo-time progression of islet immune responses and β-cell dysfunction. Several pathways, including extracellular matrix remodeling and mRNA processing, were identified as closely associated with progressive islet immune activation and loss of β-cell function. These findings provide robust proteome-wide evidence of the progression of islet dysfunction, offer a valuable resource for investigating early mechanisms of T1D pathogenesis-including novel candidates for functional studies-and underscore the utility of single-islet spatial proteomics for examining islet heterogeneity in T1D.

Introduction and Objective: Type 1 diabetes (T1D) is a polygenic, autoimmune disease characterized by pancreatic β-cell dysfunction and loss. We have recently demonstrated that β-cell dysfunction in T1D is independent of T cell infiltration into islets. To define β-cell changes in T1D, we employed an islet-centric approach to identify differentially expressed genes (DEGs) that change during T1D pathogenesis in insulin (INS)+ CD3- islets of at-risk cases. We hypothesize these DEGs contribute to β-cell failure. Methods: To nominate genetic drivers, human islet gene expression data from single AAb+, multiple AAb+ and T1D cases were integrated with the Diversity Outbred (DO) mice. This enabled us to identify strong cis-eQTL associated with alleles from the T1D-prone NOD mouse, one of eight founder strains of the DO. Results: We filtered 827 human DEGs to identify 142 DEGs with strong NOD-driven cis-eQTL. From those, 88 DEGs showed associations with diabetes in human genome-wide association studies. Remarkably, 49 cis-eQTL mapped within mouse diabetes susceptibility loci. Those genes were associated with transcription-translation (Gatc, Creb3, Fam133b, H1f3, Niban1, Smad4), glucose metabolism (Pgm1), mitochondrial function (Suclg2, Atp5e, Atp5g1, Pdhb, Acly, Coa5), insulin-granule biogenesis and exocytosis (Pcsk1, Rab2a, Cadps2. Atp2a2), and amino acid and metal transport and homeostasis (Slc36a4, Slc39a8, Slc4a7, Fth1). Conclusion: By leveraging genetics of DO mice, our functional genomics approach has identified genetic elements as specific genetic drivers of human T1D (in transcription/translation, glycolysis, mitochondria, and insulin secretion). By restricting the analysis to islets without immune infiltration these loci promote loss of β-cell function in the absence of T cell infiltration. A.E. Cuaycal: None. M. Keller: None. E.A. Butterworth: None. J. Chen: None. M. Campbell-Thompson: None. P. Smadbeck: None. J. Flannick: None. I.C. Gerling: None. C.E. Mathews: None. JDRF, NIH (P01 AI42288, UC4 DK104194, UC4 DK104167, UC4 DK104155)

Introduction & Objective: Reactive oxygen species (ROS), byproducts of cellular metabolism, are implicated in T1D progression, causing lipid, protein, and DNA damage, along with impaired β-cell function and apoptosis. Notably, at the onset of T1D, β-cells exhibit dysregulated glucose metabolism, resulting from decreased glucose uptake and impaired utilization. This metabolic perturbation diminishes NADPH production, a critical antioxidant cofactor from glycolysis and the pentose phosphate pathway (PPP). Consequently, islets in T1D individuals may exhibit compromised ROS-neutralizing abilities, heightening susceptibility to oxidative damage. The objective of this study is to determine glucose metabolism's role in β-cell antioxidant defense and T1D-related dysfunction. We hypothesize that insulitic islets in T1D are more vulnerable to damage from ROS compared to islets in individuals without diabetes and that diminished ROS detoxification contributes significantly to β-cell loss and dysfunction in T1D. Methods: Islets in live pancreatic tissue slices from both nondiabetic and recent-onset diabetic donors were acutely challenged with hydrogen peroxide (H2O2), and palmitate, followed by glucose stimulations. Real-time monitoring of ROS levels, utilizing ROS biosensor GRX1-roGFP2 and chemical ROS indicators, were conducted. Results & Conclusion: While it is generally the case that chronic hyperglycemia induces oxidative β-cell damage, our observations suggest that acute glucose stimulation reduces baseline ROS levels by promoting increased GSH in healthy β-cells. Following various ROS challenges including, high glucose stimulations quickly reverted their oxidative effects in healthy β-cells. These results support the concept that poor beta cell glucose responsiveness in T1D increases β-cell susceptibility to ROS. Disclosure C. Lazimi: None. C.E. Mathews: None. E. Phelps: Research Support; Immunocore, Ltd, MESO SCALE DIAGNOSTICS, LLC.