Biology subjects

Sai, S.

Publications and source records attributed to Sai, S..

9 recordsLinked to original sources

bioRxiv2026

Distinct macrophage and T cell programs shape pancreatic inflammation during metabolic stress and aging

Type 2 diabetes is linked to systemic inflammation driven by metabolic stress and aging. Although pancreatic inflammation associated with these factors is well documented, the dynamics of immune cell populations and their molecular changes remain poorly understood. We characterized immune cell alterations in the pancreas and pancreatic islets during Western diet (WD) feeding and aging using imaging mass cytometry (IMC) and single-cell RNA sequencing (scRNA-seq). Spatial and transcriptional analyses were performed to define immune cell subtype composition, activation states, and inferred cell-cell communication programs under metabolic and age-related stress conditions. Our analyses identified expansion of an F4/80low macrophage subtype and activated effector-like CD8+ T cells throughout the pancreas during WD feeding and aging. Within pancreatic islets, single-cell RNA sequencing identified a type I interferon-responsive macrophage population with low F4/80 expression that expanded during overnutrition. Notably, the type I interferon responses elicited by these stressors diverged: aging was associated with a more canonical type I interferon response, whereas overnutrition induced a broader response that included STAT3-associated transcriptional programs. We further provide evidence for enhanced cytokine-mediated communication between macrophages and a CD8+ cytotoxic T-cell population under overnutrition and aging. These findings show that metabolic stress and aging remodel pancreatic inflammation through overlapping but distinct immune mechanisms, involving expansion of F4/80low macrophages, activation of divergent type I interferon programs, and enhanced macrophage-CD8+ T-cell communication. Together, these findings suggest that distinct therapeutic approaches may be required to preserve islet function in type 2 diabetes driven by metabolic stress versus aging. Article HighlightsO_LIMetabolic stress and aging remodel pancreatic inflammation through overlapping but distinct immune mechanisms. C_LIO_LISpatial and single-cell analyses identified conserved remodeling of pancreatic macrophage populations accompanied by activation of cytotoxic T-cell responses during metabolic stress and aging. C_LIO_LIIn pancreatic islets, macrophages exhibited distinct type I interferon-associated transcriptional programs, with aging showing a more canonical interferon response and metabolic stress eliciting a broader inflammatory program that included STAT3-associated transcriptional signatures. C_LIO_LIThese findings provide a framework for understanding how metabolic stress and aging differentially shape pancreatic inflammation. C_LI

cell biology↗

bioRxiv2026

Workload-induced changes to cell state contribute to β-cell failure in diabetes

Insufficient insulin secretion relative to insulin demand is a key feature of type 2 diabetes (T2D). While the defects of insulin-producing {beta}-cells in T2D are well defined, little is known about how {beta}-cells progress from the functionally normal state to the decompensated state during the natural history of this disease. Here, we provide evidence that workload-induced {beta}-cell overstimulation precipitates {beta}-cell failure in T2D. We employ scRNA-seq to define workload-induced changes to {beta}-cell transcriptional states, identifying a novel compensating state that is distinct from the stressed state of decompensated {beta}-cells. We demonstrate a key role for the chromatin-modifying enzyme Lysine-specific demethylase 1 (Lsd1) in restraining workload-induced {beta}-cell state transitions, indicating epigenomic control of {beta}-cell state. Experimental manipulations that promote the compensating state accelerate {beta}-cell failure in mouse models of diabetes. Altogether, these findings show that the compensatory response of the {beta}-cell to increased workload becomes maladaptive over time and contributes to the pathogenesis of T2D.

physiology↗

bioRxiv2026

Single Cell Transcriptomics and Surface Protein Expression Reveal Distinct Cellular and Molecular Phenotypes in Human RPESC-RPE and PSC-RPE

Current retinal pigment epithelium (RPE) cell replacement strategies in trials for age-related macular degeneration (AMD) are based on either pluripotent stem cell (PSC) or adult RPE stem cell (RPESC) sources. We used Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) to simultaneously assess single-cell transcriptomic and surface protein information, comparing these two RPE sources. Both RPESC-RPE and PSC-RPE expressed key RPE markers and exhibited cellular heterogeneity. However, RPESC-RPE had higher expression of genes related to mature retinal functions, whereas PSC-RPE had greater expression of genes involved in stem cell development and differentiation. We identified two surface proteins that distinguished the cell types. The "dont eat me" signal, CD24, was detected robustly on adult RPESC-RPE cells, while CD57 was detected on most PSC-RPE cells. The differences in gene and surface protein expression suggest that the two RPE sources differ in functional, adhesion, and immunomodulatory properties, which may impact transplantation outcomes.

cell biology↗

bioRxiv2024

Metabolic control of adaptive β-cell proliferation by the protein deacetylase SIRT2

Selective and controlled expansion of endogenous {beta}-cells has been pursued as a potential therapy for diabetes. Ideally, such therapies would preserve feedback control of {beta}-cell proliferation to avoid excessive {beta}-cell expansion and an increased risk of hypoglycemia. Here, we identified a regulator of {beta}-cell proliferation whose inactivation results in controlled {beta}-cell expansion: the protein deacetylase Sirtuin 2 (SIRT2). Sirt2 deletion in {beta}-cells of mice increased {beta}-cell proliferation during hyperglycemia with little effect in homeostatic conditions, indicating preservation of feedback control of {beta}-cell mass. SIRT2 restrains proliferation of human islet {beta}-cells cultured in glucose concentrations above the glycemic set point, demonstrating conserved SIRT2 function. Analysis of acetylated proteins in islets treated with a SIRT2 inhibitor revealed that SIRT2 deacetylates enzymes involved in oxidative phosphorylation, dampening the adaptive increase in oxygen consumption during hyperglycemia. At the transcriptomic level, Sirt2 inactivation has context-dependent effects on {beta}-cells, with Sirt2 controlling how {beta}-cells interpret hyperglycemia as a stress. Finally, we provide proof-of-principle that systemic administration of a GLP1-coupled Sirt2-targeting antisense oligonucleotide achieves {beta}-cell selective Sirt2 inactivation and stimulates {beta}-cell proliferation under hyperglycemic conditions. Overall, these studies identify a therapeutic strategy for increasing {beta}-cell mass in diabetes without circumventing feedback control of {beta}-cell proliferation.

physiology↗

bioRxiv2023

In situ Patch-seq analysis of microglia reveals a lack of stress genes as found in FACS-isolated microglia

We applied the patch-seq technique to harvest transcripts from individual microglial cells from cortex, hippocampus and corpus callosum of acute brain slices from adult mice. After recording membrane currents with the patch-clamp technique, the cytoplasm was collected via the pipette and underwent adapted SMART-seq2 preparation with subsequent sequencing. On average, 4138 genes were detected in 113 cells from hippocampus, corpus callosum and cortex, including microglia markers such as Tmem119, P2ry12 and Siglec-H. Comparing our dataset to previously published single cell mRNA sequencing data from FACS-isolated microglia indicated that two clusters of cells were absent in our patch-seq dataset. Pathway analysis of marker genes in FACS-specific clusters revealed association with microglial activation and stress response. This indicates that under normal conditions microglia in situ lack transcripts associated with a stress-response, and that the microglia-isolation procedure by mechanical dissociation and FACS triggers the expression of genes related to activation and stress.

immunology↗

bioRxiv2022

Engineered vasculature induces functional maturation of pluripotent stem cell-derived islet organoids

Blood vessels play a critical role in pancreatic islet health and function, yet current culture methods to generate islet organoids from human pluripotent stem cells (SC-islets) lack a vascular component. Here, we engineered 3D vascularized SC-islet organoids by assembling SC-islet cells, human primary endothelial cells (ECs) and fibroblasts both in a non-perfused model and a microfluidic device with perfused vessels. Vasculature improved stimulus-dependent Ca2+ influx into SC-{beta}-cells, a hallmark of {beta}-cell function that is blunted in non-vascularized SC-islets. We show that an islet-like basement membrane is formed by vasculature and contributes to the functional improvement of SC-{beta}-cells. Furthermore, cell-cell communication networks based on scRNA-seq data predicted BMP2/4-BMPR2 signaling from ECs to SC-{beta}-cells. Correspondingly, BMP4 augmented the SC-{beta}-cell Ca2+ response and insulin secretion. These vascularized SC-islet models will enable further studies of crosstalk between {beta}-cells and ECs and can serve as in vivo-mimicking platforms for disease modeling and therapeutic testing.

cell biology↗

bioRxiv2022

Cell Atlas at Single-Nuclei Resolution of the Adult Human Adrenal Gland and Adrenocortical Adenomas

The human adrenal gland is a complex endocrine tissue. Developmental studies on this tissue have been limited to animal models or human foetus. Here, we present a cell atlas analysis of the adult human normal adrenal gland, combining single-nuclei RNA sequencing and spatial transcriptome data to reconstruct adrenal gland development and tumourigenesis. We identified two populations of potential progenitor cells resident within the adrenal cortex: adrenocortical progenitors NR2F2+-ID1+ cells, located within and underneath the capsule, and medullary progenitors SYT1+-CHGA- cells, located in islets in the subcapsular region. Using pseudotime analyses, we provided evidence of the centripetal nature of adrenocortical cell development and of the essential role played by the Wnt/{beta}-catenin pathway in the adrenocortical self-renewal. By comparing transcriptional profiles of cells of normal adrenal glands and adrenocortical adenomas we revealed a high heterogeneity with six adenoma-specific clusters. Overall, our results give insights into adrenal plasticity and mechanisms underlying adrenocortical tumourigenesis.

physiology↗

bioRxiv2021

TCAIM controls effector T cell generation by preventing Mitochondria-Endoplasmic Reticulum Contact Site-initiated Cholesterol Biosynthesis

T cells need to adapt their cellular metabolism for effector cell differentiation. This relies on alterations in mitochondrial physiology. Which signals and molecules regulate those alterations remains unclear. We recently reported, that the mitochondrial protein TCAIM inhibits activation-induced changes in mitochondrial morphology and function and thus, CD4+ effector T cell formation. Using conditional TCAIM knock-in (KI) and knockout (KO) mice, we now show that it also applies to CD8+ T cells and more importantly, delineate the molecular processes in mitochondria by which TCAIM controls effector cell differentiation. TCAIM KI resulted in reduced activation-induced HIF1 protein expression. Metabolomics and transcriptional data in combination with mathematical flux modeling revealed an impaired induction of anabolic pathways, especially of the mevalonate pathway and cholesterol biosynthesis in TCAIM KI CD8+ T cells. Addition of cholesterol completely rescued HIF1 protein expression, activation and proliferation of TCAIM KI CD8+ T cells. At the molecular level, TCAIM delayed activation-induced mitochondria-ER contact (MERC) formation by binding to MERC promoting proteins such as RMD3 and VDAC2. In summary, we demonstrate that TCAIM suppresses effector cell differentiation by inhibiting MERC formation, which induce HIF1-mediated increase in cellular metabolism and cholesterol biosynthesis.

immunology↗

bioRxiv2020

Dissecting HSV-1-induced host shut-off at RNA level

Herpes simplex virus 1 (HSV-1) installs a profound host shut-off during lytic infection. The virion host shut-off (vhs) protein plays a key role in this process by efficiently cleaving both host and viral mRNAs in a translation-initiation-dependent manner. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in transcriptional activity of the host genome. Both mechanisms have tremendous impact on the RNA expression profile of the infected cells. To dissect their relative contributions and elucidate gene-specific host transcriptional responses throughout the first 8h of lytic HSV-1 infection, we here employed RNA-seq of total, newly transcribed (4sU-labelled) and chromatin-associated RNA in wild-type (WT) and {Delta}vhs infection of primary human fibroblasts. Following virus entry, vhs activity rapidly plateaued at an elimination rate of around 30% of cellular mRNAs per hour until 8h p.i. In parallel, host transcriptional activity dropped down to 10-20%. While the combined effects of both phenomena dominated infection-induced changes in total RNA, extensive gene-specific transcriptional regulation was observable in chromatin-associated RNA. This was surprisingly concordant between WT HSV-1 and its {Delta}vhs mutant and at least in parts mediated by the embryonic transcription factor DUX4. Furthermore, both WT and {Delta}vhs infection induced strong transcriptional up-regulation of a small subset of genes. Most of these were either poorly or not at all expressed prior to infection but already primed by H3K4me3 histone marks at their promoters. Most interestingly, analysis of chromatin-associated RNA revealed vhs-nuclease-activity-dependent transcriptional down-regulation of at least 150 cellular genes, in particular of many genes encoding integrin adhesome and extracellular matrix components. This was accompanied by a vhs-dependent reduction in protein levels by 8h p.i. for many of these genes. In summary, our study provides a comprehensive picture of the molecular mechanisms that govern cellular RNA metabolism during the first 8h of lytic HSV-1 infection. Author SummaryThe HSV-1 virion host shut-off (vhs) protein efficiently cleaves both host and viral mRNAs in a translation-dependent manner. In this study, we model and quantify changes in vhs activity as well as the virus-induced global loss of host transcriptional activity during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were found to be predominantly shaped by these two global processes rather than gene-specific regulation. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and{Delta} vhs infection, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection and depicted vhs-dependent, transcriptional down-regulation of the integrin adhesome and extracellular matrix. The latter explained some of the gene-specific effects previously attributed to vhs-mediated mRNA degradation and resulted in a concordant loss in protein levels by 8h p.i. for many of the respective genes.

microbiology↗